Sputum metagenomics of people with bronchiectasis.

Background: The microbiota in the sputum of people with bronchiectasis has repeatedly been investigated in cohorts of different geographic origin, but so far has not been studied to the species level in comparison to control populations including healthy adults and smokers without lung disease. Methods: The microbial metagenome from sputa of 101 European Bronchiectasis Registry (EMBARC) study participants was examined by using whole-genome shotgun sequencing. Results: Our analysis of the metagenome of people with bronchiectasis revealed four clusters characterised by a predominance of Haemophilus influenzae, Pseudomonas aeruginosa or polymicrobial communities with varying compositions of nonpathogenic commensals and opportunistic pathogens. The metagenomes of the severely affected patients showed individual profiles characterised by low alpha diversity. Importantly, nearly 50% of patients with severe disease were grouped in a cluster characterised by commensals. Comparisons with the sputum metagenomes of healthy smokers and healthy nonsmokers revealed a gradient of depletion of taxa in bronchiectasis, most often Neisseria subflava, Fusobacterium periodonticum and Eubacterium sulci. Conclusion: The gradient of depletion of commensal taxa found in healthy airways is a key feature of bronchiectasis associated with disease severity.

Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region.

BACKGROUND: Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing. METHODS: We compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment. RESULTS: We could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region). CONCLUSION: Based on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d'être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment.

Longitudinal development of the airway metagenome of preterm very low birth weight infants during the first two years of life.

Preterm birth is accompanied with many complications and requires severe therapeutic regimens at the neonatal intensive care unit. The influence of the above-mentioned factors on the premature-born infants' respiratory metagenome or more generally its maturation is unknown. We therefore applied shotgun metagenome sequencing of oropharyngeal swabs to analyze the airway metagenome development of 24 preterm infants from one week postpartum to 15 months of age. Beta diversity analysis revealed a distinct clustering of airway microbial communities from hospitalized preterms and samples after hospital discharge. At nine and 15 months of age, the preterm infants lost their hospital-acquired individual metagenome signatures towards a common taxonomic structure. However, ecological network analysis and Random Forest classification of cross-sectional data revealed that by this age the preterm infants did not succeed in establishing the uniform and stable bacterial community structures that are characteristic for healthy full-term infants.

Structural genome variants of Pseudomonas aeruginosa clone C and PA14 strains.

Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.

Wochenende - modular and flexible alignment-based shotgun metagenome analysis.

BACKGROUND: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. RESULTS: Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. CONCLUSION: Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .

Changes in the Leptin to Adiponectin Ratio Are Proportional to Weight Loss After Meal Replacement in Adults With Severe Obesity.

Hypocaloric diets are known to induce changes in adipokine secretion, but the influence of a low energy liquid diet (LELD) on the leptin: adiponectin ratio (LAR), a measure of insulin resistance and cardiovascular risk, has not previously been investigated in patients with severe obesity. We conducted a prospective, single-center cohort study of adults with severe obesity (defined as body mass index (BMI) ≥40 kgm-2, or ≥35 kgm-2 with co-morbidities) who completed a 24-week milk-based LELD. We measured leptin, adiponectin and LAR at the start and on completion of the programme. Of 120 patients who started, 52 (43.3 %) completed the programme. Their mean age was 50.3 ± 11.2 (range 18-74) years, 29 (55.8 %) were female and 20 (38.5 %) had type 2 diabetes mellitus (T2DM). Weight decreased from 148.2 ± 39.6 to 125.4 ± 34.8 kg and BMI decreased from 52.4 ± 11.1 to 44.3 ± 9.8 kgm-2, respectively (all p < 0.001). In patients with T2DM, HbA1c decreased from 60.0 ± 17.4 to 47.5 ± 15.5 mmol/mol (p < 0.001). Leptin decreased (from 87.2 [48.6, 132.7] to 39.1 [21.0, 76.4] ng/ml) and adiponectin increased (from 5.6 [4.5, 7.5] to 7.1 [5.5, 8.5] µg/ml), with a reduction in LAR from 15 [8.4, 22.4] to 5.7 [3.0, 9.1] ng/µg (all p < 0.001), indicating decreased insulin resistance. The percentage weight lost was associated with the percentage reduction in LAR (ß = 2.9 [1.7, 4.1], p < 0.001) and this association was stronger in patients with T2DM. Patients with severe obesity who completed a milk-based LELD had a substantial reduction in LAR, consistent with decreased insulin resistance and cardiovascular risk, proportional to weight loss.

Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication.

To identify cellular mechanisms responsible for pressure overload triggered heart failure, we isolated cardiomyocytes, endothelial cells, and fibroblasts as most abundant cell types from mouse hearts in the subacute and chronic stages after transverse aortic constriction (TAC) and performed RNA-sequencing. We detected highly cell-type specific transcriptional responses with characteristic time courses and active intercellular communication. Cardiomyocytes after TAC exerted an early and sustained upregulation of inflammatory and matrix genes and a concomitant suppression of metabolic and ion channel genes. Fibroblasts, in contrast, showed transient early upregulation of inflammatory and matrix genes and downregulation of angiogenesis genes, but sustained induction of cell cycle and ion channel genes during TAC. Endothelial cells transiently induced cell cycle and extracellular matrix genes early after TAC, but exerted a long-lasting upregulation of inflammatory genes. As we found that matrix production by multiple cell types triggers pathological cellular responses, it might serve as a future therapeutic target.

Direct RNA Nanopore Sequencing of Pseudomonas aeruginosa Clone C Transcriptomes.

The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that-if replicated in other species-will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.

Improved Quality of Life, Fitness, Mental Health and Cardiovascular Risk Factors with a Publicly Funded Bariatric Lifestyle Intervention for Adults with Severe Obesity: A Prospective Cohort Study.

BACKGROUND: Lifestyle modification is the cornerstone of management for patients with severe and complicated obesity, but the effects of structured lifestyle programmes on quality of life, anxiety and depression scores and cardiovascular risk factors are not well-described. We sought to describe changes in self-reported quality of life and mental health-related outcomes as well as cardiovascular risk factors in patients completing a 10-week multidisciplinary lifestyle-modification programme. METHODS: We conducted a prospective cohort study of all patients referred from our bariatric service who completed the programme between 2013 and 2019. In addition to weight, body mass index (BMI), blood pressure, HbA1c, lipid profile and functional capacity, we quantified health-related quality of life using the Dartmouth COOP Questionnaire and the European Quality of Life Questionnaire Visual Analogue Scale (EQVAS) and mental health using the Hospital Anxiety and Depression Scale (HADS). RESULTS: Of 1122 patients who started the programme, 877 (78.2%) completed it and were included in per protocol analyses. Mean age was 47.3 ± 11.9 years, 66.9% were female, 34.8% were in full- or part-time employment and 69.4% were entitled to state-provided medical care. BMI decreased from 47.0 ± 7.8 to 46.2 ± 7.8 kg m-2 and weight decreased from 131.6 ± 25.5 to 129.5 ± 25.4 kg (both p < 0.001). There were significant reductions in anxiety and depression scores and improvements in all Dartmouth COOP domains. The EQVAS score increased from 52 ± 22 to 63 ± 19 (p < 0.001). Small but statistically significant reductions in LDL cholesterol, systolic blood pressure and HBA1c were also observed. CONCLUSIONS: Adults with severe and complicated obesity completing a specialised bariatric lifestyle-modification programme showed significant improvements in self-reported mental health and quality of life, in addition to reductions in cardiovascular risk factors.

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome.

Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.

Genome-Wide Methylation Mapping Using Nanopore Sequencing Technology Identifies Novel Tumor Suppressor Genes in Hepatocellular Carcinoma.

Downregulation of multiple tumor suppressor genes (TSGs) plays an important role in cancer formation. Recent evidence has accumulated that cancer progression involves genome-wide alteration of epigenetic modifications, which may cause downregulation of the tumor suppressor gene. Using hepatocellular carcinoma (HCC) as a system, we mapped 5-methylcytosine signal at a genome-wide scale using nanopore sequencing technology to identify novel TSGs. Integration of methylation data with gene transcription profile of regenerated liver and primary HCCs allowed us to identify 10 potential tumor suppressor gene candidates. Subsequent validation led us to focus on functionally characterizing one candidate-glucokinase (GCK). We show here that overexpression of GCK inhibits the proliferation of HCC cells via induction of intracellular lactate accumulation and subsequently causes energy crisis due to NAD+ depletion. This suggests GCK functions as a tumor suppressor gene and may be involved in HCC development. In conclusion, these data provide valuable clues for further investigations of the process of tumorigenesis in human cancer.

Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes.

Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

The human respiratory tract microbial community structures in healthy and cystic fibrosis infants.

The metagenome development of the human respiratory tract was investigated by shotgun metagenome metagenomic sequencing of cough swabs from healthy children and children with cystic fibrosis (CF) between 3 weeks and 6 years of age. A healthy microbial community signature was associated with increased absolute abundances in terms of bacterial-human cell ratios of core and rare species across all age groups, with a higher diversity of rare species and a tightly interconnected species co-occurrence network, in which individual members were found in close proximity to each other and negative correlations were absent. Even without typical CF pathogens, the CF infant co-occurrence network was found to be less stable and prone to fragmentation due to fewer connections between species, a higher number of bridging species and the presence of negative species correlations. Detection of low-abundant DNA of the CF hallmark pathogen Pseudomonas aeruginosa was neither disease- nor age-associated in our cohort. Healthy and CF children come into contact with P. aeruginosa on a regular basis and from early on.

Long-Term Changes in Weight in Patients With Severe and Complicated Obesity After Completion of a Milk-Based Meal Replacement Programme.

Introduction: Even with very significant short term weight loss with intensive dietary restriction, subsequent weight regain remains a challenge for most patients. We sought to assess long-term weight change in patients with obesity following completion of a 24-week milk-based meal replacement programme. Methods: We conducted a retrospective cohort study of bariatric patients who completed our milk-based meal replacement programme. This programme started with an 8-week weight loss phase, followed by weight stabilization (8 weeks) and weight maintenance (8 weeks) phases, after which patients were followed up in the bariatric outpatient clinics. A paired sample t-test was used to compare mean differences in weight at the start and the end of the programme and at follow-up. Linear regression was used to identify predictors of weight regain. Results: In total, 78 patients had long term follow-up data at a mean of 34.4 ± 19.8 months after the start of the milk diet and were included in this analysis. Mean body mass index at baseline was 50.5 ± 7.6 kg m-2, 41 (52.6%) were female and the mean age was 51.6 ± 12.0 (range 18.0-71.5) years. Weight decreased from144 ± 26 kg at the start of the milk diet to 121.2 ± 24 kg at completion (P < 0.001), with a non-significant trend upwards in the 1st and 2nd years of follow-up to 129.0 ± 27.7 (P = 0.07 compared to nadir) and 123.4 ± 29.0kg (P = 0.17), respectively. Although regains in the 3rd and 4th follow-up years were substantial to 131.0 ± 22.3 (P < 0.001), and 139.8 ± 35.4 kg (P < 0.001), there was still a moderate net weight loss of 4.7 [9.5, 0.21] and 7.0 [13.9, 0.26] kg (both P = 0.04) between the start and the 3rd and 4th follow-up years, respectively. The amount of weight regain was inversely associated with weight loss at completion of the programme, age, and directly associated with the duration of follow up in months (ß = 1.2 [0.46, 1.9] P = 0.002). Conclusion: In patients with severe obesity who completed a milk-based meal replacement programme and lost a large amount of weight, over 4 years of follow-up there was very substantial weight regain. Greater initial weight loss and older age were associated with less subsequent weight regain.

Changes in alanine aminotransferase in adults with severe and complicated obesity during a milk-based meal replacement programme.

INTRODUCTION: Excess adiposity is associated with fat accumulation within the liver, and non-alcoholic steatohepatitis (NASH) is highly prevalent in bariatric patients. Elevated alanine aminotransferase (ALT) is associated with prevalent NASH. We sought to determine the influence of a milk-based meal replacement weight-loss programme on ALT levels in adults with severe and complicated obesity. METHODS: We conducted a retrospective cohort study of patients who completed a 24-week meal replacement programme, comprised of a weight loss phase followed by weight stabilisation and maintenance phases, each 8 weeks long. ALT was quantified using an enzymatic assay with spectrophotometric detection. We examined changes over time in ALT using the non-parametric Wilcoxon singed-rank test and the Friedman test. RESULTS: Of 105 patients, 56 were female, mean age was 51.2 ± 11.2 (range 18.0-71.6) years. There was an unanticipated but transient increase in ALT from 28.0 [20.0, 40.5] iu/L at baseline to 40.0 [26.0, 55.0] iu/L after 2 weeks (p < 0.0005), followed by a gradual reduction to 21.0 [17.0, 28.3] iu/L by 24 weeks (p < 0.0005). The overall reductions in ALT were more pronounced in patients who had elevated levels at baseline. Body weight decreased from 144.2 ± 28.0 kg at baseline to 121.6 ± 25.4 kg at 24 weeks (p < 0.0005) and body mass index (BMI) decreased from 50.7 ± 8.1 kg m-2 at baseline to 43.0 ± 7.6 kg m-2 by 24 weeks (p < 0.0005). CONCLUSION: In adults with severe and complicated obesity undergoing a milk-based meal replacement programme, there was an initial unanticipated rise in ALT in the first 2 weeks, followed by a gradual overall reduction by 24 weeks. These findings suggest that rapid weight loss secondary to significant caloric restriction might induce a transient deterioration in hepatic steatosis prior to an ultimate overall improvement.

TRAIL inhibits oxidative stress in human aortic endothelial cells exposed to pro-inflammatory stimuli.

Studies suggest that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has vasoprotective potential, as low levels of TRAIL cause accelerated vascular calcification, whereas exogenous TRAIL administration exhibits anti-atherosclerotic activity. The mechanism of TRAIL-mediated vasoprotection remains unclear. We studied the effects of TRAIL (100 ng/ml) on human aortic endothelial cells (HAECs) exposed to pro-atherogenic conditions; (a) oscillatory shear stress (±10 dynes/cm2 ) using the ibidi µ-slide fluidic system; (b) pro-inflammatory injury, that is, tumor necrosis factor alpha (TNF-α, 100 ng/ml) and hyperglycemia (30 mM d-glucose). End-points examined included inflammatory gene expression and reactive oxygen species (ROS) formation. TRAIL shifted the net gene expression toward an antioxidant phenotype in HAECs exposed to oscillatory shear stress. TRAIL significantly reduced ROS formation in HAECs exposed to both TNF-α and hyperglycemia. Therefore, TRAIL appears to confer atheroprotective effects on the endothelium, at least in part, by reducing oxidative stress.

Novel candidate genes for ECT response prediction-a pilot study analyzing the DNA methylome of depressed patients receiving electroconvulsive therapy.

BACKGROUND: Major depressive disorder (MDD) represents a serious global health concern. The urge for efficient MDD treatment strategies is presently hindered by the incomplete knowledge of its underlying pathomechanism. Despite recent progress (highlighting both genetics and the environment, and thus DNA methylation, to be relevant for its development), 30-50% of MDD patients still fail to reach remission with standard treatment approaches. Electroconvulsive therapy (ECT) is one of the most powerful options for the treatment of pharmacoresistant depression; nevertheless, ECT remission rates barely reach 50% in large-scale naturalistic population-based studies. To optimize MDD treatment strategies and enable personalized medicine in the long- term, prospective indicators of ECT response are thus in great need. Because recent target-driven analyses revealed DNA methylation baseline differences between ECT responder groups, we analyzed the DNA methylome of depressed ECT patients using next-generation sequencing. In this pilot study, we did not only aim to find novel targets for ECT response prediction but also to get a deeper insight into its possible mechanism of action. RESULTS: Longitudinal DNA methylation analysis of peripheral blood mononuclear cells isolated from a cohort of treatment-resistant MDD patients (n = 12; time points: before and after 1st and last ECT, respectively) using a TruSeq-Methyl Capture EPIC Kit for library preparation, led to the following results: (1) The global DNA methylation differed neither between the four measured time points nor between ECT responders (n = 8) and non-responders (n = 4). (2) Analyzing the DNA methylation variance for every probe (=1476812 single CpG sites) revealed eight novel candidate genes to be implicated in ECT response (protein-coding genes: RNF175, RNF213, TBC1D14, TMC5, WSCD1; genes encoding for putative long non-coding RNA transcripts: AC018685.2, AC098617.1, CLCN3P1). (3) In addition, DNA methylation of two CpG sites (located within AQP10 and TRERF1) was found to change during the treatment course. CONCLUSIONS: We suggest ten novel candidate genes to be implicated in either ECT response or its possible mechanism. Because of the small sample size of our pilot study, our findings must be regarded as preliminary.

Effects of a Milk-Based Meal Replacement Program on Weight and Metabolic Characteristics in Adults with Severe Obesity.

OBJECTIVE: Low energy meal replacement regimens can induce short-term weight loss in patients with severe obesity, but usually require specially formulated dietary supplements. We sought to determine the effects of a milk-based meal replacement program on anthropometric and metabolic characteristics in adults with severe obesity. METHODS: We conducted a retrospective cohort study of patients attending our hospital-based bariatric medicine service who completed a 24-week program consisting of eight weeks of milk-based meal replacement followed by weight stabilisation and maintenance phases. Patients were seen fortnightly by the bariatric physician, nurse and dietitian. We assessed changes in anthropometric and metabolic outcomes in completers at 0, 8, 16 and 24 weeks. RESULTS: Of 105 program completers available for follow-up, 53.3% were female. Mean age was 51.1±11.2 years. Body weight decreased from 144.0±27.6 kg at baseline to 121.1±25.0 kg at 24 weeks (P<0.001), a mean total body weight loss of 15.9±6.0%, with a reduction in body mass index from 50.6±8.0 to 42.6±7.6 kg m-2 (P<0.001). In patients with diabetes, haemoglobin A1c decreased from 66.3±13.0 to 48.3±13.5 mmol/mol (P<0.001) and diabetes medication use decreased significantly. There were significant improvements also in lipid profiles and reductions in antihypertensive medication use. CONCLUSION: These preliminary findings suggest that completion of a 24-week milk-based meal replacement program has large effects on important outcomes in adults with severe obesity. However, attrition was high. Prospective assessment of the efficacy, safety, durability and cost-effectiveness of this intervention seems warranted.

Implementation of RNA sequencing and array CGH in the diagnostic workflow of the AIEOP-BFM ALL 2017 trial on acute lymphoblastic leukemia.

Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.