Ox-LDL-induced CD80+ macrophages expand pro-atherosclerotic NKT cells via CD1d in atherosclerotic mice and hyperlipidemic patients.

Atherosclerosis is an inflammatory disease of blood vessels involving the immune system. Natural killer T (NKT) cells, as crucial components of the innate and acquired immune systems, play critical roles in the development of atherosclerosis. However, the mechanism and clinical relevance of NKT cells in early atherosclerosis are largely unclear. The study investigated the mechanism influencing NKT cell function in apoE deficiency-induced early atherosclerosis. Our findings demonstrated that there were higher populations of NKT cells and interferon-gamma (IFN-γ)-producing NKT cells in the peripheral blood of patients with hyperlipidemia and in the aorta, blood, spleen, and bone marrow of early atherosclerotic mice compared with the control groups. Moreover, we discovered that the infiltration of CD80+ macrophages and CD1d expression on CD80+ macrophages in atherosclerotic mice climbed remarkably. CD1d expression increased in CD80+ macrophages stimulated by oxidized low-density lipoprotein (ox-LDL) ex vivo and in vitro. Ex vivo coculture of macrophages with NKT cells revealed that ox-LDL-induced CD80+ macrophages presented lipid antigen α-Galcer (alpha-galactosylceramide) to NKT cells via CD1d, enabling NKT cells to express more IFN-γ. Furthermore, a greater proportion of CD1d+ monocytes and CD1d+CD80+ monocytes were found in peripheral blood of hyperlipidemic patients compared with that of healthy donors. Positive correlations were found between CD1d+CD80+ monocytes and NKT cells or IFN-γ+ NKT cells in hyperlipidemic patients. Our findings illustrated that CD80+ macrophages stimulated NKT cells to secrete IFN-γ via CD1d-presenting α-Galcer, which may accelerate the progression of early atherosclerosis. Inhibiting lipid antigen presentation by CD80+ macrophages to NKT cells may be a promising immune target for the treatment of early atherosclerosis.NEW & NOTEWORTHY This work proposed the ox-LDL-CD80+ monocyte/macrophage-CD1d-NKT cell-IFN-γ axis in the progression of atherosclerosis. The proinflammatory IFN-γ+ NKT cells are closely related to CD1d+CD80+ monocytes in hyperlipidemic patients. Inhibiting CD80+ macrophages to present lipid antigens to NKT cells through CD1d blocking may be a new therapeutic target for atherosclerosis.

Oridonin suppresses the growth of glioblastoma cells via inhibiting Hippo/YAP axis.

Glioma is a brain tumor that originates from brain or spine glial cells. Despite alternative treatments, the overall survival rate remains low. Oridonin (ORI) is purified from the Chinese herb Rabdosia rubescens, which has exhibited positive effects on tumors. This study aimed to investigate the effect of ORI on U87MG glioblastoma cells and whether the Hippo/YAP-related signaling pathway was involved. Malignant glioblastoma U87MG cells and male athymic nude mice (BALB/cnu/nu) were used as the experimental models. The YAP inhibitor Verteporfin (VP) and the overexpression of YAP were used to investigate its potential relation with glioma. Here, we found that ORI inhibited cell proliferation and promoted cell apoptosis in a dose-dependent manner in U87MG cells. Moreover, ORI inhibited Bcl-2, YAP, and c-Myc protein expression but increased Bax, caspase-3, and p-YAP protein expression. Furthermore, the effect of ORI was also confirmed in a mouse model bearing glioma. ORI reversed the effect of overexpression of YAP. Collectively, oridonin suppressed glioblastoma oncogenesis via the Hippo/YAP signaling pathway and could be a potential therapeutic target in the treatment of glioblastoma.

Effect of RNA-binding proteins on osteogenic differentiation of bone marrow mesenchymal stem cells.

Tissue regeneration mediated by mesenchymal stem cells (MSCs) is an ideal way to repair bone defects. RNA-binding proteins (RBPs) can affect cell function through post-transcriptional regulation. Exploring the role of RBPs in the process of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is helpful to find a key method to promote the osteogenic efficiency of BMSCs. By reviewing the literature, we obtained a differentially expressed mRNA dataset during the osteogenic differentiation of BMSCs and a human RBP dataset. A total of 82 differentially expressed RBPs in the osteogenic differentiation of BMSCs were screened by intersection of the two datasets. Functional analysis showed that the differentially expressed RBPs were mainly involved in RNA transcription, translation and degradation through the formation of spliceosomes and ribonucleoprotein complexes. The top 15 RBPs determined by degree score were FBL, NOP58, DDX10, RPL9, SNRPD3, NCL, IFIH1, RPL18A, NAT10, EXOSC5, ALYREF, PA2G4, EIF5B, SNRPD1 and EIF6. The results of this study demonstrate that the expression of many RBPs changed during osteogenic differentiation of BMSCs.

Daidzein protects endothelial cells against high glucose-induced injury through the dual-activation of PPARα and PPARγ.

Endothelial damage caused by persistent glucose and lipid metabolism disorders is the main reason of diabetic vascular diseases. Daidzein exerts positive effects on vascular dysfunction. Peroxisome proliferator-activated receptors (PPARs) regulate critically glucose and lipid metabolism. However, the interaction of daidzein to PPARs is still insufficiently explored. In this study, the cell proliferation was detected by EdU. The intrinsic activity and binding affinity of daidzein for human PPARs (hPPARs) were estimated by transactivation reporter gene test and HPLC-UV method, respectively. Daidzein significantly reversed high glucose (HG, at 30 mmol/l)-induced injury in HUVECs, which was inhibited by both PPARα and PPARγ antagonist, but no PPARß antagonist. Daidzein selectively activated hPPARα and hPPARγ1, but weakly hPPARß. Additionally, daidzein also bound to both hPPARα and hPPARγ1. The findings suggested that daidzein may be a PPARα and PPARγ dual-agonist. The amelioration of daidzein on HUVECs from hyperglycemia may be mediated by the activation of PPARα and PPARγ receptors.

Effect of margin designs and loading conditions on the stress distribution of endocrowns: a finite element analysis.

BACKGROUND: Margin designs and loading conditions can impact the mechanical characteristics and survival of endocrowns. Analyzing the stress distribution of endocrowns with various margin designs and loading conditions can provide evidence for their clinical application. METHODS: Three finite element analysis models were established based on the margin designs: endocrown with a butt-joint type margin (E0), endocrown with a 90° shoulder (E90), and endocrown with a 135° shoulder (E135). The E0 group involved lowering the occlusal surface and preparing the pulp chamber. The E90 group created a 90° shoulder on the margin of model E0, measuring 1.5 mm high and 1 mm wide. The E135 group featured a 135° shoulder. The solids of the models were in fixed contact with each other, and the materials of tooth tissue and restoration were uniform, continuous, isotropic linear elasticity. Nine static loads were applied, with a total load of 225 N, and the maximum von Mises stresses and stress distribution were calculated for teeth and endocrowns with different margin designs. RESULTS: Compared the stresses of different models under the same loading condition. In endocrowns, when the loading points were concentrated on the buccal side, the maximum von Mises stresses were E0 = E90 = E135, and when there was a lingual loading, they were E0 < E90 = E135. In enamel, the maximum von Mises stresses under all loading conditions were E0 > E90 > E135. In dentin, the maximum von Mises stresses of the three models were basically similar except for load2, load5 and load9. Compare the stresses of the same model under different loading conditions. In endocrowns, stresses were higher when lingual loading was present. In enamel and dentin, stresses were higher when loaded obliquely or unevenly. The stresses in the endocrowns were concentrated in the loading area. In enamel, stress concentration occurred at the cementoenamel junction. In particular, E90 and E135 also experienced stress concentration at the shoulder. In dentin, the stresses were mainly concentrated in the upper section of the tooth root. CONCLUSION: Stress distribution is similar among the three margin designs of endocrowns, but the shoulder-type designs, especially the 135° shoulder, exhibit reduced stress concentration.

Gastrodin protects endothelial cells against high glucose-induced injury through up-regulation of PPARß and alleviation of nitrative stress.

In diabetes mellitus (DM), high glucose can result in endothelial cell injury, and then lead to diabetic vascular complications. Gastrodin, as the mainly components of Chinese traditional herb Tianma (Gastrodia elata Bl.), has been widely used for cardiovascular diseases. However, the known of the effect of gastrodin on endothelial cell injury is still limited. In this study, we aimed to investigate the effect and possible mechanism of gastrodin on high glucose-injured human umbilical vein endothelial cells (HUVEC). High glucose (30 mmol/L) treatment caused HUVEC injury. After gastrodin (0.1, 1, 10 µmol/L) treatment, compared with the high glucose group, the cell proliferation ability increased in a dose-dependent manner. Meanwhile, gastrodin (10 µmol/L) up-regulated the mRNA and protein expressions of PPARß and eNOS, decreased the expressions of iNOS, also reduced the protein expression of 3-nitrotyrosine, and lowed the level of ONOO-, increased NO content. Both the PPARß antagonist GSK0660 (1 µmol/L) and the eNOS inhibitor L-NAME (10 µmol/L) were able to block the above effects of gastrodin. In conclusion, gastrodin protectes vascular endothelial cells from high glucose injury, which may be, at least partly, mediated by up-regulating the expression of PPARß and negatively regulating nitrative stress.

Regiospecificity C(sp2)-C(sp3) Bond Construction between Purines and Alkenes to Synthesize C6-Alkylpurines and Purine Nucleosides Using O2 as the Oxidant.

A highly site-selective and Markovnikov-type radical C6-H alkylation of purines with alkenes is achieved, allowing fast construction of the C(sp2)-C(sp3) bond at the C-6-position of purines and purine nucleosides using O2 as a green oxidant and alkenes as cheap alkylation reagents. The route was also a radical route to synthesize C6-alkyl-N7-substituted purines with potential steric hindrance between C6-alkyl groups and N7-substituted groups. This reaction is easily scaled up and has excellent functional group compatibility and broad substrate scopes. Moreover, the unstable intermediate was also separated, which was the key evidence for the reaction mechanism.

Metal-free direct C-6-H alkylation of purines and purine nucleosides enabled by oxidative homolysis of 4-alkyl-1,4-dihydropyridines at room temperature.

Herein we report the application of 4-alkyl-1,4-dihydropyridines (DHPs), which are easily prepared from inexpensive aldehydes in one step, for the direct site-specific C-H alkylation of purines and purine nucleosides. Despite there being three active C(sp2)-H bonds (C-2-H, C-6-H, and C-8-H) in the structure, the reactions still show high regioselectivity at the purinyl C-6-H position. Importantly, the reactions successfully avoid the use of transition metal catalysts and additional acids. Meanwhile, the protocols are not sensitive to moisture and require only persulfate as an oxidant. Besides, this method displays broad functional group compatibility and is easy to scale up. Notably, pharmaceutical purines, e.g. the natural product 6-hydroxymethyl nebularine isolated from basidiomycetes, can be smoothly prepared using this protocol.

Tooth loss impairs cognitive function in SAMP8 mice via the NLRP3/Caspase-1 pathway.

OBJECTIVE: Loss of occlusal support due to tooth loss has been indicated as one of the risk factors for Alzheimer's disease. This study aimed to investigate the relationship between tooth loss and cognitive dysfunction and illustrate the role of neuroinflammation in advancing Alzheimer's disease. MATERIALS AND METHODS: Male 5-month-old senescence-accelerated mouse strain P8 (SAMP8) mice were divided into three groups (n = 7): the C (control), S (sham-operated), and TL (tooth loss) groups. The Morris water maze (MWM) test was performed to assess spatial memory. Additionally, histopathological and molecular assessments of hippocampal tissues were performed. RESULTS: The TL groups exhibited impaired spatial memory in the water maze. Tooth loss induced higher protein expression levels of the neuroinflammation cytokine interleukin-1ß (IL-1ß) in the hippocampus than in the S and C groups. Tooth loss activated the NLRP3 inflammasome and increased the expression of Caspase-1 in the hippocampus. CONCLUSIONS: The findings indicated that tooth loss impairs cognitive function in SAMP8 mice and is closely related to the activation of NLRP3/Caspase-1 in the hippocampus.

Pyruvate Dehydrogenase Kinase 1 inhibition mediated oxidative phosphorylation enhancement in cartilage promotes osteoarthritis progression.

Osteoarthritis (OA) is a common disease characterized by cartilage degradation. Growing evidence showed that glucose metabolism impacts joint homeostasis and an imbalance between glycolysis and oxidative phosphorylation (OXPHOS) may exacerbate OA progression, however, a definitive link is yet to be established. Here, we report that pyruvate metabolism and oxidative phosphorylation pathway is enriched in OA cartilage through gene set enrichment analysis (GSEA) and expression of Pyruvate Dehydrogenase Kinase 1 (PDK1), an enzyme that can phosphorylate Pyruvate Dehydrogenase (PDH), and inhibit pyruvate fluxes into the tricarboxylic acid (TCA) cycle and to OXPHOS, in articular cartilage is notably reduced through destabilization of medial meniscus (DMM). Moreover, by inhibiting PDK1, cartilage loss is markedly accelerated in DMM-induced OA through extracellular matrix (ECM) degradation and apoptosis of chondrocytes. These results indicate that PDK1 is involved in the progression of OA through accelerating cartilage matrix degradation and synovium inflammation to ameliorate cartilage degeneration.

Characteristics of oral microbiota and oral health in the patients treated with clear aligners: a prospective study.

OBJECTIVES: In this prospective clinical study, the effect of clear aligners on periodontal health and oral hygiene was examined. As the same time, microbial changes of the aligner tray and subgingival microbiota community were investigated. METHODS: The study recruited fifteen patients, and clinical parameters were recorded at three different time points: before the initiation of aligner treatment (T0), 1 month after treatment onset (T1), and 3 months after treatment onset (T3). Plaque samples were collected from the inner surface of aligners and subgingival sulcus at each of these time points. The microbial composition of the samples was analyzed using 16S rRNA gene sequencing, and changes were evaluated based on the abundance of amplicon sequence variants (ASVs). RESULTS: Reduction in plaque index and improvement in periodontal health were observed. In aligner tray plaque samples, the relative abundance of Streptococcus increased significantly, as well as the richness and diversity of microbiota decreased substantially as the duration of treatment time. In subgingival plaque samples, alpha and beta diversity of microbiota did not change significantly. CONCLUSIONS: During the clear aligner treatment, the patients' periodontium remained in a healthy condition, and clear aligner treatment had no significant impact on the composition of subgingival microbiota. The structure of the aligner tray microbiota altered significantly at both phylum and genus levels and attracted a unique and less diverse microbiota community. CLABSINABSICAL RELEVANCE: Clear aligner treatment has no significant impact on periodontal health and subgingival microbiota composition of patients.

The effects of aligner anchorage preparation on mandibular first molars during premolar-extraction space closure with clear aligners: A finite element study.

INTRODUCTION: This study aimed to determine the effectiveness of different aligner anchorage preparations on mandibular first molars during premolar-extraction space closure with clear aligners and to assess the effects of different modes of Class II elastics on mandibular first molars. METHODS: Finite element models were constructed on the basis of cone-beam computed tomography data from an orthodontic patient. The models comprised maxilla, mandible, maxillary and mandibular teeth without first premolars, periodontal ligaments, attachments and aligners. Tooth displacement tendencies were calculated using different aligner anchorage preparations and Class II elastics on the models from the same patient. Three group sets were designed on the basis of the positions of aligner cutouts and buttons (mesiobuccal, distobuccal and lingual). Four groups were established in each of the 3 group sets. Four groups were created: (1) no elastic traction + no anchorage preparation, (2) anchorage preparation only, (3) elastic traction only, and (4) elastic traction + anchorage preparation. Different aligner anchorage preparations (0°, 1°, 2°, 3°) were applied on mandibular second premolars and molars. The Class II traction force was set to 100 g. RESULTS: With clear aligners, mandibular first molars were subject to mesial tipping, lingual tipping and intrusion. In the condition of no elastic traction, aligner anchorage preparation resulted in distal tipping, buccal tipping, and extrusion effect on mandibular first molars. Aligner anchorage preparation was more effective in the distal and lingual cutout groups than in the mesial cutout group. In the condition of Class II elastic traction, the bodily movement of mandibular first molars was achieved with a 3° anchorage preparation for the mesial cutout group and a 1.7° anchorage preparation for distal and lingual cutout groups. Absolute maximal anchorage was achieved with a 2° anchorage preparation for distal and lingual cutout groups. CONCLUSIONS: Clear aligner therapy caused mesial tipping, lingual tipping and intrusion of mandibular first molars during premolar-extraction space closure. Aligner anchorage preparation effectively prevented mesial and lingual tipping of mandibular molars. Distal and lingual cutout modes were more effective than mesial cutout modes in aligner anchorage preparation. For each aligner stage (0.25 mm), 1.7° aligner anchorage preparation and Class II elastics with distal or lingual cutouts led to the bodily movement of mandibular first molars, whereas 2° anchorage preparation reached absolute maximal anchorage.

PPARß down-regulation is involved in high glucose-induced endothelial injury via acceleration of nitrative stress.

Endothelial injury plays a vital role in vascular lesions from diabetes mellitus (DM). Therapeutic targets against endothelial damage may provide critical venues for the treatment of diabetic vascular diseases. Peroxisome proliferator-activated receptor ß (PPARß) is a crucial regulator in DM and its complications. However, the molecular signal mediating the roles of PPARß in DM-induced endothelial dysfunction is not fully understood. The impaired endothelium-dependent relaxation and destruction of the endothelium structures appeared in high glucose incubated rat aortic rings. A high glucose level significantly decreased the expression of PPARß and endothelial nitric oxide synthase (eNOS) at the mRNA and protein levels, and reduced the concentration of nitric oxide (NO), which occurred in parallel with an increase in the expression of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine. The effect of high glucose was inhibited by GW0742, a PPARß agonist. Both GSK0660 (PPARß antagonist) and NG-nitro-l-arginine-methyl ester (NOS inhibitor) could reverse the protective effects of GW0742. These results suggest that the activation of nitrative stress may, at least in part, mediate the down-regulation of PPARß in high glucose-impaired endothelial function in rat aorta. PPARß-nitrative stress may hold potential in treating vascular complications from DM.

14,15-EET involved in the development of diabetic cardiac hypertrophy mediated by PPARs.

Cardiac hypertrophy is a key structural change in diabetic cardiomyopathy, which mechanism is unknown. 14,15-Epoxyeicosatrienoic acid (14,15-EET) generated from arachidonic acid by CYP2J2 has beneficial effects in metabolic syndrome, which also plays vital roles in inflammatory response. Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily and have three subtypes of α, ß (or δ) and γ. Studies have found that 14,15-EET can perform various biological functions by activating PPARs, but its role in diabetic cardiac hypertrophy is unknown. This study aimed to investigate the role of 14,15-EET-PPARs signaling pathway in the development of diabetic cardiac hypertrophy. Diabetic cardiac hypertrophy was developed by high-fat diet feeding combined with streptozotocin (40 mg/kg/d for 5 days, i.p.) in mice and was induced by glucose at 25.5 mmol/L (high glucose, HG) in H9c2 cells. The decreased level of 14,15-EET and the down-regulated expression of PPARα, PPARß and PPARγ were found following diabetic cardiac hypertrophy in mice. Similarly, both the level of 14,15-EET and the PPARs expression were also reduced in HG-induced hypertrophic cardiomyocytes. Supplementation with 14,15-EET improved the cardiomyocyte hypertrophy and up-regulated PPARs expression, which were nullified by 14,15-EEZE, a 14,15-EET antagonist. Taken together, we conclude that the decreased 14,15-EET is involved in the development of diabetic cardiac hypertrophy through the down-regulation of PPARs.

WDR5 promotes the tumorigenesis of oral squamous cell carcinoma via CARM1/ß-catenin axis.

Oral squamous cell carcinoma (OSCC) is a malignancy all over the world. WD repeat domain 5 (WDR5) is involved in cancer progression. In addition, it was reported that WDR5 is upregulated in head and neck cancer, while its role in OSCC is unknown. First, the expression of WDR5 in oral cancer tissues and cells was examined by qRT-PCR, IHF and western blot. CCK-8 assay was performed to test the cell viability. Cell migration was assessed by transwell assay. Knocking down WDR5 or CARM1 in oral cancer cells to detect its function on cancer growth, WDR5 and CARM1 were significantly upregulated in OSCC. Silencing WDR5 suppressed OSCC cell viability and migration. CARM1 level in OSCC cells was significantly inhibited by WDR5 downregulation, and CARM1 elevation could rescue the effect of WDR5 knockdown on tumorigenesis of OSCC. Moreover, silencing of WDR5 notably inactivated ß-catenin signaling pathway, while this phenomenon was restored by CARM1 overexpression. Silencing of WDR5 attenuated the tumorigenesis of OSCC via CARM1/ß-catenin axis. Thus, WDR5 might be a target for OSCC treatment.

Impact of different axial wall designs on the fracture strength and stress distribution of ceramic restorations in mandibular first molar.

BACKGROUND: The purpose of this study was to investigate the fracture strength and stress distribution of four ceramic restorations. METHODS: Forty human mandibular first molars were collected and randomized into four groups after establishing the distal defect: full crown group with 4 mm axial wall height (AWH) (FC4); short AWH crown group with 2 mm AWH (SC2); occlusal veneer group with 0 mm AWH (OV0); occlusal distal veneer group with only the distal surface prepared, and 4 mm AWH (OD4). The teeth were prepared according to the groups and the ceramic restorations were completed using celtra duo ceramic blocks. The ceramic thickness of the occlusal surface is about 1.5 mm and the edge is about 1 mm. The failure load values and fracture modes of each group were detected by mechanical test in vitro. According to the groups to establish three-dimensional finite element analysis (FEA) models, a 600 N loading force was applied vertically using a hemispherical indenter with a diameter of 6 mm. and compare the stress distribution under the condition of different restorations. RESULTS: In vitro mechanical tests showed that the failure load values were SC2 (3232.80 ± 708.12 N) > OD4 (2886.90 ± 338.72 N) > VO0 (2133.20 ± 376.15 N) > FC4(1635.40 ± 413.05 N). The failure load values of the short AWH crown and occlusal distal veneer were significantly higher than that of occlusal veneer and full crown (P<0.05). The fracture modes of the full crown and occlusal veneer groups were mainly ceramic fractures and some were restorable tooth fractures. The short AWH crown and occlusal distal veneer groups presented with three fracture modes, the proportion of non-restorable tooth fracture was higher. The results of FEA show that under the spherical loading condition, the stress of ceramic was concentrated in the contact area of the loading head, the maximum von Mises stress values were FC4 (356.2 MPa) > VO0 (214.3 MPa) > OD4 (197.9 MPa) > SC2 (163.1 MPa). The stress of enamel was concentrated in the area where the remaining enamel was thinner, the maximum von Mises stress values was OD4 (246.2 MPa) ≈ FC4 (212.4 MPa) > VO0 (61.8 MPa) ≈ SC2 (45.81 MPa). The stress of dentin is concentrated in the root furcation and the upper third region of the root. However, stress concentration was observed at the tooth cervix in the full crown. CONCLUSION: Under certain conditions, the occlusal distal veneer shows better performance than the full crown.

Inhibitory effects of sodium new houttuyfonate on growth and biofilm formation of Streptococcus mutans.

The present study aimed to assess the impact of sodium new houttuyfonate (SNH) on growth and biofilm formation of Streptococcus mutans, and the combinatorial effects of SNH with cariostatic agents. The effects of SNH on S. mutans planktonic cultures were assessed by growth curve assay. The effects of SNH on S. mutans biofilm and extracellular polysaccharides (EPS) production were observed via crystal violet (CV) assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, colony-forming unit (CFU) counting assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Quantitative real-time polymerase chain reaction (qPCR) was applied to investigate the regulatory effects of SNH on the expression of virulence genes of S. mutans. Checkerboard microdilution assay was performed to investigate the combinatorial effects of SNH with two common cariostatic agents. SNH acted as an inhibitor on planktonic cell growth, biofilm formation and EPS production of S. mutans. SNH also downregulated the expression of gtfBCD and comDE systems and exhibited synergism with chlorhexidine (CHX). In conclusion, this study indicated a possibility for SNH to become an anticaries agents by its antimicrobial activity and synergistic effects with CHX against S. mutans.

d-Alanine metabolic pathway, a potential target for antibacterial drug designing in Enterococcus faecalis.

Enterococcus faecalis (E. faecalis) is associated with persistent root canal infection because of its biofilm and various virulence factors. However, E. faecalis exhibits extensive drug resistance. d-Alanine (D-Ala) metabolism is essential for bacterial peptidoglycan biosynthesis. d-cycloserine (DCS), a second line drug used in the treatment of Mycobacterium tuberculosis infection, can inhibit two key enzymes in D-Ala metabolism: alanine racemase and d-alanine-d-alanine ligase. The aim of this study was to evaluate the effect of D-Ala metabolism on E. faecalis growth, cell wall integrity, biofilm formation and virulence gene expression by additional DCS with or without D-Ala. The results showed that DCS inhibited the planktonic growth and biofilm formation of E. faecalis in a dose-dependent manner. Both the minimum inhibitory concentration (MIC) and minimum biofilm inhibition concentration (MBIC) of DCS against E. faecalis were 200 µg/ml, whereas 50 µg/ml of DCS could inhibit planktonic growth and biofilm formation effectively. The addition of DCS also resulted in bacterial cell wall damage, biofilm surface roughness increase and biofilm adhesion force reduction. Moreover, the treatment of DCS downregulated the expression of asa1, esp, efaA, gelE, sprE, fsrB and ace genes. However, all of these inhibitory effects of DCS could be rescued by the addition of exogenous D-Ala. Meanwhile, DCS exhibited no toxicity to HGEs and HOKs. Therefore, D-Ala metabolic pathway in E. faecalis is a potential target for drug designing.

The variation in the levels of 18 amino acids in the cortex and plasma of cerebral ischemia C57BL/6 mice.

Emerging evidence suggests that amino acid (AA) neurotransmitters play important roles in the pathophysiological processes of cerebral ischemia. In this work, an HPLC with fluorescence detection (HPLC-FLR) method was developed for the simultaneous determination of 18 AAs in the cortex and plasma after cerebral ischemia in mice. The ischemia model was prepared by bilateral common carotid artery occlusion, and then the cortex and plasma of the sham, ischemia, and naringenin groups were collected. Based on the protein precipitation method, a simple and effective sample preparation method was developed. The treated sample contained minimal proteins and lipids. The analysis of the sample was performed by the proposed HPLC-FLR method in combination with o-phthalaldehyde. The results showed a statistically significant increase in excitatory AAs (aspartic acid and glutamic acid), inhibitory AAs (glycine and 4-aminobutyric acid), phenylalanine, citrulline, isoleucine, and leucine levels, and a decrease of glutathione and phenylalanine levels when compared with the sham group in the cortex. Besides, the administration of naringenin had significant effects on excitatory AAs, inhibitory AA (glycine), glutamine, tyrosine, phenylalanine, and leucine levels when compared with the sham group in the cortex. These findings could be utilized in studying and clarifying the mechanisms of ischemia.

The immunogenic reaction and bone defect repair function of ε-poly-L-lysine (EPL)-coated nanoscale PCL/HA scaffold in rabbit calvarial bone defect.

Tissue engineering is a promising strategy for bone tissue defect reconstruction. Immunogenic reaction, which was induced by scaffolds degradation or contaminating microorganism, influence cellular activity, compromise the efficiency of tissue engineering, or eventually lead to the failure of regeneration. Inhibiting excessive immune response through modulating scaffold is critical important to promote tissue regeneration. Our previous study showed that ε-poly-L-lysine (EPL)-coated nanoscale polycaprolactone/hydroxyapatite (EPL/PCL/HA) composite scaffold has enhanced antibacterial and osteogenic properties in vitro. However, the bone defect repair function and immunogenic reaction of EPL/PCL/HA scaffolds in vivo remains unclear. In the present study, three nanoscale scaffolds (EPL/PCL/HA, PCL and PCL/HA) were transplanted into rabbit paraspinal muscle pouches, and T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), and macrophage infiltration were analyzed after 1 week and 2 weeks to detect their immunogenic reaction. Then, the different scaffolds were transplanted into rabbit calvarial bone defect to compare the bone defect repair capacities. The results showed that EPL/PCL/HA composite scaffolds decreased pro-inflammatory Th1, Th17, and type I macrophage infiltration from 1 to 2 weeks, and increased anti-inflammatory Th2 infiltration into the regenerated area at 2 weeks in vivo, when compared to PCL and PCL/HA. In addition, EPL/PCL/HA showed an enhanced bone repair capacity compared to PCL and PCL/HA when transplanted into rabbit calvarial bone defects at both 4 and 8 weeks. Hence, our results suggest that EPL could regulate the immunogenic reaction and promote bone defect repair function of PCL/HA, which is a promising agent for tissue engineering scaffold modulation.