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

Interpretable Sequence Classification via Discrete Optimization

Sequence classification is the task of predicting a class label given a sequence of observations. In many applications such as healthcare monitoring or intrusion detection, early classification is crucial to prompt intervention. In this work, we learn sequence classifiers that favour early classification from an evolving observation trace. While many state-of-the-art sequence classifiers are neural networks, and in particular LSTMs, our classifiers take the form of finite state automata and are learned via discrete optimization. Our automata-based classifiers are interpretable---supporting explanation, counterfactual reasoning, and human-in-the-loop modification---and have strong empirical performance. Experiments over a suite of goal recognition and behaviour classification datasets show our learned automata-based classifiers to have comparable test performance to LSTM-based classifiers, with the added advantage of being interpretable

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

CYP-450 Epoxygenase Derived Epoxyeicosatrienoic Acid Contribute To Reversal of Heart Failure in Obesity-Induced Diabetic Cardiomyopathy via PGC-1 alpha Activation

We have previously shown that an Epoxyeicosatrienoic Acid (EET) -agonist has pleiotropic effects and reverses cardiomyopathy by decreasing inflammatory molecules and increasing antioxidant signaling. We hypothesized that administration of an EET agonist would increase Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1alpha), which controls mitochondrial function and induction of HO-1 and negatively regulates the expression of the proinflammatory adipokines CCN3/NOV in cardiac and pericardial tissues. This pathway would be expected to further improve left ventricular (LV) systolic function as well as increase insulin receptor phosphorylation. Measurement of the effect of an EET agonist on oxygen consumption, fractional shortening, blood glucose levels, thermogenic and mitochondrial signaling proteins was performed. Control obese mice developed signs of metabolic syndrome including insulin resistance, hypertension, inflammation, LV dysfunction, and increased NOV expression in pericardial adipose tissue. EET agonist intervention decreased pericardial adipose tissue expression of NOV, while normalized FS, increased PGC-1alpha, HO-1 levels, insulin receptor phosphorylation and improved mitochondrial function, theses beneficial effect were reversed by deletion of PGC-1alpha. These studies demonstrate that an EET agonist increases insulin receptor phosphorylation, mitochondrial and thermogenic gene expression, decreased cardiac and pericardial tissue NOV levels, and ameliorates cardiomyopathy in an obese mouse model of the metabolic syndrome

Monoamines, BDNF, Dehydroepiandrosterone, DHEA-Sulfate, and Childhood Depression—An Animal Model Study

Basal levels of monoamines and DHEA in four main limbic brain regions were measured in prepubertal Wistar Kyoto (WKY) rats (a putative animal model of childhood depression). Basal levels of “Brain-Derived Neurotrophic Factor (BDNF)” were also determined in two regions in the hippocampus, compared with Wistar strain controls. In the second phase, we examined the responsiveness of prepubertal WKY rats to different types of chronic antidepressant treatments: Fluoxetine, Desipramine, and dehydroepiandrosterone sulfate (DHEAS). WKY prepubertal rats exhibited different monoamine levels in the limbic system, reduced DHEA levels in the VTA and lower levels of BDNF in the hippocampus CA3 region compared to controls. In prepubertal WKY rats, only treatment with DHEAS produced a statistically significant decrease in immobility, compared to saline-administered controls in the forced swim test. Wistar controls were not affected by any antidepressant. The results imply that DHEA(S) and BDNF may be involved in the pathophysiology and pharmacotherapy of childhood depression