Online Detection of HCN in Humid Exhaled Air by Gas Flow-Assisted Negative Photoionization Mass Spectrometry.

Hydrogen cyanide (HCN) is a well-known toxic compound in many fields. The trace amount of endogenous HCN in human exhalation has been associated with the presence of Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) patients. Online monitoring of HCN profile is promising to screen PA infection rapidly and accurately. In this study, a gas flow-assisted negative photoionization (NPI) mass spectrometry method was developed for monitoring the single-exhalation HCN profile. The sensitivity could be optimized by introducing helium to eliminate the humidity influence and reduce the low mass cutoff effect, with improvements of a factor 150 observed. By employing a purging gas procedure and minimizing the length of the sample line, the residual and response time were greatly reduced. The limit of detection (LOD) of 0.3 ppbv and time resolution of 0.5 s were achieved. HCN profiles of exhalations from different volunteers before or after gargling with water were detected to show the performance of the method. All profiles showed a sharp peak and a stable end-tidal plateau, representing the concentration of oral cavity and end-tidal gas, respectively. The HCN concentration based on the plateau of the profile showed better reproducibility and accuracy, which indicates this method has potential application in the detection of PA infection in CF patients.

Rapid Determination of Ethyl Carbamate in Chinese Liquor via a Direct Injection Mass Spectrometry with Time-Resolved Flash-Thermal-Vaporization and Acetone-Assisted High-Pressure Photoionization Strategy.

Ethyl carbamate (EC), a carcinogenic compound, is naturally produced in fermented foods and alcoholic beverages. Rapid and accurate measurement of EC is necessary and important for quality control and safety evaluation of Chinese liquor, a traditionally distilled spirit with the highest consumption in China, but it remains a great challenge. In this work, a direct injection mass spectrometry (DIMS) with time-resolved flash-thermal-vaporization (TRFTV) and acetone-assisted high-pressure photoionization (HPPI) strategy has been developed. EC was rapidly separated from the main matrix components, ethyl acetate (EA) and ethanol, by the TRFTV sampling strategy due to the retention time difference of these three compounds with large boiling point differences on the inner wall of a poly(tetrafluoroethylene) (PTFE) tube. Therefore, the matrix effect of EA and ethanol was effectively eliminated. The acetone-assisted HPPI source was developed for efficient ionization of EC through a photoionization-induced proton transfer reaction between EC molecules and protonated acetone ions. The accurate quantitative analysis of EC in liquor was achieved by introducing an internal standard method (ISM) using deuterated EC (d5-EC). As a result, the limit of detection (LOD) for EC was 8.88 µg/L with the analysis time of only 2 min, and the recoveries ranged from 92.3 to 113.1%. Finally, the prominent capability of the developed system was demonstrated by rapid determination of trace EC in Chinese liquors with different flavor types, exhibiting wide potential applications in online quality control and safety evaluation of not only Chinese liquors but also other liquor and alcoholic beverages.

Rapid and highly sensitive measurement of trimethylamine in seawater using dynamic purge-release and dopant-assisted atmospheric pressure photoionization mass spectrometry.

Trimethylamine (TMA) is ubiquitous in the marine systems and may affect atmospheric chemistry as a precursor and strong stabilizer of atmospheric secondary aerosol, influencing cloud formation. Rapid and accurate measurement of the concentration of TMA in seawater is challenging due to their polarity, aqueous solubility, volatility and existence at low concentrations in marine environments. In this study, a dopant-assisted atmospheric pressure photoionization time-of-flight mass spectrometry (DA-APPI-TOFMS) coupled with a dynamic purge-release method was developed for rapid and sensitive analysis of TMA in seawater. A novel three-zones ionization source has been developed for improving the ionization efficiency of analyte molecules and minimizing the influence of high-humidity of the sample gas, which allowed direct analysis of high-humidity (RH> 90%) gas samples from microbubble purging process by the mass spectrometer. At atmospheric pressure, the three-zones ionization source allows the use of high-speed purge gas to quickly purge all organic amines dissolved in the water into the gas phase, ensuring quantitative accuracy. The limit of quantification (LOQ) for TMA down to 0.1 µg L-1 was obtained in less than 2 min by consuming only 2 mL seawater sample. This method was applied for the determination of the concentrations of TMA in the coastal seawater to validate its practicability and reliability for analysis of trace amines in marine environments.

IkappaB kinase alpha kinase activity is required for self-renewal of ErbB2/Her2-transformed mammary tumor-initiating cells.

NF-kappaB is constitutively active in many solid tumors, including breast cancer. However, the role of NF-kappaB in breast carcinogenesis is unknown. Ikkalpha(AA/AA) "knockin" mice in which activation of IkappaB kinase alpha (IKKalpha) is prevented by replacement of activation loop serines with alanines exhibit delayed mammary gland growth during pregnancy, because IKKalpha activity is required for cyclin D1 induction and proliferation of lobuloalveolar epithelial cells. Given the role of cyclin D1 in breast and mammary cancer, we examined involvement of IKKalpha in mammary carcinogenesis induced by oncogenes or a chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). The Ikkalpha(AA) mutation retarded tumor development in response to either 7,12-dimethylbenzaanthracene or the MMTV-c-neu (ErbB2/Her2) transgene but had no effect on MMTV-v-Ha-ras-induced cancer, although both oncogenes rely on cyclin D1. Strikingly, primary Ikkalpha(AA/AA)/MMTV-c-neu carcinoma cells exhibited diminished self-renewal capacity, resulting in the inability to establish secondary tumors. Ikkalpha(AA/AA)/MMTV-c-neu carcinoma cells underwent premature senescence when cultured under conditions used for propagation of mammary gland stem cells. Thus, IKKalpha is not only a regulator of mammary epithelial proliferation, but is also an important contributor to ErbB2-induced oncogenesis, providing signals that maintain mammary tumor-initiating cells. IKKalpha may represent a novel and specific target for treatment of ErbB2-positive breast cancer.

I{kappa}B kinase (IKK){beta}, but not IKK{alpha}, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss.

Transcription factor, nuclear factor kappaB (NF-kappaB), is required for osteoclast formation in vivo and mice lacking both of the NF-kappaB p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the IkappaB kinase (IKK) complex that mediate NF-kappaB activation, IKKalpha and IKKbeta, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKbeta subunit as a transducer of signals from receptor activator of NF-kappaB (RANK) to NF-kappaB. Although IKKalpha is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKbeta is required for osteoclastogenesis in vitro and in vivo. IKKbeta also protects osteoclasts and their progenitors from tumor necrosis factor alpha-induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.

Activation of IKKalpha target genes depends on recognition of specific kappaB binding sites by RelB:p52 dimers.

IkappaB Kinase (IKK)alpha is required for activation of an alternative NF-kappaB signaling pathway based on processing of the NF-kappaB2/p100 precursor protein, which associates with RelB in the cytoplasm. This pathway, which activates RelB:p52 dimers, is required for induction of several chemokine genes needed for organization of secondary lymphoid organs. We investigated the basis for the IKKalpha dependence of the induction of these genes in response to engagement of the lymphotoxin beta receptor (LTbetaR). Using chromatin immunoprecipitation, we found that the promoters of organogenic chemokine genes are recognized by RelB:p52 dimers and not by RelA:p50 dimers, the ubiquitous target for the classical NF-kappaB signaling pathway. We identified in the IKKalpha-dependent promoters a novel type of NF-kappaB-binding site that is preferentially recognized by RelB:p52 dimers. This site links induction of organogenic chemokines and other important regulatory molecules to activation of the alternative pathway.

NF-kappaB in mammary gland development and breast cancer.

Nuclear factor of kappaB (NF-kappaB) is a group of sequence-specific transcription factors that is best known as a key regulator of the inflammatory and innate immune responses. Recent studies of genetically engineered mice have clearly indicated that NF-kappaB is also required for proper organogenesis of several epithelial tissues, including the mammary gland. Mice have shown severe lactation deficiency when NF-kappaB activation is specifically blocked in the mammary gland. In addition, there are strong suggestions that NF-kappaB may play an important role in the etiology of breast cancer. Elevated NF-kappaB DNA-binding activity is detected in both mammary carcinoma cell lines and primary human breast cancer tissues.

Proteotyping of mammary tissue from transgenic and gene knockout mice with immunohistochemical markers: a tool to define developmental lesions.

Through the use of transgenic and gene knockout mice, several studies have identified specific genes required for the functional development of mammary epithelium. Although histological and milk protein gene analyses can provide useful information regarding functional differentiation, they are limited in their ability to precisely define the molecular lesions. For example, mice that carry a mutation in one of the subunits of the IkappaB kinase, IKKalpha, cannot lactate despite the presence of histologically normal alveolar compartment and the expression of milk protein genes. To further define and understand such lesions on a molecular level, we sought evidence for proteins that are differentially expressed during mammary gland development with a view to generating a tissue proteotype. Using database screens and immunohistochemical analyses, we have identified three proteins that exhibit distinct profiles. Here, using mouse models as test biological systems, we demonstrate the development and application of mammary tissue proteotyping and its use in the elucidation of specific developmental lesions. We propose that the technique of proteotyping will have wide applications in the analyses of defects in other mouse models.

The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-kappaB pathways.

The lymphotoxin-beta receptor (LTbetaR) plays critical roles in inflammation and lymphoid organogenesis through activation of NF-kappaB. In addition to activation of the classical NF-kappaB, ligation of this receptor induces the processing of the cytosolic NF-kappaB2/p100 precursor to yield the mature p52 subunit, followed by translocation of p52 to the nucleus. This activation of NF-kappaB2 requires NIK and IKKalpha, while NEMO/IKKgamma is dispensable for p100 processing. IKKbeta-dependent activation of canonical NF-kappaB is required for the expression but not processing of p100 and for the expression of proinflammatory molecules including VCAM-1, MIP-1beta, and MIP-2 in response to LTbetaR ligation. In contrast, IKKalpha controls the induction by LTbetaR ligation of chemokines and cytokines involved in lymphoid organogenesis, including SLC, BLC, ELC, SDF1, and BAFF.

NF-kappaB in cancer: from innocent bystander to major culprit.

Nuclear factor of kappaB (NF-kappaB) is a sequence-specific transcription factor that is known to be involved in the inflammatory and innate immune responses. Although the importance of NF-KB in immunity is undisputed, recent evidence indicates that NF-kappaB and the signalling pathways that are involved in its activation are also important for tumour development. NF-kappaB should therefore receive as much attention from cancer researchers as it has already from immunologists.