Osteogenic mechanism of deciduous teeth periodontal ligament stem cells in inflammatory environment.

This study aimed to illustrate the biological behavior and changes in cell function during the progression of apical periodontitis in deciduous teeth and to explore the underlying molecular mechanism. Deciduous teeth periodontal ligament stem cells (DePDLSCs) were derived and their identity was confirmed. The viability, inflammation, and osteogenic ability of cells were tested by exposing them to various concentrations of lipopolysaccharide (LPS) (0-100 µg/mL) using the cell counting kit-8 (CCK-8) assay, reverse transcription polymerase chain reaction (real-time PCR), alkaline phosphatase (ALP) staining, and ALP activity assay. In addition, osteogenic-induced cells with and without 10 µg/mL LPS were harvested for high-throughput sequencing. Based on sequencing data, proinflammatory factors and ALP expression were measured after interference with the PI3K-AKT signaling pathway activator, 740Y-P. LPS biphasically affected the proliferation and osteogenesis of DePDLSCs. Low concentrations of LPS showed stimulatory effects, whereas inhibitory effects were observed at high concentrations. Sequencing analysis showed that the PI3K-AKT signaling pathway was significantly downregulated when DePDLSCs were treated with 10 µg/mL LPS. The LPS-induced inflammation and osteogenesis inhibition of DePDLSCs were partially rescued by 740Y-P treatment. In conclusion, LPS affected DePDLSCs proliferation and osteogenesis in a biphasic manner. Moderate activation of PI3K-AKT signaling pathway was beneficial for osteogenic differentiation and anti-inflammatory effect in DePDLSCs. This research may provide etiological probes for apical periodontitis and its treatment.

Osteogenic mechanism of deciduous teeth periodontal ligament stem cells in inflammatory environment

This study aimed to illustrate the biological behavior and changes in cell function during the progression of apical periodontitis in deciduous teeth and to explore the underlying molecular mechanism. Deciduous teeth periodontal ligament stem cells (DePDLSCs) were derived and their identity was confirmed. The viability, inflammation, and osteogenic ability of cells were tested by exposing them to various concentrations of lipopolysaccharide (LPS) (0-100 μg/mL) using the cell counting kit-8 (CCK-8) assay, reverse transcription polymerase chain reaction (real-time PCR), alkaline phosphatase (ALP) staining, and ALP activity assay. In addition, osteogenic-induced cells with and without 10 μg/mL LPS were harvested for high-throughput sequencing. Based on sequencing data, proinflammatory factors and ALP expression were measured after interference with the PI3K-AKT signaling pathway activator, 740Y-P. LPS biphasically affected the proliferation and osteogenesis of DePDLSCs. Low concentrations of LPS showed stimulatory effects, whereas inhibitory effects were observed at high concentrations. Sequencing analysis showed that the PI3K-AKT signaling pathway was significantly downregulated when DePDLSCs were treated with 10 μg/mL LPS. The LPS-induced inflammation and osteogenesis inhibition of DePDLSCs were partially rescued by 740Y-P treatment. In conclusion, LPS affected DePDLSCs proliferation and osteogenesis in a biphasic manner. Moderate activation of PI3K-AKT signaling pathway was beneficial for osteogenic differentiation and anti-inflammatory effect in DePDLSCs. This research may provide etiological probes for apical periodontitis and its treatment.

Knockdown of ZEB1 Inhibits Hypertrophic Scarring through Suppressing the Wnt/ß-catenin Signaling Pathway in a Mouse Model.

BACKGROUND: Hypertrophic scars (HS) cause functional impairment and cosmetic deformities following surgeries or burns (30% to 94%). There is no target therapy yet because the pathogenesis of HS progression is not well-known. In tissue fibrosis, Zinc finger E-box binding homeobox 1 (ZEB1) abnormal upregulation is an important cause for extracellular matrix (ECM) overexpression, which is the main molecular change in HS. Therefore, we hypothesized that ZEB1-knockdown inhibits HS formation. METHODS: ZEB1 expression in human HS and TGF-ß1-induced fibroblasts were identified by PCR and western blotting. ZEB1 was knockdown by siRNA in HS fibroblasts (HSFs) and mouse HS model (C57/BL6, male, 8-12 weeks). After 8-hour-transfection, HSFs were subjected to PCR, western blotting and CCK-8, apoptosis, migration and contraction assays. Mice HS were analyzed by HE staining, PCR and western blotting after 56 days. RESULTS: ZEB1 was upregulated in HS tissue (2.0-fold; p < 0.001). ZEB1 knockdown inhibited HSFs activity (0.6 to 0.7-fold; p < 0.001), the expression of fibrotic markers (0.4 to 0.6-fold; p < 0.001) and ß-catenin, cyclinD1 and c-Myc expression (0.5-fold; p < 0.001). In mouse HS models, HS skin thickness was thinner (1.60 ± 0.40 mm vs. 4.04 ± 0.36 mm; p < 0.001) after ZEB1 knockdown. CONCLUSIONS: Knockdown of ZEB1 inhibits HS formation both in vitro and in vivo. However, this is an in vitro/mouse model and more validation is needed. CLINICAL RELEVANCE STATEMENT: The discovery of ZEB1 as a mediator of HS formation might be a potential therapeutic target in HS treatment.

The risk of postoperative complications is higher in stage I-III colorectal cancer patients with previous abdominal surgery: a propensity score matching analysis.

PURPOSE: The aim of this study is to investigate whether previous abdominal surgery (PAS) affected stage I-III colorectal cancer (CRC) patients who underwent radical resection. METHODS: Stage I-III CRC patients who received surgery at a single clinical center from Jan 2014 to Dec 2022 were retrospectively included in this study. Baseline characteristics and short-term outcomes were compared between the PAS group and the non-PAS group. Univariate and multivariate logistic regression analyses were used to find risk factors for overall complications and major complications. A 1:1 ratio propensity score matching (PSM) was used to minimize the selection bias between the two groups. Statistical analysis was performed using SPSS (version 22.0) software. RESULTS: A total of 5895 stage I-III CRC patients were included according to the inclusion and exclusion criteria. The PAS group had 1336 (22.7%) patients, and the non-PAS group had 4559 (77.3%) patients. After the PSM, there were 1335 patients in each group, and no significant difference was found in all baseline characteristics between the two groups (P > 0.05). After comparing the short-term outcomes, the PAS group had a longer operation time (before PSM, P < 0.01; after PSM, P < 0.01) and more overall complications (before PSM, P = 0.027; after PSM, P = 0.022) whether before or after PSM. In univariate and multivariate logistic regression analyses, PAS was an independent risk factor for overall complications (univariate analysis, P = 0.022; multivariate analysis, P = 0.029) but not for major complications (univariate analysis, P = 0.688). CONCLUSION: Stage I-III CRC patients with PAS might experience longer operation time and have a higher risk of postoperative overall complications. However, it did not appear to significantly affect the major complications. Surgeons should take steps to improve surgical outcomes for patients with PAS.

Electrical stimulation enhances early palatal wound healing in mice.

BACKGROUND/OBJECTIVE: Electrical stimulation (ES) has been used to treat chronic wound and other clinical applications, showing favorable results in wound closure. It was hypothesized that ES can present a positive effect on oral mucosa healing. The aim of this study was to investigate the effects of ES during the palatal mucosa early healing process in Swiss mice. METHODS: Ninety animals were divided into two groups: Control (C; n = 45), which received Sham ES applications, and Test (ES; n = 45), which received ES (100 µA; 9 kHz; 660 mVpp) once a day for 3 days. A full thickness wound was performed with a 1.5 mm diameter biopsy punch in the hard palate. Histologically, the following parameters were evaluated: palatal wound closure and epithelial and connective wound edge distance (EED and CED). Furthermore, IL-1ß, IL-6, IL-10 TNF-α, and VEGF cytokine levels were evaluated by multiplex assay. The percentage of collagen fibers was assessed using the polarization method and the Smad proteins using the immunofluorescence method. RESULTS: Palatal wound closure presented a significant reduction on day 5 in the ES group (p = 0.01). Additionally, both EED and CED were shorter for all time points in the ES group (p < 0.05), and the inflammatory markers IL-6, IL-10, TNF-α, and VEGF were reduced (p < 0.05). There were no differences in collagen fibers and phospho-Smad2 between the groups. CONCLUSION: ES had a positive effect on early palatal wound closure outcomes, as well as on inflammatory markers.

Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers

BACKGROUND: Procambarus clarkii produces high-quality, delicious meat that is high in protein, low in fat, and rich in calcium and phosphorus. It has become an important aquatic resource in China. Our objectives are (i) to analyze the level of genetic diversity of P. clarkii populations; (ii) to explore the genetic differentiation (Gst); and (iii) to propose appropriate strategies for the conservation. RESULTS: In this study, Shannon's index (I) and Nei's gene diversity index (H) for P. clarkii were high (I = 0.3462 and H = 0.2325 on average and I = 0.6264, H = 0.4377 at the species level) based on the SSR markers. The expected heterozygosity value of 17 microsatellite loci in 25 crayfish populations was 0.9317, the observed heterozygosity value was 0.9121, and the observed number of alleles per locus was 2.000; and the effective number of alleles per locus was 1.8075. Among the P. clarkii populations, the inbreeding coefficient within populations (Fis) was 0.2315, overall inbreeding coefficient (Fit) was 0.4438, genetic differentiation coefficient among populations (Fst) was 0.3145 and gene differentiation (Gst) was 0.4785 based on SSR analyses. The cluster analysis results obtained by unweighted pair-group method with arithmetic mean (UPGMA) analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were similar. A mantel test showed that the isolation-by-distance pattern was not significant. CONCLUSIONS: The high Gst among P. clarkii populations is attributed to genetic drift and geographic isolation. The results indicated that more P. clarkii populations should be collected when formulating conservation and aquaculture strategies.

Worldwide tendency and perspectives in traumatic dental injuries: A bibliometric analysis over two decades (1999-2018).

BACKGROUND/AIMS: Traumatic dental injuries (TDIs) are considered to be a public dental health problem worldwide. The aim of the current study was to provide the worldwide tendency and perspectives in TDIs in the last two decades via bibliometric analysis. METHODS: ''Tooth injuries'' was searched as the Medical Subject Headings term within PubMed with the date range from 1999 to 2018. Two investigators perused information in the articles according to the inclusion and exclusion criteria. The articles were independently categorized according to the following aspects: (a) annual scholarly output; (b) leading countries or regions; (c) leading journals; (d) productive authors; (e) citations; (f) study design; (f) distribution of topics; and (g) the type of dentition and TDIs. VOSviewer 1.6.7 and Citespace 5.2 were used for analyzing and visualizing bibliometric networks. RESULTS: A total of 2627 articles about traumatic dental injuries were published and indexed in PubMed during the two decades, and the number of publications on traumatic dental injuries was rising in general. The research outputs were mainly concentrated in developed countries and affiliated hospitals of universities. Brazil was the most productive country. The journal Dental Traumatology had the most contributions to the scientific research of traumatic dental injuries. "Case report" was the most frequent type of article (36.50%), followed by cross-sectional studies (19.57%) and case-control studies (13.67%). Most studies focused on the treatment of TDIs (38.94%), especially for avulsion (21.01%), crown fracture (9.71%), and intrusion (5.25%). Permanent teeth (66%) were the dominant dentition. CONCLUSION: There is a lack of high-quality well-designed studies such as cohort studies. The number of publications on prevention and the primary dentition is disproportionate in relation to their significance.

Activated Plasmonic Nanoaggregates for Dark-Field in Situ Imaging for HER2 Protein Imaging on Cell Surfaces.

Dark-field microscopy (DFM) based on localized surface plasmon resonance (LSPR) was used for observation of experimental phenomena, which is a hopeful nondamaging and non-photobleaching biological imaging technique. In this strategy, plasma nanoaggregates with stronger scattering efficiency were formed in the presence of the target, causing a "turn-on" phenomenon, when asymmetry modified AuNPs were introduced as probes with zero LSPR background. First, Au1-N3 probe and Au2-C≡C probe were designed for the cycloaddition between azide and alkyne to form AuNP dimers under catalytic action by Cu+, which was obtained from the reduction of Cu2+ by sodium ascorbate. The two kinds of probes were successfully used for the detection of Cu2+ in rat serum. Then, to apply this concept to protein on cells, DNA and antibody were modified on the probes. DNA1/Au1-N3 probe and anti-HER2/Au2-C≡C probe were proposed for HER2 protein DFM on cells. By designing an aptamer sequence in primer, the rolling circle amplification (RCA) was introduced in HER2 DFM on cells, and the image signal was much brighter than that from no-RCA. The unique design made it easier to discriminate the target signal from background noise in cell DFM. This method might be used in the fields of molecular diagnostics and cell imaging.

Triptolide Reduces the Required Dose of Tacrolimus by Attenuating Inflammation, Enhancing Immunosuppression, and Increasing Donor Chimerism in a Heterotopic Hindlimb Transplantation Model.

BACKGROUND: Induction of tolerance and minimizing the toxicity of immunosuppression are two fundamental goals in vascularized composite allotransplantation. Accumulating data indicate that triptolide is an agent that may have the capacity to suppress inflammation and immunologic rejection. METHODS: A heterotopic hindlimb allotransplantation model from Brown Norway to Lewis rats was established and treated with different doses of tacrolimus combined with or without triptolide. Mean survival time of the transplants was monitored, and histopathologic examination of the skin was performed. The level of inflammatory cytokine interleukin-1ß, interleukin-6, and tumor necrosis factor-á in peripheral blood was assayed. The percentage of T lymphocytes and its subsets was measured using flow cytometry. The level of recipient peripheral chimerism and the apoptosis of donor bone marrow cells were evaluated. The apoptotic related genes bcl-2 and Bax were detected by real-time polymerase chain reaction. RESULTS: The authors' results showed that triptolide not only reduces the dose of tacrolimus required for immunosuppression, but also decreased drug side effects in terms of weight gain and diarrhea. Triptolide had an obvious effect on proinflammatory cytokine expression and T-lymphocyte proliferation in the peripheral blood. Interestingly, triptolide could increase the mixed chimerism level of recipients, possibly by inhibiting the apoptosis of transplanted bone marrow cells by means of regulation of the apoptotic genes bcl-2 and Bax. CONCLUSIONS: Triptolide reduces the dose of tacrolimus required for immunosuppression by attenuating inflammation and by T-cell suppression. Furthermore, triptolide increases the chimerism level, which might contribute to acceptance of the allografts.

MicroRNA-146b regulates hepatic stellate cell activation via targeting of KLF4.

 Background. We previously identified miR-146b as being up-regulated during the development of hepatic fibrosis using deep sequencing technology and gene expression analysis. However, the roles and related mechanisms of miR-146b in hepatic stellate cells (HSCs), which are involved in fibrogenesis and fibrosis, have not been elucidated. RESULTS: We report that miR-146b expression was increased in TGF-ß1-treated HSCs. TGF- ß1 enhanced α-SMA and COL1A1 protein expression in HSCs and stimulated proliferation of these cells compared with cells transfected with inhibitor NC. Conversely, miR-146b knock-down decreased α-SMA and COL1A1 expression and inhibited HSC proliferation. In addition, we found that miR-146b specifically regulated the translation of Krüppel-like factor 4 (KLF4) by targeting its 3' untranslated region. Forced expression of KLF4 inhibited TGF- ß1-induced enhancement of α-SMA and COL1A1 expression in HSCs, as well as proliferation of these cells. Moreover, miR-146b expression was negatively associated with KLF4 expression but positively associated with expression of α-SMA and COL1A1 during hepatic fibrosis. CONCLUSIONS: Our findings demonstrate the participation of miR-146b as a novel upstream effector of HSC activation via direct targeting of KLF4. Thus, targeted transfer of miR-146b into HSCs could be a useful strategy for the treatment of hepatic fibrosis.

Identification of reference genes for gene expression normalization in safflower (Carthamus tinctorius)

ABSTRACT Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant. Gene expression analysis is gaining importance in the research of safflower. Quantitative PCR has become a powerful method for gene study. Reference genes are one of the major qualification requirements of qPCR because they can reduce the variability. To identify the reference genes in safflower, nine candidate genes of the housekeeping genes were selected from the EST library of safflower constructed by our lab: CtACT (actin), CtGAPDH (glyceraldehyde 3-phosphate dehydrogenase), CtE1F4A (elongation factor 1 alpha), CtTUA (alpha-tubulin), CtTUB (beta-tubulin), CtPP2A (serine/threonine-protein phosphatase), CtE1F4A (eukaryotic initiation factor 4A), CtUBI (Ubiquitin), and Ct60S (60S acidic ribosomal protein). Expression stability was examined by qPCR across 54 samples, representing tissues at different flowering stages and two chemotype of safflower lines. We assessed the expression stability of these candidate genes by employing four different algorithms (geNorm, NormFinder, ΔCt approach, and BestKeeper) and found that CtUBI and Ct60S were the highly ranked candidate genes. CtUBI and Ct60S were used as reference genes to evaluate the expression of CtFAD2-10 and CtKASII. Our data suggest CtUBI and Ct60S could be used as internal controls to normalize gene expression in safflower.

Development and validation of an efficient real-time quantitative reverse transcription polymerase chain reaction assay of Chinese goose CD4 and CD8

Background: CD4+ T cells, which are often referred as T-helper cells, play a central role through secreting various cytokinesto enhance immune defense to pathogen. CD8+ T cells, which are called cytotoxic T lymphocytes (CTLs), provide potentdefenses against virus infection and intracellular pathogens by killing the targets cells directly. In our previous researches,the conventional and semi-quantitative PCR were used to detect the goose CD4 and CD8α. However, the semi-quantitativeRT-PCR only detect the relative amount of gene transcription. Quantitative PCR assay was more sensitive than conventionalPCR assay, and quantitative PCR assay has a lower limit of sensitivity.Materials, Methods & Results: Contrast to conventional assays, the detection of amplicons by quantitative RT-PCR couldbe visualized as the amplifi cation progressed. This effect has provided a great deal of insight into the kinetics of the reaction and it is the foundation of kinetic of real-time qPCR. The analysis of gene transcription by qPCR has proven to bean attractive method due to its potential for increasing laboratory throughput, simultaneous processing of several samplesas well as more reliable instrumentation. With those in mind, the real-time quantitative reverse transcription PCR (qRTPCR) methods for the detection of goose CD4 and CD8α transcripts were reported here for the fi rst time. With this assay,it is possible to carry out a rapid quantitative analysis of goose CD4 and CD8α transcripts over a wide linear range, withan unknown template.CD8 is expressed on the membrane of T cells either as an αα-homodimer or αβ-heterodimer. Sinceboth forms of CD8 have α chain, the transcription levels of CD8 can be monitored by detecting CD8α mRNA expression.Assays were based on the DNA sequence of goose CD4 [GenBank: JX902315], CD8α [GenBank: KC476104], and β-actin[GenBank: M26111]. qPCR was carried out in quadruplicates in a total volume of 20 µL containing...

Deep sequencing analysis of microRNA expression in porcine serum-induced hepatic fibrosis rats.

AIM: Recent studies have suggested miRNA dysregulation in liver tissue mediates the pathogenesis of various liver diseases especially liver fibrosis, but the microRNA changes during PS-induced hepatic fibrosis are still unknown. The purpose of this study was to screen the miRNA differences in rat liver fibrosis model and clarify the relationship of miRNAs with the development of PS-induced liver fibrosis. MATERIAL AND METHODS: Two fibrotic and two normal liver tissues from 20 Sprague-Dawley rats were collected and sequenced. MiRNA profiling results and fibrosis-related genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and bioinformatics was used to predict miRNA targets. RESULTS: In total, 48 miRNAs were detected to be aberrantly expressed in fibrosis tissue compared to normal tissue. Further functional analysis of the deregulated miRNA targets revealed the miRNAs are involved in several biological functions and pathways. In addition, the expression level of miR-27a and miR-146b and fibrosis-related genes were significantly up-regulated by using qRT-PCR in fibrotic liver tissues when compared to the normal liver tissues. CONCLUSION: PS-induced hepatic fibrosis results in up-regulation of the miR-27a and miR-146b in liver tissues, suggestingmiR-27a and miR-146b would be associated with the development of PS-induced liver fibrosis and be potential therapeutic targets during hepatic fibrosis.

Development and validation of an efficient real-time quantitative reverse transcription polymerase chain reaction assay of Chinese goose CD4 and CD8

Background: CD4+ T cells, which are often referred as T-helper cells, play a central role through secreting various cytokinesto enhance immune defense to pathogen. CD8+ T cells, which are called cytotoxic T lymphocytes (CTLs), provide potentdefenses against virus infection and intracellular pathogens by killing the targets cells directly. In our previous researches,the conventional and semi-quantitative PCR were used to detect the goose CD4 and CD8α. However, the semi-quantitativeRT-PCR only detect the relative amount of gene transcription. Quantitative PCR assay was more sensitive than conventionalPCR assay, and quantitative PCR assay has a lower limit of sensitivity.Materials, Methods & Results: Contrast to conventional assays, the detection of amplicons by quantitative RT-PCR couldbe visualized as the amplifi cation progressed. This effect has provided a great deal of insight into the kinetics of the reaction and it is the foundation of kinetic of real-time qPCR. The analysis of gene transcription by qPCR has proven to bean attractive method due to its potential for increasing laboratory throughput, simultaneous processing of several samplesas well as more reliable instrumentation. With those in mind, the real-time quantitative reverse transcription PCR (qRTPCR) methods for the detection of goose CD4 and CD8α transcripts were reported here for the fi rst time. With this assay,it is possible to carry out a rapid quantitative analysis of goose CD4 and CD8α transcripts over a wide linear range, withan unknown template.CD8 is expressed on the membrane of T cells either as an αα-homodimer or αβ-heterodimer. Sinceboth forms of CD8 have α chain, the transcription levels of CD8 can be monitored by detecting CD8α mRNA expression.Assays were based on the DNA sequence of goose CD4 [GenBank: JX902315], CD8α [GenBank: KC476104], and β-actin[GenBank: M26111]. qPCR was carried out in quadruplicates in a total volume of 20 µL containing...(AU)