Multiplexed miRNA northern blots via hybridization chain reaction

Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs

High-field vortices in Josephson junctions with alternating critical current density

We study long Josephson junctions with the critical current density alternating along the junction. New equilibrium states, which we call the field synchronized or FS states, are shown to exist if the applied field is from narrow intervals centered around equidistant series of resonant fields, $H_m$. The values of $H_m$ are much higher than the flux penetration field, $H_s$. The flux per period of the alternating critical current density, $\phi_i$, is fixed for each of the FS states. In the $m$-th FS state the value of $\phi_i$ is equal to an integer amount of flux quanta, $\phi_i =m\phi_0$. Two types of single Josephson vortices carrying fluxes $\phi_0$ or/and $\phi_0/2$ can exist in the FS states. Specific stepwise resonances in the current-voltage characteristics are caused by periodic motion of these vortices between the edges of the junction.Comment: 4 pages, 5 figure

Maximum supercurrent in Josephson junctions with alternating critical current density

We consider theoretically and numerically magnetic field dependencies of the maximum supercurrent across Josephson tunnel junctions with spatially alternating critical current density. We find that two flux-penetration fields and one-splinter-vortex equilibrium state exist in long junctions.Comment: 11 pages, 8 figure

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging – analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry – analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging – digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples

Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing

Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas, from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA, imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (PAssive CLARITY Technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH

Neuroactive steroids in depression and anxiety disorders: Clinical studies

Certain neuroactive steroids modulate ligand-gated ion channels via non-genomic mechanisms. Especially 3 alpha-reduced pregnane steroids are potent positive allosteric modulators of the gamma-aminobutyric acid type A (GABA(A)) receptor. During major depression, there is a disequilibrium of 3 alpha-reduced neuroactive steroids, which is corrected by clinically effective pharmacological treatment. To investigate whether these alterations are a general principle of successful antidepressant treatment, we studied the impact of nonpharmacological treatment options on neuroactive steroid concentrations during major depression. Neither partial sleep deprivation, transcranial magnetic stimulation, nor electroconvulsive therapy affected neuroactive steroid levels irrespectively of the response to these treatments. These studies suggest that the changes in neuroactive steroid concentrations observed after antidepressant pharmacotherapy more likely reflect distinct pharmacological properties of antidepressants rather than the clinical response. In patients with panic disorder, changes in neuroactive steroid composition have been observed opposite to those seen in depression. However, during experimentally induced panic induction either with cholecystokinine-tetrapeptide or sodium lactate, there was a pronounced decline in the concentrations of 3 alpha-reduced neuroactive steroids in patients with panic disorder, which might result in a decreased GABAergic tone. In contrast, no changes in neuroactive steroid concentrations could be observed in healthy controls with the exception of 3 alpha,5 alpha-tetrahydrodeoxycorticosterone. The modulation of GABA(A) receptors by neuroactive steroids might contribute to the pathophysiology of depression and anxiety disorders and might offer new targets for the development of novel anxiolytic compounds. Copyright (c) 2006 S. Karger AG, Basel

Regional differences in AIDS and non-AIDS related mortality in HIV-positive individuals across Europe and Argentina: the EuroSIDA study

BACKGROUND Differences in access to care and treatment have been reported in Eastern Europe, a region with one of the fastest growing HIV epidemics, compared to the rest of Europe. This analysis aimed to establish whether there are regional differences in the mortality rate of HIV-positive individuals across Europe, and Argentina. METHODS 13,310 individuals under follow-up were included in the analysis. Poisson regression investigated factors associated with the risk of death. FINDINGS During 82,212 person years of follow-up (PYFU) 1,147 individuals died (mortality rate 14.0 per 1,000 PYFU (95% confidence interval [CI] 13.1-14.8). Significant differences between regions were seen in the rate of all-cause, AIDS and non-AIDS related mortality (global p<0.0001 for all three endpoints). Compared to South Europe, after adjusting for baseline demographics, laboratory measurements and treatment, a higher rate of AIDS related mortality was observed in East Europe (IRR 2.90, 95%CI 1.97-4.28, p<.0001), and a higher rate of non-AIDS related mortality in North Europe (IRR 1.51, 95%CI 1.24-1.82, p<.0001). The differences observed in North Europe decreased over calendar-time, in 2009-2011, the higher rate of non-AIDS related mortality was no longer significantly different to South Europe (IRR 1.07, 95%CI 0.66-1.75, p = 0.77). However, in 2009-2011, there remained a higher rate of AIDS-related mortality (IRR 2.41, 95%CI 1.11-5.25, p = 0.02) in East Europe compared to South Europe in adjusted analysis. INTERPRETATIONS There are significant differences in the rate of all-cause mortality among HIV-positive individuals across different regions of Europe and Argentina. Individuals in Eastern Europe had an increased risk of mortality from AIDS related causes and individuals in North Europe had the highest rate of non-AIDS related mortality. These findings are important for understanding and reviewing HIV treatment strategies and policies across the European region