Unique molecular identifiers (UMI) can be used in forensic STR sequencing to reduce the level of analytical artifacts. Here, we perform an interlaboratory study across five independent sites where the previously developed UMI-based SiMSen-Seq STR assay is applied at each laboratory to both single-source and mixed samples. The assay showed consistent results between laboratories and more than 90% of the expected alleles were detected with 31 pg DNA of template. The assay tolerated ten times higher PCR inhibitor concentrations compared to an established commercial STR sequencing method. The combined results were used to determine stutter and noise thresholds, which were applied for allele calling, allowing an estimation of the sensitivity for minor contributor alleles in mixtures. We found that the SiMSen-Seq STR method is robust across laboratories and different types of PCR and sequencing equipment and that it allows for calling of alleles from contributors of smaller proportions compared to currently commercially available non-UMI STR sequencing methods.

Cardiovascular disease remains the leading cause of mortality worldwide. Although minimally invasive interventions have improved outcomes, open surgical reconstruction using vascular grafts is still required for many patients. Autologous vessels are preferred due to superior elastic modulus and biocompatibility; however, a substantial proportion of patients lack suitable donor vessels. Synthetic grafts perform adequately in large-diameter applications but frequently fail in small-diameter settings (≤6 mm) due to thrombosis, infection, and poor long-term patency. These limitations highlight the need for next-generation arterial grafts and robust preclinical evaluation systems. Here, we present two complementary large-animal in vivo sheep models for systematic assessment of arterial graft safety, functionality and regenerative potential. One model enables implantation of short interposition grafts (≈10 cm) in the carotid artery, while a second, novel configuration accommodates long-segment grafts (≈30 cm) in a carotid-axillary setup. Both models are integrated with a comprehensive analytical framework to evaluate patency, regeneration, biomechanical stability, immune activation and transcriptomic remodeling. Method overview: • Sheep models supporting implantation of short and long arterial grafts under physiological flow • Multimodal evaluation combining imaging, biomechanics, histology, flow cytometry, and transcriptomics • Generic platform for assessing graft functionality, safety and biological characterization.

The use of Next-Generation Sequencing (NGS) in environmental DNA (eDNA) research offers increased sensitivity in detecting rare species within vast marine ecosystems. However, conventional NGS methods often fail to accurately capture low-frequency variants, posing risks of imprecise detections. Inspired by clinical research, innovations like SiMSen-Seq (simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection using sequencing) have emerged. SiMSen-Seq utilizes molecular barcoding to track individual DNA templates, correcting polymerase-induced errors and quantification biases. In this study, we adapted the SiMSen-Seq protocol for eDNA analysis, targeting 18S rRNA metabarcoding in seawater samples to enhance marine biodiversity assessment, particularly for identifying rare non-indigenous species (NIS). Comparative analysis against an Illumina MiSeq protocol (Illumina-DADA2) and the SiMSen-Seq protocol with DADA2 bioinformatics (SiMSen-Seq-DADA2) revealed that SiMSen-Seq detected a higher number (1495) of molecular Operational Taxonomic Units (mOTUs) compared to the other two pipelines (Illumina-DADA2: 585 and SiMSen-Seq-DADA2: 578). In terms of genera, SiMSen-Seq detected the lowest number of genera, Illumina-DADA2 the highest number, and 45% of genera were detected across protocols. While all three protocols identified the predominant NIS, they differed in detecting rare taxa: SiMSen-Seq uniquely detected Arenigobius bifrenatus and Pseudopolydora paucibranchiata but did not detect some others (e.g., Asterias amurensis/A. forbesi). These findings suggest that for some species (especially Stylea plicata and Ciona savignyi), SiMSen-Seq could potentially be useful to detect intraspecific variability for population genetic studies, with uses in more precise ecological monitoring and effective marine conservation.

INTRODUCTION:Smooth muscle cells (SMCs) with an origin separate from the local vein wall contribute to formation of intimal hyperplasia (IH) in mouse vein grafts. The recruitment pathway of these cells has not been defined, but circulating progenitor cells and cells from the surrounding tissue or adjacent artery to which the vein graft is anastomosed are potential sources. The aim of this study was to clarify if cells from the adjacent artery contribute to neointimal formation in vein grafts. METHODS:Aortic segments from donor SM22α-LacZ mice were anastomosed to vein segments from wild-type (WT) C57BL/6 mice ex vivo followed by implantation of the composite grafts to the right common carotid arteries of WT recipient mice. Six weeks after surgery, the composite grafts were harvested, and histology was analyzed in longitudinal sections. SMCs with origin in the SM22α-LacZ arterial segments were identified with X-gal staining. RESULTS:LacZ-positive cells were found in the medial layer of the SM22α-LacZ arterial segments but were not found in the IH in the vein graft segment. CONCLUSION:SMCs in vein grafts are not recruited from the adjacent artery through migration across the anastomosis.

Background Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. Methods To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. Results Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. Conclusions Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1ng input DNA as well as for low-template samples at 62.5pg input DNA. For twelve challenging mixtures with minor contributions of 10pg to 150pg and ratios of 1% to 15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

Purpose (the aim of the study): Primary osteoartrosis (OA) is a painful, degenerative joint disease with no disease modifying treatment available. Secondary OA, often caused by trauma to a joint, can efficiently be prevented with Autologous Chondrocyte Implantation (ACI), one of the fist cell therapies in the world, used already back in 1996 by A. Lindahl and colleagues. There is a large clinical need for development of new treatments for the many primary OA patients. Within this EU financed project AutoCRAT, sustainable sources of cells ready for therapeutic use for treatment of primary OA are generated. Human induced pluripotent stem cells (hiPSC) show high expandability, do not engender immune reactions, and allow easy commercialization and standardization as well as eliminate ethical concerns. Combined with biomaterials IPS hold promise in the field of regenerative medicine.

BackgroundMyxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. MethodsTo study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. ResultsCharacterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. ConclusionsOur experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

BACKGROUND:Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. METHODS:To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. RESULTS:Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. CONCLUSIONS:Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Three-dimensional (3D) printing has emerged as a highly valuable tool to manufacture porous constructs. This has major advantages in, for example, tissue engineering, in which 3D scaffolds provide a microenvironment with adequate porosity for cell growth and migration as a simulation of tissue regeneration. In this study, we assessed the suitability of three cellulose nanofibrils (CNF) that were obtained through 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO)-mediated oxidation. The CNFs were obtained by applying three levels of carboxylation, i.e., 2.5, 3.8, and 6.0 mmol sodium hypochlorite (NaClO) per gram of cellulose. The CNFs exhibited different nanofibrillation levels, affecting the corresponding viscosity and 3D printability of the CNF gels (0.6 wt%). The scaffolds were manufactured by micro-extrusion and the nanomechanical properties were assessed with nanoindentation. Importantly, fibroblasts were grown on the scaffolds and the expression levels of the marker genes, which are relevant for wound healing and proliferation, were assessed in order to reveal the effect of the 3D-scaffold microenvironment of the cells.

Tissue engineering is a promising methodology to produce advanced therapy medicinal products (ATMPs). We have developed personalized tissue engineered veins (P-TEV) as an alternative to autologous or synthetic vascular grafts utilized in reconstructive vein surgery. Our hypothesis is that individualization through reconditioning of a decellularized allogenic graft with autologous blood will prime the tissue for efficient recellularization, protect the graft from thrombosis, and decrease the risk of rejection. In this study, P-TEVs were transplanted to vena cava in pig, and the analysis of three veins after six months, six veins after 12 months and one vein after 14 months showed that all P-TEVs were fully patent, and the tissue was well recellularized and revascularized. To confirm that the ATMP product had the expected characteristics one year after transplantation, gene expression profiling of cells from P-TEV and native vena cava were analyzed and compared by qPCR and sequencing. The qPCR and bioinformatics analysis confirmed that the cells from the P-TEV were highly similar to the native cells, and we therefore conclude that P-TEV is functional and safe in large animals and have high potential for use as a clinical transplant graft.

New bone repair materials are needed for treatment of trauma- and disease-related skeletal defects as they still represent a major challenge in clinical practice. Additionally, new strategies are required to combat orthopedic device-related infections (ODRI), given the rising incidence of total joint replacement and fracture fixation surgeries in increasingly elderly populations. Recently, the convergence of additive manufacturing (AM) and bone tissue engineering (BTE) has facilitated the development of bone healthcare to achieve personalized three-dimensional (3D) scaffolds. This study focused on the development of a 3D printable bone repair material, based on the biopolymers poly(lactic acid) (PLA) and chitosan. Two different types of PLA and chitosan differing in their molecular weight (MW) were explored. The novel feature of this research was the successful 3D printing using biocomposite filaments composed of PLA and 10 wt% chitosan, with clear chitosan entrapment within the PLA matrix confirmed by Scanning Electron Microscopy (SEM) images. Tensile testing of injection molded samples indicated an increase in stiffness, compared to pure PLA scaffolds, suggesting potential for improved load-bearing characteristics in bone scaffolds. However, the potential benefit of chitosan on the biocomposite stiffness could not be reproduced in compression testing of 3D printed cylinders. The antibacterial assays confirmed antibacterial activity of chitosan when dissolved in acetic acid. The study also verified the biodegradability of the scaffolds, with a process producing an acidic environment that could potentially be neutralized by chitosan. In conclusion, the study indicated the feasibility of the proposed PLA/chitosan biocomposite for 3D printing, demonstrating adequate mechanical strength, antibacterial properties and biodegradability, which could serve as a new material for bone repair.

Antimicrobial peptides (AMPs) are promising antibacterial agents in the fight against multidrug resistant pathogens. However, their application to skin infections is limited by the absence of a realizable topical delivery strategy. Herein, a hybrid hierarchical delivery system for topical delivery of AMPs is accomplished through the incorporation of AMPs into dendritic nanogels (DNGs) and their subsequent embedding into poloxamer gel. The high level of control over the crosslink density and the number of chosen functionalities makes DNGs ideal capsules with tunable loading capacity for DPK-060, a human kininogen-derived AMP. Once embedded into the poloxamer gel, DPK-060 encapsulated in DNGs displays a slower release rate compared to those entrapped directly in the gels. In vitro EpiDerm Skin Irritation Tests show good biocompatibility, while MIC and time-kill curves reveal the potency of the peptide toward Staphylococcus aureus. Anti-infection tests on ex vivo pig skin and in vivo mouse infection models demonstrate that formulations with 0.5% and 1% AMPs significantly inhibit the growth of S. aureus. Similar outcomes are observed for an in vivo mouse surgical site infection model. Importantly, when normalizing the bacteria inhibition to released/free DPK-060 at the wound site, all formulations display superior efficacy compared to DPK-060 in solution.

Breast cancer is the most common cancer among women, and even though treatments are available, efficiency varies with the patients. In vitro 2D models are commonly used to develop new treatments. However, 2D models overestimate drug efficiency, which increases the failure rate in later phase III clinical trials. New model systems that allow extensive and efficient drug screening are thus required. Three-dimensional printed hydrogels containing active components for cancer cell growth are interesting candidates for the preparation of next generation cancer cell models. Macromolecules, obtained from marine- and land-based resources, can form biopolymers (polysaccharides such as alginate, chitosan, hyaluronic acid, and cellulose) and bioactive components (structural proteins such as collagen, gelatin, and silk fibroin) in hydrogels with adequate physical properties in terms of porosity, rheology, and mechanical strength. Hence, in this study attention is given to biofabrication methods and to the modification with biological macromolecules to become bioactive and, thus, optimize 3D printed structures that better mimic the cancer cell microenvironment. Ink formulations combining polysaccharides for tuning the mechanical properties and bioactive polymers for controlling cell adhesion is key to optimizing the growth of the cancer cells.

To commercialize a biomedical product as a medical device, reproducibility of production and time-stability are important parameters. Studies of reproducibility are lacking in the literature. Additionally, chemical pre-treatments of wood fibres to produce highly fibrillated cellulose nanofibrils (CNF) seem to be demanding in terms of production efficiency, being a bottleneck for industrial upscaling. In this study, we evaluated the effect of pH on the dewatering time and washing steps of 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO)-mediated oxidized wood fibres when applying 3.8 mmol NaClO/g cellulose. The results indicate that the method does not affect the carboxylation of the nanocelluloses, and levels of approximately 1390 μmol/g were obtained with good reproducibility. The washing time of a Low-pH sample was reduced to 1/5 of the time required for washing a Control sample. Additionally, the stability of the CNF samples was assessed over 10 months and changes were quantified, the most pronounced were the increase of potential residual fibre aggregates, reduction of viscosity and increase of carboxylic acid content. The cytotoxicity and skin irritation potential were not affected by the detected differences between the Control and Low-pH samples. Importantly, the antibacterial effect of the carboxylated CNFs against S. aureus and P. aeruginosa was confirmed.

Purpose:It is known that contact of nucleus pulposus with the dorsal root ganglion may induce changes in nerve conduction and pain behavior. It has also been suggested that the behavioristic changes are caused by changes in voltage-gated ion channels, which in turn have been upregulated by TNF. Such upregulations have previously been shown for NaV 1.8 and NaV 1.9. In this investigation, we expanded the number of studied ion channels after the application of nucleus pulposus or TNF. Methods:Following removal of the left L4-5 fact joint, a disc puncture was performed and the dorsal root ganglion was exposed to nucleus pulposus (n = 5) and TNF (n = 5). Operated rats without disc puncture served as sham (n = 5) and 5 non-operated (naïve) rats were included. After 24 h, the DRGs were harvested and analyzed by quantitative PCR on validated pre-spotted primer plates displaying genes for 90 voltage-gated ion channels. Results:It was evident that the changes in operated animals were separate from the naïve rats. It was also apparent that gene expression changes in rats with nucleus pulposus or TNF application showed similar trends and were also separated from sham-operated animals. Conclusion:The application of nucleus pulposus and TNF onto the DRG in rats induces comparable changes in gene expression of several ion channels. Since the changes induced by TNF and NP are similar, one might also suspect that TNF mediates the NP-induced changes. However, such a mechanism needs further investigation.

Abstract Objectives Patients with urethral stricture due to any type of trauma, hypospadias or gender dysphoria suffer immensely from impaired capacity to urinate and are in need of a new functional urethra. Tissue engineering with decellularization of a donated organ recellularized with cells from the recipient patient has emerged as a promising alternative of advanced therapy medicinal products. The aim of this pilot study was to develop an ovine model of urethral transplantation and to produce an individualized urethra graft to show proof of function in vivo. Methods Donated urethras from ram abattoir waste were decellularized and further recellularized with autologous buccal mucosa epithelial cells excised from the recipient ram and expanded in vitro. The individualized urethral grafts were implanted by reconstructive surgery in rams replacing 2.5 ± 0.5 cm of the native penile urethra. Results After surgery optimization, three ram had the tissue engineered urethra implanted for one month and two out of three showed a partially regenerated epithelium. Conclusions Further adjustments of the model are needed to achieve a satisfactory proof-of-concept; however, we interpret these findings as a proof of principle and a possible path to develop a functional tissue engineered urethral graft with de- and recellularization and regeneration in vivo after transplantation.

Patients with cardiovascular disease often need replacement or bypass of a diseased blood vessel. With disadvantages of both autologous blood vessels and synthetic grafts, tissue engineering is emerging as a promising alternative of advanced therapy medicinal products for individualized blood vessels. By reconditioning of a decellularized blood vessel with the recipient’s own peripheral blood, we have been able to prevent rejection without using immunosuppressants and prime grafts for efficient recellularization in vivo. Recently, decellularized veins reconditioned with autologous peripheral blood were shown to be safe and functional in a porcine in vivo study as a potential alternative for vein grafting. In this study, personalized tissue engineered arteries (P-TEA) were developed using the same methodology and evaluated for safety in a sheep in vivo model of carotid artery transplantation. Five personalized arteries were transplanted to carotid arteries and analyzed for safety and patency as well as with histology after four months in vivo. All grafts were fully patent without any occlusion or stenosis. The tissue was well cellularized with a continuous endothelial cell layer covering the luminal surface, revascularized adventitia with capillaries and no sign of rejection or infection. In summary, the results indicate that P-TEA is safe to use and has potential as clinical grafts.

Breast cancer is a heterogeneous disease in terms of cellular and structural composition, and besides acquired aggressive properties in the cancer cell population, the surrounding tumor microenvironment can affect disease progression and clinical behaviours. To specifically decode the clinical relevance of the cancer promoting effects of individual tumor microenvironments, we performed a comprehensive test of 110 breast cancer samples using a recently established in vivo-like 3D cell culture platform based on patient-derived scaffolds (PDSs). Cell-free PDSs were recellularized with three breast cancer cell lines and adaptation to the different patient-based microenvironments was monitored by quantitative PCR. Substantial variability in gene expression between individual PDS cultures from different patients was observed, as well as between different cell lines. Interestingly, specific gene expression changes in the PDS cultures were significantly linked to prognostic features and clinical information from the original cancer. This link was even more pronounced when ERα-status of cell lines and PDSs matched. The results support that PDSs cultures, including a cancer cell line of relevant origin, can monitor the activity of the tumor microenvironment and reveal unique information about the malignancy-inducing properties of the individual cancer niche and serve as a future complementary diagnostic tool for breast cancer.

Topical antibiotics are a key component in the management of mild to moderate skin and soft tissue infections. There are, however, concerns about the emerging bacterial resistance against topical antibacterial agents such as fusidic acid, due to the prolonged treatment period of its marketed dosage forms. Improving the efficacy of topical formulations could potentially shorten the treatment period and avoid the resistance growth. To provide a more effective drug delivery, a water-free lipid-based formulation system (AKVANO®) which can be applied by spraying, has been developed. In the current paper, different formulations containing sodium fusidate were evaluated for their in vitro skin permeability using artificial skin mimicking membranes and antibacterial properties using ex vivo and in vivo skin wound infection models. The novel formulations containing sodium fusidate showed a much higher skin permeation (up to 60% of nominal amount) than the commercially available Fucidin® cream (3%). These formulations also gave a significantly stronger antibacterial effect than Fucidin cream showing a clear dose-response relationship for the sodium fusidate content. A spray product based on the described formulation technology would therefore require a shorter treatment time and thereby lower the risk for the development of bacterial resistance. Spray administration of these formulations provides an even layer on the skin surface from which the solvent quickly evaporates and thereby facilitates a non-touch application where no rubbing is required.