Robust visualization and discrimination of nanoparticles by interferometric imaging

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.R01 AI096159 - NIAID NIH HHSFirst author draf

Bioinformatic identification and characterization of human endothelial cell-restricted genes

<p>Abstract</p> <p>Background</p> <p>In this study, we used a systematic bioinformatics analysis approach to elucidate genes that exhibit an endothelial cell (EC) restricted expression pattern, and began to define their regulation, tissue distribution, and potential biological role.</p> <p>Results</p> <p>Using a high throughput microarray platform, a primary set of 1,191 transcripts that are enriched in different primary ECs compared to non-ECs was identified (LCB >3, FDR <2%). Further refinement of this initial subset of transcripts, using published data, yielded 152 transcripts (representing 109 genes) with different degrees of EC-specificity. Several interesting patterns emerged among these genes: some were expressed in all ECs and several were restricted to microvascular ECs. Pathway analysis and gene ontology demonstrated that several of the identified genes are known to be involved in vasculature development, angiogenesis, and endothelial function (P < 0.01). These genes are enriched in cardiovascular diseases, hemorrhage and ischemia gene sets (P < 0.001). Most of the identified genes are ubiquitously expressed in many different tissues. Analysis of the proximal promoter revealed the enrichment of conserved binding sites for 26 different transcription factors and analysis of the untranslated regions suggests that a subset of the EC-restricted genes are targets of 15 microRNAs. While many of the identified genes are known for their regulatory role in ECs, we have also identified several novel EC-restricted genes, the function of which have yet to be fully defined.</p> <p>Conclusion</p> <p>The study provides an initial catalogue of EC-restricted genes most of which are ubiquitously expressed in different endothelial cells.</p

A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen.

BACKGROUND: Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. METHODS: We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. RESULTS: We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. CONCLUSIONS: Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

Background: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons

Developmental Bias in Cleavage-Stage Mouse Blastomeres

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.Molecular and Cellular Biolog

The Influence of L-Carnitine on Oxidative Modification of LDL In Vitro

Owing to their structure and function, low-density lipoproteins (LDLs) are particularly susceptible to the oxidative modifications. To prevent against oxidative modification of LDL, L-carnitine, with endogenous small water-soluble quaternary amine possessing antioxidative properties, was used. The aim of this paper was to prove the in vitro influence of L-carnitine on the degree of oxidative modification of the lipid part (estimated by conjugated dienes, lipid hydroperoxides, and malondialdehyde levels) and the protein part (estimated by dityrosine and tryptophan levels) of LDL native and oxidized by cooper ions. The level of lipophylic LDL antioxidant—α-tocopherol was also measured

Nonlinear Time Series Analysis of Nodulation Factor Induced Calcium Oscillations: Evidence for Deterministic Chaos?

Legume plants form beneficial symbiotic interactions with nitrogen fixing bacteria (called rhizobia), with the rhizobia being accommodated in unique structures on the roots of the host plant. The legume/rhizobial symbiosis is responsible for a significant proportion of the global biologically available nitrogen. The initiation of this symbiosis is governed by a characteristic calcium oscillation within the plant root hair cells and this signal is activated by the rhizobia. Recent analyses on calcium time series data have suggested that stochastic effects have a large role to play in defining the nature of the oscillations. The use of multiple nonlinear time series techniques, however, suggests an alternative interpretation, namely deterministic chaos. We provide an extensive, nonlinear time series analysis on the nature of this calcium oscillation response. We build up evidence through a series of techniques that test for determinism, quantify linear and nonlinear components, and measure the local divergence of the system. Chaos is common in nature and it seems plausible that properties of chaotic dynamics might be exploited by biological systems to control processes within the cell. Systems possessing chaotic control mechanisms are more robust in the sense that the enhanced flexibility allows more rapid response to environmental changes with less energetic costs. The desired behaviour could be most efficiently targeted in this manner, supporting some intriguing speculations about nonlinear mechanisms in biological signaling

SDF1 in the dorsal corticospinal tract promotes CXCR4+ cell migration after spinal cord injury

<p>Abstract</p> <p>Background</p> <p>Stromal cell-derived factor-1 (SDF1) and its major signaling receptor, CXCR4, were initially described in the immune system; however, they are also expressed in the nervous system, including the spinal cord. After spinal cord injury, the blood brain barrier is compromised, opening the way for chemokine signaling between these two systems. These experiments clarified prior contradictory findings on normal expression of SDF1 and CXCR4 as well as examined the resulting spinal cord responses resulting from this signaling.</p> <p>Methods</p> <p>These experiments examined the expression and function of SDF1 and CXCR4 in the normal and injured adult mouse spinal cord primarily using CXCR4-EGFP and SDF1-EGFP transgenic reporter mice.</p> <p>Results</p> <p>In the uninjured spinal cord, SDF1 was expressed in the dorsal corticospinal tract (dCST) as well as the meninges, whereas CXCR4 was found only in ependymal cells surrounding the central canal. After spinal cord injury (SCI), the pattern of SDF1 expression did not change rostral to the lesion but it disappeared from the degenerating dCST caudally. By contrast, CXCR4 expression changed dramatically after SCI. In addition to the CXCR4+ cells in the ependymal layer, numerous CXCR4+ cells appeared in the peripheral white matter and in the dorsal white matter localized between the dorsal corticospinal tract and the gray matter rostral to the lesion site. The non-ependymal CXCR4+ cells were found to be NG2+ and CD11b+ macrophages that presumably infiltrated through the broken blood-brain barrier. One population of macrophages appeared to be migrating towards the dCST that contains SDF1 rostral to the injury but not towards the caudal dCST in which SDF1 is no longer present. A second population of the CXCR4+ macrophages was present near the SDF1-expressing meningeal cells.</p> <p>Conclusions</p> <p>These observations suggest that attraction of CXCR4+ macrophages is part of a programmed response to injury and that modulation of the SDF1 signaling system may be important for regulating the inflammatory response after SCI.</p

Sparsity-based single-shot sub-wavelength coherent diffractive imaging

We present the experimental reconstruction of sub-wavelength features from the far-field intensity of sparse optical objects: sparsity-based sub-wavelength imaging combined with phase-retrieval. As examples, we demonstrate the recovery of random and ordered arrangements of 100 nm features with the resolution of 30 nm, with an illuminating wavelength of 532 nm. Our algorithmic technique relies on minimizing the number of degrees of freedom; it works in real-time, requires no scanning, and can be implemented in all existing microscopes - optical and non-optical