Exploring differential exon usage via short- and long-read RNA sequencing strategies.

Alternative splicing produces various mRNAs, and thereby various protein products, from one gene, impacting a wide range of cellular activities. However, accurate reconstruction and quantification of full-length transcripts using short-reads is limited, due to their length. Long-reads sequencing technologies may provide a solution by sequencing full-length transcripts. We explored the use of both Illumina short-reads and two long Oxford Nanopore Technology (cDNA and Direct RNA) RNA-Seq reads for detecting global differential splicing during mouse embryonic stem cell differentiation, applying several bioinformatics strategies: gene-based, isoform-based and exon-based. We detected the strongest similarity among the sequencing platforms at the gene level compared to exon-based and isoform-based. Furthermore, the exon-based strategy discovered many differential exon usage (DEU) events, mostly in a platform-dependent manner and in non-differentially expressed genes. Thus, the platforms complemented each other in the ability to detect DEUs (i.e. long-reads exhibited an advantage in detecting DEUs at the UTRs, and short-reads detected more DEUs). Exons within 20 genes, detected in one or more platforms, were here validated by PCR, including key differentiation genes, such as Mdb3 and Aplp1. We provide an important analysis resource for discovering transcriptome changes during stem cell differentiation and insights for analysing such data.

DestVI identifies continuums of cell types in spatial transcriptomics data.

Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

Unmasking cellular response of a bloom-forming alga to viral infection by resolving expression profiles at a single-cell level.

Infection by large dsDNA viruses can lead to a profound alteration of host transcriptome and metabolome in order to provide essential building blocks to support the high metabolic demand for viral assembly and egress. Host response to viral infection can typically lead to diverse phenotypic outcome that include shift in host life cycle and activation of anti-viral defense response. Nevertheless, there is a major bottleneck to discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidic setup that captures individual algal cells and multiplex quantitate PCR. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and susceptible cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.

Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity.

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

Emission of membrane vesicles: roles in complement resistance, immunity and cancer.

Complement-mediated cell death is caused by C5b-9, the membrane attack complex (MAC) composed of the five complement proteins C5b, C6, C7, C8, and C9. Assembly of the C5b-9 complex initiates oligomerization of C9 and production of a transmembrane protein channel that inflicts damage to target cells. For protection, cells eliminate the MAC from their surface either by ectocytosis (direct emission of membrane vesicles) or by endocytosis (internalization). The process of ectosome release is rapid and involves cytosolic Ca(2+) and activation of protein kinases, such as protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK). Recently, the involvement of mortalin (also known as GRP75 and mitochondrial hsp70) in MAC elimination has been suggested. Extracellular application of antibodies directed to mortalin increases cell sensitivity to MAC-mediated lysis. Release of membrane vesicles is ubiquitous and enhanced in apoptotic or tumor cells and upon cell activation. Composition of the ectosomes (also often referred to as microparticles) membrane proteins and lipids appears to be different from those of the original plasma membrane, indicating involvement of a selective sorting process during ectosome formation. Exosomes (unlike ectosomes) are membrane vesicles generated by endocytosis, endosome sorting into perinuclear multivesicular bodies (MVB) and exocytosis of MVBs. Exosomes appear to be different in size and composition from ectosomes. Exosome-associated MAC has also been described. Although research on ectosomes and exosomes is still limited, physiological roles in coagulation, vascular functions, angiogenesis, wound healing and development have been attributed to these shed membrane vesicles. On the other hand, there are indications that elevated levels of ectosomes and exosomes may predispose to morbidity. Membrane vesicles released by cells exposed to complement MAC may play roles in health and disease beyond protection from cell death.

Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis.

The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis.

Comparative genomic hybridization analysis of craniopharyngiomas.

OBJECT: Craniopharyngioma is the most common childhood brain tumor and is thought to arise from embryonic remnants of the Rathke pouch. Some craniopharyngiomas are monoclonal in origin and hence presumably harbor somatic genetic alterations, although the precise molecular mechanisms involved in craniopharyngioma development are unknown. The goal of this study was to identify genetic alterations in craniopharyngiomas. METHODS: To gain insight into the molecular mechanisms involved in development of these tumors, the authors analyzed nine adamantinomatous craniopharyngiomas by using comparative genomic hybridization. Six tumors (67%) displayed at least one genomic alteration, and three had six or more alterations. Only two tumors displayed a decrease in DNA copy number, and in all others an increase in DNA copy number was noted. CONCLUSIONS: The authors conclude that a subset of craniopharyngiomas consists of monoclonal tumors arising from activation of oncogenes located at specific chromosomal loci.