The presence of active neurogenic niches in adult humans is controversial. We focused attention to the human olfactory neuroepithelium, an extracranial site supplying input to the olfactory bulbs of ...the brain. Using single-cell RNA sequencing analyzing 28,726 cells, we identified neural stem cell and neural progenitor cell pools and neurons. Additionally, we detailed the expression of 140 olfactory receptors. These data from the olfactory neuroepithelium niche provide evidence that neuron production may continue for decades in humans.
Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at ...single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8
T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.
Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge ...during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is "set in stone" early in tumor evolution and may explain why advances in primary treatment have not improved survival.
Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational ...inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors.
A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR.
Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1(PRAME-), 38% for Class1(PRAME+), and 71% for Class 2 tumors. Median metastasis-free survival for Class1(PRAME+) patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1(PRAME+) tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003).
PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM.
Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic ...mouse model, Asxl1Y588XTg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588XTg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography–tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588XTg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.
•Transgenic expression of ASXL1aa1-587 truncating protein in the hematopoietic system leads to diverse myeloid malignancies in mice.•ASXL1aa1-587 gains an interaction with BRD4 and Asxl1Y588XTg hematopoietic stem/progenitor cells are hypersensitive to BET bromodomain inhibitors.
Abstract
Chromatin immunoprecipitation (ChIP) has ushered in a new era of scientific discovery by allowing new insights into DNA-protein interactions. ChIP is used to quantify enriched genomic ...regions using qPCR, and more recently is combined with next generation sequencing (ChIP-seq) to obtain a genome wide profile of protein binding sites. Nevertheless, ChIP-qPCR remains an integral component of this technology for quality control purposes, before the library preparation and sequencing steps. In addition, ChIP-qPCR remains more time- and cost-effective for many focused projects in which the DNA regions of interest are already known. However, the DNA oligonucleotide primers needed for ChIP-qPCR are more challenging to design than for other qPCR projects. Here, we present the first public repository for ChIP oligonucleotides that have been verified to perform well in ChIP-qPCR experiments. ChIPprimersDB was developed by manual screening of publications to ensure primer quality and provide additional specific information on the ChIP experiments where the primers have been used. In addition to the primer sequences, the database includes information about the antibody, cells and tissues used in the experiment, information on the experimental design, and a direct link to the original publication. The database is linked at https://umiamihealth.org/bascom-palmer-eye-institute/research/clinical-and-laboratory-research/ocular-oncology-laboratory/chip-primers and hosted at https://www.chipprimers.com/.
Recent advances in single cell sequencing technologies allow for greater resolution in assessing tumor clonality using chromosome copy number variations (CNVs). While single cell DNA sequencing ...technologies are ideal to identify tumor sub-clones, they remain expensive and in contrast to single cell RNA-seq (scRNA-seq) methods are more limited in the data they generate. However, CNV data can be inferred from scRNA-seq and bulk RNA-seq, for which several tools have been developed, including inferCNV, CaSpER, and HoneyBADGER. Inferences regarding tumor clonality from CNV data (and other sources) are frequently visualized using phylogenetic plots, which previously required time-consuming and error-prone, manual analysis.
Here, we present Uphyloplot2, a python script that generates phylogenetic plots directly from inferred RNA-seq data, or any Newick formatted dendrogram file. The tool is publicly available at https://github.com/harbourlab/UPhyloplot2/ .
Uphyloplot2 is an easy-to-use tool to generate phylogenetic plots to depict tumor clonality from scRNA-seq data and other sources.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gap junction communication (GJC) mediated by connexins is critical for heart function. To gain insight into the causal relationship of molecular mechanisms of disease pathology, it is important to ...understand which mechanisms contribute to impairment of gap junctional communication. Here, we present an update on the known modulators of connexins, including various interaction partners, kinases, and signaling cascades. This gap junction network (GJN) can serve as a blueprint for data mining approaches exploring the growing number of publicly available data sets from experimental and clinical studies.
Pannexin 1 (Panx1) represents a class of vertebrate membrane channels, bearing significant sequence homology with the invertebrate gap junction proteins, the innexins and more distant similarities in ...the membrane topologies and pharmacological sensitivities with gap junction proteins of the connexin family. In the nervous system, cooperation among pannexin channels, adenosine receptors, and K(ATP) channels modulating neuronal excitability via ATP and adenosine has been recognized, but little is known about the significance in vivo. However, the localization of Panx1 at postsynaptic sites in hippocampal neurons and astrocytes in close proximity together with the fundamental role of ATP and adenosine for CNS metabolism and cell signaling underscore the potential relevance of this channel to synaptic plasticity and higher brain functions. Here, we report increased excitability and potently enhanced early and persistent LTP responses in the CA1 region of acute slice preparations from adult Panx1(-/-) mice. Adenosine application and N-methyl-D-aspartate receptor (NMDAR)-blocking normalized this phenotype, suggesting that absence of Panx1 causes chronic extracellular ATP/adenosine depletion, thus facilitating postsynaptic NMDAR activation. Compensatory transcriptional up-regulation of metabotropic glutamate receptor 4 (grm4) accompanies these adaptive changes. The physiological modification, promoted by loss of Panx1, led to distinct behavioral alterations, enhancing anxiety and impairing object recognition and spatial learning in Panx1(-/-) mice. We conclude that ATP release through Panx1 channels plays a critical role in maintaining synaptic strength and plasticity in CA1 neurons of the adult hippocampus. This result provides the rationale for in-depth analysis of Panx1 function and adenosine based therapies in CNS disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ...ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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