Probes for INS

Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Private grant from Kowa Pharmaceuticals to Brigham and Woman's Hospital Calcific aortic valve stenosis (CAVS) is the most prevent valvular heart disease in the western world increasing exponentially with age, with an 112% increase in CAVS deaths in the last three decades; however no therapeutic treatment is currently available. Recently, lipoprotein(a) [Lp(a)] has been demonstrated to be an independent and causal risk factor for CAVS, yet the understanding of its cellular uptake and catabolism is limited thus underscoring the need for further investigation. This study aimed to determine a target receptor, unique for Lp(a) on the surface of valvular interstitial cells (VICs) and ascertain the role of the receptor on the development of VIC calcification. Unbiased ligand-receptor capture mass spectrometry (TriCEPS) was used to identify target receptor, with western blotting, ELISA, qPCR, alizarin red calcium staining and immunofluorescence used to validate the targets in vitro via siRNA inhibition and overexpression. Transmission electron microscopy (TEM) was used to determine uptake of Lp(a) within excised human valves. Identification of small molecule inhibitors was assessed computationally via the L1000 dataset, with the top hit candidate validated in vitro. Genotype-phenotype studies were examined using the United Kingdom Biobank (UKBB) and the Millions Veterans Program. Linear regression was used to evaluate association between aortic stenosis and plasma Lp(a) levels, and a phenotype-wide association analysis was then performed against this generated ‘genotype’. Ligand-receptor capture mass spectrometry was used to detect novel membrane proteins with specific binding to Lp(a); MFSD5, MRC2, LDLR were identified as possible candidates. MFSD5 RNAscope demonstrated its presence in human aortic valves. Lp(a) uptake in VICs was confirmed via western blot and TEM. MFSD5 siRNA significantly reduced dil-labelled Lp(a) uptake in human VICs (p=0.003) and HEPG2 cells (p=0.0003), conversely MFSD5 overexpression increased uptake (p=0.0345, p=0.0318), whilst specificity of MFSD5 to Lp(a) alone was shown via no change in LDL uptake following MFSD5 inhibition (p=0.616, p=0.991). MFSD5 inhibition reduced RUNX2 (p=0.0124) and Osteocalcin (p<0.001) RNA expression and reduced alizarin red staining following culture in Lp(a) osteogenic media for 21 days (p<0.0033). Druggability of MFSD5 was confirmed by the L1000 database, which identified aminopurvalanol as a binding partner for MFSD5 and significantly reduced Lp(a) uptake within VICs (p=0.0091). MFSD5-loss of function within the UKBB showed no significant cardiovascular association, however 50kb +/- of the MFSD5 gene showed nominal association with hyperlipidaemia and atrial fibrillation. The current study demonstrates the novel Lp(a) receptor MFSD5 may be responsible for uptake of Lp(a) within VICs, resulting in the development of aortic valve calcification, highlighting the need for further exploration into the role of MFSD5 in aortic valve disease.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins are currently in clinical trials. They all carry H1 and H4. Here we investigated whether these two hinges are essential for micro-dystrophin function in murine DMD models. Three otherwise identical micro-dystrophins were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ∆H1 (without H1), and ∆H4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent micro-dystrophin expression in total muscle lysate. However, only H1/H4 and ∆H1 showed correct sarcolemmal localization. ∆H4 mainly existed as subsarcolemmal aggregates. H1/H4 and ∆H1, but not ∆H4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by ∆H1, but not by ∆H4. Next, we compared H1/H4 and ∆H1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months post-injection, H1/H4 significantly outperformed ∆H1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable ECG improvements. We conclude that H4 is essential for micro-dystrophin function and H1 facilitates force production. Our findings will help develop next-generation micro-dystrophin gene therapy.

Background: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to decrease the risk of myocardial infarction and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a chimeric antigen receptor (CAR) targeting malonaldehyde-modified low-density lipoprotein cholesterol (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. OxidizedLDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane, and CD28/CD3z cytoplasmic domains. CD4+ CD25+ CD127low/e Tregs were purified from human blood via fluorescent activated cell sorting and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy. Autologous ox-CAR-Tregs were analyzed for activation after ex vivo coculture with carotid endarterectomy samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising oxCAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared with controls, based on CD71 expression, cytokine expression, and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using immunohistochemistry. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex vivo co-culture with atherosclerotic plaque. Conclusions: An optimized CAR targeting MDA-LDL activates Tregs Q10 when cultured with human atherosclerotic plaque ex vivo.

Objective: Identification of the developmental steps leading to somatosensory neuron development. Background: Our sense of touch is essential for life and relies on Low-Threshold Mechanoreceptors (LTMRs). LTMR subtypes characterized by early embryonic expression of Ntrk2 (TrkB) and Ret exhibit distinct properties depending on the skin region they innervate - hairy skin or glabrous(hairless) skin. In glabrous skin, TrkB+ and Ret+ LTMRs form Meissner corpuscles, while in hairy skin they form longitudinal lanceolate endings around hair follicles. These morphological features reflect the physiological properties and specialized functions of these neurons. The developmental steps leading to glabrous and hairy skin LTMR properties are largely unknown, in particular whether they are genetically pre-specified or whether interactions with different target skin regions define their unique features. Design/Methods: Sparse genetic labeling experiments demonstrate that morphological specialization of glabrous- and hairy paw skin-innervating TrkB+ and Ret+ LTMRs arise at nearly identical times during postnatal development. Interestingly, we find that individual neurons that terminate along the border of glabrous and hairy skin, termed “border neurons”, exhibit branches that form both lanceolate endings and Meissner corpuscle endings. Additionally, transcriptomic profiling and RNAscope experiments show that neonatal glabrous skin-and hairy skin-innervating TrkB+ and Ret+ neurons are transcriptionally similar, although distinct from other DRG neuron types. Lastly, using mouse mutants that have either ectopic glabrous skin or ectopic hairy skin we find that neurons that innervate ectopic skin regions of these mutants form ending types (either lanceolate or Meissner corpuscle endings) in accordance with the ectopic skin type. Results: These findings support a model in which embryonic TrkB+ and Ret+ LTMRs are able to form either Meissner corpuscle or lanceolate endings, and that the skin target region differentially instructs morphological maturation of these LTMR types. Conclusions: This model implies that neuronal identity in the peripheral nervous system is flexibly determined by target tissue.

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites.

BACKGROUND: Medulloblastoma with extensive nodularity (MBEN) represents a rare type of cerebellar tumors of infancy comprising two histologically distinct components that differ in cell differentiation and mitotic activity. Whereas some children suffering from MBEN experience disease recurrence, MBEN can also spontaneously differentiate and discontinue to grow. The underlying mechanisms of this variable biological behavior may offer insight into how embryonal tumors develop. METHODS: Fresh frozen and FFPE tumor tissue from nine MBEN-patients was subjected to multi-omics characterization including bulk sequencing, microdissection followed by RNA sequencing, single nucleus RNA-sequencing using the 10X Genomics- and SMART Seq. V2-protocols and spatial transcriptomics via RNAscope. RESULTS: All cases were molecularly classified as Sonic Hedgehog (SHH)-MB, and harbored somatic mutations within the SHH-pathway. After quality control, a total of ~30.000 cells were subjected to downstream analysis. Several non-malignant cell types, such as glial cells, were identified. In accordance with previous studies, we found only sparse immune infiltration. Unsupervised clustering identified cell clusters that differed in differentiation state and represented a continuum from embryonal-like cells with SHH-upregulation over intermediate cell states, to neuronal-like, postmitotic cells. Mapping to a single nucleus sequencing atlas of cerebellar development indicated that tumor cells reflected various stages of normally developing cerebellar granular precursors. Interestingly, one cluster of malignant cells with tumor-specific copy number alterations showed both transcriptomic features of astrocytes and embryonal cells. Using spatial transcriptomics, we were able to correlate different clusters of MBEN cells with distinct histologic MBEN compartments, with astrocyte-like tumor cells being located in the internodular compartment and in close proximity to mitotically active cancer cells. CONCLUSION: MBEN is formed by a continuum of malignant cell differentiation along the granular precursor lineage, with a subset of cells developing into cells that may represent tumor astrocytes. This differentiation process is reflected in the bicompartmental structure of MBEN.

One of the most striking aspects of the sensory epithelium of the mammalian cochlea, the organ of Corti, is the presence of precise boundaries between sensory and non-sensory cells at its medial and lateral edges. A particular example of this precision is the single row of inner hair cells and associated supporting cells along the medial (neural) boundary. Despite the regularity of this boundary, the developmental processes and genetic factors that contribute to its specification are poorly understood. In this study we demonstrate that Leucine Rich Repeat Neuronal 1 (Lrrn1), which codes for a single-pass, transmembrane protein, is expressed prior to the development of the mouse organ of Corti in the row of cells that will form its medial border. Deletion of Lrrn1 in mice of mixed sex leads to disruptions in boundary formation that manifest as ectopic inner hair cells and supporting cells. Genetic and pharmacological manipulations demonstrate that Lrrn1 interacts with the Notch signaling pathway and strongly suggest that Lrrn1 normally acts to enhance Notch signaling across the medial boundary. This interaction is required to promote formation of the row of inner hair cells and suppress the conversion of adjacent non-sensory cells into hair cells and supporting cells. These results identify Lrrn1 as an important regulator of boundary formation and cellular patterning during development of the organ of Corti.SIGNIFICANCE STATEMENT:Patterning of the developing mammalian cochlea into distinct sensory and non-sensory regions and the specification of multiple different cell fates within those regions are critical for proper auditory function. Here, we report that the transmembrane protein LRRN1 is expressed along the sharp medial boundary between the single row of mechanosensory inner hair cells and adjacent non-sensory cells. Formation of this boundary is mediated in part by Notch signaling, and loss of Lrrn1 leads to disruptions in boundary formation similar to those caused by a reduction in Notch activity, suggesting that LRRN1 likely acts to enhance Notch signaling. Greater understanding of sensory/non-sensory cell fate decisions in the cochlea will help inform the development of regenerative strategies aimed at restoring auditory function.

Distinguishing different contexts is thought to involve a form of pattern separation that minimizes overlap between neural ensembles representing similar experiences. Theoretical models suggest that the dentate gyrus (DG) segregates cortical input patterns before relaying its discriminated output patterns to the CA3 hippocampal field. This suggests that the evaluation of neural ensembles in DG and CA3 could be an important means to investigate the process of pattern separation. In the past, measurement of entorhinal cortex (EC), DG, and CA3 ensembles was largely dependent upon in vivo electrophysiological recording, which is technically difficult. This protocol provides a method to instead measure pattern separation by a molecular method that provides direct spatial resolution at the cellular level.

Highly multiplexed protein and RNA in situ detection on a single tissue section concurrently is highly desirable for both basic and applied biomedical research. CO-detection by inDEXing (CODEX) is a new and powerful platform to visualize up to 60 protein biomarkers in situ, and RNAscope in situ hybridization (RNAscope) is a novel RNA detection system with high sensitivity and unprecedent specificity at a single-cell level. Nevertheless, to our knowledge, the combination of CODEX and RNAscope remained unreported until this study. Here, we report a simple and reproducible combination of CODEX and RNAscope. We also determined the cross-reactivities of CODEX anti-human antibodies to rhesus macaques, a widely used animal model of human disease.

Spinal cord development is precisely orchestrated by spatiotemporal gene regulatory programs. However, the underlying epigenetic mechanisms remain largely elusive. Here, we profiled single-cell chromatin accessibility landscapes in mouse neural tubes spanning embryonic days 9.5-13.5. We identified neuronal-cell-cluster-specific cis-regulatory elements in neural progenitors and neurons. Furthermore, we applied a novel computational method, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression data and identify the hub enhancers within enhancer networks. It was experimentally validated in vivo for Atoh1 that knockout of the hub enhancers, but not the non-hub enhancers, markedly decreased Atoh1 expression and reduced dp1/dI1 cells. Together, our work provides insights into the epigenetic regulation of spinal cord development and a proof-of-concept demonstration of enhancer networks as a general mechanism in transcriptional regulation.

Background and aims Graft-versus-host-disease (GvHD) is a common complication following allogeneic hematopoietic stem cell transplantation (aHCT) that typically manifests as injury response to the skin, gastrointestinal mucosa and liver. In liver, late onset acute and chronic liver GvHD are more similar to an autoimmune reaction. The identification of valid GvHD biomarker is still an unmet clinical need. In our study, we therefore aimed to identify gene expression patterns, which could be used as potential indicators for the outcome of aHCTs with regard to acute or chronic GvHD. For this purpose, we expect to uncover similar and distinct gene signatures of GvHD in comparison to non-diseased liver tissues and detect potential candidates giving information about the outcome after aHCT. Method Microarray analyses from FFPE samples of patients were performed, and the following criteria were applied: fold change> [1] and a p- value

Genomic architecture appears to play crucial roles in health and a variety of diseases. How nuclear structures reorganize over different timescales is elusive, partly because the tools needed to probe and perturb them are not as advanced as needed by the field. To fill this gap, the National Institutes of Health Common Fund started a program in 2015, called the 4D Nucleome (4DN), with the goal of developing and ultimately applying technologies to interrogate the structure and function of nuclear organization in space and time.