Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni.

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.

Cyclic pressure induced decellularization of porcine descending aortas.

The demand for decellularized xenogeneic tissues used in reconstructive heart surgery has increased over the last decades. Complete decellularization of longer and tubular aortic sections suitable for clinical application has not been achieved so far. The present study aims at analyzing the effect of pressure application on decellularization efficacy of porcine aortas using a device specifically designed for this purpose. Fresh porcine descending aortas of 8 cm length were decellularized using detergents. To increase decellularization efficacy, detergent treatment was combined with pressure application and different treatment schemes. Quantification of penetration depth as well as histological staining, scanning electron microscopy, and tensile strength tests were used to evaluate tissue structure. In general, application of pressure to aortic tissue does neither increase the decellularization success nor the penetration depth of detergents. However, it is of importance from which side of the aorta the pressure is applied. Application of intermittent pressure from the adventitial side does significantly increase the decellularization degree at the intimal side (compared to the reference group), but had no influence on the penetration depth of SDC/SDS at both sides. Although the present setup does not significantly improve the decellularization success of aortas, it is interesting that the application of pressure from the adventitial side leads to improved decellularization of the intimal side. As no adverse effects on tissue structure nor on mechanical properties were observed, optimization of the present protocol may potentially lead to complete decellularization of larger aortic segments.

RNA recording in single bacterial cells using reprogrammed tracrRNAs.

Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.

Customized 3D printed bioreactors for decellularization-High efficiency and quality on a budget.

Decellularization (DC) of biomaterials with bioreactors is widely used to produce scaffolds for tissue engineering. This study uses 3D printing to develop efficient but low-cost DC bioreactors. Two bioreactors were developed to decellularize pericardial patches and vascular grafts. Flow profiles and pressure distribution inside the bioreactors were optimized by steady-state computational fluid dynamics (CFD) analysis. Printing materials were evaluated by cytotoxicity assessment. Following evaluation, all parts of the bioreactors were 3D printed in a commercial fused deposition modeling printer. Samples of bovine pericardia and porcine aortae were decellularized using established protocols. An immersion and agitation setup was used as a control. With histological assessment, DNA quantification and biomechanical testing treatment effects were evaluated. CFD analysis of the pericardial bioreactor revealed even flow and pressure distribution in between all pericardia. The CFD analysis of the vessel bioreactor showed increased intraluminal flow rate and pressure compared to the vessel's outside. Cytotoxicity assessment of the used printing material revealed no adverse effect on the tissue. Complete DC was achieved for all samples using the 3D printed bioreactors while DAPI staining revealed residual cells in aortic vessels of the control group. Histological analysis showed no structural changes in the decellularized samples. Additionally, biomechanical properties exhibited no significant change compared to native samples. This study presents a novel approach to manufacturing highly efficient and low budget 3D printed bioreactors for the DC of biomaterials. When compared to standard protocols, the bioreactors offer a cost effective, fast, and reproducible approach, which vastly improves the DC results.

Mapping of bovine pericardium to enable a standardized acquirement of material for medical implants.

OBJECTIVES: Bovine pericardium - native, fixed as well as decellularized - is one of the most common implant materials in modern cardiovascular surgery. Although used in everyday procedures, there are no recommendations in regard to which part of the pericardium to prefer. It was the aim of this study, to identify areas of the pericardium with consistent properties and high durability. METHODS: Fresh bovine pericardia were collected from a local slaughterhouse. The native pericardia were analyzed at 140 spots in regard to thickness and fiber orientation. Based on these results, five promising areas were selected for further evaluation. The pericardia were decellularized with detergents (0.5% sodiumdesoxycholate/0.5% sodiumdodecylsulfate) and subsequently incubated in DNAse. The two investigation groups native und DC consisted of 20 samples each. The efficiency of the decellularization was evaluated by DNA quantification, as well as DAPI and H&E staining. Biomechanical properties were determined using uniaxial tensile tests. To evaluate the microstructure, scanning electron microscopy, Picrosirius Red- and Movat's Pentachrome staining were utilized. To assess the long-term durability, patches were tested in a high-cycle system for a duration equaling the stress of three months in-vivo. Commercially available, fixed pericardium patches served as control group. RESULTS: Only a limited part of the pericardium showed a homogenous and usable thickness. The decellularization removed all cell nuclei, proven by negative DAPI and H&E staining, and also significantly reduced the DNA amount by 84%. The mechanical testing revealed that two investigated areas had an inconsistent tensile strength. Microscopical observations showed that the integrity of the extracellular matrix did not suffer by the decellularization procedure. During the long-term testing, most of the pericardia slowly lost tautness, though none of them got measurably damaged. Especially one area showed no decline of tensile strength after durability testing at all. Decellularized patches and fixed patches achieved comparable results in mechanical testing and microscopical evaluation after the durability testing. CONCLUSION: We could clearly document significant, location-based differences within single pericardia. Only one area showed consistent properties and a high durability. We highly recommend taking this into account for future implant material selections.

3-dimensional printing for the diagnosis of left ventricular outflow tract obstruction after mitral valve replacement.

The objective of this study was to evaluate the use of the generation of 3D models and 3D prints of complex cases for physicians at the example of an intricate left ventricular outflow tract obstruction (LVOTO). LVOTO is a known complication of mitral valve surgery. A 38-year-old female patient with increasing dyspnoea after mitral valve replacement was referred to our centre. Echocardiography showed a strut of the bioprosthetic heart valve protruding into the left ventricular outflow tract. However, the diagnosis of a LVOTO was difficult based on echocardiography alone. Therefore, we fabricated a physical model of the left ventricular outflow tract, the mitral valve, the aortic valve and the left ventricle. With this physical model in hand, we were able to visualize the LVOTO and to discuss potential therapeutic options. Moreover, we were able to plan the subsequent redo surgery in detail using the model. This case shows the benefit of 3D printing technologies for surgeons and patients, not only for analysis, but also during the decision-making and pre-operative planning process.

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians' wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions. To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models' suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians' training as well as for pre-interventional therapy planning. Printed models showed a high printing resolution (~30 µm) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible. Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.

Generation of microbubbles in extracorporeal life support and assessment of new elimination strategies.

Occurrence of microbubbles (MB) is a major problem during venoarterial extracorporeal life support (ECLS) with partially severe clinical complications. The aim of this study was to establish an in vitro ECLS setup for the generation and detection of MB. Furthermore, we assessed different MB elimination strategies. Patient and ECLS circuit were simulated using reservoirs, a centrifugal pump, a membrane oxygenator, and an occluder (modified roller pump). The system was primed with a glycerin solution of 44%. Three different revolution speeds (2500, 3000, and 3400 rpm) were applied. For MB generation, the inflow line of the pump was either statically or dynamically (15 rpm) occluded. A bubble counter was used for MB detection. The effectiveness of the oxygenator and dynamic bubble traps (DBTs) was evaluated in regard to MB elimination capacities. MB generation was highly dependent on negative pressure at the inflow line. Increasing revolution speeds and restriction of the inflow led to increased MB activity. The significant difference between inflow and outflow MB volume identified the centrifugal pump as a main source. We could show that the oxygenator's ability to withhold larger MB is limited. The application of one or multiple DBTs leads to a significant reduction in MB count and overall gas volume. The application of DBT can significantly reduce the overall gas volume, especially at high flow rates. Moreover, large MB can effectively be broken down for faster absorption. In general, the incidence of MBs is significantly dependent on pump speed and restriction of the inflow. The centrifugal pump was identified as a major source of MB generation.

Induction of Articular Chondrogenesis by Chitosan/Hyaluronic-Acid-Based Biomimetic Matrices Using Human Adipose-Derived Stem Cells.

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically.

The cardiotomy reservoir - a preliminary evaluation of a new cell source for cardiovascular tissue engineering.

OBJECTIVES: Cell sources for cardiovascular tissue engineering (TE) are scant. However, the need for an ideal TE cardiovascular implant persists. We investigated the cardiotomy reservoir (CR) as a potential cell source that is more accessible and less ethically problematic. METHODS: CR (n = 10) were removed from the bypass system after surgery. Isolation was performed using different isolation methods: blood samples were taken from the cardiopulmonary bypass and centrifuged at low density. The venous filter screen was cut out and placed into petri dishes for cultivation. The spongelike filter was removed, washed and treated in the same way as the blood samples. After cultivation, cell lines of fibroblasts (FB) and endothelial cells (EC) were obtained for analysis. The cells were seeded on polyurethane patches and analyzed via scanning electron microscopy (SEM), Life/Dead assay and immunohistochemistry. RESULTS: No correlation between age, time of surgery and quality of cells was observed. The successful extraction of FB and was proven by positive staining results for TE-7, CD31 and vWF. Cell morphology, cytoskeleton staining and quantification of proliferation using WST-1 assay resembled the cells of the control group in all ways. The topography of a confluent and vital cell layer after cell seeding was displayed by SEM analysis, Life/Dead Assay and immunohistochemistry. The establishment of an extracellular matrix (ECM) was proven by positive staining for collagen IV, laminin, fibronectin and elastin. CONCLUSIONS: Viable FB and EC cell lines were extracted from the CR after surgery. Easy access and high availability make this cell source destined for widespread application in cardiovascular tissue engineering.

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv) of S9.6 showed high affinity (0.6 nM) for DNA-RNA and also a high affinity (2.7 nM) for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

A Pulsatile Bioreactor for Conditioning of Tissue-Engineered Cardiovascular Constructs under Endoscopic Visualization.

Heart valve disease (HVD) is a globally increasing problem and accounts for thousands of deaths yearly. Currently end-stage HVD can only be treated by total valve replacement, however with major drawbacks. To overcome the limitations of conventional substitutes, a new clinical approach based on cell colonization of artificially manufactured heart valves has been developed. Even though this attempt seems promising, a confluent and stable cell layer has not yet been achieved due to the high stresses present in this area of the human heart. This study describes a bioreactor with a new approach to cell conditioning of tissue engineered heart valves. The bioreactor provides a low pulsatile flow that grants the correct opening and closing of the valve without high shear stresses. The flow rate can be regulated allowing a steady and sensitive conditioning process. Furthermore, the correct functioning of the valve can be monitored by endoscope surveillance in real-time. The tubeless and modular design allows an accurate, simple and faultless assembly of the reactor in a laminar flow chamber. It can be concluded that the bioreactor provides a strong tool for dynamic pre-conditioning and monitoring of colonized heart valve prostheses physiologically exposed to shear stress.