SN 2 versus E2 Competition of Cyclic Ethers.

We have quantum chemically studied the influence of ring strain on the competition between the two mechanistically different SN 2 and E2 pathways using a series of archetypal ethers as substrate in combination with a diverse set of Lewis bases (F- , Cl- , Br- , HO- , H3 CO- , HS- , H3 CS- ), using relativistic density functional theory at ZORA-OLYP/QZ4P. The ring strain in the substrate is systematically increased on going from a model acyclic ether to a 6- to 5- to 4- to 3-membered ether ring. We have found that the activation energy of the SN 2 pathway sharply decreases when the ring strain of the system is increased, thus on going from large to small cyclic ethers, the SN 2 reactivity increases. In contrast, the activation energy of the E2 pathway generally rises along this same series, that is, from large to small cyclic ethers. The opposing reactivity trends induce a mechanistic switch in the preferred reaction pathway for strong Lewis bases from E2, for large cyclic substrates, to SN 2, for small cyclic substrates. Weak Lewis bases are unable to overcome the higher intrinsic distortivity of the E2 pathway and, therefore, always favor the less distortive SN 2 reaction.

Transvaginal Mesh-related Complications and the Potential Role of Bacterial Colonization: An Exploratory Observational Study.

STUDY OBJECTIVE: This study aimed to investigate the potential role of transvaginal mesh bacterial colonization in the development of mesh-related complications (MRCs). DESIGN: An observational and exploratory study. SETTING: Tertiary referral center (Amsterdam UMC, location AMC, Amsterdam, The Netherlands). PATIëNTS: 49 patients indicated for mesh removal and 20 women of whom vaginal tissue was retrieved during prolapse surgery as a reference cohort. INTERVENTIONS: collection of mesh-tissue complex (patient cohort) or vaginal tissue (reference cohort) MEASUREMENTS AND MAIN RESULTS: Homogenized samples were used for quantitative microbiological culture. Inflammation and fibrosis were semiquantitatively histologically scored; Gram staining and fluorescence in situ hybridization were used to detect bacteria and bacterial biofilms. Of the 49 patients, 44 samples (90%) were culture positive, with a higher diversity of species and more Gram-negative bacteria and polymicrobial cultures in the MRC cohort than the reference cohort, with mostly staphylococci, streptococci, Actinomyces spp., Cutibacterium acnes, and Escherichia coli. Patients with clinical signs of infection or exposure had the highest bacterial counts. Histology demonstrated moderate to severe inflammation in most samples. Gram staining showed bacteria in 57% of culture-positive samples, and in selected samples, fluorescence in situ hybridization illustrated a polymicrobial biofilm. CONCLUSION: In this study, we observed distinct differences in bacterial numbers and species between patients with MRCs and a reference cohort. Bacteria were observed at the mesh-tissue interface in a biofilm. These results strongly support the potential role of bacterial mesh colonization in the development of MRCs.

Early Versus Delayed Treatment for Gartland Type III Supracondylar Humeral Fractures in Children: A Systematic Review and Meta-analysis.

Purpose: The timing of treatment for Gartland type III supracondylar fractures has been an area of contention as it was previously thought to be a surgical emergency. The aim of this systematic review and meta-analysis is to clarify whether there is a difference in perioperative outcomes between early and delayed treatment for Gartland type III supracondylar humeral fractures in children. Methods: Literature search and study selection were performed according to the PRISMA process. The early surgery (ES) and delayed surgery (DS) groups were defined by the authors of each study included, based on the time to surgery. The primary outcome was the risk of conversion to open reduction. The secondary outcome was perioperative complication risks. Results: A total of 14 studies met the eligibility criteria (n = 1263 patients), of which 665 (52.7%) patients had undergone early surgery, while 598 (47.3%) had delayed surgery. On meta-analysis, there was no significant difference between ES and DS for the outcome of open reduction conversion risk. There was also no significant difference for the secondary outcomes of post-operative compartment syndrome, iatrogenic nerve injury, vascular injury, and surgical site infection. Conclusion: Despite the limitations in the literature, evidence exists to support the notion that a delayed approach to the surgical treatment of Gartland type III supracondylar humeral fractures in children does not result in an increased risk of converting to open reduction and perioperative complications.

The impact of bacterial contamination on the host response towards fully absorbable poly-4-hydroxybutyrate and nonabsorbable polypropylene pelvic floor implants.

Polypropylene (PP) implants for the vaginal surgical correction of pelvic organ prolapse (POP) are known for adverse events, like vaginal or visceral exposures. It is hypothesized that this is a result of a prolonged inflammatory response. One of the triggering factors of prolonged inflammation might be bacterial contamination. A possible solution might lie in an absorbable biomaterial, which provides initial mechanical support while being gradually replaced by the host tissue. With this study we aimed to compare the host response, in a subcutaneous mouse implant infection model, to delayed absorbable poly-4-hydroxybutyrate (P4HB) and a latest generation PP implant. By comparing non-infected to Staphylococcus aureus infected mice, we assessed how bacterial contamination affects the host response and its role in the development of complications. Further, we included sham surgery as a control, mimicking the wound response in native tissue repair. Despite the higher surface area of the P4HB implants, the clearance of infection was similarly delayed in the presence of a P4HB or PP implant, as compared to sham. Further, the host response towards P4HB and PP was quite comparable, yet collagen deposition was significantly increased around infected P4HB implants at early time points. Adverse event rates were similar, though implant exposures were only seen in infected mice and more often with PP (11.1%) than P4HB implants (5.6%). Infected mice overall had significantly higher levels of infiltration of inflammatory cells and lower levels of vascularization and collagen deposition compared to non-infected mice. Thus, for both P4HB and PP, bacterial contamination negatively affected mesh integration by increased inflammation and an increased adverse event rate. Altogether, our results from this subcutaneous mouse implant infection study suggest that P4HB could be a promising degradable alternative to PP, warranting further research to study its potential as a new surgical solution for women with POP.

Animal experimental research assessing urogynecologic surgical mesh implants: Outcome measures describing the host response, a systematic review and meta-analysis.

AIM: Before the introduction of new biomaterials for prolapse surgery, animal studies on the host response are required. Unfortunately, large variation in study design hampers obtaining an overview of the safety and efficacy, and translation to clinical practice. Our aim is to systematically review the literature on all outcome measures describing the host response in animal studies assessing the biocompatibility of urogynecologic surgical mesh implants for prolapse surgery. Furthermore, by meta-analysis, we aim to assess the effect of implantation and compare this to control animals receiving sham surgery or native tissue repair. METHODS: We performed a systematic search from inception to August 2020. Since this is an explorative study we included original, controlled, and noncontrolled animal studies describing any host response to the implant. Quantitative outcome measures reported ≥10 times in ≥2 articles were eligible for meta-analysis. RESULTS: Fifty articles were included in the qualitative synthesis and 36 articles were eligible for meta-analysis. In total, 154 outcome measures were defined and classified into (1) histomorphology, (2) biomechanics and, (3) macroscopic morphology. Animals with vaginal implants demonstrated significantly increased M1 and M2 macrophages, MMP-2, neovascularization, TNF-α, and stiffness, and lower vaginal contractility compared to control animals. CONCLUSION: The host response significantly differs in animals after vaginal mesh implantation compared to control animals, both pro- and anti-inflammatory. However, we observed a paucity in the uniformity of reported outcomes. For future animal studies, we propose the development of a core outcome set, which ideally predicts the host response in women.

Nicotinamide Improves Delayed Tooth Eruption in Runx2+/- Mice.

Patients with cleidocranial dysplasia (CCD) caused by mutations in RUNX2 have severe dental anomalies, including delayed or absent eruption of permanent teeth. This requires painful and expensive surgical/orthodontic intervention because of the absence of medicine for this condition. Here, we demonstrate that nicotinamide, a vitamin B3 and class III histone deacetylase inhibitor, significantly improves delayed tooth eruption in Runx2+/- mice, a well-known CCD animal model, through the restoration of decreased osteoclastogenesis. We also found that Csf1 mRNA and protein levels were significantly reduced in Runx2+/- osteoblasts as compared with wild type whereas RANKL and OPG levels had no significant difference between wild type and Runx2+/- osteoblasts. The nicotinamide-induced restoration of osteoclastogenesis of bone marrow-derived macrophages in Runx2+/- mice was due to the increased expression of RUNX2 and CSF1 and increased RANKL/OPG ratio. RUNX2 directly regulated Csf1 mRNA expression via binding to the promoter region of the Csf1 gene. In addition, nicotinamide enhanced the RUNX2 protein level and transacting activity posttranslationally with Sirt2 inhibition. Taken together, our study shows the potential and underlying molecular mechanism of nicotinamide for the treatment of delayed tooth eruption by using the Runx2+/- murine model, suggesting nicotinamide as a candidate therapeutic drug for dental abnormalities in patients with CCD.

In Vitro Bacterial Adhesion and Biofilm Formation on Fully Absorbable Poly-4-hydroxybutyrate and Nonabsorbable Polypropylene Pelvic Floor Implants.

Knitted polypropylene (PP) implants for the correction of pelvic organ prolapse have been associated with complications such as vaginal exposure, infection, and pain. Since certain complications may be linked to bacterial contamination and persistent inflammation, there is a rationale to develop a biocompatible implant that is less prone to bacterial adhesion and biofilm formation. Delayed absorbable materials could meet these requirements and poly-4-hydroxybutyrate (P4HB) might be such a new material for future pelvic floor implants. We studied in vitro bacterial adhesion and biofilm formation on P4HB in comparison to PP. We investigated the influence of both polymers using flat films and compared P4HB and PP implants with different knitting designs. P4HB flat films were demonstrated to be hydrophilic with significantly less Staphylococcus aureus and Escherichia coli cultured from P4HB films than from hydrophobic PP films after 24 h of incubation. On the implants, a higher number of E. coli were cultured after 1 h of incubation from the knitted P4HB implant with the highest density and smallest pore size, compared to other P4HB and PP implants. No differences were observed between the implants for E. coli at later time points or for S. aureus incubation. These results show that in flat films, the polymer influences biofilm formation, demonstrated by a reduced biofilm formation on P4HB compared with PP flat films. In addition, the knitting design may affect bacterial adhesion. Despite certain design and material characteristics that give the knitted P4HB implants a higher surface area, this did not result in more bacterial adhesion and biofilm formation overall. Collectively, these results warrant further (pre)clinical investigations of P4HB pelvic floor implants.

Delay-adjusted age- and sex-specific case fatality rates for COVID-19 in South Korea: Evolution in the estimated risk of mortality throughout the epidemic.

OBJECTIVES: The aim of this study was to estimate delay-adjusted case fatality rates (CFRs) for COVID-19 in South Korea, and evaluate how these estimates have evolved over time throughout the epidemic. METHODS: Public data from the Korea Centers for Disease Control and Prevention (KCDC) were used to estimate age- and sex-specific CFRs for COVID-19 in South Korea up to June 12, 2020. We applied statistical methods previously developed to adjust for the delay between diagnosis and death, and presented both delay-adjusted and crude (unadjusted) CFRs throughout the epidemic. RESULTS: The overall estimated delay-adjusted CFR was 2.39% (3.05% for males and 1.92% for females). Within each age strata where deaths were reported, males were found to have significantly higher CFRs than females. The estimated CFRs increased substantially from age 60 years in males and from 70 years in females. Both the delay-adjusted and crude CFRs were found to have evolved substantially, particularly early in the epidemic, converging only from mid-April 2020. CONCLUSIONS: The CFRs for South Korea provide an estimate of mortality risk in a setting where case ascertainment is likely to be more complete. The evolution in CFRs throughout the epidemic highlights the need for caution when interpreting CFRs calculated at a given time point.

Distinctive patterns of pulmonary function change according to baseline lung volume and diffusing capacity.

SETTING: Multicentre retrospective study in South Korea.OBJECTIVE: To longitudinally evaluate changes in lung volume and diffusing capacity for carbon monoxide (DLCO) with forced expiratory volume in 1 sec (FEV1).DESIGN: A total of 155 patients with chronic obstructive pulmonary disease (COPD), whose pulmonary function parameters were measured annually for 5 years, were selected from a prospective cohort in South Korea. A random coefficients model was used to estimate mean annual FEV1, lung volume parameter and DLCO change rates.RESULTS: Patients were classified into four groups based on baseline DLCO and residual volume/total lung capacity (RV/TLC) measurements. The annual FEV1 decline rate was greater in patients with low DLCO than in those with normal DLCO, with the greatest decline occurring in patients with low DLCO and normal RV/TLC. RV and RV/TLC declined in patients with high RV/TLC, whereas these increased in patients with normal RV/TLC. DLCO decreased longitudinally in all four groups, with the greatest decline occurring in patients with normal DLCO and normal RV/TLC.CONCLUSIONS: Different subgroups of patients with COPD exhibited distinctive pulmonary function change patterns. Baseline DLCO and RV/TLC may be used as physiological markers to predict long-term changes in pulmonary function.

Effect of heat-killed Enterococcus faecalis EF-2001 on ethanol-induced acute gastric injury in mice: Protective effect of EF-2001 on acute gastric ulcer.

Enterococcus faecalis is a facultative anaerobic gram-positive commensal bacterium common in the gastrointestinal tract of animals and humans. This study aimed to investigate the protective effects of heat-killed E. faecalis EF-2001 (EF-2001) on acute gastric ulcer using a murine model of ethanol (EtOH)-induced acute gastric injury. EF-2001 (20, 40, and 80 mg/kg/day) was administered by oral gavage for 5 days before EtOH treatment (10 mL/kg body weight). EF-2001 effectively attenuated EtOH-induced gastric mucosal injury with reduced gastric mucosal ulcer and histological damage score. Pretreatment of EF-2001 markedly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38MAPK). In addition, EF-2001 significantly inhibited phosphorylation of nuclear factor kappa B (NF-κB) and subsequently suppressed the upregulation of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 in gastric tissues. Taken together, these results suggest that EF-2001 exerts a gastroprotective effect against acute gastric injury, and the underlying mechanism might be associated with the suppression of MAPKs and NF-κB signaling and consequent reduction of pro-inflammatory mediators or cytokines.

Association between time to treatment and functional outcomes according to the Diffusion-Weighted Imaging Alberta Stroke Program Early Computed Tomography Score in endovascular stroke therapy.

BACKGROUND AND PURPOSE: The rate at which the chance of a good outcome of endovascular stroke therapy (EVT) decays with time when eligible patients are selected by baseline diffusion-weighted magnetic resonance imaging (DWI-MRI) and whether ischaemic core size affects this rate remain to be investigated. METHODS: This study analyses a prospective multicentre registry of stroke patients treated with EVT based on pretreatment DWI-MRI that was categorized into three groups: small [Diffusion-Weighted Imaging Alberta Stroke Program Early Computed Tomography Score (DWI-ASPECTS)] (8-10), moderate (5-7) and large (<5) cores. The main outcome was a good outcome at 90 days (modified Rankin Scale 0-2). The interaction between onset-to-groin puncture time (OTP) and DWI-ASPECTS categories regarding functional outcomes was investigated. RESULTS: Ultimately, 985 patients (age 69 ± 11 years; male 55%) were analysed. Potential interaction effects between the DWI-ASPECTS categories and OTP on a good outcome at 90 days were observed (Pinteraction  = 0.06). Every 60-min delay in OTP was associated with a 16% reduced likelihood of a good outcome at 90 days amongst patients with large cores, although no associations were observed amongst patients with small to moderate cores. Interestingly, the adjusted rates of a good outcome at 90 days steeply declined between 65 and 213 min of OTP and then remained smooth throughout 24 h of OTP (Pnonlinearity  = 0.15). CONCLUSIONS: Our study showed that the probability of a good outcome after EVT nonlinearly decreased, with a steeper decline at earlier OTP than at later OTP. Discrepant effects of OTP on functional outcomes by baseline DWI-ASPECTS categories were observed. Thus, different strategies for EVT based on time and ischaemic core size are warranted.

Comparison of Hot Deformation Behavior Characteristics Between As-Cast and Extruded Al-Zn-Mg-Cu (7075) Aluminum Alloys with a Similar Grain Size.

The hot compressive behavior and processing maps of as-cast and extruded 7075 aluminum alloys with a similar grain size (320-350 m) were studied and compared, which allows us to directly observe the effect of segregated phases in the as-cast microstructure on the deformation behavior and hot workability of 7075 alloys. In the as-cast alloy, the compound phases segregated along the interdendritic interfaces within the interiors of original grains provided the additional sites for continuous dynamic recrystallization via the particle stimulation nucleation mechanism. As a result, the as-cast alloy exhibited higher fractions of recrystallized grains and smaller grain sizes than the extruded alloy after compression. The stress exponent values of the as-cast alloy were smaller than those of the extruded alloy. In the processing maps, the domain associated with high power dissipation efficiencies (≥35%) occurred in a wider temperature range in the as-cast alloy compared to the extruded alloy. The segregated phases that remained undissolved in the as-cast alloy after compressive deformation could be effectively eliminated during the solid solution treatment (753 K for 2 h) for T6 aging applied after hot compression. The current results suggest the possibility and advantage of omitting the extrusion step when preparing 7xxx aluminum forging or extrusion feedstocks for hot working. The proposed method can be applied to other precipitation hardenable aluminum alloys.

PIN1 is a new therapeutic target of craniosynostosis.

Gain-of-function mutations in fibroblast growth factor receptors (FGFRs) cause congenital skeletal anomalies, including craniosynostosis (CS), which is characterized by the premature closure of craniofacial sutures. Apert syndrome (AS) is one of the severest forms of CS, and the only treatment is surgical expansion of prematurely fused sutures in infants. Previously, we demonstrated that the prolyl isomerase peptidyl-prolyl cis-trans isomerase interacting 1 (PIN1) plays a critical role in mediating FGFR signaling and that Pin1+/- mice exhibit delayed closure of cranial sutures. In this study, using both genetic and pharmacological approaches, we tested whether PIN1 modulation could be used as a therapeutic regimen against AS. In the genetic approach, we crossbred Fgfr2S252W/+, a mouse model of AS, and Pin1+/- mice. Downregulation of Pin1 gene dosage attenuated premature cranial suture closure and other phenotypes of AS in Fgfr2S252W/+ mutant mice. In the pharmacological approach, we intraperitoneally administered juglone, a PIN1 enzyme inhibitor, to pregnant Fgfr2S252W/+ mutant mice and found that this treatment successfully interrupted fetal development of AS phenotypes. Primary cultured osteoblasts from Fgfr2S252W/+ mutant mice expressed high levels of FGFR2 downstream target genes, but this phenotype was attenuated by PIN1 inhibition. Post-translational stabilization and activation of Runt-related transcription factor 2 (RUNX2) in Fgfr2S252W/+ osteoblasts were also attenuated by PIN1 inhibition. Based on these observations, we conclude that PIN1 enzyme activity is important for FGFR2-induced RUNX2 activation and craniofacial suture morphogenesis. Moreover, these findings highlight that juglone or other PIN1 inhibitors represent viable alternatives to surgical intervention for treatment of CS and other hyperostotic diseases.

Identification of chronic obstructive pulmonary disease subgroups in 13 Asian cities.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous condition that can differ in its clinical manifestation, structural changes and response to treatment. OBJECTIVE: To identify subgroups of COPD with distinct phenotypes, evaluate the distribution of phenotypes in four related regions and calculate the 1-year change in lung function and quality of life according to subgroup. METHODS: Using clinical characteristics, we performed factor analysis and hierarchical cluster analysis in a cohort of 1676 COPD patients from 13 Asian cities. We compared the 1-year change in forced expiratory volume in one second (FEV1), modified Medical Research Council dyspnoea scale score, St George's Respiratory Questionnaire (SGRQ) score and exacerbations according to subgroup derived from cluster analysis. RESULTS: Factor analysis revealed that body mass index, Charlson comorbidity index, SGRQ total score and FEV1 were principal factors. Using these four factors, cluster analysis identified three distinct subgroups with differing disease severity and symptoms. Among the three subgroups, patients in subgroup 2 (severe disease and more symptoms) had the most frequent exacerbations, most rapid FEV1 decline and greatest decline in SGRQ total score. CONCLUSION: Three subgroups with differing severities and symptoms were identified in Asian COPD subjects.

Effect of relative humidity on inactivation of foodborne pathogens using chlorine dioxide gas and its residues on tomatoes.

The effect of relative humidity (RH) on the antimicrobial efficacy of chlorine dioxide (ClO2 ) gas against foodborne pathogens on tomatoes was evaluated. Also, levels of ClO2 residues on tomatoes after exposure to ClO2 gas under different RH conditions were measured to determine the quantity of solubilized ClO2 gas on tomato surfaces. Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes were inoculated on tomatoes and exposed to ClO2 gas (5, 10, 20 and 30 ppmv) under different RH conditions (50, 70 and 90%). As ClO2 gas concentration and treatment time increased, significant differences (P < 0·05) were observed between inactivation levels under different RH conditions. Exposure to 30 ppmv of ClO2 gas (50% RH) for 20 min resulted in 1·22-1·52 log reductions of the three foodborne pathogens. Levels of the three foodborne pathogens were reduced to below the detection limit (0·48 log CFU per cm2 ) within 15 min when exposed to 30 ppmv of ClO2 gas at 70% RH and within 10 min at 90% RH. At a given ClO2 gas concentration, ClO2 residues on tomatoes significantly (P < 0·05) increased with increasing RH, and there were close correlations between log reductions of pathogens and ClO2 residues on tomatoes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported on the correlation between the amount of ClO2 residues on produce surfaces and the level of inactivation of pathogens after ClO2 gas treatment. Variations in RH have great effect on the solubilization of ClO2 gas on tomato surfaces considering that ClO2 residues on tomatoes increased with increasing RH. Also, the amount of ClO2 residues on tomatoes is positively correlated with the level of inactivation of pathogens. The results of this study provide insights for predicting inactivation patterns of foodborne pathogens by ClO2 gas for practical application by the fresh produce industry.

Comparative study of new autologous material, bone-cartilage composite graft, for ossiculoplasty with Polycel® and Titanium.

OBJECTIVE: Ossiculoplasty is a surgical procedure that recreates sound transmission of the middle ear in conductive hearing loss. Various materials have been used for ossicular reconstruction, but the most ideal material for ossiculoplasty remains controversial. The purpose of this study was to introduce a novel method of autologous ossiculoplasty, bone-cartilage composite graft (BCCG) and to compare its surgical results with different types of ossiculoplastic prostheses. STUDY DESIGN: A retrospective study was performed in a tertiary referral centre. METHODS: Data of 275 patients who received ossiculoplasty using the three different materials of BCCG, Polycel® and titanium were analysed according to type of ossiculoplasty: partial or total ossicular replacement prosthesis (PORP or TORP). Hearing results, complication rates and clinical parameters including age, sex, past history, preoperative diagnosis and surgery type were compared among different groups. RESULTS: Ossiculoplasty with BCCG showed satisfactory hearing outcomes and the lowest complication rate among the three different materials. In particular, its extrusion rate was 0%. CONCLUSION: We propose that the BCCG technique is a useful alternative method for ossiculoplasty, with proper patient selection.

Adult Living Donor Liver Transplantation for Acute-on-Chronic Liver Failure in High-Model for End-Stage Liver Disease Score Patients.

The large volume of adult living donor liver transplantations (ALDLTs) at our center affords a unique opportunity to examine the impact of acute-on-chronic liver failure (ACLF) among high-Model for End-Stage Liver Disease MELD score patients. From February 1998 to March 2010, 1958 cirrhotic recipients were analyzed to study the relationship between MELD scores and ALDLT outcomes. A total of 327 high-MELD score recipients were categorized into ACLF and non-ACLF groups, and their outcomes were compared. The 5-year graft and patient survival in the high-MELD group were 75.2% and 76.4%, respectively, which were significantly worse than the low and intermediate MELD groups. The presence of ACLF associated with higher MELD scores appeared to be the dominant factor responsible for the inferior results of patients with MELD score of 30-34 points. The 5-year graft survivals in the ACLF group was 70.5% and in the non-ACLF group it was 81.0% (p = 0.035). Therefore, ALDLT should be performed as soon as possible in high-MELD score patients prior to ACLF development. Moreover, ACLF patients should be separately categorized when analyzing the outcomes of ALDLT. ALDLT for ACLF patients should not be discouraged because favorable outcomes can be expected through timely ALDLT and comprehensive management.

Nitric oxide-induced autophagy and the activation of activated protein kinase pathway protect against apoptosis in human dental pulp cells.

AIM: To investigate the role of nitric oxide (NO)-induced autophagy in human dental pulp cells (HDPCs) and the involvement of AMP-activated protein kinase (AMPK) pathway. METHODOLOGY: The MTT assay was used to determine the cytotoxic effect of the NO donor sodium nitroprusside (SNP) in HDPCs. Apoptosis was detected by means of the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and apoptosis- or autophagy-related signal molecules were observed by Western blot analysis. Acidic autophagolysosomal vacuoles were stained with acridine orange to detect autophagy in the presence of 3-methyladenine (3MA) used to inhibit autophagy. To explore the mechanism underlying autophagy and its protective role against apoptosis, compound C, the chemical AMPK inhibitor, was used. Statistical analysis was performed using Student's t-test or analysis of variance (anova) followed by the Student-Newman-Keuls test (P < 0.05). RESULTS: SNP decreased viability of the HDPCs in a dose- and time-dependent manner. Exposing the HDPCs to SNP increased the levels of p62 and LC3-II, the typical markers of autophagy, and increased the number of acidic autophagolysosomal vacuoles, indicating the appearance of autophagy as detected by acridine orange staining (P < 0.05). Pre-treatment with 3MA decreased cell viability but increased cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3, apoptosis indicators, in the SNP-treated HDPCs (P < 0.05). SNP activated AMPK/ULK signalling, whilst the inhibition of AMPK by compound C enhanced apoptotic cell death induced by SNP in the HDPCs (P < 0.05). CONCLUSION: NO induced autophagy with AMPK activation, which plays a role in the survival of HDPCs against NO-induced apoptosis.

Simvastatin inhibits the expression of inflammatory cytokines and cell adhesion molecules induced by LPS in human dental pulp cells.

AIM: To investigate the effect of simvastatin on lipopolysaccharide (LPS)-stimulated inflammatory cytokines, cell adhesion molecules and nuclear factor-κB (NF-κB) transcription factors in human dental pulp cells (HDPCs). METHODOLOGY: The effect of LPS and simvastatin on human dental pulp cell (HDPCs) viability was measured using a 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay. The expression of inflammatory cytokines and cell adhesion molecules was evaluated by reverse-transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. NF-κB transcription factors were evaluated by Western blot analysis. Statistical analysis was performed with analysis of variance (anova). RESULTS: The viability of cells exposed to different concentrations of E. coli LPS, P. gingivalis LPS and simvastatin was not significantly different compared with that of control cells (P > 0.05). LPS significantly increased interleukin (IL)-1ß (P < 0.05) and IL-6 mRNA expression (P < 0.05) and vascular cell adhesion molecule-1 (VCAM-1) (P < 0.05) and intercellular adhesion molecule-1 (ICAM-1) protein expression (P < 0.05) in HDPCs. Treatment with simvastatin significantly attenuated LPS-stimulated production of IL-1ß, IL-6, VCAM-1 and ICAM-1 (P < 0.05). Treatment with simvastatin decreased LPS-induced expression of p65 and phosphorylation of IκB and also significantly decreased the phosphorylation of p65 and IκB in the cytoplasm and the level of p65 in the nucleus (P < 0.05). CONCLUSIONS: Simvastatin has a suppressing effect on LPS-induced inflammatory cytokine, cell adhesion molecules and NF-κB transcription factors in HDPCs. Therefore, simvastatin might be a useful candidate as a pulp-capping agent in vital pulp therapy.