Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay.

Extracellular vesicles (EVs) have attracted enormous attention for their diagnostic and therapeutic potential. However, it has proven challenging to achieve the sensitivity to detect individual nanoscale EVs, the specificity to distinguish EV subpopulations, and a sufficient throughput to study EVs among an enormous background. To address this fundamental challenge, we developed a droplet-based optofluidic platform to quantify specific individual EV subpopulations at high throughput. The key innovation of our platform is parallelization of droplet generation, processing, and analysis to achieve a throughput (∼20 million droplets/min) more than 100× greater than typical microfluidics. We demonstrate that the improvement in throughput enables EV quantification at a limit of detection = 9EVs/µL, a >100× improvement over gold standard methods. Additionally, we demonstrate the clinical potential of this system by detecting human EVs in complex media. Building on this work, we expect this technology will allow accurate quantification of rare EV subpopulations for broad biomedical applications.

PHI-DAC: protein homology database through dihedral angle conservation.

UNLABELLED: Finding related conformations in the Protein Data Bank is essential in many areas of bioscience. To assist this task, we designed a dihedral angle database for searching protein segment homologs. The search engine relies on encoding of the protein coordinates into text characters representing amino acid sequence, φ and ψ dihedral angles. The search engine is advantageous owing to its high speed and interactive nature and is expected to assist scientists in discovering conformation homologs and evolutionary kinship. The search engine is fast, with query times lasting a few seconds, and freely available at http://tarshish.md.biu.ac.il/∼samsona. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Elastic network normal mode dynamics reveal the GPCR activation mechanism.

G-protein-coupled receptors (GPCR) are a family of membrane-embedded metabotropic receptors which translate extracellular ligand binding into an intracellular response. Here, we calculate the motion of several GPCR family members such as the M2 and M3 muscarinic acetylcholine receptors, the A2A adenosine receptor, the ß2 -adrenergic receptor, and the CXCR4 chemokine receptor using elastic network normal modes. The normal modes reveal a dilation and a contraction of the GPCR vestibule associated with ligand passage, and activation, respectively. Contraction of the vestibule on the extracellular side is correlated with cavity formation of the G-protein binding pocket on the intracellular side, which initiates intracellular signaling. Interestingly, the normal modes of rhodopsin do not correlate well with the motion of other GPCR family members. Electrostatic potential calculation of the GPCRs reveal a negatively charged field around the ligand binding site acting as a siphon to draw-in positively charged ligands on the membrane surface. Altogether, these results expose the GPCR activation mechanism and show how conformational changes on the cell surface side of the receptor are allosterically translated into structural changes on the inside.

Tropomyosin 1 deficiency facilitates cell state transitions to enhance hemogenic endothelial cell specification during hematopoiesis.

Tropomyosins coat actin filaments and impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. Prior work suggested that TPM1 regulated blood cell formation in vitro, but it was unclear how or when TPM1 affected hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, TPM1 knockout was found to augment developmental cell state transitions, as well as TNFα and GTPase signaling pathways, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses showed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Indeed, analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced the formation of HE during embryogenesis. These findings illuminate novel effects of TPM1 on developmental hematopoiesis.

Spontaneous pauses in firing of external pallidum neurons are associated with exploratory behavior.

Spontaneous pauses in firing are the hallmark of external pallidum (GPe) neurons. However, the role of GPe pauses in the basal ganglia network remains unknown. Pupil size and saccadic eye movements have been linked to attention and exploration. Here, we recorded GPe spiking activity and the corresponding pupil sizes and eye positions in non-human primates. We show that pauses, rather than the GPe discharge rate per se, were associated with dilated pupils. In addition, following pause initiation there was a considerable increase in the rate of spontaneous saccades. These results suggest that pauses are a powerful mechanism by which the GPe may influence basal ganglia downstream structures and play a role in exploratory behavior.

Modified Snake α-Neurotoxin Averts ß-Amyloid Binding to α7 Nicotinic Acetylcholine Receptor and Reverses Cognitive Deficits in Alzheimer's Disease Mice.

Alzheimer's disease (AD) is the most common cause of senile dementia and one of the greatest medical, social, and economic challenges. According to a dominant theory, amyloid-ß (Aß) peptide is a key AD pathogenic factor. Aß-soluble species interfere with synaptic functions, aggregate gradually, form plaques, and trigger neurodegeneration. The AD-associated pathology affects numerous systems, though the substantial loss of cholinergic neurons and α7 nicotinic receptors (α7AChR) is critical for the gradual cognitive decline. Aß binds to α7AChR under various experimental settings; nevertheless, the functional significance of this interaction is ambiguous. Whereas the capability of low Aß concentrations to activate α7AChR is functionally beneficial, extensive brain exposure to high Aß concentrations diminishes α7AChR activity, contributes to the cholinergic deficits that characterize AD. Aß and snake α-neurotoxins competitively bind to α7AChR. Accordingly, we designed a chemically modified α-cobratoxin (mToxin) to inhibit the interaction between Aß and α7AChR. Subsequently, we examined mToxin in a set of original in silico, in vitro, ex vivo experiments, and in a murine AD model. We report that mToxin reversibly inhibits α7AChR, though it attenuates Aß-induced synaptic transmission abnormalities, and upregulates pathways supporting long-term potentiation and reducing apoptosis. Remarkably, mToxin demonstrates no toxicity in brain slices and mice. Moreover, its chronic intracerebroventricular administration improves memory in AD-model animals. Our results point to unique mToxin neuroprotective properties, which might be tailored for the treatment of AD. Our methodology bridges the gaps in understanding Aß-α7AChR interaction and represents a promising direction for further investigations and clinical development.

Intracerebroventricular Administration of L-arginine Improves Spatial Memory Acquisition in Triple Transgenic Mice Via Reduction of Oxidative Stress and Apoptosis.

Arginine is one of the most versatile semi-essential amino acids. Further to the primary role in protein biosynthesis, arginine is involved in the urea cycle, and it is a precursor of nitric oxide. Arginine deficiency is associated with neurodegenerative diseases such as Parkinson's, Huntington's and Alzheimer's diseases (AD). In this study, we administer arginine intracerebroventricularly in a murine model of AD and evaluate cognitive functions in a set of behavioral tests. In addition, the effect of arginine on synaptic plasticity was tested electrophysiologically by assessment of the hippocampal long-term potentiation (LTP). The effect of arginine on ß amyloidosis was tested immunohistochemically. A role of arginine in the prevention of cytotoxicity and apoptosis was evaluated in vitro on PC-12 cells. The results indicate that intracerebroventricular administration of arginine improves spatial memory acquisition in 3xTg-AD mice, however, without significantly reducing intraneuronal ß amyloidosis. Arginine shows little or no impact on LTP and does not rescue LTP deterioration induced by Aß. Nevertheless, arginine possesses neuroprotective and antiapoptotic properties.