NEW INSIGHTS INTO THE ROLE OF THE CENTROSOMAL MARK4 KINASE IN REGULATING THE DYNAMICS AND REMODELLING OF CYTOSKELETON FROM OVEREXPRESSION STUDIES OF ITS TWO ISOFORMS IN NORMAL AND TUMOR CELLS

MAP/Microtubule Affinity Regulating Kinase 4 (MARK4) belongs to a highly conserved family of serine\u2013threonine kinases (MARKs) that are able to phosphorylate the microtubule associated proteins (MAPs), and cause these proteins to detach from microtubules (MTs) increasing microtubules dynamics. MARKs kinases represent the mammalian homologues of PAR-1, a protein involved in establishment of the cell shape and polarity in lower eukaryotes. The MARK4 gene is located at 19q13.2 and encodes at least two alternatively spliced isoforms, MARK4L and MARK4S, which have an identical protein structure apart from the C-terminal region. The two isoforms are differentially expressed in human tissues, particularly in the central nervous system (CNS). Several studies reported that MARK4S is expressed in normal brain tissue and neurons, suggesting that this isoform has a role in neuronal differentiation. Conversely, MARK4L is up-regulated in glioma and neural progenitor cells, pointing to a possible role of this isoform in cell proliferation. Recently, we highlighted an increasingly subverted MARK4L/MARK4S ratio, with prevalence of MARK4L, in glioblastoma and glioblastoma-derived cancer stem cells, that recapitulate the expression profiling of neural stem cells. These findings suggest that the expression of the two MARK4 isoforms is tightly regulated during the proliferation/differentiation of neural stem cells and changes in their expression levels may be a molecular marker of tumour transformation. Unlike the other members of the family (MARK1, MARK2 and MARK3), that exhibit uniform cytoplasmic localisation, both MARK4 isoforms localise at the centrosomes and in the midbody, supporting their involvement in mitotic division and cytokinesis. To elucidate the role played by MARK4 isoforms in cell cycle progression and in the regulation of the cytoskeleton, we monitored the activation status of MARK4 during the cell cycle and performed overexpression experiments in fibroblasts and glioma cell lines. We showed that despite MARK4 is expressed across all the cell cycle phases, its active form, phosphorylated at the Thr214 residue, is prevalent in mitosis. Phospho-MARK4 is detected in centrosomes at all mitotic stages and in the midbody during cytokinesis. Conversely, only a fraction of interphase centrosomes show phospho-MARK4 positive signals. Overexpression experiments on fibroblasts and glioma cell lines demonstrated the role of MARK4 in the regulation of cytoskeleton dynamics. Indeed overexpression of MARK4L or MARK4S led to a sharp decrease in microtubule density in both the cell systems, as monitored by immunofluorescence experiments. By contrast, overexpression of catalytically inactive MARK4L/MARK4S mutants, did not affect the microtubule network, indicating that the effects on MTs are dependent on the kinase activity of MARK4 and likely linked to MAPs phosphorylation. Besides the effect on MT array, overexpressed MARK4L in fibroblasts showed a filamentous staining pattern, co-localising with vimentin, the core component of cytoskeleton intermediate filaments. In contrast, overexpressed MARK4S co-localised with vimentin to a lesser extent and only in few cells. The MARK4L-vimentin co-localisation was particularly evident in the perinuclear zone and in some overexpressing cells, the filaments appeared reshaped as compared to those in GFP-transfected cells, and showed the formation of bundle structures. These alterations seem to be due to the kinase activity of MARK4L since overexpression of kinase dead mutants did not remodel the intermediate filaments. The overall data highlight MARK4 as a key component in the regulation of microtubules dynamics, and indicate vimentin as a plausible target of MARK4L activity, suggesting a wide-ranging influence of MARK4 on cytoskeleton. Moreover the dynamic involvement of active MARK4 in structure like centrosomes and midbody, crucial for mitosis and cytokinesis, point to a fundamental role for this kinase in the cell cycle

Profound alterations of the chromatin architecture at chromosome 11p15.5 in cells from Beckwith-Wiedemann and Silver-Russell syndromes patients

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting-related disorders associated with genetic/epigenetic alterations of the 11p15.5 region, which harbours two clusters of imprinted genes (IGs). 11p15.5 IGs are regulated by the methylation status of imprinting control regions ICR1 and ICR2. 3D chromatin structure is thought to play a pivotal role in gene expression control; however, chromatin architecture models are still poorly defined in most cases, particularly for IGs. Our study aimed at elucidating 11p15.5 3D structure, via 3C and 3D FISH analyses of cell lines derived from healthy, BWS or SRS children. We found that, in healthy cells, IGF2/H19 and CDKN1C/KCNQ1OT1 domains fold in complex chromatin conformations, that facilitate the control of IGs mediated by distant enhancers. In patient-derived cell lines, we observed a profound impairment of such a chromatin architecture. Specifically, we identified a cross-talk between IGF2/H19 and CDKN1C/KCNQ1OT1 domains, consisting in in cis, monoallelic interactions, that are present in healthy cells but lost in patient cell lines: an inter-domain association that sees ICR2 move close to IGF2 on one allele, and to H19 on the other. Moreover, an intra-domain association within the CDKN1C/KCNQ1OT1 locus seems to be crucial for maintaining the 3D organization of the region

Generation of the Becker muscular dystrophy patient derived induced pluripotent stem cell line carrying the DMD splicing mutation c.1705-8 T>C

Becker Muscular dystrophy (BMD) is an X-linked syndrome characterized by progressive muscle weakness. BMD is generally less severe than Duchenne Muscular Dystrophy. BMD is caused by mutations in the dystrophin gene that normally give rise to the production of a truncated but partially functional dystrophin protein. We generated an induced pluripotent cell line from dermal fibroblasts of a BMD patient carrying a splice mutation in the dystrophin gene (c.1705-8 T>C). The iPSC cell-line displayed the characteristic pluripotent-like morphology, expressed pluripotency markers, differentiated into cells of the three germ layers and had a normal karyotype

Standardized Methods for Enhanced Quality and Comparability of Tuberculous Meningitis Studies

Tuberculous meningitis (TBM) remains a major cause of death and disability in tuberculosis-endemic areas, especially in young children and immunocompromised adults. Research aimed at improving outcomes is hampered by poor standardization, which limits study comparison and the generalizability of results. We propose standardized methods for the conduct of TBM clinical research that were drafted at an international tuberculous meningitis research meeting organized by the Oxford University Clinical Research Unit in Vietnam. We propose a core dataset including demographic and clinical information to be collected at study enrollment, important aspects related to patient management and monitoring, and standardized reporting of patient outcomes. The criteria proposed for the conduct of observational and intervention TBM studies should improve the quality of future research outputs, can facilitate multicenter studies and meta-analyses of pooled data, and could provide the foundation for a global TBM data repository

Generation of the Rubinstein-Taybi syndrome type 2 patient-derived induced pluripotent stem cell line (IAIi001-A) carrying the EP300 exon 23 stop mutation c.3829A > T, p.(Lys1277*)

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutation in either the CREBBP (RSTS1) or EP300 (RSTS2) genes. We generated an induced pluripotent stem cell line from an RSTS2 patient's blood mononuclear cells by Sendai virus non integrative reprogramming method. The iPSC line (IAIi001RSTS2-65-A) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was stable by karyotyping. Mutation and western blot analyses demonstrated in IAIi001RSTS2-65-A the patient's specific non sense mutation in exon 23 c.3829A > T, p.(Lys 1277*) and showed reduced quantity of wild type p300 protein

Epigenetic effects of chromatin remodeling agents on organotypic cultures

Background: Tumor epigenetic defects are of increasing relevance to clinical practice, because they are 'druggable' targets for cancer therapy using chromatin-remodeling agents (CRAs). New evidences highlight the importance of the microenvironment on the epigenome regulation and the need to use culture models able to preserve tissue morphology, to better understand the action of CRAs. Methods & methods: We studied the epigenetic response induced by culturing and CRAs in a preclinical model, preserving ex vivo the original tissue microenvironment and morphology, assessing different epigenetic signatures. Our overall findings suggest that culturing and CRAs cause heterogeneous effects on the genes methylation; CRAs affect the global DNA methylation and can trigger an active DNA demethylation; the culture induces alterations in the histone deacetylase expression. Conclusion: Despite the limited number of cases, these findings can be considered a proof of concept of the possibility to test CRAs epigenetic effects on ex vivo tissues maintained in their native tissue architecture

Determination of circulating Mycobacterium tuberculosis strains and transmission patterns among pulmonary TB patients in Kawempe municipality, Uganda, using MIRU-VNTR

<p>Abstract</p> <p>Background</p> <p>Mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) genotyping is a powerful tool for unraveling clonally complex <it>Mycobacterium tuberculosis </it>(MTB) strains and detection of transmission patterns. Using MIRU-VNTR, MTB genotypes and their transmission patterns among patients with new and active pulmonary tuberculosis (PTB) in Kawempe municipality in Kampala, Uganda was determined.</p> <p>Results</p> <p>MIRU-VNTR genotyping was performed by PCR-amplification of 15 MTB-MIRU loci from 113 cultured specimens from 113 PTB patients (one culture sample per patient). To determine lineages, the genotypes were entered into the MIRU-VNTR<it>plus </it>database [<url>http://www.miru-vntrplus.org/</url>] as numerical codes corresponding to the number of alleles at each locus. Ten different lineages were obtained: Uganda II (40% of specimens), Uganda I (14%), LAM (6%), Delhi/CAS (3%), Haarlem (3%), Beijing (3%), Cameroon (3%), EAI (2%), TUR (2%) and S (1%). Uganda I and Uganda II were the most predominant genotypes. Genotypes for 29 isolates (26%) did not match any strain in the database and were considered unique. There was high diversity of MIRU-VNTR genotypes, with a total of 94 distinct patterns. Thirty four isolates grouped into 15 distinct clusters each with two to four isolates. Eight households had similar MTB strains for both index and contact cases, indicating possible transmission.</p> <p>Conclusion</p> <p>MIRU-VNTR genotyping revealed high MTB strain diversity with low clustering in Kawempe municipality. The technique has a high discriminatory power for genotyping MTB strains in Uganda.</p

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G\u2009>\u2009C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3'-phosphate, 5'-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here

Differential signature of the centrosomal MARK4 isoforms in glioma

Background: MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation. Methods: We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs) and mouse neural stem cells (NSCs) by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence. Results: Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells. Conclusions: The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker