A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p < .05), dose-dependent increase in tumor necrosis factor (TNF)-α following SWCNT and MWCNT exposure at concentrations up to 0.02mg/ml to MDM, MDDC, and the TCC-C was found. The concentration of TNF-α released by the MDM and MDDC was significantly higher (p < .05) than the TCC-C. Significant increases (p < .05) in interleukin (IL)-8 were also found for both 16HBE14o- epithelial cells and the TCC-C after SWCNTs and MWCNTs exposure up to 0.02mg/ml. The TCC-C, however, elicited a significantly (p < .05) higher IL-8 release than the epithelial cells. The oxidative potential of both SWCNTs and MWCNTs (0.005-0.02mg/ml) measured by reduced glutathione (GSH) content showed a significant difference (p < .05) between each monoculture and the TCC-C. It was concluded that because only the co-culture system could assess each endpoint adequately, that, in comparison with monoculture systems, multicellular systems that take into consideration important cell type-to-cell type interactions could be used as predictive in vitro screening tools for determining the potential deleterious effects associated with CNTs.

Evolution of sensory complexity recorded in a myxobacterial genome.

Myxobacteria are single-celled, but social, eubacterial predators. Upon starvation they build multicellular fruiting bodies using a developmental program that progressively changes the pattern of cell movement and the repertoire of genes expressed. Development terminates with spore differentiation and is coordinated by both diffusible and cell-bound signals. The growth and development of Myxococcus xanthus is regulated by the integration of multiple signals from outside the cells with physiological signals from within. A collection of M. xanthus cells behaves, in many respects, like a multicellular organism. For these reasons M. xanthus offers unparalleled access to a regulatory network that controls development and that organizes cell movement on surfaces. The genome of M. xanthus is large (9.14 Mb), considerably larger than the other sequenced delta-proteobacteria. We suggest that gene duplication and divergence were major contributors to genomic expansion from its progenitor. More than 1,500 duplications specific to the myxobacterial lineage were identified, representing >15% of the total genes. Genes were not duplicated at random; rather, genes for cell-cell signaling, small molecule sensing, and integrative transcription control were amplified selectively. Families of genes encoding the production of secondary metabolites are overrepresented in the genome but may have been received by horizontal gene transfer and are likely to be important for predation.

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

Genetic Control of Plant Development by Overriding a Geometric Division Rule

Formative cell divisions are critical for multicellular patterning. In the early plant embryo, such divisions follow from orienting the division plane. A major unanswered question is how division plane orientation is genetically controlled, and in particular whether this relates to cell geometry. We have generated a complete 4D map of early Arabidopsis embryogenesis and used computational analysis to demonstrate that several divisions follow a rule that uses the smallest wall area going through the center of the cell. In other cases, however, cell division clearly deviates from this rule, which invariably leads to asymmetric cell division. By analyzing mutant embryos and through targeted genetic perturbation, we show that response to the hormone auxin triggers a deviation from the ``shortest wall'' rule. Our work demonstrates that a simple default rule couples division orientation to cell geometry in the embryo and that genetic regulation can create patterns by overriding the default rule.

Standardised animal models of host microbial mutualism.

An appreciation of the importance of interactions between microbes and multicellular organisms is currently driving research in biology and biomedicine. Many human diseases involve interactions between the host and the microbiota, so investigating the mechanisms involved is important for human health. Although microbial ecology measurements capture considerable diversity of the communities between individuals, this diversity is highly problematic for reproducible experimental animal models that seek to establish the mechanistic basis for interactions within the overall host-microbial superorganism. Conflicting experimental results may be explained away through unknown differences in the microbiota composition between vivaria or between the microenvironment of different isolated cages. In this position paper, we propose standardised criteria for stabilised and defined experimental animal microbiotas to generate reproducible models of human disease that are suitable for systematic experimentation and are reproducible across different institutions.

Bovine coccygeal intervertebral discs contain multipotent Tie2+ cells which can differentiate into osteogenic and adipogenic lineages

Question: The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability [1]. IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin re- ceptor (Tie2) in the nucleus pulposus were identified [2]. Here we isolated primary cells from bovine IVD and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermorewe tested whether Tie2 expressing cells can differentiate into os- teogenic and adipogenic lineages in vitro. Methods: NP cells were obtained from 1 year old bovine tails by sequential digestion with pronase for 1 h and collagenase over- night. Sorted Tie2- and Tie2+ cells were cultured in osteogenic and adipogenic medium for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets. Colony forming units were prepared for both cell sus- pensions in methylcellulose-based medium and formed colonies ([10 cells) were analyzed macroscopically after 8 days. Results: After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by cal- cium deposition and fat droplet formation. By contrast, Tie2- cells generated a weak staining for calcium and no fat droplets were ob- tained (Fig. 1). Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape whereas those from Tie2- cells were spread and fibroblastic. Conclusion: Our data showed that Tie2+ cells of the nucleus pul- posus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage. References 1. Freemont AJ (2009) The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 48:5–10 2. Sakai D, Nakamura Y, Nakai T et al (2012) Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 3:1264 Acknowledgments: This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411.

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

Functional characterization of the trypanosome translational repressor SCD6

The storage of translationally inactive mRNAs in cytosolic granules enables cells to react flexibly to environmental changes. In eukaryotes, Scd6 (suppressor of clathrin deficiency 6)/Rap55 (RNA-associated protein 55), a member of the LSm14 (like-Sm14) family, is an important factor in the formation and activity of P-bodies, where mRNA decay factors accumulate, in stress granules that store mRNAs under adverse conditions and in granules that store developmentally regulated mRNAs. SCD6 from Trypanosoma brucei (TbSCD6) shares the same domain architecture as orthologous proteins in other organisms and is also present in cytosolic granules (equivalent to P-bodies). We show that TbSCD6 is a general repressor of translation and that its depletion by RNAi results in a global increase in protein synthesis. With few exceptions, the steady-state levels of proteins are unchanged. TbSCD6 is not required for the formation of starvation-induced granules in trypanosomes, and unlike Scd6 from yeast, Plasmodium and all multicellular organisms analysed to date, it does not form a complex with the helicase Dhh1 (DExD/H-box helicase 1). In common with Xenopus laevis RAP55, TbSCD6 co-purifies with two arginine methyltransferases; moreover, TbSCD6 itself is methylated on three arginine residues. Finally, a detailed analysis identified roles for the Lsm and N-rich domains in both protein localization and tr

Towards personalized medicine: Chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform

Cancer is responsible for millions of deaths worldwide and the variability in disease patterns calls for patient-specific treatment. Therefore, personalized treatment is expected to become a daily routine in prospective clinical tests. In addition to genetic mutation analysis, predictive chemosensitive assays using patient's cells will be carried out as a decision making tool. However, prior to their widespread application in clinics, several challenges linked to the establishment of such assays need to be addressed. To best predict the drug response in a patient, the cellular environment needs to resemble that of the tumor. Furthermore, the formation of homogeneous replicates from a scarce amount of patient's cells is essential to compare the responses under various conditions (compound and concentration). Here, we present a microfluidic device for homogeneous spheroid formation in eight replicates in a perfused microenvironment. Spheroid replicates from either a cell line or primary cells from adenocarcinoma patients were successfully created. To further mimic the tumor microenvironment, spheroid co-culture of primary lung cancer epithelial cells and primary pericytes were tested. A higher chemoresistance in primary co-culture spheroids compared to primary monoculture spheroids was found when both were constantly perfused with cisplatin. This result is thought to be due to the barrier created by the pericytes around the tumor spheroids. Thus, this device can be used for additional chemosensitivity assays (e.g. sequential treatment) of patient material to further approach the personalized oncology field.

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

Molecular and cellular basis of bone resorption.

Osteoclast research has an exciting history and a challenging future. More than 3 decades ago, it became evident that bone-resorbing osteoclasts are of hematopoietic origin and are ultimately linked to the "basic multicellular unit," where they team up with the other cell types, including bone-forming osteoblasts. Since 2 decades, we have learned about the signaling pathways controlling genes relevant for osteoclastogenesis and bone resorption. It took another decade until the hypothesized "osteoclast differentiation" factor was discovered and was translated into an approved pharmacologic strategy. Here, the focus is on another molecular target, cathepsin K, a cysteine protease being released by the osteoclast into the resorption compartment. Genetic deletion and pharmacological blocking of cathepsin K reduces bone resorption but with ongoing bone formation. This observation not only holds great promise to become a new pharmacologic strategy, but it also provides new insights into the coordinated work of cells in the "basic multicellular unit" and thus, bridges the history and future of osteoclast research. This article is a short primer on osteoclast biology for readers of the special issue on odanacatib, a cathepsin K inhibitor.

TECPR2 Associated Neuroaxonal Dystrophy in Spanish Water Dogs.

Clinical, pathological and genetic examination revealed an as yet uncharacterized juvenile-onset neuroaxonal dystrophy (NAD) in Spanish water dogs. Affected dogs presented with various neurological deficits including gait abnormalities and behavioral deficits. Histopathology demonstrated spheroid formation accentuated in the grey matter of the cerebral hemispheres, the cerebellum, the brain stem and in the sensory pathways of the spinal cord. Iron accumulation was absent. Ultrastructurally spheroids contained predominantly closely packed vesicles with a double-layered membrane, which were characterized as autophagosomes using immunohistochemistry. The family history of the four affected dogs suggested an autosomal recessive inheritance. SNP genotyping showed a single genomic region of extended homozygosity of 4.5 Mb in the four cases on CFA 8. Linkage analysis revealed a maximal parametric LOD score of 2.5 at this region. By whole genome re-sequencing of one affected dog, a perfectly associated, single, non-synonymous coding variant in the canine tectonin beta-propeller repeat-containing protein 2 (TECPR2) gene affecting a highly conserved region was detected (c.4009C>T or p.R1337W). This canine NAD form displays etiologic parallels to an inherited TECPR2 associated type of human hereditary spastic paraparesis (HSP). In contrast to the canine NAD, the spinal cord lesions in most types of human HSP involve the sensory and the motor pathways. Furthermore, the canine NAD form reveals similarities to cases of human NAD defined by widespread spheroid formation without iron accumulation in the basal ganglia. Thus TECPR2 should also be considered as candidate gene for human NAD. Immunohistochemistry and the ultrastructural findings further support the assumption, that TECPR2 regulates autophagosome accumulation in the autophagic pathways. Consequently, this report provides the first genetic characterization of juvenile canine NAD, describes the histopathological features associated with the TECPR2 mutation and provides evidence to emphasize the association between failure of autophagy and neurodegeneration.

Optical recording of calcium currents during impulse conduction in cardiac tissue

We explore the feasibility of obtaining a spatially resolved picture of Ca2+Ca2+ inward currents (ICaICa) in multicellular cardiac tissue by differentiating optically recorded Ca2+Ca2+ transients that accompany propagating action potentials. Patterned growth strands of neonatal rat ventricular cardiomyocytes were stained with the Ca2+Ca2+ indicators Fluo-4 or Fluo-4FF. Preparations were stimulated at 1 Hz, and Ca2+Ca2+ transients were recorded with high spatiotemporal resolution (50  μm50  μm, 2 kHz analog bandwidth) with a photodiode array. Signals were differentiated after appropriate digital filtering. Differentiation of Ca2+Ca2+ transients resulted in optically recorded calcium currents (ORCCs) that carried the temporal and pharmacological signatures of L-type Ca2+Ca2+ inward currents: the time to peak amounted to ∼2.1  ms∼2.1  ms (Fluo-4FF) and ∼2.4  ms∼2.4  ms (Fluo-4), full-width at half-maximum was ∼8  ms∼8  ms, and ORCCs were completely suppressed by 50  μmol/L50  μmol/LCdCl2CdCl2. Also, and as reported before from patch-clamp studies, caffeine reversibly depressed the amplitude of ORCCs. The results demonstrate that the differentiation of Ca2+Ca2+ transients can be used to obtain a spatially resolved picture of the initial phase of ICaICa in cardiac tissue and to assess relative changes of activation/fast inactivation of ICaICa following pharmacological interventions.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^