Automated measurement of cell motility and proliferation

BACKGROUND: Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data. RESULTS: We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth. CONCLUSION: These results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype

FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells

N6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA. m6A plays important roles in the regulation of post-transcriptional RNA splicing, translation, and degradation. Increasing studies have uncovered the significance of m6A in various biological processes such as stem cell fate determination, carcinogenesis, adipogenesis, stress response, etc, which put forwards a novel conception called epitranscriptome. However, functions of the fat mass and obesity-associated protein (FTO), the first characterized m6A demethylase, in spermatogenesis remains obscure. Here we reported that depletion of FTO by CRISPR/Cas9 induces chromosome instability and G2/M arrest in mouse spermatogonia, which was partially rescued by expression of wild type FTO but not demethylase inactivated FTO. FTO depletion significantly decreased the expression of mitotic checkpoint complex and G2/M regulators. We further demonstrated that the m6A modification on Mad1, Mad2, Bub1b, Cdk1, and Ccnb2 were directly targeted by FTO. Therefore, FTO regulates cell cycle and mitosis checkpoint in spermatogonia because of its m6A demethylase activity. The findings give novel insights into the role of RNA methylation in spermatogenesis

Untargeted metabolomics reveal rhizosphere metabolites mechanisms on continuous ramie cropping

Ramie is an important fiber feed dual-purpose crop in China and plays an important role in the national economy. However, ramie yield and quality can be reduced after many years of continuous cultivation. Currently, relatively little research has been conducted on rhizosphere metabolites and their pathways in continuous ramie cropping. Therefore, a healthy group (CK) and obstacle groups (XZQG, JZ, DJY, and GXD) with 8 years of continuous cultivation were selected for the study. LC-MS and GC-MS untargeted metabolomics were used to explore and analyze ramie rhizosphere metabolites and pathways. The results revealed that significant differences in the agronomic traits of ramie occurred after 8 years of continuous cultivation, with dwarfed plants and decreased yields in the obstacle groups. Metabolomic analysis identified 49 and 19 rhizosphere metabolites, including lipids, organic acids, phenols, and amino acids. In addition, four differential metabolic pathways (phenylpropanoid biosynthesis, fatty acid metabolism, amino acid metabolism, and ascorbate and aldarate metabolism) were elucidated. It was also clarified that sinapic acid, jasmonic acid, glutamine, and inositol might be the main metabolites affecting ramie continuous-cropping obstacle groups, and they were significantly correlated with ramie agronomic traits and physiological indicators. This provided important insights into the mechanisms affecting continuous ramie cropping. Accordingly, it is expected that the increase or decrease of sinapic acid, jasmonic acid, glutamine, and inositol in the soil will alleviate obstacles to continuous ramie cropping and promote the healthy development of the ramie industry in the future

The application of DEA (Data Envelopment Analysis) window analysis in the assessment of influence on operational efficiencies after the establishment of branched hospitals

Abstract Background Many large-scaled public hospitals have established branched hospitals in China. This study is to provide evidence for strategy making on the management and development of multi-branched hospitals by evaluating and comparing the operational efficiencies of different hospitals before and after their establishment of branched hospitals. Methods DEA (Data Envelopment Analysis) window analysis was performed on a 7-year data pool from five public hospitals provided by health authorities and institutional surveys. Results The operational efficiencies of sample hospitals measured in this study (including technical efficiency, pure technical efficiency and scale efficiency) had overall trends towards increase during this 7-year period of time, however, a temporary downturn occurred shortly after the establishment of branched hospitals; pure technical efficiency contributed more to the improvement of technical efficiency compared to scale efficiency. Conclusions The establishment of branched-hospitals did not lead to a long-term negative effect on hospital operational efficiencies. Our data indicated the importance of improving scale efficiency via the optimization of organizational management, as well as the advantage of a different form of branch-establishment, merging and reorganization. This study brought an insight into the practical application of DEA window analysis on the assessment of hospital operational efficiencies

EFFECT OF OTUD3 KNOCKOUT ON THE EXPRESSION OF CONNEXIN 43 IN NEURAL STEM CELLS

Objective To investigate the effect of OTUD3 knockout on the expression of connexin 43 (Cx43) in neural stem cells (NSCs). Methods Brain cortex tissue protein was isolated and extracted from wild-type (WT) and OTUD3-/- embryonic mice on day 14 of gestation, and WT-NSCs and OTUD3-/--NSCs were cultured in vitro and subcultured according to cell status. After subculture to the third generation, an inverted microscope was used to observe the growth state of OTUD3-/--NSCs and WT-NSCs on days 1 and 3. Immunofluorescent staining was used to measure the expression of Ki67, Nestin, and β-tubulin Ⅲ in WT-NSCs and OTUD3-/--NSCs, and Western blotting was used to measure the changes in the expression of OTUD3 and Cx43 in WT-NSCs,OTUD3-/--NSCs, and the cerebral cortex of WT and OTUD3-/- embryonic mice. Results WT-NSCs and OTUD3-/--NSCs cultured and subcultured to the third generation showed a good growth status, and immunofluorescent staining showed positive staining of Ki67, Nestin, and β-tubulin Ⅲ. Compared with WT-NSCs,OTUD3-/--NSCs had significant reductions in the expression levels of OTUD3 and Cx43 (t=16.82,3.21,P<0.05), and compared with WT embryonic mice,OTUD3-/- embryonic mice also had significant reductions in the expression levels of OTUD3 and Cx43 in the cerebral cortex (t=10.04, 20.41,P<0.05). Conclusion The expression of Cx43 in NSCs is significantly downregulated after OTUD3 knockout, and Cx43 may play a regulatory role in promoting the proliferation of NSCs and neurodevelopment