TY - JOUR A1 - Schjeide, Brit-Maren A1 - Schenke, Maren A1 - Seeger, Bettina A1 - Püschel, Gerhard T1 - Validation of a novel double control quantitative copy number PCR method to quantify off-target transgene integration after CRISPR-induced DNA modification JF - Methods and protocols : M&Ps N2 - In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations. KW - CRISPR editing validation KW - copy number analyses KW - homology-directed repair KW - homologous recombination deficiency Y1 - 2022 U6 - https://doi.org/10.3390/mps5030043 SN - 2409-9279 VL - 5 IS - 3 SP - 1 EP - 14 PB - MDPI CY - Basel, Schweiz ER - TY - JOUR A1 - Ramm, Franziska A1 - Jack, Lena A1 - Kaser, Danny A1 - Schloßhauer, Jeffrey L. A1 - Zemella, Anne A1 - Kubick, Stefan T1 - Cell-Free Systems Enable the Production of AB(5) Toxins for Diagnostic Applications JF - Toxins N2 - Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB(5) toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB(5) toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB(5) toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA's active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin's mechanism of action. KW - cholera toxin KW - heat-labile enterotoxin KW - AB(5) toxins KW - eukaryotic KW - cell-free systems KW - orthogonal systems Y1 - 2022 U6 - https://doi.org/10.3390/toxins14040233 SN - 2072-6651 VL - 14 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Ghafarian, Fatemeh A1 - Wieland, Ralf A1 - Nendel, Claas T1 - Estimating the Evaporative Cooling Effect of Irrigation within and above Soybean Canopy JF - Water N2 - Vegetation with an adequate supply of water might contribute to cooling the land surface around it through the latent heat flux of transpiration. This study investigates the potential estimation of evaporative cooling at plot scale, using soybean as example. Some of the plants' physiological parameters were monitored and sampled at weekly intervals. A physics-based model was then applied to estimate the irrigation-induced cooling effect within and above the canopy during the middle and late season of the soybean growth period. We then examined the results of the temperature changes at a temporal resolution of ten minutes between every two irrigation rounds. During the middle and late season of growth, the cooling effects caused by evapotranspiration within and above the canopy were, on average, 4.4 K and 2.9 K, respectively. We used quality indicators such as R-squared (R-2) and mean absolute error (MAE) to evaluate the performance of the model simulation. The performance of the model in this study was better above the canopy (R-2 = 0.98, MAE = 0.3 K) than below (R-2 = 0.87, MAE = 0.9 K) due to the predefined thermodynamic condition used to estimate evaporative cooling. Moreover, the study revealed that canopy cooling contributes to mitigating heat stress conditions during the middle and late seasons of crop growth. KW - canopy cooling effects KW - shading cooling KW - canopy-air temperature KW - energy KW - balance KW - the Penman-Monteith equation Y1 - 2022 U6 - https://doi.org/10.3390/w14030319 SN - 2073-4441 VL - 14 IS - 3 PB - MDPI CY - Basel ER - TY - JOUR A1 - Wetzel, Anna-Jasmin A1 - Koch, Roland A1 - Preiser, Christine A1 - Müller, Regina A1 - Klemmt, Malte A1 - Ranisch, Robert A1 - Ehni, Hans-Jörg A1 - Wiesing, Urban A1 - Rieger, Monika A. A1 - Henking, Tanja A1 - Joos, Stefanie T1 - Ethical, legal, and social implications of symptom checker Apps in primary Health Care (CHECK.APP) BT - protocol for an interdisciplinary mixed methods study JF - JMIR Research Protocols N2 - Background: Symptom checker apps (SCAs) are accessible tools that provide early symptom assessment for users. The ethical, legal, and social implications of SCAs and their impact on the patient-physician relationship, the health care providers, and the health care system have sparsely been examined. This study protocol describes an approach to investigate the possible impacts and implications of SCAs on different levels of health care provision. It considers the perspectives of the users, nonusers, general practitioners (GPs), and health care experts. Objective: We aim to assess a comprehensive overview of the use of SCAs and address problematic issues, if any. The primary outcomes of this study are empirically informed multi-perspective recommendations for different stakeholders on the ethical, legal, and social implications of SCAs. Methods: Quantitative and qualitative methods will be used in several overlapping and interconnected study phases. In study phase 1, a comprehensive literature review will be conducted to assess the ethical, legal, social, and systemic impacts of SCAs. Study phase 2 comprises a survey that will be analyzed with a logistic regression. It aims to assess the user degree of SCAs in Germany as well as the predictors for SCA usage. Study phase 3 will investigate self-observational diaries and user interviews, which will be analyzed as integrated cases to assess user perspectives, usage pattern, and arising problems. Study phase 4 will comprise GP interviews to assess their experiences, perspectives, self-image, and concepts and will be analyzed with the basic procedure by Kruse. Moreover, interviews with health care experts will be conducted in study phase 3 and will be analyzed by using the reflexive thematical analysis approach of Braun and Clark. Results: Study phase 1 will be completed in November 2021. We expect the results of study phase 2 in December 2021 and February 2022. In study phase 3, interviews are currently being conducted. The final study endpoint will be in February 2023. Conclusions: The possible ethical, legal, social, and systemic impacts of a widespread use of SCAs that affect stakeholders and stakeholder groups on different levels of health care will be identified. The proposed methodological approach provides a multifaceted and diverse empirical basis for a broad discussion on these implications. KW - symptom checker apps KW - self-diagnosis KW - self-triage KW - digitalization in KW - primary care KW - general practitioners KW - symptom checker KW - app KW - mobile app; Y1 - 2022 U6 - https://doi.org/10.2196/34026 SN - 1929-0748 VL - 11 IS - 5 PB - JMIR Research Protocols CY - Toronto ER - TY - JOUR A1 - Hoan, Tran Viet A1 - Richter, Karl-Gerd A1 - Borsig, Nicolas A1 - Bauer, Jonas A1 - Ha, Nguyen Thi A1 - Norra, Stefan T1 - An improved groundwater model framework for aquifer structures of the quaternary-formed sediment body in the southernmost parts of the Mekong Delta, Vietnam JF - Hydrology : open access journal N2 - The Ca Mau peninsula (CMP) is a key economic region in southern Vietnam. In recent decades, the high demand for water has increased the exploitation of groundwater, thus lowering the groundwater level and leading to risks of degradation, depletion, and land subsidence, as well as salinity intrusion in the groundwater of the whole Mekong Delta region. By using a finite element groundwater model with boundary expansion to the sea, we updated the latest data on hydrogeological profiles, groundwater levels, and exploitation. The basic model setup covers seven aquifers and seven aquitards. It is determined that the inflow along the coastline to the mainland is 39% of the total inflow. The exploitation of the study area in 2019 was 567,364 m(3)/day. The most exploited aquifers are the upper-middle Pleistocene (qp(2-3)) and the middle Pliocene (n(2)(2)), accounting for 63.7% and 24.6%, respectively; the least exploited aquifers are the upper Pleistocene and the upper Miocene, accounting for 0.35% and 0.02%, respectively. In the deeper aquifers, qp(2-3) and n(2)(2), the change in storage is negative due to the high exploitation rate, leading to a decline in the reserves of these aquifers. These groundwater model results are the calculations of groundwater reserves from the coast to the mainland in the entire system of aquifers in the CMP. This makes groundwater decision managers, stakeholders, and others more efficient in sustainable water resources planning in the CMP and Mekong Delta (MKD). KW - groundwater modeling KW - hydrogeology KW - aquifers system KW - water balance; KW - validation of model KW - Ca Mau peninsula KW - Kien Giang KW - Soc Trang KW - Hau Giang KW - Bac Lieu Y1 - 2022 U6 - https://doi.org/10.3390/hydrology9040061 SN - 2306-5338 VL - 9 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Li, Jianghong A1 - Bünning, Mareike A1 - Kaiser, Till A1 - Hipp, Lena T1 - Who suffered most? BT - parental stress and mental health during the COVID-19 pandemic in Germany JF - Journal of family research N2 - Objective: This study examines gender and socioeconomic inequalities in parental psychological wellbeing (parenting stress and psychological distress) during the COVID-19 pandemic in Germany. Background: The dramatic shift of childcare and schooling responsibility from formal institutions to private households during the pandemic has put families under enormous stress and raised concerns about caregivers' health and wellbeing. Despite the overwhelming media attention to families’ wellbeing, to date limited research has examined parenting stress and parental psychological distress during the COVID-19 pandemic, particularly in Germany. Method: We analyzed four waves of panel data (N= 1,771) from an opt-in online survey, which was conducted between March 2020 and April 2021. Multivariable OLS regressions were used to estimate variations in the pandemic's effects on parenting stress and psychological distress by various demographic and socioeconomic characteristics. Results: Overall, levels of parenting stress and psychological distress increased during the pandemic. During the first and third wave of the COVID-19 pandemic, mothers, parents with children younger than 11 years, parents with two or more children, parents working from home as well as parents with financial insecurity experienced higher parenting stress than other sociodemographic groups. Moreover, women, respondents with lower incomes, single parents, and parents with younger children experienced higher levels of psychological distress than other groups. Conclusion: Gender and socioeconomic inequalities in parents' psychological wellbeing increased among the study participants during the pandemic. KW - COVID-19 KW - parenting stress KW - gender inequality KW - mental health KW - psychological distress KW - Germany KW - children Y1 - 2022 U6 - https://doi.org/10.20377/jfr-704 SN - 2699-2337 VL - 34 IS - 1 SP - 281 EP - 309 PB - University of Bamberg Press CY - Bamberg ER - TY - JOUR A1 - Haueis, Lisa A1 - Stech, Marlitt A1 - Kubick, Stefan T1 - A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies JF - Frontiers in Bioengineering and Biotechnology N2 - Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, similar to 15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation. KW - cell-free protein synthesis KW - In vitro transcription KW - translation KW - nanobody KW - VHH KW - camelid KW - CHO cell lysate Y1 - 2022 U6 - https://doi.org/10.3389/fbioe.2022.896763 SN - 2296-4185 VL - 10 PB - Frontiers Media CY - Lausanne ER - TY - JOUR A1 - Procyk, Roman A1 - Lovejoy, Shaun A1 - Hébert, Raphaёl T1 - The fractional energy balance equation for climate projections through 2100 JF - Earth system dynamics / European Geosciences Union N2 - We produce climate projections through the 21st century using the fractional energy balance equation (FEBE): a generalization of the standard energy balance equation (EBE). The FEBE can be derived from Budyko-Sellers models or phenomenologically through the application of the scaling symmetry to energy storage processes, easily implemented by changing the integer order of the storage (derivative) term in the EBE to a fractional value. The FEBE is defined by three parameters: a fundamental shape parameter, a timescale and an amplitude, corresponding to, respectively, the scaling exponent h, the relaxation time tau and the equilibrium climate sensitivity (ECS). Two additional parameters were needed for the forcing: an aerosol recalibration factor alpha to account for the large aerosol uncertainty and a volcanic intermittency correction exponent upsilon. A Bayesian framework based on historical temperatures and natural and anthropogenic forcing series was used for parameter estimation. Significantly, the error model was not ad hoc but rather predicted by the model itself: the internal variability response to white noise internal forcing. The 90 % credible interval (CI) of the exponent and relaxation time were h = [0.33, 0.44] (median = 0.38) and tau = [2.4, 7.0] (median = 4.7) years compared to the usual EBE h = 1, and literature values of tau typically in the range 2-8 years. Aerosol forcings were too strong, requiring a decrease by an average factor alpha = [0.2, 1.0] (median = 0.6); the volcanic intermittency correction exponent was upsilon = [0.15, 0.41] (median = 0.28) compared to standard values alpha = upsilon = 1. The overpowered aerosols support a revision of the global modern (2005) aerosol forcing 90 % CI to a narrower range [ -1.0, -0.2] W m(-2). The key parameter ECS in comparison to IPCC AR5 (and to the CMIP6 MME), the 90 % CI range is reduced from [1.5, 4.5] K ([2.0, 5.5] K) to [1.6, 2.4] K ([1.5, 2.2] K), with median value lowered from 3.0 K (3.7 K) to 2.0 K (1.8 K) Similarly we found for the transient climate response (TCR), the 90 % CI range shrinks from [1.0, 2.5] K ([1.2, 2.8] K) to [1.2, 1.8] K ([1.1, 1.6] K) and the median estimate decreases from 1.8 K (2.0 K) to 1.5 K (1.4 K). As often seen in other observational-based studies, the FEBE values for climate sensitivities are therefore somewhat lower but still consistent with those in IPCC AR5 and the CMIP6 MME.
Using these parameters, we made projections to 2100 using both the Representative Concentration Pathway (RCP) and Shared Socioeconomic Pathway (SSP) scenarios, and compared them to the corresponding CMIP5 and CMIP6 multi-model ensembles (MMEs). The FEBE historical reconstructions (1880-2020) closely follow observations, notably during the 1998-2014 slowdown ("hiatus"). We also reproduce the internal variability with the FEBE and statistically validate this against centennial-scale temperature observations. Overall, the FEBE projections were 10 %-15 % lower but due to their smaller uncertainties, their 90 % CIs lie completely within the GCM 90 % CIs. This agreement means that the FEBE validates the MME, and vice versa. Y1 - 2022 U6 - https://doi.org/10.5194/esd-13-81-2022 SN - 2190-4979 SN - 2190-4987 VL - 13 IS - 1 SP - 81 EP - 107 PB - Copernicus CY - Göttingen ER - TY - JOUR A1 - Mani, Deepak A1 - Kupsch, Andreas A1 - Müller, Bernd R. A1 - Bruno, Giovanni T1 - Diffraction Enhanced Imaging Analysis with Pseudo-Voigt Fit Function JF - Journal of imaging : open access journal N2 - Diffraction enhanced imaging (DEI) is an advanced digital radiographic imaging technique employing the refraction of X-rays to contrast internal interfaces. This study aims to qualitatively and quantitatively evaluate images acquired using this technique and to assess how different fitting functions to the typical rocking curves (RCs) influence the quality of the images. RCs are obtained for every image pixel. This allows the separate determination of the absorption and the refraction properties of the material in a position-sensitive manner. Comparison of various types of fitting functions reveals that the Pseudo-Voigt (PsdV) function is best suited to fit typical RCs. A robust algorithm was developed in the Python programming language, which reliably extracts the physically meaningful information from each pixel of the image. We demonstrate the potential of the algorithm with two specimens: a silicone gel specimen that has well-defined interfaces, and an additively manufactured polycarbonate specimen. KW - diffraction enhanced imaging KW - analyzer-based imaging KW - X-ray refraction; KW - non-destructive evaluation KW - Pseudo-Voigt fit function KW - Python Y1 - 2022 U6 - https://doi.org/10.3390/jimaging8080206 SN - 2313-433X VL - 8 IS - 8 PB - MDPI CY - Basel ER - TY - JOUR A1 - Bufe, Aaron A1 - Cook, Kristen L. A1 - Galy, Albert A1 - Wittmann, Hella A1 - Hovius, Niels T1 - The effect of lithology on the relationship between denudation rate and chemical weathering pathways BT - evidence from the eastern Tibetan Plateau JF - Earth surface dynamics N2 - The denudation of rocks in mountain belts exposes a range of fresh minerals to the surface of the Earth that are chemically weathered by acidic and oxygenated fluids. The impact of the resulting coupling between denudation and weathering rates fundamentally depends on the types of minerals that are weathering. Whereas silicate weathering sequesters CO2, the combination of sulfide oxidation and carbonate dissolution emits CO2 to the atmosphere. Here, we combine the concentrations of dissolved major elements in stream waters with Be-10 basin-wide denudation rates from 35 small catchments in eastern Tibet to elucidate the importance of lithology in modulating the relationships between denudation rate, chemical weathering pathways, and CO2 consumption or release. Our catchments span 3 orders of magnitude in denudation rate in low-grade flysch, high-grade metapelites, and granitoid rocks. For each stream, we estimate the concentrations of solutes sourced from silicate weathering, carbonate dissolution, and sulfide oxidation using a mixing model. We find that for all lithologies, cation concentrations from silicate weathering are largely independent of denudation rate, but solute concentrations from carbonates and, where present, sulfides increase with increasing denudation rate. With increasing denudation rates, weathering may therefore shift from consuming to releasing CO2 in both (meta)sedimentary and granitoid lithologies. For a given denudation rate, we report dissolved solid concentrations and inferred weathering fluxes in catchments underlain by (meta)sedimentary rock that are 2-10 times higher compared to catchments containing granitoid lithologies, even though climatic and topographic parameters do not vary systematically between these catchments. Thus, varying proportions of exposed (meta)sedimentary and igneous rocks during orogenesis could lead to changes in the sequestration and release of CO2 that are independent of denudation rate. Y1 - 2022 U6 - https://doi.org/10.5194/esurf-10-513-2022 SN - 2196-6311 SN - 2196-632X VL - 10 IS - 3 SP - 513 EP - 530 PB - Copernicus CY - Göttingen ER -