Towards reliable spike-train recordings from thousands of neurons with multielectrodes

The new generation of silicon-based multielectrodes comprising hundreds or more electrode contacts offers unprecedented possibilities for simultaneous recordings of spike trains from thousands of neurons. Such data will not only be invaluable for finding out how neural networks in the brain work, but will likely be important also for neural prosthesis applications. This opportunity can only be realized if efficient, accurate and validated methods for automatic spike sorting are provided. In this review we describe some of the challenges that must be met to achieve this goal, and in particular argue for the critical need of realistic model data to be used as ground truth in the validation of spike-sorting algorithms.     

Statistical challenges in null model analysis

This review identifies several important challenges in null model testing in ecology: 1) developing randomization algorithms that generate appropriate patterns for a specified null hypothesis; these randomization algorithms stake out a middle ground between formal Pearson–Neyman tests (which require a fully‐specified null distribution) and specific process‐based models (which require parameter values that cannot be easily and independently estimated); 2) developing metrics that specify a particular pattern in a matrix, but ideally exclude other, related patterns; 3) avoiding classification schemes based on idealized matrix patterns that may prove to be inconsistent or contradictory when tested with empirical matrices that do not have the idealized pattern; 4) testing the performance of proposed null models and metrics with artificial test matrices that contain specified levels of pattern and randomness; 5) moving beyond simple presence–absence matrices to incorporate species‐level traits (such as abundance) and site‐level traits (such as habitat suitability) into null model analysis; 6) creating null models that perform well with many sites, many species pairs, and varying degrees of spatial autocorrelation in species occurrence data. In spite of these challenges, the development and application of null models has continued to provide valuable insights in ecology, evolution, and biogeography for over 80 years.     

DLK1(PREF1) is a negative regulator of adipogenesis in CD105⁺/CD90⁺/CD34⁺/CD31⁻/FABP4⁻ adipose-derived stromal cells from subcutaneous abdominal fat pats of adult women

The main physiological function of adipose-derived stromal/progenitor cells (ASC) is to differentiate into adipocytes. ASC are most likely localized at perivascular sites in adipose tissues and retain the capacity to differentiate into multiple cell types. Although cell surface markers for ASC have been described, there is no complete consensus on the antigen expression pattern that will precisely define these cells. DLK1(PREF1) is an established marker for mouse adipocyte progenitors which inhibits adipogenesis. This suggests that DLK1(PREF1) could be a useful marker to characterize human ASC. The DLK1(PREF1) status of human ASC is however unknown. In the present study we isolated ASC from the heterogeneous stromal vascular fraction of subcutaneous abdominal fat pats of adult women. These cells were selected by their plastic adherence and expanded to passage 5. The ASC were characterized as relatively homogenous cell population with the capacity to differentiate in vitro into adipocytes, chondrocytes, and osteoblasts and the immunophenotype CD105?/CD90?/CD34?/CD31?/FABP4?. The ASC were positive for DLK1(PREF1) which was well expressed in proliferating and density arrested cells but downregulated in the course of adipogenic differentiation. To investigate whether DLK1(PREF1) plays a role in the regulation of adipogenesis in these cells RNAi-mediated knockdown experiments were conducted. Knockdown of DLK1(PREF1) in differentiating ASC resulted in a significant increase of the expression of the adipogenic key regulator PPARγ2 and of the terminal adipogenic differentiation marker FABP4. We conclude that DLK1(PREF1) is well expressed in human ASC and acts as a negative regulator of adipogenesis. Moreover, DLK1(PREF1) could be a functional marker contributing to the characterization of human ASC.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

Validation study of existing gene expression signatures for anti-TNF treatment in patients with rheumatoid arthritis

So far, there are no means of identifying rheumatoid arthritis (RA) patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF), prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non) response.     

Estrogens can disrupt amphibian mating behavior

The main component of classical contraceptives, 17α-ethinylestradiol (EE2), has high estrogenic activity even at environmentally relevant concentrations. Although estrogenic endocrine disrupting compounds are assumed to contribute to the worldwide decline of amphibian populations by adverse effects on sexual differentiation, evidence for EE2 affecting amphibian mating behaviour is lacking. In this study, we demonstrate that EE2 exposure at five different concentrations (0.296 ng/L, 2.96 ng/L, 29.64 ng/L, 2.96 μg/L and 296.4 μg/L) can disrupt the mating behavior of adult male Xenopus laevis. EE2 exposure at all concentrations lowered male sexual arousal, indicated by decreased proportions of advertisement calls and increased proportions of the call type rasping, which characterizes a sexually unaroused state of a male. Additionally, EE2 at all tested concentrations affected temporal and spectral parameters of the advertisement calls, respectively. The classical and highly sensitive biomarker vitellogenin, on the other hand, was only induced at concentrations equal or higher than 2.96 μg/L. If kept under control conditions after a 96 h EE2 exposure (2.96 μg/L), alterations of male advertisement calls vanish gradually within 6 weeks and result in a lower sexual attractiveness of EE2 exposed males toward females as demonstrated by female choice experiments. These findings indicate that exposure to environmentally relevant EE2 concentrations can directly disrupt male mate calling behavior of X. laevis and can indirectly affect the mating behavior of females. The results suggest the possibility that EE2 exposure could reduce the reproductive success of EE2 exposed animals and these effects might contribute to the global problem of amphibian decline.     

A heterogeneous computing environment to solve the 768-bit RSA challenge

In December 2009 the 768-bit, 232-digit number RSA-768 was factored using the number field sieve. Overall, the computational challenge would take more than 1700 years on a single, standard core. In the article we present the heterogeneous computing approach, involving different compute clusters and Grid computing environments, used to solve this problem.     

Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.