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We apply hierarchical clustering (HC) of DNA k-mer counts on multiple Fastq files. The tree structures produced by HC may reflect experimental groups and thereby indicate experimental effects, but clustering of preparation groups indicates the presence of batch effects. Hence, HC of DNA k-mer counts may serve as a diagnostic device. In order to provide a simple applicable tool we implemented sequential analysis of Fastq reads with low memory usage in an R package (seqTools) available on Bioconductor. The approach is validated by analysis of Fastq file batches containing RNAseq data. Analysis of three Fastq batches downloaded from ArrayExpress indicated experimental effects. Analysis of RNAseq data from two cell types (dermal fibroblasts and Jurkat cells) sequenced in our facility indicate presence of batch effects. The observed batch effects were also present in reads mapped to the human genome and also in reads filtered for high quality (Phred > 30). We propose, that hierarchical clustering of DNA k-mer counts provides an unspecific diagnostic tool for RNAseq experiments. Further exploration is required once samples are identified as outliers in HC derived trees.

Background Preclinical and proof-of-concept studies suggest a cardioprotective effect of remote ischemic preconditioning (RIPC). However, two major clinical trials (ERICCA and RIPHeart) failed to show cardioprotection by RIPC. Aging and gender might be confounding factors of RIPC affecting the inter-organ signalling. Theoretically, confounding factors might prevent the protective potency of RIPC by interfering with cardiac signalling pathways, i.e. at the heart, and/or by affecting the release of humoral factor(s) from the remote organ, e.g. from the upper limb. This study investigated the effect of age and sex on the release of cardioprotective humoral factor(s) after RIPC in humans. Methods Blood samples were taken from young and aged, male and female volunteers before (control) and after RIPC (RIPC). To investigate the protective potency of the different plasma groups obtained from the human volunteers, isolated perfused hearts of young rats were used as bioassay. For this, hearts were perfused with the volunteer plasma (0.5% of coronary flow) before hearts underwent global ischemia and reperfusion. In addition, to characterize the protective potency of humoral factor(s) after RIPC to initiate protection not only in young but also aged hearts, plasma from young male volunteers were transferred to isolated hearts of aged rats. At the end of the experimental protocol, infarct sizes were determined by TTC-staining (expressed as % of left ventricle). Results RIPC plasma of young male volunteers reduced infarct size in young rat hearts from 47 ± 5 to 31 ± 10% (p = 0.02). In contrast, RIPC plasma of aged male volunteers had no protective effect. Infarct size after application of control plasma of young female volunteers was 33 ± 10%, and female RIPC plasma did not lead to an infarct size reduction. RIPC plasma of old female initiated no cardioprotection. RIPC plasma of young male volunteers reduced infarct size in isolated hearts from aged rats (41 ± 5% vs. 51 ± 5%; p < 0.001). Conclusions The release of humoral factor(s) into the blood after RIPC in humans is affected by both age and sex. In addition, these blood borne factor(s) are capable to initiate cardioprotection within the aged heart.

Ageing, the progressive functional decline of virtually all tissues, affects numerous living organisms. Main phenotypic alterations of human skin during the ageing process include reduced skin thickness and elasticity which are related to extracellular matrix proteins. Dermal fibroblasts, the main source of extracellular fibrillar proteins, exhibit complex alterations during in vivo ageing and any of these are likely to be accompanied or caused by changes in gene expression. We investigated gene expression of short term cultivated in vivo aged human dermal fibroblasts using RNA-seq. Therefore, fibroblast samples derived from unaffected skin were obtained from 30 human donors. The donors were grouped by gender and age (Young: 19 to 25 years, Middle: 36 to 45 years, Old: 60 to 66 years). Two samples were taken from each donor, one from a sun-exposed and one from a sun-unexposed site. In our data, no consistently changed gene expression associated with donor age can be asserted. Instead, highly correlated expression of a small number of genes associated with transforming growth factor beta signalling was observed. Also, known gene expression alterations of in vivo aged dermal fibroblasts seem to be non-detectable in cultured fibroblasts.

The metabolic syndrome is a worldwide problem mainly caused by obesity. FTO was found to be a obesity-risk gene in humans and FTO deficiency in mice led to reduction in adipose tissue. Thus, FTO is an important factor for the development of obesity. Leptin-deficient mice are a well characterized model for analysing the metabolic syndrome. To determine the relevance of FTO for the development of the metabolic syndrome we analysed different parameters in combined homozygous deficient mice (Lep(ob/ob);Fto(-/-)). Lep(ob/ob);Fto(-/-) mice showed an improvement in analysed hallmarks of the metabolic syndrome in comparison to leptin-deficient mice wild type or heterozygous for Fto. Lep(ob/ob);Fto(-/-) mice did not develop hyperglycaemia and showed an improved glucose tolerance. Furthermore, extension of beta-cell mass was prevented in Lep(ob/ob);Fto(-/-)mice and accumulation of ectopic fat in the liver was reduced. In conclusion this study demonstrates that FTO deficiency has a protective effect not only on the development of obesity but also on the metabolic syndrome. Thus, FTO plays an important role in the development of metabolic disorders and is an interesting target for therapeutic agents.

Purpose We evaluated the current performance of diagnostic ultrasound (US) for detecting cervical lymph node (LN) metastases based on objective measures and subjective findings in comparison to the gold standard, histopathological evaluation. Patients and methods From 2007 to 2016, we prospectively included patients with head and neck cancer who were scheduled for surgical therapy including neck dissection. LNs were examined by multimodal US by a level III head and neck sonologist and individually assigned to a map containing six AAO-HNS neck LN levels preoperatively. During the operation, LNs were dissected and then assessed by routine histopathology, with 86% of them examined individually and the remaining LNs (14%) per AAO-HNS neck LN level. The optimal cutoff points (OCPs) of four defined LN diameters and 2D and 3D roundness indices per AAO-HNS neck LN level were determined. Results In total, 235 patients were included, and 4539 LNs were analyzed by US, 7237 by histopathology and 2684 by both methods. Of these, 259 (9.65%) were classified as suspicious for metastasis by US, whereas 299 (11.14%) were found to be positive by histopathology. Subjective US sensitivity and specificity were 0.79 and 0.99, respectively. The OCPs of the individual LN diameters and the 2D and 3D roundness index were determined individually for all AAO-HNS neck LN levels. Across all levels, the OCP for the 2D index was 1.79 and the 3D index was 14.97. The predictive performance of all distances, indices, and subjective findings improved with increasing metastasis size. Anticipation of pN stage was best achieved with subjective US findings and the smallest diameter (Cohen’s κ = 0.713 and 0.438, respectively). Conclusion Our LN mapping and meticulous 1:1 node-by-node comparison reveals the usefulness of US for detecting metastatic involvement of neck LNs in head and neck carcinomas as compared to histopathology. The predictive ability for small tumor deposits less than 8 mm in size remains weak and urgently needs improvement.

Clinical documentation has undergone a change due to the usage of electronic health records. The core element is to capture clinical findings and document therapy electronically. Health care personnel spend a significant portion of their time on the computer. Alternatives to self-typing, such as speech recognition, are currently believed to increase documentation efficiency and quality, as well as satisfaction of health professionals while accomplishing clinical documentation, but few studies in this area have been published to date. This study describes the effects of using a Web-based medical speech recognition system for clinical documentation in a university hospital on (1) documentation speed, (2) document length, and (3) physician satisfaction. Reports of 28 physicians were randomized to be created with (intervention) or without (control) the assistance of a Web-based system of medical automatic speech recognition (ASR) in the German language. The documentation was entered into a browser's text area and the time to complete the documentation including all necessary corrections, correction effort, number of characters, and mood of participant were stored in a database. The underlying time comprised text entering, text correction, and finalization of the documentation event. Participants self-assessed their moods on a scale of 1-3 (1=good, 2=moderate, 3=bad). Statistical analysis was done using permutation tests. The number of clinical reports eligible for further analysis stood at 1455. Out of 1455 reports, 718 (49.35%) were assisted by ASR and 737 (50.65%) were not assisted by ASR. Average documentation speed without ASR was 173 (SD 101) characters per minute, while it was 217 (SD 120) characters per minute using ASR. The overall increase in documentation speed through Web-based ASR assistance was 26% (P=.04). Participants documented an average of 356 (SD 388) characters per report when not assisted by ASR and 649 (SD 561) characters per report when assisted by ASR. Participants' average mood rating was 1.3 (SD 0.6) using ASR assistance compared to 1.6 (SD 0.7) without ASR assistance (P<.001). We conclude that medical documentation with the assistance of Web-based speech recognition leads to an increase in documentation speed, document length, and participant mood when compared to self-typing. Speech recognition is a meaningful and effective tool for the clinical documentation process.

Merging data from multiple samples is required to detect low expressed transcripts or splicing events that might be present only in a subset of samples. However, the exact number of required replicates enabling the detection of such rare events often remains a mystery but can be approached through probability theory. Here, we describe a probabilistic model, relating the number of observed events in a batch of samples with observation probabilities. Therein, samples appear as a heterogeneous collection of events, which are observed with some probability. The model is evaluated in a batch of 54 transcriptomes of human dermal fibroblast samples. The majority of putative splice-sites (alignment gap-sites) are detected in (almost) all samples or only sporadically, resulting in an U-shaped pattern for observation probabilities. The probabilistic model systematically underestimates event numbers due to a bias resulting from finite sampling. However, using an additional assumption, the probabilistic model can predict observed event numbers within a <10% deviation from the median. Single samples contain a considerable amount of uniquely observed putative splicing events (mean 7122 in alignments from TopHat alignments and 86,215 in alignments from STAR). We conclude that the probabilistic model provides an adequate description for observation of gap-sites in transcriptome data. Thus, the calculation of required sample sizes can be done by application of a simple binomial model to sporadically observed random events. Due to the large number of uniquely observed putative splice-sites and the known stochastic noise in the splicing machinery, it appears advisable to include observation of rare splicing events into analysis objectives. Therefore, it is beneficial to take scores for the validation of gap-sites into account.

Genomic alignments of sequenced cellular messenger RNA contain gapped alignments which are interpreted as consequence of intron removal. The resulting gap-sites, genomic locations of alignment gaps, are landmarks representing potential splice-sites. As alignment algorithms report gap-sites with a considerable false discovery rate, validations are required. We describe two quality scores, gap quality score (gqs) and weighted gap information score (wgis), developed for validation of putative splicing events: While gqs solely relies on alignment data wgis additionally considers information from the genomic sequence. FASTQ files obtained from 54 human dermal fibroblast samples were aligned against the human genome (GRCh38) using TopHat and STAR aligner. Statistical properties of gap-sites validated by gqs and wgis were evaluated by their sequence similarity to known exon-intron borders. Within the 54 samples, TopHat identifies 1,000,380 and STAR reports 6,487,577 gap-sites. Due to the lack of strand information, however, the percentage of identified GT-AG gap-sites is rather low. While gap-sites from TopHat contain ≈89% GT-AG, gap-sites from STAR only contain ≈42% GT-AG dinucleotide pairs in merged data from 54 fibroblast samples. Validation with gqs yields 156,251 gap-sites from TopHat alignments and 166,294 from STAR alignments. Validation with wgis yields 770,327 gap-sites from TopHat alignments and 1,065,596 from STAR alignments. Both alignment algorithms, TopHat and STAR, report gap-sites with considerable false discovery rate, which can drastically be reduced by validation with gqs and wgis.

To examine corneal biomechanics in healthy and keratoconic eyes, with or without crosslinking obtained by ultrahigh-speed Scheimpflug measurements (Corvis ST). One hundred and seventeen eyes were studied in three groups: group 1 (n=39) contained keratoconic eyes without crosslinking. Group 2 (CXL; n=28) comprised keratoconic eyes after crosslinking. These were compared with a control group (n=50 matched healthy eyes). In addition, 10 keratoconus patients, before and after CXL treatment, respectively, were examined. The novel parameter A1L-A2L demonstrated highly significant differences between crosslinked corneas and untreated keratoconic or healthy corneas. Velocity during second applanation (A2V) and deformation amplitude (DA) were significantly increased in crosslinked keratoconic eyes both compared with untreated keratoconic eyes and with healthy controls. Radius at highest curvature also was significant among all groups. Inward applanation length (A1L) was significantly increased in controls, whereas outward applanation length (A2L) was significantly reduced in crosslinked keratoconic eyes compared with both other groups. The follow-up analysis revealed statistically significant changes in pachymetry and intraocular pressure and showed tendencies towards significance in applanation times 1 and 2 and in DA. Both A2V and A2L are viable parameters to discriminate healthy from keratoconic but also crosslinked from non-crosslinked keratoconic corneas. The difference of A1L-A2L could reliably discriminate crosslinked from non-crosslinked and healthy corneas. Follow-up examination in a small cohort allows distinction between crosslinked and untreated keratoconus in follow-up examinations. The difference of A1L-A2L could reliably discriminate crosslinked from non-crosslinked and healthy corneas. Measurements of corneal deformation using dynamic ultrahigh-speed Scheimpflug technology are reproducible and provide useful information about keratoconus assessment and biomechanics. Therefore, the Corvis ST seems to provide useful technology to monitor therapeutic success of crosslinking treatment.

Introduction Proper management of the clinically involved neck in OSCC patients continues to be a matter of debate. Our aim was to analyze the accuracy of computerized tomography (CT) and ultrasound (US) in anticipating the exact location of lymph node (LN) metastases of OSCC patients across the AAO-HNS (American Academy of Otolaryngology—Head and Neck Surgery) levels ipsi- and contralaterally. Furthermore, we wanted to assess the suitability of therapeutic selective neck dissection (SND) in patients with one or two ipsilateral positive nodes upon clinical staging (cN1/cN2a and cN2b(2/x) patients). Methods We prospectively analyzed the LN status of patients with primary OSCC using CT and US from 2007 to 2013. LNs were individually assigned to a map containing the AAO-HNS levels; patients bearing a single or just two ipsilateral positive nodes (designated cN1/cN2a or cN2b(2/x) patients either by CT (CT group) or US alone (US group) or in a group combining findings of CT and US (CTUS group)) received an ipsi-ND (I–V) and a contra-ND (I–IV). 78% of the LNs were sent individually for routine histopathological examination; the remaining were dissected and analyzed per neck level. Results Upon the analysis of 1.670 LNs of 57 patients, the exact location of pathology proven LN metastases in cN1 patients was more precisely predicted by US compared to CT with confirmed findings only in levels IA, IB und IIA. Clearly decreasing the number of missed lesions, the findings in the CTUS group nearly kept the spatial reliability of the US group. The same analysis for patients with exactly two supposed ipsilateral lesions (cN2b(2/x)) yielded confirmed metastases from levels I to V for both methods individually and in combination and, therefore, render SND insufficient for these cases. Conclusion Our findings stress the importance of conducting both, CT and US, in patients with primary OSCC. Only the combination of their findings warrants the application of therapeutic SND in patients with a single ipsilateral LN metastasis (cN1/cN2a patients) but not in patients with more than one lesion upon clinical staging (≥ cN2b).