From heterogeneous healthcare data to disease-specific biomarker networks: A hierarchical Bayesian network approach.

In this work, we introduce an entirely data-driven and automated approach to reveal disease-associated biomarker and risk factor networks from heterogeneous and high-dimensional healthcare data. Our workflow is based on Bayesian networks, which are a popular tool for analyzing the interplay of biomarkers. Usually, data require extensive manual preprocessing and dimension reduction to allow for effective learning of Bayesian networks. For heterogeneous data, this preprocessing is hard to automatize and typically requires domain-specific prior knowledge. We here combine Bayesian network learning with hierarchical variable clustering in order to detect groups of similar features and learn interactions between them entirely automated. We present an optimization algorithm for the adaptive refinement of such group Bayesian networks to account for a specific target variable, like a disease. The combination of Bayesian networks, clustering, and refinement yields low-dimensional but disease-specific interaction networks. These networks provide easily interpretable, yet accurate models of biomarker interdependencies. We test our method extensively on simulated data, as well as on data from the Study of Health in Pomerania (SHIP-TREND), and demonstrate its effectiveness using non-alcoholic fatty liver disease and hypertension as examples. We show that the group network models outperform available biomarker scores, while at the same time, they provide an easily interpretable interaction network.

Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection.

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process.

Discovery and Design of Family†VIII Carboxylesterases as Highly Efficient Acyltransferases.

Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".

Sequence-Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases.

Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.

Parameter-based patient-specific restoration of physiological knee morphology for optimized implant design and matching.

Total knee arthroplasty (TKA) patients may present with genetic deformities, such as trochlear dysplasia, or deformities related to osteoarthritis. This pathologic morphology should be corrected by TKA to compensate for related functional deficiencies. Hence, a reconstruction of an equivalent physiological knee morphology would be favorable for detailed preoperative planning and the patient-specific implant selection or design process. A parametric database of 673 knees, each described by 36 femoral parameter values, was used. Each knee was classified as pathological or physiological based on cut-off values from literature. A clinical and a mathematical classification approach were developed to distinguish between affected and unaffected parameters. Three different prediction methods were used for the restoration of physiological parameter values: regression, nearest neighbor search and artificial neural networks. Several variants of the respective prediction model were considered, such as different network architectures. Regarding all methods, the model variant chosen resulted in a prediction error below the parameters' standard deviation, while the regression yielded the lowest errors. Future analyses should consider other deformities, also of tibia and patella. Furthermore, the functional consequences of the parameter changes should be analyzed.

Correction: Analysis of epidemiological association patterns of serum thyrotropin by combining random forests and Bayesian networks.

[This corrects the article DOI: 10.1371/journal.pone.0271610.].

Short-Term Drop in Antibody Titer after the Third Dose of SARS-CoV-2 BNT162b2 Vaccine in Adults.

Little is known about the longevity of antibodies after a third dose of the mRNA-based SARS-CoV-2 vaccine BNT162b2 (BioNTech/Pfizer, Mainz, Germany). Therefore, serum antibody levels were evaluated after a third dose of BNT162b2 in healthy adult healthcare workers in Germany. These antibody levels dropped significantly within a short period of 11 weeks from 4155.59 ± 2373.65 BAU/mL to 2389.10 ± 1433.90 BAU/mL, p-value < 0.001 but remained higher than after the second dose (611.92 ± 450.31 BAU/mL). To evaluate the quality of the humoral immune response, we additionally measured neutralizing antibodies, which also showed a small but significant decrease within this short period. These data underline the positive effect of a third dose of BNT162b2 concerning antibody re-induction but also shows a drop of Anti-SARS-CoV-2-IgG within a short span of time.

Persistence of Immune Response in Health Care Workers After Two Doses BNT162b2 in a Longitudinal Observational Study.

Background: The mRNA-based vaccine BNT162b2 of BioNTech/Pfizer has shown high efficacy against SARS-CoV-2 infection and a severe course of the COVID-19 disease. However, little is known about the long-term durability of the induced immune response resulting from the vaccination. Methods: In a longitudinal observational study in employees at a German hospital we compared the humoral and cellular immune response in 184 participants after two doses of the BioNTech/Pfizer vaccine (BNT162b2) with a mid-term follow-up after 9 months. Anti-SARS-CoV-2 binding antibodies were determined using both a quantitative and a semi-quantitative assay. For a qualitative assessment of the humoral immune response, we additionally measured neutralizing antibodies. Cellular immune response was evaluated by measuring Interferon-gamma release after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay. Results: In the first analysis, a 100% humoral response rate was described after two doses of BNT162b2 vaccine with a mean antibody ratio of 8.01 ± 1.00. 9 months after the second dose of BNT162b2, serological testing showed a significant decreased mean antibody ratio of 3.84 ± 1.69 (p < 0.001). Neutralizing antibodies were still detectable in 96% of all participants, showing an average binding inhibition value of 68.20% ± 18.87%. Older age (p < 0.001) and obesity (p = 0.01) had a negative effect on the antibody persistence. SARS-CoV-2 specific cellular immune response was proven in 75% of individuals (mean Interferon-gamma release: 579.68 mlU/ml ± 705.56 mlU/ml). Conclusion: Our data shows a declining immune response 9 months after the second dose of BNT162b2, supporting the potentially beneficial effect of booster vaccinations, the negative effect of obesity and age stresses the need of booster doses especially in these groups.

Cellular and Humoral Immune Response to a Third Dose of BNT162b2 COVID-19 Vaccine - A Prospective Observational Study.

Background: Since the introduction of various vaccines against SARS-CoV-2 at the end of 2020, infection rates have continued to climb worldwide. This led to the establishment of a third dose vaccination in several countries, known as a booster. To date, there has been little real-world data about the immunological effect of this strategy. Methods: We compared the humoral- and cellular immune response before and after the third dose of BioNTech/Pfizer vaccine BNT162b2, following different prime-boost regimen in a prospective observational study. Humoral immunity was assessed by determining anti-SARS-CoV-2 binding antibodies using a standardized quantitative assay. In addition, neutralizing antibodies were measured using a commercial surrogate ELISA-assay. Interferon-gamma release was measured after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay to evaluate the cellular immune response. Results: We included 243 health-care workers who provided blood samples and questionnaires pre- and post- third vaccination. The median antibody level increased significantly after the third vaccination dose to 2663.1 BAU/ml vs. 101.4 BAU/ml (p < 0.001) before administration of the booster dose. This was also detected for neutralizing antibodies with a binding inhibition of 99.68% ± 0.36% vs. 69.06% ± 19.88% after the second dose (p < 0.001). 96.3% of the participants showed a detectable T-cell-response after the booster dose with a mean interferon-gamma level of 2207.07 mIU/ml ± 1905 mIU/ml. Conclusion: This study detected a BMI-dependent antibody increase after the third dose of BNT162b2 following different vaccination protocols. All participants showed a significant increase in their immune response. This, in combination with the low rate of post-vaccination-symptoms underlines the potential beneficial effect of a BNT162b2-booster dose.

Analysis of epidemiological association patterns of serum thyrotropin by combining random forests and Bayesian networks.

BACKGROUND: Approaching epidemiological data with flexible machine learning algorithms is of great value for understanding disease-specific association patterns. However, it can be difficult to correctly extract and understand those patterns due to the lack of model interpretability. METHOD: We here propose a machine learning workflow that combines random forests with Bayesian network surrogate models to allow for a deeper level of interpretation of complex association patterns. We first evaluate the proposed workflow on synthetic data. We then apply it to data from the large population-based Study of Health in Pomerania (SHIP). Based on this combination, we discover and interpret broad patterns of individual serum TSH concentrations, an important marker of thyroid functionality. RESULTS: Evaluations using simulated data show that feature associations can be correctly recovered by combining random forests and Bayesian networks. The presented model achieves predictive accuracy that is similar to state-of-the-art models (root mean square error of 0.66, mean absolute error of 0.55, coefficient of determination of R2 = 0.15). We identify 62 relevant features from the final random forest model, ranging from general health variables over dietary and genetic factors to physiological, hematological and hemostasis parameters. The Bayesian network model is used to put these features into context and make the black-box random forest model more understandable. CONCLUSION: We demonstrate that the combination of random forest and Bayesian network analysis is helpful to reveal and interpret broad association patterns of individual TSH concentrations. The discovered patterns are in line with state-of-the-art literature. They may be useful for future thyroid research and improved dosing of therapeutics.

SARS-CoV-2-antibody response in health care workers after vaccination or natural infection in a longitudinal observational study.

BACKGROUND: Following a year of development, several vaccines have been approved to contain the global COVID-19 pandemic. Real world comparative data on immune response following vaccination or natural infection are rare. METHODS: We conducted a longitudinal observational study in employees at a secondary care hospital affected by the COVID-19 pandemic. Comparisons were made about the presence of anti-SARS-CoV-2 immunglobulin G (IgG) antibody ratio after natural infection, or vaccination with one or two doses of BioNTech/Pfizer (BNT162b2), or one dose of AstraZenca (Vaxzevria) vaccine. RESULTS: We found a 100% humoral response rate in participants after 2 doses of BNT162b2 vaccine. The antibody ratio in participants with one dose BNT162b2 and Vaxzevria did not differ significantly to those with previous PCR-confirmed infection, whereas this was significantly lower in comparison to two doses of BioNTech/Pfizer. We could not identify a correlation with previous comorbidities, obesity or age within this study. Smoking showed a negative effect on the antibody response (p = 0.006) CONCLUSION: Our data provide an overview about humoral immune response after natural SARS-CoV-2 infection or following vaccination, and supports the usage of booster vaccinations, especially in patients after a natural SARS-CoV-2 infection.

Glioma with Leptomeningeal Spread Mimics Chronic Meningoencephalitis in a Young Adult.

The most malignant type of intrinsic brain tumor is glioblastoma (WHO grade IV). Primary leptomeningeal spread is rare and leads to a variety of differential considerations, as there is no typical clinical or imaging pattern. Here we present a rare and uncommon case of a primary leptomeningeal glioblastoma in combination with a low-grade glioma in a 21-year-old male, initially presenting with only headache and lower back pain. The presented case illustrates the challenging differential considerations and the severe course of leptomeningeal glioblastomas.

Half-Year Longitudinal Seroprevalence of SARS-CoV-2-Antibodies and Rule Compliance in German Hospital Employees.

COVID-19, which is caused by SARS-CoV-2, is an occupational health risk, especially for healthcare employees due to their higher exposure and consequently higher risk of symptomatic and asymptomatic infections. This study was designed to determine the longitudinal seroprevalence of specific immunoglobulin-G (IgG) antibodies in employees in a hospital setting. All employees in a secondary care hospital, including healthcare and non-healthcare workers, were invited to participate in this single-center study. After an initial screening, a 6-month follow-up was carried out, which included serological examination for SARS-CoV-2 IgG antibodies and a questionnaire for self-reported symptoms, self-perception, and thoughts about local and national hygiene and pandemic plans. The seroprevalence of SARS-CoV-2 IgG antibodies was 0.74% among 406 hospital employees (0.75% in healthcare workers, 0.72% in non-healthcare workers), initially recruited in April 2020, in their follow-up blood specimens in October 2020. In this study, 30.54% of the participants reported using the official German coronavirus mobile application and the majority were content with the local and national rules in relation to coronavirus-related restrictions. At the 6-month follow-up, the 0.74% seroprevalence was below the reported seroprevalence of 1.35% in the general German population. The prevalence in healthcare workers in direct patient care compared with that in workers without direct patient contact did not differ significantly. Further follow-up to monitor the seroprevalence in the high-risk healthcare sector during the ongoing global pandemic is essential.

Prospective Sero-epidemiological Evaluation of SARS-CoV-2 among Health Care Workers in a German Secondary Care Hospital.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the pulmonary disease coronavirus disease 2019 (COVID-19, which has challenged health care facilities worldwide. The sustainability of health care systems is largely reliant on the health status of their health care workers (HCW). This study aimed to detect the SARS-CoV-2 virus and specific antibodies among HCWs in a German hospital as a model system for the potential spread of the pandemic. METHODS: Between March and June 2020, we used a combination of RT-PCR testing to detect SARS-CoV-2 RNA and an enzyme-linked immunosorbent assay to detect the presence of anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies among HCWs in a German hospital based on repetitive oropharyngeal swabs (OPSs) and blood samples. RESULTS: In total, 871/1081 employees participated in this prospective longitudinal study. During the study period of 9 weeks, 5329 OPSs and 2136 blood samples were analyzed. SARS-CoV-2 RNA was detected in three participants (0.34%). Anti-SARS-CoV-2 IgG antibodies were detected in 38 (4.36%) participants. CONCLUSION: Our study determined a low prevalence of COVID-19 in HCW, which may reflect the effectiveness of hygiene protocols. However, it could also indicate a low prevalence of SARS CoV-2 in hospital employees. Our study protocol may serve as an instructive example for future pandemic containment protocols in hospitals.

Interaction of Staphylococcus aureus and Host Cells upon Infection of Bronchial Epithelium during Different Stages of Regeneration.

The primary barrier that protects our lungs against infection by pathogens is a tightly sealed layer of epithelial cells. When the integrity of this barrier is disrupted as a consequence of chronic pulmonary diseases or viral insults, bacterial pathogens will gain access to underlying tissues. A major pathogen that can take advantage of such conditions is Staphylococcus aureus, thereby causing severe pneumonia. In this study, we investigated how S. aureus responds to different conditions of the human epithelium, especially nonpolarization and fibrogenesis during regeneration using an in vitro infection model. The infective process was monitored by quantification of the epithelial cell and bacterial populations, fluorescence microscopy, and mass spectrometry. The results uncover differences in bacterial internalization and population dynamics that correlate with the outcome of infection. Protein profiling reveals that, irrespective of the polarization state of the epithelial cells, the invading bacteria mount similar responses to adapt to the intracellular milieu. Remarkably, a bacterial adaptation that was associated with the regeneration state of the epithelial cells concerned the early upregulation of proteins controlled by the redox-responsive regulator Rex when bacteria were confronted with a polarized cell layer. This is indicative of the modulation of the bacterial cytoplasmic redox state to maintain homeostasis early during infection even before internalization. Our present observations provide a deeper insight into how S. aureus can take advantage of a breached epithelial barrier and show that infected epithelial cells have limited ability to respond adequately to staphylococcal insults.

Implantable loop recorders in patients with unexplained syncope: Clinical predictors of pacemaker implantation.

BACKGROUND: Implantable loop recorders (ILR) are a valuable tool for the investigation of unexplainedsyncopal episodes. The aim of this retrospective single center study was to identify predictive factors for pacemaker implantation in patients with unexplained syncope who underwent ILR insertion. METHODS: One hundred six patients were retrospectively analyzed (mean age 59.1 years; 47.2% male) with unexplained syncope and negative conventional testing who underwent ILR implantation. The pri- mary study endpoint was detection of symptomatic or asymptomatic bradycardia requiring pacemaker implantation. RESULTS: The average follow-up period after ILR implantation was 20 ± 15 months. Pacemaker im- plantation according to current guidelines was necessary in 22 (20.8%) patients, mean duration until index bradycardia was 81 ± 88 (2-350) days. Ten (45.5%) patients received a pacemaker due to sinus arrest, 7 (31.8%) patients due to third-degree atrioventricular block, 2 (9.1%) patients due to second- degree atrioventricular block and 1 (4.5%) patient due to atrial fibrillation with a slow ventricular rate. Three factors remained significant in multivariate analysis: obesity, which defined by a body mass index above 30 kg/m2 (OR: 7.39, p = 0.014), a right bundle branch block (OR: 9.40, p = 0.023) and chronic renal failure as defined by a glomerular filtration rate of less than 60 mL/min (OR: 6.42, p = 0.035). CONCLUSIONS: Bradycardia is a frequent finding in patients undergoing ILR implantation due to un- explained syncope. Obesity, right bundle branch block and chronic renal failure are independent clinical predictors of pacemaker implantation.

Comparison of Cell Arrays and Multi-Well Plates in Microscopy-Based Screening.

Multi-well plates and cell arrays enable microscopy-based screening assays in which many samples can be analysed in parallel. Each of the formats possesses its own strengths and weaknesses, but reference comparisons between these platforms and their application rationale is lacking. We aim to fill this gap by comparing two RNA interference (RNAi)-mediated fluorescence microscopy-based assays, namely epidermal growth factor (EGF) internalization and cell cycle progression, on both platforms. Quantitative analysis revealed that both platforms enabled the generation of data with the appearance of the expected phenotypes significantly distinct from the negative controls. The measurements of cell cycle progression were less variable in multi-well plates. The result can largely be attributed to higher cell numbers resulting in less data variability when dealing with the assay generating phenotypic cell subpopulations. The EGF internalization assay with a uniform phenotype over nearly the whole cell population performed better on cell arrays than in multi-well plates. The result was achieved by scoring five times less cells on cell arrays than in multi-well plates, indicating the efficiency of the cell array format. Our data indicate that the choice of the screening platform primarily depends on the type of the cellular assay to achieve a maximum data quality and screen efficiency.