Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation.

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases

Computed Tomography Measurement of Rib Cage Morphometry in Emphysema

Background: Factors determining the shape of the human rib cage are not completely understood. We aimed to quantify the contribution of anthropometric and COPD-related changes to rib cage variability in adult cigarette smokers. Methods: Rib cage diameters and areas (calculated from the inner surface of the rib cage) in 816 smokers with or without COPD, were evaluated at three anatomical levels using computed tomography (CT). CTs were analyzed with software, which allows quantification of total emphysema (emphysema%). The relationship between rib cage measurements and anthropometric factors, lung function indices, and %emphysema were tested using linear regression models. Results: A model that included gender, age, BMI, emphysema%, forced expiratory volume in one second (FEV1)%, and forced vital capacity (FVC)% fit best with the rib cage measurements (R2  = 64% for the rib cage area variation at the lower anatomical level). Gender had the biggest impact on rib cage diameter and area (105.3 cm2; 95% CI: 111.7 to 98.8 for male lower area). Emphysema% was responsible for an increase in size of upper and middle CT areas (up to 5.4 cm2; 95% CI: 3.0 to 7.8 for an emphysema increase of 5%). Lower rib cage areas decreased as FVC% decreased (5.1 cm2; 95% CI: 2.5 to 7.6 for 10 percentage points of FVC variation). Conclusions: This study demonstrates that simple CT measurements can predict rib cage morphometric variability and also highlight relationships between rib cage morphometry and emphysema

Obstructive Sleep Apnea Syndrome Phenotyping by Cluster Analysis: Typical Sleepy, Obese Middle-aged Men with Desaturating Events are A Minority of Patients in A Multi-ethnic Cohort of 33% Women

ObjectiveSeveral clinical obstructive sleep apnea syndrome (OSAS) phenotypes associated with heterogeneous cardiovascular risk profiles have been recently identified. The purpose of this study was to identify clusters amongst these profiles that allow for the differentiation of patients.MethodsThis retrospective study included all moderate-to-severe OSAS patients referred to the sleep unit over a 5-year period. Demographic, symptom, comorbidity, polysomnographic, and continuous positive airway pressure (CPAP) adherence data were collected. Statistical analyses were performed to identify clusters of patients.ResultsA total of 567 patients were included (67% men, 54±13 years, body mass index: 32±7 kg/m2, 65% Caucasian, 32% European African). Five clusters were identified: less severe OSAS (n=172); healthier severe OSAS (n=160); poorly sleeping OSAS patients with cardiometabolic comorbidities (n=87); younger obese men with sleepiness at the wheel (n=94); sleepy obese men with very severe desaturating OSAS and cardiometabolic comorbidities (n=54). Patients in clusters 3 and 5 were older than those in clusters 2 and 4 (P=0.034). Patients in clusters 4 and 5 were significantly more obese than those in the other clusters (P=0.04). No significant differences were detected in terms of symptoms and comorbidities. Polysomnographic profiles were very discriminating between clusters. CPAP adherence was similar in all clusters but, among adherent patients, daily usage was more important in cluster 1 (less severe patients) than in cluster 5.ConclusionThis study highlights that the typical sleepy obese middle-aged men with desaturating events represent only a minority of patients in our multi-ethnic moderate-to-severe OSAS cohort of 33% females.info:eu-repo/semantics/publishe

African ethnicity is associated with a higher prevalence of diabetes in obstructive sleep apnea patients ::results of a retrospective analysis

Purpose : Obstructive sleep apnea (OSA) syndrome is a well-recognized independent risk factor for cardiovascular disease and its prevalence is increasing. OSA symptomology, polysomnographic features, and comorbidities are heterogeneous among patients. Ethnicity is thought to influence OSA phenotypes, but extensive knowledge of OSA ethnic patterns is lacking. The primary aim of the present study was to compare comorbidities in Caucasian and African OSA. Secondary aims were to observe OSA symptomatology, polysomnographic characteristics, and CPAP adherence in these two ethnic groups. Methods : In this retrospective study, 1717 patients suffering from moderate/severe OSA were included between 2013 and 2017. Data on demographics, symptomatology, comorbidities, polysomnographic characteristics, and CPAP adherence were collected. Data were analyzed to identify potential differences between Caucasians and Africans. Results : Despite healthier lifestyles and lower BMI, a higher prevalence of diabetes but less cardiac comorbidities and dyslipidemia was observed in the African population. Younger African patients (< 56 years) suffered more from cognitive impairment than Caucasians and both younger and older Africans complained more of nighttime choking than Caucasians. In analysis of polysomnographic data, Africans had higher apnea-hypopnea index (AHI) in REM sleep, lower supine AHI, lower desaturation time, and lower periodic leg movements index. Conclusions : Compared with Caucasians, African OSA showed a particular comorbidity profile. There are younger patients who exhibit more diabetes but less cardiac comorbidities than the Caucasians. African diabetics should be more promptly referred for OSA testing. Moreover, as they suffer more often from choking and cognitive impairment, OSA treatment could positively impact their quality of life

Cardiometabolic comorbidities in obstructive sleep apnea patients are related to disease severity, nocturnal hypoxemia, and decreased sleep quality

Background: Obstructive sleep apnea syndrome (OSA) is currently recognized as an independent risk factor for hypertension, arrhythmia, coronary heart disease, stroke, and metabolic disorders (e.g. diabetes, dyslipidemia). In clinical practice, apnea-hypopnea index (AHI) is the marker used to classify disease severity and guide treatment. However, AHI alone does not sufficiently identify OSA patients at risk for cardiometabolic comorbidities. With this in mind, the aim of this retrospective study was to determine whether some polysomnographic parameters (e.g. apnea-hypopnea duration, sleep structure, nocturnal hypoxemia) are specifically associated with cardiometabolic comorbidities in OSA. Methods: In this retrospective study, 1717 patients suffering from moderate/severe OSA were included between 2013 and 2017. Data on demographics, comorbidities, and polysomnographic characteristics were collected and analyzed to identify factors associated with cardiometabolic complications. Results: The medical files of 1717 patients (68% male) were reviewed. The mean AHI was 43.1 +/- 27.7 with 57.3% of patients suffering from severe OSA, and 52% from at least one cardiovascular comorbidity (CVCo). Diabetes affected 22% of the patients and 27% exhibited dyslipidemia. Patients affected by CVCos were older, and more often women and non-smokers. These patients also had worse sleep quality, and a more marked intermittent/global nocturnal hypoxemia. With regard to diabetes, diabetics were older, more often non-smoker, non-drinker women, and were more obese. These patients also exhibited more severe OSA, especially in non-REM (NREM) sleep, worse sleep quality, and a more marked intermittent/global nocturnal hypoxemia. Dyslipidemia was more frequent in the absence of alcohol consumption, and was associated with OSA severity, decreased sleep quality, and longer AH in REM sleep. Conclusions: This study identifies demographic and polysomnographic factors associated with cardiometabolic comorbidities. Patients (especially women) suffering from more severe OSA, longer sleep apneas and hypopneas, worse sleep quality, and marked intermittent/global nocturnal hypoxemia are more likely to develop cardiometabolic comorbidities. This should stimulate clinicians to obtain adequate treatment in this population.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Rapid ligand fishing for identification of acetylcholinesterase-binding peptides in snake venom reveals new properties of dendrotoxins.

Acetylcholinesterase (AChE) from Electrophorus electricus (eel) was immobilized on the surface of amino-modified paramagnetic beads to serve as a model for the development, validation and application of a new affinity-based ligand-fishing assay for the discovery of bioactive peptides from complex protein mixtures such as venoms. Nano liquid chromatography-mass spectrometry (nanoLC-MS) was used for the analysis of trapped peptides. Using enzyme-functionalized beads, the ligand-fishing assay was evaluated and optimized using a peptide reference mixture composed of one acetylcholinesterase binder (fasciculin-II) and five non-binders (mambalgin-1, angiotensin-II, bradykinin, cardiotoxin and α-bungarotoxin). As proof of concept, snake venom samples spiked with fasciculin-II demonstrated assay selectivity and sensitivity, fishing the peptide binder from complex venom solutions at concentrations as low as 1.0 μg/mL. As negative controls for method validation, venoms of four different snake species, not known to harbor AChE binding peptides, were screened and no AChE binders were detected. The applicability of the ligand fishing assay was subsequently demonstrated with venom from the black mamba, Jameson's mamba and western green mamba (Dendroaspis spp.), which have previously been reported to contain the AChE binding fasciculins. Unknown peptides (i.e. not fasciculins) with affinity to AChE were recovered from all mamba venoms tested. Tryptic digestion followed by nano-LC-MS analysis of the material recovered from black mamba venom identified the peptide with highest AChE-binding affinity as dendrotoxin-I, a pre-synaptic neurotoxin previously not known to interact with AChE. Co-incubation of AChE with various dendrotoxins in vitro revealed reduced inactivation of AChE activity over time, thus demonstrating that these toxins stabilise AChE. [Abstract copyright: Copyright © 2018. Published by Elsevier Ltd.

Human fecal samples microbial 16S rRNA gene sequence

16S rRNA gene of 16 human fecal samples were sequenced and correlated to microbial fructoselysine degradation studies in vitro

Chiral Discrimination of DL-Amino Acids by Trapped Ion Mobility Spectrometry after Derivatization with (+)-1-(9-Fluorenyl)ethyl Chloroformate

A novel analytical method based on hybrid trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has been developed to achieve fast enantiomeric separation of amino acids (AAs). Resolution of chiral AAs was achieved by forming diastereomers through derivatization with the chiral agent (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), avoiding the use of reference compounds. Electrospray ionization (ESI) in positive mode yielded sodiated FLEC-AAs ions of which the diastereomers could be separated by TIMS. The effect of other alkali metal ions (such as Li and K) on the enantioselectivity was studied, but chiral discrimination was only observed for Na. TIMS conditions, including voltage ramp, ramp time, and accumulation time were optimized for each AA, and collision cross sections (CCSs) were determined for all diastereomers. The migration order of the DL enantiomers was found to be dependent on the structure of the AA. The resulting TIMS resolution (K0/ΔK0) for the FLEC-AA diastereomers on average was 115, requiring a mobility (K0) difference of about 0.009 cm 2 /(V s) to achieve 50%-valley separation. From the 21 AAs studied, enantiomer separation was achieved for 17 AAs with mobility differences ranging from 0.009 for lysine up to 0.061 cm 2 /(V s) for asparagine. Moreover, the presented methodology provided mutual separation of various AAs, allowing chiral analysis of multiple AAs simultaneously which may be challenging with previous enantioselective IMS approaches. It appeared possible to fully resolve all studied DL-AAs using three distinct TIMS methods, resulting in a total MS run time of about 3 min (1 min per method) and a total analysis time (including derivatization) of less than 15 min. The method demonstrated capable to determine enantiomeric ratios down to 2.5% with detection limits for the D enantiomers in the nanomolar range. This new TIMS-based methodology opens up possibilities for easy and fast analysis of AA enantiomers

Human fecal samples microbial 16S rRNA gene sequence

16S rRNA gene of 16 human fecal samples were sequenced and correlated to microbial fructoselysine degradation studies in vitro

Nanofractionation Platform with Parallel Mass Spectrometry for Identification of CYP1A2 Inhibitors in Metabolic Mixtures

With early assessment of inhibitory properties of drug candidates and their circulating metabolites toward cytochrome P450 enzymes, drug attrition, especially later in the drug development process, can be decreased. Here we describe the development and validation of an at-line nanofractionation platform, which was applied for screening of CYP1A2 inhibitors in Phase I metabolic mixtures. With this platform, a metabolic mixture is separated by liquid chromatography (LC), followed by parallel nanofractionation on a microtiter well plate and mass spectrometry (MS) analysis. After solvent evaporation, all metabolites present in the nanofractionated mixture are assayed utilizing a fluorescence CYP1A2 inhibition bioassay performed on the plate. Next, a bioactivity chromatogram is constructed from the bioassay results. By peak shape and retention time correlation of the bioactivity peaks with the obtained MS data, CYP1A2-bioactive inhibiting metabolites can be identified. The method correctly evaluated the potency of five CYP1A2 inhibitors. Mixtures comprising potent inhibitors of CYP1A2 or in vitro–generated metabolites of ellipticine were evaluated for their inhibitory bioactivities. In both cases, good LC separation of all compounds was achieved and bioactivity data could be accurately correlated with the parallel recorded MS data. Generation and evaluation of Phase II metabolites of hydroxylated ellipticine was also pursued