Quality Assessment of Cardiovascular Cells and Tissues by Raman Microspectroscopy and Imaging

The increasing lifespan of the human population has been accompanied by a higher prevalence of cardiovascular diseases. It has been more than 50 years since the first heart valve was transplanted in a human patient and many new approaches in cardiovascular transplantation and tissue engineering (TE) have been evolving ever since. However, the availability of human donor tissues is limited. Ideal, vital, durable, nonimmunogenic heart valve or cardiovascular replacements are not yet commercially available. Thus, a better understanding of developmental and regulating mechanisms of cardiovascular tissues is essential to develop new implant materials. Moreover, cardiovascular tissue transplants or tissue-engineered grafts need to be monitored before transplantation. This thesis aimed to establish Raman microspectroscopy and Raman imaging as marker-independent, non-destructive technique for quality assessment of cardiovascular transplants and tissue-engineered products. Towards this aim, the influence of an ice-free cryopreservation technique (IFC) on tissue integrity and immunogenicity of heart valves was analyzed. The extracellular matrix (ECM) structures of standard cryopreserved (FC) and IFC allograft leaflets were compared to native leaflets after longterm implantation in sheep. Moreover, the mid-term immunogenic effects on IFC treated xenografts were assessed. Quantitative monitoring of interstitial cryoprotectant (CPA) concentrations was performed for quality control of cryopreserved heart valves. Furthermore, phenotype and tissue origin of human smooth muscle cells (SMCs) that are applied in cardiovascular TE, were analyzed. The ECM remodeling of SMC ring constructs under different culture conditions was monitored. In addition to Raman measurements, routine techniques such as immunocytochemistry, quantitative polymerase chain reaction and histological staining were performed. The results demonstrate the superiority of Raman microspectroscopy and Raman imaging as marker-independent, non-destructive and sensitive method, which is also time- and cost efficient when compared to routine techniques. Raman analysis combined with multivariate data analysis tools allowed for the determination and characterization of structural ECM changes in FC heart valves and real-time quantification of residual CPAs. These techniques enabled the identification and discrimination of single human SMCs based on their tissue origin and phenotype. Moreover, ECM remodeling in tissue-engineered SMC rings was non-invasively monitored. This work affirms the potential of Raman techniques for future applications in in situ quality assessment in cardiovascular research

Broad betacoronavirus neutralization by a stem helix–specific human antibody

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection

Extensive alterations of the whole-blood transcriptome are associated with body mass index: results of an mRNA profiling study involving two large population-based cohorts

Background: Obesity, defined as pathologically increased body mass index (BMI),is strongly related to an increased risk for numerous common cardiovascular and metabolic diseases. It is particularly associated with insulin resistance, hyperglycemia, and systemic oxidative stress and represents the most important risk factor for type 2 diabetes (T2D). However, the pathophysiological mechanisms underlying these associations are still not completely understood. Therefore, in order to identify potentially disease-relevant BMI-associated gene expression signatures, a transcriptome-wide association study (TWAS) on BMI was performed. Methods: Whole-blood mRNA levels determined by array-based transcriptional profiling were correlated with BMI in two large independent population-based cohort studies (KORA F4 and SHIP-TREND) comprising a total of 1977 individuals. Results: Extensive alterations of the whole-blood transcriptome were associated with BMI: More than 3500 transcripts exhibited significant positive or negative BMI-correlation. Three major whole-blood gene expression signatures associated with increased BMI were identified. The three signatures suggested: i) a ratio shift from mature erythrocytes towards reticulocytes, ii) decreased expression of several genes essentially involved in the transmission and amplification of the insulin signal, and iii) reduced expression of several key genes involved in the defence against reactive oxygen species (ROS). Conclusions: Whereas the first signature confirms published results, the other two provide possible mechanistic explanations for well-known epidemiological findings under conditions of increased BMI, namely attenuated insulin signaling and increased oxidative stress. The putatively causative BMI-dependent down-regulation of the expression of numerous genes on the mRNA level represents a novel finding. BMI-associated negative transcriptional regulation of insulin signaling and oxidative stress management provide new insights into the pathogenesis of metabolic syndrome and T2D

Continuous Feature-Based Tracking of the Inner Ear for Robot-Assisted Microsurgery

Robotic systems for surgery of the inner ear must enable highly precise movement in relation to the patient. To allow for a suitable collaboration between surgeon and robot, these systems should not interrupt the surgical workflow and integrate well in existing processes. As the surgical microscope is a standard tool, present in almost every microsurgical intervention and due to it being in close proximity to the situs, it is predestined to be extended by assistive robotic systems. For instance, a microscope-mounted laser for ablation. As both, patient and microscope are subject to movements during surgery, a well-integrated robotic system must be able to comply with these movements. To solve the problem of on-line registration of an assistance system to the situs, the standard of care often utilizes marker-based technologies, which require markers being rigidly attached to the patient. This not only requires time for preparation but also increases invasiveness of the procedure and the line of sight of the tracking system may not be obstructed. This work aims at utilizing the existing imaging system for detection of relative movements between the surgical microscope and the patient. The resulting data allows for maintaining registration. Hereby, no artificial markers or landmarks are considered but an approach for feature-based tracking with respect to the surgical environment in otology is presented. The images for tracking are obtained by a two-dimensional RGB stream of a surgical microscope. Due to the bony structure of the surgical site, the recorded cochleostomy scene moves nearly rigidly. The goal of the tracking algorithm is to estimate motion only from the given image stream. After preprocessing, features are detected in two subsequent images and their affine transformation is computed by a random sample consensus (RANSAC) algorithm. The proposed method can provide movement feedback with up to 93.2 μm precision without the need for any additional hardware in the operating room or attachment of fiducials to the situs. In long term tracking, an accumulative error occurs