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The present study was designed to assess genetic divergence between commercial bread wheat genotypes based on yield and its related traits and to carry out phenotypic correlation. Twenty bread hexaploid wheat varieties were assessed for mean performance, correlation analysis and genetic distance. Randomized complete block design was used with three replications during growing season, 2018-2019 at Wheat and Barley Research Institute, Tandojam. The mean squares depicted significant differences (P

The demand for high-quality forages is increasing in tropical regions, and could be filled with legume species of the genus Grona, which have good nutritive value. In this study, a comparison of the forage production and nutritive value of 10 accessions of Grona spp. was carried out in the field at Danzhou, Hainan from 2016 to 2018. Yield, plant height, survival rate, leaf:stem ratio and concentrations of crude protein, crude fiber, crude fat (ether extract), nitrogen free extract, crude ash, calcium and phosphorus were measured. Results showed that Grona strigillosa (syn. Desmodium strigillosum) cv. Reyan No. 27 and G. heterocarpa subsp. ovalifolia (syn. Desmodium ovalifolium) cv. Maquenque displayed the best performance, owing to their 261.3% and 235.6% higher dry matter yields, respectively, compared with the Control germplasm, G. heterocarpa subsp. ovalifolia cv. Reyan No. 16 in 2018. Cultivar Maquenque had a higher survival rate than the Control (P

Salivary exosomes contain various components and play an important role in oral diseases. We found that the expression of miR-223-3p in salivary exosomes was down regulated in inflammatory gingival tissue, and NLRP3 was the target of miR-223-3p. It has been reported that NLRP3 was involved in the formation of inflammasome and induced a type of cell death by cleaving gsdermin D (GSDMD), which is called pyroptosis. The purpose of this study was to investigate the role of miR-223-3p in NLRP3 inflammasome activation and pyroptosis. We found that miR-223-3p down regulated the activation of NLRP3, IL-1 β and caspase-1, and then released the pyroptosis of THP-1-derived macrophages inducing by Porphyromonas gingivalis -LPS ( P. gingivalis -LPS). In addition, NLRP3, and GSDMD was highly active in inflammatory gingival tissue compared with healthy controls. In summary, we hypothesized that miR-223-3p in salivary exosomes regulates GSDMD-mediated pyroptosis by targeting NLRP3. Detection of miR-223-3p expression in salivary exosomes could be used as an important non-invasive method to diagnose and evaluate the severity of periodontitis.

Objective:We examined whether breakfast frequency was associated with chronic inflammatory, as assessed by high-sensitivity C-reactive protein (CRP) concentration.Design:Cross-sectional study.Setting:Kailuan community, China.Participants:Included were 70 092 Chinese adults without CVD and cancer in 2014 with CRP concentrations Results:Breakfast frequency was associated with CRP concentration (P-trend < 0·001). The adjusted mean CRP was 1·33 mg/l (95 % CI 1·23, 1·44) for the ‘no breakfast’ group and 1·07 mg/l (95 % CI 1·0, 1·14) for the ‘breakfast everyday’ group (P-difference < 0·001), adjusting for age, sex, diet quality, total energy, obesity, education, occupation, marital status, smoking, alcohol consumption, blood pressure, sleep parameters, fasting blood glucose and lipid profiles. Consistently, the adjusted OR for CRP ≥ 1·0 mg/l and CRP ≥ 3·0 mg/l were 1·86 (95 % CI 1·73, 2·00) and 1·27 (95 % CI 1·15, 1·40), respectively, when comparing these two breakfast consumption groups (P-trend < 0·001 for both). The associations were more pronounced among older adults, relative to those who were younger (P-interaction < 0·001). Significant association between breakfast skipping and elevated CRP concentration was observed in those with poor diet quality, but not those with good diet quality.Conclusions:Habitually skipping breakfast is associated with elevated concentrations of CRP. Future prospective studies including repeated assessment of inflammatory biomarkers and a collection of detailed information on type and amount of breakfast foods are warranted.

The recovery of impaired periodontium is still a challenge to the treatment of periodontitis. This study was the first to apply the mesoporous hydroxyapatites/chitosan (mHA/CS) composite scaffold to periodontal regeneration. The aim of our study is to evaluate the biological effects of mesoporous hydroxyapatite/chitosan (mHA/CS) loaded with recombinant human amelogenin (rhAm) on periodontal regeneration. The physicochemical properties of mHA/CS scaffolds were examined by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and Brunauer–Emmett–Teller (BET) analysis. Then, the biological effects of the mHA/CS loaded with rhAm were evaluated, including antibacterial effect, controlled-release capacity, osteogenic and cementogenic effects in vitro and in vivo. The antibacterial effect was tested on 1.5 mg/mL CS; 3 mg/mL mHA; 2.25 mg/mL mHA/CS; 4.5 mg/mL mHA/CS and 20 μg/mL rhAm. Tryptic Soy Broth culture medium was used as a baseline control. Osteogenic effect of rhAm (20 μg/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 μg/mL rhAm) on human periodontal ligament cells (hPDLCs) was evaluated in osteogenic media. The hPDLCs treated either with osteogenic media or Dulbecco's modified Eagle's medium (DMEM) alone were used as the baseline control. In the animal model, 4-week-old nude mice (BALB/c) (n = 6) implanted with root slices subcutaneously were used to observe the cementogenic effect in vivo. The root slices were treated with rhAm (20 μg/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 μg/mL rhAm). The root slices treated with osteogenic medium alone were used as the baseline control. The analyses showed that the mHA/CS particles were 2 μm in diameter and had a uniform pore size. The mesoporous structure was 7 nm in diameter and its surface area was 33.95 m2/g. The scaffold exhibited antibacterial effects against Fusobacterium nucleatum and Porphyromonas gingivalis. The mHA/CS scaffold sustainably released rhAm. The mHA/CS loaded with 20 μg/mL rhAm upregulated ALP activity, the expression levels of osteogenesis-related genes and proteins in vitro. Additionally, it promoted the formation of cementum-like tissue in vivo. Our findings suggest that mHA/CS loaded with 20 μg/mL rhAm could inhibit the growth of periodontal pathogens and promote the formation of bone and cementum-like tissue.

Background Visceral obesity and fatty liver are prevalent in postmenopausal women. The stilbene-rich extract of Cajanus cajan (L.) Millsp. has been reported to prevent ovariectomy-induced and diet-induced weight gain in animal models, and stilbenoids from C. cajan are thought to have the potential to prevent postmenopausal obesity and fatty liver. Purpose Cajanolactone A (CLA) is the main stilbenoid from C. cajan with osteoblastogenic promoting activity. This study investigated the potential of CLA to prevent postmenopausal obesity and fatty liver. Underlying mechanisms were also investigated. Method Ovariectomized C57BL/6 mice fed a regular diet were used as mimics of postmenopausal women and given 10, 20, or 40 mg/kg/d of CLA, 0.1 mg/kg/d of estradiol valerate (EV, positive control), or vehicle (OVX) orally for 16 weeks. Mice of the same age subjected to a sham operation were used as control (Sham). Body weights were recorded every 2 weeks for 16 weeks. Body compositions were analyzed via micro-CT. Serum levels of lipids, adipocytokines and aminotransferases were measured using the relevant kits. mRNA levels of genes of interest were detected by RT-qPCR. Proteomic study of perigonadal white adipose tissue (pWAT) was performed using tandem-mass-tags-based proteomic technology combined with Parallel-Reaction-Monitoring (PRM) validation. Results CLA showed potential equivalent to that of EV to prevent ovariectomy-induced overweight, obesity, dyslipidemia, liver steatosis and liver dysfunction, but did not prevent uterine atrophy. In the liver, CLA significantly inhibited ovariectomy-induced upregulation in expression of lipogenic genes SREBP-1c and ChREBP, and stimulated the mRNA expression of apolipoprotein B gene ApoB. In pWAT, CLA reversed, or partially reversed ovariectomy-induced downregulation in the expression of a number of metabolism- and mitochondrial-function-related proteins, including Ndufa3, Pcx, Pdhb, Acly, Acaca, Aldh2, Aacs and Echs1. In addition, ovariectomy-inhibited mRNA expression of Pdhb, Aacs, Acsm5, Echs1, and Aldh2 genes in pWAT was also reversed. Conclusion CLA was demonstrated to be a potential non-estrogen-like drug candidate for prevention of postmenopausal obesity and fatty liver. The underlying mechanism might involve the inhibition of lipogenesis and promotion of triglycerides output in the liver, and the promotion of metabolism and mitochondrial functions of visceral white adipose tissue.

The overall aim of this research was to investigate the differences in the expression of programmed death ligand 1 (PD-L1) in human gingival fibroblasts (HGFs) between a periodontal healthy group and a periodontal inflammatory group. and explore the possible mechanism involved.Differences in PD-L1 mRNA and protein expression in HGFs from a periodontal healthy group and a periodontal inflammatory group were examined by qPCR and western blotting, respectively, and were further tested after lipopolysaccharide (LPS) stimulation in both groups. The effects of a p38 pathway inhibitor on the changes in p38 phosphorylation levels and PD-L1 expression after LPS stimulation were investigated in both groups.PD-L1 mRNA and protein levels in HGFs in the periodontal inflammatory group were significantly higher than those in the periodontal healthy group (p0.05). After 10 μg/mL LPS stimulation, PD-L1 mRNA levels in HGFs from both groups increased significantly (p0.05), peaking at 4 h, and the peak was significantly higher in the periodontal inflammatory group than in the periodontal healthy group (p0.05). However, PD-L1 protein expression was upregulated only in the inflammatory group (p0.05). Inhibition of the p38 pathway in HGFs decreased p38 phosphorylation in both groups (p0.05) but this treatment reversed the LPS-induced increase in PD-L1 mRNA and protein levels only in the inflammatory group (p0.05).In the periodontal inflammatory state, the expression of PD-L1 in HGFs is more easily activated, and may be influenced by the p38 pathway.

Large numbers of viable cells cannot be obtained from periodontal ligament tissues of patients with periodontitis. Therefore, it is imperative to establish an ex vivo environment that can support cell proliferation and delay senescence. Here, we have successfully reconstructed a native extracellular matrix (ECM), derived from early-passage human periodontal ligament cells (PDLCs) using the NH4 OH/Triton X-100 protocol. The ECM was investigated by scanning electron microscopy and immunostaining for specific ECM proteins (collagen I and fibronectin). Late-passage ECM-expanded PDLCs exhibited a much higher proliferation index and lower levels of reactive oxygen species (ROS), confirmed by the increased expression of pluripotent markers and enhanced osteogenic capacity. Interestingly, the Wnt pathway was suppressed during the ECM expansion-mediated increase in pluripotency, but was activated in an osteogenic differentiation environment, as confirmed by treatment with the XAV-939 β-catenin inhibitor or the SP600125 c-Jun N-terminal kinase (JNK) inhibitor. Cell sheets formed by ECM-expanded PDLCs exhibited an enhanced periodontal tissue regeneration capacity compared to those formed on tissue culture polystyrene (TCP) surfaces in vivo. Taken together, the cell-free ECM provides a tissue-specific cell niche for the ex vivo expansion of PDLCs while retaining stemness and osteogenic potential, partially via the Wnt pathway. This represents a promising matrix for future applications in periodontal tissue regeneration therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 272-284, 2018.

A lot of previous studies have recently reported that the gut microbiota influences the development of colorectal cancer (CRC) in Western countries, but the role of the gut microbiota in Chinese population must be investigated fully. The goal of this study was to determine the role of the gut microbiome in the initiation and development of CRC. We collected fecal samples of 206 Chinese individuals: 59 with polyp (group P), 54 with adenoma (group A), 51 with colorectal cancer (group CC), and 42 healthy controls (group HC).16S ribosomal RNA (rRNA) was used to compare the microbiota community structures among healthy controls, patients with polyp, and those with adenoma or colorectal cancer. Our study proved that intestinal flora, as a specific indicator, showed significant differences in its diversity and composition. Sobs, Chao, and Ace indexes of group CC were significantly lower than those of the healthy control group (CC group: Sobs, Chao, and Ace indexes were 217.3 ± 69, 4265.1 ± 80.7, and 268.6 ± 78.1, respectively; HC group: Sobs, Chao, and Ace indexes were 228.8 ± 44.4, 272.9 ± 58.6, and 271.9 ± 57.2, respectively). When compared with the healthy individuals, the species richness and diversity of intestinal flora in patients with colorectal cancer were significantly reduced: PCA and PCoA both revealed that a significant separation in bacterial community composition between the CC group and HC group (with PCA using the first two principal component scores of PC1 14.73% and PC2 10.34% of the explained variance, respectively; PCoA : PC1 = 14%, PC2 = 9%, PC3 = 6%). Wilcox tests was used to analyze differences between the two groups, it reveals that Firmicutes (P=0.000356), Fusobacteria (P=0.000001), Proteobacteria (P=0.000796), Spirochaetes (P=0.013421), Synergistetes (P=0.005642) were phyla with significantly different distributions between cases and controls. The proportion of microorganism composition is varying at different stages of colon cancer development: Bacteroidetes (52.14%) and Firmicutes (35.88%) were enriched in the healthy individuals; on the phylum level, the abundance of Bacteroidetes (52.14%-53.92%-52.46%–47.06%) and Firmicutes (35.88%-29.73%-24.27%–25.36%) is decreasing with the development of health-polyp-adenomas-CRC, and the abundance of Proteobacteria (9.33%-12.31%-16.51%–22.37%) is increasing. PCA and PCOA analysis showed there was no significant (P<0.05) difference in species similarity between precancerous and carcinogenic states. However, the composition of the microflora in patients with precancerous lesions (including patients with adenoma and polyp) was proved to have no significant disparity (P<0.05). Our study provides insights into new angles to dig out potential biomarkers in diagnosis and treatment of colorectal cancer and to provide scientific advice for a healthy lifestyle for the sake of gut microbiota.

OBJECTIVES: Previous studies have shown that regular breakfast consumption was associated with lower risk of cardiovascular disease (CVD), but this remains controversial. Chronic inflammation is a well-established risk factor for cardiovascular disease. We, thus, examined whether breakfast frequency was associated with inflammation, assessed by high sensitivity C-reactive protein (CRP) concentration, among individuals without CVD. METHODS: Included were 71,748 participants of the Kailuan Study, an ongoing Chinese cohort, who were free of CVD and cancer.Breakfast frequency was assessed via questionnaire in 2014, and participants were categorized into four groups in the current analysis –no breakfast, 1–2 times/week, 3–5 times/week, or breakfast every day. Plasma CRP concentration was measured using a high-sensitivity, particle-enhanced immunonephelometry assay. General linear models were used to calculate adjusted means with 95% confidence intervals (CIs) for CRP, and logistic regression models were used to calculate odds ratios (ORs) of chronic inflammation (CRP concentration ≥1.0 mg/L or ≥3.0 mg/L), across the four breakfast groups. We adjusted for age, sex, diet quality score, body mass index, education level, occupation type, marital status, smoking status, systolic blood pressure, fasting blood glucose, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in the models. RESULTS: Greater breakfast frequency was associated with lower CRP concentration (P-trend

The aging periodontium may be vulnerable to periodontal pathogens and poor response to inflammation and susceptible to tumorigenesis. Human gingival fibroblasts (hGFs) through continuously replicative culture served as an in vitro surrogate for aging. To investigate the effects of the mechanistic target of rapamycin (mTOR) inhibition on the aging gingiva, we stimulated the high-passage hGFs with rapamycin (20 nmol/L) for 3 days and 30 days. The cellular and biological changes were examined by immunofluorescence, real-time PCR, ELISA, Western blotting, and flow cytometry. The data demonstrated that the inhibition of mTOR signaling led to fewer senescence-associated beta-galactosidase- (SA-β-Gal-) positive cells, delayed the onset of senescence, preserved the capability of proliferation, and lowered the expression levels of relevant senescence-associated markers, such as p16INK4a, p21CIP1a, interleukin-6 (IL-6), and IL-8. In addition, when infected by prominent periodontal pathogens, Porphyromonas gingivalis (ATCC 33277), rapamycin-pretreated groups decreased the expression of inflammatory cytokines (IL-6 and IL-8) compared with the control group. mTOR inhibition upregulated the gene expression of antioxidant components (Cat, Sod2, and Prdx3; P<0.05) and consequently neutralized the excessive reactive oxygen species (ROS). In conclusion, our results indicated that mTOR inhibition might rejuvenate the aging gingiva to some extent and relieve inflammation through eliminating oxidative stress.

The maternal or paternal dietary composition can have important effects on various aspects of their offspring's physiology. Studies from animal models and humans showed that a maternal high-fiber diet protected offspring against fat accumulation. However, little is known about how a maternal low-fiber diet modifies the metabolism of offspring in herbivorous rodents. We hypothesized that a maternal low-fiber diet would confer long-lasting beneficial effects on offspring metabolic phenotypes in herbivorous Brandt's vole (Lasiopodomys brandtii). Female voles were fed either a control (12.4% fiber) or a low-fiber (3.5% fiber) diet throughout pregnancy and lactation, and all offspring were fed the control diet after weaning till 14 wk old. Offspring were sampled from each litter at 18 d and 14 wk of age. Another subset of adult offspring at 15 wk of age was fed a high-fat diet for 8 wk. We found that there was no difference in litter size, litter mass, or pup mass before weaning between the two maternal diet groups. Offspring from the maternal low-fiber diet increased energy intake, body mass, and lean mass; suppressed fat accumulation; and improved glucose tolerance compared with those from the control diet. Moreover, the maternal low-fiber diet alleviated high-fat diet-induced obesity in the adult offspring. Serum leptin concentration and uncoupling protein 1 content in brown adipose tissue of offspring were not affected by a maternal low-fiber diet. We demonstrate that herbivorous females fed a low-fiber diet during pregnancy and lactation may predispose their offspring to accelerated growth of lean tissue, which may increase the opportunity for survival and reproduction in offspring.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 μM and 200 μM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 μM, and a respective minimum bactericidal concentration (MBC) of 12.5 μM and 25 μM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 μM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 μM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 μM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 μM EPA. For exponential-phase bacteria, 100 μM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 μM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of periodontal diseases.

Hybrid poplars are important economic plants and sources of biofuel all over the world. Poplars are subjected to a wide range of diseases (Cellerino 1999). In September 2019, powdery mildew was observed on the hybrid poplar Populus davidiana × P. alba var. pyramidalis, a fast-growing elite poplar cultivar widely planted in the northeast of China, in one poplar nursery room in the Northeast Forestry University in China (45.7266°N, 126.6380°E). Disease incidence during September and October was only 5 to 15% but gradually increased to 100% during November and December. The symptom of white circular powdery spots with diameters of up to 12 mm appeared on the upper surface of the mature leaves of poplar plants over 40 days old. Under nursery conditions (50 to 65% humidity, 20 to 25°C, and 16-h/8-h day/night cycles), the diameter of a mildew spot could develop from merely visible to 10 mm in 3 days. The mildew site was observed with a stereomicroscope (Leica DVM6S, Germany). A single fungal species demonstrating characteristics of the obligate pathogens Erysiphe spp. was observed. The conidiophores grew straight upward on the leaf surface until reaching 102 to 165 μm in length (n = 30). Then, even-looking, ellipse-shaped conidia were developed, forming a singular, curved string of up to tens of conidia, on top of each conidiophore. Conidia (n = 30) measured 29.4 to 35.1 × 18.1 to 21.0 μm. Appressoria were lobed or nipple-shaped and appeared in pairs or singular. The germ tubes were slightly curly unbranched tubes pointing at one direction near the long axis of the conidium. We carefully collected the mildew by scraping it off the upper surfaces of fresh diseased leaves with a scalpel and extracted the genomic DNA. The internal transcribed spacer rDNA region was amplified according to the method of Siahmard et al. (2017). The resulting nucleotide sequence (deposited in GenBank as MN998572) displayed 99.84% identity with that of Erysiphe adunca (KY653179.1). Hence, we identified the causal pathogen of this poplar powdery mildew as an E. adunca strain. Furthermore, an Mcm7 sequence (deposited as MN998573) and a Chs sequence (deposited as MN998574) were cloned according to the method of Ellingham et al. (2019). A voucher specimen and the sequence information have been deposited in the Northeast Forestry University Culture Collection (NECC) and named as NECCFP001. Pathogenicity was tested in the nursery, by merely placing healthy 40-day-old plants next to diseased plants (n = 10, with a 15 cm distance between plants), and by pressing the mildew sites of diseased leaves onto each mature leaf of the healthy plants (n = 10, also with 15 cm distance). The same symptoms appeared on heathy plants after 10 to 15 days, and 6 to 8 days, respectively. The induced mildew was confirmed morphologically to be NECCFP001 by microscopic examination. E. adunca occurs on aspen, willow, and Populus spp. throughout temperate regions of the Northern Hemisphere and forms fruiting bodies (chasmothecia) to overwinter (Horst 2001; Sinclair and Lyon 2005). However, chasmothecia were not observed in our case, suggesting no sexual morph under the nursery conditions. Incidents of powdery mildew on hybrid poplars caused by Phyllactinia populi had been reported in Jiangsu and Shandong provinces of China (Zhao et al. 2013). This is the first report of E. adunca causing poplar powdery mildew in Heilongjiang, China.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune condition. Because the immune system develops early in life, it is possible that exposure to adversity like famine in utero or during early childhood may have lifelong impacts on risk of autoimmune disease. We thus investigated whether exposure (in utero or in early life) to the Great Chinese Famine of 1959–1961 was associated with risk of RA in adulthood. METHODS: Included were 101,510 participants of the Kailuan Study that joined the study at baseline (2006). RA cases were confirmed by medical record review. We used logistic regression to calculate the odds ratio (OR) and 95% confidence interval (95% CI) for RA, according to famine exposure status (exposed in utero, between 0 and 3 years, between 3 and 6 years, or at 6 years or older), in comparison to participants born after 1961 (not exposed to famine). Potential confounders (e.g., sex, body mass index, smoking status, and plasma concentrations of c-reactive protein, low-density lipoprotein and high-density lipoprotein) were adjusted in the model. RESULTS: During 12 years of follow-up (2006–2018), we identified 187 RA cases. Individuals exposed to the Famine in utero or in ages 0–3 years, had a higher prevalence of RA, relative to other groups (0.24%–0.35% vs 0.08–0.20%). After adjustment for potential confounders, the OR of RA was 2.70 (95% CI: 1.40 to 5.21) for in utero famine exposed individuals, 4.44 (95% CI: 2.66 to 7.39) for those exposed in age 0–3 y, 2.50 (95% CRI: 1.40 to 4.47) for those exposed in age 3–6 y, and 2.61 (95% CI: 1.63 to 4.18) for those exposed after at least 6 years old versus individuals who were born after 1961. A similar association was observed for men and women (P-interaction = 0.86). CONCLUSIONS: Individuals with exposure to famine in utero or early childhood (0–3 years), were more likely to suffer from RA in adulthood. This study reflects the importance of early life as a key developmental period for the immune system, and demonstrates that exposure to famine during this time results in increased risk of RA in adulthood. FUNDING SOURCES: Study funded by start-up grant from the College of Health and Human Development and the Department of Nutritional Sciences, The Pennsylvania State University, and Natural Science Foundation of Hebei Province (H2018209318).

Food quality can affect many physiological characteristics in small mammals. Reproduction is a highly energy-demanding period especially for the females to produce and feed their offspring. We hypothesized that energy intake was constrained at different levels in nonreproductive and reproductive females and thus they adopted diverse energy strategies in response to diet changes. Here, we tested the effects of low fiber diet (3.5% vs. 12.4%) on energy intake and thermogenesis in nonreproductive and reproductive Brandt’s voles (Lasiopodomys brandtii (Radde, 1861)), a herbivorous species. We found that the voles decreased food intake while keeping a stable digestible energy intake (DEI) in response to the low fiber diet, but DEI was increased in reproductive voles at peak lactation. Uncoupling protein 1 content in brown adipose tissue decreased in nonreproductive voles, but was stable in reproductive voles on the low fiber diet. Litter mass on day 18 of age tended to increase in the low fiber group compared with that in the control group. Our findings demonstrate that the voles have a target intake to maintain energy balance when diet composition changes and energy intake may be constrained at a high level for the reproductive voles to improve their offspring’s fitness.

Hypoxia is a crucial microenvironment for inflamed periodontal tissue and periodontal wound healing. Enamel matrix proteins (EMPs) potentially can promote the formation of new periodontium. The effects of EMPs on periodontal ligament cells under hypoxia, however, remain unclear. We investigated the effects of EMPs on cellular biobehavior and osteogenic differentiation of human periodontal ligament cells (hPDLCs) under hypoxia. Under cobalt chloride (CoCl2)-induced hypoxia, cellular biobehavior of hPDLCs, including proliferation, attachment, spreading, and migration with or without EMPs, was evaluated by 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), cell counting, spreading area measurement and wound scratch assay. The osteogenic activity of hPDLCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining (ARS). The expressions of osteogenic genes including runt related transcription factor 2 (Runx2), ALP, osteocalcin (OCN) and collagen type I (Col-I) were detected using real time quantitative PCR, western blot and immunocytochemistry assays. The biobehavior and osteogenic differentiation of hPDLCs were inhibited significantly under hypoxia. EMPs have no effect on cell proliferation under mimicked hypoxia. EMPs partly reversed the inhibitory effects of hypoxia, however, for other cellular biobehavior including attachment, spreading and migration, and markedly up-regulated osteogenic differentiation activities including ALP, mineralization ability and the expressions of osteogenic genes such as Runx2, ALP, osteocalcin, and collagen type I in hPDLCs under hypoxia. EMPs attenuate the hypoxic injury to cellular biobehavior and osteogenic differentiation in hPDLCs under hypoxia.

Background In gingival tissues, lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) is the most critical stimulator for inducing inflammatory response. Human gingival fibroblasts (HGFs) are the major constituents of gingival connective tissues. The aim of this study was to investigate P. gingivalis LPS induced whole transcriptional profile in HGFs and the potential crosstalk between microRNAs (miRNAs) and inflammatory cytokines. Methods RNA-seq was performed on HGFs with and without P. gingivalis LPS treatment. The gene expression of selected inflammatory cytokines and miRNAs induced by LPS at different time points was evaluated by quantitative RT-PCR. The protein expression of chemokines was further confirmed by ELISA. Results Interestingly, most of the significantly changed genes (198/204) were up-regulated at 4 h after 10 μg/ml LPS stimulation, including inflammatory cytokines and miRNAs. Confirmed by quantitative RT-PCR, the mRNA levels of IL-1β, IL-6 and IL-8 showed single up-regulation peak (4 h/6 h) after 1 μg/ml and 10 μg/ml LPS treatment. Similarly, 1 μg/ml LPS induced single up-regulation peak (8 h) of miRNA-146a, −146b and −155 expression. However, 10 μg/ml LPS induced the increased expression of miRNA-146a and −155 at both early stage (2 h/4 h) and late stage (24 h). Conclusion Taken together, we investigated P. gingivalis LPS induced whole transcriptional profile, and the different behaviors of miRNA expression induced by different doses of LPS in HGFs.

The aim of this study was to evaluate the potential antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm modes of Streptococcus mutans (S. mutans). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The effects on planktonic growth and biofilm metabolic activity were evaluated by growth curve determination and MTT assay, respectively. Then, colony forming unit (CFU) counting, scanning electron microscopy (SEM) and real-time PCR were performed to further investigate the actions of DHA and EPA on exponential phase-S. mutans. Confocal laser scanning microscopy (CLSM) was used to detect the influences on mature biofilms. The MICs of DHA and EPA against S. mutans were 100 μM and 50 μM, respectively; the MBC of both compounds was 100 μM. In the presence of 12.5 μM–100 μM DHA or EPA, the planktonic growth and biofilm metabolic activity were reduced in varying degrees. For exponential-phase S. mutans, the viable counts, the bacterial membranes and the biofilm-associated gene expression were damaged by 100 μM DHA or EPA treatment. For 1-day-old biofilms, the thickness was decreased and the proportion of membrane-damaged bacteria was increased in the presence of 100 μM DHA or EPA. These results indicated that, DHA and EPA possessed antibacterial activities against planktonic and biofilm growing S. mutans.

Objective Chronic periodontitis is a chronic inflammatory disease of the periodontal tissues and is caused by invasion of certain types of bacteria and Archaea , with Methanobrevibacter oralis as the predominant archaeon. In this study, we investigated the prevalence and quantity of the newly discovered Archaea phylotype Thermoplasmata in patients with chronic periodontitis. Methods Subgingival plaque samples were obtained from 49 patients with chronic periodontitis and 45 periodontally healthy subjects. Qualitative analyses of Archaea and class Thermoplasmata were carried out by amplification of 16S rRNA genes in DNA extracts from plaque samples, and all the samples were quantitatively analyzed by real-time polymerase chain reaction (PCR). Results The prevalence of Archaea in patients with chronic periodontitis was 69.4% according to the conventional PCR results, but was 87.8% according to real-time PCR. In the control group, three samples were detected as positive, but none of these were confirmed in qualitative analyses. The prevalence of class Thermoplasmata was 18.4% by nested PCR and 24.5% by quantitative PCR in the chronic periodontitis group. The prevalence of Thermoplasmata was significantly lower than that of total Archaea. The relative abundances of Archaea and Thermoplasmata varied among samples. Thermoplasmata were not the predominant archaeons in the subgingival dental plaque. Among the clinical parameters of patients with periodontitis, probing depth was positively associated with Archaea detection. Conclusions The existence of Archaea was correlated closely with the presence of chronic periodontitis. Thermoplasmata represented a minor archaeon in periodontal infection.

The aim of this study was to develop new Prevotella intermedia-specific PCR primers based on the 16S rRNA. The new primer set, Pi-192 and Pi-468, increased the accuracy of PCR-based P. intermedia identification and could be useful in the detection of P. intermedia as well as epidemiological studies on periodontal disease.

For the first time, we used Tullgren method made a study on vertical migrating and cluster analysis of the soil mesofauna in Dongying Halophytes Garden in the Yellow River Delta (YRD), Shandong Province. The results showed that the soil mesofauna tended to gather on soil surface in most samples at most times, but the vertical migrating greatly varied in different seasons or environment conditions. Acari was the dominant group. The index of diversity of the soil fauna was correlated with the index of evenness. The Acari's number of individuals infected other species and numbers. Dominant group-Acari made greater contribution to the result of cluster analysis, and there were significant differences between communities in different habitats by cluster analysis with both Bray-Curtis and Jaccard similarity coefficient.

This study aims to investigate the nitrous oxide (N2O) emission in an aerobic granulation sequencing batch airlift reactor (SBAR) and the associated microbial community of aerobic granular sludge at ambient temperature (18 ± 3)°C. After 48 days of operation, 1–2 mm granules were obtained and excellent chemical oxygen demand (COD) and ammonium ( NH 4 + − N ) removal efficiencies were stably achieved. N2O concentration in the off gas was maximal at the beginning of the aerobic period and stabilized at a lower concentration after an initial peak. (0.60 ± 0.17, n = 3) % of the total nitrogen load to the SBAR was emitted as N2O. A dramatic change in the microbial community structure was noted between the initial seed sludge and the final mature aerobic granular sludge. Nitrosospira was identified to be the dominant ammonium oxidizing bacteria (AOB) which was attributed as the dominant source of N2O production in aerobic granular sludge by analysis of 16S rDNA sequences.

Aims: The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone-like DNA binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity. Methods and Results: Formed biofilm mass was measured with 0.1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7-hydoxyl-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and anti-HLP antibody without fixation, respectively. DNase I treatment (200 U ml-1) markedly decreased biofilm formation and cell density in biofilms. Co-localization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml-1) purified from S. intermedius, other Gram-positive, –negative bacteria, or human KB cells into the S. intermedius culture increased the biofilm mass of all tested strains of S. intermedius, wild-type, HLP down-regulated strain, and control strains. In contrast, the addition of eDNA (> 1 μg ml-1) decreased the biofilm mass of all S. intermedius strains. Conclusions: These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity. Significance and Impact of the Study: eDNA- and HLP-targeting strategies may be applicable to novel treatments for bacterial biofilm-related infectious diseases.

Objectives Amelogenins are proposed to be responsible for enamel matrix derivative (EMD)-induced periodontal regeneration; however, heterogeneity of amelogenins makes it challenging to purify the full-length proteins. This study has been carried out to express and purify a recombinant full-length human amelogenin protein (rHhAm175) in the eukaryotic yeast Pichia pastoris, and further compare biological responses of human periodontal ligament fibroblasts (PDLFs) to rHhAm175 and porcine EMD (pEMD). Materials and methods Human cDNA encoding a 175-amino acid amelogenin was subcloned into the pPIC3.5K vector. The rHhAm175 expressed in P. pastoris GS115 (Mut+) was purified and characterized. We examined cell attachment, migration and proliferation responses of human PDLFs to rHhAm175 and pEMD respectively, and characterized associated changes of proliferation-related intracellular signalling molecules, including extracellular signal response kinase (ERK) and Akt kinases/protein kinase B (Akt/PKB) kinases. Results The purified rHhAm175 was confirmed to be molecular mass 22 021.13 Da, phosphorylated human amelogenin, and alone significantly promoted proliferation and migration of human PDLFs to an extent comparable to that of pEMD. Cell attachment was increased over the first 60 min incubation with rHhAm175 or pEMD. Both rHhAm175 and pEMD induced PDLF mitogenesis via extracellular signal response kinase (ERK1/2), but not by Akt kinases/protein kinase B (Akt/PKB). Conclusions rHhAm175 modulated cell activities of human PDLFs, to a comparable extent as porcine EMD. These data suggest that rHhAm175 might be used to induce periodontal tissue regeneration.

Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) has been reported to induce the expression of vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelial cells. This finding suggests the potential roles for Pg in the pathogenesis of atherosclerosis. However, the mechanism involved in Pg LPS-induced VCAM-1 production in endothelial cells remains unclear.Quantitative real-time polymerase chain reaction and Western blotting were used, respectively, to investigate the mRNA expression and protein production of VCAM-1 in human aortic endothelial cells (HAECs) induced by Pg LPS. The involvement of the p38 mitogen-activated protein kinase (p38 MAPK) cell signaling pathway in VCAM-1 expression was investigated by assays with specific inhibitors.Pg LPS-induced expression in HAECs of VCAM-1 occurred in a dose- and time-dependent manner. In addition, the p38 MAPK inhibitor (SB 203580) significantly attenuated Pg LPS-induced VCAM-1 expression.Activation of p38 MAPK is at least partially involved in Pg LPS-induced VCAM-1 expression in HAECs, which may contribute to the acceleration of atherosclerosis.

NF-κB plays a major role in the pathology of multiple myeloma. Here, we intended to investigate the regulating effect of cardamonin on NF-κB in myeloma cells. We found for the first time that cardamonin suppressed viability and induced apoptosis of myeloma cells. Cardamonin activated caspase-3 and PARP and suppressed the expression of various anti-apoptotic proteins. We discovered that NF-κB was repressed by cardamonin through suppression of IKK expression and IκBα phosphorylation. Furthermore, the expression of NF-κB-regulated gene products ICAM-1, COX-2 and VEGF was down-regulated by cardamonin. These results suggest that cardamonin blocks NF-κB pathway in human multiple myeloma cells.

X-linked retinitis pigmentosa (XLRP) is the most severe type of retinitis pigmentosa (RP), with patients consistently showing early onset and rapid deterioration. Obtaining a genetic diagnosis for a family with XLRP is important for counseling purposes. In this study, we aimed to identify disease-causing mutations in two unrelated XLRP families. Genetic analysis was performed on two unrelated XLRP families. Genomic DNA was extracted from peripheral blood or amniotic fluid samples. The coding regions and intron/exon boundaries of the Retinitis Pigmentosa GTPase Regulator (RPGR) and RP2 genes were amplified by PCR and then sequenced directly. A clinically unaffected pregnant female and the four month old fetus were found to have a hemizygous 2 base pair deletion (g.ORF15+484_485delAA) in the exon ORF15 of RPGR gene. In another XLRP family, a nonsense mutation (g.ORF15+810G>T) was identified. Neither mutation has been reported previously. Both are predicted to cause premature termination of the protein. In conclusion, we identified a micro-deletion through prenatal genetic diagnosis and another novel nonsense mutation in RPGR-ORF15. Identifying a disease-causing mutation facilitated early diagnosis and genetic counseling for the patients. Discovery of novel mutations also broadens knowledge of XLRP and the spectrum of its pathogenic genotypes.

Objectives: Enamel matrix proteins (EMPs) have been demonstrated to promote periodontal regeneration. However, effects of EMPs on human alveolar osteoblasts (hAOBs), up to now, have still been unclear. The purpose of this study was to investigate influence of EMPs on proliferation, differentiation and attachment of hAOBs in vitro. Materials and methods: EMPs were extracted using the acetic acid method, hAOBs were obtained and cultured in vitro. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of osteogenic markers and cell attachment were measured in the absence and in the presence of EMPs (50, 100 and 200 μg/ml). Results: EMPs increased proliferation of hAOBs; however, they inhibited ALP activity and mRNA expression of osteogenic markers (collagen I, ALP, runt-related protein 2, osteocalcin, bone sialoprotein and osteopontin). Meanwhile, EMPs hindered hAOBs’ attachment. These effects occurred in EMPs concentration-dependent manner. Conclusions: These results indicate that EMPs may inhibit osteoblastic differentiation and attachment to prevent ankylosis and allow other cell types to regenerate periodontal tissues.

DGGE of 16S rDNA is one of the most frequently used methods to study microbial communities. In this study, the DGGE profiles of different 16S rDNA regions of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella nigrescens were investigated. The results suggested that V3-V5 and V6-V8 fragments may be suitable for community analysis of subgingival bacteria. Further analysis of subgingival samples with V3-V5 and V6-V8 regions as target fragments suggested that, in chronic periodontitis, re-colonization by periodontal bacteria with a population very similar to the baseline may occur by 6 weeks after mechanical debridement.

Objectives: Age-related changes are common in many tissues and organs. However, cell-related causes in human alveolar bone remain unclear. This study has been carried out to explore the possibility that advancing age might change the biological characteristics of alveolar osteoblasts (AOBs) in women. Materials and methods: Alveolar osteoblasts from women donors (five women aged 33–38 years and five women aged 62–68 years) were cultured in vitro. The cells were serially passaged and maximal lifespan evaluated. Cell viability, ultramicrostructure and osteogenic differentiation ability were determined respectively, using MTT assay, transmission electron microscopy, alkaline phosphatase (ALP) activity assay and von Kossa staining assay. These parameters of the two groups of AOBs were evaluated. Results: When compared with cells from young adult donors, AOBs from elderly women exhibited lower maximal lifespan (P

Oral Diseases (2010) 16, 343–350 Objective: Toll-like receptors (TLRs) are the essential components in the innate and adaptive immune systems. Colony stimulating factor 2 (CSF2) is a cytokine that may prevent endotoxin tolerance, and LY64 has the ability to interfere with the recognition of bacteria via TLR4. The aim of this study was to explore the in vivo expressions of TLR2, TLR4, CSF2 and LY64 in Chinese chronic periodontitis patients. Methods: Gingival biopsies were collected from 24 chronic periodontitis patients and 19 healthy controls. The gene expression profiles of TLR2, TLR4, CSF2 and LY64 were investigated by real-time polymerase chain reaction, and the protein expressions of TLR2 and TLR4 were detected by immunohistochemistry. In addition, the levels of CSF2 in gingival crevicular fluid (GCF) were determined by ELISA. Results: The higher mRNA expressions of TLR2, TLR4 and CSF2, and the lower mRNA expression of LY64 were detected in chronic periodontitis patients. And the increased protein expressions of TLR2 and TLR4 were confirmed by immunohistochemistry. In addition, the increase of total amount of CSF2 in GCF was observed in chronic periodontitis patients. Conclusions: Our results suggest that TLR2 and TLR4 may play a role in periodontal pathogenesis. In addition, CSF2 and LY64 may contribute to the regulation of inflammatory response and maintaining periodontal homeostasis.

Objectives: The aim of this study was to investigate biological effects and gene expression profiles of enamel matrix proteins (EMPs), on human bone marrow stromal cells (HBMSCs), for preliminary understanding of mechanisms involved in promoting periodontal regeneration by EMPs. Materials and methods: EMPs were extracted using the acetic acid method, and HBMSCs from human bone marrow aspirates were cultured. Attachment levels, level of cells morphologically attenuated, cell proliferation, alkaline phosphatase (ALP) activity and staining of HBMSCs were measured in the absence and in the presence of EMPs. Microarray analysis was performed to detect gene profiles of HBMSCs by treatment with 200 μg/ml EMPs, for 5 days. Four differential genes were selected for validation of the microarray data using real-time PCR. Results: EMPs promoted proliferation and ALP activity of HBMSCs in a time- and dose-dependent manner, and at a concentration of 200 μg/ml significantly enhanced proliferation and ALP expression. However, there were no significant changes between EMP-treated groups and the control group in cell attachment and cell process attenuation levels. Twenty-seven genes were differentially expressed by HBMSCs in the presence of EMPs. Expressions of 18 genes were upregulated and expressions of nine genes were found to be downregulated. There was good consistency between data obtained from the validation group and microarray results. Conclusions: EMPs promoted cell proliferation and differentiation and gene expression profiles of HBMSCs were affected. This may help elucidation of mechanisms involved in promoting regeneration of periodontal tissues by EMPs.

Background and Objective: Salivary proteomics technology can be used to evaluate the disease progession of periodontitis and the systemic screening of proteomes of saliva from subjects with aggressive periodontitis has not been available. The objective of this preliminary study was to compare the proteomic profile of whole unstimulated saliva of subjects with generalized aggressive periodontitis (GAgP) with that of healthy volunteers to identify proteins, the levels of which were significantly altered between the two groups. Material and Methods: Whole unstimulated saliva was obtained from five subjects with GAgP and five healthy subjects, and proteins were separated using two-dimensional gel electrophoresis. Proteins, the levels of which were significantly different between the two groups, were identified by computer image analyses and subsequent electrospray ionization tandem mass spectrometry. Results: Eleven proteins that exhibited a different level in the GAgP group vs. the control group were identified. Compared with whole saliva of healthy control subjects, the levels of serum albumin, immunoglobulin (Ig) γ2 chain C region, Ig α2 chain C region, vitamin D-binding protein, salivary α-amylase and zinc-α2 glycoprotein were increased in whole unstimulated saliva of GAgP subjects, while those of lactotransferrin, elongation factor 2, 14-3-3 sigma, short palate, lung and nasal epithelium carcinoma-associated protein 2 precursor and carbonic anhydrase 6 were decreased. Conclusion: Comparison of the proteomic profile of whole unstimulated saliva of GAgP subjects with that of healthy control subjects revealed at least 11 differential proteins. The approach applied herein might be helpful to aid understanding of the etiology of GAgP.

Current epidemiologic data suggest that a localized infection (periodontitis) can disseminate into the distant tissues, and subgingival bacteria can migrate in the bloodstream, thereby contributing to independent systemic disease processes. To test this hypothesis, we investigated the effect of repeated systemic inoculations with Porphyromonas gingivalis (Pg) on intimal hyperplasia in iliac arteries in a rabbit model of balloon injury.One week after single balloon injury to the iliac artery, 30 male New Zealand rabbits were randomly assigned to intravenous inoculation with 100 microl live Pg (10(7) colony-forming units; n = 15) or vehicle (n = 15) once weekly for 4, 8, or 12 consecutive weeks. Arteries were fixed by perfusion and removed for analysis of neointimal lesion formation. We measured intimal and medial lesion areas in iliac artery cross-sections as well as the intimal/medial ratio (I/M). We also analyzed Pg 16S ribosomal DNA amplification with polymerase chain reaction, systemic proinflammatory mediators with enzyme-linked immunosorbent assay, and immunolocalization of macrophages in the balloon-injured arteries.At 12 weeks, iliac intimal hyperplasia was accelerated, and I/M was significantly increased in Pg-inoculated animals (I/M 3.961 +/- 0.536 in the Pg group versus 3.585 +/- 0.353 in the control animals; P0.01). Pg-inoculated animals also had significant increases in macrophage infiltration at 12 weeks, C-reactive protein levels at all time points, and interleukin-6 levels at 12 weeks. Moreover, Pg ribosomal DNA was found in the injured arteries of Pg-inoculated animals, but only after 12 weeks.Long-term systemic challenge with Pg, an oral pathogen, may accelerate intimal hyperplasia in balloon-injured iliac arteries.

In this study, avoidance behavior of the freshwater cladoceran Daphnia magna Straus was used as indicator to assess the early stress of accidental organophosphorus pesticide (OP) contamination. The movement behavior was detected by a multi-species biomonitoring system. There was obvious concentration–response relationship between the OP stress and the behavioral response even at sublethal exposure. A rising OP stress resulted in a significant decrease of response time to escape (RTE; p

Objective In human gingival fibroblasts (HGFs), TLR4 recognises Pathogen-associated molecular patterns (PAMPs), such as LPS, and subsequently activates downstream signals that lead to the production of pro-inflammatory cytokines. The aim of this study was to explore the mechanisms of LPS-induced miRNA-146 regulation of TLR4 signals in HGFs. Materials and methods HGFs were treated with Porphyromonas gingivalis (P.g) LPS, the cells were harvested, and kinase phosphorylation levels were detected by western blot. Selective pharmacological inhibitors and agonists were used to block or activate the relevant kinases, miRNA-146a/b expression levels were detected by real-time PCR, and IL-1, IL-6, and TNF-α production were measured by enzyme-linked immunosorbent assays (ELISA). A luciferase reporter plasmid containing miRNA-146a/b promoter was tested in terms of p50/p65 regulation. Results After P.g LPS treatment, NF-κB and Erk1/2 were strongly activated in HGFs. miRNA-146a/b, IL-1, IL-6 and TNF-α levels were down-regulated when NF-κB inhibitor was used. p50/p65 strongly activated miRNA-146a/b promoter as measured with the luciferase assay. Conclusion In TLR4 signalling in HGFs, both miRNA-146a and miRNA-146b are downstream targets of NF-κB, but not of AP-1 signalling. miRNA-146a/b expression was specifically dependent on NF-κB but not Erk1/2 or JNK signalling.

Maternal under- or over-nutrition not only alters neonatal body mass but also increases the risk of metabolic disorders in adulthood. Little is known about how maternal dietary protein affects offspring fitness in wild rodents. The present study was conducted to test the hypothesis that maternal dietary protein supplement has a long-term beneficial effect on offspring fitness in Brandt's vole (Lasiopodomys brandtii), a herbivorous rodent model. The vole dams were fed either a control (18% protein) or high-protein (36% protein) diet throughout pregnancy and lactation. After weaning, all offspring received a control diet till 14 weeks old. Energetic parameters, serum leptin concentration and glucose tolerance were measured. The adult offspring were fed high-fat diet for 8 weeks, and body weight and food intake were measured. No difference was observed in litter size, litter mass or pup mass before weaning. Maternal protein supplement increased body mass and the mass of reproductive organ but decreased digestibility and fat deposition and alleviated HFD-induced obesity especially in the males. Glucose tolerance was elevated in the offspring from maternal protein supplement, especially in the females. The accelerated growth may be associated with high serum leptin concentration at weaning, a state of leptin resistance, and the low digestibility may predispose obesity resistance especially in male offspring from maternal high-protein diet. These data demonstrate that maternal protein supplement confers the long-term sex-specific beneficial consequences of accelerated growth and improved obesity resistance and glucose tolerance of their offspring.

Temporal control of p27 Kip1 (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF Skp2 and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF Skp2 also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27 Kip1 , independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.

NF-κB is a transcription factor required for inducible expression of a number of proinflammatory mediators including cytokines, chemokines, and leukocyte adhesion molecules (6). In addition, NF-κB regulates the expression of survival genes which prevent cell death in response to tumor necrosis factor alpha (TNF-α) (7, 37, 59, 62). NF-κB is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 subunits. In quiescent cells, NF-κB is retained in the cytosol bound to IκB, a family of inhibitory proteins which mask the nuclear localization and DNA binding sequences on NF-κB (5, 22). Stimulation of these cells with various cytokines, lipopolysaccharide, viruses, antigens, or oxidants triggers signaling events that ultimately lead to the phosphorylation and degradation of IκB, allowing NF-κB to translocate into the nucleus and activate target genes (3, 21, 38, 54). Phosphorylation of Ser32 and Ser36 has been shown to target IκB for ubiquitination and subsequent proteolysis by the ubiquitin-proteasome pathway (UPP) of protein degradation (2, 8, 45, 49). The UPP is the principal pathway for intracellular protein turnover, including regulatory proteins (9). Protein substrates that enter the UPP are first marked by the covalent ligation of polyubiquitin chains mediated by a cascade of enzymes called E1 (ubiquitin activation enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase) (9). In a reaction requiring ATP, ubiquitin is activated by E1 and charged onto an E2 through a thioester formed between the active-site cysteine residue in the E2 and the C-terminal glycine of ubiquitin. The E3 then directs the transfer of ubiquitin from the E2 onto lysine residues within specific substrate proteins, ultimately resulting in the formation of a ubiquitin-protein conjugate. Polyubiquitinated proteins are then recognized and degraded by the 26S proteasome complex to yield small peptides and monomeric ubiquitin. Recently, the receptor component of the IκB E3 was identified as a member of the βTrCP (beta-transducin repeat-containing protein) family of proteins called E3RSIκB (39, 53, 63, 65) or HOS (11). βTrCP is a member of a much larger family of F-box domain containing proteins which form SCF complexes. The core components of SCF complexes include Skp-1, which interacts with the F-box domain, and Cul-1, which is linked to the F-box protein via binding to Skp-1 (4, 10, 35, 46, 47, 51). At least two additional SCF components have been described: (i) Rbx1, which is thought to stabilize the interaction between Cul-1 and the E2s, Cdc34, and Ubc5 (25, 26, 43, 50, 52, 56), and (ii) Sgt1, a protein which interacts with Skp-1 (27). SCF complexes were initially described in yeast to function as E3 ligases for a variety of phosphorylated proteins, including the cell cycle regulator, Sic1 (10, 51). In addition to an F-box domain, βTrCP also contains a WD40 repeat domain that specifically recognizes IκBα only when IκBα is phosphorylated on Ser32 and Ser36. Similarly, at least two other proteins are recognized by βTrCP in a phosphorylation-dependent manner, β-catenin (16, 31, 36, 63) and human immunodeficiency virus type 1 Vpu (40). βTrCP in which the F-box is deleted (ΔF-βTrCP) retains its specificity for phosphorylated IκBα but fails to interact with Skp-1 and no longer supports the ubiquitination reaction. Thus, the interaction of the F-box protein with other SCF components is essential for function. The core components of SCFβTrCP alone, however, are not sufficient to support the ubiquitination of phosphorylated IκBα (53, 63, 65). Additional components, supplied by the addition of crude cellular extracts (63) or recombinant proteins (including UbcH5 [43, 53, 65], Cdc34 [56], and Rbx1 [56]), are required for activity, suggesting that essential proteins and/or modifications to existing proteins are needed to support ubiquitination of IκBα by SCFβTrCP. To date, modifications of the cellular components in SCFβTrCP have not been characterized. In an effort to understand the requirements for SCFβTrCP-mediated ubiquitination of IκBα, we examined SCF core components that associate with βTrCP as well as with IκBα. Remarkably, we observed that endogenous phosphorylated IκBα associated exclusively with a form of Cul-1 that is singly modified by the ubiquitin-like protein Nedd8. Along this line, we found that optimal ubiquitination of IκBα in vitro required the presence of Nedd8 and the Nedd8-conjugating enzyme, Ubc12, as well as two ubiquitin-conjugating enzymes, UbcH5A and Cdc34. Moreover, a Cul-1 point mutant which retains the ability to associate with other SCF components, but lacks the site of the Nedd8 modification, showed a greatly reduced ability to ubiquitinate IκBα in vitro. It is well established that a small percentage of cellular Cul-1 and related cullin proteins form conjugates containing single molecules of Nedd8 in yeast and mammalian cells (30, 44, 60), and the Nedd8 homologue, Rub1, has been genetically linked to SCF components in yeast (30) and plants (14). However, a functional role for Nedd8 in any ubiquitination reaction or cellular process has not been demonstrated. Here we show that the Nedd8 modification of Cul-1 is necessary for the function of SCFβTrCP, linking the ubiquitin and Nedd8 pathways in the regulation of targeted protein degradation.

In neonatal rats, precocious introduction of dietary fructose significantly enhances brush-border fructose transport rates and GLUT-5 mRNA levels during early weaning. In this study, these rates and levels were more than two times higher in the anastomosed intestine compared with those in the bypassed loop of weaning pups that underwent Thiry-Vella surgery and consumed high-fructose (HF) diets. In Thiry-Vella pups fed fructose-free (NF) diets, uptake rates and mRNA levels in the anastomosed intestine were very low and similar to those in the bypassed loop. In sham-operated littermates, transport rates and mRNA levels were similar between intestinal regions that corresponded to anastomosed and bypassed loops in Thiry-Vella pups and were two to three times greater in pups fed HF than in those fed NF diet. In contrast, rates of brush-border glucose transport and levels of SGLT-1 and of GLUT-2 mRNA were independent of diet and were similar between bypassed and anastomosed regions. Changes in GLUT-5 expression did not follow a distinct diurnal rhythm. When pups were fed HF diet after 12 h of starvation to empty the intestinal lumen, fructose transport rates increased with feeding duration and reached a plateau 12–24 h after feeding; in contrast, GLUT-5 mRNA levels were highest within 4 h after arrival of chyme in the jejunum and then decreased gradually and returned to baseline levels 24 h later. In littermates fed NF diet, mRNA levels and uptake rates were each independent of feeding duration. Luminal, and not endocrine, signals regulate GLUT-5 expression in weaning pups.

Cleft palate represents one of the most common congenital birth defects in humans. TGFβ signaling, which is mediated by Smad-dependent and Smad-independent pathways, plays a crucial role in regulating craniofacial development and patterning, particularly in palate development. However, it remains largely unknown whether the Smad-independent pathway contributes to TGFβ signaling function during palatogenesis. In this study, we investigated the function of TGFβ activated kinase 1 (Tak1), a key regulator of Smad-independent TGFβ signaling in palate development. We show that Tak1 protein is expressed in both the epithelium and mesenchyme of the developing palatal shelves. Whereas deletion of Tak1 in the palatal epithelium or mesenchyme did not give rise to a cleft palate defect, inactivation of Tak1 in the neural crest lineage using the Wnt1-Cre transgenic allele resulted in failed palate elevation and subsequently the cleft palate formation. The failure in palate elevation in Wnt1-Cre;Tak1(F/F) mice results from a malformed tongue and micrognathia, resembling human Pierre Robin sequence cleft of the secondary palate. We found that the abnormal tongue development is associated with Fgf10 overexpression in the neural crest-derived tongue tissue. The failed palate elevation and cleft palate were recapitulated in an Fgf10-overexpressing mouse model. The repressive effect of the Tak1-mediated noncanonical TGFβ signaling on Fgf10 expression was further confirmed by inhibition of p38, a downstream kinase of Tak1, in the primary cell culture of developing tongue. Tak1 thus functions to regulate tongue development by controlling Fgf10 expression and could represent a candidate gene for mutation in human PRS clefting.

Birc5 (previously known as survivin) is a cancer-specific protein. Due to the upregulation of its expression in various human malignancies and its key role in apoptosis, proliferation and angiogenesis, Birc5 has attracted attention as a target for anticancer therapies. In this study, when Birc5 was silenced in HepG2 cells, 29.7±3.3% cells underwent apoptosis as expected. It was found that the expression levels of glucose-regulated protein 78 (Hspa5, previously known as Grp78) was increased by almost 3-fold in Birc5-silenced HepG2 cells. Hspa5, a master regulator of the anti-apoptotic unfolded protein response signalling network, can also promote tumor proliferation, survival and metastasis. Hence, we hypothesized that the co-silencing of Birc5 and Hspa5 may exert a stronger apoptosis-inducing effect than single gene interference. To verify this, the expression levels of Birc5 and Hspa5 in human hepatocellular carcinoma tissues were determined. Immunohistochemical staining showed that the expression of Birc5 and Hspa5 was elevated in 28 out of 31 samples. Additionally, plasmid-based siRNA against Birc5 and/or Hspa5 were constructed and transfected into the human hepatocellular liver carcinoma cell line, HepG2. Compared with the HepG2 cells, in which Birc5 or Hspa5 were silenced alone, only 44.2±3.4% of the co-silenced cells proliferated, and 40.3±3.7% co-silenced cells underwent apoptosis (p

Objectives Oxygen deficiency caused by occlusal trauma and smoking can be present in patients with periodontitis. However, biochemical events important in periodontal tissues during hypoxia remain unclear. The aim of this study was to investigate effects of hypoxia on apoptosis and autophagy of human periodontal ligament cells (PDLCs) in vitro. Materials and methods Human PDLCs were obtained and cultured in vitro. Cell viability, apoptosis, autophagy and gene and protein expression were measured in presence and absence of cobalt chloride (CoCl2). Results CoCl2 induced cytotoxicity of human PDLCs in a concentration-dependent manner dependent on macromolecular synthesis, and resulted in apoptosis and mitochondrial dysfunction. CoCl2 also induced redistribution of autophagy marker LC3, increased ratio of LC3-IIto LC3-Iand function of lysosomes. Furthermore, CoCl2 promoted expression of HIF-1α following upregulation of expressions of Bnip3. Significant increases in expression of IL-1β and MMP-8 were also observed. All these results were reversed by pre-treatment with antioxidant N-acetylcysteine. Conclusions Our data showed that CoCl2 could induce cytotoxicity through mitochondria- apoptotic and autophagic pathways involved in HIF-1α. CoCl2-treated PDLCs may serve as an in vitro model for studies of molecular mechanisms in periodontitis.

MicroRNAs (miRNAs) have been demonstrated to play an important role in regulation of the immuno-inflammatory response; however, the function of miRNAs in periodontal inflammation has not been investigated. The objective of this study was to explore the properties of miRNAs in periodontal inflammation by comparing miRNA profiles of inflamed and healthy gingival tissues. Gingival tissues were obtained from 10 periodontitis patients and 10 healthy subjects. After RNA extraction, miRNA profiles were analyzed by microarray, and expression levels of selected miRNAs were confirmed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Analyses using two computational methods, Targetscan and MicroRNA.org, were combined to identify common targets of these miRNAs. Finally, the individual miRNA expression levels of three toll-like receptor (TLR)-related miRNAs from inflamed and healthy gingival tissues were evaluated by RT-PCR. Ninety-one miRNAs were found to be upregulated and thirty-four downregulated over two-fold in inflamed gingival tissue compared with those in healthy gingival tissue. Twelve selected inflammatory-related miRNAs, hsa-miR-126*, hsa-miR-20a, hsa-miR-142-3p, hsa-miR-19a, hsa-let-7f, hsa-miR-203, hsa-miR-17, hsa-miR-223, hsa-miR-146b, hsa-miR-146a, hsa-miR-155, and hsa-miR-205 showed comparable expression levels by microarray and real-time quantitative RT-PCR analyses. In addition, the putative inflammation targets of these miRNAs were predicted, and three that were tested (hsa-miRNA-146a, hsa-miRNA-146b, and hsa-miRNA-155), showed significant differences between inflamed and healthy gingiva. This remarkable difference in miRNA profiles between periodontal diseased and healthy gingiva implicates a probable close relationship between miRNAs and periodontal inflammation. The data also suggest that the regulation of TLRs in periodontal inflammation may involve miRNA pathways.

Periodontitis is a bacterially induced chronic inflammatory disease. Toll-like receptors (TLRs), which could recognize microbial pathogens, are important components in the innate and adaptive immune systems. Both qualitatively and quantitatively distinct immune responses might result from different bacteria stimulation and the triggering of different TLRs. This study explores the interaction of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) with TLR2 and TLR4.We studied the gene expression changes of TLR2 and TLR4 and cytokine production (interleukin-1β, -6, -8, -10, and tumor necrosis factor-alpha) in human periodontal ligament cells (HPDLCs) stimulated with heat-killed bacteria or P. gingivalis lipopolysaccharide (LPS) in the presence or absence of monoclonal antibodies to TLR2 or TLR4 (anti-TLR2/4 mAb).Both test bacteria and 10 microg/ml P. gingivalis LPS treatment increased the gene expression of TLR2 and TLR4 and cytokine production in HPDLCs. In addition, these upregulations could be blocked by anti-TLR2/4 mAb. However, the expression of TLR4 mRNA in HPDLCs stimulated with 1 microg/ml P. gingivalis LPS was not increased. No differences were found in the cytokine production caused by 1 microg/ml P. gingivalis LPS treatment in the presence or absence of anti-TLR4 mAb.These patterns of gene expression and cytokine production indicate that Gram-negative periodontal bacteria or their LPS might play a role in triggering TLR2 and/or TLR4, and be of importance for the immune responses in periodontitis.

Two arrowhead proteinase inhibitors (inhibitors A and B) were characterized and their primary structures were determined. Both inhibitors A and B are double-headed and multifunctional protease inhibitors. Inhibitor A inhibits an equimolar amount of trypsin and chymotrypsin simultaneously and weakly inhibits kallikrein. Inhibitor B inhibits two molecules of trypsin simultaneously and inhibits kallikrein more strongly than does inhibitor A. The amino acid sequences of inhibitors A and B were determined by sequencing the reduced and S-carboxamidomethylated proteins and their peptides produced by cyanogen bromide or proteolytic lysylendopeptidase or Staphylococcus aureus V8 protease cleavage. Inhibitors A and B consist of 150 amino acid residues with three disulfide bonds (Cys 43-Cys 89, Cys 110-Cys 119, and Cys 112-Cys 115) and share 90% sequence identity, with 13 different residues. Since the primary structures are totally different from those of all other serine protease inhibitors so far known, these inhibitors might be classified into a new protease inhibitor family.

Toll-like receptors (TLRs) are a group of sensors on the surface of antigen-presenting cells, such as dendritic cells and macrophages, which recognize microbial pathogens and induce innate and adaptive immune responses. Periodontitis is an inflammatory disease characterized by the destruction of tooth-supporting structures. In order to address whether TLR4 signaling plays a role in periodontitis, we studied the gene expression change in human periodontal ligament cells (HPDLCs) in response to TLR4 ligand, lipopolysaccharide treatment by microarray analysis. Expression of TLR4 was detected in HPDLCs. Lipopolysaccharide treatment increased the expression of 12 genes (more than twofold), including TLR4, TLR5, TLR7, Pellino 1, colony stimulating factor 2 (CSF2) and IL-6. In addition, the expression of 15 genes (less than equal to twofold) was decreased, including Fos, LY64 and LY86. In addition, real-time PCR was used to confirm the change of gene expression of TLR4, IL-6 and Fos. We also showed that the upregulation of IL-6 by lipopolysaccharide treatment was TLR4-dependent. This pattern of gene expression indicates that pathogens may trigger TLR4 signaling and cause periodontitis. Manipulating TLR4 signaling may potentially become one of the recognized therapies for periodontitis.

Behavior of an organism is affected by exposure to toxic chemicals. However, less has been known about behavioral responses of an organism to stresses of toxic chemicals with different toxic characteristics. In present work, Daphnia magna Straus was exposed to gradient concentrations of deltamethrin, chlorothalonil and nitrofen and the behavioral changes of Daphnia magna under different stress were examined. The results showed that the behavioral responses of Daphnia magna to the tested chemicals were affected in general by exposure concentration, rather than toxic characteristics of the chemicals. The duration of avoidance response (DAR) was in a power regression relationship with the toxic unit (TU), defined as the ratio of exposure concentration of the tested chemical to its LC(50-48). DAR was independent of the toxic characteristics of chemicals. However, significant behavior adjustment could be observed after exposure to deltamethrin while only step-by-step decrease in behavior strength could be observed when exposed to chlorothalonil and nitrofen. It was suggested from the observation that avoidance behaviors of Daphnia magna to exposures of chemicals with different toxic characteristics could be similar, while their specific response could be different.

Carex procumbens, a new species of Carex sect. Rhomboidales from Hainan, China, is described and illustrated. The new species is similar to C. longipetiolata but differs in culms soft and usually prostrate on the ground, having narrower leaves with the leaf base gradually narrowed, with 2–3 spikes, terminal spike 8–12 mm long, lateral spikes 5–15 mm long and loosely flowered, staminate glumes ovate with green middle vein and apex rounded, pistillate glumes ovate-lanceolate ca. 6 mm and middle vein excurrent into a short awn for ca. 2 mm, perigynia fusiform and glabrous, nutlet brown to dark brown and ovate-rhomboid, nutlet ca. 7 mm long and with a short curved beak.