Analysis, Design, and Construction of Nucleic Acid Devices

Nucleic acids present great promise as building blocks for nanoscale devices. To achieve this potential, methods for the analysis and design of DNA and RNA need to be improved. In this thesis, traditional algorithms for analyzing nucleic acids at equilibrium are extended to handle a class of pseudoknots, with examples provided relevant to biologists and bioengineers. With these analytical tools in hand, nucleic acid sequences are designed to maximize the equilibrium probability of a desired fold. Upon analysis, it is concluded that both affinity and specificity are important when choosing a sequence; this conclusion holds for a wide range of target structures and is robust to random perturbations to the energy model. Applying the intuition gained from these studies, a process called hybridization chain reaction (HCR) is invented, and sequences are chosen that experimentally verify this phenomenon. In HCR, a small number of DNA or RNA molecules trigger a system wide configurational change, allowing the amplification and detection of specific, nucleic acid sequences. As an extension, HCR is combined with a pre-existing aptamer domain to successfully construct an ATP sensor, and the groundwork is laid for the future development of sensors for other small molecules. In addition, recent studies on multi-stranded algorithms and improvements to HCR are included in the appendices. Not only will these advancements increase our understanding of biological RNAs, but they will also provide valuable tools for the future development of nucleic acid nanotechnologies

Amino acid residues that are important for Hyal2 function as a receptor for jaagsiekte sheep retrovirus

BACKGROUND: Infection by jaagsiekte sheep retrovirus (JSRV) and by enzootic nasal tumor virus (ENTV) depends on cell-surface expression of the virus entry receptor, hyaluronidase 2 (Hyal2). Human Hyal2 binds the envelope (Env) proteins of these viruses and is functional as a receptor, but Hyal2 from mice does not bind Env nor does it mediate entry of either virus. Here we have explored the amino acid determinants that account for the difference in receptor function. RESULTS: Analysis of human-mouse Hyal2 chimeric proteins showed that amino acid differences responsible for the difference in Hyal2 receptor activity were localized to the central third of Hyal2. Human Hyal2 mutants containing single or double amino acid replacements with the respective mouse amino acids were generated across this region and were assayed for activity. None of the single or double mutation reduced the receptor activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. While the 3-dimensional structures of mammalian Hyal2 proteins are unknown, bee venom hyaluronidase shows significant amino acid similarity to human and mouse Hyal2 and its structure has been determined. Many mutations having the largest negative effects on human Hyal2 function mapped to a small region of the bee venom hyaluronidase close to but not overlapping the active site of the enzyme, suggesting that this site represents the binding site for Env. Analysis of synonymous and non-synonymous nucleotide substitutions in the coding sequences of multiple mammalian Hyal2 proteins shows that the proteins are undergoing strong selection for amino acid conservation. We found no evidence for positive selection of amino acid changes that might reflect evolution of mammalian hosts to resist JSRV or ENTV infection. CONCLUSION: These results show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert. In particular, no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure, which may represent the binding site for Env

Dynamics of Bacterial Root Endophytes of Malus domestica Plants Grown in Field Soils Affected by Apple Replant Disease

Apple replant disease (ARD) is a worldwide problem for tree nurseries and orchards leading to reduced plant growth and fruit quality. The etiology of this complex phenomenon is poorly understood, but shifts of the bulk soil and rhizosphere microbiome seem to play an important role. Since roots are colonized by microbes from the rhizosphere, studies of the endophytic microbiome in relation to ARD are meaningful. In this study, culture-independent and culture-dependent approaches were used in order to unravel the endophytic root microbiome of apple plants 3, 7, and 12 months after planting in ARD-affected soil and ARD-unaffected control soil at two different field sites. Next to a high diversity of Pseudomonas in roots from all soils, molecular barcoding approaches revealed an increase in relative abundance of endophytic Actinobacteria over time in plants grown in ARD and control plots. Furthermore, several amplicon sequence variants (ASVs) linked to Streptomyces, which had been shown in a previous greenhouse ARD biotest to be negatively correlated to shoot length and fresh mass, were also detected in roots from both field sites. Especially in roots of apple plants from control soil, these Streptomyces ASVs increased in their relative abundance over time. The isolation of 150 bacterial strains in the culture-dependent approach revealed a high diversity of members of the genus Pseudomonas, confirming the data of the molecular barcoding approach. However, only partial overlaps were found between the two approaches, underlining the importance of combining these methods in order to better understand this complex disease and develop possible countermeasures. Overall, this study suggests a key role of Streptomyces in the etiology of ARD in the field. Copyright © 2022 Mahnkopp-Dirks, Radl, Kublik, Gschwendtner, Schloter and Winkelmann

Immunization of preterm infants: current evidence and future strategies to individualized approaches

Preterm infants are at particularly high risk for infectious diseases. As this vulnerability extends beyond the neonatal period into childhood and adolescence, preterm infants beneft greatly from infection-preventive measures such as immunizations. However, there is an ongoing discussion about vaccine safety and efcacy due to preterm infants’ distinct immunological features. A signifcant proportion of infants remains un- or under-immunized when discharged from primary hospital stay. Educating health care professionals and parents, promoting maternal immunization and evaluating the potential of new vaccination tools are important means to reduce the overall burden from infectious diseases in preterm infants. In this narrative review, we summarize the current knowledge about vaccinations in premature infants. We discuss the specifcities of early life immunity and memory function, including the role of polyreactive B cells, restricted B cell receptor diversity and heterologous immunity mediated by a cross-reactive T cell repertoire. Recently, mechanistic studies indicated that tissue-resident memory (Trm) cell populations including T cells, B cells and macrophages are already established in the fetus. Their role in human early life immunity, however, is not yet understood. Tissue-resident memory T cells, for example, are diminished in airway tissues in neonates as compared to older children or adults. Hence, the ability to make specifc recall responses after secondary infectious stimulus is hampered, a phenomenon that is transcriptionally regulated by enhanced expression of T-bet. Furthermore, the microbiome establishment is a dominant factor to shape resident immunity at mucosal surfaces, but it is often disturbed in the context of preterm birth. The proposed function of Trm T cells to remember benign interactions with the microbiome might therefore be reduced which would contribute to an increased risk for sustained infammation. An improved understanding of Trm interactions may determine novel targets of vaccination, e.g., modulation of T-bet responses and facilitate more individualized approaches to protect preterm babies in the future

Modified Blalock Taussig shunt: a not-so-simple palliative procedure

OBJECTIVES Thirty-two consecutive isolated modified Blalock Taussig (BT) shunts performed in infancy since 2004 were reviewed and analysed to identify the risk factors for shunt intervention and mortality. METHODS Sternotomy was the only approach used. Median age and weight were 10.5 (range 1-74) days and 2.9 (1.9-4.4) kg, respectively. Shunt palliation was performed for biventricular hearts (Tetralogy of Fallot/double outlet right ventricle/transposition of great arteries_ventricular septal defect_pulmonary stenosis/pulmonary atresia_ventricular septal defect/others) in 21, and univentricular hearts in 11, patients. Hypoplastic left heart syndrome patients were excluded. Two procedures required cardiopulmonary bypass. Median shunt size was 3.5 (3-4) mm and median shunt size/kg body weight was 1.2 (0.9-1.7) mm/kg. Reduction in shunt size was necessary in 5 of 32 (16%) patients. RESULTS Three of 32 (9%) patients died after 3 (1-15) days due to cardiorespiratory decompensation. Lower body weight (P = 0.04) and bigger shunt size/kg of body weight (P = 0.004) were significant risk factors for mortality. Acute shunt thrombosis was observed in 3 of 32 (9%), none leading to death. Need for cardiac decongestive therapy was associated with univentricular hearts (P < 0.001), bigger shunt size (P = 0.054) and longer hospital stay (P = 0.005). Twenty-eight patients have undergone a successful shunt takedown at a median age of 5.5 (0.5-11.9) months, without late mortality. CONCLUSIONS Palliation with a modified BT shunt continues to be indicated despite increased thrust on primary corrective surgery. Though seemingly simple, it is associated with significant morbidity and mortality. Effective over-shunting and acute shunt thrombosis are the lingering problems of shunt therap

Outcome of acute respiratory distress syndrome in university and non- university hospitals in Germany

Background This study investigates differences in treatment and outcome of ventilated patients with acute respiratory distress syndrome (ARDS) between university and non-university hospitals in Germany. Methods This subanalysis of a prospective, observational cohort study was performed to identify independent risk factors for mortality by examining: baseline factors, ventilator settings (e.g., driving pressure), complications, and care settings—for example, case volume of ventilated patients, size/type of intensive care unit (ICU), and type of hospital (university/non-university hospital). To control for potentially confounding factors at ARDS onset and to verify differences in mortality, ARDS patients in university vs non-university hospitals were compared using additional multivariable analysis. Results Of the 7540 patients admitted to 95 ICUs from 18 university and 62 non-university hospitals in May 2004, 1028 received mechanical ventilation and 198 developed ARDS. Although the characteristics of ARDS patients were very similar, hospital mortality was considerably lower in university compared with non- university hospitals (39.3% vs 57.5%; p = 0.012). Treatment in non-university hospitals was independently associated with increased mortality (OR (95% CI): 2.89 (1.31–6.38); p = 0.008). This was confirmed by additional independent comparisons between the two patient groups when controlling for confounding factors at ARDS onset. Higher driving pressures (OR 1.10; 1 cmH2O increments) were also independently associated with higher mortality. Compared with non- university hospitals, higher positive end-expiratory pressure (PEEP) (mean ± SD: 11.7 ± 4.7 vs 9.7 ± 3.7 cmH2O; p = 0.005) and lower driving pressures (15.1 ± 4.4 vs 17.0 ± 5.0 cmH2O; p = 0.02) were applied during therapeutic ventilation in university hospitals, and ventilation lasted twice as long (median (IQR): 16 (9–29) vs 8 (3–16) days; p < 0.001). Conclusions Mortality risk of ARDS patients was considerably higher in non-university compared with university hospitals. Differences in ventilatory care between hospitals might explain this finding and may at least partially imply regionalization of care and the export of ventilatory strategies to non-university hospitals

Towards Composable GPU Programming: Programming GPUs with Eager Actions and Lazy Views

In this paper, we advocate a composable approach to programming systems with Graphics Processing Units (GPU): programs are developed as compositions of generic, reusable patterns. Current GPU programming approaches either rely on low-level, monolithic code without patterns (CUDA and OpenCL), which achieves high performance at the cost of cumbersome and error-prone programming, or they improve the programmability by using pattern-based abstractions (e.g., Thrust) but pay a performance penalty due to inefficient implementations of pattern composition. We develop an API for GPUs based programming on C++ with STL-style patterns and its compiler-based implementation. Our API gives the application developers the native C++ means (views and actions) to specify precisely which pattern compositions should be automatically fused during code generation into a single efficient GPU kernel, thereby ensuring a high target performance. We implement our approach by extending the range-v3 library which is currently being developed for the forthcoming C++ standards. The composable programming in our approach is done exclusively in the standard C++14, with STL algorithms used as patterns which we re-implemented in parallel for GPU. Our compiler implementation is based on the LLVM and Clang frameworks, and we use advanced multi-stage programming techniques for aggressive runtime optimizations. We experimentally evaluate our approach using a set of benchmark applications and a real-world case study from the area of image processing. Our codes achieve performance competitive with CUDA monolithic implementations, and we outperform pattern-based codes written using Nvidia’s Thrust

Прикладна механіка і основи конструювання: навчально-методичний посібник

Розроблено відповідно до навчальної програми і призначено для виконання розрахунково-графічної роботи з дисципліни «Прикладна механіка і основи конструювання» студентам напряму підготовки 6.050202 «Автоматизація та компютерно-ігрегровані технології» денної та заочної форм навчання. Посібник рекомендовано також для самостійної роботи студентів, оскільки він вміщує короткі теоретичні викладки основного матеріалу дисципліни «Прикладна механіка і основи конструювання», умови завдань, приклади їх розв’язування, необхідні довідкові дані