Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method.

BACKGROUND: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). METHOD: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. RESULTS: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. CONCLUSION: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.

Peripheral-neuron-like properties of differentiated human dental pulp stem cells (hDPSCs).

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.

Improvement of classification performance of Parkinson's disease using shape features for machine learning on dopamine transporter single photon emission computed tomography.

OBJECTIVE: To assess the classification performance between Parkinson's disease (PD) and normal control (NC) when semi-quantitative indicators and shape features obtained on dopamine transporter (DAT) single photon emission computed tomography (SPECT) are combined as a feature of machine learning (ML). METHODS: A total of 100 cases of both PD and normal control (NC) from the Parkinson's Progression Markers Initiative database were evaluated. A summed image was generated and regions of interests were set to the left and right striata. Area, equivalent diameter, major axis length, minor axis length, perimeter and circularity were calculated as shape features. Striatum binding ratios (SBRputamen and SBRcaudate) were used as comparison features. The classification performance of the PD and NC groups according to receiver operating characteristic analysis of the shape features was compared in terms of SBRs. Furthermore, we compared the classification performance of ML when shape features or SBRs were used alone and in combination. RESULTS: The shape features (except minor axis length) and SBRs indicated significant differences between the NC and PD groups (p < 0.05). The top five areas under the curves (AUC) were as follows: circularity (0.972), SBRputamen (0.972), major axis length (0.945), SBRcaudate (0.928) and perimeter (0.896). When classification was done using ML, AUC was as follows: circularity and SBRs (0.995), circularity alone (0.990), and SBRs (0.973). The classification performance was significantly improved by combining SBRs and circularity than by SBRs alone (p = 0.018). CONCLUSION: We found that the circularity obtained from DAT-SPECT images could help in distinguishing NC and PD. Furthermore, the classification performance of ML was significantly improved using circularity in SBRs together.