A high-content platform to characterise human induced pluripotent stem cell lines.

Induced pluripotent stem cells (iPSCs) provide invaluable opportunities for future cell therapies as well as for studying human development, modelling diseases and discovering therapeutics. In order to realise the potential of iPSCs, it is crucial to comprehensively characterise cells generated from large cohorts of healthy and diseased individuals. The human iPSC initiative (HipSci) is assessing a large panel of cell lines to define cell phenotypes, dissect inter- and intra-line and donor variability and identify its key determinant components. Here we report the establishment of a high-content platform for phenotypic analysis of human iPSC lines. In the described assay, cells are dissociated and seeded as single cells onto 96-well plates coated with fibronectin at three different concentrations. This method allows assessment of cell number, proliferation, morphology and intercellular adhesion. Altogether, our strategy delivers robust quantification of phenotypic diversity within complex cell populations facilitating future identification of the genetic, biological and technical determinants of variance. Approaches such as the one described can be used to benchmark iPSCs from multiple donors and create novel platforms that can readily be tailored for disease modelling and drug discovery.

Vascular inflammaging: Endothelial CEACAM1 expression is upregulated by TNF-α via independent activation of NF-κB and ß-catenin signaling.

Chronic inflammation with progressive age, called inflammaging, contributes to the pathogenesis of cardiovascular diseases. Previously, we have shown increased vascular expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in aged mice and humans, presumably via mutual upregulation with the pro-inflammatory cytokine TNF-α. CEACAM1 is critical for aging-associated vascular alterations like endothelial dysfunction, fibrosis, oxidative stress, and sustained inflammation and can be regarded as a main contributor to vascular inflammaging. This study was conducted to elucidate the mechanisms underlying endothelial CEACAM1 upregulation by TNF-α in detail. Using wildtype (WT) and TNF-α knockout (Tnf-/-) mice, we confirmed that the aging-related upregulation of endothelial CEACAM1 critically depends on TNF-α. The underlying mechanisms were analyzed in an endothelial cell culture model. TNF-α time-dependently upregulated CEACAM1 in vitro. In pharmacological experiments, we identified an early NF-κB- and a delayed ß-catenin-mediated response. Involvement of ß-catenin was further substantiated by siRNA-mediated knockdown of the ß-catenin-targeted transcription factor TCF4. Both signaling pathways acted independent from each other. Elucidating the delayed response, co-immunoprecipitation analysis revealed release of ß-catenin from adherens junctions by TNF-α. Finally, TNF-α activated Akt kinase by increasing its Ser473 phosphorylation. Consequently, Akt kinase facilitated ß-catenin signaling by inhibiting its degradation via phosphorylation of GSK3ß at Ser9 and by increased phosphorylation of ß-catenin at Ser552 that augments its transcriptional activity. Taken together, our study provides novel mechanistic insights into the aging-related, inflammation-mediated endothelial upregulation of CEACAM1. Beyond the pathogenesis of cardiovascular diseases, these findings may be significant to all fields of inflammaging.

Therapeutic drug monitoring of linezolid: HPLC-based assays for routine quantification of linezolid in human serum and cerebrospinal fluid.

OBJECTIVE: Therapeutic drug monitoring (TDM) of linezolid can prevent over- and under-dosing in critically ill patients and can be crucial to successful antibiotic treatment. Quick and simple high-performance liquid chromatography (HPLC) assays for the detection of linezolid in human serum and cerebrospinal fluid (CSF) were developed in this study. METHODS: The methods used an Atlantis T3 5.0 µm stationary phase. The mobile phase A contained water (99.4% m/m) and formic acid (0.6% m/m) (pH 2.30). The mobile phase B contained acetonitrile (93.6% m/m), water (6% m/m) and formic acid (0.4% m/m). The methods were isocratic, using 23% of mobile phase B and 77% of mobile phase A. Ultraviolet absorbance detection at 252 nm was used. For sample preparation an internal standard was added, and acetonitrile/methanol was added for protein precipitation. RESULTS: The methods were investigated for linearity, specificity, accuracy, and precision. Stability of linezolid and internal standard was assessed. The retention times of linezolid were 8.5 min and 8.1 min, and the single run time was 15 min. Linezolid was quantified from the lower limit of quantification (0.2 mg/L) to the upper limit of quantification (50 mg/L, 75 mg/L, and 100 mg/L). In routine analysis a high variability of serum and CSF levels was observed and the mean CSF/serum ratio was 0.71±0.16. CONCLUSION: The developed assays enable the study of correlations between the applied dosage, serum concentration and CSF concentration. Additionally, studies with a higher number of samples can be performed to investigate the penetration of linezolid into the central nervous system.

DNA- and RNA-based bacterial communities and geochemical zonation under changing sediment porewater dynamics on the Aldabra Atoll.

The remote Aldabra Atoll, Seychelles, provides the rare opportunity to study bacterial communities in pristine carbonate sediments across an entire biome. The four sampled sites cover sand with high porewater exchange, bioturbated silt and mud with intermediate exchange, as well as a seasonally and episodically desiccated landlocked pool. As sediments harbour dead cells and environmental DNA alongside live cells, we used bacterial 16S rRNA gene and transcript analysis to distinguish between past and present inhabitants. Previously described laminated sediments mirroring past conditions in the Cerin, France could not be retrieved. Thus, the aim was adjusted to determine whether bacterial community composition and diversity follow typical geochemical zonation patterns at different locations of the atoll. Our data confirm previous observations that diversity decreases with depth. In the lagoon, the bacterial community composition changed from Pseudomonas dominating in the sand to diverse mixed surface and sulphate reduction zones in the anaerobic mud with strongly negative Eh. The latter correlated with high total alkalinity, ammonia, and total sulphide, alongside a decrease in SO42-/Cl- and high relative abundances of sulphate reducing (Halo-) Desulfovibrio, sulphur oxidizing Arcobacteraceae, photo(hetero)troph Cyanobacteria, Alphaproteobacteria, and fermenting Propionigenium. In contrast to expectations, deeper mud and pool sediments harboured high abundances of Halomonas or Alphaproteobacteria alongside high C/N and increased salinity. We believe that this atypical community shift may be driven by a change in the complexity of available organic matter.

Cyanobacterial Mats in Calcite-Precipitating Serpentinite-Hosted Alkaline Springs of the Voltri Massif, Italy.

(1) Background: Microbial communities in terrestrial, calcifying high-alkaline springs are not well understood. In this study, we investigate the structure and composition of microbial mats in ultrabasic (pH 10-12) serpentinite springs of the Voltri Massif (Italy). (2) Methods: Along with analysis of chemical and mineralogical parameters, environmental DNA was extracted and subjected to analysis of microbial communities based upon next-generation sequencing. (3) Results: Mineral precipitation and microbialite formation occurred, along with mat formation. Analysis of the serpentinite spring microbial community, based on Illumina sequencing of 16S rRNA amplicons, point to the relevance of alkaliphilic cyanobacteria, colonizing carbonate buildups. Cyanobacterial groups accounted for up to 45% of all retrieved sequences; 3-4 taxa were dominant, belonging to the filamentous groups of Leptolyngbyaceae, Oscillatoriales, and Pseudanabaenaceae. The cyanobacterial community found at these sites is clearly distinct from creek water sediment, highlighting their specific adaptation to these environments.

Bacterial succession along a sediment porewater gradient at Lake Neusiedl in Austria.

We provide bacterial 16S rRNA community and hydrochemical data from water and sediments of Lake Neusiedl, Austria. The sediments were retrieved at 5 cm intervals from 30-40 cm push cores. The lake water community was recovered by filtration through a 3.0/0.2 µm filter sandwich. For 16S rRNA gene amplicon-based community profiling, DNA was extracted from the sediment and filters and the bacterial V3-V4 regions were amplified and sequenced using a MiSeq instrument (Illumina). The reads were quality-filtered and processed using open source bioinformatic tools, such as PEAR, cutadapt and VSEARCH. The taxonomy was assigned against the SILVA SSU NR 132 database. The bacterial community structure was visualised in relation to water and porewater chemistry data. The bacterial community in the water column is distinct from the sediment. The most abundant phyla in the sediment shift from Proteobacteria to Chloroflexota (formerly Chloroflexi). Ammonium and total alkalinity increase while sulphate concentrations in the porewater decrease. The provided data are of interest for studies targeting biogeochemical cycling in lake sediments.

Metabolic microenvironmental control by photosynthetic biofilms under changing macroenvironmental temperature and pH conditions.

Ex situ microelectrode experiments, using cyanobacterial biofilms from karst water creeks, were conducted under various pH, temperature, and constant-alkalinity conditions to investigate the effects of changing environmental parameters on cyanobacterial photosynthesis-induced calcification. Microenvironmental chemical conditions around calcifying sites were controlled by metabolic activity over a wide range of photosynthesis and respiration rates, with little influence from overlying water conditions. Regardless of overlying water pH levels (from 7.8 to 8.9), pH at the biofilm surface was approximately 9.4 in the light and 7.8 in the dark. The same trend was observed at various temperatures (4 degrees C and 17 degrees C). Biological processes control the calcium carbonate saturation state (Omega) in these and similar systems and are able to maintain Omega at approximately constant levels over relatively wide environmental fluctuations. Temperature did, however, have an effect on calcification rate. Calcium flux in this system is limited by its diffusion coefficient, resulting in a higher calcium flux (calcification and dissolution) at higher temperatures, despite the constant, biologically mediated pH. The ability of biological systems to mitigate the effects of environmental perturbation is an important factor that must be considered when attempting to predict the effects of increased atmospheric partial CO(2) pressure on processes such as calcification and in interpreting microfossils in the fossil record.

ß-Catenin Stabilization in Skin Fibroblasts Causes Fibrotic Lesions by Preventing Adipocyte Differentiation of the Reticular Dermis.

The Wnt/ß-catenin pathway plays a central role in epidermal homeostasis and regeneration, but how it affects fibroblast fate decisions is unknown. We investigated the effect of targeted ß-catenin stabilization in dermal fibroblasts. Comparative gene expression profiling of stem cell antigen 1(-) (Sca1(-)) and Sca1(+) neonatal fibroblasts from upper and lower dermis, respectively, confirmed that Sca1(+) cells had a preadipocyte signature and showed differential expression of Wnt/ß-catenin-associated genes. By targeting all fibroblasts or selectively targeting Dlk1(+) lower dermal fibroblasts, we found that ß-catenin stabilization between developmental stages E16.5 and P2 resulted in a reduction in the dermal adipocyte layer with a corresponding increase in dermal fibrosis and an altered hair cycle. The fibrotic phenotype correlated with a reduction in the potential of Sca1(+) fibroblasts to undergo adipogenic differentiation ex vivo. Our findings indicate that Wnt/ß-catenin signaling controls adipogenic cell fate within the lower dermis, which potentially contributes to the pathogenesis of fibrotic skin diseases.

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

Macrophage Infiltration and Alternative Activation during Wound Healing Promote MEK1-Induced Skin Carcinogenesis.

Macrophages are essential for the progression and maintenance of many cancers, but their role during the earliest stages of tumor formation is unclear. To test this, we used a previously described transgenic mouse model of wound-induced skin tumorigenesis, in which expression of constitutively active MEK1 in differentiating epidermal cells results in chronic inflammation (InvEE mice). Upon wounding, the number of epidermal and dermal monocytes and macrophages increased in wild-type and InvEE skin, but the increase was greater, more rapid, and more sustained in InvEE skin. Macrophage ablation reduced tumor incidence. Furthermore, bioluminescent imaging in live mice to monitor macrophage flux at wound sites revealed that macrophage accumulation was predictive of tumor formation; wounds with the greatest number of macrophages at day 5 went on to develop tumors. Gene expression profiling of flow-sorted monocytes, macrophages, and T cells from InvEE and wild-type skin showed that as wound healing progressed, InvEE macrophages altered their phenotype. Throughout wound healing and after wound closure, InvEE macrophages demonstrated sustained upregulation of several markers implicated in alternative macrophage activation including arginase-1 (ARG1) and mannose receptor (CD206). Notably, inhibition of ARG1 activity significantly reduced tumor formation and epidermal proliferation in vivo, whereas addition of L-arginase to cultured keratinocytes stimulated proliferation. We conclude that macrophages play a key role in early, inflammation-mediated skin tumorigenesis, with mechanistic evidence suggesting that ARG1 secretion drives tumor development by stimulating epidermal cell proliferation. These findings highlight the importance of cancer immunotherapies aiming to polarize tumor-associated macrophages toward an antitumor phenotype.

Topical treatment with propranolol gel as a supplement to the existing treatment of hemangiomas.

BACKGROUND: Systemic treatment with propranolol is proven to be effective for patients with hemangiomas with less side-effect. We used a propranolol gel for topical use on hemangiomas. METHODS: In this retrospective study, we analyzed 148 patients who had been treated topically with propranolol gel for 12 weeks. We analyzed the data of patients and clinically gave each hemangioma a "hemangioma score" to determine the treatment success. RESULTS: In 147 of the 148 patients, strong signs of resolution under treatment included lightening, paling, and less vascularization. The hemangioma score showed a significant decrease during the treatment. Relevant serum levels of propranolol were not found. Adverse effects were rare and not related to propranolol. CONCLUSION: Topical treatment with propranolol gel is suitable for specific hemangiomas in addition to cryotherapy and systemic treatment with propranolol.

Stenomaps: Shorthand for shapes.

We address some of the challenges in representing spatial data with a novel form of geometric abstraction-the stenomap. The stenomap comprises a series of smoothly curving linear glyphs that each represent both the boundary and the area of a polygon. We present an efficient algorithm to automatically generate these open, C1-continuous splines from a set of input polygons. Feature points of the input polygons are detected using the medial axis to maintain important shape properties. We use dynamic programming to compute a planar non-intersecting spline representing each polygon's base shape. The results are stylised glyphs whose appearance may be parameterised and that offer new possibilities in the 'cartographic design space'. We compare our glyphs with existing forms of geometric schematisation and discuss their relative merits and shortcomings. We describe several use cases including the depiction of uncertain model data in the form of hurricane track forecasting; minimal ink thematic mapping; and the depiction of continuous statistical data.

Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.

Reprint of: Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.

Phylogenetic analysis of a microbialite-forming microbial mat from a hypersaline lake of the Kiritimati atoll, Central Pacific.

On the Kiritimati atoll, several lakes exhibit microbial mat-formation under different hydrochemical conditions. Some of these lakes trigger microbialite formation such as Lake 21, which is an evaporitic, hypersaline lake (salinity of approximately 170‰). Lake 21 is completely covered with a thick multilayered microbial mat. This mat is associated with the formation of decimeter-thick highly porous microbialites, which are composed of aragonite and gypsum crystals. We assessed the bacterial and archaeal community composition and its alteration along the vertical stratification by large-scale analysis of 16S rRNA gene sequences of the nine different mat layers. The surface layers are dominated by aerobic, phototrophic, and halotolerant microbes. The bacterial community of these layers harbored Cyanobacteria (Halothece cluster), which were accompanied with known phototrophic members of the Bacteroidetes and Alphaproteobacteria. In deeper anaerobic layers more diverse communities than in the upper layers were present. The deeper layers were dominated by Spirochaetes, sulfate-reducing bacteria (Deltaproteobacteria), Chloroflexi (Anaerolineae and Caldilineae), purple non-sulfur bacteria (Alphaproteobacteria), purple sulfur bacteria (Chromatiales), anaerobic Bacteroidetes (Marinilabiacae), Nitrospirae (OPB95), Planctomycetes and several candidate divisions. The archaeal community, including numerous uncultured taxonomic lineages, generally changed from Euryarchaeota (mainly Halobacteria and Thermoplasmata) to uncultured members of the Thaumarchaeota (mainly Marine Benthic Group B) with increasing depth.

Reprogramming to iPS cells and their subsequent hematopoietic differentiation is more efficient from MEFs than from preB cells.

Efficiencies of the generation of induced pluripotent stem (iPS) cells from either mouse embryonic fibroblasts (MEF) or from mouse fetal liver (FL) derived preB cells and their hematogenic potencies were compared. In 10 days approximately 2% of the MEFs transduced with Sox-2, Oct-4 and Klf-4 developed to iPS cells, while only 0.01% of transduced FL-preB cells yielded iPS cells, and only after around 3 weeks. Subsequently, the generated iPS cells were induced to differentiate into hematopoietic cells in vitro. On day 5 of differentiation MEF-iPS yielded numbers and percentages of Flk-1(+) mesodermal-like cells comparable to those developed from embryonic stem (ES) cells. Compared to ES cells further differentiation to hematopoietic and lymphopoietic cells was reduced, possibly because of persistent expression of the reprogramming factors. By contrast, FL-iPS cells developed lower numbers and percentages of Flk-1(+) cells, and no significant further development to hematopoietic or lymphopoietic cells could be induced. These results indicate that the efficiencies of iPS generation and subsequent hematopoietic development depends on the type of differentiated cell from which iPS cells are generated.

Epidemiological dynamics of hepatitis C virus among 747 German individuals: new subtypes on the advance.

This study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.