Miracle-dz/UniFAR-FTUs huggingface dataset:
Implementing decentralised management in Ghana: The experience of the Sekyere West District health administration

Purpose – The purpose of this paper is to examine the barriers to the implementation of management decentralisation of health services and programmes at district level in Ghana.Design/methodology/approach – Data for the study were collected through a qualitative technique in the Sekyere West district.Findings – The findings are: transfer of authority for management decision making and planning was rarely operational at district level; and control over financial and personnel decisions remained centralised. The paper identified lack of staff capacity, lack of commitment and inter‐organisational conflict between health managers and local government officials as factors obstructing the implementation of management decentralisation in the district.Originality/value – The lesson from the study is that the implementation of health care management decentralisation at district level has stalled because of the combined effect of internal and external constraints. The paper suggests that given the enormity of the p.
Proteasomes tether to two distinct sites at the nuclear pore complex

Significance This study compares the native structures of cytosolic and nuclear proteasomes, visualized directly within cells. The assembly states and functional states of proteasomes in each compartment were similar, indicating comparable levels of proteolytic activity per proteasome. Nuclear proteasomes were tethered to two different sites at the nuclear pore complex (NPC): the inner nuclear membrane and the NPC basket. Structural analysis revealed mechanistic details of the two tethering interactions. These results present direct evidence that proteasomes bind at NPCs, establishing a cellular hub for protein degradation at the gateway between the nucleus and cytoplasm. This work demonstrates how cryo-electron tomography can reveal biological mechanisms by directly observing the interactions between molecular complexes within the native cellular environment. The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.
ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations

Significance The symptoms of neurological diseases such as autism and schizophrenia are often attributed to a loss of excitatory/inhibitory balance of neural network function. By showing that ATR and ATM impact inhibitory and excitatory vesicle trafficking differently, our work expands the known repertoire of cytoplasmic functions for the two kinases and provides a new perspective on the origins of the symptoms of ataxia-telangiectasia (A-T) and Seckel syndrome (ATM and ATR deficiency, respectively). While these findings have their most immediate implications for the neurologic and cognitive symptoms of A-T and Seckel syndrome, they have potential relevance to a much broader range of neurologic conditions. ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) are large PI3 kinases whose human mutations result in complex syndromes that include a compromised DNA damage response (DDR) and prominent nervous system phenotypes. Both proteins are nuclear-localized in keeping with their DDR functions, yet both are also found in cytoplasm, including on neuronal synaptic vesicles. In ATM- or ATR-deficient neurons, spontaneous vesicle release is reduced, but a drop in ATM or ATR level also slows FM4-64 dye uptake. In keeping with this, both proteins bind to AP-2 complex components as well as to clathrin, suggesting roles in endocytosis and vesicle recycling. The two proteins play complementary roles in the DDR; ATM is engaged in the repair of double-strand breaks, while ATR deals mainly with single-strand damage. Unexpectedly, this complementarity extends to these proteins’ synaptic function as well. Superresolution microscopy and coimmunoprecipitation reveal that ATM associates exclusively with excitatory (VGLUT1+) vesicles, while ATR associates only with inhibitory (VGAT+) vesicles. The levels of ATM and ATR respond to each other; when ATM is deficient, ATR levels rise, and vice versa. Finally, blocking NMDA, but not GABA, receptors causes ATM levels to rise while ATR levels respond to GABA, but not NMDA, receptor blockade. Taken together, our data suggest that ATM and ATR are part of the cellular “infrastructure” that maintains the excitatory/inhibitory balance of the nervous system. This idea has important implications for the human diseases resulting from their genetic deficiency.
dCas9-targeted locus-specific protein isolation method identifies histone gene regulators

Significance Identifying proteins selectively associated with a genomic locus provides an important entry point toward understanding how a specific gene is regulated. Over the years, there have been several reports describing targeted chromatin-purification methods. However, none has been widely adopted due to the complexity and investment required for such protocols. Here, we present an adaptable chromatin purification system, CLASP, that capitalizes on the versatility of purified dCas9 RNA/protein complexes. We deployed CLASP to purify and identify proteins associated with telomere sequences in human cells as a proof of concept. Next, we targeted a different genomic locus, the Drosophila melanogaster histone cluster, and identified several regulators of the essential histone locus and validated their functional association with genes within the locus. Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called “CLASP” (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors. Our CLASP method, coupled to mass spectrometry and functional screens, can be efficiently adapted for isolating associated regulatory factors in an unbiased manner targeting multiple genomic loci across different cell types. Here, we applied our method to isolate the Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3′ UTRs.
S4–S5 linker movement during activation and inactivation in voltage-gated K+ channels

Significance Excitability in heart and nervous system is based on sensing and propagation of the membrane potential by voltage-gated ion channels. Despite the increasing availability of high-resolution structures of voltage-gated ion channels, key questions about their dynamics remain elusive. Here we followed the movements of the gating machinery on the cytosolic surface of the Shaker KV channel by introducing a fluorescent noncanonical amino acid at different positions along the linker between voltage sensor and pore. We can thus map the movement of each position and reconstruct the dynamics of the gating machinery during activation and inactivation. Considering that the Shaker channel serves as a model system, the mechanisms are likely conserved among those KV channels containing a sizeable S4–S5 linker. The S4–S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4–S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker KV channel. Using two-color voltage-clamp fluorometry, we compared S4–S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4–S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4–S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4–S5 linker and pore opening disappears.
Three classes of recurrent DNA break clusters in brain progenitors identified by 3D proximity-based break joining assay

Significance Human brain neuron genomes can differ from one another, giving rise to brain mosaicism. We developed a sensitive DNA break joining assay that uses “bait” DNA breaks introduced on different chromosomes to detect endogenous “prey” DNA breaks across the mouse brain progenitor cell genome. This approach revealed 27 recurrently breaking sites, many of which occur in long neural-specific genes associated with mental illnesses and cancer. We have exploited the finding that bait and prey DSB join more frequently when on the same chromosome to increase assay sensitivity. This approach confirms previously identified breaking neural genes and identifies new ones, often with the same intriguing characteristics. Our study offer potential insights into brain diversification and disease. We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to “bait” DSBs in specific chromosomal locations. Cellular heterogeneity in 3D genome organization allowed unequivocal identification of RDCs on 14 different chromosomes using HTGTS baits on three mouse chromosomes. Additional candidate RDCs were also implicated, however, suggesting that some RDCs were missed. To more completely identify RDCs, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome. Thus, we used CRISPR/Cas9 to introduce specific DSBs into each mouse chromosome in NSPCs that were used as bait for HTGTS libraries. This analysis confirmed all 27 previously identified RDCs and identified many new ones. NSPC RDCs fall into three groups based on length, organization, transcription level, and replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support the idea that the latter treatment augments an already active endogenous process.
Carbon‐Quantum‐Dots‐Loaded Ruthenium Nanoparticles as an Efficient Electrocatalyst for Hydrogen Production in Alkaline Media

Highly active, stable, and cheap Pt‐free catalysts for the hydrogen evolution reaction (HER) are facing increasing demand as a result of their potential use in future energy‐conversion systems. However, the development of HER electrocatalysts with Pt‐like or even superior activity, in particular ones that can function under alkaline conditions, remains a significant challenge. Here, the synthesis of a novel carbon‐loaded ruthenium nanoparticle electrocatalyst (Ru@CQDs) for the HER, using carbon quantum dots (CQDs), is reported. Electrochemical tests reveal that, even under extremely alkaline conditions (1 m KOH), the as‐formed Ru@CQDs exhibits excellent catalytic behavior with an onset overpotential of 0 mV, a Tafel slope of 47 mV decade−1, and good durability. Most importantly, it only requires an overpotential of 10 mV to achieve the current density of 10 mA cm−2. Such catalytic characteristics are superior to the current commercial Pt/C and most noble metals, non‐noble metals, and nonmetallic catalysts under basic conditions. These findings open a new field for the application of CQDs and add to the growing family of metal@CQDs with high HER performance.
Leptin levels are associated with decreased depressive symptoms in women across the weight spectrum, independent of body fat

Objective Leptin is anorexigenic, and levels are markedly decreased in women with low body weight and high in women with obesity. Ghrelin opposes leptin effects on appetite and is negatively associated with body mass index. These appetite‐regulating hormones may have opposing effects on mood and stress pathways. Women with anorexia nervosa (AN), hypothalamic amenorrhoea (HA) and obesity are at increased risk of depression and anxiety. It is unknown whether dysregulation of leptin or ghrelin contributes to the development of depression and/or anxiety in these disorders. We investigated the relationship between leptin and ghrelin levels and symptoms of depression, anxiety and perceived stress in women across the weight spectrum.
Ligand metathesis as rational strategy for the synthesis of cubane-type heteroleptic iron–sulfur clusters relevant to the FeMo cofactor

Significance The biosynthesis and mechanism of action of nitrogenase, an enzyme that converts dinitrogen to ammonia under ambient conditions, are problems of prominent significance in metallobiochemistry. Because the active centers of the enzyme are metal–ligand clusters, it is feasible that they are attainable by synthesis and as such are primary goals in the field of biomimetic inorganic chemistry. Here we present a ligand metathesis strategy utilizing the periodic near-identity of molybdenum and tungsten when incorporated into analogous compounds. The approach provides a pathway for constructing heterometal heteroleptic Fe–S clusters of presumed relevance to the active site. Based on cubane-type stereochemistry, clusters have been prepared allowing alterations in structure and ligand binding, and inclusion of a light core atom. Molybdenum-dependent nitrogenases catalyze the transformation of dinitrogen into ammonia under ambient conditions. The active site (FeMo cofactor) is the structurally and electronically complex weak-field metal cluster [MoFe7S9C] built of Fe4S3 and MoFe3S3C portions connected by three sulfur bridges and containing an interstitial carbon atom centered in an Fe6 trigonal prism. Chemical synthesis of this cluster is a major challenge in biomimetic inorganic chemistry. One synthetic approach of core ligand metathesis has been developed based on the design and synthesis of unprecedented incomplete ([(Tp*)WFe2S3Q3]−) and complete ([(Tp*)WFe3S3Q4]2−) cubane-type clusters containing bridging halide (Q = halide). These clusters are achieved by template-assisted assembly in the presence of sodium benzophenone ketyl reductant; products are controlled by reaction stoichiometry. Incomplete cubane clusters are subject to a variety of metathesis reactions resulting in substitution of a μ2-bridging ligand with other bridges such as N3−, MeO−, and EtS−. Reactions of complete cubanes with Me3SiN3 and S8 undergo a redox metathesis process and lead to core ligand displacement and formation of [(Tp*)WFe3S3(μ3-Q)Cl3]− (Q = Me3SiN2−, S2−). This work affords entry to a wide variety of heteroleptic clusters derivable from incomplete and complete cubanes; examples are provided. Among these is the cluster [(Tp*)WFe3S3(μ3-NSiMe3)Cl3]−, one of the very few instances of a synthetic Fe–S cluster containing a light atom (C, N, O) in the core, which constitutes a close mimic of the [MoFe3S3C] fragment in FeMo cofactor. Superposition of them and comparison of metric information disclose a clear structural relationship [Tp* = tris(3,5-dimethyl-1-pyrazolyl)hydroborate(1−)].
Mutations in EID1 and LNK2 caused light-conditional clock deceleration during tomato domestication

Significance Internal timekeepers, called circadian clocks, are prevalent in all domains of life. Variation in circadian rhythms allows fine-tuning of an organism to its specific environment. Here we show that a mutation in LNK2, in addition to the already described mutation in EID1, was responsible for the deceleration of circadian rhythms in cultivated tomatoes. We show that the mutant alleles of both genes arose in the earliest cultivated types and were selected during the domestication process. Notably, both mutant alleles specifically affect light input to the clock, leading to a light-conditional clock deceleration. Such light-conditionality may be a widespread means to enhance resonance with changed day–night cycles at higher latitudes, despite the fixed 24-h period of the Earth. Circadian period and phase of cultivated tomato (Solanum lycopersicum) were changed during domestication, likely adapting the species to its new agricultural environments. Whereas the delayed circadian phase is mainly caused by allelic variation of EID1, the genetic basis of the long circadian period has remained elusive. Here we show that a partial deletion of the clock gene LNK2 is responsible for the period lengthening in cultivated tomatoes. We use resequencing data to phylogenetically classify hundreds of tomato accessions and investigate the evolution of the eid1 and lnk2 mutations along successive domestication steps. We reveal signatures of selection across the genomic region of LNK2 and different patterns of fixation of the mutant alleles. Strikingly, LNK2 and EID1 are both involved in light input to the circadian clock, indicating that domestication specifically targeted this input pathway. In line with this, we show that the clock deceleration in the cultivated tomato is light-dependent and requires the phytochrome B1 photoreceptor. Such conditional variation in circadian rhythms may be key for latitudinal adaptation in a variety of species, including crop plants and livestock.
Fork pausing allows centromere DNA loop formation and kinetochore assembly

Significance There is an epigenetic requirement for de novo kinetochore assembly in budding yeast. Perturbations that delay S phase suppress assembly defects. We propose a role for replication fork progression in allowing DNA to adopt configurations conducive for centromere function. The function of replication fork stalling upon encounters with DNA damage or other blockades is to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex. De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.
Ultrafast Spin‐to‐Charge Conversion at the Surface of Topological Insulator Thin Films

Strong spin–orbit coupling, resulting in the formation of spin‐momentum‐locked surface states, endows topological insulators with superior spin‐to‐charge conversion characteristics, though the dynamics that govern it have remained elusive. Here, an all‐optical method is presented, which enables unprecedented tracking of the ultrafast dynamics of spin‐to‐charge conversion in a prototypical topological insulator Bi2Se3/ferromagnetic Co heterostructure, down to the sub‐picosecond timescale. Compared to pure Bi2Se3 or Co, a giant terahertz emission is observed in the heterostructure that originates from spin‐to‐charge conversion, in which the topological surface states play a crucial role. A 0.12 ps timescale is identified that sets a technological speed limit of spin‐to‐charge conversion processes in topological insulators. In addition, it is shown that the spin‐to‐charge conversion efficiency is temperature independent in Bi2Se3 as expected from the nature of the surface states, paving the way for designing next‐generation high‐speed optospintronic devices based on topological insulators at room temperature.
Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

Significance The efficiency of genome editing with CRISPR-Cas9 can vary widely at different targets and in different cells. Some of this variability may be due to the inherent quality of different guide RNAs, but it may also depend on the cellular context of the genomic target DNA. In this report, we demonstrate that targets bound by nucleosomes are cut much less efficiently than targets from which nucleosomes are absent or have been depleted. This information can inform target selection, particularly in cases where cells are quiescent or nucleosome mobility is limited. Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.