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.

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.

[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.

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.

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.

Synthetic cannabinoid induced ocular self-injury.

Synthetic cannabinoids are a relatively new and increasingly popular recreational drug. While used for their hallucinogenic properties similar to natural cannabis, they have a greater and more serious side effect profile, including potentially severe neuropsychiatric toxicity. We report the cases of two patients with untreated schizophrenia who presented after ocular self-injury while intoxicated on K2. Both patients hallucinated that a bug was behind their eye, and in their attempts at removing the bug, damaged the periocular soft tissue. To our knowledge these are the first reports of ocular self-injury from synthetic cannabinoid intoxication.

An experimental study to test the efficacy of Mesenchymal Stem Cells in reducing corneal scarring in an ex-vivo organ culture model.

In this study, we evaluated the effect of placenta-derived Mesenchymal Stem Cells (MSCs) versus placebo in improving corneal transparency following experimental injury in an ex-vivo organ culture model of post-mortem human corneas. We also compared the influence of MSCs on the basic histopathology of the cornea and the immunohistochemistry markers of fibrotic corneal scarring. Mesenchymal Stem Cells extracted from the placenta were isolated and expanded in-vitro. Five pairs of post-mortem human corneas harvested for the corneal transplant of equal grade were included in the study. Corneas of the same pair were randomly assigned to either the case arm or the control arm. All corneas underwent a standardized superficial keratectomy, 4 mm in diameter. The case and control arm corneas received an intrastromal injection of MSCs and placebo respectively. The corneal button was maintained in an organ culture system for 28 days under the standard protocol. Laser light was passed through the corneas mounted on a self-styled modified artificial anterior chamber. Image analysis was used to quantify corneal transparency. Haematoxylin & Eosin staining and Immunohistochemistry was done for Alpha SMA (Smooth Muscle Actin). Laser scatter measurements were measured using Image Analysis (Image J Software). The difference in the mean of Full-Width Half Maximum (FWHM), Max intensity and Red pixel intensity between the cases and the controls was 101.5, 16.3 and 11.4 respectively which was found to be statistically significant (P < 0.05). Histopathology showed a disorganized Bowman's layer in the controls as compared to the cases. Alpha Smooth Muscle Actin at the injury site stained 3 + in all controls as compared to 1 + in the cases, showing a statistically significant difference (p = 0.005). Based on our findings, we consider that placenta-derived Mesenchymal Stem Cells can alter evolving corneal scarring into a more favourable outcome with better corneal transparency and lesser fibrotic corneal scarring.

Prevalence and properties of intragenic copy-number variation in Mendelian disease genes.

PURPOSE: We investigated the frequencies and characteristics of intragenic copy-number variants (CNVs) in a deep sampling of disease genes associated with monogenic disorders. METHODS: Subsets of 1507 genes were tested using next-generation sequencing to simultaneously detect sequence variants and CNVs in >143,000 individuals referred for genetic testing. We analyzed CNVs in gene panels for hereditary cancer syndromes and cardiovascular, neurological, or pediatric disorders. RESULTS: Our analysis identified 2844 intragenic CNVs in 384 clinically tested genes. CNVs were observed in 1.9% of the entire cohort but in a disproportionately high fraction (9.8%) of individuals with a clinically significant result. CNVs accounted for 4.7-35% of pathogenic variants, depending on clinical specialty. Distinct patterns existed among CNVs in terms of copy number, location, exons affected, clinical classification, and genes affected. Separately, analysis of de-identified data for 599 genes unrelated to the clinical phenotype yielded 4054 CNVs. Most of these CNVs were novel rare events, present as duplications, and enriched in genes associated with recessive disorders or lacking loss-of-function mutational mechanisms. CONCLUSION: Universal intragenic CNV analysis adds substantial clinical sensitivity to genetic testing. Clinically relevant CNVs have distinct properties that distinguish them from CNVs contributing to normal variation in human disease genes.

Cytomorphometric Neutrophil and Monocyte Markers May Strengthen the Diagnosis of Sepsis.

BACKGROUND:: The diagnosis of sepsis is challenging in the absence of a gold standard test. Recent studies have explored the role of neutrophil and monocyte volume, conductivity, and scatter (VCS), derived from automated hematology analyzers, in diagnosing sepsis. We assessed the diagnostic accuracy of VCS parameters in critically ill patients with sepsis. METHODOLOGY:: In this prospective study, VCS parameters, procalcitonin, and C-reactive protein (CRP) were assessed in patients with proven sepsis (cases) and 2 control groups (intensive care unit [ICU] patients without sepsis and healthy blood donors). The diagnostic property of each test was explored by calculating sensitivity, specificity, negative and positive predictive values, and area under the curve (AUC). RESULTS:: The study included 65 patients with sepsis, 58 nonseptic ICU controls, and 98 blood donors. Procalcitonin and CRP were not significantly different ( P > .06) between patients with sepsis and nonseptic patients. Mean (95% confidence interval [CI]) neutrophil volume (MNV) was significantly higher ( P < .001) in patients with sepsis (165.5; 95%CI 161.6-169.4) than in nonseptic (157.3; 95%CI 154.6-160.1) patients and donors (148.9; 95%CI 147.9-150). A similar pattern was seen with mean monocyte volume (MMoV). Neutrophil and monocyte conductivity and scatter parameters were variably associated. The AUC was highest for MMoV (0.74) and lowest for CRP (0.62). Among all parameters, MNV and MMoV had the highest specificity of 85% and 80%, respectively. CONCLUSION:: In critically ill patients with suspected sepsis, VCS parameters may help strengthen the diagnostic probability of sepsis. Future studies may explore the role of serial monitoring of VCS to track response to antimicrobial therapy.

A PROSPECTIVE, RANDOMIZED TRIAL COMPARING PLAIN GUT TO POLYGLACTIN 910 (VICRYL) SUTURES FOR SCLEROTOMY CLOSURE AFTER 23-GAUGE PARS PLANA VITRECTOMY.

PURPOSE: To report a prospective, randomized comparative study assessing clinical outcomes of plain gut versus polyglactin 910 (PG910) sutures for sclerotomy closure after 23-gauge pars plana vitrectomy. METHODS: A single-masked, randomized, prospective study was undertaken with 49 eyes of 49 patients undergoing 23-gauge pars plana vitrectomy randomized to sclerotomy closure with either plain gut suture, PG910 (Vicryl) suture or a combination of the two. Assessment was based on both a postoperative pain scale and a standardized assessment of scleral inflammation at each suture site. RESULTS: No wound leakage was noted postoperatively in any patient. Across all groups, scleral inflammation was significantly higher at the PG910 suture sites compared with the plain gut suture sites at both the 1-week (P = 0.04) and 1-month postoperative visits (P < 0.001). Patients with PG910 sutures reported greater pain at the 1-month postoperative visit than those with plain gut sutures (P = 0.018). CONCLUSION: This prospective study suggests improved tolerability and reduced inflammation using plain gut suture compared with an 8-0 PG910 suture to close 23-gauge sclerotomies.

Topology-Scaling Identification of Layered Solids and Stable Exfoliated 2D Materials.

The Materials Project crystal structure database has been searched for materials possessing layered motifs in their crystal structures using a topology-scaling algorithm. The algorithm identifies and measures the sizes of bonded atomic clusters in a structure's unit cell, and determines their scaling with cell size. The search yielded 826 stable layered materials that are considered as candidates for the formation of two-dimensional monolayers via exfoliation. Density-functional theory was used to calculate the exfoliation energy of each material and 680 monolayers emerge with exfoliation energies below those of already-existent two-dimensional materials. The crystal structures of these two-dimensional materials provide templates for future theoretical searches of stable two-dimensional materials. The optimized structures and other calculated data for all 826 monolayers are provided at our database (https://materialsweb.org).

Genotype and SNP calling from next-generation sequencing data.

Meaningful analysis of next-generation sequencing (NGS) data, which are produced extensively by genetics and genomics studies, relies crucially on the accurate calling of SNPs and genotypes. Recently developed statistical methods both improve and quantify the considerable uncertainty associated with genotype calling, and will especially benefit the growing number of studies using low- to medium-coverage data. We review these methods and provide a guide for their use in NGS studies.

Transcription elongation factor ELL2 drives Ig secretory-specific mRNA production and the unfolded protein response.

Differentiation of B cells into Ab-secreting cells induces changes in gene transcription, IgH RNA processing, the unfolded protein response (UPR), and cell architecture. The transcription elongation factor eleven nineteen lysine-rich leukemia gene (ELL2) stimulates the processing of the secreted form of the IgH mRNA from the H chain gene. Mice (mus musculus) with the ELL2 gene floxed in either exon 1 or exon 3 were constructed and crossed to CD19-driven cre/CD19(+). The B cell-specific ELL2 conditional knockouts (cKOs; ell2(loxp/loxp) CD19(cre/+)) exhibit curtailed humoral responses both in 4-hydroxy-3-nitrophenyl acetyl-Ficoll and in 4-hydroxy-3-nitrophenyl acetyl-keyhole limpet hemocyanin immunized animals; recall responses were also diminished. The number of immature and recirculating B cells in the bone marrow is increased in the cKOs, whereas plasma cells in spleen are reduced relative to control animals. There are fewer IgG1 Ab-producing cells in the bone marrow of cKOs. LPS ex vivo-stimulated B220(lo)CD138(+) cells from ELL2-deficient mouse spleens are 4-fold less abundant than from control splenic B cells; have a paucity of secreted IgH; and have distended, abnormal-appearing endoplasmic reticulum. IRE1α is efficiently phosphorylated, but the amounts of Ig κ, ATF6, BiP, Cyclin B2, OcaB (BOB1, Pou2af1), and XBP1 mRNAs, unspliced and spliced, are severely reduced in ELL2-deficient cells. ELL2 enhances the expression of BCMA (also known as Tnfrsf17), which is important for long-term survival. Transcription yields from the cyclin B2 and the canonical UPR promoter elements are upregulated by ELL2 cDNA. Thus, ELL2 is important for many aspects of Ab secretion, XBP1 expression, and the UPR.

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.

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.

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.

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.

Blockwise HMM computation for large-scale population genomic inference.

MOTIVATION: A promising class of methods for large-scale population genomic inference use the conditional sampling distribution (CSD), which approximates the probability of sampling an individual with a particular DNA sequence, given that a collection of sequences from the population has already been observed. The CSD has a wide range of applications, including imputing missing sequence data, estimating recombination rates, inferring human colonization history and identifying tracts of distinct ancestry in admixed populations. Most well-used CSDs are based on hidden Markov models (HMMs). Although computationally efficient in principle, methods resulting from the common implementation of the relevant HMM techniques remain intractable for large genomic datasets. RESULTS: To address this issue, a set of algorithmic improvements for performing the exact HMM computation is introduced here, by exploiting the particular structure of the CSD and typical characteristics of genomic data. It is empirically demonstrated that these improvements result in a speedup of several orders of magnitude for large datasets and that the speedup continues to increase with the number of sequences. The optimized algorithms can be adopted in methods for various applications, including the ones mentioned above and make previously impracticable analyses possible. AVAILABILITY: Software available upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: yss@eecs.berkeley.edu.