Rapid detection of Bacillus spore aerosol particles by direct in situ analysis using MALDI-TOF mass spectrometry.

UNLABELLED: Accurate and rapid analytical methods are essential for the detection and identification of biological warfare agents (BWA). Although various studies have investigated the use of MALDI-TOF MS for bacterial classification, only a few studies have examined the applicability of method for the identification of BWAs. This study aimed to generate, collect and analyse Bacillus spore aerosol particles of 2-10 µm, the optimal size of a BWA. Bacillus spore aerosol particles of 2-10 µm were rapidly analysed using direct in situ MALDI-TOF MS without pretreatment processes. A mass spectrum of Bacillus spore aerosol particles was successfully detected. For real-time detection and identification, a mass spectral database of Bacillus spores was constructed, and an algorithm was developed and applied. Bacillus spore particles were rapidly detected and identified by MS, which can be used for the detection and inspection of BWAs. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods that permit accurate and fast detection and identification are essential in the case of pathogens such as Bacillus anthracis, which can be used as biological warfare agents or bioterrorism materials. Here, direct in situ MALDI-TOF MS allowed the high-throughput detection and identification of aerosolized Bacillus spore particles, and this method could be effectively applied for the detection and inspection of BWAs. In this context, we believe that this method may contribute to the realization of real-time and on-site identification of BWAs.

Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex.

Skp2 (S-phase kinase-associated protein-2) SCF complex displays E3 ligase activity and oncogenic activity by regulating protein ubiquitination and degradation, in turn regulating cell cycle entry, senescence and tumorigenesis. The maintenance of the integrity of Skp2 SCF complex is critical for its E3 ligase activity. The Skp2 F-box protein is a rate-limiting step and key factor in this complex, which binds to its protein substrates and triggers ubiquitination and degradation of its substrates. Skp2 is found to be overexpressed in numerous human cancers, which has an important role in tumorigenesis. The molecular mechanism by which the function of Skp2 and Skp2 SCF complex is regulated remains largely unknown. Here we show that Foxo3a transcription factor is a novel and negative regulator of Skp2 SCF complex. Foxo3a is found to be a transcriptional repressor of Skp2 gene expression by directly binding to the Skp2 promoter, thereby inhibiting Skp2 protein expression. Surprisingly, we found for the first time that Foxo3a also displays a transcription-independent activity by directly interacting with Skp2 and disrupting Skp2 SCF complex formation, in turn inhibiting Skp2 SCF E3 ligase activity and promoting p27 stability. Finally, we show that the oncogenic activity of Skp2 is repressed by Foxo3a overexpression. Our results not only reveal novel insights into how Skp2 SCF complex is regulated, but also establish a new role for Foxo3a in tumor suppression through a transcription-dependent and independent manner.

Characterization of DDRI-18 (3,3'-(1H,3'H-5,5'-bibenzo[d]imidazole-2,2'-diyl)dianiline), a novel small molecule inhibitor modulating the DNA damage response.

BACKGROUND AND PURPOSE: Recently, the DNA damage response (DDR) has emerged as a promising target for anticancer drug development. In our previous study, we identified several DDR-inhibiting compounds via high-content screening of a small molecule library using γH2AX foci as a biomarker. Here, we studied the effects of the DNA damage response inhibitor DDRI-18 (3,3'-(1H,3'H-5,5'-bibenzo[d]imidazole-2,2'-diyl)dianiline) on DDR. EXPERIMENTAL APPROACH: Osteosarcoma U2OS cells were treated with etoposide to induce DDR. The nuclear foci of γH2AX and other signalling molecules in DDR were visualized by immunofluorescence and quantified using an IN Cell Analyzer. The DNA repair capacity of cells was analysed using the comet assay and in vivo DNA end-joining assay. Cell survival after drug treatment was quantified using the MTT assay, and apoptotic cell death was analysed by Annexin V staining and flow cytometry. KEY RESULTS: DDRI-18 inhibited the non-homologous end-joining (NHEJ) DNA repair process and delayed the resolution of DNA damage-related proteins (γH2AX, ATM and BRCA1) from DNA lesions at a later phase of DDR. Furthermore, DDRI-18 enhanced the cytotoxic effects of anticancer DNA-damaging drugs, including etoposide, camptothecin, doxorubicin and bleomycin. This synergistic effect on cell death was shown to be due to caspase-dependent apoptosis. CONCLUSIONS AND IMPLICATIONS: We identified a chemical compound, DDRI-18, that has chemosensitization activity. Although the target molecule and mechanism of action of DDRI-18 remain unknown, DDRI-18 is an effective chemosensitizing agent and may improve the therapy with classical anticancer drugs.

Association between interleukin-4 gene polymorphisms and intracerebral haemorrhage in Korean population.

Interleukin-4 (IL4) polymorphisms (rs2243250, rs2070874) were analysed in Korean patients with ischaemic stroke (IS) (n=119) and intracerebral haemorrhage (ICH) (n=79), and age-matched controls (n =267, IS; n=401, ICH) using direct sequencing. Both single nucleotide polymorphisms and their haplotypes were associated with ICH, but not IS.

Alfentanil attenuates phenylephrine-induced contraction in rat aorta.

BACKGROUND AND OBJECTIVES: Alfentanil was reported to relax the rat aorta by direct action on the vascular smooth muscle. The aims of this in vitro study were to examine the effect of alfentanil on phenylephrine-induced contractions in the rat aorta and to determine the cellular mechanism associated with this process. METHODS: Endothelium-denuded aortic rings were suspended in order to record isometric tension. In the rings with or without 10(-6) mol naloxone or 10(-5) mol verapamil, the concentration-response curves for phenylephrine and potassium chloride were generated in the presence or absence of alfentanil (10(-6), 5 x 10(-5), 10(-4) mol). In the rings exposed to a calcium-free isotonic depolarizing solution, the contractile response induced by the addition of calcium was assessed in the presence or absence of alfentanil (5 x 10(-5), 10(-4) mol). RESULTS: Alfentanil (5 x 10(-5), 10(-4) mol) attenuated (P < 0.05) the phenylephrine-induced contraction in the ring with or without 10(-6) mol naloxone but had no effect on the phenylephrine-induced contraction in the rings pretreated with verapamil. Alfentanil (5 x 10(-5), 10(-4) mol) produced a significant rightward shift (P < 0.01) in the potassium chloride dose-response curve, and attenuated the contractile response (P < 0.001) induced by calcium in the calcium-free isotonic depolarizing solution in a dose-dependent manner. CONCLUSIONS: A supraclinical dose of alfentanil attenuates the phenylephrine-induced contraction via an inhibitory effect on calcium influx by blocking the l-type calcium channels in the rat aortic vascular smooth muscle.

Highly polarization-dependent periodic coupling in mechanically induced long period grating over air-silica fibers.

A very flexible and versatile tunable mechanical grating platform is introduced, with which highly polarization-dependent mode coupling is observed for three types of air-silica microstructured fibers: hollow core fiber, hexagonal-boundary holey fiber (HHF), and circular-boundary holey fiber. The resonances of gratings showed highly polarization-dependent broadband coupling compared with conventional single-mode fibers due to their unique beat-length dispersions between the core and the cladding modes, which could find applications in wideband polarization-dependent loss compensation. We further present significance of the spatial symmetry of HHF in distinct mode coupling for different rotation angles around the fiber axis.

Targeted degradation of proteins by small molecules: a novel tool for functional proteomics.

A novel strategy that targets protein for degradation has recently been developed by exploiting a protein-targeting chimeric molecule ('Protac'). Typically, the chimeric Protac is composed of a small-molecule ligand ('bait') on one end and a synthetic octapeptide on the other. This octapeptide is recognized by E3 ubiquitin ligase pVHL (von Hippel Lindau tumor suppressor protein), thereby recruiting a small molecule-bound protein ('prey') to pVHL for ubiquitination and degradation. Since selective degradation of a cellular protein generates a "loss of function" mutation, this protein knock-out strategy may be useful to study the function of a given protein or to evaluate whether a cellular protein is a potential target for drug intervention, in a manner reminiscent of gene knock-out or siRNA approaches. Herein, we show that a synthetic pentapeptide is sufficient to interact with pVHL E3 ligase, and that the pentapeptide-based Protac efficiently induces ubiquitination and degradation of target protein. Our results also demonstrate that the pentapeptide-based Protac can enter cells efficiently to exerts its biological activity effectively. These results suggest that the synthetic pentapeptide can be used either directly in the preparation of cell-permeable Protacs or as a template to develop peptidomimetic or non-peptide Protacs.

Short report: genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate.

We determined the full-length genome sequence of Japanese encephalitis virus (JEV) K94P05 isolated in Korea. Sequence analysis showed that the 10,963-nucleotide-long RNA genome of K94P05 was 13 or 14 nucleotides shorter than the genome of other JEV isolates because of a deletion in the 3' noncoding region of K94P05. Compared with sequences of other JEV isolates, the full-length nucleotide sequence showed 89.0-89.6% homology, and the deduced amino acid sequence showed between 96.4-97.3% homology. A region of approximately 60 nucleotides immediately downstream of the open reading frame stop codon of K94P05 showed high sequence variability as compared with other JEV isolates. K94P05 formed a distinct group within a phylogenetic tree established with the full-length genome sequences. Cross-neutralization studies showed that polyclonal antibodies to Korean isolates were 3 times better at neutralizing the Korean isolates than antibodies to Nakayama-NIH. These findings suggest that Korean JEV K94P05 is genetically and antigenically distinct from other Asian JEV isolates.

Risk factors for infection and role of C-reactive protein in Korean patients with systemic lupus erythematosus.

To evaluate risk factors for infection and the role of C-reactive protein (CRP) in the diagnosis of infection, a retrospective case control study was performed among Korean systemic lupus erythematosus patients. Of 120 proven infections, 31 episodes (25.8%) occurred in patients taking no corticosteroids (CS). The risk of infection was lower in patients taking low-dose CS (< 300 mg prednisolone/month) than no CS (odds ratio (OR) 0.36). In patients receiving high-dose CS (> 1000 mg prednisolone/month), however, the risk increased (OR 2.9). In patients taking no CS, disease activity manifested as increased SLEDAI, anemia and active urinary sediment, was associated with infection. The CRP was higher in the patients with infection than controls and the CRP levels over 50 mg/l were observed only in infection. These results suggest that CS have a bimodal influence on infection depending on dose. Disease activity is an important risk factor for infection in patients taking no CS. Finally, CRP levels greater than 50 mg/l suggest the presence of infection.

Comparison of the efficacy and safety of different formulations of omeprazole-based triple therapies in the treatment of Helicobacter pylori-positive peptic ulcer.

Little is known about the efficacy and safety of different formulations of omeprazole-based triple therapy regimens for the treatment of Helicobacter pylori-positive peptic ulcer. We compared the efficacy and safety of two formulations of omeprazole used in triple therapies in patients with H. pylori-positive active peptic ulcer. Seventy-four patients with endoscopically proven H. pylori-positive active peptic ulcer were randomized to two groups, each with 37 patients, to receive either OAC-I (6 weeks of "A" formulation of omeprazole [20 mg twice daily] plus 2 weeks of amoxicillin [1.0 g twice daily] and clarithromycin [500 mg twice daily] or OAC-II (6 weeks of "B" formulation of omeprazole [20 mg twice daily] plus 2 weeks of the same antibiotics. The H. pylori and ulcer healing status were assessed at the baseline and at the 6-week endpoint of therapy. Gastrointestinal symptoms, documentation of adverse events, and standard laboratory examinations were assessed at each visit. Eradication of H. pylori (intention to treat [n = 74]/per protocol [n = 66]) and healing of the ulcer were successful in 83.8%/96.9% and 93.8%, respectively, of the OAC-I group patients, and in 91.9%/100% and 97.1%, respectively, of the OAC-II group patients (P = 0.477; P = 0.608). The OAC-I group experienced rapid resolution of symptoms, but no significant differences were found between the two groups for number of days taken for resolution of gastrointestinal symptoms, adverse events, and laboratory findings. The two different formulations of omeprazole used in triple therapy regimens produced similar efficacy and safety results after 6 weeks of treatment in patients with H. pylori-positive active peptic ulcer.

A new guaiane type sesquiterpene from Torilis japonica.

A new guaiane type sesquiterpene was isolated from the fruit of Torilis japonica (Umbelliferae). Based on NMR, IR and mass spectroscopy its structure was confirmed as deangeloyloxy torilin, 1beta, 7alpha, 10alphaH-11-acetoxy-guaia-4-en-3-one (1). This is the first report showing that this compound can be isolated from Torilis japonica.

Bioconversion of cellulose into ethanol by nonisothermal simultaneous saccharification and fermentation.

The kinetic characteristics of cellulase and beta-glucosidase during hydrolysis were determined. The kinetic parameters were found to reproduce experimental data satisfactorily and could be used in a simultaneous saccharification and fermentation (SSF) system by coupling with a fermentation model. The effects of temperature on yeast growth and ethanol production were investigated in batch cultures. In the range of 35-45 degrees C, using a mathematical model and a computer simulation package, the kinetic parameters at each temperature were estimated. The appropriate forms of the model equation for the SSF considering the effects of temperature were developed, and the temperature profile for maximizing the ethanol production was also obtained. Briefly, the optimum temperature profile began at a low temperature of 35 degrees C, which allows the propagation of cells. Up to 10 h, the operating temperature increased rapidly to 39 degrees C, and then decreased slowly to 36 degrees C. In this nonisothermal SSF system with the above temperature profile, a maximum ethanol production of 14.87 g/L was obtained.

Successful treatment of protein-losing enteropathy due to AA amyloidosis with somatostatin analogue and high dose steroid in ankylosing spondylitis.

Secondary amyloidosis is an occasional complication of ankylosing spondylitis (AS) and in most cases renal amyloidosis presents with proteinuria, nephrotic syndrome and decreased renal function. We describe a 32-year-old male patient with AS manifested by frequent diarrhea, intermittent abdominal pain and low serum albumin levels. He has suffered from severe inflammatory back pain for 14 years with multiple peripheral joint involvement. Protein-losing enteropathy due to gastrointestinal amyloidosis was diagnosed with 99mTc-human albumin scintigraphy, fecal alpha-1 antitrypsin clearance and colonoscopic biopsy with Congo red staining. Somatostatin analogue octreotide and prednisolone were introduced with successful result.

A new bisabolene epoxide from Tussilago farfara, and inhibition of nitric oxide synthesis in LPS-activated macrophages.

A new bisabolene epoxide was isolated from the flower buds of Tussilago farfara, and the structure was determined by spectroscopic methods to be 1alpha,5alpha-bisacetoxy-8-angeloyloxy-3beta, 4beta-epoxy-bisabola-7(14),10-dien-2-one (1). Compound 1 showed inhibition of nitric oxide synthesis in lipopolysaccharide-activated macrophages.

Yomogin, an inhibitor of nitric oxide production in LPS-activated macrophages.

In activated macrophages the inducible form of nitric oxide synthase (i-NOS) generates high amounts of toxic mediator, nitric oxide (NO) which contributes to the circulatory failure associated with septic shock. A sesquiterpene lactone compound (yomogin) isolated from medicinal plant Artemisia princeps Pampan inhibited the production of NO in LPS-activated RAW 264.7 cells by suppressing i-NOS enzyme expression. Thus, yomogin may be a useful candidate for the development of new drugs to treat endotoxemia and inflammation accompanied by the overproduction of NO.

Coronavirus transcription mediated by sequences flanking the transcription consensus sequence.

In our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. Using our subgenomic DI system, we have studied the effects of sequences flanking the MHV transcription consensus sequence on subgenomic RNA transcription. We obtained the following results. (i) Insertion of a 12-nucleotide-long sequence including the UCUAAAC transcription consensus sequence at different locations of the DI RNA resulted in different efficiencies of subgenomic DI RNA synthesis. (ii) Differences in the amount of subgenomic DI RNA were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the DI RNA. (iii) Naturally occurring flanking sequences of intergenic sequences at gene 6-7, but not at genes 1-2 and 2-3, contained a transcription suppressive element(s). (iv) Each of three naturally occurring flanking sequences of an MHV genomic cryptic transcription consensus sequence from MHV gene 1 also contained a transcription suppressive element(s). These data showed that sequences flanking the transcription consensus sequence affected MHV transcription.

Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques.

Adult macaques do not develop disease after infection with a nef deletion mutant of the simian immunodeficiency virus (SIV) and are protected against challenge with pathogenic virus. This finding led to the proposal to use nef-deleted viruses as live, attenuated vaccines to prevent human acquired immunodeficiency syndrome (AIDS). In contrast, neonatal macaques developed persistently high levels of viremia after oral exposure to and SIV nef, vpr, and negative regulatory element (NRE) deletion mutant. Severe hemolytic anemia, thrombocytopenia, and CD4+ T cell depletion were observed, indicating that neither nef nor vpr determine pathogenicity in neonates. Because such constructs have retained their pathogenic potential, they should not be used as candidate live, attenuated virus vaccines against human AIDS.

Evidence for coronavirus discontinuous transcription.

Coronavirus subgenomic mRNA possesses a 5'-end leader sequence which is derived from the 5' end of genomic RNA and is linked to the mRNA body sequence. This study examined whether coronavirus transcription involves a discontinuous transcription step; the possibility that a leader sequence from mouse hepatitis virus (MHV) genomic RNA could be used for MHV subgenomic defective interfering (DI) RNA transcription was examined. This was tested by using helper viruses and DI RNAs that were easily distinguishable. MHV JHM variant JHM(2), which synthesizes a subgenomic mRNA encoding the HE gene, and variant JHM(3-9), which does not synthesize this mRNA, were used. An MHV DI RNA, DI(J3-9), was constructed to contain a JHM(3-9)-derived leader sequence and an inserted intergenic region derived from the region preceding the MHV JHM HE gene. DI(J3-9) replicated efficiently in JHM(2)- or JHM(3-9)-infected cells, whereas synthesis of subgenomic DI RNAs was observed only in JHM(2)-infected cells. Sequence analyses demonstrated that the 5' regions of both helper virus genomic RNAs and genomic DI RNAs maintained their original sequences in DI RNA-replicating cells, indicating that the genomic leader sequences derived from JHM(2) functioned for subgenomic DI RNA transcription. Replication and transcription of DI(J3-9) were observed in cells infected with an MHV A59 strain whose leader sequence was similar to that of JHM(2), except for one nucleotide substitution within the leader sequence. The 5' region of the helper virus genomic RNA and that of the DI RNA were the same as their original structures in virus-infected cells, and the leader sequence of DI(J3-9) subgenomic DI RNA contained the MHV A59-derived leader sequence. The leader sequence of subgenomic DI RNA was derived from that of helper virus; therefore, the genomic leader sequence had a trans-acting property indicative of a discontinuous step in coronavirus transcription.