Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

COVID-19, the disease caused by the novel Coronavirus, SARS-CoV-2, is increasingly being recognized as a systemic thrombotic and microvascular injury syndrome that may have its roots in complement activation. We had the opportunity to study the placental pathology of five full-term births to COVID-19 patients. All five exhibited histology indicative of fetal vascular malperfusion characterized by focal avascular villi and thrombi in larger fetal vessels. Vascular complement deposition in the placentas was not abnormal, and staining for viral RNA and viral spike protein was negative. While all cases resulted in healthy, term deliveries, these findings indicate the systemic nature of COVID-19 infection. The finding of vascular thrombosis without complement deposition may reflect the systemic nature of COVID-19's procoagulant effects unrelated to systemic complement activation

The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 and 2 (GAD1, GAD2), and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe and nucleus incertus, and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus, and ventral tegmental area. Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.SIGNFICANCE STATEMENT The lateral habenula provides a major link between subcortical forebrain areas and the dopamine (DA) and serotonin (5HT) systems of the midbrain and pons, and it has been implicated in reward mechanisms and the regulation of mood states. Few markers have been available for the specific cell types and complex output pathways of the lateral habenula. Here we examined the expression of genes mediating GABAergic and glutamatergic transmission in the mouse and rat LHb, where no neurons in either species expressed a full complement of GABAergic markers, and all expressed the glutamatergic marker VGluT2. Consistent with this, in mice the LHb GAD2 neurons are excitatory and appear to use only glutamate for fast synaptic transmission.

The treatment of melanoma has remained a difficult challenge. Targeting the tumor stroma has recently attracted attention for developing novel strategies for melanoma therapy. Activating transcription factor 3 (ATF3) plays a crucial role in regulating tumorigenesis and development, but whether the expression of ATF3 in human dermal fibroblasts (HDFs) can affect melanoma development hasn't been studied. Our results show that ATF3 expression is downregulated in stromal cells of human melanoma. HDFs expressing high levels of ATF3 suppressed the growth and migration of melanoma cells in association with downregulation of different cytokines including IL-6 in vitro. In vivo, HDFs with high ATF3 expression reduced tumor formation. Adding recombinant IL-6 to melanoma cells reversed those in vitro and in vivo effects, suggesting that ATF3 expression by HDFs regulates melanoma progression through the IL-6/STAT3 pathway. More importantly, HDFs pretreated with cyclosporine A or phenformin to induce ATF3 expression inhibited melanoma cell growth in vitro and in vivo. In summary, our study reveals that ATF3 suppresses human melanoma growth and that inducing the expression of ATF3 in HDFs can inhibit melanoma growth, a new potential melanoma therapeutic approach

The tumor suppressor RNA-binding motif 5 (RBM5) regulates the expression levels and cassette exon-definition (i.e. splicing) of a select set of mRNAs in a tissue-specific manner. Most RBM5-regulated targets were identified in oncological investigations and frequently involve genes which mediate apoptotic cell death. Little is known about the role of RBM5 in the brain. Also, it is unclear if a brain injury may be required to detect RBM5 mediated effects on pro-apoptotic genes due to their low expression levels in the healthy adult CNS at baseline. Conditional/floxed (brain-specific) gene deleter mice were generated to elucidate CNS-specific RBM5 mRNA targets. Male/female mice were subjected to a severe controlled cortical impact (CCI) traumatic brain injury (TBI) in order to increase the background expression of pro-death mRNAs and facilitate testing of the hypothesis that RBM5 inhibition decreases post-injury upregulation of caspases/FAS in the CNS. As expected, a CCI increased caspases/FAS mRNA in the injured cortex. RBM5 KO did not affect their levels or splicing. Surprisingly, KO increased the mRNA levels of novel targets including casein kinase 2 alpha prime interacting protein (Csnka2ip/CKT2) - a gene not thought to be expressed in the brain, contrary to findings here. Twenty-two unique splicing events were also detected in KOs including increased block-inclusion of cassette exons 20-22 in regulating synaptic membrane exocytosis 2 (Rims2). In conclusion, here we used genome-wide transcriptomic analysis on healthy and injured RBM5 KO mouse brain tissue to elucidate the first known gene targets of this enigmatic RBP in this CNS

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p?