Imaging of dense cell cultures by multiwavelength lens-free video microscopy.

They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm2 ) over a large field of view of 29.4 mm2 . Combined with the compactness of the system which fits altogether inside an incubator, lens-free microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry.

Apoptosis-modulatory miR-361-3p as a novel treatment target in endocrine-responsive and endocrine-resistant breast cancer.

Breast cancer (BC) is the most diagnosed cancer in women worldwide. In estrogen receptor (ER)-positive disease, anti-estrogens and aromatase inhibitors (AI) improve patient survival; however, many patients develop resistance. Dysregulation of apoptosis is a common resistance mechanism; thus, agents that can reinstate the activity of apoptotic pathways represent promising therapeutics for advanced drug-resistant disease. Emerging targets in this scenario include microRNAs (miRs). To identify miRs modulating apoptosis in drug-responsive and -resistant BC, a high-throughput miR inhibitor screen was performed, followed by high-content screening microscopy for apoptotic markers. Validation demonstrated that miR-361-3p inhibitor significantly increases early apoptosis and reduces proliferation of drug-responsive (MCF7), plus AI-/antiestrogen-resistant derivatives (LTED, TamR, FulvR), and ER- cells (MDA-MB-231). Importantly, proliferation-inhibitory effects were observed in vivo in a xenograft model, indicating the potential clinical application of miR-361-3p inhibition. RNA-seq of tumour xenografts identified FANCA as a direct miR-361-3p target, and validation suggested miR-361-3p inhibitor effects might be mediated in part through FANCA modulation. Moreover, miR-361-3p inhibition resulted in p53-mediated G1 cell cycle arrest through activation of p21 and reduced BC invasion. Analysis of publicly available datasets showed miR-361-3p expression is significantly higher in primary breast tumours vspaired normal tissue and is associated with decreased overall survival. In addition, miR-361-3p inhibitor treatment of BC patient explants decreased levels of miR-361-3p and proliferation marker, Ki67. Finally, miR-361-3p inhibitor showed synergistic effects on BC growth when combined with PARP inhibitor, Olaparib. Together, these studies identify miR-361-3p inhibitor as a potential new treatment for drug-responsive and -resistant advanced BC.

Transcriptome analysis of muscle in horses suffering from recurrent exertional rhabdomyolysis revealed energetic pathway alterations and disruption in the cytosolic calcium regulation.

Recurrent exertional rhabdomyolysis (RER) is frequently observed in race horses like trotters. Some predisposing genetic factors have been described in epidemiological studies. However, the exact aetiology is still unknown. A calcium homeostasis disruption was suspected in previous experimental studies, and we suggested that a transcriptome analysis of RER muscles would be a possible way to investigate the pathway disorder. The purpose of this study was to compare the gene expression profile of RER vs. control muscles in the French Trotter to determine any metabolic or structural disruption. Total RNA was extracted from the gluteal medius and longissimus lumborum muscles after biopsies in 15 French Trotter horses, including 10 controls and 5 RER horses affected by 'tying-up' with high plasmatic muscular enzyme activities. Gene expression analysis was performed on the muscle biopsies using a 25K oligonucleotide microarray, which consisted of 24,009 mouse and 384 horse probes. Transcriptome analysis revealed 191 genes significantly modulated in RER vs. control muscles (P < 0.05). Many genes involved in fatty acid oxidation (CD36/FAT, SLC25A17), the Krebs cycle (SLC25A11, SLC25A12, MDH2) and the mitochondrial respiratory chain were severely down-regulated (tRNA, MT-ND5, MT-ND6, MT-COX1). According to the down-regulation of RYR1, SLC8A1 and UCP2 and up-regulation of APP and HSPA5, the muscle fibre calcium homeostasis seemed to be greatly affected by an increased cytosolic calcium and a depletion of the sarcoplasmic reticulum calcium. Gene expression analysis suggested an alteration of ATP synthesis, with severe mitochondrial dysfunction that could explain the disruption of cytosolic calcium homeostasis and inhibition of muscular relaxation.

Signaling Pathways, Chemical and Biological Modulators of Nucleotide Excision Repair: The Faithful Shield against UV Genotoxicity.

The continuous exposure of the human body's cells to radiation and genotoxic stresses leads to the accumulation of DNA lesions. Fortunately, our body has several effective repair mechanisms, among which is nucleotide excision repair (NER), to counteract these lesions. NER includes both global genome repair (GG-NER) and transcription-coupled repair (TC-NER). Deficiencies in the NER pathway underlie the development of several DNA repair diseases, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). Deficiencies in GG-NER and TC-NER render individuals to become prone to cancer and neurological disorders, respectively. Therefore, NER regulation is of interest in fine-tuning these risks. Distinct signaling cascades including the NFE2L2 (NRF2), AHR, PI3K/AKT1, MAPK, and CSNK2A1 pathways can modulate NER function. In addition, several chemical and biological compounds have proven success in regulating NER's activity. These modulators, particularly the positive ones, could therefore provide potential treatments for genetic DNA repair-based diseases. Negative modulators, nonetheless, can help sensitize cells to killing by genotoxic chemicals. In this review, we will summarize and discuss the major upstream signaling pathways and molecules that could modulate the NER's activity.

A large-scale RNAi screen identifies LCMR1 as a critical regulator of Tspan8-mediated melanoma invasion.

Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3ß, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.

A large-scale RNAi screen identifies LCMR1 as a critical regulator of Tspan8-mediated melanoma invasion.

This corrects the article DOI: 10.1038/onc.2016.219.

Lensfree diffractive tomography for the imaging of 3D cell cultures.

New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup.

The proton-translocating ATPase of Candida tropicalis plasma membrane.

Proton translocation activity of Candida tropicalis plasma membrane ATPase has been demonstrated using a fluorescent delta pH probe (ACMA) and by direct pH measurements. Modifications in fluorescence intensity and H+ transport are highly specific for Mg2+ and ATP, and are sensitive to the well-known inhibitors of the plasma membrane ATPase, vanadate and DCCD. A H+/ATP ratio of 0.54 is found.

The tonoplast proton-translocating ATPase of higher plants as a third class of proton-pumps.

Taken together, all the data reported recently in the literature suggest that tonoplast ATPase belongs to a new class of proton pumps. To date, the most studied system is the proton-pumping ATPase from the tonoplast of Hevea latex. Its main characteristics are presented. It resembles the mitochondrial ATPase in its specificity, its substrate affinity, and its sensitivity to different inhibitors. However, for some aspects, it resembles the plasma membrane system in its response to other inhibitors tested (quercetin for example). It differs from both ATPases in its sensitivity to nitrate as well as by its molecular structure, i.e. a complex exhibiting a least 4 or 5 polypeptides. These results favor the existence of a third class of proton pumps, intermediate between the F1F0-class and the E1E2-class.

Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells.

Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.

Statistical modeling and analyses of a base-specific Salmonella mutagenicity assay.

Statistical features of a base-specific Salmonella mutagenicity assay are considered in detail, following up on a previous report comparing responses of base-specific Salmonella (Ames II) strains with those of traditional tester strains. In addition to using different Salmonella strains, the new procedure also differs in that it is performed as a microwell fluctuation test, as opposed to the standard plate or preincubation test. This report describes the statistical modeling of data obtained from the use of these new strains in the microwell test procedure. We emphasize how to assess any significant interactions between replicate cultures and exposure doses, and how to identify a significant increase in the mutagenic response to a series of concentrations of a test substance.

Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens: the results of a validation study.

The ability of a TA7000 series of Salmonella his- mutant tester strains to detect mutagens as classified by the traditional tester strains (TA100, TA98, TA1535, TA1537, TA97, TA102 and TA104) was evaluated using 30 coded chemicals, 5 of which were duplicates with different code numbers. The TA7000 series of tester strains were TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006, each of which reverts by a specific base substitution. In addition, each chemical was tested in a mixture of the base-specific strains (the Mix), plus the traditional strains, TA98 and TA1537. A liquid version of the Salmonella mutagenicity assay was performed in microtiter plates to allow partial automation for increased throughput. The results were compared to those in the National Toxicology Program (NTP) database, which were obtained from the traditional strains in the preincubation assay. In the two strains common to both protocols, TA98 and TA1537, the agreement was 80% and 85%, respectively. When compared to the NTP results for TA100, the Mix gave a 72% concordance, while the addition of the frameshift tester strain, TA98, increased the agreement to 76%. The overall agreement on positive or negative classifications of mutagenicity was 88% for the 25 chemicals tested. There were three notable exceptions to the overall agreement. Benzaldehyde was detected as a mutagen in TA7005 in contrast to its classification as a non-mutagen in the NTP database. This does not necessarily contradict the NTP results because the base-specific strains may respond to different mutagens. Two weak mutagens in the NTP database, 1-chloro-2-propanol and isobutyl nitrite, were not detected as mutagens in the base-specific new strains in the liquid protocol. While there are a number of major differences in the two assays, it was concluded that the results from each procedure are comparable.

An EWOD-based microfluidic chip for single-cell isolation, mRNA purification and subsequent multiplex qPCR.

Single cell analysis circumvents the need to average data from large populations by observing each cell individually, thus enabling the analysis of cell-to-cell variability. The ability to work on this scale presents many new opportunities for the life sciences and biomedical applications. Microfluidics has become a tool of choice for such studies and electrowetting on dielectric (EWOD) technology is well adapted for samples with reduced size and biological studies at the single cell level. In the present manuscript, for the first time, we present an integrated and automated system based on EWOD that can process the complete workflow on a single device, from the isolation of a single cell to mRNA purification and gene expression analysis.

Continuous microcarrier-based cell culture in a benchtop microfluidic bioreactor.

Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.

High-throughput monitoring of major cell functions by means of lensfree video microscopy.

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.

Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8.

The exposure of calreticulin (CRT) on the surface of stressed and dying cancer cells facilitates their uptake by dendritic cells and the subsequent presentation of tumor-associated antigens to T lymphocytes, hence stimulating an anticancer immune response. The chemotherapeutic agent mitoxantrone (MTX) can stimulate the peripheral relocation of CRT in both human and yeast cells, suggesting that the CRT exposure pathway is phylogenetically conserved. Here, we show that pheromones can act as physiological inducers of CRT exposure in yeast cells, thereby facilitating the formation of mating conjugates, and that a large-spectrum inhibitor of G protein-coupled receptors (which resemble the yeast pheromone receptor) prevents CRT exposure in human cancer cells exposed to MTX. An RNA interference screen as well as transcriptome analyses revealed that chemokines, in particular human CXCL8 (best known as interleukin-8) and its mouse ortholog Cxcl2, are involved in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. MTX stimulated the production of CXCL8 by human cancer cells in vitro and that of Cxcl2 by murine tumors in vivo. The knockdown of CXCL8/Cxcl2 receptors (CXCR1/Cxcr1 and Cxcr2) reduced MTX-induced CRT exposure in both human and murine cancer cells, as well as the capacity of the latter-on exposure to MTX-to elicit an anticancer immune response in vivo. Conversely, the addition of exogenous Cxcl2 increased the immunogenicity of dying cells in a CRT-dependent manner. Altogether, these results identify autocrine and paracrine chemokine signaling circuitries that modulate CRT exposure and the immunogenicity of cell death.

Gene expression profiling in blood cells of endurance horses completing competition or disqualified due to metabolic disorder.

REASONS FOR PERFORMING STUDY: Genomics using cDNA microarrays could provide useful information about physiological adaptations and metabolic disorders in endurance horses. OBJECTIVES: In order to show that genes are modulated in leucocytes in relationship with performance and clinical status of the horses, gene expression in leucocytes, haematological and biochemical parameters were compared between successful and disqualified endurance horses. METHODS: Blood samples were collected at rest (TO) and just after a 140-160 km endurance race (T1) in 2 groups of horses: 10 continuing successful (S) and 10 disqualified horses stopped at a vet-gate for metabolic disorders (D). Total RNA was extracted from the blood cells (leucocytes), checked for purity, amplified and hybridised using mouse cDNA microarrays including 15,264 unique genes. Differential gene expressions were studied by hybridisation of each sample T1 vs. a control sample collected at TO (pool of 20 sound horses). RESULTS: Some significant differences were observed in the haematology and biochemistry of the 2 groups (S vs. D). In Group D, rhadomyolysis was confirmed with CK 13,124 u/l and AST 1242 u/l. The list of 726 (including 603 annotated genes) significant genes was filtered according to a high P-value cut-off (P<0.00001). Among them, 130 were upregulated (expression ratio>1.5) and 288 were down-regulated (<1/1.5). Analysis of variance revealed 62 genes differentially expressed (P<0.05) in Groups D and S. The expression levels of 28 and 50 genes were significantly correlated (r>0.75) with CK and AST level in Group D, respectively. The gene ontology classification showed that more genes were up-regulated in S than in the D. More genes were down-regulated in the disqualified horses. CONCLUSIONS: Long exercise induced many significant gene modulations in leucocytes. Some genes were expressed in relationship with the clinical phenotype observed in Group D: rhabdomyolysis and haemolysis. POTENTIAL RELEVANCE: Some of these genes could be candidates to explain poor performance or pathologies. Further association studies with a greater number of genes should be conducted.

Gene expression profiling in equine muscle tissues using mouse cDNA microarrays.

REASONS FOR PERFORMING STUDY: Progress could be achieved by using microarrays to understand metabolic adaptations and disorders in equine muscle in response to exercise. OBJECTIVES: To test the feasibility of using mouse cDNA microarrays to analyse gene expression profile in normal equine muscles. METHODS: Muscular biopsies of dorsal gluteus medius and longissimus lumborum were done in 4 healthy Standardbreds. Total RNA was extracted from the muscle samples. The concentration and quality of RNA were measured before and after amplification. Gene expression profiles were measured using mouse cDNA microarrays including 15,264 unique genes representing about 11,000 documented genes. Three hybridisation tests were performed to check interspecificity, reproducibility and to compare gene expression in these muscles. For each test, a dye-swap hybridisation with Cy3 and Cy5 fluoromarkers were done and the gene list filtered according the signal level. RESULTS: According to the specificity test, the mouse cDNA microarrays were correctly hybridised by equine muscle cDNA. All positive control genes (GAPDH, HPRT and beta-Actin) and no negative control gene (yeast, plant) hybridised. The reproducibility test demonstrated a good linearity between the duplicate hybridisations: 99.99% of the significant expressed genes have an expression ratio between 1.4 and 1/1.4 = 0.71. These limits can be considered as the thresholds to qualify as up-regulated (ratio >1.4) or downregulated (ratio <0.71). In the muscle comparison test between gluteus medius vs. longissimus lumborum, 63 genes were found up-regulated and 8 genes down-regulated. The range of gene expression ratios in the gluteus medius was 0.61-8.31 x the longissimus lumborum. This list of modulated genes was classified by functions using a gene ontology data basis. CONCLUSION: Mouse microarrays could be used to hybridise equine RNA extracted from muscle tissues. For many genes there are large sequence identities that allowed interspecific cDNA hybridisation. The sensitivity of the method allowed quantification of up- and down-regulated genes after applying appropriate filters. POTENTIAL RELEVANCE: Expression profiling could be used to explore the muscle metabolism changes related to exercise, training, pathology and illegal medication in horses.

Interaction of a redox-sensitive DNA-binding factor with the 5'-flanking region of the micF gene in Escherichia coli.

The product of the micF gene is an endogenous antisense RNA which down-regulates the expression of a major outer membrane protein, OmpF, in E. coli. We report here that two DNA-binding factors compete for the same site in the promoter region of the micF gene: RSBF, a high-affinity redox-sensitive DNA-binding factor that responds to an active oxygen species other than hydrogen peroxide or superoxide anions; and HRBF a heat-resistant DNA-binding factor. Both RSBF and HRBF bind to the same DNA sequence, 5'-TTAAAATCAATAACTTATTCTTAA3-', located upstream of the transcription start site of the micF gene. We present evidence that RSBF could be the controlling factor of a novel regulon involved in the response to oxidative stress in E. coli.

Chloride-ion stimulation of the tonoplast H+-translocating ATPase from Hevea brasiliensis (rubber tree) latex. A dual mechanism.

The effect of Cl- and other anions on the tonoplast H+-translocating ATPase (H+-ATPase) from Hevea brasiliensis (rubber tree) latex was investigated. Cl- and other anions stimulated the ATPase activity of tightly sealed vesicles prepared from Hevea tonoplast, with the following decreasing order of effectiveness: Cl- greater than Br- greater than SO4(2-) greater than NO3-. As indicated by the changes of the protonmotive potential difference, anion stimulation of tonoplast H+-ATPase was caused in part by the ability of these anions to dissipate the electrical potential. This interpretation assumes not a channelling of these anions against a membrane potential, negative-inside, but a modification of the permeability of these ions through the tonoplast membrane. In addition, Cl- and the other anions stimulated the ATPase activity solubilized from the tonoplast membrane. Consequently, the tonoplast H+-pumping ATPase can be considered as an anion-stimulated enzyme. These results are discussed in relation to various models described in the literature for the microsomal H+-ATPase systems claimed as tonoplast entities.