Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems

BACKGROUND: The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface. RESULTS: A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated. CONCLUSION: The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale

A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome

Halo mass - concentration relation from weak lensing

We perform a statistical weak lensing analysis of dark matter profiles around tracers of halo mass from galactic- to cluster-size halos. In this analysis we use 170,640 isolated ~L* galaxies split into ellipticals and spirals, 38,236 groups traced by isolated spectroscopic Luminous Red Galaxies (LRGs) and 13,823 MaxBCG clusters from the Sloan Digital Sky Survey (SDSS) covering a wide range of richness. Together these three samples allow a determination of the density profiles of dark matter halos over three orders of magnitude in mass, from 10^{12} M_{sun} to 10^{15} M_{sun}. The resulting lensing signal is consistent with an NFW or Einasto profile on scales outside the central region. We find that the NFW concentration parameter c_{200b} decreases with halo mass, from around 10 for galactic halos to 4 for cluster halos. Assuming its dependence on halo mass in the form of c_{200b} = c_0 [M/(10^{14}M_{sun}/h)]^{\beta}, we find c_0=4.6 +/- 0.7 (at z=0.22) and \beta=0.13 +/- 0.07, with very similar results for the Einasto profile. The slope (\beta) is in agreement with theoretical predictions, while the amplitude is about two standard deviations below the predictions for this mass and redshift, but we note that the published values in the literature differ at a level of 10-20% and that for a proper comparison our analysis should be repeated in simulations. We discuss the implications of our results for the baryonic effects on the shear power spectrum: since these are expected to increase the halo concentration, the fact that we see no evidence of high concentrations on scales above 20% of the virial radius suggests that baryonic effects are limited to small scales, and are not a significant source of uncertainty for the current weak lensing measurements of the dark matter power spectrum. [ABRIDGED]Comment: 17 pages, 5 figures, accepted to JCAP pending minor revisions that are included in v2 here on arXi

A robust lower limit on the amplitude of matter fluctuations in the universe from cluster abundance and weak lensing

Cluster abundance measurements are among the most sensitive probes of the amplitude of matter fluctuations in the universe, which in turn can help constrain other cosmological parameters, like the dark energy equation of state or neutrino mass. However, difficulties in calibrating the relation between the cluster observable and halo mass, and the lack of completeness information, make this technique particularly susceptible to systematic errors. Here we argue that a cluster abundance analysis using statistical weak lensing on the stacked clusters leads to a robust lower limit on the amplitude of fluctuations. The method compares the average weak lensing signal measured around the whole cluster sample to a theoretical prediction, assuming that the clusters occupy the centers of all of the most massive halos above some minimum mass threshold. If the amplitude of fluctuations is below a certain limiting value, there are too few massive clusters in this model and the theoretical prediction falls below the observations. Since any effects that modify the model assumptions can only decrease the theoretical prediction, the limiting amplitude becomes a robust lower limit. Here, we apply it to a volume limited sample of 16,000 group/cluster candidates identified from isolated luminous red galaxies (LRGs) in the Sloan Digital Sky Survey (SDSS). We find $\sigma_8 (\Omega_m/0.25)^{0.5}>0.62$ at the 95% c.l. after taking into account observational errors in the lensing analysis. While this is a relatively weak constraint, both the scatter in the LRG luminosity-halo mass relation and the lensing errors are large; the constraints could improve considerably in the future with more sophisticated cluster identification algorithms and smaller errors in the lensing analysis. [Abridged]Comment: 10 pages, 5 figures; new version has only very minor revisions to match published versio

Hubble Space Telescope STIS Observations of GRB 000301C: CCD Imaging and Near-Ultraviolet MAMA Spectroscopy

We present Space Telescope Imaging Spectrograph observations of the optical transient (OT) counterpart of the γ-ray burster GRB 000301C obtained 5 days after the burst, on 2000 March 6. CCD clear-aperture imaging reveals a R ≃ 21.50 ± 0.15 source with no apparent host galaxy. An 8000 s, 1150 Å 18 on the line of sight to the OT. This measured redshift is conservatively a lower limit to the GRB redshift. However, as all other GRBs that have deep Hubble Space Telescope images appear to lie on the stellar field of a host galaxy, and as the large H I column density measured here and in later ground-based observations is unlikely on a random line of sight, we believe we are probably seeing absorption from H I in the host galaxy. In any case, this represents the largest direct redshift determination of a γ-ray burster to date. Our data are compatible with an OT spectrum represented by a power law with an intrinsic index α = 1.2 (f_ν ∝ ν^(-α)) and no extinction in the host galaxy, or with α = 0.5 and extinction by SMC-like dust in the OT rest frame with A_V = 0.15. The large N_(H I) and the lack of a detected host are similar to the situation for damped Lyα absorbers at z > 2

Effect of a pneumococcal whole cell vaccine on influenza A-induced pneumococcal otitis media in infant mice.

The pneumococcus remains a common cause of otitis media (OM) despite the widespread introduction of pneumococcal conjugate vaccines. In mice, a pneumococcal whole cell vaccine (WCV) induces serotype-independent protection against pneumococcal colonisation and invasive disease via TH17- and antibody-mediated immunity, respectively. We investigated the effect of WCV on influenza A-induced pneumococcal OM in an infant mouse model. C57BL/6 mice were immunised subcutaneously with a single dose of WCV or adjuvant at 6 days of age, infected with pneumococci (EF3030 [serotype 19F] or PMP1106 [16F]) at 12 days of age, and given influenza A virus (A/Udorn/72/307 [H3N2], IAV) at 18 days of age to induce pneumococcal OM. Pneumococcal density in middle ear and nasopharyngeal tissues was determined 6 and 12 days post-virus. Experiments were repeated in antibody (B6.μMT-/-)- and CD4+ T-cell-deficient mice to investigate the immune responses involved. A single dose of WCV did not prevent the development of pneumococcal OM, nor accelerate pneumococcal clearance compared with mice receiving adjuvant alone. However, WCV reduced the density of EF3030 in the middle ear at 6 days post-viral infection (p = 0.022), and the density of both isolates in the nasopharynx at 12 days post-viral infection (EF3030, p = 0.035; PMP1106, p = 0.011), compared with adjuvant alone. The reduction in density in the middle ear required antibodies and CD4+ T cells: WCV did not reduce EF3030 middle ear density in B6.μMT-/- mice (p = 0.35) nor in wild-type mice given anti-CD4 monoclonal antibody before and after IAV inoculation (p = 0.91); and WCV-immunised CD4+ T cell-deficient GK1.5 mice had higher levels of EF3030 in the middle ear than their adjuvant-immunised counterparts (p = 0.044). A single subcutaneous dose of WCV reduced pneumococcal density in the middle ears of co-infected mice in one of two strains tested, but did not prevent OM from occurring in this animal model

HST/STIS observations of GRB000301C: CCD imaging and NUV MAMA spectroscopy

We present HST/STIS observations of the optical counterpart (OT) of the gamma-ray burster GRB 000301C obtained on 2000 March 6, five days after the burst. CCD clear aperture imaging reveals a R ~ 21.50+/-0.15 source with no apparent host galaxy. An 8000 s, 1150 < lambda/A < 3300 NUV-MAMA prism spectrum shows a relatively flat continuum (in f_lambda) between 2800 and 3300 A, with a mean flux 8.7 (+0.8,-1.6)+/- 2.6 10^(-18) ergs/s/cm^2/A, and a sharp break centered at 2797+/-25 A. We interpret it as HI Lyman break at z = 2.067+/-0.025 indicating the presence of a cloud with a HI column density log(HI) > 18 on the line-of-sight to the OT. This value is conservatively a lower limit to the GRB redshift. However, the facts that large N(HI) system are usually considered as progenitors of present day galaxies and that other OTs are found associated with star forming galaxies strongly suggest that it is the GRB redshift. In any case, this represents the largest direct redshift determination of a gamma-ray burster to date. Our data are compatible with an OT spectrum represented by a power-law with an intrinsic index \alpha = 1.2((f_nu \propto nu^-alpha) and no extinction in the host galaxy or with alpha = 0.5 and extinction by a SMC-like dust in the OT rest-frame with A_V = 0.15. The large N(HI) and the lack of detected host is similar to the situation for damped Ly-alpha absorbers at z > 2.Comment: Replaced by final version. 10 p., 2 fig. Scheduled to appear in ApJ 555 n2 Jul 10, 2001. Minor changes, both redshift and mean near UV flux are revised with slightly larger values, due to a wrong offset sign in the wavelength calibratio

Plasmodium–Helminth Coinfection and Its Sources of Heterogeneity Across East Africa

Background. Plasmodium–helminth coinfection can have a number of consequences for infected hosts, yet our knowledge of the epidemiology of coinfection across multiple settings is limited. This study investigates the distribution and heterogeneity of coinfection with Plasmodium falciparum and 3 major helminth species across East Africa

Combating subclonal evolution of resistant cancer phenotypes

Metastatic breast cancer remains challenging to treat, and most patients ultimately progress on therapy. This acquired drug resistance is largely due to drug-refractory sub-populations (subclones) within heterogeneous tumors. Here, we track the genetic and phenotypic subclonal evolution of four breast cancers through years of treatment to better understand how breast cancers become drug-resistant. Recurrently appearing post-chemotherapy mutations are rare. However, bulk and single-cell RNA sequencing reveal acquisition of malignant phenotypes after treatment, including enhanced mesenchymal and growth factor signaling, which may promote drug resistance, and decreased antigen presentation and TNF-α signaling, which may enable immune system avoidance. Some of these phenotypes pre-exist in pre-treatment subclones that become dominant after chemotherapy, indicating selection for resistance phenotypes. Post-chemotherapy cancer cells are effectively treated with drugs targeting acquired phenotypes. These findings highlight cancer's ability to evolve phenotypically and suggest a phenotype-targeted treatment strategy that adapts to cancer as it evolves

Impact of Common Variation in Bone-Related Genes on Type 2 Diabetes and Related Traits

Exploring genetic pleiotropy can provide clues to a mechanism underlying the observed epidemiological association between type 2 diabetes and heightened fracture risk. We examined genetic variants associated with bone mineral density (BMD) for association with type 2 diabetes and glycemic traits in large well-phenotyped and -genotyped consortia. We undertook follow-up analysis in ∼19,000 individuals and assessed gene expression. We queried single nucleotide polymorphisms (SNPs) associated with BMD at levels of genome-wide significance, variants in linkage disequilibrium (r2 > 0.5), and BMD candidate genes. SNP rs6867040, at the ITGA1 locus, was associated with a 0.0166 mmol/L (0.004) increase in fasting glucose per C allele in the combined analysis. Genetic variants in the ITGA1 locus were associated with its expression in the liver but not in adipose tissue. ITGA1 variants appeared among the top loci associated with type 2 diabetes, fasting insulin, β-cell function by homeostasis model assessment, and 2-h post–oral glucose tolerance test glucose and insulin levels. ITGA1 has demonstrated genetic pleiotropy in prior studies, and its suggested role in liver fibrosis, insulin secretion, and bone healing lends credence to its contribution to both osteoporosis and type 2 diabetes. These findings further underscore the link between skeletal and glucose metabolism and highlight a locus to direct future investigations