Peptides from the Variable Region of Specific Antibodies Are Shared among Lung Cancer Patients

Late diagnosis of lung cancer is still the main reason for high mortality rates in lung cancer. Lung cancer is a heterogeneous disease which induces an immune response to different tumor antigens. Several methods for searching autoantibodies have been described that are based on known purified antigen panels. The aim of our study is to find evidence that parts of the antigen-binding-domain of antibodies are shared among lung cancer patients. This was investigated by a novel approach based on sequencing antigen-binding-fragments (Fab) of immunoglobulins using proteomic techniques without the need of previously known antigen panels. From serum of 93 participants of the NELSON trial IgG was isolated and subsequently digested into Fab and Fc. Fab was purified from the digested mixture by SDS-PAGE. The Fab containing gel-bands were excised, tryptic digested and measured on a nano-LC-Orbitrap-Mass-spectrometry system. Multivariate analysis of the mass spectrometry data by linear canonical discriminant analysis combined with stepwise logistic regression resulted in a 12-antibody-peptide model which was able to distinguish lung cancer patients from controls in a high risk population with a sensitivity of 84% and specificity of 90%. With our Fab-purification combined Orbitrap-mass-spectrometry approach, we found peptides from the variable-parts of antibodies which are shared among lung cancer patients.     

3-[(3-Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate-Solubilized Binding Sites for 2-[125I]Iodomelatonin in Chick Brain Retain Sensitivity to Guanine Nucleotides

Binding of 2-[125I]iodomelatonin to 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized sites from chick forebrain was rapid. reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2-[125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 +/- 22 (n = 4) and 302 +/- 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 +/- 2.0 and 49.5 +/- 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than prazosin greater than N-acetylserotonin much greater than serotonin greater than metergoline greater than ketanserin greater than propranolol greater than phentolamine greater than cyproheptadine. Guanyl nucleotides inhibited binding of 2-[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high-affinity to a low-affinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane-bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.     

Ultrasound guidance improves the adequacy of our preoperative thyroid cytology but not its accuracy

AIMS: Our thyroid cytology audit results of 1990-1995 showed an unsatisfactory rate of 43.1% and prediction of neoplasia with a sensitivity of 86.8%. Increasingly, ultrasound scan (USS)-guided core sampling for cytology is proving a valuable tool instead of freehand fine needle aspiration (FNA) or following unsatisfactory freehand FNA. We present the results of freehand FNA and USS-guided core samples for cytology in two separate patient groups in our centre. METHODS: Patients who had a thyroid resection and preoperative thyroid cytology in our institution between 1996 and 2002 were included. The histological diagnoses were correlated with the preceding cytology results. RESULTS: A total of 450 FNAs were performed on 394 patients. Freehand FNAs were performed for 348 (77.3%) samples and USS-guided core for 102 (22.7%) samples; 121 (26.8%) were repeat aspirates performed on 45 patients. Using aspiration cytology (AC) grading, freehand FNA was cytologically inadequate (AC0 or AC1) in 34.8% cases whereas USS-guided core was inadequate in 17.6% cases (P = 0.001). Freehand FNA (AC3, AC4, AC5) predicted neoplasia with a sensitivity of 83.2%, specificity of 46.6%, accuracy of 63.0%, positive predictive value of 56.0% and negative predictive value of 77.1%. USS-guided core sample for cytology (AC3, AC4, AC5) predicted neoplasia with a sensitivity of 93.5%, specificity of 26.0%, accuracy of 51.9%, positive predictive value of 43.9% and negative predictive value of 86.7%. CONCLUSIONS: Although USS-guided core provides more satisfactory specimens than freehand FNA, in our centre it does not provide increased accuracy.     

Two in a bed: The influence of couple sleeping and chronotypes on relationship and sleep. An overview

Objective: To summarize research on couple sleeping with respect to gender-specific differences and chronotype. Methods: Systematic review of the literature. Results: Millions of adults around the world share their beds with a partner. This may be an expression of intimacy and attachment and tends to intensify romantic relationships. Yet, couple sleeping still has underestimated implications for the quality of the relationship, quality of sleep and for physical and psychological health which are not consistently positive. Implications for research and therapy are discussed. Conclusions: Despite the people involved perhaps not even being aware of their nocturnal interactions, it is important that sleeping together becomes a subject of discussion.

Abbreviations: REM: rapid eye movement; QOL: quality of life; OSA: obstructive sleep apnea; CPAP: continuous positive airway pressure     

MALDI-TOF mass spectrometry analysis of cerebrospinal fluid tryptic peptide profiles to diagnose leptomeningeal metastases in patients with breast cancer

Leptomeningeal metastasis (LM) is a devastating complication that occurs in 5% of patients with breast cancer. Early diagnosis and initiation of treatment are essential to prevent neurological deterioration. However, early diagnosis of LM remains challenging because 25% of cerebrospinal fluid (CSF) samples produce false-negative results at first cytological examination. We developed a new, MS-based method to investigate the protein expression patterns present in the CSF from patients with breast cancer with and without LM. CSF samples from 106 patients with active breast cancer (54 with LM and 52 without LM) and 45 control subjects were digested with trypsin. The resulting peptides were measured by MALDI-TOF MS. Then, the mass spectra were analyzed and compared between patient groups using newly developed bioinformatics tools. A total of 895 possible peak positions was detected, and 164 of these peaks discriminated between the patient groups (Kruskal-Wallis, p<0.01). The discriminatory masses were clustered, and a classifier was built to distinguish patients with breast cancer with and without LM. After bootstrap validation, the classifier had a maximum accuracy of 77% with a sensitivity of 79% and a specificity of 76%. Direct MALDI-TOF analysis of tryptic digests of CSF gives reproducible peptide profiles that can assist in diagnosing LM in patients with breast cancer. The same method can be used to develop diagnostic assays for other neurological disorders.     

Role of Conjugative Elements in the Evolution of the Multidrug-Resistant Pandemic Clone Streptococcus pneumoniaeSpain23F ST81

Streptococcus pneumoniae is a human commensal and pathogen able to cause a variety of diseases that annually result in over a million deaths worldwide. The S. pneumoniae^Spain23F sequence type 81 lineage was among the first recognized pandemic clones and was responsible for almost 40% of penicillin-resistant pneumococcal infections in the United States in the late 1990s. Analysis of the chromosome sequence of a representative strain, and comparison with other available genomes, indicates roles for integrative and conjugative elements in the evolution of pneumococci and, more particularly, the emergence of the multidrug-resistant Spain 23F ST81 lineage. A number of recently acquired loci within the chromosome appear to encode proteins involved in the production of, or immunity to, antimicrobial compounds, which may contribute to the proficiency of this strain at nasopharyngeal colonization. However, further sequencing of other pandemic clones will be required to establish whether there are any general attributes shared by these strains that are responsible for their international success.Streptococcus pneumoniae (the pneumococcus) is a human commensal and pathogen that represents a major cause of otitis media, pneumonia, and meningitis (8). Worldwide, pneumococcal disease is thought to be responsible for over a million fatalities annually, including more than 800,000 deaths in children under 5 years of age living in developing countries (64). While the introduction of the heptavalent polysaccharide conjugate vaccine (PCV7) has dramatically reduced the incidence of pneumococcal disease in some areas (37), limited serotype coverage, strain replacement, and capsule switching have resulted in a smaller, and decreasing, impact in other communities (66).S. pneumoniae is a naturally competent, genetically diverse species, with less than half of the pan-genome conserved between all strains thus far studied (33). The pneumococcal population is normally confined to the human nasopharynx, with rates of asymptomatic carriage varying with demographics, region, and season: surveys of colonization in healthy children generally estimate between 20 and 97% of younger individuals carry pneumococci (9, 32), with levels falling with age. Epidemiological data and animal models of infection indicate that strains exhibit differing propensities for causing invasive disease (13, 29, 63). The invasive disease potential odds ratio, which takes into account the relative frequencies of invasive disease and asymptomatic carriage observed in the human population, varies 80-fold between serotypes (13). However, the functional genetic variation to which this differing ability to cause disease is attributable remains largely unknown. Genome sequencing efforts have mainly focused on clinical pneumococcal isolates; the complete genomes of two highly invasive strains, TIGR4 (70) and D39 (38) (and the laboratory-adapted D39 derivative R6) (34), have been published, along with draft sequences for serotype 19F strain G54 (26) and eight clinical isolates from a hospital in Pittsburgh (65). However, in order to understand the bacterial population structure, and the reasons underlying the variation in pathogenicity, genomic studies of strains that only rarely invade past the mucosal surfaces are required.S. pneumoniae^Spain23F sequence type 81 (ST81) was one of the first pandemic penicillin-resistant clones identified (47). Initially characterized among isolates from Spain in the 1980s, it spread globally, and by the late 1990s it was estimated to constitute almost 40% of penicillin-resistant disease isolates in the United States (21). The clone is also resistant to chloramphenicol and tetracycline and is one of those most frequently associated with the emergence of fluoroquinolone and macrolide resistance (58, 60). This lack of susceptibility to the major classes of antimicrobial chemotherapies used to treat pneumococcal infections has undoubtedly aided the spread of the strain and lead to the inclusion of the 23F serotype in PCV7. This has resulted in a reduction in the prevalence of the S. pneumoniae^Spain23F ST81 clone in some regions (35). However, the lineage has undergone capsule switching to alternative capsule types on at least three occasions (from serotype 23F to 14 (36), 19A (20), and 19F (19), suggesting it is liable to eventually evade the vaccine. Despite its high carriage prevalence (22), it has a low propensity for causing invasive disease (67) (odds ratio of 0.4 [13]), suggesting its intercontinental distribution has been facilitated by adaptations to colonization of, and survival within, the human nasopharynx. Here we report our analysis of the complete genome of S. pneumoniae ATCC 700669, a member of the serotype 23F ST81 lineage that was isolated in a hospital in Barcelona in 1984 (18).     

The relationship between impulsive choice and impulsive action: a cross-species translational study

Maladaptive impulsivity is a core symptom in various psychiatric disorders. However, there is only limited evidence available on whether different measures of impulsivity represent largely unrelated aspects or a unitary construct. In a cross-species translational study, thirty rats were trained in impulsive choice (delayed reward task) and impulsive action (five-choice serial reaction time task) paradigms. The correlation between those measures was assessed during baseline performance and after pharmacological manipulations with the psychostimulant amphetamine and the norepinephrine reuptake inhibitor atomoxetine. In parallel, to validate the animal data, 101 human subjects performed analogous measures of impulsive choice (delay discounting task, DDT) and impulsive action (immediate and delayed memory task, IMT/DMT). Moreover, all subjects completed the Stop Signal Task (SST, as an additional measure of impulsive action) and filled out the Barratt impulsiveness scale (BIS-11). Correlations between DDT and IMT/DMT were determined and a principal component analysis was performed on all human measures of impulsivity. In both rats and humans measures of impulsive choice and impulsive action did not correlate. In rats the within-subject pharmacological effects of amphetamine and atomoxetine did not correlate between tasks, suggesting distinct underlying neural correlates. Furthermore, in humans, principal component analysis identified three independent factors: (1) self-reported impulsivity (BIS-11); (2) impulsive action (IMT/DMT and SST); (3) impulsive choice (DDT). This is the first study directly comparing aspects of impulsivity using a cross-species translational approach. The present data reveal the non-unitary nature of impulsivity on a behavioral and pharmacological level. Collectively, this warrants a stronger focus on the relative contribution of distinct forms of impulsivity in psychopathology.