Precise correction of a spectrum of ß-thalassemia mutations in coding and non-coding regions by base editors.

ß-thalassemia/HbE results from mutations in the ß-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of ß-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of ß-thalassemia mutations that were integrated into the genome as fragments of HBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with ß-thalassemia/HbE. These ß-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional ß-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe ß0/ßE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.

Crafting Ethnographic Relationships During the COVID-19 Pandemic in Germany Using Voice-Based Technologies.

Drawing on a two-year ethnography of care practices during the COVID-19 pandemic in Germany, we discuss the affordances of voice-based technologies (smartphones, basic mobile phones, and landline telephones) in collecting ethnographic data and crafting relationships with participants. We illustrate how such technologies allowed us to move with participants, eased data collection through the social expectations around their use, and reoriented our attention to the multiple qualities of sound. Adapting research on the performativity of technology, we argue that voice-based technologies integrated us into participants' everyday lives while also maintaining physical distance in times of infectious sociality.

Hybrid Photoiniferter and Ring-Opening Polymerization Yields One-Pot Anisotropic Nanorods.

Polymerization-induced self-assembly (PISA) has emerged as a scalable one-pot technique to prepare block copolymer (BCP) nanoparticles. Recently, a PISA process, that results in poly(l-lactide)-b-poly(ethylene glycol) BCP nanoparticles coined ring-opening polymerization (ROP)-induced crystallization-driven self-assembly (ROPI-CDSA), was developed. The resulting nanorods demonstrate a strong propensity for aggregation, resulting in the formation of 2D sheets and 3D networks. This article reports the synthesis of poly(N,N-dimethyl acrylamide)-b-poly(l)-lactide BCP nanoparticles by ROPI-CDSA, utilizing a two-step, one-pot approach. A dual-functionalized photoiniferter is first used for controlled radical polymerization of the acrylamido-based monomer, and the resulting polymer serves as a macroinitiator for organocatalyzed ROP to form the solvophobic polyester block. The resulting nanorods are highly stable and display anisotropy at higher molecular weights (>12k Da) and concentrations (>20% solids) than the previous report. This development expands the chemical scope of ROPI-CDSA BCPs and provides readily accessible nanorods made with biocompatible materials.

Macrophakia: The characterization of a novel lens condition.

Purpose: To present a novel characterization of a lens condition termed macrophakia which includes large or proportionally large lenses with shallow anterior chamber dimensions that are statistically deviant of a normal population. Observations: We identified five eyes from three cases to have significantly large lens parameters and small anterior chamber depths. In all five eyes, the anterior chamber depth was less than 2 mm and the anterior chamber depth to lens thickness ratio was two standard deviations outside the normative range of lens and chamber measurements. These large lenses were all observed in the absence of typical pathology and other biometric abnormalities. Conclusions and importance: A novel lens characterization termed macrophakia is proposed to describe large or proportionately large lenses. Naming this condition is clinically relevant and will enhance cataract evaluations and patient education.

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

CryoEM reveals the complex self-assembly of a chemically driven disulfide hydrogel.

Inspired by the adaptability of biological materials, a variety of synthetic, chemically driven self-assembly processes have been developed that result in the transient formation of supramolecular structures. These structures form through two simultaneous reactions, forward and backward, which generate and consume a molecule that undergoes self-assembly. The dynamics of these assembly processes have been shown to differ from conventional thermodynamically stable molecular assemblies. However, the evolution of nanoscale morphologies in chemically driven self-assembly and how they compare to conventional assemblies has not been resolved. Here, we use a chemically driven redox system to separately carry out the forward and backward reactions. We analyze the forward and backward reactions both sequentially and synchronously with time-resolved cryogenic transmission electron microscopy (cryoEM). Quantitative image analysis shows that the synchronous process is more complex and heterogeneous than the sequential process. Our key finding is that a thermodynamically unstable stacked nanorod phase, briefly observed in the backward reaction, is sustained for ∼6 hours in the synchronous process. Kinetic Monte Carlo modeling show that the synchronous process is driven by multiple cycles of assembly and disassembly. The collective data suggest that chemically driven self-assembly can create sustained morphologies not seen in thermodynamically stable assemblies by kinetically stabilizing transient intermediates. This finding provides plausible design principles to develop and optimize supramolecular materials with novel properties.

A transparent synthetic peptide hydrogel as a haemostatic agent in athermal nerve sparing robot-assisted radical prostatectomy: an observational study.

Background: PuraStat® is a new synthetic haemostatic agent constituting peptides that self-assemble into sheets when exposed to ionic charges. The objective of this submission is to assess the perioperative, functional and user-reported outcomes of PuraStat® as an athermal topical haemostatic agent for use on the neurovascular bundle (NVB) in robot-assisted radical prostatectomy (RARP), and to inform further research into this developing field. Methods: Demographic and disease data for 29 consecutive patients undergoing RARP were recorded. PuraStat® was used as the primary haemostatic agent to the NVB, without thermal or suture haemostasis, unless necessary. Preoperative, 1-h postoperative and 24-h postoperative haemoglobin (Hb) were measured. Operative data including postoperative complications up to 30 days were noted. Urinary function, continence and erectile function (EF) were measured pre- and postoperatively with the International Prostate Symptom Score (IPSS), patient reporting of pad usage, and International Index of Erectile Function (IIEF)-5 respectively. A qualitative assessment of PuraStat® was made intraoperatively by the surgical assistant in the following categories: transparency, haemostatic efficacy, ready-to-use, handling, and overall satisfaction. Results: Twenty-nine males aged between 49 and 75 years underwent a nerve-sparing RARP under a single surgeon for clinically significant prostate cancer with PuraStat® used as the primary haemostatic agent at the NVB. One patient required an additional haemostatic suture. The median prostate volume was 36 mL. Mean blood loss was 363 mL. The mean Hb at 1 and 24 h postoperative was 135.2 and 125.1 mg/dL. Median Hb change from 1-24 h postoperative was 11 mg/dL. No transfusions were required, and there were no postoperative complications. Urinary function and continence were preserved. EF in our series was lower than published data. Conclusions: Our observational study suggests that PuraStat® is a safe haemostatic agent in RARP with similar perioperative bleeding outcomes, comparable long-term urinary outcomes and a high level of intraoperative user satisfaction. The effects on EF requires further investigation. PuraStat® appears to be a useful therapeutic tool for the urologist performing RARPs.

van der Waals Epitaxy, Superlubricity, and Polarization of the 2D Ferroelectric SnS.

Tin monosulfide (SnS) is a two-dimensional layered semiconductor that exhibits in-plane ferroelectric order at very small thicknesses and is of interest in highly scaled devices. Here we report the epitaxial growth of SnS on hexagonal boron nitride (hBN) using a pulsed metal-organic chemical vapor deposition process. Lattice matching is observed between the SnS(100) and hBN{11̅0} planes, with no evidence of strain. Atomic force microscopy reveals superlubricity along the commensurate direction of the SnS/hBN interface, and first-principles calculations suggest that friction is controlled by the edges of the SnS islands, rather than interface interactions. Differential phase contrast imaging detects remnant polarization in SnS islands with domains that are not dictated by step-edges in the SnS. The growth of ferroelectric SnS on high quality hBN substrates is a promising step toward electrically switchable ferroelectric semiconducting devices.

Social health: rethinking the concept through social practice theory and feminist care ethics.

The social sciences have long shown that health is not born of pure biology, empirically (re)centred the social and material causes of disease, and affirmed the subjective experiences of disease. Disputed both in popular and academic discourses, social health has variously attempted to stress the social aspects of health. Existing conceptions remain analytically limited as they are predominantly used as descriptors for populational health. This article theorises social health as an analytical lens for making sense of the relations, affects and events where health unfolds and comes into expression. Drawing on social practice theory, feminist care ethics and posthumanism this conceptual paper re-imagines how social health might be conceived as lived social practices anchored in care. Care within our framework acknowledges the unavoidable interdependency foundational to the existence of beings and stresses the 'know how' and embodied practices of care in the mundane in order to emphasise that care itself is absolutely integral to the maintenance of social health. The article argues that health needs to be understood as a verb intrinsically (re)made in and through social contexts and structures and comprised of meaningful, human-human and human-non-human interactions. Ultimately, in theorising social health through mundane care practices, we hope to open up research to making sense of how the doing of health unfolds inside often banal, patterned forms of social activity. Such taken-for-granted social practices exemplify the often overlooked lived realities that comprise our health. To understand health in its own right, we argue, these everyday practices need to be interrogated.

Why comply? Risk and efficacy perceptions drive compliance in mass marketing scams.

Using a mix-method design, we examined participants' willingness to respond to mass marketing scams (MMS). In Experiment 1, we examined the effect of age (young versus older) and letter style ("hot" versus "cold") on the intention to respond. The intention of responding was negatively associated with risk (p < .001) and having at least a high school education was positively associated with perception of benefits (b = .684, p < .001). In Experiment 2, we examined reward sensitivity on the intention to respond by manipulating reward amounts (low versus high) and the presence of an activation fee. The presence of an activation fee decreased intent to contact, but percentages remained high (25.75%). Analyses of qualitative data indicated that risk and benefit were both predicted by perceived self-efficacy. The results indicate that consumers' beliefs about their ability to control the outcomes of future interactions affected how they behaved when provided with MMS materials.

[Pandemic Management in the Context of Everyday Care and the need for Good Care: Results and Recommendations for Action from the Coronacare Workshops]. / Pandemie-Management unter Berücksichtigung des Pflegealltags und dem Bedürfnis nach guter Pflege. Ergebnisse und Handlungsempfehlungen aus den CoronaCare-Workshops.

The Covid-19 pandemic, officially declared in March 2020 by the WHO, poses major challenges to public, private, and occupational life. CoronaCare is an ethnographic research project that investigates the everyday life of people during the Covid-19 pandemic in Germany with a particular focus on social health. The aim of the project was to develop recommendations for pandemic preparedness planning focusing on expanding social health care. Through a series of workshops conducted between June and November 2021 with stakeholders from the fields of science, health and social administration drawn from both local and state levels, care institutions and social associations, the research team developed specific recommendations for pandemic preparedness and response on the basis of empirically substantiated vignettes demonstrating key tensions in caring practices. These tensions illustrate that pandemic management must be understood as a so-called wicked problem in which there are only relational rather than clear-cut, ultimate solutions. As such, the recommendations developed in the workshops point to the imperative to 1. recognize the irresolvable tension between measures to contain the pandemic in planning pandemic management and the human desire to care and be cared for; 2. understand and manage pandemics at the community level; 3. aim for close collaboration between actors at the local health, social, and family level; and 4. create spaces for ethical reflection on good care during a pandemic and develop context-specific strategies for action. For pandemic preparedness and management as a 'wicked problem', this means that measures should be disseminated as recommendations rather than regulations in order to have some leeway that enables care to be tailored to individual needs. They should be accompanied by stable guidance for action as well as regular training for staff. The workshop formats can be understood as an example of multidimensional knowledge transfer in a socially challenging situation.

Gaining Structural Control by Modification of Polymerization Rate in Ring-Opening Polymerization-Induced Crystallization-Driven Self-Assembly.

Polymerization-induced self-assembly (PISA) has become an important one pot method for the preparation of well-defined block copolymer nanoparticles. In PISA, morphology is typically controlled by changing molecular architecture and polymer concentration. However, several computational and experimental studies have suggested that changes in polymerization rate can lead to morphological differences. Here, we demonstrate that catalyst selection can be used to control morphology independent of polymer structure and concentration in ring-opening polymerization-induced crystallization-driven self-assembly (ROPI-CDSA). Slower rates of polymerization give rise to slower rates of self-assembly, resulting in denser lamellae and more 3D structures when compared to faster rates of polymerization. Our explanation for this is that the fast samples transiently exist in a nonequilibrium state as self-assembly starts at a higher solvophobic block length when compared to the slow polymerization. We expect that subsequent examples of rate variation in PISA will allow for greater control over morphological outcome.

Scalable detection of technically challenging variants through modified next-generation sequencing.

BACKGROUND: Some clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high- similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions). METHODS: We customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes. RESULTS: Our adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats. CONCLUSIONS: NGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.

MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state.

In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.

Ingrained: An Automated Framework for Fusing Atomic-Scale Image Simulations into Experiments.

To fully leverage the power of image simulation to corroborate and explain patterns and structures in atomic resolution microscopy, an initial correspondence between the simulation and experimental image must be established at the outset of further high accuracy simulations or calculations. Furthermore, if simulation is to be used in context of highly automated processes or high-throughput optimization, the process of finding this correspondence itself must be automated. In this work, "ingrained," an open-source automation framework which solves for this correspondence and fuses atomic resolution image simulations into the experimental images to which they correspond, is introduced. Herein, the overall "ingrained" workflow, focusing on its application to interface structure approximations, and the development of an experimentally rationalized forward model for scanning tunneling microscopy simulation are described.

Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

Visualizing Teixobactin Supramolecular Assemblies and Cell Wall Damage in B. Subtilis Using CryoEM.

The antibiotic teixobactin targets bacterial cell walls. Previous research has proposed that the active form of teixobactin is a nano-/micron-sized supramolecular assembly. Here, we use cryogenic transmission electron microscopy to show that at 1 mg/mL, teixobactin forms sheet-like assemblies that selectively act upon the cell wall. At 4 µg/mL, teixobactin is active, and aggregates are formed either transiently or sparingly at the cell surface.

CoronaCare study protocol: an ethnographic study of the risks to and potential for social health during the COVID-19 pandemic.

INTRODUCTION: German government regulations such as physical distancing and limited group numbers, designed to curb the spread of COVID-19, have had far-reaching consequences for the very foundations of social life. They have, to name only a few, transformed greetings and goodbyes, blurred private and public worlds, and complicated basic communication with mandatory mask wearing. The ethnographic study CoronaCare investigates how these sociopolitical measures affect social health, a form of health which unfolds through and across social relations. It explores how caring as a fundamental human activity and one integral to sustaining social health is impacted when in-person and person-to-person contacts are restricted and everyone is radically redefined as at risk from others and a risk to others. It explores care relationships, relationships involving the giving or receiving of care in everyday life, institutional settings such as an assisted living facility, and informal settings, such as a housing block. Inside of the pandemic, relationships are a pivotal site at which the negotiation of caring and risk is intensified and where the consequences for social health and social life more generally are pronounced. METHODS AND ANALYSIS: This ethnographic project aims to understand the tensions that arise in the lives of individuals and communities living under the sociopolitical regulations and to analyse the tacit forms of practice that individuals and communities develop to uphold social health. Fueled by citizen science, the ethnography uses a variety of methods namely telephone and video interviews with 60-70 research participants, the collection of ethnographic material including video and audio diaries, storyboards, first-person camera footage, photographs and a survey to enrich the sample description based on the Copenhagen Psychosocial Questionnaire. The analysis will draw on elements of grounded theory and through the aid of the qualitative software MAXQDA it will rigorously document and explain how the social regulations are (re)shaping our ability to be cared for and to care for one another. The survey data will be analysed through the use of the quantitative software programme R. ETHICS AND DISSEMINATION: The ethics committee of the Brandenburg Medical School Theodor Fontane has approved the project (E-01-20200605). The dissemination strategy includes publications in medical, sociological and research methods journals, as well as a stakeholder discussion with political and civil society leaders where the research team will present its recommendations for future pandemic preparedness.