Airway microecology in rifampicin-resistant and rifampicin-sensitive pulmonary tuberculosis patients.

BACKGROUND: Pulmonary tuberculosis is a chronic infectious disease of the respiratory system. It is still one of the leading causes of death from a single infectious disease, but it has been stuck in the study of a single pathogen. Recent studies have shown that many diseases are associated with disruption of the native microbiota. In this study we investigated the occurrence of tuberculosis and the correlation between drug resistance and respiratory flora. High-throughput 16 S rRNA gene sequencing was used to characterize the respiratory microbiota composition of 30 tuberculosis (TB) affected patients and compared with 30 healthy (H) controls. According to their Gene Xpert results, 30 pulmonary tuberculosis patients were divided into 12 persons in the drug-sensitive group (DS0) and 18 persons in the drug-resistant group (DR0). The microbial flora of the two were compared with the H group. RESULTS: The data generated by sequencing showed that Firmicutes, Proteus, Bacteroides, Actinomyces and Fusobacterium were the five main bacterial phyla detected, and they constituted more than 96% of the microbial community. The relative abundances of Fusobacterium, Haemophilus, Porphyromonas, Neisseria, TM7, Spirochetes, SR1, and Tenericutes in the TB group was lower than that of the H group, and Granulicatella was higher than the H group. The PcoA diagrams of the two groups had obvious clustering differences. The Alpha diversity of the TB group was lower than that of the H group, and the Beta diversity was higher than that of the H group (P < 0.05). The relative abundance of Streptococcus in the DS0 group was significantly higher than that in the DR0 group (P < 0.05). CONCLUSION: Pulmonary tuberculosis can cause disorders of the respiratory tract microbial flora, in which the relative abundance of Streptococcus was significantly different between rifampicin-sensitive and rifampicin-resistant patients.

Effects of BMP9 and pulsed electromagnetic fields on the proliferation and osteogenic differentiation of human periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) have been confirmed to have self-renewal capacity and multidifferentiation potential and are good candidates for periodontal tissue regeneration. Pulsed electromagnetic field (PEMF) has been demonstrated to promote osteogenesis in non-union fractures, partly by regulating mesenchymal stem cells or osteoblast activity. However, there is no report about the osteo-inductive effect of PEMF stimulation on human PDLSCs (hPDLSCs). Thus, we tested the hypothesis that PEMF biophysical stimulation alone has an influence on the proliferation and osteogenic differentiation of hPDLSCs. To detect the osteo-inductive potential of bone morphogenetic protein (BMP9), we transfected the STRO-1+ /CD146+ hPDLCSs with BMP9-expressing recombinant adenoviruses. We examined the proliferation and osteogenic differentiation of hPDLSCs treated with either PEMF (15 Hz, 1 h daily, different intensities), or BMP9, or both stimuli. Cell counting kit-8 (CCK-8) assay showed that PEMF of different intensities had no effect on the proliferation of hPDLSCs and did not enhance the proliferative capability of BMP9-transfected cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting showed that the combination of both PEMFs (1.8 or 2.4 mT) and BMP9 stimulation had a synergistic effect on early and intermediate osteogenic genes and protein expressions of runt-related transcription factor 2, alkaline phosphatase, osteopontin, and late mineralized extracellular matrix formation in hPDLSCs. Bioelectromagnetics. 38:63-77, 2017. © 2016 Wiley Periodicals, Inc.

Controlled release of minocycline in hydroxyapatite/chitosan composite for periodontal bone defect repair.

The aim was to obtain bone repair materials with sustained release of minocycline and evaluate the effect in periodontal bone defect repair. Two complex material, hydroxyapatite/chitosan (HA/CS) and minocycline-hydroxyapatite/chitosan (Mino-HA/CS), were prepared by the co-precipitation method. The physical and chemical property, cytotoxicity, release of minocycline and the bacteriostasis examination of the materials were evaluated, they were applied to the rabbit model of mandible bone defect to evaluate their effects on the regeneration of periodontal bone defect. After minocycline was added to HA/CS, the setting time of the material was prolonged, the compressive strength was reduced and the pore size and porosity were increased significantly. The pH value did not change obviously and stayed in the neutral range. Mino-HA/CS could promote the growth of osteoblasts effectively compared with control medium. In vivo, Mino-HA/CS material showed better effect of promoting periodontal bone formation.

Rapid visualized assessment of drug efficacy in live mice with a selectable marker-free autoluminescent Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that is responsible for community acquired infections and nosocomial infections. Antibiotic-resistant K. pneumoniae and/or hypervirulent K. pneumoniae are emerging as a serious threat to public health. For the sake of alleviating and conquering current dilemma, discovery of effective new drugs against K. pneumoniae is a tough challenge. However, traditional anti-K. pneumoniae drug discovery methods cost considerable amount of time, animals, labor and so on. So an efficient technique for in vitro and in vivo drug screening with the least time duration, animals and labor cost is highly needed for the discovery of new effective compounds. Hence, in this study we constructed a selectable marker-free autoluminescent K. pneumoniae (SfAlKp) harboring luxCDABE by combining Tn7 transposon and Xer-dif system. SfAlKp can be used for discovery of new drugs via detecting luminescence intensity as a surrogate marker. The energy-consuming autoluminescent reaction catalyzed by the LuxAB enzymes which use the substrates produced by LuxCDE using the metabolites of the bacteria. Tn7 can insert exogenous genes into the bacterial genome and the DNA fragment in between dif sequences can be recognized and removed by endogenous XerCD recombinases of K. pneumoniae. The drug susceptibility and growth rate of SfAlKp are identical to its parent strain, meanwhile the luminescence intensity and stability are also significant characteristics of SfAlKp. Compared to conventional techniques, the autoluminescence-based measurement is more applicable to high throughput screening for compounds both in vitro as well as in vivo in animal model.

Rapid, serial, non-invasive quantification of Pseudomonas aeruginosa in live mice with a selectable marker-free autoluminescent strain.

Pseudomonas aeruginosa is an increasingly prevalent pathogen that has become a serious health concern due to an increasing incidence of multidrug-resistant (MDR) hospital-acquired infections. The emergence of MDR-P. aeruginosa coupled with shrinking antibiotic pipelines has increased the demand for new antimicrobials and therapeutics. An effective tool for drug screening both in vitro and in vivo can facilitate the discovery of drugs and regimens for treating P. aeruginosa infection. Here, for the first time, we combined the mini-Tn7 system and Xer/dif recombinase system to construct a stable and selectable marker-free autoluminescent P. aeruginosa (SfAlPa) by one step. Afterwards, in vitro and in vivo activities of several antibiotics including amikacin, biapenem, levofloxacin and polymyxin B were assessed using SfAlPa. This study demonstrated that the use of SfAlPa could significantly facilitate rapid real-time evaluating the activities of compounds. Compared to prevailing methods, this method reduces the time, effort, animals and costs consumed in the discovery of new drugs against P. aeruginosa. Additionally, the methodology described in this study could be easily modified for construction of selectable marker-free reporter strain in other Gram-negative bacteria.

One-Step Engineering of a Stable, Selectable Marker-Free Autoluminescent Acinetobacter baumannii for Rapid Continuous Assessment of Drug Activity.

The rising cases of multidrug-resistant Acinetobacter baumannii (Ab) and the lack of effective drugs call for quick attention. Here, based on a Tn7 transposon and Xer/dif system, we constructed a stable, selectable marker-free autoluminescent Ab capable of producing visible light without extra substrates. Utilization of this autoluminescent reporter strain has the potential to reduce the time, effort and costs required for the evaluation of activities of anti-Ab drug candidates in vitro.

Calcitonin induces collagen synthesis and osteoblastic differentiation in human periodontal ligament fibroblasts.

BACKGROUND: Extracellular matrix (ECM) secretion and osteogenic differentiation in periodontal ligament fibroblasts (PDLF) facilitate the neogenesis of alveolar bone, which is the cellular basis for alveolar bone repair. Calcitonin (CT) has been reported to play an important role in promoting ECM expression and inducing osteogenic differentiation in osteoblast, but its effects on PDLFs remain obscure. METHODS: The expression of CT, transforming growth factor-beta 1(TGF-ß1) and bone morphogenetic protein (BMP) in gingival crevicular fluid (GCF) was measured by ELISA. The effects of CT on collagen synthesis and osteogenic differentiation in hPDLFs were investigated by using the primarily cultured hPDLFs infected with adenovirus carrying the CT gene. Gene expression was measured by quantitative PCR and western blot. RESULTS: The expression of CT in gingival crevicular fluid (GCF) of patients with periodontitis was significantly higher than that of healthy subjects. In addition, CT expression correlated with the clinical indexes including probing pocket depth (PPD), clinical attachment level (CAL), and gingival index (GI). The in vitro study demonstrated that overexpression of CT by adenovirus infection increased the expression of TGF-ß1, collagen type I and III, and osteoblastic markers including BMP-2/-4, alkaline phosphatase and osteocalcin in human PDLFs. Moreover, CT-enhanced collagen synthesis was abrogated in hPDLFs transfected with TGF-ß1 siRNA, and CT-induced osteoblastic differentiation was blocked in hPDLFs by BMPs inhibitor noggin. CONCLUSIONS: These results suggest that CT promotes collagen synthesis and osteogenic differentiation in hPDLFs via the TGF-ß1 and BMPs signaling pathways, respectively.

Bone Morphogenetic Protein-9 Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells via the JNK Pathway.

Bone morphogenetic protein-9 (BMP9) shows great osteoinductive potential in bone regeneration. Periodontal ligament stem cells (PDLSCs) with multi-differentiation capability and low immunogenicity are increasingly used as seed cells for periodontal regenerative therapies. In the present study, we investigated the potent osteogenic activity of BMP9 on human PDLSCs (hPDLSCs), in which the c-Jun N-terminal kinase (JNK) pathway is possibly involved. Our results showed that JNK inhibition by the specific inhibitor SP600125 or adenovirus expressing small interfering RNA (siRNA) targeting JNK (AdR-si-JNK) significantly decreased BMP9-induced gene and protein expression of early and late osteogenic markers, such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN), in hPDLSCs. We also confirmed the in-vivo positive effect of JNKs on ectopic bone formation induced by hPDLSCs injected into the musculature of athymic nude mice and BMP9 ex vivo gene delivery. For the cellular mechanism, we found that BMP9 activated the phosphorylation of JNKs and Smad2/3, and that JNKs may engage in cross-talk with the Smad2/3 pathway in BMP9-mediated osteogenesis.

Does matching relation exist between the length and the tilting angle of terminal implants in the all-on-four protocol? stress distributions by 3D finite element analysis.

PURPOSE: To explore whether there is matching relation between the length and the tilting angle of terminal implants in the All-on-Four protocol by studying the effects of different implant configurations on stress distributions of implant, bone, and framework. MATERIALS AND METHODS: Four implants were employed to support a full-arch fixed prosthesis and five three-dimensional finite element models were established with CT images, based on the length (S and L) and distal tilt angle (0°, 30° and 45°) of terminal implants for an edentulous mandible, which named: Tilt0-S, Tilt30-S, Tilt30-L, Tilt45-S and Tilt45-L. An oblique 240 N was loaded at second molar. The von Mises Stresses were analyzed. The implants were consecutively named #1 to #4 from the loading point. RESULTS: 1) Tilt0-S had the greatest stress on the implants, with the other groups exhibiting variable reductions; the four implants of Tilt45-L demonstrated the greatest reduction in stress. 2) Tilt0-S had the greatest stress at bone around #1 implant neck, and Tilt45-L exhibited the least stress, which was a 36.3% reduction compared to Tilt0-S. 3) The greatest stress in the framework was found on the cantilevers distal to #1 implant. Tilt45-S exhibited the least stress. CONCLUSION: Matching different length and tilting angle of the terminal implants led to variable stress reductions on implants, bone and the superstructure. By optimizing implant configuration, the reduction of stress on implants and surrounding bone could be maximized. Under the present condition, Tilt45-L was the preferred configuration. Further clinical testings are required.

Health status and burden of health care costs among urban elderly in China.

This study evaluates health status among elderly residents in urban China and medical insurance schemes for them. A total of 931 urban elderly individuals aged 60 years or above in 3 cities were interviewed. The survey gathered data on individual sociodemographic characteristics, self-reported health status, chronic disease diagnoses, use of and expenditure related to health care services, satisfaction with medical insurance, and so on. The prevalence of chronic disease was 79.1% among the respondents, and medical expenditure on chronic disease per patient was 4656 Yuan in half a year. Over the 2 earlier weeks, 27.4% of respondents had felt sick, and 63.5% of them had had outpatient visits; 29.5% of respondents had been hospitalized in the past year. Among different groups, the inpatient reimbursement rate ranged from 45.5% to 81.2%, and the outpatient reimbursement rate was between 4.1% and 100%. The health care demand had not been met for many elderly individuals because of the lack of general outpatient coverage in the basic medical insurance scheme.

A novel controlled-release system for antibacterial enzyme lysostaphin delivery using hydroxyapatite/chitosan composite bone cement.

In this work, a lysostaphin-loaded, control-released, self-setting and injectable porous bone cement with efficient protein delivery was prepared by a novel setting method using hydroxyapatite/chitosan (HA/CS) composite scaffold. The cement samples were made through cementitious reactions by mixing solid powder, a mixture of HA/CS composite particles, lysostaphin, Ca(OH)2, CaCO3 and NaHCO3, with setting liquid containing citric acid, acetic acid, NaH2PO4, CaCl2 and poloxamer. The setting parameters of the cement samples were determined. The results showed that the final setting time was 96.6±5.2 min and the pH value increased from approximately 6.2 to nearly 10 during the setting process and the porosity was 34% at the end. And the microstructure and composition were detected by scanning electron microscopy (SEM), x-ray diffraction and Fourier transform-infrared spectroscopy. For the release behavior of lysostaphin loaded in the cement sample, the in vitro cement extract experiment indicated that about 94.2±10.9% of the loaded protein was released before day 8 and the in vivo Qdot 625 fluorescence tracking experiment showed that the loaded protein released slower than the free one. Then the biocompatibility of the cement samples was evaluated using the methylthiazol tetrazolium assay, SEM and hematoxylin-eosin staining, which suggested good biocompatibility of cement samples with MC 3T3-E1 cells and subcutaneous tissues of mice. Finally the antibacterial activity assay indicated that the loaded lysostaphin had good release ability and strong antibacterial enzymatic activity against methicillin-resistant Staphylococcus aureus. Collectively, all the results suggested that the lysostaphin-loaded self-setting injectable porous bone cement released the protein in a controlled and effective way and the protein activity was well retained during the setting and releasing process. Thus this bone cement can be potentially applied as a combination of artificial bone substitute and controlled-release system for delivery of lysostaphin to treat bone defects and infections.

[Management of apicectomy and root canal obturation completed one time under direct vision].

PURPOSE: To evaluate the clinical effect of apicectomy and root canal obturation completed one time under direct vision on chronic periapical periodontitis of anterior teeth. METHODS: 34 cases(40 teeth) with RCT failing or larger periodontal lesion, which couldn't be cured depending on RCT only, were chosen for this clinical study. They were randomly divided into two groups with 20 teeth in each group. Root canal obturation was done after apicectomy under direct vision(group A),or apicectomy was done after RCT(group B).The time of filling process, the ratio of postobturation pain, the obturation quality and short-term efficiency of the treatment were assessed. The data were separately subjected to t test, Chi-square test, Mann-Whitney U test with SPSS12.0 software package. RESULTS: The time of filling process in group A was significantly less than group B (P<0.05); No statistically significant difference was found between group A and group B in the ratio of postobturation pain, obturation quality and clinical therapeutic efficiency. CONCLUSIONS: The method of apicectomy and root canal obturation completed one time under direct vision is rapid, simple and effective in treating chronic periapical periodontitis of anterior teeth.The short-term clinical therapeutic efficiency is similar to that of apicectomy done after RCT.