Deep-Learning-Based Segmentation of the Shoulder from MRI with Inference Accuracy Prediction

Three-dimensional (3D)-image-based anatomical analysis of rotator cuff tear patients has been proposed as a way to improve repair prognosis analysis to reduce the incidence of postoperative retear. However, for application in clinics, an efficient and robust method for the segmentation of anatomy from MRI is required. We present the use of a deep learning network for automatic segmentation of the humerus, scapula, and rotator cuff muscles with integrated automatic result verification. Trained on N = 111 and tested on N = 60 diagnostic T1-weighted MRI of 76 rotator cuff tear patients acquired from 19 centers, a nnU-Net segmented the anatomy with an average Dice coefficient of 0.91 ± 0.06. For the automatic identification of inaccurate segmentations during the inference procedure, the nnU-Net framework was adapted to allow for the estimation of label-specific network uncertainty directly from its subnetworks. The average Dice coefficient of segmentation results from the subnetworks identified labels requiring segmentation correction with an average sensitivity of 1.0 and a specificity of 0.94. The presented automatic methods facilitate the use of 3D diagnosis in clinical routine by eliminating the need for time-consuming manual segmentation and slice-by-slice segmentation verification

Molecular characterisation of the response regulators ArsR, HP1043, HP1021 of Helicobacter pylori

Bakterien müssen ständig in der Lage sein auf Veränderungen in ihrer Umwelt reagieren zu können. Zur Wahrnehmung dieser Veränderungen haben sich unterschiedliche Signaltransduktionssysteme entwickelt. Ein weit verbreiteter und gut charakterisierter Mechanismus zur Signaltransduktion sind die so genannten Zwei-Komponentensysteme. Im Genom von H. pylori konnten nur wenige Bestandteile von Zwei-Komponentensystemen identifiziert werden. Dazu zählen neben dem Chemotaxis-System lediglich drei Histidin-Kinasen, ArsS, CrdS und FlgS, und fünf Response-Regulatoren HP1021, HP1043, ArsR, CrdR und FlgR, die vermutlich Transkriptions-regulatorische Funktionen haben. Zwei der Response-Regulatoren, HP1043 und ArsR als essentiell für das Überleben von H. pylori, während HP1021 einen deutlichen Einfluss auf das Zellwachstum hat, da ein Wachstums-Defekt zu erkennen ist, wenn das entsprechende Gen hp1021 deletiert wird. Eine Deletion von arsS, dem Gen der zugehörigen Histidin-Kinase von ArsR, hat unter Standard-Wachstumsbedingungen keine Auswirkung auf das Zellwachstum von H. pylori. Diese Beobachtung spricht für die Hypothese, dass der Response-Regulator ArsR die Transkription zweier unterschiedlicher Sets von Zielgenen kontrolliert. Demzufolge reguliert der Response-Regulator ArsR nach Säure-induzierter Phosphorylierung durch ArsS die Transkription von Genen, die zur Säureresistenz beitragen, während ArsR im nicht-phosphorylierten Zustand die Transkription von weiteren Zielgenen kontrolliert, von denen mindestens eines für das Zellwachstum essentiell sein sollte. Das durch ArsR~P kontrollierte Regulon konnte bereits weitgehend charakterisiert werden allerdings sind die Zielgene des unphosphorylierten Regulators bislang unbekannt. In der vorliegenden Arbeit konnte die zuvor beschriebene Hypothese bestätigt werden, da gezeigt wurde, dass ein Derivat von ArsR, mit einer Mutation der Phosphorylierungsstelle D52 zu N52, das wildtypische Protein bezüglich des Zellwachstums unter Standardbedingungen funktionell ersetzen kann. Für die Response-Regulatoren HP1021 und HP1043 konnte bislang keine zugehörige Histidin-Kinase identifiziert werden und interessanterweise findet man in der Receiver-Domäne dieser Response-Regulatoren atypische Abweichungen von der Konsensus-Sequenz. Um die Bedeutung dieser atypischen Primärsequenzen für die Funktion dieser Response-Regulatoren zu untersuchen wurden mutierte H. pylori-Stämme konstruiert, die ausschließlich Derivate von HP1021 bzw. HP1043 exprimieren, die in ihrer Receiver-Sequenz der Konsensus-Sequenz entsprachen. Da diese Mutanten sich bezüglich ihres Zellwachstums nicht vom Wildtyp unterscheiden, konnte nachgewiesen werden, dass die atypischen Receiver-Sequenzen der beiden Response-Regulatoren nicht entscheidend für die Funktionen der Response-Regulatoren sind. Weiterhin konnten Indizien dafür gesammelt werden, dass HP1021 und HP1043 hinsichtlich ihrer Aktivierung vermutlich vom üblichen Zwei-Komponentenparadigma abweichen. Derivate von HP1021 und HP1043 mit Mutationen ihrer putativen atypischen Phosphorylierungsstelle sind in der Lage ihre wildtypischen Pendants hinsichtlich der bekannten Phänotypen funktionell zu ersetzen. Somit ist eine Phosphorylierung der Receiver-Domäne dieser Response-Regulatoren keine Voraussetzung für ein normales Zellwachstum von H. pylori. Diese Hypothese wird gestützt durch die Beobachtung, dass ein Ortholog von HP1043 aus C. jejuni CJ0355, das natürlicherweise an der potentiellen Phosphorylierungsstelle einen nicht phosphorylierbaren Aminosäurerest trägt, HP1043 in seiner Funktion ersetzen kann. Es konnte gezeigt werden, dass in vitro keine Phosphorylierung durch radioaktiv markiertes Acetylphosphat stattfindet und dass ein H. pylori-Stamm mit einer Deletion der Gene pta und ackA, welche Proteine kodieren, die bei der Synthese von zellulärem Acetylphosphat benötigt werden, einen normalen Wachstums-Phänotyp zeigt. Zusätzlich konnten in einer massenspektrometrischen Analyse des Proteins HP1021, welches nach Zweidimensionaler Gelelektrophorese von Gesamtzellproteinlysaten aus H. pylori isoliert wurde, keine Hinweise auf eine Serinphosphorylierung entdeckt werden. Es ist daher fraglich ob in vivo eine funktionell relevante Phosphorylierung stattfindet. Die Mechanismen zur Modulation der Regulator-Aktivität von HP1043 und HP1021 bleiben unklar. In der vorliegenden Arbeit konnte demonstriert werden, dass eine strikte Transkriptionskontrolle nicht für die Zellwachstums-assoziierten Funktionen von HP1021 von Bedeutung ist. Dagegen wurden Hinweise darauf erzielt, dass die Expression von HP1043 auf einem posttranskriptionellen und/oder auf einem posttranslationellen Level reguliert wird. Es waren bislang keine Zielgene von HP1021 bekannt. Durch vergleichende Zweidimensionale Gelelektrophorese der H. pylori Stämme 26695 und 26695 1021Δ konnten einige potentielle Zielgene des Response-Regulators HP1021 identifiziert werden.Bacteria need to react instantaneously on changes in their environment. For the sensing of environmental changes many different signaltransduction-systems have been evolved and the most widespread and well-characterised mechanism for signal transduction among the bacteria are the so called two-component systems. The genome of Helicobacter pylori encodes only a few proteins which are part of a two-component system. In addition to the chemotaxis-system there are just three histidine kinases, ArsS (HP0165), CrdS (HP1364) and HP0244, and five response regulators HP1021, HP1043, ArsR (HP0166), CrdR (HP1365) and HP0703, which are probably involved in transcriptional regulation (Alm et al., 1999; Tomb et al., 1997). Interestingly two of the response regulators, HP1043 and ArsR (HP0166) proved to be essential for the survival of H. pylori, while the response regulator HP1021 has a distinct influence on the cell-growth, as indicated by a severe growth defect when hp1021 is deleted (Beier & Frank, 2000; McDaniel et al., 2001; Schär, 2001). Under standard growth-conditions, the deletion of arsS (hp0165), encoding the cognate histidine kinase of ArsR (HP0166), has no effect on the cell-growth of H. pylori. This observation argues for the hypothesis that the response regulator ArsR (HP0166) controls the transcription of two different sets of target-genes. Upon acid-induced phosphorylation via ArsS (HP0165), the response regulator ArsR (HP0166) regulates the transcription of genes which are involved in acid resistance mechanisms, while in an unphosphorylated state ArsR (HP0166) controls other target genes, of which at least one is essential for cell-growth. The regulon controlled by ArsR~P (HP0166~P) has been extensively studied (Dietz, 2002; Forsyth et al., 2002; Pflock et al., 2004; Pflock et al., 2006; Pflock et al., 2005), while target genes of the unphosphorylated response regulator are so far unknown. In this work this hypothesis could be proven. It was shown that a derivative of ArsR (HP0166), with a mutation of the phosphorylation site D52 to N52, is able to substitute the wildtype protein functionally with respect to cell-growth. In the case of the response regulators HP1021 and HP1043 no cognate histidine kinases have been identified so far (Beier & Frank, 2000). Interestingly the receiver domains of the response regulators differ from the consensus sequence at highly conserved aminoacid residues. To investigate the importance of the atypical primary sequences for the function of HP1021 and HP1043, mutated H. pylori strains expressing exclusively derivatives of HP1021 or HP1043 with receiver sequences matching the consensus sequence were constructed. As these mutants did not show differences in cell-growth in comparison to the wildtype strain, it was proven that the atypical receiver sequences are not crucial for cell growth. Furthermore, indications could be found that HP1021 and HP1043 presumably differ from the common two component paradigm regarding their activation. Derivatives of HP1021 and HP1043 with mutations in their putative phosphorylation sites could functionally complement their wildtype counterparts with regards to cell growth. Therefore the phosphorylation of the receiver domain of these response regulators is not a pre-requisite for normal cell growth of H. pylori. In line with this hypothesis it could be demonstrated that there is no in vitro phosphorylation of the receiver domain of HP1021 and HP1043 with radioactive labelled acetylphosphate and that a H. pylori strain which a deletion of the genes pta and ackA, encoding proteins involved in the synthesis of acetylphosphate in the cell, showed a normal growth-phenotype. Moreover, when the protein HP1021, which was isolated by two-dimensional gelelectrophoreses, was analysed by mass spectrometry no evidence of a serine phosphorylation was obtained. Aditionally the observation that deletion of hp1043 can be complemented by the C. jejuni ortholog cj0355 encoding a response regulator protein with an asparagine residue replacing the consensus phosphate-accepting aspatic acid residue supports this hypothesis. Therefore it is questionable whether in vivo there is a phosphorylation which is functional relevant at all at the receiver domain. The mechanisms by which the activity of the Response-Regulators HP1021 and HP1043 is modulated still remain unclear. Here it could be demonstrated that strict transcriptional control of its expression is not relevant for the cell-growth associated function of HP1021. In contrast there are hints that the expression of HP1043 is controlled on a post-transcriptional and/or a post-translational level. Up to now no target genes of HP1021 were known. Using comperative two-dimensional gelelectrophoresis some potential target genes have been identified, among others HP0695, FadA and the Catalase

Phosphorylation-Independent Activity of Atypical Response Regulators of Helicobacter pylori

The genome of the gastric pathogen Helicobacter pylori harbors a remarkably low number of regulatory genes, including three and five open reading frames encoding two-component histidine kinases and response regulators, respectively, which are putatively involved in transcriptional regulation. Two of the response regulator genes, hp1043 and hp166, proved to be essential for cell growth, and inactivation of the response regulator gene hp1021 resulted in a severe growth defect, as indicated by a small-colony phenotype. The sequences of the receiver domains of response regulators HP1043 and HP1021 differ from the consensus sequence of the acidic pocket of the receiver domain which is involved in the phosphotransfer reaction from the histidine kinase to the response regulator. Using a genetic complementation system, we demonstrated that the function of response regulator HP166, which is essential for cell growth, can be provided by a mutated derivative carrying a D52N substitution at the site of phosphorylation. We found that the atypical receiver sequences of HP1043 and HP1021 are not crucial for the function of these response regulators. Phosphorylation of the receiver domains of HP1043 and HP1021 is not needed for response regulator function and may not occur at all. Thus, the phosphorylation-independent action of these regulators differs from the well-established two-component paradigm

Die Unhintergehbarkeit des Körpers in der ethnografischen Forschung. Praktiken der Grafie von Praktiken

Carnin J. Die Unhintergehbarkeit des Körpers in der ethnografischen Forschung. Praktiken der Grafie von Praktiken. In: Schär C, Ganterer J, Grosse M, eds. Erfahren – Widerfahren – Verfahren. Körper und Leib als analytische und epistemologische Kategorien Sozialer Arbeit. Wiesbaden: Springer Fachmedien Wiesbaden; 2021: 141-152.Der Beitrag legt eine analytische und method(olog)ische Untersuchung von (Forschenden-)Körpern vor. Darin fungiert der Körper als Materialität und Soziales ethnografischer Forscher*innen. Von einem praxistheoretischen Körperverständnis ausgehend argumentiert er, dass der Körper Ort von Objektivierungs- und Subjektivierungsprozessen ist, die von den Akteur*innen leiblich gespürt, miterfahren und inkorporiert werden. Die „Unhintergehbarkeit des Körpers in der ethnografischen Forschung“ wird an der (sozialpädagogischen) Kindheitsforschung expliziert

Genome-wide QTL mapping of nine body composition and bone mineral density traits in pigs

Background: Since the pig is one of the most important livestock animals worldwide, mapping loci that are associated with economically important traits and/or traits that influence animal welfare is extremely relevant for efficient future pig breeding. Therefore, the purpose of this study was a genome-wide mapping of quantitative trait loci (QTL) associated with nine body composition and bone mineral traits: absolute (Fat, Lean) and percentage (FatPC, LeanPC) fat and lean mass, live weight (Weight), soft tissue X-ray attenuation coefficient (R), absolute (BMC) and percentage (BMCPC) bone mineral content and bone mineral density (BMD). Methods: Data on the nine traits investigated were obtained by Dual-energy X-ray absorptiometry for 551 pigs that were between 160 and 200 days old. In addition, all pigs were genotyped using Illumina's PorcineSNP60 Genotyping BeadChip. Based on these data, a genome-wide combined linkage and linkage disequilibrium analysis was conducted. Thus, we used 44 611 sliding windows that each consisted of 20 adjacent single nucleotide polymorphisms (SNPs). For the middle of each sliding window a variance component analysis was carried out using ASReml. The underlying mixed linear model included random QTL and polygenic effects, with fixed effects of sex, housing, season and age. Results: Using a Bonferroni-corrected genome-wide significance threshold of P < 0.001, significant peaks were identified for all traits except BMCPC. Overall, we identified 72 QTL on 16 chromosomes, of which 24 were significantly associated with one trait only and the remaining with more than one trait. For example, a QTL on chromosome 2 included the highest peak across the genome for four traits (Fat, FatPC, LeanPC and R). The nearby gene, ZNF608, is known to be associated with body mass index in humans and involved in starvation in Drosophila, which makes it an extremely good candidate gene for this QTL. Conclusions: Our QTL mapping approach identified 72 QTL, some of which confirmed results of previous studies in pigs. However, we also detected significant associations that have not been published before and were able to identify a number of new and promising candidate genes, such as ZNF608

The Orphan Response Regulator HP1021 of Helicobacter pylori Regulates Transcription of a Gene Cluster Presumably Involved in Acetone Metabolism▿ †

Helicobacter pylori is a gastric pathogen for which no nonhuman reservoir is known. In accordance with the tight adaptation to its unique habitat, the human stomach, H. pylori is endowed with a very restricted repertoire of regulatory proteins. Nevertheless, the three complete two-component systems of H. pylori were shown to be involved in the regulation of important virulence traits like motility and acid resistance and in the control of metal homeostasis. HP1021 is an orphan response regulator with an atypical receiver domain whose inactivation has a considerable impact on the growth of H. pylori. Here we report the identification of HP1021-regulated genes by whole-genome transcriptional profiling. We show that the transcription of the essential housekeeping genes nifS and nifU, which are required for the assembly of Fe-S clusters, is activated by HP1021. Furthermore, we demonstrate that the expression of a gene cluster comprising open reading frames hp0690 to hp0693 and hp0695 to hp0697 which is probably involved in acetone metabolism is strongly upregulated by HP1021. Evidence is provided for a direct regulation of the hp0695-to-hp0697 operon by the binding of HP1021 to its promoter region

Genome-wide QTL mapping of nine body composition and bone mineral density traits in pigs

[Background] Since the pig is one of the most important livestock animals worldwide, mapping loci that are associated with economically important traits and/or traits that influence animal welfare is extremely relevant for efficient future pig breeding. Therefore, the purpose of this study was a genome-wide mapping of quantitative trait loci (QTL) associated with nine body composition and bone mineral traits: absolute (Fat, Lean) and percentage (FatPC, LeanPC) fat and lean mass, live weight (Weight), soft tissue X-ray attenuation coefficient (R), absolute (BMC) and percentage (BMCPC) bone mineral content and bone mineral density (BMD).[Methods] Data on the nine traits investigated were obtained by Dual-energy X-ray absorptiometry for 551 pigs that were between 160 and 200 days old. In addition, all pigs were genotyped using Illumina’s PorcineSNP60 Genotyping BeadChip. Based on these data, a genome-wide combined linkage and linkage disequilibrium analysis was conducted. Thus, we used 44 611 sliding windows that each consisted of 20 adjacent single nucleotide polymorphisms (SNPs). For the middle of each sliding window a variance component analysis was carried out using ASReml. The underlying mixed linear model included random QTL and polygenic effects, with fixed effects of sex, housing, season and age.[Results] Using a Bonferroni-corrected genome-wide significance threshold of P < 0.001, significant peaks were identified for all traits except BMCPC. Overall, we identified 72 QTL on 16 chromosomes, of which 24 were significantly associated with one trait only and the remaining with more than one trait. For example, a QTL on chromosome 2 included the highest peak across the genome for four traits (Fat, FatPC, LeanPC and R). The nearby gene, ZNF608, is known to be associated with body mass index in humans and involved in starvation in Drosophila, which makes it an extremely good candidate gene for this QTL.[Conclusions] Our QTL mapping approach identified 72 QTL, some of which confirmed results of previous studies in pigs. However, we also detected significant associations that have not been published before and were able to identify a number of new and promising candidate genes, such as ZNF608

Glycerol Metabolism and PrfA Activity in Listeria monocytogenes▿ †

Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF2) and a second putative glycerol kinase (GlpK2) were less enhanced under these conditions. GlpK1 but not GlpK2 was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK1 affected replication in Caco-2 cells less than did the loss of GlpK2 and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity

Pyruvate Carboxylase Plays a Crucial Role in Carbon Metabolism of Extra- and Intracellularly Replicating Listeria monocytogenes▿

The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the 13C isotopologue perturbation method in a defined minimal medium containing [U-13C6]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-13C6]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model