Sequencing-free Analysis of Multiple Methylations on Gene-Specific mRNAs.

RNA epigenetics is a new layer of mechanism to regulate gene expression, but limited techniques are available to profile the status of mRNA modifications. Here, we describe a molecule proximity-based technique for simultaneous analysis of multiple types of mRNA methylation with specific gene information in living cells. N6-methyladenosine (m6A) or N1-methyladenosine (m1A) modifications on multiple mRNAs can be individually or simultaneously analyzed. A chip fabricated with vertically aligned, high-aspect-ratio diamond nanoneedles was used to access the intracellular domain in a minimum-invasive format and to isolate the mRNAs out of the cell cytoplasm while keeping cells alive. In the subsequent on-chip analytical procedures, the isolated RNAs were encoded, amplified, and visualized to derive a quantitative measurement of the associated gene-specific m6A or m1A modifications. Notably, a proximity ligation approach was developed to resolve dual methylation on an individual mRNA segment. Using this method, we investigated the dynamics of mRNA methylation in mammalian cells under physical or chemical stimuli and showed that m6A and m1A in mRNAs are heavily involved in the cellular stress response. Our results also suggested the common existence of single m6A modification in the basigin (BSG) mRNA but a rare occurrence of m6A and m1A dual methylation in the same BSG transcript.

Biohybrid Triboelectric Nanogenerator for Label-Free Pharmacological Fingerprinting in Cardiomyocytes.

The development of new drugs requires high-throughput and cost-effective pharmacological assessment in relevant biological models. Here, we introduce a novel pharmacological screening platform that combines a biohybrid triboelectric nanogenerator (TENG) and informatic analysis for self-powered, noninvasive, and label-free biosensing in cardiac cells. The cyclic mechanical activity of functional cardiomyocytes is dynamically captured by a specially designed biohybrid TENG device and is analyzed by a custom-made machine learning algorithm to reveal distinctive fingerprints in response to different pharmacological treatment. The core of the TENG device is a multilayer mesh substrate with microscale-gapped triboelectric layers, which are induced to generate electrical outputs by the characteristic motion of cardiomyocytes upon pharmaceutical treatment. Later bioinformatic extraction from the recorded TENG signal is sufficient to predict a drug's identity and efficacy, demonstrating the great potential of this platform as a biocompatible, low-cost, and highly sensitive drug screening system.

Core-Shell-Shell Upconversion Nanoparticles with Enhanced Emission for Wireless Optogenetic Inhibition.

Recent advances in upconversion technology have enabled optogenetic neural stimulation using remotely applied optical signals, but limited success has been demonstrated for neural inhibition by using this method, primarily due to the much higher optical power and more red-shifted excitation spectrum that are required to work with the appropriate inhibitory opsin proteins. To overcome these limitations, core-shell-shell upconversion nanoparticles (UCNPs) with a hexagonal phase are synthesized to optimize the doping contents of ytterbium ions (Yb3+) and to mitigate Yb-associated concentration quenching. Such UCNPs' emission contains an almost three-fold enhanced peak around 540-570 nm, matching the excitation spectrum of a commonly used inhibitory opsin protein, halorhodopsin. The enhanced UCNPs are utilized as optical transducers to develop a fully implantable upconversion-based device for in vivo tetherless optogenetic inhibition, which is actuated by near-infrared (NIR) light irradiation without any electronics. When the device is implanted into targeted sites deep in the rat brain, the electrical activity of the neurons is reliably inhibited with NIR irradiation and restores to normal level upon switching off the NIR light. The system is further used to perform tetherless unilateral inhibition of the secondary motor cortex in behaving mice, achieving control of their motor functions. This study provides an important and useful supplement to the upconversion-based optogenetic toolset, which is beneficial for both basic and translational neuroscience investigations.

The role of terminations and coordination atoms on the pseudocapacitance of titanium carbonitride monolayers.

Nowadays, MXenes have received extensive concern as a prominent electrode material of electrochemical capacitors. As two important factors to the capacitance, the influence of the intrinsical terminations (F, O and OH) and coordination atoms (C and N) is investigated using first-principles calculations. According to the density of states aligned with the standard hydrogen electrode, it turns out that a Ti3CNO2 monolayer is proven to show an obvious pseudocapacitive behavior, while the bare, F and OH terminated Ti3CN monolayers may only present electrochemical double layer characters in an aqueous electrolyte. Moreover, the illustration of molecular orbitals over the Fermi level are mainly contributed by the d-orbitals of Ti atoms coordinated with O and N atoms, indicating that the redox pseudocapacitance of the Ti3CNO2 monolayer is promoted by the coordination N atoms. Then the superiority of N bonded Ti atoms in accepting charges can be visualized through the charge population. Further, the larger ratio of C/N in the coordination environment of Ti atoms indicates that more electrons can be stored. Our investigation can give an instructional advice in the MXenes-electrode production.

Self-Healing Electronics for Prognostic Monitoring of Methylated Circulating Tumor DNAs.

Methylated circulating DNAs (ctDNAs) have recently been reported as a promising biomarker for early cancer diagnostics, but limited tools are currently available for continuous and dynamic profiling of ctDNAs and their methylation levels, especially when such assays need to be conducted in point-of-care (POC) scenarios. Here, a self-healing bioelectronic patch (iMethy) is developed that combines transdermal interstitial fluid (ISF) extraction and field effect transistor-based (FET-based) biosensing for dynamic monitoring of methylated ctDNAs as a prognostic approach for cancer risk management. The projection micro-stereolithography-based 3D patterning of an Eutectic Gallium-Indium (EGaIn) circuit with an unprecedented 10 µm resolution enables the construction of self-healing EGaIn microfluidic circuits that remain conductive under 100% strain and self-healing under severe destruction. In combination with continuous transdermal ISF sampling of methylated ctDNAs, iMethy can detect ctDNAs as low as 10-16  m in cellular models and is capable of phenotypic analysis of tumor growth in rodent animals. As the first demonstration of a wearable device for real-time in vivo analysis of disease-indicative biomarkers, this proof-of-concept study well demonstrated the potential of the iMethy platform for cancer risk management based on dynamic transdermal surveillance of methylated ctDNAs via a painless and self-administrable procedure.

Discrete single-cell microRNA analysis for phenotyping the heterogeneity of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogenous cancer in hematopoiesis, and its subtype specification is greatly important in the clinical practice for AML diagnosis and prognosis. Increasing evidence has shown the association between microRNA (miRNA) phenotype and AML therapeutic outcomes, emphasizing the need for novel techniques for convenient, sensitive, and efficient miRNA profiling in clinical practices. Here, we describe a nanoneedle-based discrete single-cell microRNA profiling technique for multiplexed phenotyping of AML heterogeneity without the requirement of sequencing or polymerase chain reaction (PCR). In virtue of a biochip-based and non-destructive nature of the assay, the expression of nine miRNAs in large number of living AML cells can be simultaneously analyzed with discrete single-cell level information, thus providing a proof-of-concept demonstration of an AML subtype classifier based on the multidimensional miRNA data. We showed successful analysis of subtype-specific cellular composition with over 90% accuracy and identified drug-responsive leukemia subpopulations among a mixed suspension of cells modeling different AML subtypes. The adoption of machine learning algorithms for processing the large-scale nanoneedle-based miRNA data shows the potential for powerful prediction capability in clinical applications to assist therapeutic decisions. We believe that this platform provides an efficient and cost-effective solution to move forward the translational prognostic usage of miRNAs in AML treatment and can be readily and advantageously applied in analyzing rare patient-derived clinical samples.

Organic electrochemical transistor arrays for real-time mapping of evoked neurotransmitter release in vivo.

Though neurotransmitters are essential elements in neuronal signal transduction, techniques for in vivo analysis are still limited. Here, we describe an organic electrochemical transistor array (OECT-array) technique for monitoring catecholamine neurotransmitters (CA-NTs) in rat brains. The OECT-array is an active sensor with intrinsic amplification capability, allowing real-time and direct readout of transient CA-NT release with a sensitivity of nanomolar range and a temporal resolution of several milliseconds. The device has a working voltage lower than half of that typically used in a prevalent cyclic voltammetry measurement, and operates continuously in vivo for hours without significant signal drift, which is inaccessible for existing methods. With the OECT-array, we demonstrate simultaneous mapping of evoked dopamine release at multiple striatal brain regions in different physiological scenarios, and reveal a complex cross-talk between the mesolimbic and the nigrostriatal pathways, which is heterogeneously affected by the reciprocal innervation between ventral tegmental area and substantia nigra pars compacta.

Cells in the nervous system pass messages using a combination of electrical and chemical signals. When an electrical impulse reaches the end of one cell, it triggers the release of chemicals called neurotransmitters, which pass the message along. Neurotransmitters can be either activating or inhibitory, determining whether the next cell fires its own electrical signal or remains silent. Currently, researchers lack effective methods for measuring neurotransmitters directly. Instead, methods mainly focus on electrical recordings, which can only tell when cells are active. One new approach is to use miniature devices called organic electrochemical transistors. Transistors are common circuit board components that can switch or amplify electrical signals. Organic electrochemical transistors combine these standard components with a semi-conductive material and a flexible membrane. When they interact with certain biological molecules, they release electrons, inducing a voltage. This allows organic electrochemical transistors to detect and measure neurotransmitter release. So far, the technology has been shown to work in tissue isolated from a brain, but no-one has used it to detect neurotransmitters inside a living brain. Xie, Wang et al. now present a new device that can detect the release of the neurotransmitter, dopamine, in real-time in living rats. The device is a miniature microarray of transistors fixed to a blade-shaped film. Xie, Wang et al. implanted this device into the brain of an anaesthetised rat and then stimulated nearby brain cells using an electrode. The device was able to detect the release of the neurotransmitter dopamine, despite there being a range of chemicals released inside the brain. It was sensitive to tiny amounts of the neurotransmitter and could distinguish bursts that were only milliseconds apart. Finally, Xie, Wang et al. also implanted the array across two connected brain areas to show that it was possible to watch different brain regions at the same time. This is the first time that transistor arrays have measured neurotransmitter release in a living brain. The new device works at low voltage, so can track brain cell activity for hours, opening the way for brand new neuroscience experiments. In the future, adaptations could extend the technology even further. More sensors could give higher resolution results, different materials could detect different neurotransmitters, and larger arrays could map larger brain areas.

High-throughput three-dimensional chemotactic assays reveal steepness-dependent complexity in neuronal sensation to molecular gradients.

Many cellular programs of neural development are under combinatorial regulation by different chemoattractive or chemorepulsive factors. Here, we describe a microfluidic platform that utilizes well-controlled three-dimensional (3D) diffusion to generate molecular gradients of varied steepness in a large array of hydrogel cylinders, allowing high-throughput 3D chemotactic assays for mechanistic dissection of steepness-dependent neuronal chemotaxis. Using this platform, we examine neuronal sensitivity to the steepness of gradient composed of netrin-1, nerve growth factor, or semaphorin3A (Sema3A) proteins, and reveal dramatic diversity and complexity in the associated chemotactic regulation of neuronal development. Particularly for Sema3A, we find that serine/threonine kinase-11 and glycogen synthase kinase-3 signaling pathways are differentially involved in steepness-dependent chemotactic regulation of coordinated neurite repellence and neuronal migration. These results provide insights to the critical role of gradient steepness in neuronal chemotaxis, and also prove the technique as an expandable platform for studying other chemoresponsive cellular systems.

Regeneration of cortical tissue from brain injury by implantation of defined molecular gradient of semaphorin 3A.

Despite great efforts in the exploration of therapeutic strategies for treating brain injuries, it is still challenging to regenerate neural tissues and to restore the lost function within an injured brain. In this report, we employed a tissue engineering approach to regenerate cortical tissue from brain injury by implantation of defined semaphorin 3A (Sema3A) gradient packaged in a hydrogel based device. Over a thirty-day recovery period, the implanted Sema3A gradient was sufficient to induce substantial migration of neural progenitor cells to the hydrogel and to promote differentiation of these cells for neuroregeneration at the injury site. As revealed by molecular characterization and RNA transcriptome analysis, the regenerated tissues induced by Sema3A gradient exhibited significant similarity to normal cortical tissues. Many genes associated with neuronal migration and stem cell differentiation were significantly up-regulated. In addition, our result suggested a crosstalk between Sema3A and Wnt/ß-catenin pathways in course of induced brain regeneration. This study demonstrated an innovative strategy to regenerate brain tissue after traumatic injury by controlling the in vivo chemotactic environment with unprecedented sophistication, and also resolved new insights about Sema3A's role in adult neurogenesis.