Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch.

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2ß2) disorders, sickle cell disease, and ß-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to ß- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

Between Life and Death: How do Muslim Terminal Patients in Pakistan cope with Hepatitis C utilizing their Beliefs and Social Support?

Life-threatening events including terminal illness intensify the search for meaning and incite individuals to get closer to religion. Terminal patients can often find religious practices as helpful as medical therapy for bettering both physical and mental health. The present research aims to explain the interaction between religion, spirituality, and social support in coping with terminal illness among Muslim hepatitis C patients in Pakistan. A semi-structured open-ended interview guide was utilized to collect the data. Participants expressed that the deployment of religious and spiritual beliefs along with socio-emotional support during illness fostered medical therapy. Participants also revealed that belief in God provided them the strength to be steadfast during the terminal stage of the disease. Religious beliefs enabled terminal participants to accept death as an eventual reality and a normal part of their lives. Furthermore, participants put forward their longing for those kinds of religious practices that terminal diseases usually restrained them from receiving. The emotional support stemming from social relationships also improved resilience to cope with the terminal stage of illness. The study concludes that the interplay of religion, spirituality, and social support normalizes the fear of death, lessens pain, and improves resilience among Muslim hepatitis C patients in Pakistan.

Ba2-xBixCoRuO6 (0.0 ≤ x ≤ 0.6) Hexagonal Double-Perovskite-Type Oxides as Promising p-Type Thermoelectric Materials.

A new series of Ba2-xBixCoRuO6 (0.0 ≤ x ≤ 0.6) hexagonal double perovskite oxides have been synthesized by a solid-state reaction method by substituting Ba with Bi. The polycrystalline materials are structurally characterized by the laboratory X-ray diffraction, synchrotron X-ray, and neutron powder diffraction. The lattice parameters are found to increase with increasing Bi doping despite the smaller ionic radius of Bi3+ compared to Ba2+. The expansion is attributed to the reduction of Co/Ru-site cations. Scanning electron microscopy further shows that the grain size increases with the Bi content. All Ba2-xBixCoRuO6 (0.0 ≤ x ≤ 0.6) samples exhibit p-type behavior, and the electrical resistivity (ρ) is consistent with a small polaron hopping model. The Seebeck coefficient (S) and thermal conductivity (κ) are improved significantly with Bi doping. High values of the power factor (PF ∼ 6.64 × 10-4 W/m·K2) and figure of merit (zT ∼ 0.23) are obtained at 618 K for the x = 0.6 sample. These results show that Bi doping is an effective approach for enhancing the thermoelectric properties of hexagonal Ba2-xBixCoRuO6 perovskite oxides.

Investigation of the Crystal Structure and Ionic Pathways of the Hexagonal Perovskite Derivative Ba3-xVMoO8.5-x.

The hexagonal perovskite derivatives Ba3NbMoO8.5, Ba3NbWO8.5, and Ba3VWO8.5 have recently been reported to exhibit significant oxide ion conductivity. Here, we report the synthesis and crystal structure of the hexagonal perovskite derivative Ba3-xVMoO8.5-x. Rietveld refinement from neutron and X-ray diffraction data show that the cation vacancies are ordered on the M2 site, leading to a structure consisting of palmierite-like layers of M1Ox polyhedra separated by vacant octahedral layers. In contrast to other members of the Ba3M'M″O8.5 family, Ba3-xVMoO8.5-x is not stoichiometric and both barium and oxygen vacancies are present. Although synthesized in air at elevated temperatures, Ba3-xVMoO8.5-x is unstable at lower temperatures, as illustrated by the formation of BaCO3 and BaMoO4 by heat treatment in air at 400 °C. This precludes measurement of the electrical properties. However, bond-valence site energy (BVSE) calculations strongly suggest that oxide ion conductivity is present in Ba3-xVMoO8.5-x.

BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

Coping Through Religiosity, Spirituality and Social Support Among Muslim Chronic Hepatitis Patients.

Academics acknowledge religiosity, spirituality and social support as socio-behavioral factors that influence patients' ability to deal with chronic illness. This study has attempted to describe empirical reality of how these factors influence patients. The sample of this study was 500 chronically ill hepatitis patients and was selected through the multistage sampling techniques. Through structured interview schedule, data were collected during the period of September 2016 to March 2017 from five most populated cities of Punjab (Pakistan). Data were analyzed through descriptive (frequency and percentage) and inferential statistics (Cronbach's alpha, Pearson correlation, and structural equation modeling). The study suggests some recommendations and suggestions to policy makers regarding the significance of religiosity, spirituality and social support as coping strategies during chronic illness. The findings illustrate that social support has more association with coping than religiosity and spirituality of the patients.

Strict in vivo specificity of the Bcl11a erythroid enhancer.

BCL11A, a repressor of human fetal (γ-)globin expression, is required for immune and hematopoietic stem cell functions and brain development. Regulatory sequences within the gene, which are subject to genetic variation affecting fetal globin expression, display hallmarks of an erythroid enhancer in cell lines and transgenic mice. As such, this enhancer is a novel, attractive target for therapeutic gene editing. To explore the roles of such sequences in vivo, we generated mice in which the orthologous 10-kb intronic sequences were removed. Bcl11a enhancer-deleted mice, Bcl11a(Δenh), phenocopy the BCL11A-null state with respect to alterations of globin expression, yet are viable and exhibit no observable blood, brain, or other abnormalities. These preclinical findings provide strong in vivo support for genetic modification of the enhancer for therapy of hemoglobin disorders.

Absence of colossal magnetoresistance in the oxypnictide PrMnAsO0.95F0.05.

We have recently reported a new mechanism of colossal magnetoresistance (CMR) in electron doped manganese oxypnictides NdMnAsO1-xFx. Magnetoresistances of up to -95% at 3 K have been observed. Here we show that upon replacing Nd for Pr, the CMR is surprisingly no longer present. Instead a sizable negative magnetoresistance is observed for PrMnAsO0.95F0.05 below 35 K (MR7T (12 K) = -13.4% for PrMnAsO0.95F0.05). A detailed neutron and synchrotron X-ray diffraction study of PrMnAsO0.95F0.05 has been performed, which shows that a structural transition, Ts, occurs at 35 K from tetragonal P4/nmm to orthorhombic Pmmn symmetry. The structural transition is driven by the Pr 4f electrons degrees of freedom. The sizable -MR observed below the transition most likely arises due to a reduction in magnetic and/or multipolar scattering upon application of a magnetic field.

Renewable energy consumption - financial globalization-tourism nexus with nonlinearity and cross-sectional dependence during Asia's carbon-free transition.

Adopting renewable energy consumption is one of the most important aspects of international efforts to combat climate change, improve energy security, and encourage the shift to a more robust and sustainable energy system. Therefore, the empirics and policymakers worldwide are searching for factors that can promote renewable energy consumption. This analysis intends to investigate the role of financial globalization and tourism on renewable energy consumption in Asia and sub-regions such as Central Asia, East Asia, South Asia, Southeast Asia, and West Asia. The analysis utilized the linear and nonlinear CS-ARDL methods. Long-run outcomes of the linear and nonlinear models confirm that a rise in financial globalization and tourism promotes renewable energy consumption in Asia and all sub-regions. However, the nonlinear model highlights that a fall in financial globalization hurts renewable energy consumption in Central and South Asian regions, and tourism only hurts renewable energy consumption in Asia. In addition, ICT, GDP, and GHG emissions help promote renewable energy consumption. These results suggest that Asian policymakers must increase collaboration in the financial sectors and promote sustainable tourism in the regions to promote renewable energy consumption.

An integrated toolkit for human microglia functional genomics.

BACKGROUND: Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9. METHODS: Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step, 20-day process, producing iMG with a normal karyotype. We confirmed the iMG's authenticity and quality through single-cell RNA sequencing, chromatin accessibility profiles (ATAC-Seq), proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further, we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . RESULTS: Our method generates iMG that closely align with human primary microglia in terms of transcriptomic, proteomic, and chromatin accessibility profiles. Functionally, these iMG exhibit Ca2 + transients, cytokine driven migration, immune responses to inflammatory signals, and active phagocytosis of CNS related substrates including synaptosomes, amyloid beta and myelin. Significantly, the toolkit facilitates repeated iMG harvesting, essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia, positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. CONCLUSIONS: Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform, our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism, the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement.

Improved High-Temperature Thermoelectric Properties of Dual-Doped Ca3Co4O9.

Layered structured Ca3Co4O9 has displayed great potential for thermoelectric (TE) renewable energy applications, as it is nontoxic and contains abundantly available constituent elements. In this work, we study the crystal structure and high-temperature TE properties of Ca3-2y Na2y Co4-y Mo y O9 (0 ≤ y ≤ 0.10) polycrystalline materials. Powder X-ray diffraction (XRD) analysis shows that all samples are single-phase samples and without any noticeable amount of the secondary phase. X-ray photoelectron spectroscopic (XPS) measurements depict the presence of a mixture of Co3+ and Co4+ valence states in these materials. The Seebeck coefficient (S) of dual-doped materials is significantly enhanced, and electrical resistivities (ρ) and thermal conductivities (κ) are decreased compared to the pristine compound. The maximum thermoelectric power factor (PF = S 2/ρ) and dimensionless figure of merit (zT) obtained for the y = 0.025 sample at 1000 K temperature are ∼3.2 × 10-4 W m-1 K-2 and 0.27, respectively. The zT value for Ca2.95Na0.05Co3.975Mo0.025O9 is about 2.5 times higher than that of the parent Ca3Co4O9 compound. These results demonstrate that dual doping of Na and Mo cations is a promising strategy for improving the high-temperature thermoelectric properties of Ca3Co4O9.

Bioluminescence imaging of Olig2-neural stem cells reveals improved engraftment in a demyelination mouse model.

A major issue in the potential application of neural stem cell (NSC)-based cell replacement therapy for demyelinating diseases is the question of the survival, functional behavior, and stability of implanted NSC-derived oligodendrocyte precursor cells (OPCs) over an extended period. To address this issue, we employed bioluminescence imaging (BLI) as a noninvasive longitudinal in vivo monitoring technique and followed the fate of NSCs isolated from luciferase-green fluorescent protein-actin transgenic mice after stereotactic implantation in the demyelinated corpus callosum of cuprizone-fed mice. We compared normal NSCs with NSCs that were primed to become OPCs by the induction of Olig2 overexpression (Olig2-NSCs). BLI, validated by immunohistochemistry, revealed that, after a steep cell loss after implantation during the first 3 weeks, approximately 10% of the Olig2-NSCs stably survived for 2 months after implantation, in contrast to <1% of the normal NSCs. Immunohistochemistry, at the light and electron microscopic levels, revealed that the majority of the surviving Olig2-NSCs had differentiated into an oligodendrocytic cell lineage and contributed to remyelination of axons in the corpus callosum. The number of axons remyelinated by the implanted cells, however, was a small fraction of the total number of axons remyelinated by endogenous oligodendrocytes. Apparently, most of the implanted NSCs did not survive the transition into an inappropriate non-neurogenic niche, compressed by surrounding host tissue, in hostile, inflammatory conditions created by activated microglia. Only the ones that managed to differentiate rapidly into a mature neural cell type and become functionally integrated survived.

Epigenetic mechanisms facilitating oligodendrocyte development, maturation, and aging.

The process of oligodendrocyte differentiation is regulated by a dynamic interaction between a genetic and an epigenetic program. Recent studies, addressing nucleosomal histone modifications have considerably increased our knowledge regarding epigenetic regulation of gene expression during oligodendrocyte development and aging. These results have generated new hypotheses regarding the mechanisms underlying the decreased efficiency of endogenous remyelination in response to demyelinating injuries with increasing age. In this review, we present an overview of the epigenetic mechanisms regulating gene expression at specific stages of oligodendrocyte differentiation and maturation as well as the changes that occur with aging.

Differentiation of neural stem cells into oligodendrocytes: involvement of the polycomb group protein Ezh2.

The mechanisms underlying the regulation of neural stem cell (NSC) renewal and maintenance of their multipotency are still not completely understood. Self-renewal of stem cells in general implies repression of genes that encode for cell lineage differentiation. Enhancer of zeste homolog 2 (Ezh2) is a Polycomb group protein involved in stem cell renewal and maintenance by inducing gene silencing via histone methylation and deacetylation. To establish the role of Ezh2 in the maintenance and differentiation of NSCs, we have examined the expression of Ezh2 in NSCs isolated from embryonic (embryonic day 14) mice during proliferation and differentiation in vitro. Our results show that Ezh2 is highly expressed in proliferating NSCs. In accordance with its suggested role as a transcription repressor, the expression of Ezh2 decreased when the NSCs differentiated into neurons and was completely suppressed during differentiation into astrocytes. Surprisingly, Ezh2 remained highly expressed in NSCs that differentiated into an oligodendrocytic cell lineage, starting from oligodendrocyte precursor cells (OPCs) up to the immature (premyelinating) oligodendrocyte stage. To further establish the role of Ezh2 in NSC differentiation, we silenced and induced overexpression of the Ezh2 gene in NSCs. High levels of Ezh2 in differentiating NSCs appeared to be associated with an increase in oligodendrocytes and a reduction in astrocytes, whereas low levels of Ezh2 led to completely opposite effects. The increase in the number of oligodendrocytes induced by enhanced expression of Ezh2 could be ascribed to stimulation of OPC proliferation although stimulation of oligodendrocyte differentiation cannot be excluded. Disclosure of potential conflicts of interest is found at the end of this article.

Electrical, magnetic and magnetotransport properties of Na and Mo doped Ca3Co4O9 materials.

We report the electrical, magnetic and magnetotransport properties of Na and Mo dual doped Ca3-2x Na2x Co4-x Mo x O9 (0 ≤ x ≤ 0.15) polycrystalline samples. The results indicate that the strength of ferrimagnetic interaction decreases with increase in doping, as is evident from the observed decrease in Curie temperatures (T C). The substitution of non-magnetic Mo6+ ions (4d0) in CoO2 layers and the presence of oxygen vacancies are responsible for decrease in ligand field strength, which results in an enhanced magnetization in the low doped x = 0.025 sample due to a change from the low spin to partial high spin electron configuration. The electrical resistivity of samples exhibits a semiconducting-like behavior in the low temperature range, a strongly correlated Fermi liquid-like behavior in the intermediate temperature range, and an incoherent metal-like behavior in the temperature range 210-300 K. All the samples show a large negative magnetoresistance (MR) at low temperature with a maximum MR value of -59% for the x = 0.025 sample at 2 K and 16 T applied field. The MR values follow the observed trend in magnetization at 5 K and sharply increase below the Curie temperatures of the samples, suggesting that the ferrimagnetic interactions are mainly responsible for the decrease in electrical resistivity under an applied magnetic field.

Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of ß-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.

High-temperature thermoelectric properties of Na- and W-Doped Ca3Co4O9 system.

The detailed crystal structures and high temperature thermoelectric properties of polycrystalline Ca3-2x Na2x Co4-x W x O9 (0 ≤ x ≤ 0.075) samples have been investigated. Powder X-ray diffraction data show that all samples are phase pure, with no detectable traces of impurity. The diffraction peaks shift to lower angle values with increase in doping (x), which is consistent with larger ionic radii of Na+ and W6+ ions. X-ray photoelectron spectroscopy data reveal that a mixture of Co2+, Co3+ and Co4+ valence states are present in all samples. It has been observed that electrical resistivity (ρ), Seebeck coefficient (S) and thermal conductivity (κ) are all improved with dual doping of Na and W in Ca3Co4O9 system. A maximum power factor (PF) of 2.71 × 10-4 W m-1 K-2 has been obtained for x = 0.025 sample at 1000 K. The corresponding thermoelectric figure of merit (zT) for x = 0.025 sample is calculated to be 0.21 at 1000 K, which is ∼2.3 times higher than zT value of the undoped sample. These results suggest that Na and W dual doping is a promising approach for improving thermoelectric properties of Ca3Co4O9 system.

CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro .