Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype.

Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner. PAPERCLIP.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Corrigendum: Landscape of X chromosome inactivation across human tissues.

This corrects the article DOI: 10.1038/nature24265.

Loss of sarcospan exacerbates pathology in mdx mice, but does not affect utrophin amelioration of disease.

The dystrophin-glycoprotein complex (DGC) is a membrane adhesion complex that provides structural stability at the sarcolemma by linking the myocyte's internal cytoskeleton and external extracellular matrix. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to the loss of the DGC at the sarcolemma, resulting in sarcolemmal instability and progressive muscle damage. Utrophin (UTRN), an autosomal homolog of dystrophin, is upregulated in dystrophic muscle and partially compensates for the loss of dystrophin in muscle from patients with DMD. Here, we examine the interaction between Utr and sarcospan (SSPN), a small transmembrane protein that is a core component of both UTRN-glycoprotein complex (UGC) and DGC. We show that additional loss of SSPN causes an earlier onset of disease in dystrophin-deficient mdx mice by reducing the expression of the UGC at the sarcolemma. In order to further evaluate the role of SSPN in maintaining therapeutic levels of Utr at the sarcolemma, we tested the effect of Utr transgenic overexpression in mdx mice lacking SSPN (mdx:SSPN -/-:Utr-Tg). We found that overexpression of Utr restored SSPN to the sarcolemma in mdx muscle but that the ablation of SSPN in mdx muscle reduced Utr at the membrane. Nevertheless, Utr overexpression reduced central nucleation and improved grip strength in both lines. These findings demonstrate that high levels of Utr transgenic overexpression ameliorate the mdx phenotype independently of SSPN expression but that loss of SSPN may impair Utr-based mechanisms that rely on lower levels of Utr protein.

Single-Cell Transcriptomics Reveal Disrupted Kidney Filter Cell-Cell Interactions after Early and Selective Podocyte Injury.

The health of the kidney filtration barrier requires communication among podocytes, endothelial cells, and mesangial cells. Disruption of these cell-cell interactions is thought to contribute to disease progression in chronic kidney diseases (CKDs). Podocyte ablation via doxycycline-inducible deletion of an essential endogenous molecule, CTCF [inducible podocyte-specific CTCF deletion (iCTCFpod-/-)], is sufficient to drive progressive CKD. However, the earliest events connecting podocyte injury to disrupted intercellular communication within the kidney filter remain unclear. Single-cell RNA sequencing of kidney tissue from iCTCFpod-/- mice after 1 week of doxycycline induction was performed to generate a map of the earliest transcriptional effects of podocyte injury on cell-cell interactions at single-cell resolution. A subset of podocytes had the earliest signs of injury due to disrupted gene programs for cytoskeletal regulation and mitochondrial function. Surviving podocytes up-regulated collagen type IV ɑ5, causing reactive changes in integrin expression in endothelial populations and mesangial cells. Intercellular interaction analysis revealed several receptor-ligand-target gene programs as drivers of endothelial cell injury and abnormal matrix deposition. This analysis reveals the earliest disruptive changes within the kidney filter, pointing to new, actionable targets within a therapeutic window that may allow us to maximize the success of much needed therapeutic interventions for CKDs.

Landscape of X chromosome inactivation across human tissues.

X chromosome inactivation (XCI) silences transcription from one of the two X chromosomes in female mammalian cells to balance expression dosage between XX females and XY males. XCI is, however, incomplete in humans: up to one-third of X-chromosomal genes are expressed from both the active and inactive X chromosomes (Xa and Xi, respectively) in female cells, with the degree of 'escape' from inactivation varying between genes and individuals. The extent to which XCI is shared between cells and tissues remains poorly characterized, as does the degree to which incomplete XCI manifests as detectable sex differences in gene expression and phenotypic traits. Here we describe a systematic survey of XCI, integrating over 5,500 transcriptomes from 449 individuals spanning 29 tissues from GTEx (v6p release) and 940 single-cell transcriptomes, combined with genomic sequence data. We show that XCI at 683 X-chromosomal genes is generally uniform across human tissues, but identify examples of heterogeneity between tissues, individuals and cells. We show that incomplete XCI affects at least 23% of X-chromosomal genes, identify seven genes that escape XCI with support from multiple lines of evidence and demonstrate that escape from XCI results in sex biases in gene expression, establishing incomplete XCI as a mechanism that is likely to introduce phenotypic diversity. Overall, this updated catalogue of XCI across human tissues helps to increase our understanding of the extent and impact of the incompleteness in the maintenance of XCI.

HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.

Resolving the full spectrum of human genome variation using Linked-Reads.

Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as "Linked-Reads". This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2 Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

Structure of the C1r-C1s interaction of the C1 complex of complement activation.

The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody-antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r2C1s2 as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r-C1s interaction that forms the core of C1. Both fragments are L-shaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r2C1s2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.

Clinical or gimmickal: The use and effectiveness of mobile mental health apps for treating anxiety and depression.

OBJECTIVES: The increase in ownership of smartphones and tablet devices has seen a worldwide government push, championed by the World Health Organization, towards digital healthcare services generally. Mental health has been a strong presence in the digitisation of healthcare because of the potential to solve some of the difficulties in accessing face-to-face services. This review summarises the recent history of e-mental health services and illuminates two very different paths. The first is the considerable amount of research that has proven the effectiveness of many online mental health programmes for personal computers and laptops, resulting in widespread acceptance of their ability to make a contribution in an individual's recovery from anxiety and depression. The second is associated with the more recent development of apps for smartphones and tablet devices and the contrasting paucity of research that has accompanied this burgeoning area of e-mental health. This review also outlines the current state of play for research into the effectiveness of mobile mental health apps for anxiety and depression, including issues associated with methodology, and offers sources of practical advice for clinicians wanting more information about these new digital tools. CONCLUSION: Research into the effectiveness of mental health apps is lacking, and the majority have no evidence of efficacy. Clinicians need to be aware of what apps have such evidence and should exercise caution when recommending apps to patients. Suggestions are offered on the direction of future research, including an appeal to further include clinicians in the development and efficacy testing of mental health apps.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.

Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle weakness, ultimately leading to early mortality in affected teenagers and young adults. Previous work from our lab has shown that a small transmembrane protein called sarcospan (SSPN) can enhance the recruitment of adhesion complex proteins to the cell surface. When human SSPN is expressed at three-fold levels in mdx mice, this increase in adhesion complex abundance improves muscle membrane stability, preventing many of the histopathological changes associated with DMD. However, expressing higher levels of human SSPN (ten-fold transgenic expression) causes a severe degenerative muscle phenotype in wild-type mice. Since SSPN-mediated stabilization of the sarcolemma represents a promising therapeutic strategy in DMD, it is important to determine whether SSPN can be introduced at high levels without toxicity. Here, we show that mouse SSPN (mSSPN) can be overexpressed at 30-fold levels in wild-type mice with no deleterious effects. In mdx mice, mSSPN overexpression improves dystrophic pathology and sarcolemmal stability. We show that these mice exhibit increased resistance to eccentric contraction-induced damage and reduced fatigue following exercise. mSSPN overexpression improved pulmonary function and reduced dystrophic histopathology in the diaphragm. Together, these results demonstrate that SSPN overexpression is well tolerated in mdx mice and improves sarcolemma defects that underlie skeletal muscle and pulmonary dysfunction in DMD.

Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin-glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7ß1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan 'rescue' of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7ß1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7ß1 integrin.

A History of Social Work in Public Health.

Social work is a core health profession with origins deeply connected to the development of contemporary public health in the United States. Today, many of the nation's 600 000 social workers practice broadly in public health and in other health settings, drawing on a century of experience in combining clinical, intermediate, and population approaches for greater health impact. Yet, the historic significance of this long-standing interdisciplinary collaboration-and its current implications-remains underexplored in the present era. This article builds on primary and contemporary sources to trace the historic arc of social work in public health, providing examples of successful collaborations. The scope and practices of public health social work practice are explored, and we articulate a rationale for an expanded place for social work in the public health enterprise.

Health in All Social Work Programs: Findings From a US National Analysis.

OBJECTIVES: To establish a baseline of health content in 4 domains of US social work education-baccalaureate, master's, doctoral, and continuing education programs-and to introduce the Social Work Health Impact Model, illustrating social work's multifaceted health services, from clinical to wide-lens population health approaches. METHODS: We analyzed US social work programs' Web site content to determine amount and types of health content in mission statements, courses, and specializations. Coding criterion determined if content was (1) health or health-related (HHR) and (2) had wide-lens health (WLH) emphasis. A second iteration categorized HHR and WLH courses into health topics. RESULTS: We reviewed 4831 courses. We found broad HHR content in baccalaureate, master's, and continuing education curricula; doctoral programs had limited health content. We identified minimal WLH content across all domains. Topical analysis indicated that more than 50% of courses concentrated on 3 areas: mental and behavioral health, abuse and violence, and substance use and addictions. CONCLUSIONS: As a core health profession, social work must strengthen its health and wide-lens content to better prepare graduates for integrated practice and collaboration in the changing health environment.

Advancing Social Work Education for Health Impact.

Social work education plays a critical role in preparing social workers to lead efforts that improve health. Because of the dynamic health care landscape, schools of social work must educate students to facilitate health care system improvements, enhance population health, and reduce medical costs. We reviewed the existing contributions of social work education and provided recommendations for improving the education of social workers in 6 key areas: aging, behavioral health, community health, global health, health reform, and health policy. We argue for systemic improvement in the curriculum at every level of education, including substantive increases in content in health, health care, health care ethics, and evaluating practice outcomes in health settings. Schools of social work can further increase the impact of the profession by enhancing the curricular focus on broad content areas such as prevention, health equity, population and community health, and health advocacy.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation.

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca(2+)-coordinating residues. The data explain the Ca(2+)-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome.

BACKGROUND: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca(2+)-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly(204)Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. RESULTS: In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca(2+) binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca(2+) during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. CONCLUSIONS: We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca(2+) binding during biosynthesis.