Recently, a bio-electrochemical technique known as CLARITY was reported for three-dimensional phenotype mapping within transparent tissues, allowing clearer whole-body and organ visualization with CB-perfusion (CUBIC) and leading to the development of whole-body clearing and transparency of intact tissues with the PACT (passive clarity technique) and PARS (perfusion-assisted agent release in situ) methodologies. We evaluated the structure–function relationships in circuits of the whole central nervous system (CNS) and various internal organs using improved methods with optimized passive clarity. Thus, in the present study, we aimed to improve the original PACT procedure and passive clearing protocols for different intact rodent tissues. We determined the optimal conditions for the passive clarity method that allowed the production of a transparent whole CNS by clearing the brain and spinal cord, as well as various organs. We also improved the tissue transparency using mPACT (modified PACT), a method for direct passive clearing, and whole perfusion-based PARS-mPACT, a method for fusion clearing, and we identified the appropriate experimental conditions. These optimized methods can be used for easy and economical high-resolution mapping and phenotyping of normal and pathological elements within intact tissues.

a b s t r a c tThe SARS-CoV nucleocapsid (N) protein serves multiple functions in viral replication, transcription, and assembly of the viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, and in SARS-CoV, it is reported that this process is driven by an interaction between the Nprotein and a packaging signal encoded within the viral RNA. While recent studies have uncovered the sequence of this packaging signal, little is known about the specific interaction between the N-protein and the packaging signal sequence, and the mechanisms by which this interaction drives viral genome packaging. In this study, we developed a novel in vivo cell-based assay for examining this interaction between the N-protein and packaging signal RNA for SARS-CoV, as well as other viruses within the coronaviridae family. Our results demonstrate that the N-protein specifically recognizes the SARS-CoV packaging signal with greater affinity compared to signals from other coronaviruses or noncoronavirus species. We also use deletion mapping to identify a 151-nt region within the packaging signal sequence that is critical for N-protein-RNA binding, and conversely, we show that both the Nterminal and C-terminal domains of the N protein are necessary for recognizing the packaging RNA. These results describe, for the first time, in vivo evidence for an interaction between the SARS-CoV Nprotein and its packaging signal RNA, and demonstrate the feasibility of using this cell-based assay to further probe viral RNA-protein interactions in future studies.

Dental gels and rinses for caries prophylactic contain fluoride at concentrations ranging from 0.1 to 1%. In addition, many types of fluoride-releasing materials have been used in dental applications. The purpose of the study was to investigate the addition effect of fluoride into artificial saliva on the corrosion resistance of pure titanium and titanium-silver alloys. Titanium and titanium-silver alloys were arc melted, homogenized at 950 degrees C for 72 h, hot rolled, and solution heat treated and quenched. In order to investigate the effect of the fluoride ions on the corrosion resistance, potentiodynamic polarization testing, potentiostatic testing, and open-circuit potential measurements were performed in plain artificial saliva and 0.1 and 1% NaF-added artificial saliva. The passive current densities of titanium and titanium-silver alloys increased with increasing fluoride-ion concentration. Ti2.0Ag and Ti3.0Ag exhibited a low current density relatively and showed a stable behavior compared to titanium. The open-circuit potential of titanium decreased and current density at 250 mV (SCE) potentiostatic testing reacted sensitively with increasing fluoride concentration. On the other hand, the open-circuit potential of titanium-silver alloys with a high silver content (3.0-4.0 at %) reacted less sensitively to the fluoride-ion concentration. Among titanium-silver alloys, Ti3.0Ag alloy had a higher resistance against the attack of fluoride ions and showed a more stable open-circuit potential and current density than titanium in the fluoride-containing solution. It is concluded that they are electrochemically stable and maintained good corrosion resistance in fluoride-containing artificial saliva.

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Titanium and its alloys are widely used in biomedical and dental fields because of their excellent corrosion resistance and biocompatibility. It is well known that titanium is protected from corrosion because of the stability of the passive film that controls and determines the corrosion resistance and biocompatibility of titanium and its alloys. The purpose of this study was to evaluate the electrochemical properties of titanium-silver alloys and the surface characteristics of passive film in artificial saliva.We designed titanium-silver alloys with silver contents ranging from 0 to 5 at.%, in 1% increments. These alloys were arc-melted, homogenized, hot-rolled to 2 mm thickness, and finally solution heat-treated for 1 h and quenched. Potentiostatic testing was performed, and the open circuit potentials of the alloys were measured in artificial saliva, at 37°C. The passive films of the titanium-silver alloys were analyzed via XPS.Titanium-silver alloys maintained low current density and showed stable passive region and also had high open circuit potential as compared with pure titanium. The open circuit potential of titanium-silver alloys increased as silver addition increased. With regard to the fraction of oxygen species, a component of over 80% was found to be comprised of oxide. Therefore, the titanium surface mainly consisted of titanium oxide and, on the titanium-silver alloys, this film was composed of TiO 2 , Ti 2 O 3 , and TiO. As silver content increased, the TiO 2 fraction also increased, as did the thickness of the titanium oxide layer formed.

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.

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