An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids

The Pseudomonas aeruginosa accessory genome elements influence virulence towards Caenorhabditis elegans

BACKGROUND: Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans to provide evolutionary insights into host-pathogen relationships. RESULTS: Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identify accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we find a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence while the presence of CRISPR-Cas associates with higher virulence. CONCLUSIONS: Our analysis demonstrates substantial strain variation in P. aeruginosa virulence, mediated by specific accessory genome elements that promote increased or decreased virulence. We exemplify that viral-like accessory genome elements that decrease virulence can be restricted by bacterial CRISPR-Cas immune defense systems, and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in virulence maintenance

Unbiased Shape Compactness for Segmentation

We propose to constrain segmentation functionals with a dimensionless, unbiased and position-independent shape compactness prior, which we solve efficiently with an alternating direction method of multipliers (ADMM). Involving a squared sum of pairwise potentials, our prior results in a challenging high-order optimization problem, which involves dense (fully connected) graphs. We split the problem into a sequence of easier sub-problems, each performed efficiently at each iteration: (i) a sparse-matrix inversion based on Woodbury identity, (ii) a closed-form solution of a cubic equation and (iii) a graph-cut update of a sub-modular pairwise sub-problem with a sparse graph. We deploy our prior in an energy minimization, in conjunction with a supervised classifier term based on CNNs and standard regularization constraints. We demonstrate the usefulness of our energy in several medical applications. In particular, we report comprehensive evaluations of our fully automated algorithm over 40 subjects, showing a competitive performance for the challenging task of abdominal aorta segmentation in MRI.Comment: Accepted at MICCAI 201

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation

Glutamate-mediated blood-brain barrier opening. implications for neuroprotection and drug delivery

The blood-brain barrier is a highly selective anatomical and functional interface allowing a unique environment for neuro-glia networks. Blood-brain barrier dysfunction is common in most brain disorders and is associated with disease course and delayed complications. However, the mechanisms underlying blood-brain barrier opening are poorly understood. Here we demonstrate the role of the neurotransmitter glutamate in modulating early barrier permeability in vivo Using intravital microscopy, we show that recurrent seizures and the associated excessive glutamate release lead to increased vascular permeability in the rat cerebral cortex, through activation of NMDA receptors. NMDA receptor antagonists reduce barrier permeability in the peri-ischemic brain, whereas neuronal activation using high-intensity magnetic stimulation increases barrier permeability and facilitates drug delivery. Finally, we conducted a double-blind clinical trial in patients with malignant glial tumors, using contrast-enhanced magnetic resonance imaging to quantitatively assess blood-brain barrier permeability. We demonstrate the safety of stimulation that efficiently increased blood-brain barrier permeability in 10 of 15 patients with malignant glial tumors. We suggest a novel mechanism for the bidirectional modulation of brain vascular permeability toward increased drug delivery and prevention of delayed complications in brain disorders. SIGNIFICANCE STATEMENT: In this study, we reveal a new mechanism that governs blood-brain barrier (BBB) function in the rat cerebral cortex, and, by using the discovered mechanism, we demonstrate bidirectional control over brain endothelial permeability. Obviously, the clinical potential of manipulating BBB permeability for neuroprotection and drug delivery is immense, as we show in preclinical and proof-of-concept clinical studies. This study addresses an unmet need to induce transient BBB opening for drug delivery in patients with malignant brain tumors and effectively facilitate BBB closure in neurological disorders

Autoresonance in a Dissipative System

We study the autoresonant solution of Duffing's equation in the presence of dissipation. This solution is proved to be an attracting set. We evaluate the maximal amplitude of the autoresonant solution and the time of transition from autoresonant growth of the amplitude to the mode of fast oscillations. Analytical results are illustrated by numerical simulations.Comment: 22 pages, 3 figure

Perturbative analysis of wave interactions in nonlinear systems

This work proposes a new way for handling obstacles to asymptotic integrability in perturbed nonlinear PDEs within the method of Normal Forms - NF - for the case of multi-wave solutions. Instead of including the whole obstacle in the NF, only its resonant part is included, and the remainder is assigned to the homological equation. This leaves the NF intergable and its solutons retain the character of the solutions of the unperturbed equation. We exploit the freedom in the expansion to construct canonical obstacles which are confined to te interaction region of the waves. Fo soliton solutions, e.g., in the KdV equation, the interaction region is a finite domain around the origin; the canonical obstacles then do not generate secular terms in the homological equation. When the interaction region is infifnite, or semi-infinite, e.g., in wave-front solutions of the Burgers equation, the obstacles may contain resonant terms. The obstacles generate waves of a new type, which cannot be written as functionals of the solutions of the NF. When an obstacle contributes a resonant term to the NF, this leads to a non-standard update of th wave velocity.Comment: 13 pages, including 6 figure