The importance of murine phospho-MLKL-S345 in situ detection for necroptosis assessment in vivo.

Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl-/- or Ripk3-/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.

Insights into the structure and function of the RNA ligase RtcB.

To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.

Illuminating the role of lncRNAs ROR and MALAT1 in cancer stemness state of anaplastic thyroid cancer: An exploratory study.

Background: Anaplastic thyroid cancer (ATC) is one of the most aggressive malignancies in humans that accounts for a considerable rate of cancer-associated mortality. Since conventional therapies are lacking sufficient efficacy, new treatment approaches are required. This goal could be achieved through a better understanding of the molecular pathogenesis of ATC. Thyroid tumorigenesis is initiated by a subpopulation of cells known as cancer stem cells (CSCs) with specific markers such as CD133 that confers to processes such as self-renewal and metastasis. Besides, some long non-coding RNAs (lncRNAs) promote tumorigenesis by mediating the aforementioned processes. Methods: Here, we designed an exploratory study to investigate the role of lncRNAs ROR and MALAT1 and their related genes in CSC stemness. Using magnetic-activated cell sorting (MACS), the CD133- and CD133+ subpopulations were separated in SW1736 and C643 ATC cell lines. Next, the expression profiles of the CD133 marker, MALAT1, and its associated genes (CCND1, NESTIN, MYBL2, MCL1, IQGAP1), as well as ROR and its related genes (POU5F1, SOX2, NANOG), were explored by qRT-PCR. Results: We found significant up-regulation of ROR, POU5F1, SOX2, NANOG, CD133, MALAT1, IQGAP1, and MCL1 in CD133+ SW1736 cells compared to CD133- cells. As for CD133+ C643 cells, CCND1, IQGAP1, POU5F1, SOX2, NANOG, and NESTIN were significantly up-regulated compared to CD133- cells. Conclusions: This study suggests that these lncRNAs in CD133-positive SW1736 and C643 cells might regulate stemness behaviors in ATC.

Network analysis reveals essential proteins that regulate sodium-iodide symporter expression in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most rare and lethal form of thyroid cancer and requires effective treatment. Efforts have been made to restore sodium-iodide symporter (NIS) expression in ATC cells where it has been downregulated, yet without complete success. Systems biology approaches have been used to simplify complex biological networks. Here, we attempt to find more suitable targets in order to restore NIS expression in ATC cells. We have built a simplified protein interaction network including transcription factors and proteins involved in MAPK, TGFß/SMAD, PI3K/AKT, and TSHR signaling pathways which regulate NIS expression, alongside proteins interacting with them. The network was analyzed, and proteins were ranked based on several centrality indices. Our results suggest that the protein interaction network of NIS expression regulation is modular, and distance-based and information-flow-based centrality indices may be better predictors of important proteins in such networks. We propose that the high-ranked proteins found in our analysis are expected to be more promising targets in attempts to restore NIS expression in ATC cells.

A meta-analysis of gene expression data highlights synaptic dysfunction in the hippocampus of brains with Alzheimer's disease.

Since the world population is ageing, dementia is going to be a growing concern. Alzheimer's disease is the most common form of dementia. The pathogenesis of Alzheimer's disease is extensively studied, yet unknown remains. Therefore, we aimed to extract new knowledge from existing data. We analysed about 2700 upregulated genes and 2200 downregulated genes from three studies on the CA1 of the hippocampus of brains with Alzheimer's disease. We found that only the calcium signalling pathway enriched by 48 downregulated genes was consistent between all three studies. We predicted miR-129 to target nine out of 48 genes. Then, we validated miR-129 to regulate six out of nine genes in HEK cells. We noticed that four out of six genes play a role in synaptic plasticity. Finally, we confirmed the upregulation of miR-129 in the hippocampus of brains of rats with scopolamine-induced amnesia as a model of Alzheimer's disease. We suggest that future research should investigate the possible role of miR-129 in synaptic plasticity and Alzheimer's disease. This paper presents a novel framework to gain insight into potential biomarkers and targets for diagnosis and treatment of diseases.