Carbon nanofiber-based multiplexed immunosensor for the detection of survival motor neuron 1, cystic fibrosis transmembrane conductance regulator and Duchenne Muscular Dystrophy proteins.

Simultaneous and point-of-care detection of multiple protein biomarkers has significant impact on patient care. Spinal Muscular Atrophy (SMA), Cystic Fibrosis (CF) and Duchenne Muscular Dystrophy (DMD) are well known progressive hereditary disorders associated with increased morbidity as well as mortality. Therefore, rapid detection of biomarkers specific for these three disorders in newborns offers new opportunities for early diagnosis, delaying symptoms and effective treatment. Here, we report the development of a disposable carbon nanofiber (CNF)-based electrochemical immunosensor for simultaneous detection of survival motor neuron 1 (SMN1), cystic fibrosis transmembrane conductance regulator (CFTR) and DMD proteins. The CNF-modified array electrodes were first functionalized by electroreduction of carboxyphenyl diazonium salt. Then, the immunosensor was fabricated by the covalent immobilization of the three antibodies on the working electrodes of the array sensor via carbodiimide (EDC/NHS) chemistry. Simultaneous detection of CFTR, DMD and SMN1 was achieved with high sensitivity and detection limits of 0.9 pg/ml, 0.7 pg/ml and 0.74 pg/ml, respectively. The multiplexed immunosensor has also shown strong selectivity against non-specific proteins. Moreover, high recovery percentage was obtained when the immunosensor was applied in spiked whole blood samples. This voltammetric immunosensor offers cost effective, easy to use, rapid and high throughput potential screening method for these three hereditary disorders using only few drops of blood.

Electrochemical immunosensors for the detection of survival motor neuron (SMN) protein using different carbon nanomaterials-modified electrodes.

Spinal muscular atrophy is an untreatable potentially fatal hereditary disorder caused by loss-of-function mutations in the survival motor neuron (SMN) 1 gene which encodes the SMN protein. Currently, definitive diagnosis relies on the demonstration of biallelic pathogenic variants in SMN1 gene. Therefore, there is an urgent unmet need to accurately quantify SMN protein levels for screening and therapeutic monitoring of symptomatic newborn and SMA patients, respectively. Here, we developed a voltammetric immunosensor for the sensitive detection of SMN protein based on covalently functionalized carbon nanofiber-modified screen printed electrodes. A comparative study of six different carbon nanomaterial-modified electrodes (carbon, graphene (G), graphene oxide (GO), single wall carbon nanotube (SWCNT), multi-wall carbon nanotube (MWCNT), and carbon nanofiber (CNF)) was performed. 4-carboxyphenyl layers were covalently grafted on the six electrodes by electroreduction of diazonium salt. Then, the terminal carboxylic moieties on the electrodes surfaces were utilized to immobilize the SMN antibody via EDC/NHS chemistry and to fabricate the immunosensors. The electrochemical characterization and analytical performance of the six immunosensors suggest that carbon nanofiber is a better electrode material for the SMN immunosensor. The voltammetric SMN carbon nanofiber-based immunosensor showed high sensitivity (detection limit of 0.75pg/ml) and selectivity against other proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and dystrophin (DMD). We suggest that this novel biosensor is superior to other developed assays for SMN detection in terms of lower cost, higher sensitivity, simplicity and capability of high throughput screening.

[The construction of urine-derived cell lines from patients with spinal muscular atrophy].

Spinal muscular atrophy (SMA) is a common neurodegenerative disease in childhood and infancy, clinically characterized by progressive and symmetric muscular weakness and atrophy. Few effective therapies are available now, and SMA is one of the most common genetic causes of infantile mortality. SMA patient-derived cells are beneficial in basic research on this disease, but the most common model cell, fibroblasts can only be obtained through invasive procedures such as muscle or skin biopsy, which are unwelcome to patients and their families. In this study, fresh urine from SMA patients and healthy controls was collected and centrifuged, and the urine sediment was cultured in vitro. The growth characteristics of urine-derived cells were observed, and the survival of motor neuron (SMN) gene, and the amount and localization of SMN protein in different urine cell lines were investigated. In total, 25 urine cell lines from 11 SMA patients and 14 healthy controls were established. These urine-derived cells expand robustly in vitro with stable cell morphological characteristics. The urine cell lines derived from patients carry the SMN1 gene defect and express a low level of SMN protein, while the intracellular localization of SMN protein is normal. Urine-derived cell culture technology is simple, non-invasive and highly reproducible, a way of obtaining and storing rare cell samples from SMA patients with which to study the pathogenesis of SMA.

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds.

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA.

Laminin induced local axonal translation of ß-actin mRNA is impaired in SMN-deficient motoneurons.

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of ß-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the ß-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of ß-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that ß-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the ß-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of ß-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn(-/-);SMN2). Taken together our findings suggest that local translation of ß-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.

Towards the mammalian interactome: Inference of a core mammalian interaction set in mouse.

Protein-protein interactions (PPIs) underpin key biological processes in cells. Describing and interpreting this network of interactions is a key focus of computational systems biology. While mouse is commonly used as a model system for mammalian biology, the description of mouse PPIs available in public interaction databases is remarkably poor. Collectively, public resources such as BIND, IntACT and MINT contain only a few thousand mouse PPIs, far behind the many tens or hundreds of thousands likely to exist. To supplement this lack and to take advantage of other high-throughput omic data sets in mouse, here we identify that portion of the human interactome with orthologs in mouse, and from that infer a mouse interolog network. By inferring interactions in mouse based on only the most closely related species with abundant PPI data (human), we create a view of mouse interactions enriched for shared mammalian biological processes. We also demonstrate that available methods for determining orthologs between even closely related species produce distinctly different results, and we propose an integrated view of mouse-human orthology from which to infer a broader interolog network.

Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy.

We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.