Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease

Trends in microRNA detection

MicroRNAs (miRNAs) are short, approximately 22 nucleotide length RNAs that perform gene regulation. Recently, miRNA has been shown to be linked with the onset of cancer and other diseases based on miRNA expression levels. It is important, therefore, to understand miRNA function as it pertains to disease onset; however, in order to fully understand miRNA's role in a disease, it is necessary to detect the expression levels of these small molecules. The most widely used miRNA detection method is Northern blotting, which is considered as the standard of miRNA detection methods. This method, however, is time-consuming and has low sensitivity. This has led to an increase in the amount of detection methods available. These detection methods are either solid phase, occurring on a solid support, or solution phase, occurring in solution. While the solid-phase methods are adaptable to high-throughput screening and possess higher sensitivity than Northern blotting, they lack the ability for in vivo use and are often time-consuming. The solution-phase methods are advantageous in that they can be performed in vivo, are very sensitive, and are rapid; however, they cannot be applied in high-throughput settings. Although there are multiple detection methods available, including microarray technology, luminescence-based assays, electrochemical assays, etc., there is still much work to be done regarding miRNA detection. The current gaps of miRNA detection include the ability to perform multiplex, sensitive detection of miRNA with single-nucleotide specificity along with the standardization of these new methods. Current miRNA detection methods, gaps in these methods, miRNA therapeutic options, and the future outlook of miRNA detection are presented here

Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization

Reassembly of split reporter proteins, also referred to as protein complementation, is utilized in the detection of protein-protein or protein-nucleic acid interactions. In this strategy, a reporter protein is fragmented into two inactive polypeptides to which interacting/binding partners are fused. The interaction between fused partners leads to the formation of a reassembled, active reporter. In this Communication, we have presented a proof-of-concept for the detection of a target nucleic acid sequence based on the reassembly of the bioluminescent reporter Renilla luciferase (Rluc), which is driven by DNA hybridization. Although, reassembly of Rluc though protein interactions has been demonstrated by others, the Rluc reassembly through DNA hybridization has not been shown yet, which is the novelty of this work. It is well established that bioluminescence detection offers significant advantages due to the absence of any background signal. In our study, two rationally designed fragments of Rluc were conjugated to complementary oligonucleotide probes. Hybridization of the two probes with fused Rluc fragments resulted in the reassembly of the fragments, generating active Rluc, measurable by the intensity of light given off upon addition of coelenterazine. Our study also shows that the reassembly of Rluc can be inhibited by an oligonucleotide probe that competes to bind to the hybridized probe-Rluc fragment complex, indicating a potential strategy for the quantitative detection of target nucleic acid. We were able to achieve the reassembly of Rluc fused to oligonucleotide probes using femtomole amounts of the probe-fragment protein conjugate. This concentration is approximately 4 orders of magnitude less than that reported using green fluorescent protein (GFP) as the reporter. A DNA-driven Rluc reassembly study performed in a cellular matrix did not show any interference from the matrix

Molecular biosensing system based on intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219-498) was utilized in the detection of p53 (residues 311-393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311-393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219-498). Dansyl-X, SE-conjugated BRCA1 (219-498) detected p53 (311-393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change

Bioluminescence-based detection of microRNA, miR21 in breast cancer cells

A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death. miRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the determination of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell extracts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early molecular markers and their detection methods in cancer diagnosis is tremendous. The characteristics of the assay developed in this work show its suitability for early cancer diagnosis based on miRNA as a biomarker

Clinical translation of stem cells: insight for cartilage therapies.

The limited regenerative capacity of articular cartilage and deficiencies of current treatments have motivated the investigation of new repair technologies. In vitro cartilage generation using primary cell sources is limited by cell availability and expansion potential. Pluripotent stem cells possess the capacity for chondrocytic differentiation and extended expansion, providing a potential future solution to cell-based cartilage regeneration. However, despite successes in producing cartilage using adult and embryonic stem cells, the translation of these technologies to the clinic has been severely limited. This review discusses recent advances in stem cell-based cartilage tissue engineering and the major current limitations to clinical translation of these products. Concerns regarding appropriate animal models and studies, stem cell manufacturing, and relevant regulatory processes and guidelines will be addressed. Understanding the significant hurdles limiting the clinical use of stem cell-based cartilage may guide future developments in the fields of tissue engineering and regenerative medicine