Search

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and affects boys in infancy or early childhood. Current methods for diagnosing DMD are often laborious, expensive, invasive, and typically diagnose the disease late in its progression. In an effort to improve the accuracy and ease of diagnosis, this study focused on developing a novel method for diagnosing DMD which combines Raman hyperspectroscopic analysis of blood serum with advanced statistical analysis. Partial least squares discriminant analysis was applied to the spectral dataset acquired from blood serum of a mouse model of Duchenne muscular dystrophy (mdx) and control mice. Cross-validation showed 95.2% sensitivity and 94.6% specificity for identifying diseased spectra. These results were verified via external validation, which achieved 100% successful classification accuracy at the donor level. This proof-of-concept study presents Raman hyperspectroscopic analysis of blood serum as an easy, fast, non-expensive, and minimally invasive detection method for distinguishing control and mdx model mice, with a strong potential for clinical diagnosis of DMD.

MicroRNAs are involved in the crucial processes of development and diseases and have emerged as a new class of biomarkers. The field of DNA nanotechnology has shown great promise in the creation of novel microRNA biosensors that have utility in lab-based biosensing and potential for disease diagnostics. In this Survey and Summary, we explore and review DNA nanotechnology approaches for microRNA detection, surveying the literature for microRNA detection in three main areas of DNA nanostructures: DNA tetrahedra, DNA origami, and DNA devices and motifs. We take a critical look at the reviewed approaches, advantages and disadvantages of these methods in general, and a critical comparison of specific approaches. We conclude with a brief outlook on the future of DNA nanotechnology in biosensing for microRNA and beyond.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy, typically affecting males in infancy. The disease causes progressive weakness and atrophy of skeletal muscles, with approximately 20,000 new cases diagnosed yearly. Currently, methods for diagnosing DMD are invasive, laborious, and unable to make accurate early detections. While there is no cure for DMD, there are limited treatments available for managing symptoms. As such, there is a crucial unmet need to develop a simple and non-invasive method for accurately detecting DMD as early as possible. Raman spectroscopy with chemometric analysis is shown to have the potential to fill this diagnostic need.

The genetics of skeletal muscle lineage commitment are deceptively complicated. MyoD overexpression is sufficient to convert fibroblasts into skeletal muscle myotubes. In vivo, there are a number of different steps of differentiation that require a large network of transcription factors that control differentiation and homeostasis of skeletal muscle progenitors. Each transcription factor has been shown to have the ability to promote the next factor in the cascade, but the mechanisms regulating the transitions remain incomplete. Recently, microRNAs have been shown to be important for a large number of developmental and oncogenic processes. In this review, we will discuss recent advances in the understanding of how microRNA is critical for skeletal muscle development by interacting with protein-coding genes that had previously been shown to be important for myogenesis.

MicroRNAs play important roles in many cell processes, including the differentiation process in several different lineages. For example, microRNAs can promote differentiation by repressing negative regulators of transcriptional activity. These regulated transcription factors can further up-regulate levels of the microRNA in a feed-forward mechanism. Here we show that MyoD up-regulates miR-378 during myogenic differentiation in C2C12 cells. ChIP and high throughput sequencing analysis shows that MyoD binds in close proximity to the miR-378 gene and causes both transactivation and chromatin remodeling. Overexpression of miR-378 increases the transcriptional activity of MyoD, in part by repressing an antagonist, MyoR. The 3' untranslated region of MyoR contains a direct binding site for miR-378. The presence of this binding site significantly reduces the ability of MyoR to prevent the MyoD-driven transdifferentiation of fibroblasts. MyoR and miR-378 were anticorrelated during cardiotoxin-induced adult muscle regeneration in mice. Taken together, this shows a feed-forward loop where MyoD indirectly down-regulates MyoR via miR-378.

We report the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein disconnected (disco) as a bait. DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD). Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays. DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors. In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression. Later in development DIP1 expression is decreased in dividing cells in different tissues. Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule. A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed.

The Drosophila disconnected (disco) gene encodes a transcription factor containing two C2H2 zinc-fingers that functions in the development of both embryonic and adult structures. The disco gene was initially identified from a mutation in which adult photoreceptor axons fail to connect with their target cells in the larval optic lobe (Steller et al., 1987). The optic lobes of disco mutants are poorly developed and lack optic lobe neurons, including the lateral neurons. Abnormal circadian rhythmicity has been attributed to the absence or aberrant differentiation of these lateral neurons (Hardin et al., 1992; Helfrich-Forster et al., 1998). Additionally, disco mutants show defects in thoracic and abdominal sensory axon development (Glossop and Shepherd, 1998). More recently, disco was found to have a broader role in embryonic pattern formation, functioning as a cofactor for the Hox proteins Deformed (Dfd) and Sex Combs Reduced (Scr) in the gnathocephalic segments (mandibular, maxillary, and labial); this more extensive role was masked by the partially redundant gene, disco-related (disco-r) (Mahaffey et al., 2001). Lack of both Disco and Disco-r causes embryonic lethality and the absence of some larval mouth structures arising from the gnathocephalic segments. The disco gene is also expressed in the antennal and leg imaginal discs and likely has a role in the development of these structures (Lee et al., 1991). The disco gene is located in region 14B of the X chromosome and consists of two exons separated by a single intron. The first exon is untranslated and comprises the majority of the disco 5 UTR. The second exon contains the complete open reading frame (ORF) encoding the 568 amino acid protein. The two C2H2 zinc finger regions, defining the DNA binding domain, are located between amino acids 89 and 149 (Heilig et al., 1991). As tools for further study of Disco function, we generated three UAS-disco constructs—two encoding wildtype proteins and one producing an altered protein like that encoded by the disco null allele. The UAS-disco wild-type construct was generated by the sequential insertion of two disco fragments into the Drosophila transformation vector pUAST (Brand and Perrimon, 1993). A 1,270-bp BglII-XbaI fragment representing the majority of the disco ORF (a subclone from 542.2; Surdej et al., 1990) was inserted into BglII-XbaI digested pUAST. Next, a 473-bp fragment, with an artificial EcoRI site immediately upstream of the initiation codon, was obtained by an EcoRI-BglII digestion of a 1,276-bp PCR product generated with the following primers: Forward primer, 5 GGA TCC GAATTC ATG GAG CAC ATA ATG 3 ; Reverse Primer, 5 ATG TAG GCA CTG TCG CTA TCC 3 . This fragment was inserted into the EcoRI-BglII sites of the initial BglII-XbaI pUAST construct. The final construct includes a 1,743-bp EcoRI-XbaI insert encoding the entire disco ORF with some 3 UTR. The second wild-type construct (further referred to as UAS-disco) and the mutant construct (further referred to as UAS-disco) were generated by PCR amplification of genomic DNA. A 1,704-bp fragment containing the complete disco ORF was generated using primers containing EcoRI sites: Forward primer, 5 CCC ACC ACA GAATTC ATG GAG CAC 3 ; Reverse Primer, 5 CCA TGG ATC GAATTC TGG ACA TTA 3 . After EcoRI digestion, the resulting PCR product was inserted into an EcoRI digested pUAST. For the UAS-disco construct, Oregon R genomic DNA was used as a template, while homozygous disco genomic DNA was used as a template for the mutant UAS-disco construct. This form of the protein contains a single amino acid change— Cysteine 127 is substituted with a Serine—which disrupts the first zinc finger. Prior analysis indicated that this mutant form of the Disco protein is nonfunctional (Heilig et al., 1991; Mahaffey et al., 2001). The sequence and reading frame of all constructs were verified by DNA sequence analysis. Integration of UAS-disco constructs into the Drosophila genome was carried out using P-element-mediated germ-line transformation essentially as described by Rubin and Spradling (1982). Two UAS-disco lines were obtained—a heterozygous line carrying UAS-disco on the second chromosome balanced over CyO (UAS-disco(II)), and a homozygous line with the insert on the