RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems ...caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.
CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine ...receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.
We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.
The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.
DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We ...developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm 9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.
Carers of people with eating disorders report high levels of distress. In addition, carers' responses to the illness may perpetuate eating disorder symptoms. A cognitive interpersonal maintenance ...model of eating disorders is proposed and interventions for carers may improve well-being in both carers and patients. Aims To examine an interpersonal maintenance model of eating disorders, using a self-help intervention for carers.
A pre-test-post-test design was used with carers randomised into self-help or guided self-help, which included the Expert Carers Helping Others (ECHO) intervention. Carers' distress, well-being, proposed maintenance factors, and carer reports on the status of the patient were measured.
Carers' distress reduced and secondary outcomes improved. Improvement in carers' status and perceived improvements in patients were associated with reductions in expressed emotion and in accommodating and enabling behaviours. Self-help and guided self-help versions were comparable.
Changes in maintenance factors from the theoretical model were associated with a reduction in carers' distress and improvement in perceived patient functioning. Interventions which specifically target maintaining factors may be of benefit.
Objectives. Contemporary models of eating disorders (EDs) argue that both cognitive style (weak coherence and poor set shifting) and social emotional difficulties are involved in the maintenance of ...EDs. This study aimed to explore the factor structure of cognitive and social emotional functioning and to investigate whether a particular cognitive or social emotional profile was associated with a more severe and chronic form of illness.
Design. A cross‐sectional design was used to investigate cognitive and social emotional functioning in people with EDs compared to healthy controls (HCs) and those recovered from an ED.
Methods. Two hundred twenty‐five participants were assessed (100 with an ED, 35 recovered from an ED, and 90 HCs) using a battery of set shifting, coherence, and social emotional measures.
Results. There were no significant correlations between the cognitive or social emotional variables. A principal components analysis (PCA) identified three components: a fragmented perseverative cognitive style, for which the ED group scored highly, a global flexible cognitive style, for which HCs scored highly, and a social emotional difficulties profile, for which those with EDs scored highly. Individuals in recovery from an ED did not differ from the acute group, suggesting this cognitive and social emotional profile may be a trait associated with EDs. ED participants scoring highest for the fragmented perseverative cognitive style and social emotional difficulties had a more severe and chronic form of illness.
Conclusions. The findings provide empirical support for Schmidt and Treasure's (2006) maintenance model of EDs and suggest both cognition and emotional functioning should be considered in treatment.
Axonal regeneration and related functional recovery following axonal injury in the adult central nervous system are extremely limited, due to a lack of neuronal intrinsic competence and the presence ...of extrinsic inhibitory signals. As opposed to what occurs during nervous system development, a weak proregenerative gene expression programme contributes to the limited intrinsic capacity of adult injured central nervous system axons to regenerate. Here we show, in an optic nerve crush model of axonal injury, that adenoviral (cytomegalovirus promoter) overexpression of the acetyltransferase p300, which is regulated during retinal ganglion cell maturation and repressed in the adult, can promote axonal regeneration of the optic nerve beyond 0.5 mm. p300 acetylates histone H3 and the proregenerative transcription factors p53 and CCAAT-enhancer binding proteins in retinal ganglia cells. In addition, it directly occupies and acetylates the promoters of the growth-associated protein-43, coronin 1 b and Sprr1a and drives the gene expression programme of several regeneration-associated genes. On the contrary, overall increase in cellular acetylation using the histone deacetylase inhibitor trichostatin A, enhances retinal ganglion cell survival but not axonal regeneration after optic nerve crush. Therefore, p300 targets both the epigenome and transcription to unlock a post-injury silent gene expression programme that would support axonal regeneration.
Abstract
The intermediate-conductance calcium-activated potassium channel K
Ca
3.1 has been proposed to be a new potential target for glioblastoma treatment. This study analyzed the effect of ...combined irradiation and K
Ca
3.1-targeting with TRAM-34 in the syngeneic, immune-competent orthotopic SMA-560/VM/Dk glioma mouse model. Whereas neither irradiation nor TRAM-34 treatment alone meaningfully prolonged the survival of the animals, the combination significantly prolonged the survival of the mice. We found an irradiation-induced hyperinvasion of glioma cells into the brain, which was inhibited by concomitant TRAM-34 treatment. Interestingly, TRAM-34 did neither radiosensitize nor impair SMA-560’s intrinsic migratory capacities in vitro. Exploratory findings hint at increased TGF-β1 signaling after irradiation. On top, we found a marginal upregulation of
MMP9
mRNA, which was inhibited by TRAM-34. Last, infiltration of CD3
+
, CD8
+
or FoxP3
+
T cells was not impacted by either irradiation or K
Ca
3.1 targeting and we found no evidence of adverse events of the combined treatment. We conclude that concomitant irradiation and TRAM-34 treatment is efficacious in this preclinical glioma model.
Astrocytes are the most abundant glial cells in the central nervous system (CNS) with the capacity to sense and react to injury and inflammatory events. While it has been widely documented that ...astrocytes can exert tissue-degenerative functions, less is known about their protective and disease-limiting roles. Here, we report the upregulation of pleiotrophin (PTN) by mouse and human astrocytes in multiple sclerosis (MS) and its preclinical model experimental autoimmune encephalomyelitis (EAE). Using CRISPR-Cas9-based genetic perturbation systems, we demonstrate
that astrocyte-derived PTN is critical for the recovery phase of EAE and limits chronic CNS inflammation. PTN reduces pro-inflammatory signaling in astrocytes and microglia and promotes neuronal survival following inflammatory challenge. Finally, we show that intranasal administration of PTN during the late phase of EAE successfully reduces disease severity, making it a potential therapeutic candidate for the treatment of progressive MS, for which existing therapies are limited.
Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple ...sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.
PDF
