Exonic Variants Associated with Development of Aspirin Exacerbated Respiratory Diseases

Aspirin-exacerbated respiratory disease (AERD) is one phenotype of asthma, often occurring in the form of a severe and sudden attack. Due to the time-consuming nature and difficulty of oral aspirin challenge (OAC) for AERD diagnosis, non-invasive biomarkers have been sought. The aim of this study was to identify AERD-associated exonic SNPs and examine the diagnostic potential of a combination of these candidate SNPs to predict AERD. DNA from 165 AERD patients, 397 subjects with aspirin-tolerant asthma (ATA), and 398 normal controls were subjected to an Exome BeadChip assay containing 240K SNPs. 1,023 models (2¹⁰-1) were generated from combinations of the top 10 SNPs, selected by the p-values in association with AERD. The area under the curve (AUC) of the receiver operating characteristic (ROC) curves was calculated for each model. SNP Function Portal and PolyPhen-2 were used to validate the functional significance of candidate SNPs. An exonic SNP, exm537513 in HLA-DPB1, showed the lowest p-value (p = 3.40×10⁻⁸) in its association with AERD risk. From the top 10 SNPs, a combination model of 7 SNPs (exm537513, exm83523, exm1884673, exm538564, exm2264237, exm396794, and exm791954) showed the best AUC of 0.75 (asymptotic p-value of 7.94×10⁻²¹), with 34% sensitivity and 93% specificity to discriminate AERD from ATA. Amino acid changes due to exm83523 in CHIA were predicted to be “probably damaging” to the structure and function of the protein, with a high score of ‘1’. A combination model of seven SNPs may provide a useful, non-invasive genetic marker combination for predicting AERD.     

Selection of Aptamers for Mature White Adipocytes by Cell SELEX Using Flow Cytometry

Background

Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited.

Methods and Results

We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes.

Conclusions

These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity.     

Combination Effects of Peramivir and Favipiravir against Oseltamivir-Resistant 2009 Pandemic Influenza A(H1N1) Infection in Mice

Antiviral drugs are being used for therapeutic purposes against influenza illness in humans. However, antiviral-resistant variants often nullify the effectiveness of antivirals. Combined medications, as seen in the treatment of cancers and other infectious diseases, have been suggested as an option for the control of antiviral-resistant influenza viruses. Here, we evaluated the therapeutic value of combination therapy against oseltamivir-resistant 2009 pandemic influenza H1N1 virus infection in DBA/2 mice. Mice were treated for five days with favipiravir and peramivir starting 4 hours after lethal challenge. Compared with either monotherapy, combination therapy saved more mice from viral lethality and resulted in increased antiviral efficacy in the lungs of infected mice. Furthermore, the synergism between the two antivirals, which was consistent with the survival outcomes of combination therapy, indicated that favipiravir could serve as a critical agent of combination therapy for the control of oseltamivir-resistant strains. Our results provide new insight into the feasibility of favipiravir in combination therapy against oseltamivir-resistant influenza virus infection.     

Expression of Vesicular Glutamate Transporters VGLUT1 and VGLUT2 in the Rat Dental Pulp and Trigeminal Ganglion following Inflammation

Background

There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund’s adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis.

Results

The density of VGLUT2− immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group.

Conclusions

These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.     

Genome sequence of the chromate-resistant bacterium Leucobacter salsicius type strain M1-8T

Leucobacter salsicius M1-8^T is a member of the Microbacteriaceae family within the class Actinomycetales. This strain is a Gram-positive, rod-shaped bacterium and was previously isolated from a Korean fermented food. Most members of the genus Leucobacter are chromate-resistant and this feature could be exploited in biotechnological applications. However, the genus Leucobacter is poorly characterized at the genome level, despite its potential importance. Thus, the present study determined the features of Leucobacter salsicius M1-8^T, as well as its genome sequence and annotation. The genome comprised 3,185,418 bp with a G+C content of 64.5%, which included 2,865 protein-coding genes and 68 RNA genes. This strain possessed two predicted genes associated with chromate resistance, which might facilitate its growth in heavy metal-rich environments.     

Establishment and characterization of bortezomib-resistant U266 cell line: Constitutive activation of NF-κB-mediated cell signals and/or alterations of ubiquitylation-related genes reduce bortezomib-induced apoptosis

Bortezomib has been known as the most promising anti-cancer drug for multiple myeloma (MM). However, recent studies reported that not all MM patients respond to bortezomib. To overcome such a stumbling-block, studies are needed to clarify the mechanisms of bortezomib resistance. In this study, we established a bortezomib-resistant cell line (U266/velR), and explored its biological characteristics. The U266/velR showed reduced sensitivity to bortezomib, and also showed crossresistance to the chemically unrelated drug thalidomide. U266/velR cells had a higher proportion of CD138 negative subpopulation, known as stem-like feature, compared to parental U266 cells. U266/velR showed relatively less inhibitory effect of prosurvival NF-κB signaling by bortezomib. Further analysis of RNA microarray identified genes related to ubiquitination that were differentially regulated in U266/velR. Moreover, the expression level of CD52 in U266 cells was associated with bortezomib response. Our findings provide the basis for developing therapeutic strategies in bortezomib-resistant relapsed and refractory MM patients. [BMB Reports 2014; 47(5): 274-279]     

AMD3100 improves ovariectomy-induced osteoporosis in mice by facilitating mobilization of hematopoietic stem/progenitor cells

Inhibition of an increase of osteoclasts has become the most important treatment for osteoporosis. The CXCR4 antagonist, AMD3100, plays an important role in the mobilization of osteoclast precursors within bone marrow (BM). However, the actual therapeutic impact of AMD3100 in osteoporosis has not yet been ascertained. Here we demonstrate the therapeutic effect of AMD3100 in the treatment of ovariectomy-induced osteoporosis in mice. We found that treatment with AMD3100 resulted in direct induction of release of SDF-1 from BM to blood and mobilization of hematopoietic stem/progenitor cells (HSPCs) in an osteoporosis model. AMD3100 prevented bone density loss after ovariectomy by mobilization of HSPCs, suggesting a therapeutic strategy to reduce the number of osteoclasts on bone surfaces. These findings support the hypothesis that treatment with AMD3100 can result in efficient mobilization of HSPCs into blood through direct blockade of the SDF-1/CXCR4 interaction in BM and can be considered as a potential new therapeutic intervention for osteoporosis. [BMB Reports 2014; 47(8): 439-444]     

A novel antagonist to the inhibitors of apoptosis (IAPs) potentiates cell death in EGFR-overexpressing non-small-cell lung cancer cells

In the effort to develop an efficient chemotherapy drug for the treatment of non-small-cell lung cancer (NSCLC), we analyzed the anti-tumorigenic effects of a novel small molecule targeting the inhibitor of apoptosis (IAPs), HM90822B, on NSCLC cells. HM90822B efficiently decreased IAP expression, especially that of XIAP and survivin, in several NSCLC cells. Interestingly, cells overexpressing epidermal growth factor receptor (EGFR) due to the mutations were more sensitive to HM90822B, undergoing cell cycle arrest and apoptosis when treated. In xenograft experiments, inoculated EGFR-overexpressing NSCLC cells showed tumor regression when treated with the inhibitor, demonstrating the chemotherapeutic potential of this agent. Mechanistically, decreased levels of EGFR, Akt and phospho-MAPKs were observed in inhibitor-treated PC-9 cells on phosphorylation array and western blotting analysis, indicating that the reagent inhibited cell growth by preventing critical cell survival signaling pathways. In addition, gene-specific knockdown studies against XIAP and/or EGFR further uncovered the involvement of Akt and MAPK pathways in HM90822B-mediated downregulation of NSCLC cell growth. Together, these results support that HM90822B is a promising candidate to be developed as lung tumor chemotherapeutics by targeting oncogenic activities of IAP together with inhibiting cell survival signaling pathways.Resistance to apoptosis is a hallmark of many solid tumors, including lung cancer, and is, therefore, an important target mechanism for controlling cancer proliferation. The inhibitor of apoptosis (IAP) is a family of proteins containing one or more conserved cysteine and histidine-rich baculoviral IAP repeat (BIR) in their N-terminal domains and a C-terminal RING (really interesting new gene) domain. The BIR domains of IAPs form zinc figure-like structures that bind to active caspases to block caspase activity, while the RING domain acts as an ubiquitin ligase to facilitate proteasome degradation of caspases. Several IAPs have been identified in mammals, including X-linked IAP (XIAP), cellular IAP-1 and -2 (cIAP-1 and cIAP-2) and survivin. Among these IAP proteins, XIAP is a central regulator of both the death receptor- and mitochondria-mediated apoptosis pathways. Consistent with their role in the inhibition of apoptosis, XIAP and survivin are highly expressed in a diverse array of tumors and are often associated with resistance to apoptosis and low sensitivity to chemotherapy drugs in some tumor types.^{1, 2, 3}Recent studies have shown that inhibition of the expression level or function of survivin and/or XIAP with anti-sense RNA, short interfering RNA (siRNA), dominant-negative mutants, or small molecules induces apoptotic cell death in tumor cells but not in normal cells.⁴ Several chemical IAP antagonists, such as AT-406, LCL-161, GDC-0152, TL-32711, LBW242 and HGS-1029, which mimic the interactions of IAP proteins with secondary mitochondria-derived activator of caspase (SMAC) N-terminal peptide (an endogenous antagonist of IAP proteins), have been developed and are currently being evaluated in clinical settings.^{5, 6, 7, 8} The elucidation of the mechanism of antagonism and identification of biomarkers that indicate apoptotic cell death in tumors are key issues in the development of IAP antagonists. As such, the role of IAPs in regulating the apoptotic response and as molecular targets for achieving selective therapeutic effects in tumor cells has attracted great attention in an effort to identify peptide antagonists or small-molecule inhibitors.Lung cancer is the leading cause of cancer-related death worldwide, with more than one million mortalities each year. Almost 85% of all lung cancer cases are diagnosed as non-small-cell lung cancers (NSCLC), which are further classified histologically as adenocarcinoma, squamous cell carcinoma or large cell carcinoma. Platinum-based chemotherapy represents the recommended standard first-line systemic treatment for advanced NSCLC, although the results of this approach are limited to a modest increase in survival rates. Epidermal growth factor receptor (EGFR) is often hyper-activated in many lung cancers due to the presence of a mutation in the kinase domain, causing the activation of multiple cell survival signals, especially Akt and mitogen-activated protein kinase (MAPK) pathways. This finding has led to the development of targeted therapeutics against the kinase, such as erlotinib and gefitinib, which becomes one of the most promising strategies for cancer treatment. The targeted therapeutics has often failed, however, due to the development of resistance through multiple mechanisms, indicating that additional adjuvants are necessary to achieve effective results.In this study, we investigated the therapeutic potential of HM90822B, originally synthesized to inhibit IAP activity, on NSCLC cells and in a xenograft mouse model and analyzed the cellular effects of the drug to elucidate its mechanism of action. Our results showed that HM90822B inhibits cell growth resulting in cell cycle arrest and apoptosis by targeting XIAP and survivin in conjunction with the inhibition of EGFR-MAPK pathway, primarily AKT, p38 and c-jun phosphorylation. These results indicate that the IAP inhibitor HM90822B is a promising therapeutics for the treatment of NSCLC.     

pH-Sensitive Polymeric Micelle-Based pH Probe for Detecting and Imaging Acidic Biological Environments

To overcome the limitations of monomeric pH probes for acidic tumor environments, this study designed a mixed micelle pH probe composed of polyethylene glycol (PEG)-b-poly(l-histidine) (PHis) and PEG-b-poly(l-lactic acid) (PLLA), which is well-known as an effective antitumor drug carrier. Unlike monomeric histidine and PHis derivatives, the mixed micelles can be structurally destabilized by changes in pH, leading to a better pH sensing system in nuclear magnetic resonance (NMR) techniques. The acidic pH-induced transformation of the mixed micelles allowed pH detection and pH mapping of 0.2-0.3 pH unit differences by pH-induced "on/off"-like sensing of NMR and magnetic resonance spectroscopy. The micellar pH probes sensed pH differences in nonbiological phosphate buffer and biological buffers such as cell culture medium and rat whole blood. In addition, the pH-sensing ability of the mixed micelles was not compromised by loaded doxorubicin. In conclusion, PHis-based micelles could have potential as a tool to simultaneously treat and map the pH of solid tumors in vivo.