Faculty Bibliography

Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5' leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.

OBJECTIVE: To compare clinical and radiological outcome between acetabular fractures with marginal impaction that were treated with either cancellous bone graft (CBG) or tricalcium phosphate cement (TPC) as bone void filler. DESIGN: Retrospective study. PATIENTS: Forty-three patients with acetabular fractures with marginal impaction. INTERVENTION: Eighteen patients received cancellous bone graft and 25 patients received tricalcium phosphate cement as bone void filler. MAIN OUTCOME MEASUREMENT: Clinical outcome was assessed using the Merle d'Aubigne score and Short-form-36. Radiographs were evaluated for postoperative reduction, arthritis grade and development of heterotopic ossification. RESULTS: Forty-three patients met the inclusion criteria. There was no significant difference in the demographics, laterality, fracture type, associated injuries, surgical approach and postoperative quality of reduction between the groups. At final follow-up, a significantly higher number of patients in the cancellous bone graft group exhibited signs of moderate to severe post-traumatic arthritis (CBG: 6 (33%) vs. TPC: 4 (20%), p=0.007) and required a total hip arthroplasty (CBG: 4 (22.2%) vs. TPC: 1 (5%), p=0.08). There was no significant difference between the two groups in the SF-36 score and the modified Merle d'Aubigne score. CONCLUSION: Patients with acetabular fractures with marginal impaction treated with tricalcium phosphate cement exhibit a significantly lower incidence of post-traumatic arthritis when compared to patients treated with cancellous bone graft. LEVEL OF EVIDENCE: III.

Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a 'Brainbow' of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish.

During wound healing, stem cells provide functional mature cells to meet acute demands for tissue regeneration. Simultaneously, the tissue must maintain a pool of stem cells to sustain its future regeneration capability. However, how these requirements are balanced in response to injury is unknown. Here we demonstrate that after wounding or ultraviolet type B irradiation, melanocyte stem cells (McSCs) in the hair follicle exit the stem cell niche before their initial cell division, potentially depleting the pool of these cells. We also found that McSCs migrate to the epidermis in a melanocortin 1 receptor (Mc1r)-dependent manner and differentiate into functional epidermal melanocytes, providing a pigmented protective barrier against ultraviolet irradiation over the damaged skin. These findings provide an example in which stem cell differentiation due to injury takes precedence over stem cell maintenance and show the potential for developing therapies for skin pigmentation disorders by manipulating McSCs.

OBJECTIVES: Triazole resistance in Aspergillus fumigatus has been increasing. We explored the A. fumigatus azole resistance profiles in bronchoalveolar lavage (BAL) fluid samples from Danish patients examined for aspergillosis. METHODS: A total of 94 BAL samples from 87 patients were evaluated by galactomannan (GM) test and A. fumigatus CYP51A profiling by PCR. RESULTS: Aspergillus spp. were isolated from 27/48 (56.3%) cultured samples, including 23 A. fumigatus with one resistant strain (4.3%). Samples were classified into GM-positive (>/=3.0), GM-intermediate (0.5 to <3.0) and GM-negative (<0.5) groups, where the CYP51A PCR was positive in 81.8% (36/44), 56.3% (18/32) and 38.9% (7/18) of samples, respectively. Nine CYP51A PCR-positive samples (9/61, 14.8%) were found to have mutations resulting in amino acid substitutions. M220V was detected from a sample culture positive for susceptible A. fumigatus and P216L was found in a culture-negative BAL sample. Conversely, no mutation was found in one sample culture positive for azole-resistant A. fumigatus. The tandem repeat/L98H mutation was not detected. CONCLUSIONS: Our study shows that azole resistance in A. fumigatus can be cryptic and may go undiagnosed. The combination of improved culture/susceptibility tests and the direct molecular detection of resistance markers will facilitate prompt institution of appropriate antifungal therapy.

Drosophila eye development has been extensively studied, due to the ease of genetic screens for mutations disrupting this process. The eye imaginal disc is specified during embryonic and larval development by the Pax6 homolog Eyeless and a network of downstream transcription factors. Expression of these factors is regulated by signaling molecules and also indirectly by growth of the eye disc. Differentiation of photoreceptor clusters initiates in the third larval instar at the posterior of the eye disc and progresses anteriorly, driven by the secreted protein Hedgehog. Within each cluster, the combined activities of Hedgehog signaling and Notch-mediated lateral inhibition induce and refine the expression of the transcription factor Atonal, which specifies the founding R8 photoreceptor of each ommatidium. Seven additional photoreceptors, followed by cone and pigment cells, are successively recruited by the signaling molecules Spitz, Delta, and Bride of sevenless. Combinations of these signals and of intrinsic transcription factors give each ommatidial cell its specific identity. During the pupal stages, rhodopsins are expressed, and the photoreceptors and accessory cells take on their final positions and morphologies to form the adult retina. Over the past few decades, the genetic analysis of this small number of cell types arranged in a repetitive structure has allowed a remarkably detailed understanding of the basic mechanisms controlling cell differentiation and morphological rearrangement. WIREs Dev Biol 2012, 2:545-557. doi: 10.1002/wdev.100 For further resources related to this article, please visit the WIREs website.

Bone morphogenetic proteins (BMPs) are effective for bone regeneration, and are used clinically. However, supraphysiological doses are required, which limits their use. Cartilage oligomeric matrix protein is an extracellular matrix protein, which we have previously shown can bind to growth factors of the TGFs family, suggesting that COMP may also bind to BMP-2. Rather than being a passive component of the matrix, COMP may serve as an "instructive matrix" component capable of increasing local growth factor concentration, slowing the diffusion of growth factors, and promoting their biological activity. The purpose of this investigation was to determine whether COMP binds to BMP-2, and whether it promotes the biological activity of BMP-2 with respect to osteogenesis. We found that COMP binds BMP-2, and characterized the biochemical nature of the binding interaction. COMP binding enhanced BMP-2-induced intracellular signaling through Smad proteins, increased the levels of BMP receptors, and up-regulated the luciferase activity from a BMP-2-responsive reporter construct. COMP binding enhanced BMP-2-dependent osteogenesis in vitro, in the C2C12 cell line and in primary human bone mesenchymal stem cells, as measured by alkaline phosphatase activity, matrix mineralization, and gene expression. Finally, we found that COMP enhanced BMP-2-dependent ectopic bone formation in a rat model assessed histologically, by alkaline phosphatase activity, gene expression, and micro-CT. In summary, this study demonstrates that COMP enhances the osteogenic activity of BMP-2, both in-vitro and in-vivo.

The development of nanocarriers that selectively release guest molecules on sensing a particular biological signal is being actively pursued in nanomedicine for diagnostic and therapeutic purposes. Here we report a protein-based nanocarrier that opens in the presence of intracellular adenosine-5'-triphosphate (ATP). The nanocarrier consists of multiple barrel-shaped chaperonin units assembled through coordination with Mg(2+) into a tubular structure that protects guest molecules against biological degradation. When its surface is functionalized with a boronic acid derivative, the nanocarrier is able to enter cells. The hydrolysis of intracellular ATP into adenosine-5'-diphosphate (ADP) induces conformational changes of the chaperonin units, which in turns generate a mechanical force that leads to the disassembly of the tube and release of the guests. This scission occurs with a sigmoidal dependence on ATP concentration, which means that the nanocarrier can differentiate biological environments in terms of the concentration of ATP for selective guest release. Furthermore, biodistribution tests reveal preferential accumulation of the nanocarriers in a tumour tissue.

Hyperlipidemia is a risk factor for various cardiovascular and metabolic disorders. Overproduction of lipoproteins, a process that is dependent on microsomal triglyceride transfer protein (MTP), can contribute to hyperlipidemia. We show that microRNA-30c (miR-30c) interacts with the 3' untranslated region of MTP mRNA and induces its degradation, leading to reductions in MTP activity and in apolipoprotein B (APOB) secretion. miR-30c also reduces lipid synthesis independently of MTP. Hepatic overexpression of miR-30c reduced hyperlipidemia in Western diet-fed mice by decreasing lipid synthesis and the secretion of triglyceride-rich ApoB-containing lipoproteins and decreased atherosclerosis in Apoe(-/-) mice. Furthermore, inhibition of hepatic miR-30c by anti-miR-30c increased hyperlipidemia and atherosclerosis. Therefore, miR-30c coordinately reduces lipid biosynthesis and lipoprotein secretion, thereby regulating hepatic and plasma lipid concentrations. Raising miR-30c levels might be useful in treating hyperlipidemias and associated disorders.