Background: Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results: The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions: Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

P53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML.

The aim of this pilot study was to compare spirometric values obtained with different types of spirometers, spirometers of same type, and repeated measurements with the same spirometer in a pulmonary function laboratory setting. 12 healthy volunteers performed spirometry on four hot-wire (SensorMedics), two ultrasonic (Spirare) and one wedge-bellows (Vitalograph S) spirometers, according to ATS/ERS (American Thoracic Society/European Respiratory Society) guidelines. Spirometric values were compared using linear mixed models analysis with a random intercept for subjects and a fixed effect for type of spirometer used. Confidence intervals and p values were adjusted for multiple comparisons. Mean ± SD (L) values for hot-wire, ultrasonic and wedge-bellows spirometers for FVC (forced vital capacity) were 4.02 ± 0.66, 3.69 ± 0.61 and 3.93 ± 0.69, and for FEV1 (forced expiratory volume in one second) 3.06 ± 0.44, 2.95 ± 0.44 and 3.10 ± 0.49. Significant differences were found between hot-wire and ultrasonic and between wedge-bellows and ultrasonic spirometers for FVC and FEV1, and between hot-wire and wedge-bellows spirometers for FVC but not for FEV1. There were no significant differences between spirometers of same type, and low mean differences in repeated measurements for all spirometers included. In conclusion, the pilot study shows systematically higher values for FVC and FEV1 for hot-wire and wedge-bellows compared to ultrasonic spirometers.

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.

Background: Improved knowledge of molecular mechanisms involved during acute exacerbations of COPD (AECOPD) is needed, in particular for different subsets of COPD. Aims: To perform quantitative proteomics analysis of induced sputum from stable and exacerbated COPD patients, and study proteome changes during exacerbations for COPD subgroups. Methods: We used paired induced sputum samples from 15 COPD patients (stable and exacerbation state). Proteins in the cell-free supernatant were digested into peptides, labeled with tandem mass tags, fractionated by mixed-mode chromatography and analyzed by liquid chromatography-tandem mass spectrometry. Results: More than 2000 proteins were identified in the sputum samples, and proteins were considered differently abundant between stable and exacerbated COPD or between different subgroups when p1.5. Differences were found in exacerbated compared to stable COPD, including elevated levels of c-reactive protein, dickkopf-related protein-3, ferritin heavy chain, ficolin-1, perilipin-3, serine/arginine-rich splicing factor 1, and ubiquitin carboxyl-terminal hydrolase 15. Furthermore, differences in changes during AECOPD for patients with GOLD stage III/IV compared to II were demonstrated by clustering analysis (Figure 1). Conclusions: We found an altered sputum proteome during AECOPD, with differences in changes according to GOLD stage. These findings need to be validated in larger cohorts.

Background: Although several inflammatory markers are increased in sputum of COPD patients, inflammatory responses during exacerbations for different subsets of COPD patients are less studied. Aims: To investigate changes in sputum inflammatory markers IL-6, IL-8, IL-18, IP-10 (CXCL10), MIG (CXCL9) and TNF-α between stable state and exacerbations for COPD patients with different stable state levels of the same markers. Methods: Paired induced sputum samples were collected from 39 COPD patients (stable and exacerbation state) in the Bergen COPD Cohort and Bergen COPD Exacerbation Studies. Inflammatory markers were analyzed by a bead-based immunoassay. Wilcoxon rank sum test, Wilcoxon signed rank test and Spearman’s rank correlation were used for statistical analyses. Results: There was a significant negative correlation between sputum stable state levels and changes during COPD exacerbations for most of the markers (changes are defined as exacerbation minus stable state levels). Spearman’s rho (with CI) for IL-6 was -0.72(-0.85, -0.51),p<0.001, for IL-8 -0.61(-0.78, -0.35),p<0.001, for IL-18 -0.35(-0.60, -0.03),p=0.030, for IP-10 -0.31(-0.58, 0.02),p=0.055, for MIG -0.38(-0.93, -0.07),p=0.016, and for TNF-α -0.44(-0.67, -0.13),p=0.006. Some markers showed significantly higher levels in GOLD stage III/IV compared to GOLD stage II patients in stable state sputum samples, but decreased upregulation (change) during exacerbations for GOLD stage III/IV compared to GOLD stage II. Conclusions: Our results suggest that COPD patients with high levels of sputum inflammatory markers in the stable state have less potential for upregulation of the markers during exacerbations.