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Abstract: NEMO/IKKγ gene, which is responsible of two allelic diseases in human, EDA-ID and IP, encodes for a protein with a central regulatory role in the activation of the NF-kB pathway. We here provide insights into the molecular mechanism governing NEMO/IKKγ expression. We mapped 4 distinctive NEMO/IKKγ transcription start sites each corresponding to an alternative first exon, controlled by two conserved promoters. A distal promoter, named promoter A, located 10 kb upstream of the coding region and a proximal promoter, promoter B, with strong bi-directional activity driving also the transcription of G6PD gene in the opposite direction. The promoter B is housekeeping, it is embedded in a CpG island, required for proper expression and it is down-regulated by methylation. The promoter A is active in cells of hepatic origin and it directs transcription of the main NEMO/IKKγ 5′ UTR alternative transcript in liver, which starts at a tissue-specific site. Qualitative and quantitative expression analysis revealed that each NEMO/IKKγ 5′ UTR alternative transcript has different expression profiles indicating that the control of NEMO/IKKγ expression is mediated through tissue-specific transcription initiation sites and multiple regulatory regions. [Copyright &y& Elsevier]

Membrane-associated guanylate kinase (MAGUK) proteins are cell–cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein–protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein–protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31–33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa. [Copyright &y& Elsevier]

• Cryopreserved primary mouse cortical cells can be used for single-cell RNA sequencing. • High reproducible single-cell data can be obtained from E15.5 mouse embryos. • Neurostore-cryoconserved samples can be shipped to other laboratories in dry-ice for further perspective studies. The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas. Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing. We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity. By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis. Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations. [ABSTRACT FROM AUTHOR]