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Like for many enteric bacteria, flagella are a crucial virulence factor for the foodborne pathogenCampylobacter jejuni, allowing the bacteria to move through the viscous mucus of the human intestine. Assembly of the complex flagellar machinery and filament requires hierarchical regulation via transcriptional control of each component. InC. jejuni, class I genes are transcribed from σ70-dependent promoters and class II/III genes with the help of the alternative sigma factors RpoN (σ54) and FliA (σ28). In contrast to transcriptional control, less is known about post-transcriptional regulation of flagellar biosynthesis cascades via small regulatory RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on the cascade that fine-tuneC. jejuniflagellar filament assembly and thereby impact motility. We demonstrate that the highly conservedCampylobactersRNA CJnc230 (FlmE, flagellar length andmotilityenhancer), encoded downstream of the flagellar hook structural protein FlgE, is dependent on RpoN and that RNase III processes CJnc230 from theflgEmRNA, while RNase Y and PNPase mature its 3’ end. We identify mRNAs encoding a regulator of flagella-flagella interactions and the anti-σ28factor FlgM as direct targets of CJnc230 repression. Overexpression of CJnc230 de-represses FliA activity and upregulates class III flagellar genes, such as the major flagellinflaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR, flagellar length andmotilityrepressor) reduces flagellar length and motility. Overall, our study demonstrates sRNA-mediated post-transcriptional regulation fine-tunesC. jejuniflagellar biosynthesis through balancing of the hierarchically expressed components.

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.

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.

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.

Multiple efforts have been made to develop small-diameter tissue engineered vascular grafts using a great variety of bioreactor systems at different steps of processing. Nevertheless, there is still an extensive need for a compact all-in-one system providing multiple and simultaneous processing. The aim of this project was to develop a new device to fulfill the major requirements of an ideal system that allows simultaneous seeding, conditioning, and perfusion. The newly developed system can be actuated in a common incubator and consists of six components: a rotating cylinder, a pump, a pulse generator, a control unit, a mixer, and a reservoir. Components that are in direct contact with cell media, cells, and/or tissue allow sterile processing. Proof-of-concept experiments were performed with polyurethane tubes and collagen tubes. The scaffolds were seeded with fibroblasts and endothelial cells that were isolated from human saphenous vein segments. Scanning electron microscopy and immunohistochemistry showed better seeding success of polyurethane scaffolds in comparison to collagen. Conditioning of polyurethane tubes with 100 dyn/cm2 resulted in cell detachments, whereas a moderate conditioning program with stepwise increase of shear stress from 10 to 40 dyn/cm2 induced a stable and confluent cell layer. The new bioreactor is a powerful tool for quick and easy testing of various scaffold materials for the development of tissue engineered vascular grafts. The combination of this bioreactor with native tissue allows testing of medical devices and medicinal substances under physiological conditions that is a good step towards reduction of animal testing. In the long run, the bioreactor could turn out to produce tissue engineered vascular grafts for human applications “at the bedside”.