Unraveling RNA contribution to the molecular origins of bacterial surface-enhanced Raman spectroscopy (SERS) signals.

Surface-enhanced Raman spectroscopy (SERS) is widely utilized in bacterial analyses, with the dominant SERS peaks attributed to purine metabolites released during sample preparation. Although adenosine triphosphate (ATP) and nucleic acids are potential molecular origins of these metabolites, research on their exact contributions remains limited. This study explored purine metabolite release from E. coli and RNA integrity following various sample preparation methods. Standard water washing generated dominant SERS signals within 10 s, a duration shorter than the anticipated RNA half-lives under starvation. Evaluating RNA integrity indicated that the most abundant ribosomal RNA species remained intact for hours post-washing, whereas messenger RNA and transfer RNA species degraded gradually. This suggests that bacterial SERS signatures observed after the typical washing step could originate from only a small fraction of endogenous purine-containing molecules. In contrast, acid depurination led to degradation of most RNA species, releasing about 40 times more purine derivatives than water washing. Mild heating also instigated the RNA degradation and released more purine derivatives than water washing. Notably, differences were also evident in the dominant SERS signals following these treatments. This work provides insights into SERS-based studies of purine metabolites released by bacteria and future development of methodologies.

[The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in human periodontal ligament stem cells].

PURPOSE: To explore the cytotoxic effect of a menthol-favored E-liquid on human periodontal ligament stem cells (hPDLSCs), as well as the underlying mechanism of electronic cigarette (E-cig)-induced cell apoptosis. METHODS: PDLSCs were isolated and cultured from periodontal ligament tissues of healthy premolars extracted for orthodontic reasons. Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. Then the cells were exposed to different doses of menthol-favored E-liquid (at 59 mg/L nicotine concentration) in the culture median (the final nicotine concentrations were 0.1 µg/mL, 1.0 µg/mL, 10 µg/mL, 50 µg/mL, 0.1 mg/mL, 0.2 mg/mL and 0.5 mg/mL, respectively) for different period of times (24, 48 and 72 h). The cell viability was analyzed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry (7-AAD and Annexin V staining) and TUNEL assay. Reactive oxygen species (ROS) production was detected with fluorescence probe DCFH-DA by confocal microscopy and flow cytometry. The protein expression levels associated with ROS/JNK/caspase 3 axis(p-JNK, JNK, c-Jun, p-c-Jun, Bcl-2, Bax and cleaved-caspase 3) were analyzed by Western blot. Immunocytofluorescense staining was applied to evaluate the expression level of p-JNK. After addition of NAC, a ROS scavenger, and MAPK/JNK specific blocker SP600125, their effects on E-cig-induced cell apoptosis were evaluated. Statistical analysis was performed with Graph Pad 5.0 software package. RESULTS: Human PDLSCs were successfully isolated and cultured and flow cytometry assay showed the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. CCK8 assay indicated cell viability was significantly(P＜0.001) different among all concentration groups at various time points (24, 48 or 72 h), and the difference in apoptosis rate among all concentration groups was also statistically significant (P＜0.001). After exposure to E-liquid with nicotine concentration ≥50 µg/mL, cell viability was significantly reduced, and the proportion of apoptotic cells and the cellular ROS level was significantly increased in a dose-dependent manner as compared with the control group(0.0 mg/mL). Western blot assay showed E-cig exposure could promote MAPK/JNK phosphorylation in a dose-dependent and time-dependent manner. Either NAC or SP600125 could partially rescue the E-cig-induced cell apoptosis via reversing up-regulation of p-JNK and cleaved caspase 3. CONCLUSIONS: ROS/JNK/caspase 3 axis is involved in menthol-favored E-liquid-induced apoptosis of hPDLSCs.

Micro-computed tomographic evaluation of the shaping ability of three nickel-titanium rotary systems in the middle mesial canal of mandibular first molars: an ex vivo study based on 3D printed tooth replicas.

BACKGROUND: The preparation of the middle mesial (MM) canal of mandibular molars represents a challenge because it is often curved, narrow, and close to the root concave. The purpose of this study was to evaluate the ex vivo shaping ability of 3 nickel-titanium (NiTi) rotary systems in the MM canal using 3D printed resin tooth replicas. METHODS: A permanent mandibular first molar with a MM canal was acquired from a pool of extracted teeth and reproduced by a 3D printer. The resin tooth replicas (n = 18) were equally assigned to 3 groups for the evaluation of the shaping abilities of 3 NiTi rotary systems (OneShape [OS], Twisted Files [TF], and ProTaper Gold [PTG]) according to the manufacturer's recommendations. The tooth replicas were scanned by micro-computed tomography (micro-CT) twice before and after instrumentation of the mesiobuccal (MB), mesiolingual (ML), and MM root canals. After 3D reconstruction, the canal straightening, change of root canal volume and surface area, the mesial and distal canal wall thickness and canal transportation at the levels of 1, 2, and 3 mm below furcation were assessed. One-way variance analysis and Turkey's post hoc test were used for comparisons of the means among different groups, and paired-t test was used to compare the mesial and distal sides of the mesial roots. RESULTS: As compared with OS and TF, the use of PTG in preparation of MM canals resulted in significantly more straightening of canal curvature (p < 0.05), greater post-instrumentation canal volume and surface area, and thinner mesial and distal remaining canal wall thickness at 1, 2 and 3 mm below furcation (all p < 0.05). Regarding the root canal transportation in the mesiodistal direction, there was no significant difference among the 3 instruments (all p > 0.05) after the preparation of the MB and ML canals. However, in the MM canal, more pronounced transportation was detected in the PTG group at 2 mm below furcation, and in the TF group at 3 mm below furcation as compared with the other 2 systems (both p < 0.05). CONCLUSIONS: 3D printed tooth replicas have the advantages of consistency and can be an ideal model to evaluate the shaping ability of different instruments in the MM canal. OS and TF files performed similarly and both are appropriate for shaping the MM canal, while PTG may cause excessive and uneven resin removal, especially near the furcation, and may lead to root fragility and procedural errors.

Rapid detection of nicotine and benzoic acid in e-liquids with surface-enhanced Raman scattering and artificial intelligence-assisted spectrum interpretation.

Electronic cigarettes have rapidly gained acceptance recently. Nicotine-containing electronic cigarette liquids (e-liquids) are prohibited in some countries, but are permitted and simply available online in others. A rapid detection method is therefore required for on-site inspection or screening of a large amount of samples. Our previous study demonstrated a surface-enhanced Raman scattering (SERS)-based approach to identify nicotine-containing e-liquids; without any pre-treatment, e-liquid can be directly tested on our solid-phase SERS substrates, made of silver nanoparticle arrays embedded in anodic aluminium oxide nanochannels (Ag/AAO). However, this approach required manual determination of spectral signatures and negative samples should be validated in the second round detection. Here, after examining 406 commercial e-liquids, we refined this approach by developing artificial intelligence (AI)-assisted spectrum interpretations. We also found that nicotine and benzoic acid can be simultaneously detected in our platform. This increased test sensitivity because benzoic acid is usually used in nicotine salts. Around 64% of nicotine-positive samples in this study showed both signatures. Using either cutoffs of nicotine and benzoic acid peak intensities or a machine learning model based on the CatBoost algorithm, over 90% of tested samples can be correctly discriminated with only one round of SERS measurement. False negative and false positive rates were 2.5-4.4% and 4.4-8.9%, respectively, depending on the interpretation method and thresholds applied. The new approach takes only 1 microliter of sample and can be performed in 1-2 min, suitable for on-site inspection with portable Raman detectors. It could also be a complementary platform to reduce samples that need to be analyzed in the central labs and has the potential to identify other prohibited additives.

Transcriptome characterization of human gingival mesenchymal and periodontal ligament stem cells in response to electronic-cigarettes.

The potential toxicities and threats of electronic cigarettes (E-cigs) on periodontal health remain elusive. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs) contribute to cell differentiation and regeneration for periodontium as well as inflammatory modulation. However, the effects of E-cig exposure on periodontal tissues, particularly GMSCs and PDLSCs, and the underlying epigenetic mechanisms remain largely unknown. In this study, we conducted RNA-seq analysis to examine the transcriptome of human GMSCs and PDLSCs exposed to four types of E-cigs (aerosol and liquid with tobacco and menthol flavor) and conventional tobacco smoke in vitro. Our results showed that E-cig exposure primarily impacted the immunoregulation and inflammatory responses to pathogenic microorganisms in GMSCs, and the microenvironment, differentiation and response to corticosteroid in PDLSCs, which were significantly different from the damage effects caused by tobacco smoke. Additionally, we discovered a large number of differentially expressed non-coding RNAs among the different E-cig exposure methods and flavors. We also noticed that in GMSCs, CXCL2 was especially down-regulated by E-cig aerosol exposure whereas up-regulated by E-liquid exposure compared to control. Of note, the enhancer elements near CXCL2 and other genes located at Chromosome 4 contributed to the transcription activity of these genes, and KDM6B was remarkably elevated in response to E-liquid exposure. Lastly, we conducted ChIP-seq analysis to confirm that the elevated gene transcription by E-liquids was due to the weakened H3K27me3 at genome-wide enhancer elements in GMSCs, but not at promoter regions. Taken together, our results characterized the diverse gene expression profiles of GMSCs and PDLSCs in response to E-cigs with different exposure methods and flavors in vitro, and indicated a novel mechanism of KDM6B-mediated H3K27me3 on enhancers for gene transcription regulation. Our data could be served as a resource for emphasizing the understanding of E-cigs in periodontal health.

Genomic distribution of signal transducer and activator of transcription (STAT) family in colorectal cancer.

JAK/STAT pathway has been widely acknowledged in the development of human cancers. However, the role of different phosphorylated STAT proteins translocating into nucleus in transcription activation of target genes is not fully understood. In present research, ChIP-seq was carried on to investigate the genome-wide distribution of the activated STAT1, STAT2, STAT3, STAT5 and STAT6 in colorectal cancer HCT-116 cells. Our observations indicated that the homodimers rather than heterodimers of STAT protein predominantly occupied on genomic DNA. STAT3 accounted for the largest proportion among all STAT proteins HCT-116 cells. Furthermore, the biased binding motif targeted by different STAT homodimers suggested the distinct biological functions. Here, we noticed that NR5A2 was a specific co-activator of STAT3 by DNA motif analysis. Co-IP assay determined that NR5A2 indeed interacted with STAT3 homodimer rather than other homodimers or heterodimers. NR5A2 knockdown resulted in a reduced binding affinity of STAT3 homodimer in the original regions. Taken together, we characterize the genome-wide landscape of activated STAT proteins, and reveal the differences of binding patterns as well as the target genes and associated functions between homodimer and heterodimer of STAT proteins in HCT-116 cells. We also present some new findings and possible mechanisms regarding the role of NR5A2 on STAT3 in CRC. Our findings may provide new insights into the design of STAT inhibitors to treat CRC and other diseases.

Finding appropriate nickel-titanium instruments for lingual canals in mandibular first premolars with two canals: A micro-CT study.

This study aimed to find appropriate nickel-titanium instruments for lingual canals in mandibular first premolars with two canals. Forty-two extracted mandibular first premolars with lingual canals (Vertucci type V) verified by micro-CT scanning were selected. The teeth were randomly divided into three groups, and their lingual canals were instrumented by M3, HyFlex CM and XP-endo Shaper, respectively. After instrumentation, the canal morphology was scanned again by micro-CT. The canal morphologies of pre- and post-instrumentation were reconstructed and aligned. Morphological changes of the lingual canals were evaluated. No instrument breakages occurred during the procedure of root canal instrumentation. HyFlex CM and XP-endo Shaper performed better than M3 files in preparation of lingual canals (Vertucci type V) of mandibular first premolars in terms of apical transportation and unprepared surface area.

Analysis of second mesiobuccal root canal instrumentation in maxillary first molars with three nickel-titanium rotary instruments: a micro-computed tomographic study.

The aim of this study was to evaluate the shaping characteristics of Protaper Universal (PTU; Dentsply Tulsa Dental Specialties, Johnson City, TN), Hero Shaper (HS; MicroMega, Besacon, France) and Hyflex CM (HCM; Coltene-Whaledent, Allstetten, Switzerland) nickel-titanium systems with various apical sizes and tapers in second mesiobuccal (MB2) canal instrumentation using micro-computed tomographic imaging. A total of 27 maxillary first molars with independent patent MB2 canals were selected and randomly assigned to three groups according to the 3-dimensional morphologic aspects obtained from preoperative micro-computed tomographic scans. Canals were first negotiated with a size 8 K-file and finally prepared to F1, F2, and F3 with PTU and to sizes 20.04 taper, 25.04 taper, and 30.04 taper with HS and HCM. Postoperative scans were performed after each instrumentation with the same parameters used in the initial scan. The canal volume, canal transportation, untouched canal surface and wall thickness were measured and calculated using Mimics 10.01 software (Image Works, Materialise, Belgium). Statistical analysis was performed using one-way analysis of variance post hoc LSD tests. PTU removed more dentin than HS and HCM in all sections when instrumented to the same apical size (P < 0.05). HS and HCM presented a lower mean value of canal transportation than PTU in all measured sizes and sections. PTU presented a lower mean value of distal wall thickness than HS and HCM at the level of 1 and 2 mm below the furcation region in all measured sizes. In conclusion, for MB2 canal instrumentation, HS and HCM of 0.04 taper are safer than PTU.

Rapid identification of nicotine in electronic cigarette liquids based on surface-enhanced Raman scattering.

Nicotine-containing electronic cigarette liquid (e-liquid) is prohibited in many countries, creating requirements for rapid detection approaches for on-site inspection or screening for large amounts of samples. Here, we demonstrate a simple way to identify nicotine using surface-enhanced Raman scattering (SERS) with substrates made of silver nanoparticle arrays imbedded in anodic aluminum oxide nanochannels (Ag/AAO). Compared with the reported colloidal nanoparticle-based SERS, that required serial dilutions to enable colloid aggregation in the viscous e-liquid, a small amount of undiluted e-liquid sample can be directly added onto our solid-phase Ag/AAO substrate without any pre-treatment. The sensitivity of our SERS measurements is 2-3 orders of magnitude higher than that required for identification of nicotine in e-liquid, which is typically around 1000-18,000 ppm. Using such nanoparticle array-based SERS, we have tested 22 commercially available e-liquid products, using the corresponding gas chromatography-mass spectrometry (GC-MS) reports as the reference. The SERS measurements were done within one hour and successfully identified 20 samples. Only 2 samples showed SERS interference from ingredients that were not suitable for SERS analysis.

Oestrogen receptor α regulates the odonto/osteogenic differentiation of stem cells from apical papilla via ERK and JNK MAPK pathways.

OBJECTIVES: Oestrogen receptor (ER) is a common nucleus receptor that is essential for the regulation of cell growth, proliferation and differentiation. This study was to examine whether ERα can affect the proliferation and odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs). MATERIALS AND METHODS: Stem cells from apical papillas were isolated, purified and then transfected with ERα lentiviruses. The proliferation capacity was investigated by cell counting kit-8 (CCK-8) assay and flow cytometry. The odonto/osteogenic differentiation ability was analysed by alkaline phosphatase (ALP) activity, alizarin red staining, western blot assay (WB) and real-time RT-PCR. MAPK pathway and its downstream transcriptional factors were explored by WB assay. RESULTS: As indicated by CCK-8 assay and flow cytometry, ERα had no significant effect on the proliferation of SCAPs. When ERα was overexpressed, the ALP activity and the formation of calcified nodules were significantly enhanced in SCAPs. Moreover, the odonto/osteogenic markers (DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OCN/OCN) in SCAPs were significantly up-regulated at both mRNA and protein levels. On the contrary, the odonto/osteogenic differentiation ability of SCAPs was remarkably inhibited after suppression of ERα. Mechanistically, the protein levels of phosphorylated ERK and JNK significantly increased after ERα overexpression. Moreover, some downstream transcriptional factors of MAPK pathway were simultaneously activated by ERα overexpression. CONCLUSIONS: Together, the data accumulated here indicated that ERα can enhance the odonto/osteogenic differentiation of SCAPs via ERK and JNK MAPK pathways.

Receptor-Interacting Protein 3/Caspase-8 May Regulate Inflammatory Response and Promote Tissue Regeneration in the Periodontal Microenvironment.

BACKGROUND Periodontal ligament stem cells (PDLSCs) possess characteristics of multi-potential differentiation and immuno-modulation, and PDLSCs-mediated periodontal tissue regeneration is regarded as a hopeful method for periodontitis treatment. Recent studies demonstrated that RIP3 and caspase8 regulate bacteria-induced innate immune response and programmed necrosis, which is also called necroptosis. This study aimed to determine the role of the RIP3/Caspase8 signal pathway on necroptosis of PDLSCs under the inflammatory microenvironment, both [i]in vitro[/i] and [i]in vivo[/i]. MATERIAL AND METHODS PDLSCs were cultured, and transmission electron microscopy and flow cytometry were used to detect necroptosis. PCR, ALP, and Alizarin Red S staining were used to assess the effect of necroptosis on osteogenesis differentiation of PDLSCs [i]in vitro[/i], while HE and Masson staining were taken after the nude mouse subcutaneous transplant experiment. RESULTS Our research indicates that RIP3/caspase8 can regulate the immune response of PDLSCs, and blockade of RIP3/caspase8 can protect the biological characteristics of the PDLSCs, effectively promoting periodontal tissue regeneration in the inflammatory microenvironment. CONCLUSIONS Inhibiting RIP3/caspase8 can effectively promote periodontal tissue regeneration in the inflammatory microenvironment.

Enhancement of NK cells proliferation and function by Shikonin.

CONTEXT: Shikonin is a kind of naphthoquinone compound found mainly in Lithospermum erythrorhizon Sieb,et Zucc. Previous studies have shown that Shikonin has anti-tumor, anti-inflammatory and extensive pharmacological effects. According to new studies, Shikonin could also modulate the immune system function, but the effect to NK (nature killer) cells is yet unknown. OBJECTIVE: To investigate the effect and mechanism of Shikonin on NK cells proliferation and cytotoxicity to colon cancer cell line (Caco-2). METHODS: The proliferation and cytotoxicity of NK cells cultured with Shikonin were detected with CCK-8 assay. The expressions of perforin, GranB and IFN-γ were examined with FCM. The content of TNF-alpha was disclosed with ELISA kit. p-ERK1/2 and p-Akt expression of NK cells were detected with western blot. RESULTS: With CCK-8 assay, it is found that Shikonin could significantly enhance NK cells proliferation and cytotoxicity to colon cancer cells. With FCM assay, it is found that Shikonin could improve the expression of perforin and GranB in a dose-dependent manner. Shikonin had no effect on TNF-alpha and IFN-γ expression. In mechanism, the study shows that Shikonin could enhance the expression of p-ERK1/2 and p-Akt. CONCLUSIONS: Shikonin enhances NK cells proliferation and cytotoxicity via the improvement of perforin, GranB, p-ERK1/2 and p-Akt expression.

Transplantation of gingiva-derived mesenchymal stem cells ameliorates collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic, progressive, and inflammatory autoimmune disease which primarily affects the small arthrodial joints. The aim of this study was to test whether transplantation of mesenchymal stem cells derived from gingival tissue (GMSCs) could ameliorate collagen-induced arthritis (CIA), and to explore the role of the FasL/Fas pathway in the underlying mechanism. METHODS: DBA/1 mice with collagen II-induced arthritis were treated with GMSCs from the C57BL/6 J mouse, the B6Smn.C3-FasLgld/J mouse (FasL-/- GMSCs), and FasL overexpressed FasL-/- GMSCs (FasL TF GMSCs). Inflammation was evaluated by measuring clinical score, tumor necrosis factor (TNF)-α and anti-collagen II antibody levels, and histological analyses. The levels of CD4+ Th cell subsets in spleens and draining lymph nodes were assessed by flow cytometric analysis. RESULTS: Systemic infusion of GMSCs can significantly reduce the severity of experimental arthritis, and resume the balance of Th cell subsets. FasL-/- GMSCs failed to induce apoptosis of activated T cells in vitro and in vivo, and therefore show no therapeutic effects, whereas FasL TF GMSCs can rescue the immunosuppressant effects in the treatment of CIA. CONCLUSIONS: GMSC-based therapy induces T-cell apoptosis via the FasL/Fas pathway and results in immune tolerance and amelioration of the CIA inflammation.

Congenital tri-cavernous hemangiomas of the right buccal region, right accessory parotid gland, and masseter muscle region.

We report a rare case of congenital tri-cavernous hemangiomas of the right buccal region, right accessory parotid gland, and masseter muscle region in an adult. The patient, a 25-year-old woman, complained of 3 masses in her right midcheek. Ultrasonographic and computed tomographic findings showed an irregular-shaped mass (multiple calcifications) with a well-defined margin in the masseter muscle region, an ellipse-shaped mass (multiple calcifications) with a well-defined margin in the right buccal region, and a comma-shaped mass (no calcifications) with a well-defined margin separate from the parotid gland in the right accessory parotid gland region. These iconographic findings suggested that the masses were all hemangiomas separately originating from the parotid gland, accessory parotid gland, and masseter muscle. The masses were completely removed through a standard parotid incision without postoperative facial palsy, skin deformity, and difficulty in secreting saliva. Findings from histologic examination of the tumor revealed multiple, thin-walled, and dilated blood vessels, confirming the diagnosis of cavernous hemangiomas. Ultrasonographic and computed tomographic findings were extremely useful in diagnosing the mass/masses as hemangioma before surgery, clarifying relationships between the mass and adjacent structures, and determining the surgical approach to the mass/masses.

The frequency and distribution of caries among the Iron Age population (about 2200 years BP) buried in the Mausoleum of Emperor Qinshihuang.

OBJECTIVES: The purpose of this study was to assess the prevalence, distribution, and intensity of dental caries in the Iron Age population of northern China in order to increase knowledge about the type of food, dietary habit, and social stratification in this Iron Age people. MATERIALS AND METHODS: The samples analyzed were dental remains of 1548 permanent teeth from 69 male individuals unearthed from the Qin archaeological site of Emperor Qinshihuang's Mausoleum in Lintong (northern China). The sex and the age-at-death of the samples were estimated. RESULTS: Overall frequency of antemortem tooth loss in the samples was 0.8%. The proportion of individuals with at least one carious tooth was 65.2%, and the frequency of carious lesions was 9.4%, both showing a trend to rise as age increased. Data obtained on dental caries and antemortem tooth loss provided a corrected rate of 9.5% of teeth with caries. The most frequent carious lesions were occlusal lesions (2.6%), followed by interproximal (2.5%) and buccal/lingual lesions (1.0%). Tooth type analysis showed that molars had the highest percentage of caries (18.6%), followed by premolars (4.5%), canines (3.0%), and incisors (3.0%). The total SRCI was 1.6, increasing with age. CONCLUSIONS: These findings indicate that dental caries may be related, at least in part, to the subsistence and diet of this Iron Age population.

[Experimental study of preparation errors during instrumentation of artificial curved root canals with stainless steel K files].

PURPOSE: To investigate the features and rules of preparation errors developed from instrumentation of curved root canals. METHODS: 8 curved artificial root canals were prepared with stainless steel K files by the step-back technique,the root canals were digitally photographed,the images were analysed by the software Image-Pro Plus,the shapes and positions of the root canals were measured and analysed.SPSS 10.0 software package was used for statistical analysis, paired-samples t test was used to compare the difference of the means among sample groups. RESULTS: After root canal preparation,the mean value of the angles of canal curvature decreased from 36.21 degrees to 21.98 degrees , while the mean value of the radius of the canal curvature increased from 6.25mm to 11.35mm; the canal axes transported and intercrossed with the original axes at 2 points and created 3 intersection zones.The taper of the root canals was destroyed and a series of instrumentation defaults were created. CONCLUSION: The cause of preparation errors was the transportation of the root canal instruments leaded by the elastic force and cutting effects.