CRISPR-based biosensing is prospective for rapid and sensitive diagnosis of pediatric tuberculosis.

Pediatric tuberculosis (TB) is an important part of global TB prevention and control. Diagnosis of childhood TB still remains challenging when using conventional tests, due to the non-specific clinical manifestations and paucibacillary nature of the specimens. Thus, a sensitive, rapid and low-cost diagnostic test is of great demand. Benefiting from specific and rapid Cas-protein-based catalytic activities, CRISPR-based biosensing platforms (CRISPR platforms) are showing superiority in detecting pathogen nucleic acid traces in clinical samples. Based on their excellent sensitivity, and time and cost saved in existing research, this study aimed to highlight the potential of CRISPR platforms as a tool for diagnosing pediatric TB, and advocate for studies to evaluate its performance in specimens collected from children, especially noninvasive specimens. These platforms are also promising in identifying drug resistance and genotyping. All of the above will help early diagnosis of pediatric TB, thus guide reasonable treatment, and be significant in achieving the World Health Organization End-TB strategy.

[Osteoclast differentiation of Raw264.7 cells expressing enhanced green fluorescent protein in vitro].

OBJECTIVE: To observe the osteoclast differentiation of Raw264.7 strain stably expressing enhanced green fluorescent protein (EGFP). METHODS: Raw264.7 cells were transfected with EGFP-Lifeact gene via retrovirus. The G3 cell clone was obtained by limited dilution technique which stably expressed EGFP under the fluorescence microscope. The morphology of G3 cells were observed. The effects of transfection on receptor activator of nuclear factor kappaB ligand (RANKL)--induced osteoclastogenesis and bone resorbing function of G3 cells were examined by tartrate resistant acid phosphatase (TRAP) staining, immunoblotting detection of cathepsin K and bone pit resorption assay. The real-time images of podosome dynamics were taken by laser confocal microscopy during osteoclast differentiation of G3 cells. RESULTS: The Raw264.7 cells were successfully transfected with EGFP-Lifeact gene. The G3-EGFP cloned strain which could stably express EGFP even after 20 passages was constructed. There was no significant difference in morphology between G3-EGFP and wild Raw264.7 cells. The fusion rates of the transfection group and of the wild control group were (35±5)% and (39±5)%, respectively, which were not significantly different (P>0.05). The semi-quantitative ratio of cathepsin K/ß-actin in the wild control group and in the transfection group was 0.83±0.07 and 1.02±0.08 (P>0.05), respectively. Bone pit results showed that the total area of the bone resorption was respectively 272,252±36,193 and 262,408±23,243 (P>0.05) and the number of the bone pits was respectively 320±51 and 339±55 (P>0.05). The photos of laser confocal microscopy showed the constant cell-cell fusion during osteoclast differentiation of G3-EGFP cells. In addition, the dynamic self-organized podosome initially assembled podosome clusts, then dynamic rings, finally formed the characteristic podosome belt pattern in mature osteoclasts. CONCLUSIONS: Enhanced green fluorescent protein high effectively expressed in Raw264.7. Biological character does not change after transfection.

Controlled delivery of carvedilol nanosuspension from osmotic pump capsule: in vitro and in vivo evaluation.

This study intended to develop a novel controlled delivery osmotic pump capsule of carvedilol nanosuspension. The capsule is assembled using a semi-permeable capsule shell with contents including nanosuspension drying powder, mannitol and Plasdone S-630. The physical characteristics of semi-permeable capsule walls were compared among different coating solutions under different temperature. The composition of the coating solution and drying temperature appeared to be important for the formation of the shells. Carvedilol nanosuspension was prepared by precipitation-ultrasonication technique and was further lyophilized. Response surface methodology was used to investigate the influence of factors on the responses. The optimized formulation displayed complete drug delivery and zero-order release rate. The TEM and particle size analysis indicated that the morphology of the resultant nanoparticle in the capsule was spherical shaped with a mean size of 252±19 nm. The in vivo test in beagle dogs demonstrated that the relative bioavailability of the novel system was 203.5% in comparison to that of the marketed preparation. The capsule successfully controlled the release of carvedilol and the fluctuation of plasma concentration was minimized. The system is a promising strategy to improve the oral bioavailability for poorly soluble drugs and preparing it into elementary osmotic pump conveniently.