Near telomere-to-telomere genome assemblies of two Chlorella species unveil the composition and evolution of centromeres in green algae.

BACKGROUND: Centromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown. RESULTS: We constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and Chlorella pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated their centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromeric CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements, although they differed significantly in their composition. Species within the Chlorella genus display a more diverse centromere composition, with major constituents including members of the LTR/Copia, LINE/L1, and LINE/RTEX families. This is in contrast to green algae including Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, in which centromere composition instead has a pronounced single-element composition. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeric sequence evolves more rapidly than sequence in non-centromeric regions. CONCLUSIONS: This study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.

Expert consensus on irrigation and intracanal medication in root canal therapy.

Chemical cleaning and disinfection are crucial steps for eliminating infection in root canal treatment. However, irrigant selection or irrigation procedures are far from clear. The vapor lock effect in the apical region has yet to be solved, impeding irrigation efficacy and resulting in residual infections and compromised treatment outcomes. Additionally, ambiguous clinical indications for root canal medication and non-standardized dressing protocols must be clarified. Inappropriate intracanal medication may present side effects and jeopardize the therapeutic outcomes. Indeed, clinicians have been aware of these concerns for years. Based on the current evidence of studies, this article reviews the properties of various irrigants and intracanal medicaments and elucidates their effectiveness and interactions. The evolution of different kinetic irrigation methods, their effects, limitations, the paradigm shift, current indications, and effective operational procedures regarding intracanal medication are also discussed. This expert consensus aims to establish the clinical operation guidelines for root canal irrigation and a position statement on intracanal medication, thus facilitating a better understanding of infection control, standardizing clinical practice, and ultimately improving the success of endodontic therapy.

Homologous proteins SAPCD2X1 and SAPCD2 have significantly different carcinogenic capacities in human colorectal cancer cells based on structural prediction and functional verification.

We discussed the expression and biological functions of the SAPCD2X1 protein in the HCT116 CRC cell line by bioinformatics analysis and prediction, and biological function verification. Spatial conformation models of SAPCD2X1 and SAPCD2 were predicted using the threading method, ensemble method, and several other protein structure prediction approaches. The conformational similarity between SAPCD2X1 and SAPCD2 was studied, and their functions were predicted. The biological experiments showed that SAPCD2X1 and SAPCD2 were overexpressed in CRC cells. SAPCD2X1-specific antibodies were prepared. The expressions of SAPCD2X1 and SAPCD2 were localized in cells using the immunofluorescence assay. The SAPCD2 and SAPCD2X1 overexpression models were validated using Western Blot and RT-qPCR. We successfully predicted the structures of the SAPCD2X1 and SAPCD2 proteins, and visualized them using the VDM software. It was predicted that the tertiary structure of SAPCD2X1 changed significantly compared with SAPCD2. Alteration of the biological functions of SAPCD2X1 was also predicted due to the changes in the spatial conformation of the protein. Anti-SAPCD2X1 antibody and SAPCD2X1-EGFP and SAPCD2-EGFP recombinant plasmids were established. The overexpression of the two proteins was induced in HCT116 cells using the recombinant plasmids, and verified by RT-qPCR and Western Blot. Meanwhile, the anti-SAPCD2X1 antibody was proved to have a high specificity. The immunofluorescence assay showed that SAPCD2X1 and SAPCD2 are mainly expressed in the cytoplasm. SAPCD2X1 and SAPCD2 exhibited significantly different biological functions in HCT116 cells. SAPCD2 is a carcinogenic protein, while SAPCD2X1 does not affect the proliferation, invasion, and migration of human CRC HCT116 cells.

The effect of myofascial therapy on postpartum rectus abdominis separation, low back and leg pain, pelvic floor dysfunction: A systematic review and meta-analysis.

BACKGROUND: During pregnancy and postpartum, changes in biomechanics can cause dysfunctions in the myofascial system, such as rectus abdominis diastasis, various types of pain, and pelvic floor dysfunction. These common postpartum problems seriously threaten women's health. Myofascial therapy, as an effective means of improving biomechanics, has no unified understanding of its therapeutic effects on postpartum functional disorders. This study aims to systematically evaluate the rehabilitative effects of myofascial therapy on postpartum rectus abdominis diastasis, low back and leg pain, and pelvic floor dysfunction through a meta-analysis of published randomized controlled trials. METHODS: A systematic literature search of databases in Chinese and English was performed through May 2023. The treatment methods were randomized controlled studies using myofascial therapy in the treatment of rectus abdominis separation, lumbo-leg pain, and pelvic floor dysfunction. The main outcome indicators were abdominal circumference, rectus abdominis separation distance, visual analogue pain score, pelvic floor muscle potential, ability to live daily activities, number of events, and treatment effectiveness. RESULTS: There were 22 studies, including 2235 patients. The result showed that compared with control group, myofascial therapy demonstrated to reduce abdominal circumference and rectus abdominis separation index, improve lumbar function significantly, and decrease urinary incontinence and pelvic organ prolapse. In the myofascial therapy group, pelvic floor muscle strength was significantly enhanced, anterior/posterior resting potential of pelvic floor muscle was significantly decreased, and pelvic floor muscle potential was enhanced. Compared with the control group, the number of patients with various types of pain and pain scores were significantly reduced after myofascial therapy. When myofascial therapy lasted <4 weeks, pain relief was greater. In the myofascial therapy group, the ability to perform daily activities was significantly improved. An analysis of the effectiveness of the treatment showed that after myofascial therapy, the patient's symptoms improved significantly. There also saw low heterogeneity among all outcomes. CONCLUSION: The results suggested that myofascial therapy could effectively reduce rectus abdominis separation, relieve pelvic floor muscle dysfunction, enhance lumbar function, relieve pain, and improve the ability of daily living activities. All the data demonstrated that myofascial therapy had a good therapeutic effect on postpartum dysfunction.

Identifying the Potential of miRNAs in Houttuynia cordata-Derived Exosome-Like Nanoparticles Against Respiratory RNA Viruses.

Introduction: Pathogenic respiratory RNA viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), and SARS-CoV-2, are major causes of causes of acute respiratory infection globally. Plant-derived exosome-like nanoparticles containing miRNAs have shown substantial cross-kingdom regulatory effects on both viral and human transcripts. Houttuynia cordata (H. cordata), a traditional Chinese medicine frequently used to treat respiratory diseases. However, the role of H. cordata-derived exosome-like nanoparticles (HELNs) and the miRNA they encapsulated are unclear. Methods: HELNs were isolated from fresh underground roots (uHELNs) and above ground stems and leaves (aHELNs) using differential centrifugation. The HELNs were identified using transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Small RNA sequencing and RT-PCR were employed to determine the miRNA expression in uHELNs and aHELNs. All genomes were sourced from the NCBI database. Target prediction of viral genomes was performed using RNAhybrid, while human target prediction was conducted using both RNAhybrid and Miranda. Functional enrichment analysis was applied to the predicted human targets to explore the hub targets and their roles in antiviral effects. The accessibility of miRNA target sites was determined through the MFOLD web server, and customized dual-luciferase reporter assays were administered to validate the computational findings. Results: A total of 12 highly enriched miRNAs were identified in both uHELNs and aHELNs. Upon prediction and verification, miR858a and miR858b were shown to target the NP gene in H1N1, while miR166a-3p targeted the ORF1ab in SARS-CoV-2. However, no valid miRNA targets were found for RSV. Regarding human transcripts, miR168a-3p, miR168b-3p, and miR8175 were found to inhibit MAPK3 expression, and novel_mir2 could suppress both AKT1 and MAPK3 expression. Discussion: This study sheds light on the collaborative antiviral mechanism of miRNAs in HELNs across two species and explores the potential antiviral scopes of both H. cordata miRNAs and HELNs.

Ginger: a representative material of herb-derived exosome-like nanoparticles.

Edible plant-derived exosome-like nanoparticles (PELNs) provide numerous benefits, including high yield, low cost, ethical compatibility, and multiple health benefits, which enable them to address technical constraints associated with mammalian nanoparticles. Herbs, known for their abundant bioactive components, are considered the primary source of natural medicines within the plant kingdom. Recently, a number of herbaceous sources have been investigated for the isolation and functionality of exosome-like nanoparticles (ELNs). However, they are commonly referred to as PELNs, and their distinct pharmacological properties are overlooked. In this review, these herb-derived ELNs are designated as HELNs, a novel herbal product that may also exhibit superior pharmacological activity compared to other types of PELNs. Among the documented HELNs, ginger-derived exosome-like nanoparticles (GELNs) are the most extensively studied. This review employs GELNs as an exemplar to delineate the process of extraction and purification, together with their physical and biochemical characteristics and therapeutic potential. The aim of this review is to promote the development and application of HELNs, and future research is encouraged to uncover their additional properties, extending beyond those of GELNs.

Effect of Various Lasers With or Without Systemp.desensitizer on Dentine Tubules: An In Vitro Scanning Electron Microscopy Analysis.

Objective: The current study was carried out to evaluate the effects of laser and Systemp.desensitizer therapy. Further, scanning electron microscopy (SEM) was used to determine the effects of individual or combined desensitizers on human dentinal tubules. Background: The most common clinical condition that makes people uncomfortable is dentin hypersensitivity (DH). Both lasers and drugs that reduce sensitivity have been used to treat DH. Materials and methods: A total of 100 dentinal samples were taken from newly extracted third molars (affected) and divided into 10 groups (A to J), that is, control (A); Systemp.desensitizer (B); diode laser (980 nm) (C); Nd:YAG laser (D); Er:YAG laser (E); Er,Cr:YSGG laser (F); Systemp.desensitizer + diode laser (G); Systemp.desensitizer + Nd:YAG laser (H); Systemp.desensitizer + Er:YAG laser (I); and Systemp.desensitizer + Er,Cr:YSGG laser (J). SEM was used to evaluate the dentinal specimens in each group (longitudinal and transverse portions), and then images of each sample were captured (20 images/sample). In addition, the number of open dentinal tubules was counted and then the occlusion depth in dentinal tubules was measured. Mann-Whitney and Kruskal-Wallis tests were employed to analyze the obtained data. Results: All treatment procedures and protocols were effective in blocking dentinal tubules (p < 0.05). Compared with the other groups, dentinal tubules in the laser and laser combination therapy groups were significantly obstructed (p < 0.05). Diode and Nd:YAG lasers with or without Systemp.desensitizer showed significantly more tubule occlusion and greater sealing depth than Er:YAG and Er,Cr:YSGG lasers with or without Systemp.desensitizer (p < 0.05). Conclusions: In summary, lasers alone or in combination can play a significant role in occluding the dentinal tubules. However, combining the diode or Nd:YAG laser with Systemp.desensitizers is a more effective treatment strategy and may have immediate and long-lasting effects.

The Generation and Application of Monoclonal Antibodies to Detect SAPCD2 Expression in Precancerous and Malignant Gastric Lesions.

Background: Suppressor APC domain containing 2 (SAPCD2) is involved in cell cycle regulation and its mRNA levels are higher in cancer tissues. But, the function of SAPCD2 in cancer development remains unclear. Objective: To generate mouse monoclonal antibodies (mAbs) specific to SAPCD2 and thus clarify the function of SAPCD2 in the development of gastric carcinoma (GC). Methods: Purified SAPCD2-GST immunized BALB/c mouse spleen cells were collected and fused with myeloma cells to obtain monoclonal antibody hybridoma. A group of monoclonal antibodies exhibiting high specificity and sensitivity against SAPCD2 has been generated and characterized by IHC, WB, IP, IF, and ELISA. By immunohistochemical analysis, the SAPCD2 expression was evaluated in 228 clinical samples of gastric mucosal lesions, including precancerous lesions and GC samples. Results: We identified a highly specific and sensitive clone of s12 in eukaryotic cells and performed an IHC analysis. We found that 81.3% of 107 GC tissues were SAPCD2-positive, compared with the 26.2% in the matched adjacent normal-appearing tissues (P<0.001). Furthermore, among the 121 gastritis tissues, SAPCD2 was overexpressed in precancerous gastric lesions such as dysplasia (Dys, 78.9%), intestinal metaplasia (IM, 44.7%), and chronic atrophic gastritis (CAG, 6.1%) compared with that in chronic non-atrophic gastritis (CNAG, 3.2%) (P<0.001). The SAPCD2-positivity rate was 81.3% in GC, suggesting that the expression of SAPCD2 increased with the severity of the lesion (P<0.001). Conclusion: In summary, we have described novel monoclonal antibodies against SAPCD2, which are highly expressed in GC tissues and may serve as the basis for an early clinical marker for GC development.

Selenium Biofortification Enhanced miR167a Expression in Broccoli Extracellular Vesicles Inducing Apoptosis in Human Pancreatic Cancer Cells by Targeting IRS1.

Purpose: Pancreatic adenocarcinoma (PAAD) presents an extremely high morbidity and mortality rate. Broccoli has excellent anti-cancer properties. However, the dosage and serious side effects still limit the application of broccoli and its derivatives for cancer therapy. Recently, extracellular vesicles (EVs) derived from plants are emerging as novel therapeutic agents. Thus, we conducted this study to determine the effectiveness of EVs isolated from Se-riched broccoli (Se-BDEVs) and conventional broccoli (cBDEVs) for the treatment of PAAD. Methods: In this study, we first isolated Se-BDEVs and cBDEVs by a differential centrifugation method, and characterized them by using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Then, miRNA-seq was combined with target genes prediction, and functional enrichment analysis to reveal the potential function of Se-BDEVs and cBDEVs. Finally, the functional verification was conducted in PANC-1 cells. Results: Se-BDEVs and cBDEVs exhibited similar characteristics in size and morphology. Subsequent miRNA-seq revealed the expression of miRNAs in Se-BDEVs and cBDEVs. Using a combination of miRNA target prediction and KEGG functional analysis, we found miRNAs in Se-BDEVs and cBDEVs may play an important role in treating pancreatic cancer. Indeed, our in vitro study showed that Se-BDEVs had greater anti-PAAD potency than cBDEVs due to increased bna-miR167a_R-2 (miR167a) expression. Transfection with miR167a mimics significantly induced apoptosis of PANC-1 cells. Mechanistically, further bioinformatics analysis showed that IRS1, which is involved in the PI3K-AKT pathway, is the key target gene of miR167a. Conclusion: This study highlights the role of miR167a transported by Se-BDEVs which could be a new tool for counteracting tumorigenesis.

Effect of full pulpotomy using a calcium silicate-based bioactive ceramic in adult permanent teeth with symptoms indicative of irreversible pulpitis: A retrospective study.

BACKGROUND: The authors studied the treatment effect of full pulpotomy using a calcium silicate-based bioactive ceramic in adult permanent teeth with symptoms indicative of irreversible pulpitis. METHODS: Eighty-one adult permanent teeth with symptoms indicative of irreversible pulpitis in 78 patients aged 18 through 72 years were evaluated for inclusion in the study. After caries excavation, the pulp was amputated to the level of the canal orifices. After hemostasis was achieved, calcium silicate-based bioactive ceramic was placed as the capping agent. The cavity was sealed temporarily with a glass ionomer cement and then restored with flowable resin and composite resin after 2 weeks if no positive symptoms were reported or detected. Postoperative evaluation was performed by means of clinical and radiographic examination at 2 weeks and 3, 6, and 12 months. RESULTS: Overall success rates of the procedure were 96.3% (78 of 81), 93.8% (76 of 81), 92.6% (75 of 81), and 92.6% (75 of 81) at the 2-week, 3-month, 6-month, and 12-month recall visits, respectively. Six of the 81 teeth failed and required root canal therapy. In these 6 teeth, 3 exhibited severe cold stimuli pain and spontaneous pain at the 2-week follow-up, 2 had no response to electric pulp testing with apical percussion pain and periapical rarefaction at the 3-month follow-up, and 1 tooth exhibited periapical rarefaction and labial mucosal fistula at the 6-month follow-up. CONCLUSIONS: Under the conditions of this study, full pulpotomy using a calcium silicate-based bioactive ceramic was a successful option for the treatment of adult permanent teeth with carious originated symptoms indicative of irreversible pulpitis. PRACTICAL IMPLICATIONS: Vital pulp therapy is no longer impossible for adult permanent teeth with carious originated symptoms indicative of irreversible pulpitis.

Interdisciplinary management of combined periodontal-endodontic lesions with palatogingival grooves of the maxillary lateral incisors: a case report.

A palatogingival groove of the maxillary lateral incisor is an anatomic malformation, which always predisposes the tooth to pulpal and periodontal disease. The diagnosis and treatment planning become complicated, with uncertain prognosis. Herein, we present an effective interdisciplinary management of a case of combined periodontal-endodontic lesions caused by palatogingival grooves. A series of treatment modalities were undertaken to preserve the two teeth, including root canal treatment, periodontal initial therapy, splinting the mobile teeth, occlusal adjustment, apical microsurgery, grinding and sealing grooves, and guided tissue regeneration. An apparent healing of the lesions was visible after 12 months. Therefore, interdisciplinary management of combined periodontal-endodontic lesions with palatogingival grooves of the maxillary lateral incisors is necessary for a favourable long-term outcome.

Transforming growth factor-ß1 promotes early odontoblastic differentiation of dental pulp stem cells via activating AKT, Erk1/2 and p38 MAPK pathways.

Background/purpose: TGF-ß1 (Transforming growth factor-ß1) plays an important role in the regeneration and repair of pulp-dentin complex. However, the biological function of TGF-ß1 on odontoblastic differentiation remains unclear, mainly due to the processes of differentiation were controlled by complex signaling pathways. This study aimed to investigate the signaling pathways involved in regulating the early differentiation of dental pulp stem cells (DPSCs) by TGF-ß1 and their functional role. Materials and methods: DPSCs were treated with 1 ng/mL TGF-ß1 and Western blotting was conducted to examine the activation of protein kinase B (AKT), small mothers against decapentaplegic 3 (Smad3), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2). DPSCs were exposed to mineralization medium contained TGF-ß1 with/without the specific signaling pathway inhibitors, and early odontogenic differentiation was evaluated by assessing the expression of alkaline phosphatase (ALP), collagen type 1 alpha 1 (COL1A), dentin matrix protein 1 (DMP-1) and runt-related transcription factor 2 (Runx2). Results: TGF-ß1 stimulated AKT, Smad3, p38 MAPK, Erk1/2 and JNK phosphorylation in DPSCs within 120 min. TGF-ß1 enhanced ALP activity and elevated levels of COL1A, DMP-1 and Runx2. LY294002, U0126 and SB203580 attenuated the effect of TGF-ß1 on DPSCs, however, the SIS3 and SP600125 treated groups had no significant effect. Conclusion: TGF-ß1 promotes the early stage of odontoblastic differentiation in DPSCs by activating AKT, Erk1/2 and p38 MAPK signaling pathways, but not by Smad3 and JNK.

Calculus Bovis Sativus alleviates estrogen cholestasis-induced gut and liver injury in rats by regulating inflammation, oxidative stress, apoptosis, and bile acid profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Natural Calculus Bovis (NCB) is a traditional Chinese medicine used for anti-inflammation, treating fever, pain, sedation, and recovering hepatobiliary function. Calculus Bovis Sativus (CBS), produced from in vitro artificial cultivation by bioengineering techniques, acts as an ideal substitute for NCB when treating various diseases. AIM OF THE STUDY: Gut-liver injury is an important pathological feature of several cholestatic liver diseases, including estrogen-induced cholestasis (EIC). The strong link between cholestatic liver injury and intestinal damage emphasizes the need of considering gut-liver integrity during treatment. The purpose of this study is to look into the pharmacological activities of CBS on EIC-induced gut and liver damage. MATERIALS AND METHODS: EIC-induced cholestatic rats were given oral gavage daily for five days with or without CBS (150 mg/kg). The liver/body weight, serum biochemistry, and tissue histopathology were then evaluated. Quantitative real-time PCR, Western blot analyses, and immunofluorescence were used to determine the gene expression associated with pathological alterations of the liver and intestine in EIC-induced cholestatic rats. Bile acid profiles within enterohepatic circulation were detected by liquid chromatography-mass spectrometry. RESULTS: CBS significantly reduced relative liver weight, restored serum biochemistry levels, and improved the hepatic and intestinal pathological damage in EIC model rats. CBS reduced EIC-induced hepatic inflammation by inactivation of the NF-κB signaling and inhibition of TNFα, IL-1ß, and IL-6 expression. CBS alleviated EIC-induced hepatic and intestinal oxidative stress by regulating Nrf2-GCLM/GCLC and Nrf2-HO-1 pathways, respectively. CBS treatment upregulated Bcl-2 and downregulated Bax and cleaved caspase3 to improve EIC-induced hepatic and intestinal cell apoptosis. Additionally, CBS reversed the disorders of bile acid profiles in the enterohepatic circulation by reducing bile acid accumulation in the liver and plasma and increasing bile excretion and intestinal reabsorption of bile acids. CONCLUSION: CBS alleviates EIC-induced hepatic and intestinal injury through regulating inflammation, oxidative stress, apoptosis, and bile acid profiles. These results suggest that CBS or drugs targeting the gut-liver axis may be effective therapeutic agents for cholestasis.

Stage-Dependent Regulation of Dental Pulp Stem Cell Odontogenic Differentiation by Transforming Growth Factor-ß1.

Transforming growth factor-ß1 (TGF-ß1) is an important multifunctional cytokine with dual effects on stem cell differentiation. However, the role of TGF-ß1 on odontogenic differentiation of dental pulp stem cells (DPSCs) remains to be entirely elucidated. In the present study, we initially investigated the effect of TGF-ß1 at a range of concentrations (0.1-5 ng/mL) on the proliferation, cell cycle, and apoptosis of DPSCs. Subsequently, to determine the effect of TGF-ß1 on odontogenic differentiation, alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining assays at different concentrations and time points were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were used to determine the levels of odonto-/osteo-genic differentiation-related gene and protein expression, respectively. For in vivo studies, newly formed tissue was assessed by Masson's trichrome and von Kossa staining. Data indicated that TGF-ß1 inhibited DPSCs proliferation in a concentration-and time-dependent manner (p < 0.05) and induced cell cycle arrest but did not affect apoptosis. ALP activity was enhanced, while ARS reduced gradually with increasing TGF-ß1 concentrations, accompanied by increased expression of early marker genes of odonto-/osteo-genic differentiation and decreased expression of late-stage mineralization marker genes (p < 0.05). ALP expression was elevated in the TGF-ß1-treatment group until 14 days, and the intensity of ARS staining was attenuated at days 14 and 21 (p < 0.05). Compared with the control group, abundant collagen but no mineralized tissues were observed in the TGF-ß1-treatment group in vivo. Overall, these findings indicate that TGF-ß1 promotes odontogenic differentiation of DPSCs at early-stage while inhibiting later-stage mineralization processes.

MicroRNA-22 suppresses NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokine production by targeting the HIF-1α and NLRP3 in human dental pulp fibroblasts.

AIM: To investigate the synergetic regulatory effect of miR-22 on HIF-1α and NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1ß and IL-18 in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis. METHODOLOGY: Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to determine the localization of miR-22-3p, NLRP3 and HIF-1α in human dental pulp tissues (HDPTs). The miR-22 mimics and inhibitor or plasmid of NLRP3 or HIF-1α were used to upregulate or downregulate miR-22 or NLRP3 or HIF-1α in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay were conducted to confirm target association. The mRNA and protein expression of HIF-1α, NLRP3, caspase-1, IL-1ß and IL-18 were determined by qRT-PCR and western blotting, respectively. The release of IL-1ß and IL-18 was analysed by ELISA. The significance of the differences between the experimental and control groups was determined by one-way analysis of variance, p < .05 indicated statistical significance. RESULTS: A decrease in miR-22 and an increase in HIF-1α and NLRP3 in HDPTs occurred during the transformation of reversible pulpitis into irreversible pulpitis compared with that in the healthy pulp tissues (p < .05). In the normal HDPTs, miR-22-3p was extensively expressed in dental pulp cells. HIF-1α and NLRP3 were mainly expressed in the odontoblasts and vascular endothelial cells. Whereas in the inflamed HDPTs, the odontoblast layers were disrupted. HDPFs were positive for miR-22-3p, HIF-1α and NLRP3. Computational prediction via TargetScan 5.1 and luciferase reporter assays confirmed that both NLRP3 and HIF-1α were direct targets of miR-22 in HDPFs. The miR-22 inhibitor further promoted the activation of NLRP3/CASP1 inflammasome pathway induced by ATP plus LPS and hypoxia (p < .05). In contrast, the miR-22 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS and hypoxia (p < .05). CONCLUSION: MiR-22, as a synergetic negative regulator, is involved in controlling the secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3 and HIF-1α. These results provide a novel function and mechanism of miR-22-HIF-1α-NLRP3 signalling in the control of proinflammatory cytokine secretion, thus indicating a potential therapeutic strategy for future endodontic treatment.

Amphiregulin regulates odontogenic differentiation of dental pulp stem cells by activation of mitogen-activated protein kinase and the phosphatidylinositol 3-kinase signaling pathways.

BACKGROUND: Human dental pulp stem cells (hDPSCs) have received widespread attention in the fields of tissue engineering and regenerative medicine. Although amphiregulin (AREG) has been shown to play a vital function in the biological processes of various cell types, its effects on DPSCs remain largely unknown. The aim of this study was to explore the specific role of AREG as a biologically active factor in the regeneration of dental pulp tissue. METHODS: The growth of hDPSCs, together with their proliferation and apoptosis, in response to AREG was examined by CCK-8 assay and flow cytometry. We explored the effects of AREG on osteo/odontogenic differentiation in vitro and investigated the regeneration and mineralization of hDPSCs in response to AREG in vivo. The effects of AREG gain- and loss-of-function on DPSC differentiation were investigated following transfection using overexpression plasmids and shRNA, respectively. The involvement of the mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the mineralization process and the expression of odontoblastic marker proteins after AREG induction were investigated by using Alizarin Red S staining and Western blotting, respectively. RESULTS: AREG (0.01-0.1 µg/mL) treatment of hDPSCs from 1 to 7 days increased hDPSCs growth and affected apoptosis minimally compared with negative controls. AREG exposure significantly promoted hDPSC differentiation, shown by increased mineralized nodule formation and the expression of odontoblastic marker protein expression. In vivo micro-CT imaging and quantitative analysis showed significantly greater formation of highly mineralized tissue in the 0.1 µg/mL AREG exposure group in DPSC/NF-gelatin-scaffold composites. AREG also promoted extracellular matrix production, with collagen fiber, mineralized matrix, and calcium salt deposition on the composites, as shown by H&E, Masson, and Von Kossa staining. Furthermore, AREG overexpression boosted hDPSC differentiation while AREG silencing inhibited it. During the differentiation of hDPSCs, AREG treatment led to phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and PI3K/Akt. Notably, a specific inhibitor of ERK, JNK, and PI3K/Akt signaling markedly reduced AREG-induced differentiation, as well as levels of phosphorylated ERK and JNK in hDPSCs. CONCLUSIONS: The data indicated that AREG promoted odontoblastic differentiation and facilitated regeneration and mineralization processes in hDPSCs.

One-Step Genotyping Method in loxP-Based Conditional Knockout Mice Generated by CRISPR-Cas9 Technology.

With the development of CRISPR-Cas9 gene editing and in vitro fertilization (IVF) technology, we can now easily construct genetically modified mouse strains with indels, especially for loxP-based strategy. However, the general genotyping methods are time-consuming and unreliable given the loxP site is only 34 bp long. Here, based on the tetra primer-paired PCR amplification, we describe an efficient genotyping method which can simultaneously generate the internal control band, wild type (wt)-genotype band, and/or loxP-genotype band through one single PCR amplification. It is easy to interpret the mouse genotypes from the pattern of the bands. Further, the results could also help to exclude the possibility of minor cross-contamination, since the ratio between the bands' quantity in wt/wt, wt/loxP, and loxP/loxP mice are relatively constant, which makes the genotyping more reliable when it is performed in a large amount.

Aloperine protects beta-cells against streptozocin-induced injury to attenuate diabetes by targeting NOS1.

Type 1 diabetes (T1D) is a metabolic dysfunction characterized by the selective destruction of islet ß-cells, with oxidative stress playing an essential role in the manifestation of this disease state. Aloperine (ALO) represents the main active alkaloid extracted from the traditional Chinese herbal Sophora alopecuroides L. and features outstanding antioxidative properties. In this study, T1D was induced by a single high dose streptozotocin (STZ, 150 mg/kg, intraperitoneal) in mice. Diabetic animals were intragastrically administered ALO at a dose of 50 mg/kg/day. Notably, treatment of ALO (50 mg/kg/day) for seven consecutive days could observably reverse the onset of diabetes induced by STZ accompanied by weight gain, lower blood glucose levels, and relief of ß-cells damage. Our in vitro study further demonstrated that ALO protected ß-cells from STZ/hydrogen peroxide-induced oxidative damage as manifested by increased expression of MnSOD and CAT. Furthermore, a network pharmacology study revealed that NOS1 represented the main target of ALO. Mechanistic studies subsequently showed that treatment of ALO increased the expression of NOS1, whereas NOS2 was decreased. Moreover, a docking study carried out suggested that ALO could fit into the binding pocket of human NOS1 and molecular dynamics simulation further validated this docking event. Collectively, the administration of ALO prior to diabetes could be a viable approach to the prevention of ß-cell injury. This study may offer a novel potential herbal medicine against T1D and may further help improve the understanding of the underlying molecular mechanisms of ALO-mediated protection against oxidative stress.

Effect of Various Laser-Activated Irrigation Protocols on Endodontic Sealer Penetration into the Dentinal Tubules: A Confocal Laser Scanning Microscopy Study.

Objective: To study the effect of various laser-activated irrigation protocols on endodontic sealer penetration into the dentinal tubules. Background data: Laser-activated irrigation is helpful for smear layer removal and thus might increase the penetration of endodontic sealer into the dentinal tubules. Methods: Forty-eight mandibular premolars with straight canals were instrumented and divided into four groups: C-I group, conventional needle irrigation with sodium hypochlorite (NaOCl) followed by ethylenediaminetetraacetic acid (EDTA); Er:YAG-I, Er,Cr:YSGG-I, and Nd:YAP-I groups, NaOCl and EDTA irrigations activated by erbium:yttrium-aluminum-garnet (Er:YAG), erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG), and neodymium:yttrium-aluminum-perovskite (Nd:YAP) lasers, respectively. The cleanness of the treated canals was evaluated by scanning electron microscopy (SEM). Then, canals were obturated with AH-Plus sealer labeled with rhodamine B (0.1%) and combined with gutta-percha. After setting, canals were sectioned at 2, 5, and 8 mm short from the root apex, and the penetration area of the AH-Plus into the dentinal tubules was observed using confocal laser scanning microscopy (CLSM). Results: SEM results indicated that the Er:YAG-I and Er,Cr:YSGG-I lasers were more effective than the Nd:YAP-I and C-I in removing the smear layer. CLSM results indicated that the average AH-Plus penetration areas among the tested groups were in ascending order: C-I < Nd:YAP-I < Er:YAG-I, Er,Cr:YSGG-I (p < 0.05). Conclusions: Laser-activated irrigations significantly increased the penetration of the endodontic sealer into the dentinal tubules, among which Er:YAG-I and Er,Cr:YSGG-I lasers were the most effective and may be considered routine protocols for endodontic treatment.

IL-6 downregulates hepatic carboxylesterases via NF-κB activation in dextran sulfate sodium-induced colitis.

Ulcerative colitis (UC) is associated with increased levels of inflammatory factors, which is attributed to the abnormal expression and activity of enzymes and transporters in the liver, affecting drug disposition in vivo. This study aimed to examine the impact of intestinal inflammation on the expression of hepatic carboxylesterases (CESs) in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Two major CESs isoforms, CES1 and CES2, were down-regulated, accompanied by decreases in hepatic microsomal metabolism of clopidogrel and irinotecan. Meanwhile, IL-6 levels significantly increased compared with other inflammatory factors in the livers of UC mice. In contrast, using IL-6 antibody simultaneously reversed the down-regulation of CES1, CES2, pregnane X receptor (PXR), and constitutive androstane receptor (CAR), as well as the nuclear translocation of NF-κB in the liver. We further confirmed that treatment with NF-κB inhibitor abolished IL-6-induced down-regulation of CES1, CES2, PXR, and CAR in vitro. Thus, it was concluded that IL-6 represses hepatic CESs via the NF-κB pathway in DSS-induced colitis. These findings indicate that caution should be exercised concerning the proper and safe use of therapeutic drugs in patients with UC.