Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

Introduction

Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens.

Materials and Methods

Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens.

Results

Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin.

Discussion

Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin.     

Kudi: A free open-source python library for the analysis of properties along reaction paths

With increasing computational capabilities, an ever growing amount of data is generated in computational chemistry that contains a vast amount of chemically relevant information. It is therefore imperative to create new computational tools in order to process and extract this data in a sensible way. Kudi is an open source library that aids in the extraction of chemical properties from reaction paths. The straightforward structure of Kudi makes it easy to use for users and allows for effortless implementation of new capabilities, and extension to any quantum chemistry package. A use case for Kudi is shown for the tautomerization reaction of formic acid. Kudi is available free of charge at www.github.com/stvogt/kudi     

Varying foraging patterns in response to competition? A multicolony approach in a generalist seabird

Reducing resource competition is a crucial requirement for colonial seabirds to ensure adequate self‐ and chick‐provisioning during breeding season. Spatial segregation is a common avoidance strategy among and within species from neighboring breeding colonies. We determined whether the foraging behaviors of incubating lesser black‐backed gulls (Larus fuscus) differed between six colonies varying in size and distance to mainland, and whether any differences could be related to the foraging habitats visited. Seventy‐nine incubating individuals from six study colonies along the German North Sea coast were equipped with GPS data loggers in multiple years. Dietary information was gained by sampling food pellets, and blood samples were taken for stable isotope analyses. Foraging patterns clearly differed among and within colonies. Foraging range increased with increasing colony size and decreased with increasing colony distance from the mainland, although the latter might be due to the inclusion of the only offshore colony. Gulls from larger colonies with consequently greater density‐dependent competition were more likely to forage at land instead of at sea. The diets of the gulls from the colonies furthest from each other differed, while the diets from the other colonies overlapped with each other. The spatial segregation and dietary similarities suggest that lesser black‐backed gulls foraged at different sites and utilized two main habitat types, although these were similar across foraging areas for all colonies except the single offshore island. The avoidance of intraspecific competition results in colony‐specific foraging patterns, potentially causing more intensive utilization of terrestrial foraging sites, which may offer more predictable and easily available foraging compared with the marine environment.     

Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Evidence of Inbreeding in Hodgkin Lymphoma

Genome-wide association studies (GWASs) have identified several, mainly co-dominantly acting, single-nucleotide polymorphisms (SNPs) associated with Hodgkin lymphoma (HL). We searched for recessively acting disease loci by performing an analysis of runs of homozygosity (ROH) based on windows of homozygous SNP-blocks and by calculating genomic inbreeding coefficients on a SNP-wise basis. We used data from a previous GWAS with 906 cases and 1217 controls from a population with a long history of no matings between relatives. Ten recurrent ROHs were identified among 25 055 ROHs across all individuals but their association with HL was not genome-wide significant. All recurrent ROHs showed significant evidence for natural selection. As a novel finding genomic inbreeding among cases was significantly higher than among controls (P = 2.11*10⁻¹⁴) even after correcting for covariates. Higher inbreeding among the cases was mainly based on a group of individuals with a higher average length of ROHs per person. This result suggests a correlation of higher levels of inbreeding with higher cancer incidence and might reflect the existence of recessive alleles causing HL. Genomic inbreeding may result in a higher expression of deleterious recessive genes within a population.     

Methicillin-Resistant Staphylococcus aureus in Saarland,Germany: The Long-Term Care Facility Study

BackgroundMultiresistant organisms pose a threat for patients and care recipients. Control interventions need to be tailored to region, the type of institution considered, and risk factors. The German state of Saarland is ideally suited to study colonisation epidemiology throughout its various health and care institutions. After conclusion of a large admission prevalence study in acute care hospitals, we now performed a methicillin-resistant Staphylococcus aureus (MRSA) point prevalence study in Saarland long term care facilities (LTCF), allowing for a direct comparison with respect of MRSA prevalence and associated risk factors between these two institutional types located within a confined region.ConclusionAs already known from previous studies, colonisation with MRSA is common in LTCF residents even in an area with relatively low MRSA prevalence. This found prevalence can now be related to the acute care admission prevalence (2.2%) as well as to the admission prevalence in acute care geriatric departments (7.6%). The common clonal attribution (spa type) of MRSA isolates prevalent in the LTCF population as well as in the acute care admission population points towards a close relationship between both types of institutions. However, the ostensible absence of risk factors such as “previous hospitalisation” in conjunction with newly identified factors such as “multiple decolonisation cycles” refers to MRSA colonisation risks independent of contact with acute care facilities. Overall, this large LTCF point prevalence study allows data-based, region-tailored decisions on MRSA screening policies and provides a basis for additional preventative measures.