A Targeted Gene Panel That Covers Coding, Non-coding and Short Tandem Repeat Regions Improves the Diagnosis of Patients With Neurodegenerative Diseases.
ABSTRACT: Genetic testing for neurodegenerative diseases (NDs) is highly challenging because of genetic heterogeneity and overlapping manifestations. Targeted-gene panels (TGPs), coupled with next-generation sequencing (NGS), can facilitate the profiling of a large repertoire of ND-related genes. Due to the technical limitations inherent in NGS and TGPs, short tandem repeat (STR) variations are often ignored. However, STR expansions are known to cause such NDs as Huntington's disease and spinocerebellar ataxias type 3 (SCA3). Here, we studied the clinical utility of a custom-made TGP that targets 199 NDs and 311 ND-associated genes on 118 undiagnosed patients. At least one known or likely pathogenic variation was found in 54 patients; 27 patients demonstrated clinical profiles that matched the variants; and 16 patients whose original diagnosis were refined. A high concordance of variant calling were observed when comparing the results from TGP and whole-exome sequencing of four patients. Our in-house STR detection algorithm has reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort. This study also uncovered a trove of novel and recurrent variants that may enrich the repertoire of ND-related genetic markers. We propose that a combined comprehensive TGPs-bioinformatics pipeline can improve the clinical diagnosis of NDs.
Project description:Genetic testing for diagnosis of neurodegenerative diseases (NDs) is highly challenging because of genotype heterogeneity and overlapping clinical manifestations. Targeted-gene panels (TGPs), coupled with next-generation sequencing (NGS), can facilitate the profiling of a large repertoire of ND-related genes and help with differential diagnosis. Due to the technical limitations inherent in NGS and TGPs, short tandem repeat (STR) variations are often ignored. However, STR expansions are known to cause such NDs as Huntington’s disease and several types of spinocerebellar ataxias, such as SCA type 3 (SCA3).Here, we studied the clinical utility of a custom-made TGP that targets 199 NDs and 311 ND-associated genes on 118 undiagnosed patients. At least one known pathogenic or likely pathogenic variation was found in 54 (45.8%) patients; 27 (22.9%) of the 118 patients demonstrated clinical profiles that matched the variants. Based on the findings from the TGP, we refined the original diagnosis of 16 patients. A high concordance of single-nucleotide polymorphisms (99.9%) and small insertions and deletions (93.2%) were observed when comparing the results from TGP and whole-exome sequencing of four patients. We tested an in-house STR detection algorithm, and it reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort. This study also uncovered a trove of novel and recurrent variants that may enrich the repertoire of ND-related genetic markers. We propose that a combination of comprehensive TGPs and the bioinformatics analysis pipeline can improve the clinical diagnosis and accelerate the diagnosis of NDs.
Project description:While the central common feature of the neurodegenerative diseases (NDs) is the accumulation of misfolded proteins, they share other pathogenic mechanisms. However, we miss an explanation for the onset of the NDs. The mechanisms through which genetic mutations, present from conception are expressed only after several decades of life are unknown. We aim to find clues on the complexity of the disease onset trigger of the different NDs expressed in the number of steps of factors related to a disease. We collected brain autopsies on diseased patients with NDs, and found a dynamic increase of the ND multimorbidity with the advance of age. Together with the observation that the NDs accumulate multiple misfolded proteins, and the same misfolded proteins are involved in more than one ND, motivated us to propose a model for a genealogical tree of the NDs. To collect the dynamic data needed to build the tree, we used a Big-data approach that searched automatically epidemiological datasets for age-stratified incidence of NDs. Based on meta-analysis of over 400 datasets, we developed an algorithm that checks whether a ND follows a multistep model, finds the number of steps necessary for the onset of each ND, finds the number of common steps with other NDs and the number of specific steps of each ND, and builds with these findings a parsimony tree of the genealogy of the NDs. The tree discloses three types of NDs: the stem NDs with less than 3 steps; the trunk NDs with 5 to 6 steps; and the crown NDs with more than 7 steps. The tree provides a comprehensive understanding of the relationship across the different NDs, as well as a mathematical framework for dynamic adjustment of the genealogical tree of the NDs with the appearance of new epidemiological studies and the addition of new NDs to the model, thus setting the basis for the search for the identity and order of these steps. Understanding the complexity, or number of steps, of factors related to disease onset trigger is important prior deciding to study single factors for a multiple steps disease.
Project description:<h4>Background</h4>This study investigated the use of nanodiamond particles (NDs) as a promising material for drug delivery in vivo and in vitro.<h4>Methods</h4>HepG2 cells (a human hepatic carcinoma cell line) were used to determine the characteristics of a nanodiamond-doxorubicin complex (ND-DOX) when taken up by cells in vitro using laser scanning confocal microscopy and dialysis experiments. We also compared the survival rate and histopathology of tumor-bearing mice after treatment with NDs or ND-DOX in vivo.<h4>Results</h4>In vitro investigation showed that ND-DOX has slow and sustained drug release characteristics compared with free doxorubicin. In vivo, the survival rate of tumor-bearing mice treated with ND-DOX was four times greater than that of mice treated with free doxorubicin. Interestingly, the survival rate in mice treated with NDs alone was close to that of mice treated with free doxorubicin. This indicates that treatment with ND-DOX can prolong the lifespan of tumor-bearing mice significantly compared with conventional doxorubicin and that NDs can have this effect as well. Histopathological analysis showed that neither the NDs nor ND-DOX were toxic to the kidney, liver, or spleen in contrast with the well-known toxic effects of free doxorubicin on the kidney and liver. Further, both the bare NDs and ND-DOX could suppress tumor growth effectively.<h4>Conclusion</h4>NDs can potentially prolong survival, and ND-DOX may act as a nanodrug with promising chemotherapeutic efficacy and safety.
Project description:Nanodiamonds (NDs) are carbon nanomaterials with a core diamond crystalline structure and crystal defects, such as graphitic carbon and heteroatoms, on their surface. For electrochemistry, NDs are promising to increase active sites and decrease fouling, but NDs have not been studied for neurotransmitter electrochemistry. Here, we optimized ND coatings on microelectrodes and found that ND increases the sensitivity for neurotransmitters with fast-scan cyclic voltammetry detection and decreases electrochemical and biofouling. Different sizes and functionalizations of NDs were tested, and ND suspensions were drop-casted onto carbon-fiber microelectrodes (CFMEs). The 5 nm ND-H and 5 nm ND-COOH formed thick coatings, while the 15 and 60 nm ND-COOH formed more sparse coatings. With electrochemical impedance spectroscopy, 5 nm ND-H and 5 nm ND-COOH had high charge-transfer resistance, while 15 and 60 nm ND-COOH had low charge-transfer resistance. ND-COOH (15 nm) was optimal, with the best electrocatalytic properties and current for dopamine. Sensitivity was enhanced 2.1 ± 0.2 times and the limit of detection for dopamine improved to 3 ± 1 nM. ND coating increased current for other cations such as serotonin, norepinephrine, and epinephrine, but not for the anion ascorbic acid. Moreover, NDs decreased electrochemical fouling from serotonin and 5-hydroxyindoleacetic acid, and they also decreased biofouling in brain slice tissue by 50%. The current at biofouled ND-coated electrodes is similar to the signal of pristine, unfouled CFMEs. The carboxylated ND-modified CFMEs are beneficial for neurotransmitter detection because of easy fabrication, improved limit of detection, and antifouling properties.
Project description:The concentration and small size of nanodiamonds (NDs) plays a crucial role in the mechanical performance of epoxy-based nanocomposites by modifying the interface strength. Herein, we systemically analyzed the relation between the high concentration and small size of ND and the fracture properties of its epoxy-based nanocomposites. It was observed that there is a two-fold increase in fracture toughness and a three-fold increase in fracture energy. Rationally, functionalized-NDs (F-NDs) showed a much better performance for the nanocomposite than pristine NDs (P-NDs) because of additional functional groups on its surface. The F-ND/epoxy nanocomposites exhibited rougher surface in contrast with the P-ND/epoxy, indicating the presence of a strong interface. We found that the interfaces in F-ND/epoxy nanocomposites at high concentrations of NDs overlap by making a web, which can efficiently hinder further crack propagation. In addition, the de-bonding in P-ND/epoxy nanocomposites occurred at the interface with the appearance of plastic voids or semi-naked particles, whereas the de-bonding for F-ND/epoxy nanocomposites happened within the epoxy molecular network instead of the interface. Because of the strong interface in F-ND/epoxy nanocomposites, at high concentrations the de-bonding within the epoxy molecular network may lead to subsequent cracks, parallel to the parent crack, via crack splitting which results in a fiber-like structure on the fracture surface. The plastic void growth, crack deflection and subsequent crack growth were correlated to higher values of fracture toughness and fracture energy in F-ND/epoxy nanocomposites.
Project description:Detonation nanodiamonds (NDs) were deposited on the surface of aligned carbon nanotubes (CNTs) by immersing a CNT array in an aqueous suspension of NDs in dimethylsulfoxide (DMSO). The structure and electronic state of the obtained CNT-ND hybrid material were studied using optical and electron microscopy and Infrared, Raman, X-ray photoelectron and near-edge X-ray absorption fine structure spectroscopy. A non-covalent interaction between NDs and CNT and preservation of vertical orientation of CNTs in the hybrid were revealed. We showed that current-voltage characteristics of the CNT-ND cathode are changed depending on the applied field; below ~3 V/µm they are similar to those of the initial CNT array and at the higher field they are close to the ND behavior. Involvement of the NDs in field emission process resulted in blue luminescence of the hybrid surface at an electric field higher than 3.5 V/µm. Photoluminescence measurements showed that the NDs emit blue-green light, while blue luminescence prevails in the CNT-ND hybrid. The quenching of green luminescence was attributed to a partial removal of oxygen-containing groups from the ND surface as the result of the hybrid synthesis.
Project description:Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.
Project description:Nanodiamonds (NDs) are emerging carbon platforms with promise as gene/drug delivery vectors for cancer therapy. Specifically, NDs functionalized with the polymer polyethylenimine (PEI) can transfect small interfering RNAs (siRNA) in vitro with high efficiency and low cytotoxicity. Here we present a modeling framework to accurately guide the design of ND-PEI gene platforms and elucidate binding mechanisms between ND, PEI, and siRNA. This is among the first ND simulations to comprehensively account for ND size, charge distribution, surface functionalization, and graphitization. The simulation results are compared with our experimental results both for PEI loading onto NDs and for siRNA (C-myc) loading onto ND-PEI for various mixing ratios. Remarkably, the model is able to predict loading trends and saturation limits for PEI and siRNA, while confirming the essential role of ND surface functionalization in mediating ND-PEI interactions. These results demonstrate that this robust framework can be a powerful tool in ND platform development, with the capacity to realistically treat other nanoparticle systems.
Project description:Detonation nanodiamonds (NDs) are promising drug delivery and imaging agents due to their uniquely faceted surfaces with diverse chemical groups, electrostatic properties, and biocompatibility. Based on the potential to harness ND properties to clinically address a broad range of disease indications, this work reports the in-human administration of NDs through the development of ND-embedded gutta percha (NDGP), a thermoplastic biomaterial that addresses reinfection and bone loss following root canal therapy (RCT). RCT served as the first clinical indication for NDs since the procedure sites involved nearby circulation, localized administration, and image-guided treatment progress monitoring, which are analogous to many clinical indications. This randomized, single-blind interventional treatment study evaluated NDGP equivalence with unmodified GP. This progress report assessed one control-arm and three treatment-arm patients. At 3-mo and 6-mo follow-up appointments, no adverse events were observed, and lesion healing was confirmed in the NDGP-treated patients. Therefore, this study is a foundation for the continued clinical translation of NDs and other nanomaterials for a broad spectrum of applications.
Project description:Nanodiamonds (NDs) have been used as drug delivery vehicles due to their low toxicity and biocompatibility. Recently, it has been reported that NDs have also osteogenic differentiation capacity. However, their capacity using NDs alone is not enough. To significantly improve their osteogenic activity, we developed icariin (ICA)-functionalized NDs (ICA-NDs) and evaluated whether ICA-NDs enhance their in vitro osteogenic capacity. Unmodified NDs and ICA-NDs showed nanosized particles that were spherical in shape. The ICA-NDs achieved a prolonged ICA release for up to 4 weeks. The osteogenic capacities of NDs, ICA (10 ?g)-NDs, and ICA (50 ?g)-NDs were demonstrated by alkaline phosphatase (ALP) activity; calcium content; and mRNA gene levels of osteogenic-related markers, including ALP, runt-related transcript factor 2 (RUNX2), collagen type I alpha 1 (COL1A1), and osteopontin (OPN). In vitro cell studies revealed that ICA (50 ?g)-ND-treated MC3T3-E1 cells greatly increased osteogenic markers, including ALP, calcium content, and mRNA gene levels of osteogenic-related markers, including ALP, RUNX2, COL1A1, and OPN compared to ICA (10 ?g)-NDs or ND-treated cells. These our data suggest that ICA-NDs can promote osteogenic capacity.