Sox9CreER-mediated deletion of ß-catenin in palatal mesenchyme results in delayed palatal elevation accompanied with repressed canonical Wnt signaling and reduced actin polymerization.

Cleft palate is a good model to pushing us toward a deeper understanding of the molecular mechanisms of spatiotemporal patterns in tissues and organisms because of the multiple-step processes such as elevation and fusion. Previous studies have shown that the epithelial ß-catenin is crucial for palatal fusion, however, the function of the mesenchymal ß-catenin remains elusive. We investigate the role of mesenchymal ß-catenin in palatal development by generating a ß-catenin conditional knockout mouse (CKO) (Sox9CreER; Ctnnb1F/F ). We found that the CKO mice exhibited delayed palatal elevation, leading to cleft palate in both in vivo and ex vivo. Abnormal cell proliferation and repressed mesenchymal canonical Wnt signaling were found in the CKO palate. Interestingly, Filamentous actin (F-actin) polymerization was significantly reduced in the palatal mesenchyme of mutant embryos. Furthermore, overexpression of adenovirus-mediated transfection with Acta1 in the mutant could help to elevate the palatal shelves but could not prevent cleft palate in ex vivo. Our results suggest that conditionally knock out ß-catenin in the palatal mesenchyme by Sox9CreER leading to delayed palatal elevation, which results in repressed mesenchymal canonical Wnt signaling, decreased cell proliferation, and reduced actin polymerization, finally causes cleft palate.

Vibration-enhanced disposable electroanalytical platform for selective analysis of tryptophan in fruits based on molecular imprinting.

Although electrochemical detection based on molecular imprinting polymers (MIP) could dramatically improve the selectivity, the procedure is time-consuming because of the essential incubation step. In addition, current MIP electrochemical detections were not suitable for analysis of microliter-level sample solutions, limiting their applications for real samples. This investigation aims at applying vibration to enhance efficiency of MIP electrochemical detection of 20 µL sample solutions. MIP analysis of Tryptophan (Trp) was used as the model with disposable MIP electrodes prepared by electrochemical polymerization of o-phenylenediamine on carbon ink coated on stainless steel sheets. The MIP electrode was integrated in a 3D-printed analytical device for vibration-enhanced electrochemical detection of Trp. Our results showed that this vibration-enhanced strategy could significantly increase electrochemical responses of Trp at the same incubation time. Such improvement might be attributed to the enhanced mass transfer at the surface of the working electrode brought by vibration. It needs to be emphasized that this strategy is suitable for analysis of sample solutions with the volume of microliters, which is superior to normal stirring in MIP electrochemical detection. Our approach could be successfully utilized for differentiation of Trp in different fruits, opening more opportunities for MIP electrochemical detection of real samples. The enhanced efficiency by vibration could pave foundation for extensive practical MIP detection of sample solutions at the level of microliters.

Three-dimensional reconstruction of systematic histological sections: application to observations on palatal shelf elevation.

Normal mammalian secondary palate development undergoes a series of processes, including palatal shelf (PS) growth, elevation, adhesion and fusion, and palatal bone formation. It has been estimated that more than 90% of isolated cleft palate is caused by defects associated with the elevation process. However, because of the rapidly completed elevation process, the entire process of elevation will never be easy to clarify. In this article, we present a novel method for three-dimensional (3D) reconstruction of thick tissue blocks from two-dimensional (2D) histological sections. We established multiplanar sections of the palate and tongue in coronal and sagittal directions, and further performed 3D reconstruction to observe the morphological interaction and connection between the two components prior to and during elevation. The method completes an imaging system for simultaneous morphological analysis of thick tissue samples using both synthetic and real data. The new method will provide a comprehensive picture of reorientation morphology and gene expression pattern during the palatal elevation process.

Liposome-mediated transient transfection reduces cholesterol-dependent coxsackievirus infectivity.

Liposome-mediated gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Coxsackievirus B3 is an important human pathogen associated with various diseases. Here we reported that liposome-mediated transient transfection of plasmid cDNA inhibited coxsackieviral replication at the levels of RNA, protein and viral progeny release. These inhibitory effects were observed in various cell types and by using different liposome reagents. We further showed that the inhibition was likely due to the lack of virus attachment. Moreover, we showed that addition of cholesterol restored, at least in part, the viral infectivity. Interestingly, we found that membrane cholesterol levels were unchanged during transfection, indicating that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Our data suggest that liposome-mediated cDNA transient transfection inhibits coxsackievirus infectivity via inhibition of viral attachment, which is likely occurring through the changes of membrane cholesterol integrity.