High throughput isolation of RNA from single-cells within an intact tissue for spatial and temporal sequencing a reality.

Single-cell transcriptomics is essential for understanding biological variability among cells in a heterogenous population. Acquiring high-quality single-cell sequencing data from a tissue sample has multiple challenges including isolation of individual cells as well as amplification of the genetic material. Commercially available techniques require the isolation of individual cells from a tissue through extensive manual manipulation before single cell sequence data can be acquired. However, since cells within a tissue have different dissociation constants, enzymatic and mechanical manipulation do not guarantee the isolation of a homogenous population of cells. To overcome this drawback, in this research we have developed a revolutionary approach that utilizes a fully automated nanopipette technology in combination with magnetic nanoparticles to obtain high quality sequencing reads from individual cells within an intact tissue thereby eliminating the need for manual manipulation and single cell isolation. With the proposed technology, it is possible to sample an individual cell within the tissue multiple times to obtain longitudinal information. Single-cell RNAseq was achieved by aspirating only1-5% of sub-single-cell RNA content from individual cells within fresh frozen tissue samples. As a proof of concept, aspiration was carried out from 22 cells within a breast cancer tissue slice using quartz nanopipettes. The mRNA from the aspirate was then selectively captured using magnetic nanoparticles. The RNAseq data from aspiration of 22 individual cells provided high alignment rates (80%) with 2 control tissue samples. The technology is exceptionally simple, quick and efficient as the entire cell targeting and aspiration process is fully automated.

Mutant Huntingtin Affects Diabetes and Alzheimer's Markers in Human and Cell Models of Huntington's Disease.

A higher incidence of diabetes was observed among family members of individuals affected by Huntington's Disease with no follow-up studies investigating the genetic nature of the observation. Using a genome-wide association study (GWAS), RNA sequencing (RNA-Seq) analysis and western blotting of Rattus norvegicus and human, we were able to identify that the gene family of sortilin receptors was affected in Huntington's Disease patients. We observed that less than 5% of SNPs were of statistical significance and that sortilins and HLA/MHC gene expression or SNPs were associated with mutant huntingtin (mHTT). These results suggest that ST14A cells derived from R. norvegicus are a reliable model of HD, since sortilins were identified through analysis of the transcriptome in these cells. These findings help highlight the genes involved in mechanisms targeted by diabetes drugs, such as glucose transporters as well as proteins controlling insulin release related to mHTT. To the best of our knowledge, this is the first GWAS using RNA-Seq data from both ST14A rat HD cell model and human Huntington's Disease.

Return to the Sea, Get Huge, Beat Cancer: An Analysis of Cetacean Genomes Including an Assembly for the Humpback Whale (Megaptera novaeangliae).

Cetaceans are a clade of highly specialized aquatic mammals that include the largest animals that have ever lived. The largest whales can have ∼1,000× more cells than a human, with long lifespans, leaving them theoretically susceptible to cancer. However, large-bodied and long-lived animals do not suffer higher risks of cancer mortality than humans-an observation known as Peto's Paradox. To investigate the genomic bases of gigantism and other cetacean adaptations, we generated a de novo genome assembly for the humpback whale (Megaptera novaeangliae) and incorporated the genomes of ten cetacean species in a comparative analysis. We found further evidence that rorquals (family Balaenopteridae) radiated during the Miocene or earlier, and inferred that perturbations in abundance and/or the interocean connectivity of North Atlantic humpback whale populations likely occurred throughout the Pleistocene. Our comparative genomic results suggest that the evolution of cetacean gigantism was accompanied by strong selection on pathways that are directly linked to cancer. Large segmental duplications in whale genomes contained genes controlling the apoptotic pathway, and genes inferred to be under accelerated evolution and positive selection in cetaceans were enriched for biological processes such as cell cycle checkpoint, cell signaling, and proliferation. We also inferred positive selection on genes controlling the mammalian appendicular and cranial skeletal elements in the cetacean lineage, which are relevant to extensive anatomical changes during cetacean evolution. Genomic analyses shed light on the molecular mechanisms underlying cetacean traits, including gigantism, and will contribute to the development of future targets for human cancer therapies.

Employment of Iron-Binding Protein from Haemophilus influenzae in Functional Nanopipettes for Iron Monitoring.

Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus influenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 µM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

Identification of novel transcripts and peptides in developing murine lens.

We previously investigated the transcriptome and proteome profiles of the murine ocular lens at six developmental time points including two embryonic (E15 and E18) and four postnatal time points (P0, P3, P6, and P9). Here, we extend our analyses to identify novel transcripts and peptides in developing  mouse lens. We identified a total of 9,707 novel transcripts and 325 novel fusion genes in developing mouse lens. Additionally, we identified 13,281 novel alternative splicing (AS) events in mouse lens including 6,990 exon skipping (ES), 2,447 alternative 3' splice site (A3SS), 1,900 alternative 5' splice site (A5SS), 1,771 mutually exclusive exons (MXE), and 173 intron retention (IR). Finally, we integrated our OMIC (Transcriptome and Proteome) datasets identifying 20 novel peptides in mouse lens. All 20 peptides were validated through matching MS/MS spectra of synthetic peptides. To the best of our knowledge, this is the first report integrating OMIC datasets to identify novel peptides in developing murine lens.

Functionalized Quartz Nanopipette for Intracellular Superoxide Sensing: A Tool for Monitoring Reactive Oxygen Species Levels in Single Living Cell.

Reactive oxygen species (ROS), including superoxide radical anions, are vital components in numerous biological functions, including cell signaling and immune responses. Since ROS react with other biomolecules and oxidize them quickly, it is essential for cells to have superoxide-scavenging enzymes and other regulating enzymes that can catalyze the dismutation of superoxide radical anions into less damaging molecules. Otherwise, ROS overproduction can cause oxidative damage to DNA, proteins, cells, and tissues, damage that is associated with the pathogenesis of a range of neurodegenerative disorders, age-related diseases, and cancer. Understanding the relationship between superoxide and these disorders can help the development of innovative therapies for combating oxidative stress and degeneration of nerve cells. Although methods to quantify ROS already exist, they are indirect, destructive, ambiguous, and/or cannot provide real-time measurements in single cells. In this paper, we report a technique for sensing superoxide radical anions in single living cells using functionalized nanopipettes. These nanopipettes allow us to enter the cell as we measure intracellular ROS concentrations over time. We observed that these devices provide precise real-time measurements that are accurate and not possible to obtain with other conventional techniques.

Single-cell nanobiopsy reveals compartmentalization of mRNAs within neuronal cells.

In highly polarized cells such as neurons, compartmentalization of mRNA and of local protein synthesis enables remarkably fast, precise, and local responses to external stimuli. These responses are highly important for neuron growth cone guidance, synapse formation, and regeneration following injury. Because an altered spatial distribution of mRNA can result in mental retardation or neurodegenerative diseases, subcellular transcriptome analysis of neurons could be a useful tool for studying these conditions, but current techniques, such as in situ hybridization, bulk microarray, and RNA-Seq, impose tradeoffs between spatial resolution and multiplexing. To obtain a comprehensive analysis of the cell body versus neurite transcriptome from the same neuron, we have recently developed a label-free, single-cell nanobiopsy platform based on scanning ion conductance microscopy that uses electrowetting within a quartz nanopipette to extract cellular material from living cells with minimal disruption of the cellular membrane and milieu. In this study, we used this platform to collect samples from the cell bodies and neurites of human neurons and analyzed the mRNA pool with multiplex RNA sequencing. The minute volume of a nanobiopsy sample allowed us to extract samples from several locations in the same cell and to map the various mRNA species to specific subcellular locations. In addition to previously identified transcripts, we discovered new sets of mRNAs localizing to neurites, including nuclear genes such as Eomes and Hmgb3 In summary, our single-neuron nanobiopsy analysis provides opportunities to improve our understanding of intracellular mRNA transport and local protein composition in neuronal growth, connectivity, and function.

FOXE3 contributes to Peters anomaly through transcriptional regulation of an autophagy-associated protein termed DNAJB1.

FOXE3 is a lens-specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole-exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in a mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, here we report DNAJB1 is a transcriptional target of FOXE3 in a novel pathway that is crucial for the development of the anterior segment of the eye.

A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells.

UNLABELLED: The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc-GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence. IMPORTANCE: Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.

Single Cell "Glucose Nanosensor" Verifies Elevated Glucose Levels in Individual Cancer Cells.

Because the transition from oxidative phosphorylation to anaerobic glycolytic metabolism is a hallmark of cancer progression, approaches to identify single living cancer cells by their unique glucose metabolic signature would be useful. Here, we present nanopipettes specifically developed to measure glucose levels in single cells with temporal and spatial resolution, and we use this technology to verify the hypothesis that individual cancer cells can indeed display higher intracellular glucose levels. The nanopipettes were functionalized as glucose nanosensors by immobilizing glucose oxidase (GOx) covalently to the tip so that the interaction of glucose with GOx resulted in a catalytic oxidation of ß-d-glucose to d-gluconic acid, which was measured as a change in impedance due to drop in pH of the medium at the nanopipette tip. Calibration studies showed a direct relationship between impedance changes at the tip and glucose concentration in solution. The glucose nanosensor quantified single cell intracellular glucose levels in human fibroblasts and the metastatic breast cancer lines MDA-MB-231 and MCF7 and revealed that the cancer cells expressed reproducible and reliable increases in glucose levels compared to the nonmalignant cells. Nanopipettes allow repeated sampling of the same cell, as cells remain viable during and after measurements. Therefore, nanopipette-based glucose sensors provide an approach to compare changes in glucose levels with changes in proliferative or metastatic state. The platform has great promise for mechanistic investigations, as a diagnostic tool to distinguish cancer cells from nonmalignant cells in heterogeneous tissue biopsies, as well as a tool for monitoring cancer progression in situ.

FOXM1 cistrome predicts breast cancer metastatic outcome better than FOXM1 expression levels or tumor proliferation index.

FOXM1 is a key transcription factor regulating cell cycle progression, DNA damage response, and a host of other hallmark cancer features, but the role of the FOXM1 cistrome in driving estrogen receptor-positive (ER+) versus estrogen receptor-negative (ER-) breast cancer clinical outcomes remains undefined. Chromatin immunoprecipitation sequencing (ChIP-Seq) coupled with RNA sequencing (RNA-Seq) analyses was used to identify FOXM1 target genes in breast cancer cells (MCF-7) where FOXM1 expression was either induced by cell proliferation or repressed by p53 upregulation. The prognostic performance of these FOXM1 target genes was assessed relative to FOXM transcript levels and a 61-gene proliferation score (PS) for their ability to dichotomize a pooled cohort of 683 adjuvant chemotherapy-naïve, node-negative breast cancer cases (447 ER+, 236 ER-). Differences in distant metastasis-free survival (DMFS) between the dichotomized expression groups were determined by Cox proportional hazard modeling. Proliferation-associated FOXM1 upregulation induced a set of 145 differentially bound and expressed genes (direct targets), and these demonstrated minimal overlap with differentially bound and expressed genes following FOXM1 repression by p53 upregulation. This proliferation-associated FOXM1 cistrome was not only better at significantly predicting metastatic outcome of ER+ breast cancers (HR: 2.8 (2.0-3.8), p = 8.13E-10), but was the only parameter trending toward significance in predicting ER- metastatic outcome (HR: 1.6 (0.9-2.9), p = 0.087). Our findings demonstrate that FOXM1 target genes are highly dependent on the cellular context in which FOXM1 expression is modulated, and a newly identified proliferation-associated FOXM1 cistromic signature best predicts breast cancer metastatic outcome.

Ancient hot and cold genes and chemotherapy resistance emergence.

We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (i) emergence of genes with high de novo substitution densities (i.e., hot genes) and (ii) genes never substituted (i.e., cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never-substituted genes were biased in age toward the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.

Single-cell intracellular nano-pH probes.

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing.

The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drug-tolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

The Sequencing Bead Array (SBA), a next-generation digital suspension array.

Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.

HLA typing from RNA-seq data using hierarchical read weighting [corrected].

Correctly matching the HLA haplotypes of donor and recipient is essential to the success of allogenic hematopoietic stem cell transplantation. Current HLA typing methods rely on targeted testing of recognized antigens or sequences. Despite advances in Next Generation Sequencing, general high throughput transcriptome sequencing is currently underutilized for HLA haplotyping due to the central difficulty in aligning sequences within this highly variable region. Here we present the method, HLAforest, that can accurately predict HLA haplotype by hierarchically weighting reads and using an iterative, greedy, top down pruning technique. HLAforest correctly predicts >99% of allele group level (2 digit) haplotypes and 93% of peptide-level (4 digit) haplotypes of the most diverse HLA genes in simulations with read lengths and error rates modeling currently available sequencing technology. The method is very robust to sequencing error and can predict 99% of allele-group level haplotypes with substitution rates as high as 8.8%. When applied to data generated from a trio of cell lines, HLAforest corroborated PCR-based HLA haplotyping methods and accurately predicted 16/18 (89%) major class I genes for a daughter-father-mother trio at the peptide level. Major class II genes were predicted with 100% concordance between the daughter-father-mother trio. In fifty HapMap samples with paired end reads just 37 nucleotides long, HLAforest predicted 96.5% of allele group level HLA haplotypes correctly and 83% of peptide level haplotypes correctly. In sixteen RNAseq samples with limited coverage across HLA genes, HLAforest predicted 97.7% of allele group level haplotypes and 85% of peptide level haplotypes correctly.

Distinctive microRNA expression signatures in proton-irradiated mice.

Proton particles comprise the most abundant ionizing radiation (IR) in outer space. These high energy particles are known to cause frequent double- and single-stranded DNA lesions that can lead to cancer and tumor formation. Understanding the mechanism of cellular response to proton-derived IR is vital for determining health risks to astronauts during space missions. Our understanding of the consequences of these high energy charged particles on microRNA (miRNA) regulation is still in infancy. miRNAs are non-coding, single-stranded RNAs of ~22 nucleotides that constitute a novel class of gene regulators. They regulate diverse biological processes, and each miRNA can control hundreds of gene targets. To investigate the effect of proton radiation on these master regulators, we examined the miRNA expression in selected mice organs that had been exposed to whole-body proton irradiation (2 Gy), and compared this to control mice (0 Gy exposure). RNA was isolated from three tissues (testis, brain, and liver) from treated and control mice and subjected to high-throughput small RNA sequencing. Bioinformatics analysis of small RNA sequencing data revealed dysregulation of (p < 0.05; 20 up- and 10 down-regulated) 14 mouse testis, 8 liver, and 8 brain miRNAs. The statistically significant and unique miRNA expression pattern found among three different proton-treated mouse tissues indicates a tissue-specific response to proton radiation. In addition to known miRNAs, sequencing revealed differential expression of 11 miRNAs in proton-irradiated mice that have not been previously reported in association with radiation exposure and cancer. The dysregulation of miRNAs on exposure to proton radiation suggest a possible mechanism of proton particles involvement in the onset of cell tumorgenesis. In summary, we have established that specific miRNAs are vulnerable to proton radiation, that such differential expression profile may depend upon the tissue, and that there are more miRNAs affected by proton radiation than have been previously observed.

Identification of radiation-induced microRNA transcriptome by next-generation massively parallel sequencing.

Gene regulation in cells exposed to ionizing radiation (IR) occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) play a significant role in post-transcriptional gene regulation in irradiated cells. miRNA are RNA molecules 18-24 nucleotides in length that are involved in negatively regulating the stability or translation of target messenger RNA. Previous studies from our laboratory have shown that the expression of various miRNA is altered in IR-treated cells. In the present study we monitored genome-wide expression changes of miRNA transcriptome by massively parallel sequencing of human cells irradiated with X-rays. The baseline expression of 402 miRNA indicated a wide range of modulation without exposure to IR. Differences in the expression of many miRNA were observed in a time-dependent fashion following radiation treatment. The Short Time-series Expression Miner (STEM) clustering tool was used to characterize 190 miRNA to six statistically significant temporal expression profiles. miR-19b and miR-93 were induced and miR-222, miR-92a, and miR-941 were repressed after radiation treatment. miR-142-3p, miR-142-5p, miR-107, miR-106b, miR-191, miR-21, miR-26a, miR-182, miR-16, miR-146a, miR-22 and miR-30e exhibited two peaks of induction: one at 8 h and the other at 24 h post-irradiation. miR-378, miR-let-7a, miR-let-7g, miR-let-7f, miR-103b, miR-486-3p, miR-423-5p, miR-4448, miR-3607-5p, miR-20b, miR-130b, miR-155, miR-181, miR-30d and miR-378c were induced only at the 8-h time-point. This catalogue of the inventory of miRNA that are modulated as a response to radiation exposure will be useful for explaining the mechanisms of gene regulation under conditions of stress.

Voltage controlled nano-injection system for single-cell surgery.

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.