Three-dimensional Structure Of DNA Research Articles

DNASCANNER v2: A Web-Based Tool to Analyze the Characteristic Properties of Nucleotide Sequences.

Throughout the process of evolution, DNA undergoes the accumulation of distinct mutations, which can often result in highly organized patterns that serve various essential biological functions. These patterns encompass various genomic elements and provide valuable insights into the regulatory and functional aspects of DNA. The physicochemical, mechanical, thermodynamic, and structural properties of DNA sequences play a crucial role in the formation of specific patterns. These properties contribute to the three-dimensional structure of DNA and influence their interactions with proteins, regulatory elements, and other molecules. In this study, we introduce DNASCANNER v2, an advanced version of our previously published algorithm DNASCANNER for analyzing DNA properties. The current tool is built using the FLASK framework in Python language. Featuring a user-friendly interface tailored for nonspecialized researchers, it offers an extensive analysis of 158 DNA properties, including mono/di/trinucleotide frequencies, structural, physicochemical, thermodynamics, and mechanical properties of DNA sequences. The tool provides downloadable results and offers interactive plots for easy interpretation and comparison between different features. We also demonstrate the utility of DNASCANNER v2 in analyzing splice-site junctions, casposon insertion sequences, and transposon insertion sites (TIS) within the bacterial and human genomes, respectively. We also developed a deep learning module for the prediction of potential TIS in a given nucleotide sequence. In the future, we aim to optimize the performance of this prediction model through extensive training on larger data sets.

Predicting DNA structure using a deep learning method

Understanding the mechanisms of protein-DNA binding is critical in comprehending gene regulation. Three-dimensional DNA structure, also described as DNA shape, plays a key role in these mechanisms. In this study, we present a deep learning-based method, Deep DNAshape, that fundamentally changes the current k-mer based high-throughput prediction of DNA shape features by accurately accounting for the influence of extended flanking regions, without the need for extensive molecular simulations or structural biology experiments. By using the Deep DNAshape method, DNA structural features can be predicted for any length and number of DNA sequences in a high-throughput manner, providing an understanding of the effects of flanking regions on DNA structure in a target region of a sequence. The Deep DNAshape method provides access to the influence of distant flanking regions on a region of interest. Our findings reveal that DNA shape readout mechanisms of a core target are quantitatively affected by flanking regions, including extended flanking regions, providing valuable insights into the detailed structural readout mechanisms of protein-DNA binding. Furthermore, when incorporated in machine learning models, the features generated by Deep DNAshape improve the model prediction accuracy. Collectively, Deep DNAshape can serve as versatile and powerful tool for diverse DNA structure-related studies.

Constructions of DNA and polypeptide cages based on plane graphs and odd crossing [formula omitted]-junctions

The constructions of three-dimensional synthetic DNA and polypeptide structures with a single closed DNA strand and polypeptide chain are mathematically based on strong traces of polyhedral graphs. However, a method developed for constructing such a DNA and polypeptide structure may impose additional restrictions on the types of strong traces and polyhedral graphs. In this paper, we show that strong traces for certain 2-connected plane graphs (allowed to have multiple edges) can be obtained using thickened graphs (sometimes called ribbon graphs) constructed with only two types of junctions : 0-crossing junction and special d(v)-crossing junction (called π-junction), where d(v) is the degree of the vertex v at which the d(v)-crossing junction is to be placed. The π-junctions are only applicable to vertices with odd degrees (≥3). We characterize the 2-connected plane graphs to which our approach can be applied and provide a brief guideline for the implementation of our method. This approach provides the theory, as well as a set of candidates, for designing and constructing stable DNA and polypeptide molecules needing only a method capable of creating the 0-crossing and π-junctions in a 2-connected plane graph.

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

Ab initio predictions for 3D structure and stability of single- and double-stranded DNAs in ion solutions.

The three-dimensional (3D) structure and stability of DNA are essential to understand/control their biological functions and aid the development of novel materials. In this work, we present a coarse-grained (CG) model for DNA based on the RNA CG model proposed by us, to predict 3D structures and stability for both dsDNA and ssDNA from the sequence. Combined with a Monte Carlo simulated annealing algorithm and CG force fields involving the sequence-dependent base-pairing/stacking interactions and an implicit electrostatic potential, the present model successfully folds 20 dsDNAs (≤52nt) and 20 ssDNAs (≤74nt) into the corresponding native-like structures just from their sequences, with an overall mean RMSD of 3.4Å from the experimental structures. For DNAs with various lengths and sequences, the present model can make reliable predictions on stability, e.g., for 27 dsDNAs with/without bulge/internal loops and 24 ssDNAs including pseudoknot, the mean deviation of predicted melting temperatures from the corresponding experimental data is only ~2.0°C. Furthermore, the model also quantificationally predicts the effects of monovalent or divalent ions on the structure stability of ssDNAs/dsDNAs.

Light-emitting crystals of aptamer-hybrid organic semiconductor signaling on human cells expressing EpCAM

In this study, we successfully performed protein-cell recognition by combining an organic light-emitting diode (OLED) material and an aptamer with a three-dimensional DNA structure. Tris (8-hydroxyquinoline) aluminum (Alq3), one of the most widely used green OLED components used in displays, was chosen as an emitter. Epithelial cell adhesion molecule (EpCAM) was selected as a target because it is well-established in cancer studies. The generated micrometer-sized particles exhibited increased luminous intensity based on the specific recognition to proteins and cells on hybrid interfaces, proving that the aptamer maintained its three-dimensional structure while binding to its target. Interestingly, only human cells expressing EpCAMs were distinguishable, whereas mouse carcinoma cells were not. Furthermore, use of graphene oxide as a quencher made these aptamer-Alq3 microrods 2-fold more effective at enhancing the fluorescence signal upon interaction with human oral squamous carcinoma cells. This new approach, which imparts biometric functionality to organic semiconductor materials, will provide a new platform for the evolution of OLED hybrid materials.

Enzyme-free catalyzed self-assembly of three-dimensional hyperbranched DNA structures for in situ SERS imaging and molecular logic operations

Nonenzymatic isothermal amplification strategy has emerged as a versatile tool for microRNA detection. However, the slow kinetics, limited sensitivity and unsatisfactory efficiency often hinder their further practical applications. Herein, we proposed a DNA tetrahedron-mediated branched catalytic hairpin assembly (DTM-bCHA), achieving the dynamic fabrication of three-dimensional hyperbranched DNA structures. Compared with traditional target-catalyzed hairpin assembly, the reaction kinetics of DTM-bCHA is accelerated by 11.1-fold through tethering the hairpin probes on DNA tetrahedron due to the increased collision probability among the reactants. Moreover, the surface enhanced Raman scattering (SERS) hot spots are generated by assembling Raman reporter DTNB-functionalized gold nanoparticles (DTNB@AuNPs) with hyperbranched DNAs, leading to the ultrasensitive detection and in situ imaging of microRNA-21 (miR-21) in different living cells. Moreover, a series of two-input molecular logic gates, including AND, OR, NOR and INHIBIT gates, are constructed in response to miR-21 and miR-155, which achieves the multiplexed analysis of biomarkers in a facile way. Overall, the proposed DTM-bCHA based isothermal amplification strategy expands the avenues for the construction of complex DNA nanostructures, which is of great significance for bioanalysis and clinical diagnosis.

Three-dimensional DNA structures in situ decorated with metal nanoclusters for dual-mode biosensing of glucose

The monitoring of blood glucose levels is essential to the diagnosis and management of diseases, especially diabetes. Thus, it is of great importance to develop a simple, accurate and sensitive method for glucose detection. Herein, the three-dimensional DNA flowers-templated Ag nanoclusters (DFs/AgNCs) with enhanced fluorescent brightness and good stability are in situ synthesized via rolling circle amplification (RCA), which are further successfully applied for the construction of fluorescent/electrochemical dual-mode glucose biosensor. In this assay, the AgNCs encapsulated in DFs can be etched by H2O2 that is generated from the process of glucose oxidase (GOx)-catalyzed glucose oxidation, resulting in the release of Ag+ to facilitate the signal-off fluorescent detection of glucose. Based on the coordination interaction of cytosine-Ag+-cytosine (C-Ag+-C), the liberated Ag+ further induces the conformation switch of ferrocene (Fc)-labeled DNA capture probe (Fc-DNA) with C-rich sequence on Au electrode, leading to the signal-on electrochemical detection of glucose. The proposed DFs/AgNCs-based fluorescent/electrochemical dual-mode biosensor has achieved the sensitive and selective detection of glucose in human serums with good accuracy and reliability, which not only provides a rapid, reliable and accurate method for glucose detection but also opens a new avenue to the development of DNA nanotechnology-based biosensing strategies.

Novel implications of a strictly monomorphic (GCC) repeat in the human PRKACB gene

PRKACB (Protein Kinase CAMP-Activated Catalytic Subunit Beta) is predominantly expressed in the brain, and regulation of this gene links to neuroprotective effects against tau and Aβ-induced toxicity. Here we studied a (GCC)-repeat spanning the core promoter and 5′ UTR of this gene in 300 human subjects, consisting of late-onset neurocognitive disorder (NCD) (N = 150) and controls (N = 150). We also implemented several models to study the impact of this repeat on the three-dimensional (3D) structure of DNA. While the PRKACB (GCC)-repeat was strictly monomorphic at 7-repeats, we detected two 7/8 genotypes only in the NCD group. In all examined models, the (GCC)7 and its periodicals had the least range of divergence variation on the 3D structure of DNA in comparison to the 8-repeat periodicals and several hypothetical repeat lengths. A similar inert effect on the 3D structure was not detected in other classes of short tandem repeats (STRs) such as GA and CA repeats. In conclusion, we report monomorphism of a long (GCC)-repeat in the PRKACB gene in human, its inert effect on DNA structure, and enriched divergence in late-onset NCD. This is the first indication of natural selection for a monomorphic (GCC)-repeat, which probably evolved to function as an “epigenetic knob”, without changing the regional DNA structure.

DNA Nanolithography Enables a Highly Ordered Recognition Interface in a Microfluidic Chip for the Efficient Capture and Release of Circulating Tumor Cells.

Microfluidic chips with nano-scale structures have shown great potential, but the fabrication and cost issues restrict their application. Herein, we propose a conceptually new "DNA nanolithography in a microfluidic chip" by using sub-10 nm three-dimensional DNA structures (TDNs) as frameworks with a pendant aptamer at the top vertex (ApTDN-Chip). The nano-scale framework ensures that the aptamer is in a highly ordered upright orientation, avoiding the undesired orientation or crowding effects caused by conventional microfluidic interface fabrication processes. Compared with a monovalent aptamer modified chip, the capture efficiency of ApTDN-Chip was enhanced nearly 60 % due to the highly precise dimension and rigid framework of TDNs. In addition, the scaffolds make DNase I more accessible to the aptamer with up to 83 % release efficiency and 91 % cell viability, which is fully compatible with downstream molecular analysis. Overall, this strategy provides a novel perspective on engineering nano-scaffolds to achieve a more ordered nano-topography of microfluidic chips.

Nanopore-Based DNA Hard Drives for Rewritable and Secure Data Storage.

Nanopores are powerful single-molecule tools for label-free sensing of nanoscale molecules including DNA that can be used for building designed nanostructures and performing computations. Here, DNA hard drives (DNA-HDs) are introduced based on DNA nanotechnology and nanopore sensing as a rewritable molecular memory system, allowing for storing, operating, and reading data in the changeable three-dimensional structure of DNA. Writing and erasing data are significantly improved compared to previous molecular storage systems by employing controllable attachment and removal of molecules on a long double-stranded DNA. Data reading is achieved by detecting the single molecules at the millisecond time scale using nanopores. The DNA-HD also ensures secure data storage where the data can only be read after providing the correct physical molecular keys. Our approach allows for easy-writing and easy-reading, rewritable, and secure data storage toward a promising miniature scale integration for molecular data storage and computation.

Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA Structure.

The translocation of double-stranded DNA through a solid-state nanopore may either decrease or increase the ionic current depending on the ionic concentration of the surrounding solution. Below a certain crossover ionic concentration, the current change inverts from a current blockade to current enhancement. In this paper, we show that the crossover concentration for bundled DNA nanostructures composed of multipleconnected DNA double-helices is lower than that of double-stranded DNA. Our measurements suggest that counterion mobility in the vicinity of DNA is reduced depending on the three-dimensional structure of the molecule. We further demonstrate that introducing neutral polymers such as polyethylene glycol into the measurement solution reduces electroosmotic outflow from the nanopore, allowing translocation of large DNA structures at low salt concentrations. Our experiments contribute to an improved understanding of ion transport in confined DNA environments, which is critical for the development of nanopore sensing techniques as well as synthetic membrane channels. Our salt-dependent measurements of model DNA nanostructures will guide the development of computational models of DNA translocation through nanopores.

Length doesn't matter-telomere damage triggers cellular senescence in the ageing heart.

Telomere shortening induces cellular senescence in proliferative cells. Yet, it is presently unclear how it is triggered in post‐mitotic cells such as cardiac myocytes. A new study by Anderson et al (2019) reports that during ageing of the heart, cellular senescence develops independently of telomere length, but is evoked by DNA damage, which preferentially accumulates at the telomere. Removal of senescent cells using senolytic drugs ameliorated cardiac hypertrophy and fibrosis and may inform novel approaches to improve the conditions for the ageing heart.

Abstract 1552: SATB1 is a novel prognostic factor in esophageal cancer patients and promotes migration and invasion of esophageal cancer cells

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with extremely poor overall survival. There is no available molecular marker in clinical application until now. SATB1(special AT-rich binding protein 1) tether genomic DNA in nucleolus and hold three-dimensional structure of genomic DNA. We evaluated SATB1 expression using clinical samples from patients of ESCC and analyzed the association with clinicopathological factors and prognosis to identify novel prognostic marker in ESCC. In addition, we investigated the function of SATB1 using ESCC cells. We focused on matrix metalloproteinase protein (MMP3), which has a SATB1 binding site in its upstream. Materials and Methods: We accumulated 71 samples of ESCC patients who underwent primary tumor resection. The relative expression levels of SATB1 were obtained by normalizing the amount of SATB1 mRNA with respect to GAPDH mRNA as an endogenous control for each sample. We divided 71 ESCC samples into 2 groups, which are SATB1 low expression group (n=35) and SATB1 high expression group (n=36) according to the median of SATB1 expression level, and performed the comparison between two groups. In order to elucidate biological significance, we generated SATB1 overexpression stable transfectants in KYSE150, and performed migration and invasion assays. Next, we knockdowned SATB1 expression of KYSE410, which are ESCC lines, using SATB1 siRNA and evaluated the capability of migration and invasion. Moreover, to validate whether SATB1 altered MMP3 genomic locus, we analyzed comparative genomic hybridization (CGH) array obtained from 54 esophageal cancer samples. We examined MMP3 expression in SATB1 overexpression cells using qRT-PCR. Result: The SATB1 high expression group showed greater tumor depth than the low expression group (P=0.003). In the overall survival curve, patients in the SATB1 high expression group had a significant poorer prognosis than those in the SATB1 low expression group (P=0.013). Multivariate analysis showed that the level of SATB1 expression was an independent prognostic predictor (OR; 1.56, 95%CI; 1.04 - 2.44, P = 0.043). SATB1 transfectants significantly increased the capability of migration and invasion compared to mock (P<0.05). KYSE410 cells transfected with SATB1 siRNA significantly decreased the capability of migration and invasion compared to control cells (P<0.05). Genomic alterations in SATB1 locus were strongly correlated with those in MMP3 locus (P<0.0001, ρ=0.5156). Overexpression of SATB1 showed the elevated MMP3 level compared to mock (P<0.05). Conclusion: We indicated that SATB1 is novel prognostic factor in ESCC and that SATB1 promote migration and invasion of ESCC cells. SATB1 may be a promising new candidate for targeted therapies for ESCC. Citation Format: Yusuke Tsuruda, Ryunosuke Kogo, Shuhei Ito, Akihiro Kitagawa, Atsushi Fujii, Shotaro Kuramitsu, Dai Simizu, Yosuke Kuroda, Hidetoshi Eguchi, Tkaaki Masuda, Shoji Natsugoe, Masaki Mori, Koshi Mimori. SATB1 is a novel prognostic factor in esophageal cancer patients and promotes migration and invasion of esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1552.

Unfolding the Genome: The Case Study of P. falciparum.

The development of new ways to probe samples for the three-dimensional (3D) structure of DNA paves the way for in depth and systematic analyses of the genome architecture. 3C-like methods coupled with high-throughput sequencing can now assess physical interactions between pairs of loci in a genome-wide fashion, thus enabling the creation of genome-by-genome contact maps. The spreading of such protocols creates many new opportunities for methodological development: how can we infer 3D models from these contact maps? Can such models help us gain insights into biological processes? Several recent studies applied such protocols to P. falciparum (the deadliest of the five human malaria parasites), assessing its genome organization at different moments of its life cycle. With its small genomic size, fairly simple (yet changing) genomic organization during its lifecyle and strong correlation between chromatin folding and gene expression, this parasite is the ideal case study for applying and developing methods to infer 3D models and use them for downstream analysis. Here, I review a set of methods used to build and analyse three-dimensional models from contact maps data with a special highlight on P. falciparum's genome organization.

Hyperstretching DNA

The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely used in biophysical/chemical assays and drug treatments. We present single-molecule experiments and a three-state statistical mechanical model that provide a quantitative understanding of the interplay between B-DNA, overstretched DNA and intercalated DNA. The predictions of this model include a hitherto unconfirmed hyperstretched state, twice the length of B-DNA. Our force-fluorescence experiments confirm this hyperstretched state and reveal its sequence dependence. These results pin down the physical principles that govern DNA mechanics under the influence of tension and biochemical reactions. A predictive understanding of the possibilities and limitations of DNA extension can guide refined exploitation of DNA in, e.g., programmable soft materials and DNA origami applications.

DNA Sequence can Pin the Position of DNA Supercoils

While DNA obviously functions as the carrier of genetic information, it may also encode the three-dimensional spatial structure of DNA in cells. Here we study the relationship between structure and sequence in supercoiled DNA, which involves a reorganization of the DNA configuration via the formation of so-called plectonemes - coiled loop structures that locally form to minimize the torsional stress within the DNA. We use a novel high-throughput single-molecule assay called ISD (‘Intercalation-induced Supercoiling of DNA’, see Ganji et al, Nano Lett. 16, 4699, 2016), where we induce supercoiling of surface-attached DNA molecules by adding an intercalating dye. This assay allows us to visualize individual plectonemes and characterize their position-dependent frequency as well as dynamics such as nucleation, termination, and diffusion. From profiles of the average plectoneme density as a function of the position along the DNA molecule, we can study whether plectonemes exhibit any preference for certain DNA sequences. We indeed find that particular local DNA sequences pin plectonemes. Specifically, we identify DNA stretches with exclusively A or T bases (which we name ‘TorA tracts’) that strongly pin plectonemes. Interestingly, TorA tracts pin plectonemes just as well as poly-A tracts. This suggests that it is not a local intrinsic bending of the DNA (such as predicted for polyA tracts) that underlies the plectoneme pinning. Instead, we propose that local melting may lead to the formation of a kink at the tip of a plectoneme that provides a pinning site. Indeed we observed strong pinning at DNA mutation sites where 1 to 10 neighboring nucleotides are mispaired such that a ssDNA bubble is formed. Our data show that the DNA sequence does not merely encode for genetic information but also for structural information that governs the three-dimensional organization of supercoiled DNA.

Phase Transition Detection in Accumulation of a Potential Anticancer Drug Cl-IPBD with DNA: Supercoiled and Linear pUC19 Plasmids

The electrochemical investigation of the plasmid DNA (pUC19) interactions with Cl-IPBD (4-chloro-6-(1H-imidazo[4,5-b]phenasine-2-yl)benzene-1,3-diol), a prospective anticancer drug, have revealed the presence of a phase transition between two orientations of the compound in the DNA layer adsorbed on a glassy carbon (GC) electrode. Except for the needed sufficient incorporation of the compound into the plasmid, these Orientations, I and II, depend significantly on the superhelicity of the plasmid, used either in a superhelical circular (sc) or linear (lin) form. Different architecture of compact layers produced by these sc and lin plasmids has been confirmed by AFM measurements. The incorporation of Cl-IPBD into the DNA layers is tested here using a dual approach: (a) Differential Pulse (DP) or Square Wave (SW), as well as Alternating Current (AC) and impedance spectroscopy is used to monitor the redox activity of Cl-IPBD and (b) Alternating Current (AC) is used to detect the Cl-IPBD condensation via capacitative/resistive (C/R) mode. Since the initial accumulation of the pUC19 plasmid in the electrode layer has been detected in a ‘label-free’ procedure, i.e. without the addition of the redox probe, then the electroactive compound under investigation, accumulated in the DNA layer, can be, in fact, regarded as an actual redox probe and tested. The condensation of such a probe, in the case of a prospective anticancer drug, depends significantly on the superhelicity of the plasmid, thus showing, additionally, the role of the three-dimensional DNA structure on the formation of the DNA-compound complex. The correlations between redox and C/R properties of a vitamin (riboflavin) and an antioxidant (rutin), different from Cl-IPBD compound, confirm the role of the condensation of the compounds in realizing their interactions with DNA, important for a possible future use of Cl-IPBD as a drug.

Transfer of molecular recognition information from DNA nanostructures to gold nanoparticles.

DNA nanotechnology offers unparalleled precision and programmability for the bottom-up organization of materials. This approach relies on pre-assembling a DNA scaffold, typically containing hundreds of different strands, and using it to position functional components. A particularly attractive strategy is to employ DNA nanostructures not as permanent scaffolds, but as transient, reusable templates to transfer essential information to other materials. To our knowledge, this approach, akin to top-down lithography, has not been examined. Here we report a molecular printing strategy that chemically transfers a discrete pattern of DNA strands from a three-dimensional DNA structure to a gold nanoparticle. We show that the particles inherit the DNA sequence configuration encoded in the parent template with high fidelity. This provides control over the number of DNA strands and their relative placement, directionality and sequence asymmetry. Importantly, the nanoparticles produced exhibit the site-specific addressability of DNA nanostructures, and are promising components for energy, information and biomedical applications.

Epigenetics and Inflammation: Exploring the Link for Chronic Human Diseases Including Periodontal Disease

Epigenetics refers to changes in gene expression that are not mediated by alterations of the genetic code itself but are caused by biochemical modifications of the nucleotide bases that alter the three-dimensional structure of DNA. The epigenome, consisting of chromatin and its modifications, functions as an interface between the inherited genome and the dynamism imposed by the environment. Over the past decade, there has been mounting evidence suggestive of associations between epigenetic modifications and various human conditions such as aging, and most common human diseases viz. cancer, cardiovascular diseases, diabetes, rheumatoid arthritis, HIV, etc., and the clearest evidence as the central mechanism for common multifactorial diseases has been identified with the factors involved in the inflammatory response. This emerging knowledge can give a great contribution to the field of medicine as it can be potentially utilized to develop newer understandings about pathogenesis, diagnostics, and therapies for major chronic human diseases, e.g., chronic metabolic diseases, immune-inflammatory disorders (including periodontal disease), and cancers, etc. The current paper aims to provide the clinicians a comprehensive review, based on the available supportive evidence to update this interesting concept capable of providing a better comprehension of the underlying disease mechanisms, so as to develop and decipher the novel methodologies to diagnose and treat these diseases.