113,817 research outputs found
Molecular bases of GLUT1 deficiency syndrome: functional in vitro and in silico studies and a workflow implementation for variants’ calling and annotation
No full textGLUT1 deficiency syndrome (GLUT1 DS, OMIM #606777) is a rare genetic disorder characterized by insufficient glucose transport into the brain due to variants in the SLC2A1 gene, which encodes the GLUT1 protein, that facilitates glucose crossing the blood-brain barrier, essential for brain energy.
Classic GLUT1 DS phenotype is characterized by seizures, movement disorders, and cognitive/behavioral disturbances, however the expanding phenotypic spectrum includes various other symptoms, such as paroxysmal non-epileptic manifestations, and mild phenotypes.
Over 300 variants in SLC2A1 have been identified, ranging from large deletions to single nucleotide variants (SNVs) and insertions/deletions (INDELs), with significant phenotypic variability and with no clear genotype-phenotype correlations. Moreover, in about 20% of patients with clinical signs of the syndrome, no SLC2A1 mutation has been identified. The molecular pathogenetic mechanism is caused by haploinsufficiency, mainly due to loss-of-function variants, however it has been suggested, and only preliminary demonstrated, that missense variants may affect GLUT1 function in multiple ways.
In this work of thesis, we investigated the functional effects of selected SLC2A1 missense variants with different and complementary approaches, both in vitro and in silico, and we implemented an integrated analysis workflow to potentially increase the diagnostic yield of GLUT1 DS genetic tests. Glucose uptake assay detected a reduction of glucose transport in some mutants and, for three of them, immunofluorescence analysis showed an alteration of GLUT1 subcellular localization and trafficking, with evident accumulations in late endosomes/lysosomes. These results were concordant with in silico analysis by Molecular Dynamics simulations, showing an important steric hindrance hampering the correct glucose pathway.
We also implemented an integrative workflow for Next Generation Sequencing (NGS) data analysis for the SLC2A1 gene, including variant call, biological annotation, and filtering criteria to support functional and clinical interpretation of potentially pathogenic variants, especially those that are more difficult to interpret, namely missense variants, which are often classified as variants of uncertain significance (VUS). This workflow also considers variants that are usually filtered out in standard analysis (i.e. intronic, synonymous, UTRs) or not called by the standard pipelines (as Copy Number Variants and somatic variants), that can still prove to be pathogenic.
The detailed knowledge of different pathogenic mechanisms caused by GLUT1 mutations, the increase of diagnostic yield of genetics tests, and the resulting improvement of genotype-phenotype correlations may have important implications in genetic counseling and patient management, as a timely diagnosis is crucial for therapy, since ketogenic diet is the standard of care treatment
Functional studies of human K+ channels KCNH1 and KCNK4 and their role in human pathogenesis
No full textPotassium (K+) channels constitute the most diversified class of ion channels with regard to structure and gating characteristics. They contribute to the maintenance and stabilization of the resting potential and are key players in regulating cell excitability and functions in response to multiple signals (Tian et al., 2014). In recent years, the aberrant function of some of these channels has been documented to affect development and underlie syndromic disorders (Bauer et al., 2018).
KCNH1 (MIM 603305) is a member of the EAG (ether-à-go-go) family of voltage-gated K+ channels (Cázares-Ordoñez and Pardo, 2017). Recent studies have demonstrated that gain-of-function mutations in KCHN1 are implicated in Zimmermann-Laband syndromes (ZLS; MIM 135500;Kortüm et al., 2015) and other forms of developmental deficits that include mental retardation and epilepsy (Simons et al., 2015;Bramswig et al., 2015; Mégarbané et al., 2016; Fukai et al., 2016). These findings suggest that KCNH1 might be important for cognitive development in human. Recently, gain-of-function mutations in KCNK4 (MIM 605720), encoding a two-pore-domain K+ channel (K2P), have been reported in subjects with a phenotype of facial dysmorphism, hypertrichosis, epilepsy, developmental delay/ID, and gingival overgrowth (FHEIG syndrome, MIM 618381; Bauer et al., 2018). The clinical features of the KCNK4-related condition are reminiscent of ZLS (Kortüm et al., 2015), providing evidence for a channelopathy caused by hyperactivation of K+ channels, including KCNH1 and KCNK4. Some ion channels are bifunctional proteins contributing to several cell functions (Hegle et al., 2006). To date, only electrophysiology experiments were performed to explore the mechanism involving KCNH1 and KCNK4 in ZLS and FHEIG related developmental processes (Kortüm et al., 2015; Simons et al.,2015; Bauer et al., 2018).
Based on these considerations, major aims of my PhD project were to better characterize the cell role of KCNH1 and KCNK4 channels and to explore the functional impact of the KCNH1-ZLS and KCNK4-FHEIG mutations using cellular and molecular biology approaches, including Immunofluorescence, Western Blot and Real-Time Quantitative PCR. To this aim, we used cell lines, control fibroblasts and primary skin fibroblasts derived from ZLS-patients carrying mutations in KCNH1 (p.Arg330Gln and p.Leu352Val) and from FHEIG-patients carrying mutations in KCNK4 (p.Ala172Glu and p.Ala244Pro). We demonstrated impaired proliferation of KCNH1 and KCNK4 mutant fibroblasts, confirming the role of KCNH1 as a regulator of cell cycle of non-transformed cells and highlight a new function for KCNK4 in regulation of cell proliferation. We also found a significant increase of cilia number and a cilium-related pathway, i.e. SHH pathway, activation in all the mutant fibroblasts, thus suggesting functional role of KCNH1 and KCNK4 in cilia regulation. Moreover, confocal microscopy analysis revealed defects in cilia morphology in fibroblasts from ZLS patients. Confocal analysis refined also the reported KCNH1 localization in the cilium (Sánchez et al., 2016) to be concentrated at the centrosome and ciliary pocket regions for both wild-type and mutant fibroblasts. Finally, immunofluorescence analysis conducted in
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wild-type fibroblasts and cells lines disclosed a nucleolar localization of KCNK4 channels, possibly indicating unrevealed roles of KCNK4.
In summary, we demonstrated that KCNH1 and KCNK4 may have specific ion channel-independent function converging on some share cellular pathways whose alteration might affect development processes. Overall, these findings highlight the importance to characterize the intracellular role of K+ to shed light on the mechanisms of pathogenesis of ZLS and ZLS related developmental processes
Comparison of different protocols for the quantification of trunk kinematics and dynamics in controlled conditions
No full textATM: An unexpected tumor-promoting factor in HER2-expressing tumors
No full textATM kinase is a gatekeeper of genome stability. However, its role in several other signaling pathways suggests that it might not always act as a tumor suppressor. Here, we discuss recent data that unveil a function of ATM as a tumor promoter in HER2-positive breast cancer
Imaging nuclear, endoplasmic reticulum and plasma membrane events in real time
No full textLive cell imaging can provide important information on cellular dynamics; however, the full utilisation of this technology has been hampered by the limitations of imaging reagents. Metal-based complexes have the potential to overcome many of the issues common to many current imaging agents. The rhenium (I)-based complex fac-[Re(CO)3 (1,10-phenanthroline)(4-pyridyltetrazolate)], herein referred to as ReZolve-ER(™) , shows promise as a live cell imaging agent with rapid cell uptake, low cytotoxicity, resistance to photobleaching and compatibility with multicolour imaging. ReZolve-ER(™) localised to the nuclear membrane/endoplasmic reticulum (ER) and allowed the detection of exocytotic events at the plasma membrane. Thus, we present a new imaging agent for monitoring live cell events in real time, which is ideal for imaging either short- or long-time courses.Christie A. Bader, Alexandra Sorvina, Peter V. Simpson, Phillip J. Wright, Stefano Stagni, Sally E. Plush, Massimiliano Massi and Douglas A. Brook
Ataxia-telangiectasia mutated kinase in the control of oxidative stress, mitochondria, and autophagy in cancer: A maestro with a large orchestra
No full textAtaxia-telangiectasia mutated kinase (ATM) plays a central role in the DNA damage response (DDR) and mutations in its gene lead to the development of a rare autosomic genetic disorder, ataxia telangiectasia (A-T) characterized by neurodegeneration, premature aging, defects in the immune response, and higher incidence of lymphoma development. The ability of ATM to control genome stability several pointed to ATM as tumor suppressor gene. Growing evidence clearly support a significant role of ATM, in addition to its master ability to control the DDR, as principle modulator of oxidative stress response and mitochondrial homeostasis, as well as in the regulation of autophagy, hypoxia, and cancer stem cell survival. Consistently, A-T is strongly characterized by aberrant oxidative stress, significant inability to remove damaged organelles such as mitochondria. These findings raise the question whether ATM may contribute to a more general hijack of signaling networks in cancer, therefore, playing a dual role in this context. Indeed, an unexpected tumorigenic role for ATM, in particular, tumor contexts has been demonstrated. Genetic inactivation of Beclin-1, an autophagy regulator, significantly reverses mitochondrial abnormalities and tumor development in ATM-null mice, independently of DDR. Furthermore, ATM sustains cancer stem cells survival by promoting the autophagic flux and ATM kinase activity is enhanced in HER2-dependent tumors. This mini-review aims to shed new light on the complexity of these new molecular circuits through which ATM may modulate cancer progression and to highlight a novel role of ATM in the control of proteostasis
Virtual organization management across middleware boundaries
No full textOne of the most important challenges in production Grids is to achieve interoperation across several heterogeneous Grid middleware platforms: eScience applications need a coordinated resource sharing among dynamic collections of individuals/institutions, independently from whatever middleware the resources are running. For this reason, there is a great effort going on to define standard interfaces, in order to implement common services that can be used to achieve cross-middlewares interoperability. In this paper, we present our modifications to the Virtual Organization Management Service (VOMS), a widely-known and used tool that acts as an Attribute Authority. We enhanced VOMS to expose the standardized interface of the Security Assertion Markup Language (SAML), and therefore to release SAML assertions. This way we want VOMS to be available on the larger possible number of Grid middleware platforms
A new player in the development of TRAIL based therapies for hepatocarcinoma treatment: ATM kinase
No full textHepatocellular carcinoma (HCC) is one of the most common cancers worldwide. HCCs are genetically and phenotypically heterogeneous tumors characterized by very poor prognosis, mainly due to the lack, at present, of effective therapeutic options, as these tumors are rarely suitable for radiotherapy and often resistant to chemotherapy protocols. In the last years, agonists targeting the Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) death receptor, has been investigated as a valuable promise for cancer therapy, based on their selectivity for malignant cells and low toxicity for healthy cells. However, many cancer models display resistance to death receptor induced apoptosis, pointing to the requirement for the development of combined therapeutic approaches aimed to selectively sensitize cancer cells to TRAIL. Recently, we identified ATM kinase as a novel modulator of the ability of chemotherapeutic agents to enhance TRAIL sensitivity. Here, we review the biological determinants of HCC responsiveness to TRAIL and provide an exhaustive and updated analysis of the molecular mechanisms exploited for combined therapy in this context. The role of ATM kinase as potential novel predictive biomarker for combined therapeutic approaches based on TRAIL and chemotherapeutic drugs will be closely discussed
Unprecedented staining of polar lipids by a luminescent rhenium complex revealed by FTIR microspectroscopy in adipocytes
No full textData source: Supplementary information, http://www.rsc.org/suppdata/c6/mb/c6mb00242k/c6mb00242k1.pdfFourier transform infrared (FTIR) microspectroscopy and confocal imaging have been used to demonstrate that the neutral rhenium(i) tricarbonyl 1,10-phenanthroline complex bound to 4-cyanophenyltetrazolate as the ancillary ligand is able to localise in regions with high concentrations of polar lipids such as phosphatidylethanolamine (PE), sphingomyelin, sphingosphine and lysophosphatidic acid (LPA) in mammalian adipocytes.C. A. Bader, E. A. Carter, A. Safitri, P. V. Simpson, P. Wright, S. Stagni, M. Massi, P. A. Lay, D. A. Brooksa and S. E. Plus
Quantitative Monostatic GPR Analysis in Interface Profiling: Multisignal Detection-Based Layer Estimation
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