Electronic cigarette use in young people in Great Britain 2013-2014.

OBJECTIVES: The recent growth in the market for electronic cigarettes (e-cigarettes) has led to concerns over their use by young people. It is therefore important to examine trends in the perception and use of e-cigarettes and conventional cigarettes in this group. STUDY DESIGN: Two-wave cross-sectional survey design. METHODS: Young people aged 11-18 in Great Britain were surveyed online by YouGov in 2013 and 2014. Use of e-cigarettes, together with perceived health harms and intention to use were assessed and compared in relation to cigarette smoking history, age and gender. RESULTS: Ever-use of e-cigarettes increased significantly from 4.6% (95% CI 3.8-5.7) in 2013 to 8.2% (95% CI 7.0-9.6) in 2014. Monthly or more use of e-cigarettes increased from 0.9% (95% CI 0.5-1.5) to 1.7 (1.2-2.4), but remained rare in never-smokers at under 0.2%. The proportion of young people who perceived e-cigarettes to be less harmful to users than cigarettes fell from 73.4% (95% CI 71.0-75.8) to 66.9% (95% CI 64.5-69.2), while the proportion who considered e-cigarettes to cause similar levels of harm increased from 11.8% (95% CI 10.0-13.5) to 18.2% (95% CI 16.3-20.1). Of the 8.2% of e-cigarette ever-users in 2014, 69.8% (95% CI 62.2%-77.3%) had smoked a cigarette prior to using an e-cigarette, while 8.2% (95% CI 4.1%-12.2%) first smoked a cigarette after e-cigarette use. CONCLUSIONS: A growing proportion of young people in Great Britain believe e-cigarettes are as harmful as smoking tobacco. Use of e-cigarettes by young people is increasing, but is largely confined to those who smoke.

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

Public health measures to reduce smoking prevalence in the UK: how many lives could be saved?

OBJECTIVE: To estimate the number of deaths that could be prevented in the UK by implementing population strategies to reduce smoking prevalence. DESIGN: A prospective analysis of future mortality using recent national smoking prevalence data and relative risks of mortality in current smokers, ex-smokers, and never-smokers. POPULATION: Smokers in the UK. INTERVENTIONS: Population measures of proven effectiveness assumed to reduce smoking prevalence by 1 percentage point per year for 10 years, or alternatively by 13% over 19 years (1 percentage point per annum for seven years, 0.5 percentage point per annum for 12 years) as considered to be achievable in a recent report to the UK Chancellor of the Exchequer. MAIN OUTCOME MEASURE: Estimated deaths from smoking prevented in the 35-75 year age group. RESULTS: Reducing the prevalence of smoking by 1 percentage point each year for 10 years would prevent 69 049 deaths at ages between 35 and 74 years during that period. The model of reduction by 13% over 19 years would prevent 54 308 and 194 493 deaths in 10 and 19 years, respectively. Continued prevalence reductions at the current rate of 0.4 percentage points each year will prevent 23 192 deaths over 10 years. CONCLUSIONS: Full implementation of simple population measures to encourage smoking cessation could prevent substantial numbers of deaths in the UK.

Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas.

A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located. Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors.

Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis.

The high resolving power of two-dimensional polyacrylamide gel electrophoresis 2D-PAGE and its full analytical and preparative potential have been described with special emphasis on reproducibility and standardization of protein spot patterns, enhanced protein detection sensitivity, and computer analysis database development. New methodologies for peptide mass fingerprinting, peptide, sequence, and fragmentation tagging have been highlighted. Major challenges associated with 2D-PAGE/mass spectrometric protein sequencing were outlined which need to be addressed in the future, including sample enrichment, use of alternative gel matrices, improvements in separation systems interfaced directly to the mass spectrometer, and design of high-sensitivity instruments with very high mass ranges. It is hoped that comparative studies to identify, quantitate, and characterize proteins differentially expressed in normal versus diseased cells would give insight into mechanisms of pathogenesis and allow the development of a way to control both the etiology and the course of diseases.

Amino acid sequence of the minor isomorph of the crustacean hyperglycemic hormone (CHH-II) of the Mexican crayfish Procambarus bouvieri (Ortmann): presence of a D-amino acid.

The primary structure of the neurohormone crustacean hyperglycemic hormone (CHH-II) was determined by means of enzymatic digestions, manual Edman degradation, and mass spectrometry. CHH-II is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. The peptide has blocked N- and C-termini, and lacks tryptophan, histidine, and methionine. The CHH-I and CHH-II of Procambarus bouvieri have identical sequences and elicit levels of hyperglycemia that are not distinguishable. The difference between the two isomorphs consists in a posttranslational modification of a L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus.

Cherubism--an initial unilateral presentation.

An unusual presentation of cherubism is reported owing to the initial unilateral nature and late onset of occurrence. A classification for cherubism is proposed and the difficulty in diagnosis of unilateral cases is discussed.

WISP-1 binds to decorin and biglycan.

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Structural and experimental evidence suggests that CCN family member activities are modulated by their interaction with sulfated glycoconjugates. To elucidate the mechanism of action for WISP-1, we characterized the specificity of its tissue and cellular interaction and identified binding factors. WISP-1 binding was restricted to the stroma of colon tumors and to cells with a fibroblastic phenotype. By using a solid phase assay, we showed that human skin fibroblast conditioned media contained WISP-1 binding factors. Competitive inhibition with different glycosaminoglycans and treatment with glycosaminoglycan lyases and proteases demonstrated that binding to the conditioned media was mediated by dermatan sulfate proteoglycans. Mass spectrometric analysis identified the isolated binding factors as decorin and biglycan. Decorin and biglycan interacted directly with WISP-1 and inhibited its binding to components in the conditioned media. Similarly, WISP-1 interaction with human skin fibroblasts was inhibited by dermatan sulfate, decorin, and biglycan or by treatment of the cell surface with dermatan sulfate-specific lyases. Together these results demonstrate that decorin and biglycan are WISP-1 binding factors that can mediate and modulate its interaction with the surface of fibroblasts. We propose that this specific interaction plays a role in the regulation of WISP-1 function.

Mass spectrometry of proteins and peptides: sensitive and accurate mass measurement and sequence analysis.

The study of proteins and peptides by mass spectrometry has been advanced by the development of matrix-assisted laser desorption/ionization and electrospray ionization. Proteins with masses > 100 kDa may be measured accurately at picomole sensitivities. Sequence analysis of peptides in mixtures at subpicomole quantities is possible by tandem mass spectrometry. Practical applications of the new technology to biological research are illustrated.

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry.

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.

Kinetics of Polychlorinated Biphenyls (Aroclor 1254) in Lactating Bovines and Their Distribution in Dairy Products.

Lactating cows were given orally, single or multiple graded doses of polychlorinated biphenyls (PCB) as Aroclor 1254 and the tissue distribution and excretion were measured. Persistence of PCB in milk was determined at all dose levels of PCB administered. A distinct predilection of PCB for tissues with high lipid content was noted. Similarly, PCB appeared in higher concentration in dairy products with high fat content.

Bacteriological Evaluation of Retail Ground Beef, Frozen Beef Patties, and Cooked Hamburger.

A total of 108 samples of fresh refrigerated ground beef, 99 samples of frozen hamburger patties, and 107 fried hamburgers, purchased from retail stores and fast-food outlets in Ontario, were analyzed for their bacteriological quality. About 44% of non-frozen ground beef samples had aerobic plate counts exceeding 50 million/g; 50 of 108 samples (46.3%) contained Staphylococcus aureus and 46 of these 50 samples (88%) exceeded 1000 organisms/g; 43 of 108 samples were positive for Escherichia coli with 38 samples (88.4%) exceeding 500 organisms/g. About 19% of frozen hamburger patties had aerobic plate counts in excess of 10 million/g; 93 of 99 samples (93.9%) contained S. aureus with 83 of these samples (89.3%) exceeding 1000 organisms/g; 28 of 99 samples were positive for E. coli with 7 of these samples (25%) exceeding 500 organisms/g. About 96.3% of fried hamburger samples had aerobic plate counts of less than 10,000/g.

Post-translational cleavage of a histone H1-like protein in the sperm of Mytilus.

Starting with total RNA from spermatogenic cells of Mytilus trossulus and using random priming, we have cloned and sequenced the c-DNAs corresponding to two variants of the sperm-specific protein PLII* (phi 2B). DNA sequencing in conjunction with mass spectrometry and protein sequence data have allowed us to establish that of the three sperm-specific proteins present in the sperm of Mytilus (PL-II*(phi 2B), PL-III (phi 1), PL-IV (phi 3)), the first and the last one are the result of post-translational cleavage of a common precursor. This common precursor is a member of the histone H1 family, and it exhibits inter- and intraspecific microheterogeneity.

An integrated approach to proteome analysis: identification of proteins associated with cardiac hypertrophy.

Hypertrophy of cardiac myocytes is a primary response of the heart to overload, and is an independent predictor of heart failure and death. Distinct cellular phenotypes are associated with hypertrophy resulting from different causes. These phenotypes have been described by others at the molecular level by analysis of gene transcription patterns. An alternative approach is the analysis of large-scale protein expression patterns (the proteome) by two-dimensional polyacrylamide gel electrophoresis. Realization of this goal requires the ability to rigorously analyze complex 2D gel images, efficiently digest individual gel isolated proteins (especially those expressed at low levels), and analyze the resulting peptides with high sensitivity for rapid database searches. We have undertaken to improve the technology and experimental approaches to these challenges in order to effectively study a cell culture model for cardiac hypertrophy. The 2D gel patterns for cell lysates from multiple samples of cardiac myocytes with or without phenylephrine-induced hypertrophy were analyzed and spots which changed in abundance with statistical significance were located. Eleven such spots were identified using improved procedures for in-gel digestion of silver-stained proteins and high-sensitivity mass spectrometry. The incorporation of low levels of sodium dodecyl sulfate into the digestion buffer improved peptide recovery. The combination of matrix-assisted laser desorption mass spectrometry for initial measurements and capillary liquid chromatography-ion trap mass spectrometry for peptide sequence determination yielded efficient protein identification. The integration of 2D gel image analysis and routine identification of proteins present in gels at the subpicomole level represents a general model for proteome studies relating genomic sequence with protein expression patterns.

Unilateral nostril breathing influences lateralized cognitive performance.

Relative nostril efficiency (nasal cycle) is related to hemispheric EEG differences and performance on cognitive tasks. We investigated how unilateral forced nostril breathing influences spatial and verbal performance. Right-handed males and females performed both tasks under either left-nostril, right-nostril, or free-breathing conditions. Unilateral breathing affects performance differently in males and females. It influences male performance ipsilaterally on both tasks: Their spatial performance is better during right-nostril breathing, and their verbal performance is better during left-nostril breathing. Unilateral breathing influences female performance contralaterally, but only on the spatial task: Their spatial performance is better during left-nostril breathing. These differences within and between sexes may exist because unilateral nostril breathing differentially activates the two hemispheres and thereby facilitates performance, or because attempts of the brain to control the nasal cycle unilaterally interfere with performance.

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes.

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.