High-Throughput Cryopreservation of Plant Cell Cultures for Functional Genomics

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model “Mitomouse” (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial

Down-Regulation of miR-92 in Human Plasma Is a Novel Marker for Acute Leukemia Patients

BACKGROUND: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs that primarily function as gene regulators. Aberrant microRNA expression has been described for several human malignancies, and this new class of small regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this knowledge, there is little information regarding microRNAs in plasma especially because microRNAs in plasma, if exist, were thought to be digested by RNase. Recent studies, however, have revealed that microRNAs exist and escape digestion in plasma. METHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA microaray to obtain insight into microRNA deregulation in the plasma of a leukemia patient. We have revealed that microRNA-638 (miR-638) is stably present in human plasmas, and microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for distinguishing leukemia patients from healthy body. CONCLUSIONS/SIGNIFICANCE: The ratio of miR-92a/miR-638 in plasma has strong potential for clinical application as a novel biomarker for detection of leukemia

Metabolomics approach for determining growth-specific metabolites based on Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) is the best MS technology for obtaining exact mass measurements owing to its great resolution and accuracy, and several outstanding FT-ICR/MS-based metabolomics approaches have been reported. A reliable annotation scheme is needed to deal with direct-infusion FT-ICR/MS metabolic profiling. Correlation analyses can help us not only uncover relations between the ions but also annotate the ions originated from identical metabolites (metabolite derivative ions). In the present study, we propose a procedure for metabolite annotation on direct-infusion FT-ICR/MS by taking into consideration the classification of metabolite-derived ions using correlation analyses. Integrated analysis based on information of isotope relations, fragmentation patterns by MS/MS analysis, co-occurring metabolites, and database searches (KNApSAcK and KEGG) can make it possible to annotate ions as metabolites and estimate cellular conditions based on metabolite composition. A total of 220 detected ions were classified into 174 metabolite derivative groups and 72 ions were assigned to candidate metabolites in the present work. Finally, metabolic profiling has been able to distinguish between the growth stages with the aid of PCA. The constructed model using PLS regression for OD600 values as a function of metabolic profiles is very useful for identifying to what degree the ions contribute to the growth stages. Ten phospholipids which largely influence the constructed model are highly abundant in the cells. Our analyses reveal that global modification of those phospholipids occurs as E. coli enters the stationary phase. Thus, the integrated approach involving correlation analyses, metabolic profiling, and database searching is efficient for high-throughput metabolomics

Accuracy assurance in binary interaction approximation for N-Body problems

Two accuracy assurance schemes are combined into the Binary Interaction Approximation (BIA) to N-body problems. The first one is a sort of variable time step (VTS) scheme for a given error tolerance. Since this scheme sometimes does not converge, an error-tolerance-adjusting (ETA) scheme is also introduced. With these two schemes combined into the original BIA, a significant improvement in terms of numerical error is obtained.This article is based on the presentation at the 21st International Toki Conference (ITC21

Noxious chemical discrimination by Tribolium castaneum TRPA1 channel in the HEK293 cell expression system

Nociception is the sensory perception of noxious chemical stimuli. Repellent behavior to avoid noxious stimuli is indispensable for survival, and this mechanism has been evolutionarily conserved across a wide range of species, from mammals to insects. The transient receptor potential ankyrin 1 (TRPA1) channel is one of the most conserved noxious chemical sensors. Here, we describe the heterologous stable expression of Tribolium castaneum TRPA1 (TcTRPA1) in human embryonic kidney (HEK293) cells. The intracellular Ca2+ influx was measured when two compounds, citronellal and l-menthol, derived from plant essential oils, were applied in vitro using a fluorescence assay. The analysis revealed that citronellal evoked Ca2+ influx dose-dependently for TcTRPA1, whereas l-menthol did not. In combination with our present and previous results of the avoidance-behavioral assay at the organism level, we suggest that TcTRPA1 discriminates between these two toxic compounds, and diversification in the chemical nociception selectivity has occurred in TRPA1 channel among insect taxa

Pathological diagnosis of gastric cancers with a novel computerized analysis system

Background: Recent studies of molecular biology have provided great advances for diagnostic molecular pathology. Automated diagnostic systems with computerized scanning for sampled cells in fluids or smears are now widely utilized. Automated analysis of tissue sections is, however, very difficult because they exhibit a complex mixture of overlapping malignant tumor cells, benign host-derived cells, and extracellular materials. Thus, traditional histological diagnosis is still the most powerful method for diagnosis of diseases. Methods: We have developed a novel computer-assisted pathology system for rapid, automated histological analysis of hematoxylin and eosin (H and E)-stained sections. It is a multistage recognition system patterned after methods that human pathologists use for diagnosis but harnessing machine learning and image analysis. The system first analyzes an entire H and E-stained section (tissue) at low resolution to search suspicious areas for cancer and then the selected areas are analyzed at high resolution to confirm the initial suspicion. Results: After training the pathology system with gastric tissues samples, we examined its performance using other 1905 gastric tissues. The system's accuracy in detecting malignancies was shown to be almost equal to that of conventional diagnosis by expert pathologists. Conclusions: Our novel computerized analysis system provides a support for histological diagnosis, which is useful for screening and quality control. We consider that it could be extended to be applicable to many other carcinomas after learning normal and malignant forms of various tissues. Furthermore, we expect it to contribute to the development of more objective grading systems, immunohistochemical staining systems, and fluorescent-stained image analysis systems

Potential Role of DEC1 in Cervical Cancer Cells Involving Overexpression and Apoptosis

Basic helix-loop-helix (BHLH) transcription factors differentiated embryonic chondrocyte gene 1 (DEC1) and gene 2 (DEC2) regulate circadian rhythms, apoptosis, epithelial mesenchymal transition (EMT), invasions and metastases in various kinds of cancer. The stem cell markers SOX2 and c-MYC are involved in the regulation of apoptosis and poor prognosis. In cervical cancer, however, their roles are not well elucidated yet. To determine the function of these genes in human cervical cancer, we examined the expression of DEC1, DEC2, SOX2 and c-MYC in human cervical cancer tissues. In immunohistochemistry, they were strongly expressed in cancer cells compared with in non-cancerous cells. Notably, the strong rate of DEC1 and SOX2 expressions were over 80% among 20 cases. We further examined the roles of DEC1 and DEC2 in apoptosis. Human cervical cancer HeLa and SiHa cells were treated with cisplatin—HeLa cells were sensitive to apoptosis, but SiHa cells were resistant. DEC1 expression decreased in the cisplatin-treated HeLa cells, but had little effect on SiHa cells. Combination treatment of DEC1 overexpression and cisplatin inhibited apoptosis and affected SOX2 and c-MYC expressions in HeLa cells. Meanwhile, DEC2 overexpression had little effect on apoptosis and on SOX2 and c-MYC expressions. We conclude that DEC1 has anti-apoptotic effects and regulates SOX2 and c-MYC expressions on apoptosis