Integration of comprehensive 3D microCT and signaling analysis reveals differential regulatory mechanisms of craniofacial bone development.

Growth factor signaling regulates tissue-tissue interactions to control organogenesis and tissue homeostasis. Specifically, transforming growth factor beta (TGFß) signaling plays a crucial role in the development of cranial neural crest (CNC) cell-derived bone, and loss of Tgfbr2 in CNC cells results in craniofacial skeletal malformations. Our recent studies indicate that non-canonical TGFß signaling is activated whereas canonical TGFß signaling is compromised in the absence of Tgfbr2 (in Tgfbr2(fl/fl);Wnt1-Cre mice). A haploinsufficiency of Tgfbr1 (aka Alk5) (Tgfbr2(fl/fl);Wnt1-Cre;Alk5(fl/+)) largely rescues craniofacial deformities in Tgfbr2 mutant mice by reducing ectopic non-canonical TGFß signaling. However, the relative involvement of canonical and non-canonical TGFß signaling in regulating specific craniofacial bone formation remains unclear. We compared the size and volume of CNC-derived craniofacial bones (frontal bone, premaxilla, maxilla, palatine bone, and mandible) from E18.5 control, Tgfbr2(fl/fl);Wnt1-Cre, and Tgfbr2(fl/fl);Wnt1-Cre;Alk5(fl/+)mice. By analyzing three dimensional (3D) micro-computed tomography (microCT) images, we found that different craniofacial bones were restored to different degrees in Tgfbr2(fl/fl);Wnt1-Cre;Alk5(fl/+) mice. Our study provides comprehensive information on anatomical landmarks and the size and volume of each craniofacial bone, as well as insights into the extent that canonical and non-canonical TGFß signaling cascades contribute to the formation of each CNC-derived bone. Our data will serve as an important resource for developmental biologists who are interested in craniofacial morphogenesis.

Statins suppress cell-to-cell propagation of α-synuclein by lowering cholesterol.

Cell-to-cell propagation of protein aggregates has been implicated in the progression of neurodegenerative diseases. However, the underlying mechanism and modulators of this process are not fully understood. Here, we screened a small-molecule library in a search for agents that suppress the propagation of α-synuclein and mutant huntingtin (mHtt). These screens yielded several molecules, some of which were effective against both α-synuclein and mHtt. Among these molecules, we focused on simvastatin and pravastatin. Simvastatin administration in a transgenic model of synucleinopathy effectively ameliorated behavioral deficits and α-synuclein accumulation, whereas pravastatin had no effect. Because only simvastatin enters the brain effectively, these results suggest that inhibition of brain cholesterol synthesis is important in simvastatin effects. In cultured cells, accumulation of intracellular cholesterol, induced by genetic ablation of the NPC1 gene or by pharmacological treatment with U18666A, increased α-synuclein aggregation and secretion. In contrast, lowering cholesterol using methyl-ß-cyclodextrin or statins reversed α-synuclein aggregation and secretion in NPC1-knockout cells. Consistent with these observations, feeding a high-fat diet aggravated α-synuclein pathology and behavioral deficits in the preformed fibril-injected mouse model, an effect that was also reversed by simvastatin administration. These results suggest that statins suppress propagation of protein aggregates by lowering cholesterol in the brain.

Modeling α-Synuclein Propagation with Preformed Fibril Injections.

The aggregation of α-synuclein (α-syn) has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Postmortem analyses of α-syn pathology, especially that of PD, have suggested that aggregates progressively spread from a few discrete locations to wider brain regions. The neuron-to-neuron propagation of α-syn has been suggested to be the underlying mechanism by which aggregates spread throughout the brain. Many cellular and animal models has been created to study cell-to-cell propagation. Recently, it has been shown that a single injection of preformed fibrils (PFFs) made of recombinant α-syn proteins into various tissues and organs of many different animal species results in widespread α-syn pathology in the central nervous system (CNS). These PFF models have been extensively used to study the mechanism by which aggregates spread throughout the brain. Here, we review what we have learned from PFF models, describe the nature of PFFs and the neuropathological features, neurophysiological characteristics, and behavioral outcomes of the models.

Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support.

BACKGROUND: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. RESULTS: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. CONCLUSION: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

BMP-SHH signaling network controls epithelial stem cell fate via regulation of its niche in the developing tooth.

During embryogenesis, ectodermal stem cells adopt different fates and form diverse ectodermal organs, such as teeth, hair follicles, mammary glands, and salivary glands. Interestingly, these ectodermal organs differ in their tissue homeostasis, which leads to differential abilities for continuous growth postnatally. Mouse molars lose the ability to grow continuously, whereas incisors retain this ability. In this study, we found that a BMP-Smad4-SHH-Gli1 signaling network may provide a niche supporting transient Sox2+ dental epithelial stem cells in mouse molars. This mechanism also plays a role in continuously growing mouse incisors. The differential fate of epithelial stem cells in mouse molars and incisors is controlled by this BMP/SHH signaling network, which partially accounts for the different postnatal growth potential of molars and incisors. Collectively, our study highlights the importance of crosstalk between two signaling pathways, BMP and SHH, in regulating the fate of epithelial stem cells during organogenesis.

Humeral shaft fracture treatment in the elite throwing athlete: a unique application of flexible intramedullary nailing.

Humeral shaft stress fractures are being increasingly recognized as injuries that can significantly impact throwing mechanics if residual malalignment exists. While minimally displaced and angulated injuries are treated nonoperatively in a fracture brace, the management of significantly displaced humeral shaft fractures in the throwing athlete is less clear. Currently described techniques such as open reduction and internal fixation with plate osteosynthesis and rigid antegrade/retrograde locked intramedullary nailing have significant morbidity due to soft tissue dissection and damage. We present a case report of a high-level baseball pitcher whose significantly displaced humeral shaft stress fracture failed to be nonoperatively managed and was subsequently treated successfully with unlocked, retrograde flexible intramedullary nailing. The athlete was able to return to pitching baseball in one year and is currently pitching in Major League Baseball. We were able to recently collect 10-year follow-up data.

Reagentless ultrasensitive specific DNA array detection based on responsive polymeric biochips.

Self-assembled molecular structures immobilized on solid substrates and composed of fluorophore-tagged oligonucleotide probes and an optical polymeric transducer were investigated for the trace level detection of DNA target molecules. Rapid and efficient energy transfer between the polymeric transducer and fluorophores within the molecular aggregates leads to a massive intrinsic amplification of the fluorescence signal and to the label-free detection of as little as 300 DNA molecules, with the specificity required for the detection of single-nucleotide mismatches. This capacity for attomolar detection levels while the sensing structures are attached onto solid supports could lead to the development of biochip platforms for fast and simple PCR-free multitarget DNA detection.

Optical sensors based on hybrid DNA/conjugated polymer complexes.

Single-stranded DNA (ss-DNA) can specifically bind to various targets, including a complementary ss-DNA, ions, proteins, drugs, and so forth. When binding takes place, the oligonucleotide probe often undergoes a conformational transition. This conformational change of the negatively charged ss-DNA can be detected by using a water-soluble, cationic polythiophene derivative, which transduces the complex formation into an optical (colorimetric or fluorometric) signal without any labeling of the probe or the target. This simple and rapid methodology has enabled the specific and sensitive detection of nucleic acids and human thrombin. This new biophotonic tool can easily be applied to the detection of various other biomolecules and is also useful in the high-throughput screening of new drugs.

Optical sensors based on hybrid aptamer/conjugated polymer complexes.

Single-stranded DNA (aptamer) can specifically bind potassium ions or human alpha-thrombin. When binding takes place, the aptamer undergoes a conformational transition from an unfolded to a folded structure. This conformational change of the negatively charged oligonucleotide can be detected by adding a water-soluble, cationic polythiophene derivative, which transduces the new complex formation into an optical (colorimetric or fluorometric) signal without any labeling of the probe or of the target. This simple and rapid methodology has enabled the detection of human thrombin in the femtomole range. This new biophotonic tool can easily be applied to the detection of various other proteins as well as being useful in the high-throughput screening of new drugs.

Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level.

We report the specific detection of a few hundred molecules of genetic material using a fluorescent polythiophene biosensor. Such recognition is based on simple electrostatic interactions between a cationic polymeric optical transducer and the negatively charged nucleic acid target and can be done in less than 1 h, simply and affordably, and without any chemical reaction. This simple system is versatile enough to detect nucleic acids of various lengths, including a segment from the RNA genome of the Influenza virus.