Corrective osteotomy of the distal radius with palmar locking plate osteosynthesis without bone grafting and without cortical contact.

The aim of this study was to assess bone healing and secondary fracture displacement after corrective osteotomy of the distal radius without any cortical contact using palmar locking plates without bone grafting. Between 2009 and 2021, 11 palmar corrective osteotomies of extra-articular malunited distal radius fractures and palmar plate fixations without the use of bone grafts and without cortical contact, were assessed. All patients showed complete osseous restoration and significant improvement in all radiographic parameters. Except for one patient, there were no secondary dislocations or loss of reduction in the postoperative follow-up. Bone grafts may not be mandatory for bone healing and prevention of secondary fracture displacement after palmar corrective osteotomy without cortical contact and fixation with palmar locking plate.Level of evidence: IV.

Mucor osteomyelitis after traumatic forearm amputation in a 38-year-old patient.

We report the case of a forest ranger who developed a polymicrobial infection with Mucor circinelloides after traumatic forearm amputation. Based on our case report we discuss epidemiology and management of this rare and potentially fatal infection.

Lung macrophages utilize unique cathepsin K-dependent phagosomal machinery to degrade intracellular collagen.

Resident tissue macrophages are organ-specialized phagocytes responsible for the maintenance and protection of tissue homeostasis. It is well established that tissue diversity is reflected by the heterogeneity of resident tissue macrophage origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using a quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneum-, lung-, and brain-resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phagosomal proteolysis and collagenolysis in lung-resident macrophages. Furthermore, profibrotic TGF-ß negatively regulated CtsK-mediated phagosomal collagen degradation independently from classical endocytic-proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cigarette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung, and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.

In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain.

Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.

Revision rate of THA in patients younger than 40 years depends on primary diagnosis - a retrospective analysis with a minimum follow-up of 10 years.

BACKGROUND: Treating osteoarthritis in elderly patients with THA is very successful. However, surgeons hesitate to recommend THA in younger patients. The spectrum of etiologies for end stage hip disease in the younger population is diverse and therefore different courses may be assumed. Our objective was to evaluate THA revision rate within a minimum follow-up period of 10 years in young patients and to analyze the difference between different primary diagnoses. METHODS: We included 144 consecutive hips in 127 patients younger than 40 years, who received a primary THA from 01/1996 to 12/2007. Operative reports, clinical and radiographic documentation were reviewed to determine primary diagnosis, prior hip surgery, component specifications and revision surgery. 111 hips in 97 patients were available for outcome analysis with a minimum follow-up of 10 years. RESULTS: The mean age was 33 years (range 15-40 years) at the time of the index THA, 68 patients were female and 59 were male. Ten years revision rate on the prosthetic components was 13%. The most common primary diagnosis was DDH. DDH was associated with a risk of 17% for requiring a reoperation on the prosthetic components because of mechanical fatigue and therefore, significantly higher than for any other primary diagnosis (p = 0.005). CONCLUSION: THA in young patients is associated with a high revision rate of 13% in 10 years. 17% of patients with DDH required revision surgery for mechanical fatigue within 10 years, which was significantly higher than for any other primary diagnosis (1.2%, OR 16.8).

Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.

The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.

Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Absolute quantification of cohesin, CTCF and their regulators in human cells.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

A noninvasive platform for imaging and quantifying oil storage in submillimeter tobacco seed.

While often thought of as a smoking drug, tobacco (Nicotiana spp.) is now considered as a plant of choice for molecular farming and biofuel production. Here, we describe a noninvasive means of deriving both the distribution of lipid and the microtopology of the submillimeter tobacco seed, founded on nuclear magnetic resonance (NMR) technology. Our platform enables counting of seeds inside the intact tobacco capsule to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. Hundreds of seeds can be simultaneously imaged at an isotropic resolution of 25 µm, sufficient to assess each individual seed. The relative contributions of the embryo and the endosperm to both seed size and total lipid content could be assessed. The extension of the platform to a range of wild and cultivated Nicotiana species demonstrated certain evolutionary trends in both seed topology and pattern of lipid storage. The NMR analysis of transgenic tobacco plants with seed-specific ectopic expression of the plastidial phosphoenolpyruvate/phosphate translocator, displayed a trade off between seed size and oil concentration. The NMR-based assay of seed lipid content and topology has a number of potential applications, in particular providing a means to test and optimize transgenic strategies aimed at the manipulation of seed size, seed number, and lipid content in tobacco and other species with submillimeter seeds.

Black cohosh hepatotoxicity: quantitative causality evaluation in nine suspected cases.

OBJECTIVE: Black cohosh (BC), synonym for Actaea racemosa and Cimicifuga racemosa, is a herbal remedy for the treatment of menopausal symptoms. Recently, worldwide discussions have emerged as to whether its use maybe associated with the risk of rare hepatotoxicity in a few susceptible women. METHODS: We have evaluated the causal relationship in nine cases with suspected hepatotoxicity by the use of BC. The updated Council for International Organizations of Medical Sciences scale was used to quantitatively assess the causality for BC. RESULTS: In eight of nine patients with liver disease, causality for BC +/- comedication was excluded (n = 4) or unlikely (n = 4). The failure to ascribe causality in these cases was mainly due to alternative diagnosis, missing temporal association and dechallenge, and presentation of low quality data. In only one case, causality was possible for a BC preparation of an unknown brand taken for 2 months with an unknown daily dose. Confounding factors in this case include symptomatic cholelithiasis and fatty liver. Comedication with synthetic drugs and herbal or other dietary supplements was reported in five of nine patients. CONCLUSIONS: In nine cases of patients with liver disease, causality for BC +/- comedication was possible (n = 1), unlikely (n = 4), or excluded (n = 4). Due to this lack of significant circumstantial evidence, the present study shows little, if any, hepatotoxic risks by the use of BC in the analyzed cases.

Leg axis after computer-navigated total knee arthroplasty: a prospective randomized trial comparing computer-navigated and manual implantation.

To compare the alignment after computer-navigated total knee arthroplasty, 52 patients were randomly allocated to 2 groups. Twenty-seven patients received a total knee arthroplasty with the aid of a kinematic computer-navigation system, and 25 patients received a total knee arthroplasty with the conventional method. Both groups were well balanced concerning demographic data and preoperative scores. At 3-month follow-up, the mechanical alignment of the leg reached the desired straight axis in more cases with the computer-navigated implantation. This difference was statistically significant. The femoral and tibial mechanical anteroposterior axis and the femoral and tibial sagittal tilt (slope) measured on sagittal x-rays were not significantly improved in this patient group.