1alpha,25-dihydroxyvitamin D3 potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: role for IL-10 and TGF-beta

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a potent inhibitor of NF-kappaB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)(2)D(3) potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-beta are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 microg. Interestingly, coadministration of 10 ng of 1,25(OH)(2)D(3) with 100 microg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-beta and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-kappaB pathway using the selective IkappaB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-beta before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)(2)D(3) potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-beta play a crucial role in the effector phase of this mouse model.     

Gas chromatographic–mass spectrometric determination of serum mexiletine concentration after derivatization with perfluorooctanoyl chloride, a new derivative

Mexiletine is an antiarrhythmic agent used in the treatment of ventricular arrhythmia. The drug has a narrow therapeutic window which necessitates monitoring its serum concentrations. We describe a gas chromatographic–mass spectrometric analysis of mexiletine using selected ion monitoring. Mexiletine was extracted from alkaline serum with dichloromethane and then derivatized with perfluorooctanoyl chloride. The derivatization reaction was completed in 20 min at 80°C. We used N-propylamphetamine as the internal standard. The ions monitored were m/z 122, 454 and 575 for the derivatized mexiletine and m/z 91, 118, 440 and 452 for the derivatized internal standard. The within-run precision at a serum mexiletine concentration of 1 mg/l was 1.9% (mean=0.98, S.D.=0.019 mg/l, n=7) and the between-run precision was 2.5% (mean=0.99, S.D.=0.025 mg/l, n=7). The assay was linear for serum mexiletine concentrations of 0.2 to 4 mg/l. The detection limit was 0.1 mg/l. The average recoveries of mexiletine and the internal standard were 80% and 84%, respectively at a mexiletine concentration of 1 mg/l. There was no carry over problem in our assay. We observed a good correlation between mexiletine concentrations measured by a reference laboratory (GC) and by our new GC–MS assay.     

An enclosed-chamber labeling system for the safe <Superscript>14</Superscript>C-enrichment of phytochemicals in plant cell suspension cultures

Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generale ¹⁴C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied with ¹⁴C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O₂ replenishment and complete capture of respired ¹⁴CO₂ throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [¹⁴C]sucrose as the carbon source, demonstrated a 20–23% efficiency of ¹⁴C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.     

The expanded human kallikrein gene family: locus characterization and molecular cloning of a new member, KLK-L3 (KLK9)

In rodents, kallikreins are encoded by a large multigene family but in humans, only three kallikrein genes were thought to exist. Based on the homology between the human and the rodent kallikrein loci, we defined a 300-kb human kallikrein gene region on chromosome 19q13. 3-q13.4. By using linear sequence information, restriction analysis, PCR, and blotting techniques, we were able to construct the first detailed map of the human kallikrein gene locus. Comparative analysis of genes located in this area enabled us to expand the human kallikrein multigene family with some recently identified serine proteases and establish common structural features. We further identified a new kallikrein-like gene, named kallikrein-like gene 3 (KLK-L3; HGMW-approved symbol KLK9). We describe the structural characterization of the KLK-L3 gene, together with its precise chromosomal localization in relation to other kallikreins and its tissue expression pattern and hormonal regulation.     

Assessment of probiotic potential and anticancer activity of newly isolated vaginal bacterium Lactobacillus plantarum 5BL

Numerous bacteria in and on its external parts protect the human body from harmful threats. This study aimed to investigate the potential beneficial effects of the vaginal ecosystem microbiota. A type of bacteria was isolated from vaginal secretions of adolescent and young adult women, cultured on an appropriate specific culture medium, and then molecularly identified through 16S rDNA gene sequencing. Results of 16S rDNA sequencing revealed that the isolate belongs to the Lactobacillus plantarum species. The isolated strain exhibited probiotic properties such as low pH and high bile salt concentration tolerance, antibiotic susceptibility and antimicrobial activity against some pathogenic bacteria. The anticancer effects of the strain on human cancer cell lines (cervical, HeLa; gastric, AGS; colon, HT‐29; breast, MCF‐7) and on a human normal cell line (human umbilical vein endothelial cells [HUVEC]) were investigated. Toxic side effects were assessed by studying apoptosis in the treated cells. The strain exhibited desirable probiotic properties and remarkable anticancer activity against the tested human cancer cell lines (P ≤ 0.05) with no significant cytotoxic effects on HUVEC normal cells (P ≤ 0.05). Overall, the isolated strain showed favorable potential as a bioactive therapeutic agent. Therefore, this strain should be subjected to the other required tests to prove its suitability for clinical therapeutic application.     

Brenneria alni,causal agent bark canker of Alnus subcordata

In 2014, bark cankers were observed on Caucasian alder (Alnus subcordata) trees in Iran. The disease was characterized by a dark watery liquid often exuding from longitudinal cankers in the bark of the tree trunks which stained the surface. Symptomatic tissue from A. subcordata was sampled from a number of sites in the Mazandaran province. Isolations were performed on nutrient agar supplemented with sucrose (SNA) and yielded bacterial colonies that were uniform, round and whitish. The bacterial strains isolated from alder trees in Iran were similar to Brenneria alni based on phenotypic and genotypic (nucleotide sequences of the 16S rRNA, and housekeeping genes gyrB, and infB) characteristics. The pathogenicity of the representative strains was determined by inoculating stem pieces of A. subcordata. All tested strains caused longitudinal necrotic lesions 30 days after inoculation and were re-isolated from this tissue. To our knowledge, this is the first report of the occurrence of B. alni in Iran, and on A. subcordata globally.     

Induced fit in guanidino kinases--comparison of substrate-free and transition state analog structures of arginine kinase

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 A with a final R of 22.3% (R(free) = 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.     

Prevalence of MTHFR C677T Single Nucleotide Polymorphism in Genetically Isolated Populations in Jordan

Methylenetetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism is a major inherited risk factor of venous thromboembolism. We sought to determine its prevalence in genetically isolated populations of Chechens and Circassians in Jordan. The MTHFR C677T mutation was analyzed from blood samples taken from 120 random unrelated Chechens and 72 Circassians. The prevalence of the MTHFR mutation in the Chechen population was 27.5% (allele frequency 15%); the prevalence among the Circassians was 50% (allele frequency 29.2%). The prevalence in the Chechen population is similar to that in Jordan and other world populations, but it is higher in the Circassian population. This study will contribute to understanding the interaction between genetic and environmental risk factors underlying thrombosis and will be useful in deciding which genetic variants should be tested in a clinical genetic testing service.     

Learning from positive examples when the negative class is undetermined- microRNA gene identification

Background

The application of machine learning to classification problems that depend only on positive examples is gaining attention in the computational biology community. We and others have described the use of two-class machine learning to identify novel miRNAs. These methods require the generation of an artificial negative class. However, designation of the negative class can be problematic and if it is not properly done can affect the performance of the classifier dramatically and/or yield a biased estimate of performance. We present a study using one-class machine learning for microRNA (miRNA) discovery and compare one-class to two-class approaches using naïve Bayes and Support Vector Machines. These results are compared to published two-class miRNA prediction approaches. We also examine the ability of the one-class and two-class techniques to identify miRNAs in newly sequenced species.

Results

Of all methods tested, we found that 2-class naive Bayes and Support Vector Machines gave the best accuracy using our selected features and optimally chosen negative examples. One class methods showed average accuracies of 70–80% versus 90% for the two 2-class methods on the same feature sets. However, some one-class methods outperform some recently published two-class approaches with different selected features. Using the EBV genome as and external validation of the method we found one-class machine learning to work as well as or better than a two-class approach in identifying true miRNAs as well as predicting new miRNAs.

Conclusion

One and two class methods can both give useful classification accuracies when the negative class is well characterized. The advantage of one class methods is that it eliminates guessing at the optimal features for the negative class when they are not well defined. In these cases one-class methods can be superior to two-class methods when the features which are chosen as representative of that positive class are well defined.

Availability

The OneClassmiRNA program is available at: [1]

     

Proteomic profiling and functional characterization of early and late shoulder osteoarthritis

Introduction

The development of effective treatments for osteoarthritis (OA) has been hampered by a poor understanding of OA at the cellular and molecular levels. Emerging as a disease of the ''whole joint’, the importance of the biochemical contribution of various tissues, including synovium, bone and articular cartilage, has become increasingly significant. Bathing the entire joint structure, the proteomic analysis of synovial fluid (SF) from osteoarthritic shoulders offers a valuable ''snapshot’ of the biologic environment throughout disease progression. The purpose of this study was to identify differentially expressed proteins in early and late shoulder osteoarthritic SF in comparison to healthy SF.

Methods

A quantitative ¹⁸O labeling proteomic approach was employed to identify the dysregulated SF proteins in early (n = 5) and late (n = 4) OA patients compared to control individuals (n = 5). In addition, ELISA was used to quantify six pro-inflammatory and two anti-inflammatory cytokines.

Results

Key results include a greater relative abundance of proteins related to the complement system and the extracellular matrix in SF from both early and late OA. Pathway analyses suggests dysregulation of the acute phase response, liver x receptor/retinoid x receptor (LXR/RXR), complement system and coagulation pathways in both early and late OA. The network related to lipid metabolism was down-regulated in both early and late OA. Inflammatory cytokines including interleukin (IL) 6, IL 8 and IL 18 were up-regulated in early and late OA.

Conclusions

The results suggest a dysregulation of wound repair pathways in shoulder OA contributing to the presence of a ''chronic wound’ that progresses irreversibly from early to later stages of OA. Protease inhibitors were downregulated in late OA suggesting uncontrolled proteolytic activity occurring in late OA. These results contribute to the theory that protease inhibitors represent promising therapeutic agents which could limit proteolytic activity that ultimately leads to cartilage destruction.