Standalone cell culture microfluidic device-based microphysiological system for automated cell observation and application in nephrotoxicity tests.

Microphysiological systems (MPS) offer an alternative method for culturing cells on microfluidic platforms to model organ functions in pharmaceutical and medical sciences. Although MPS hardware has been proposed to maintain physiological organ function through perfusion culture, no existing MPS can automatically assess cell morphology and conditions online to observe cellular dynamics in detail. Thus, with this study, we aimed to establish a practical strategy for automating cell observation and improving cell evaluation functions with low temporal resolution and throughput in MPS experiments. We developed a versatile standalone cell culture microfluidic device (SCCMD) that integrates microfluidic chips and their peripherals. This device is compliant with the ANSI/SLAS standards and has been seamlessly integrated into an existing automatic cell imaging system. This integration enables automatic cell observation with high temporal resolution in MPS experiments. Perfusion culture of human kidney proximal tubule epithelial cells using the SCCMD improves cell function. By combining the proximal tubule MPS with an existing cell imaging system, nephrotoxicity studies were successfully performed to automate morphological and material permeability evaluation. We believe that the concept of building the ANSI/SLAS-compliant-sized MPS device proposed herein and integrating it into an existing automatic cell imaging system for the online measurement of detailed cell dynamics information and improvement of throughput by automating observation operations is a novel potential research direction for MPS research.

Ferroic Order for Anisotropic Magnetic Dipole Term in Collinear Antiferromagnets of (t_{2g})

We present the possibility of x-ray magnetic circular dichroism on RuO_{2} with collinear antiferromagnetism (AFM). Given that the crystal symmetry breaks the time reversal symmetry when the antiparallel spin aligns along the [100] direction, the expectation vector of the anisotropic magnetic dipole operator ⟨t⟩ remains uncanceled along the [010] direction. Our Letter reveals that the magnetic dipole (T_{z}) term in the x-ray magnetic circular dichroism is induced by the residual ⟨t⟩. Because the features of the magnetic moment can be detected via absorption measurements even in the AFM, this technique will be useful for determining the magnetic phase, the Van Vleck-type paramagnet or the excitonic AFM in (t_{2g})^{4} system.

Automated algorithm development to assess survival of human neurons using longitudinal single-cell tracking: Application to synucleinopathy.

The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.

Electronic and crystal structures ofLnFeAsO1-xHx(Ln= La, Sm) studied by x-ray absorption spectroscopy, x-ray emission spectroscopy, and x-ray diffraction: II pressure dependence.

We examine electronic and crystal structures of iron-based superconductorsLnFeAsO1-xHx(Ln= La, Sm) under pressure by means of x-ray absorption spectroscopy (XAS), x-ray emission spectroscopy (XES), and x-ray diffraction. In LaFeAsO the pre-edge peak on high-resolution XAS at the Fe-Kabsorption edge gains in intensity on the application of pressure up to 5.7 GPa and it saturates in the higher pressure region. We found integrated-absolute difference values on XES forLn= La, corresponding to a spin state, decline on the application of pressure, and then it is minimized when theTcapproaches the maximum at around 5 GPa. In contrast, such the optimum value was not detected forLn= Sm. We reveal that the superconductivity is closely related to the lower spin state forLn= La unlike Sm case. We observed that As height from the Fe basal plane and As-Fe-As angle on the FeAs4tetrahedron forLn= La deviate from the optimum values of the regular tetrahedron in superconducting (SC) phase, which has been widely accepted structural guide to SC thus far. In contrast, the structural parameters were held near the optimum values up to ∼15 GPa forLn= Sm.

Electronic and crystal structures ofLnFeAsO1-xHx(Ln= La, Sm) studied by x-ray absorption spectroscopy, x-ray emission spectroscopy, and x-ray diffraction (part I: carrier-doping dependence).

A carrier doping by a hydrogen substitution in LaFeAsO1-xHxis known to cause two superconducting (SC) domes with the magnetic order at both end sides of the doping. In contrast, SmFeAsO1-xHxhas a similar phase diagram but shows single SC dome. Here, we investigated the electronic and crystal structures for iron oxynitrideLnFeAsO1-xHx(Ln= La, Sm) with the range ofx= 0-0.5 by using x-ray absorption spectroscopy, x-ray emission spectroscopy, and x-ray diffraction. For both compounds, we observed that the pre-edge peaks of x-ray absorption spectra near the Fe-Kedge were reduced in intensity on doping. The character arises from the weaker As-Fe hybridization with the longer As-Fe distance in the higher doped region. We can reproduce the spectra near the Fe-Kedge according to the Anderson impurity model with realistic valence structures using the local-density approximation (LDA) plus dynamical mean-field theory (DMFT). ForLn= Sm, the integrated-absolute difference (IAD) analysis from x-ray Fe-Kßemission spectra increases significantly. This is attributed to the enhancement of magnetic moment of Fe 3delectrons stemming from the localized picture in the higher doped region. A theoretical simulation implementing the self-consistent vertex-correction method reveals that the single dome superconducting phase forLn= Sm arises from a better nesting condition in comparison withLn= La.

Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons.

Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Efficiency of Gastrointestinal Cancer Detection by Nematode-NOSE (N-NOSE).

BACKGROUND/AIM: Early detection of gastrointestinal cancer may reduce mortality. Recently, Caenorhabditis elegans has been reported to be capable of differentiating patients with cancers from healthy persons by the smell of urine. This novel technique using C. elegans olfaction has been named as Nematode-NOSE (N-NOSE). MATERIALS AND METHODS: We collected 180 urine samples from patients with gastrointestinal cancer and 76 samples from healthy subjects. N-NOSE test was performed using these samples and N-NOSE index was obtained. Quantification of the olfactory behavior of C. elegans was performed as established in past studies. By receiver operating characteristic (ROC) analysis, we examined the diagnostic capability of N-NOSE. RESULTS: ROC analysis revealed that N-NOSE showed an area under the curve value of more than 0.80, even in early-stage cancers. CONCLUSION: C. elegans olfaction enabled the detection of gastrointestinal cancers from urine with high sensitivity, which can provide the basis for the development of N-NOSE as a gastrointestinal cancer screening test.

[Development of an Early Cancer Detection Method Using the Olfaction of the Nematode Caenorhabditis elegans].

Early detection and treatment are important for the successful eradication of various cancers; therefore, the development of economical, noninvasive novel cancer screening systems is critical. Previous reports using canine scent detection have demonstrated the existence of cancer-specific odors. However, it is difficult to introduce canine scent recognition into clinical practice because of the need to maintain accuracy. In this study, we developed a Nematode-Nose (N-NOSE) test using Caenorhabditis elegans to provide a novel, highly accurate cancer detection system that is economical, painless, rapid, and convenient. We demonstrated that wild-type C. elegans displayed attractive chemotaxis toward human cancer cell secretions, cancer tissues, and urine from cancer patients but avoided control urine. In parallel, C. elegans olfactory neurons showed a significantly stronger response to urine from cancer patients than to control urine. In contrast, G protein α mutants and animals with ablated olfactory neurons were not attracted to urine from cancer patients, suggesting that they sense odors in urine. We tested 242 samples to measure the performance of the N-NOSE test and found that the sensitivity was 95.8%, which is markedly higher than that of other existing tumor markers. Furthermore, the specificity was 95.0%. Importantly, this test could detect various cancer types tested at the early stage (stage 0 or 1). C. elegans scent-based analyses therefore might provide a new strategy for the detection and study of disease-associated scents.

Behavioural Response Alteration in Caenorhabditis elegans to Urine After Surgical Removal of Cancer: Nematode-NOSE (N-NOSE) for Postoperative Evaluation.

The technique used for cancer monitoring is essential for effective cancer therapy. Currently, several methods such as diagnostic imaging and biochemical markers have been used for cancer monitoring, but these are invasive and show low sensitivity. A previous study reported that Caenorhabditis elegans sensitively discriminated patients with cancer from healthy subjects, based on the smell of a urine sample. However, whether C. elegans olfaction can detect the removal of cancerous tumours remains unknown. This study was conducted to examine C. elegans olfactory behaviour to urine samples collected from 78 patients before and after surgery. The diagnostic ability of the technique termed Nematode-NOSE (N-NOSE) was evaluated by receiver operating characteristic (ROC) analysis. The ROC curve of N-NOSE was higher than those of classic tumour markers. Furthermore, we examined the change in C. elegans olfactory behaviour following exposure to preoperative and postoperative samples. The results suggest that a reduction in attraction indicates the removal of the cancerous tumour. This study may lead to the development of a noninvasive and highly sensitive tool for evaluating postoperative cancer patients.

A morphology-based assay platform for neuroepithelial-like cells differentiated from human pluripotent stem cells.

Cell morphology is recognized as an important hallmark of neural cells. During the differentiation of human pluripotent stem cells (hPSCs) into neural cells, cell morphology changes dynamically. Therefore, characterization of the morphology of cells during this period is important to improve our understanding of the differentiation and development of neural cells. General methods for the directed induction of hPSCs include the steps of multi-cellular aggregation or high-density cell culture, particularly at the early phase of neural differentiation, and therefore, the morphology of each differentiating cell is difficult to recognize. Here, we have developed a new method for the directed differentiation of neuroepithelial-like cells (NELCs) from hPSCs at a low cell density in an adherent monolayer culture, as well as an image-processing algorithm to evaluate the cell morphology of differentiating NELCs, in order to follow cell morphology during the differentiation of hPSCs into NELCs. Using these methods, the morphological transition of differentiating cells was observed in real time using phase contrast imaging and then quantified. Because cell morphology is also considered an inherent biological marker of neural cells cultured in vitro, this method is potentially useful to study the mechanisms underlying neural cell differentiation.

Using Automated Live Cell Imaging to Reveal Early Changes during Human Motor Neuron Degeneration.

Human neurons expressing mutations associated with neurodegenerative disease are becoming more widely available. Hence, developing assays capable of accurately detecting changes that occur early in the disease process and identifying therapeutics able to slow these changes should become ever more important. Using automated live-cell imaging, we studied human motor neurons in the process of dying following neurotrophic factor withdrawal. We tracked different neuronal features, including cell body size, neurite length, and number of nodes. In particular, measuring the number of nodes in individual neurons proved to be an accurate predictor of relative health. Importantly, intermediate phenotypes were defined and could be used to distinguish between agents that could fully restore neurons and neurites and those only capable of maintaining neuronal cell bodies. Application of live-cell imaging to disease modeling has the potential to uncover new classes of therapeutic molecules that intervene early in disease progression.

A-Site and B-Site Charge Orderings in an s-d Level Controlled Perovskite Oxide PbCoO3.

Perovskite PbCoO3 synthesized at 12 GPa was found to have an unusual charge distribution of Pb2+Pb4+3Co2+2Co3+2O12 with charge orderings in both the A and B sites of perovskite ABO3. Comprehensive studies using density functional theory (DFT) calculation, electron diffraction (ED), synchrotron X-ray diffraction (SXRD), neutron powder diffraction (NPD), hard X-ray photoemission spectroscopy (HAXPES), soft X-ray absorption spectroscopy (XAS), and measurements of specific heat as well as magnetic and electrical properties provide evidence of lead ion and cobalt ion charge ordering leading to Pb2+Pb4+3Co2+2Co3+2O12 quadruple perovskite structure. It is shown that the average valence distribution of Pb3.5+Co2.5+O3 between Pb3+Cr3+O3 and Pb4+Ni2+O3 can be stabilized by tuning the energy levels of Pb 6s and transition metal 3d orbitals.

Visualization of morphological categories of colonies for monitoring of effect on induced pluripotent stem cell culture status.

From the recent advances, there are growing expectations toward the mass production of induced pluripotent stem cells (iPSCs) for varieties of applications. For such type of industrial cell manufacturing, the technology which can stabilize the production efficiency is strongly required. Since the present iPSC culture is covered by delicate manual operations, there are still quality differences in produced cells from same culture protocols. To monitor the culture process of iPSCs with the quantified data to evaluate the culture status, we here introduce image-based visualization method of morphological diversity of iPSC colonies. We have set three types of experiments to evaluate the influential factors in iPSC culture technique that may disturb the undifferentiation status of iPSC colonies: (Exp. 1) technical differences in passage skills, (Exp. 2) technical differences in feeder cell preparation, and (Exp. 3) technical differences in maintenance skills (medium exchange frequency with the combination of manual removal of morphologically irregular colonies). By measuring the all existing colonies from real-time microscopic images, the heterogenous change of colony morphologies in the culture vessel was visualized. By such visualization with morphologically categorized Manhattan chart, the difference between technical skills could be compared for evaluating appropriate cell processing.

Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control.

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of 'hPSC colony morphology', permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

Voltage-dependent anion channel (VDAC-1) is required for olfactory sensing in Caenorhabditis elegans.

The Ras-MAP kinase signaling pathway plays important roles for the olfactory reception in olfactory neurons in Caenorhabditis elegans. However, given the absence of phosphorylation targets of MAPK in the olfactory neurons, the mechanism by which this pathway regulates olfactory function is unknown. Here, we used proteomic screening to identify the mitochondrial voltage-dependent anion channel VDAC-1 as a candidate target molecule of MAPK in the olfactory system of C. elegans. We found that Amphid Wing "C" (AWC) olfactory neuron-specific knockdown of vdac-1 caused severe defects in chemotaxis toward AWC-sensed odorants. We generated a new vdac-1 mutant using the CRISPR-Cas9 system, with this mutant also showing decreased chemotaxis toward odorants. This defect was rescued by AWC-specific expression of vdac-1, indicating that functions of VDAC-1 in AWC neurons are essential for normal olfactory reception in C. elegans. We observed that AWC-specific RNAi of vdac-1 reduced AWC calcium responses to odorant stimuli and caused a decrease in the quantity of mitochondria in the sensory cilia. Behavioral abnormalities in vdac-1 knockdown animals might therefore be due to reduction of AWC response, which might be caused by loss of mitochondria in the cilia. Here, we showed that the function of VDAC-1 is regulated by phosphorylation and identified Thr175 as the potential phosphorylation site of MAP kinase.

A highly accurate inclusive cancer screening test using Caenorhabditis elegans scent detection.

Early detection and treatment are of vital importance to the successful eradication of various cancers, and development of economical and non-invasive novel cancer screening systems is critical. Previous reports using canine scent detection demonstrated the existence of cancer-specific odours. However, it is difficult to introduce canine scent recognition into clinical practice because of the need to maintain accuracy. In this study, we developed a Nematode Scent Detection Test (NSDT) using Caenorhabditis elegans to provide a novel highly accurate cancer detection system that is economical, painless, rapid and convenient. We demonstrated wild-type C. elegans displayed attractive chemotaxis towards human cancer cell secretions, cancer tissues and urine from cancer patients but avoided control urine; in parallel, the response of the olfactory neurons of C. elegans to the urine from cancer patients was significantly stronger than to control urine. In contrast, G protein α mutants and olfactory neurons-ablated animals were not attracted to cancer patient urine, suggesting that C. elegans senses odours in urine. We tested 242 samples to measure the performance of the NSDT, and found the sensitivity was 95.8%; this is markedly higher than that of other existing tumour markers. Furthermore, the specificity was 95.0%. Importantly, this test was able to diagnose various cancer types tested at the early stage (stage 0 or 1). To conclude, C. elegans scent-based analyses might provide a new strategy to detect and study disease-associated scents.

A role for Ras in inhibiting circular foraging behavior as revealed by a new method for time and cell-specific RNAi.

BACKGROUND: The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. In particular, RNAi is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible RNAi or cell-specific RNAi. However, conventional RNAi is insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions. RESULTS: Here, we investigated the Ras gene for its role in exploratory behavior in C. elegans. We found that, under poor environmental conditions, mutations in the Ras-MAPK signaling pathway lead to circular locomotion instead of normal exploratory foraging. Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion. We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons. To investigate the temporal- and cell-specific profiles of the functions of Ras, we developed a new RNAi method that enables simultaneous time- and cell-specific knockdown. In this method, one RNA strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded RNA in the target cell when heat shock is induced. This technique revealed that control of GLR-1 localization in RMD neurons requires Ras at the adult stage. Further, we demonstrated the application of this method to other genes. CONCLUSIONS: We have established a new RNAi method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the Ras-MAPK pathway in the control of exploratory behavior under poor environmental conditions.

High-pressure synthesis, crystal structure, and unusual valence state of novel perovskite oxide CaCu3Rh4O12.

A novel perovskite oxide, CaCu3Rh4O12, has been synthesized under high-pressure and high-temperature conditions (15 GPa and 1273 K). Rietveld refinement of synchrotron X-ray powder diffraction data indicates that this compound crystallizes in a cubic AA'3B4O12-type perovskite structure. Synchrotron X-ray absorption and photoemission spectroscopy measurements reveal that the Cu and Rh valences are nearly trivalent. The spectroscopic analysis based on calculations suggests that the appropriate ionic model of this compound is Ca(2+)Cu(∼2.8+)3Rh(∼3.4+)4O12, as opposed to the conventional Ca(2+)Cu(2+)3Rh(4+)4O12. The uncommon valence state of this compound is attributed to the relative energy levels of the Cu 3d and Rh 4d orbitals, in which the large crystal-field splitting energy of the Rh 4d orbitals is substantial.

Screening of odor-receptor pairs in Caenorhabditis elegans reveals different receptors for high and low odor concentrations.

Olfactory systems sense and respond to various odorants. Olfactory receptors, which in most organisms are G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors, directly bind volatile or soluble odorants. Compared to the genomes of mammals, the genome of the nematode Caenorhabditis elegans contains more putative olfactory receptor genes, suggesting that in nematodes there may be combinatorial complexity to the receptor-odor relationship. We used RNA interference (RNAi) screening to identify nematode olfactory receptors necessary for the response to specific odorants. This screening identified 194 candidate olfactory receptor genes linked to 11 odorants. Additionally, we identified SRI-14 as being involved in sensing high concentrations of diacetyl. Rescue and neuron-specific RNAi experiments demonstrated that SRI-14 functioned in ASH neurons, specific chemosensory neurons, resulting in avoidance responses. Calcium imaging revealed that ASH neurons responded to high diacetyl concentrations only, whereas another class of chemosensory neurons, AWA neurons, reacted to both low and high concentrations. Loss of SRI-14 function hampered ASH responses to high diacetyl concentrations, whereas loss of ODR-10 function reduced AWA responses to low odorant concentrations. Chemosensory neurons ectopically expressing SRI-14 responded to a high concentration of diacetyl. Thus, nematodes have concentration-dependent odor-sensing mechanisms that are segregated at the olfactory receptor and sensory neuron levels.

Temporally-regulated quick activation and inactivation of Ras is important for olfactory behaviour.

Responses to environmental stimuli are mediated by the activation and inactivation of various signalling proteins. However, the temporal dynamics of these events in living animals are not well understood. Here we show real-time imaging of the activity of the key regulator of the MAP kinase pathway, Ras, in living Caenorhabditis elegans and that Ras is transiently activated within a few seconds in olfactory neurons in response to increase in the concentration of odorants. This fast activation of Ras is dependent on the olfactory signalling pathway and Ras guanyl nucleotide-releasing protein (RasGRP). A negative feedback loop then quickly leads to Ras inactivation despite the continued presence of the odorant. Phenotypes of Ras mutants suggest this rapid activation and inactivation of Ras is important for regulation of interneuron activities and olfactory behaviours. Our results reveal novel kinetics and biological implication of transient activation of Ras in olfactory systems.