Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation. The ideal anti-OA drug should have immunomodulatory effects while at the same time having limited or no toxicity. We examined the anti-inflammatory effects of Ginkgo biloba extract (EGb) in interleukin-1 (IL-1)-stimulated human chondrocytes. Chondrocytes were prepared from cartilage specimens taken from patients with osteoarthritis who had received total hip or total knee replacement. The concentrations of chemokines and the degree of cell migration were determined by ELISA and chemotaxis assays, respectively. The activation of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases (MAPKs), activator protein-1 (AP-1), and nuclear factor-kappaB (NF-κB) was determined by immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay. We found that EGb inhibited IL-1-induced production of chemokines, which in turn resulted in attenuation of THP-1 cell migration toward EGb-treated cell culture medium. EGb also suppressed IL-1-stimulated iNOS expression and release of nitric oxide (NO). The EGb-mediated suppression of the iNOS-NO pathway correlated with the attenuation of activator protein-1 (AP-1) but not nuclear factor-kappaB (NF-κB) DNA-binding activity. Of the mitogen-activated protein kinases (MAPKs), EGb inhibited only c-Jun N-terminal kinase (JNK). Unexpectedly, EGb selectively caused degradation of c-Jun protein. Further investigation revealed that EGb-mediated c-Jun degradation was preceded by ubiquitination of c-Jun and could be prevented by the proteosome inhibitor MG-132. The results imply that EGb protects against chondrocyte degeneration by inhibiting JNK activation and inducing ubiquitination-dependent c-Jun degradation. Although additional research is needed, our results suggest that EGb is a potential therapeutic agent for the treatment of OA.     

Ran GTPase-Activating Protein 1 Is a Therapeutic Target in Diffuse Large B-Cell Lymphoma

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1⁺, while reactive lymphoid hyperplasia (n = 12) was RanGAP1⁺ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin’s lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.     

Phylogenetic Diversity of the Bacillus pumilus Group and the Marine Ecotype Revealed by Multilocus Sequence Analysis

Bacteria closely related to Bacillus pumilus cannot be distinguished from such other species as B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus simply by 16S rRNA gene sequence. In this report, 76 marine strains were subjected to phylogenetic analysis based on 7 housekeeping genes to understand the phylogeny and biogeography in comparison with other origins. A phylogenetic tree based on the 7 housekeeping genes concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB was constructed and compared with trees based on the single genes. All these trees exhibited a similar topology structure with small variations. Our 79 strains were divided into 6 groups from A to F; Group A was the largest and contained 49 strains close to B. altitudinis. Additional two large groups were presented by B. safensis and B. pumilus respectively. Among the housekeeping genes, gyrB and pyrE showed comparatively better resolution power and may serve as molecular markers to distinguish these closely related strains. Furthermore, a recombinant phylogenetic tree based on the gyrB gene and containing 73 terrestrial and our isolates was constructed to detect the relationship between marine and other sources. The tree clearly showed that the bacteria of marine origin were clustered together in all the large groups. In contrast, the cluster belonging to B. safensis was mainly composed of bacteria of terrestrial origin. Interestingly, nearly all the marine isolates were at the top of the tree, indicating the possibility of the recent divergence of this bacterial group in marine environments. We conclude that B. altitudinis bacteria are the most widely spread of the B. pumilus group in marine environments. In summary, this report provides the first evidence regarding the systematic evolution of this bacterial group, and knowledge of their phylogenetic diversity will help in the understanding of their ecological role and distribution in marine environments.     

The Effects of Postmenopausal Hormone Use on Cataract: A Meta-Analysis

Background

Cataract is the leading cause of blindness worldwide. Many observational studies assessed the relationship between postmenopausal hormone replacement therapy (HRT) and risk of cataract development, but the reported results were controversial. The aim of present meta-analysis was to evaluate the association of postmenopausal hormone replacement therapy with risk of cataract development.

Methods

The eligible observational studies, including cross-sectional, case–control and cohort studies, were identified by searching PubMed and Embase during March of 2013. Either a fixed- or a random-effects model was used to calculate the pooled odds ratio (OR) with its 95% confidence interval (95%CI). Subgroup analysis on cataract types was performed.

Results

A total of four cohort and five case-control or cross-sectional studies were finally included into this meta-analysis. Overall, a significant decreased risk of developing any type of cataract was found in ever HRT group as compared with non-HRT group among cohort studies (OR 0.83; 95%CI: 0.71,0.97) and case-control or cross-sectional studies (OR 0.74; 95%CI: 0.59,0.93). Subgroup analysis on cataract types determined that the significantly decreased risk of nuclear cataract in current HRT group (OR 0.72; 95%CI: 0.61,0.85) and also a critically reduced risk of nuclear cataract in ever HRT group (OR 0.80; 95% CI: 0.64,1.01) were found among case-control or cross-sectional studies, as compared with non-HRT group. No association of HRT with risk of cortical and posterior subcapsular cataract was observed.

Conclusions

The results of present meta-analysis indicate that postmenopausal hormone use may play a protective role in cataract development.     

Molecular Epidemiology of Clostridium difficile at a Medical Center in Taiwan: Persistence of Genetically Clustering of A?B+ Isolates and Increase of A+B+ Isolates

Introduction

We investigated the changing trend of various toxigenic Clostridium difficile isolates at a 3 500-bed hospital in Taiwan. Genetic relatedness and antimicrobial susceptibility of toxigenic C. difficile isolates were also examined.

Methods

A total of 110 non-repeat toxigenic C. difficile isolates from different patients were collected between 2002 and 2007. Characterization of the 110 toxigenic isolates was performed using agar dilution method, multilocus variable-number tandem-repeat analysis (MLVA) genotyping, tcdC genotyping, and toxinotyping.

Results

Among the 110 toxigenic isolates studied, 70 isolates harbored tcdA and tcdB (A⁺B⁺) and 40 isolates harbored tcdB only (A⁻B⁺). The annual number of A⁺B⁺ isolates considerably increased over the 6-year study (P = 0.055). A total of 109 different MLVA genotypes were identified, in which A⁺B⁺ isolates and A⁻B⁺ isolates were differentiated into two genetic clusters with similarity of 17.6%. Twenty-four (60%) of the 40 A⁻B⁺ isolates formed a major cluster, MLVA-group 1, with a similarity of 85%. Seven (6.4%) resistant isolates were identified, including two metronidazole-resistant and five vancomycin-resistant isolates.

Conclusions

This study indicated a persistence of a MLVA group 1 A⁻B⁺ isolates and an increase of A⁺B⁺ isolates with diverse MLVA types. Moreover, C. difficile isolates with antimicrobial resistance to metronidazole or vancomycin were found to have emerged. Continuous surveillance is warranted to understand the recent situation and control the further spread of the toxigenic C. difficile isolates, especially among hospitalized patients.     

High Seroprevalence of Mycoplasma pneumoniae IgM in Acute Q Fever by Enzyme-Linked Immunosorbent Assay (ELISA)

Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA), a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001) and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001) of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5%) and seroconversion rate (33.3%) of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases) with those who were negative (43 cases), the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255), sore throat (8.5% vs. 16.3%, p=0.351), cough (35.6% vs. 23.3%, p=0.199), and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258), were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.     

Bayesian Phylogeography of Crimean-Congo Hemorrhagic Fever Virus in Europe

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis mainly transmitted by ticks that causes severe hemorrhagic fever and has a mortality rate of 5-60%. The first outbreak of CCHF occurred in the Crimean peninsula in 1944-45 and it has recently emerged in the Balkans and eastern Mediterranean. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach, which estimated a mean evolutionary rate of 2.96 x 10^-4 (95%HPD=1.6 and 4.7 x 10^-4) substitutions/site/year for the analysed fragment. All of the isolates segregated into seven highly significant clades that correspond to the known geographical clades: in particular the two new isolates from northern Albania clustered significantly within the Europe 1 clade. Our phylogeographical reconstruction suggests that the global CCHFV clades originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s, when a still circulating but less or non-pathogenic virus emerged in Greece and Turkey, and the second in the early 1900s, when a pathogenic CCHFV strain began to spread in eastern Europe. The most probable location for the origin of this European clade 1 was Russia, but Turkey played a central role in spreading the virus throughout Europe. Given the close proximity of the infected areas, our data suggest that the movement of wild and domestic ungulates from endemic areas was probably the main cause of the dissemination of the virus in eastern Europe.     

Uremic Pruritus,Dialysis Adequacy,and Metabolic Profiles in Hemodialysis Patients: A Prospective 5-Year Cohort Study

Background

Uremic pruritus is a common and intractable symptom in patients on chronic hemodialysis, but factors associated with the severity of pruritus remain unclear. This study aimed to explore the associations of metabolic factors and dialysis adequacy with the aggravation of pruritus.

Methods

We conducted a 5-year prospective cohort study on patients with maintenance hemodialysis. A visual analogue scale (VAS) was used to assess the intensity of pruritus. Patient demographic and clinical characteristics, laboratory parameters, dialysis adequacy (assessed by Kt/V), and pruritus intensity were recorded at baseline and follow-up. Change score analysis of the difference score of VAS between baseline and follow-up was performed using multiple linear regression models. The optimal threshold of Kt/V, which is associated with the aggravation of uremic pruritus, was determined by generalized additive models and receiver operating characteristic analysis.

Results

A total of 111 patients completed the study. Linear regression analysis showed that lower Kt/V and use of low-flux dialyzer were significantly associated with the aggravation of pruritus after adjusting for the baseline pruritus intensity and a variety of confounding factors. The optimal threshold value of Kt/V for pruritus was 1.5 suggested by both generalized additive models and receiver operating characteristic analysis.

Conclusions

Hemodialysis with the target of Kt/V ≥1.5 and use of high-flux dialyzer may reduce the intensity of pruritus in patients on chronic hemodialysis. Further clinical trials are required to determine the optimal dialysis dose and regimen for uremic pruritus.     

A Whole-Cell Biosensor for the Detection of Gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g⁻¹ or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes_(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).