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DNA methylation in neurons is directly linked to neuronal genome regulation and maturation. Unlike other tissues, vertebrate neurons accumulate high levels of atypical DNA methylation in the CH sequence context (mCH) during early postnatal brain development. Here, we investigate to what extent neurons derived in vitro from both mouse and human pluripotent stem cells recapitulate in vivo DNA methylation patterns. While human ESC-derived neurons did not accumulate mCH in either 2D culture or 3D organoid models even after prolonged culture, cortical neurons derived from mouse ESCs acquired in vivo levels of mCH over a similar time period in both primary neuron cultures and in vivo development. mESC-derived neuron mCH deposition was coincident with a transient increase in Dnmt3a, preceded by the postmitotic marker Rbfox3 (NeuN), was enriched at the nuclear lamina, and negatively correlated with gene expression. We further found that methylation patterning subtly differed between in vitro mES-derived and in vivo neurons, suggesting the involvement of additional noncell autonomous processes. Our findings show that mouse ESC-derived neurons, in contrast to those of humans, can recapitulate the unique DNA methylation landscape of adult neurons in vitro over experimentally tractable timeframes, which allows their use as a model system to study epigenome maturation over development.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1–4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization. Human fibroblasts are reprogrammed to generate blastocyst-like structures called iBlastoids, which recapitulate aspects of embryo implantation.

CRISPR-dCas9 based targeted epigenome editing tools allow precise manipulation and functional investigation of various genome modifications. However, these tools often display substantial context dependency, with highly variable efficacy between target genes and cell types, potentially due to underlying variation in the chromatin modifications present. While simultaneous recruitment of multiple distinct ‘effector’ chromatin regulators has improved efficacy, these systems typically lack control over which effectors bind and their spatial organisation. To overcome this we have created a new modular combinatorial epigenome editing platform, called SSSavi. This system acts as an interchangeable and reconfigurable docking platform fused to dCas9 to enable simultaneous recruitment of up to four different effectors, allowing precise control and reconfiguration of the effector composition and spatial ordering of their binding. We demonstrate the activity and specificity of the SSSavi system and compare it to existing multi-effector targeting systems, establishing its efficacy. Furthermore, by altering the spatial ordering of effector recruitment, across multiple target genes and cell lines, we demonstrate the importance of effector recruitment order for effective transcriptional regulation. Together, this system offers the capacity to explore effector co-recruitment to specific loci to potentially enhance the manipulation of chromatin contexts previously resistant to targeted epigenomic editing.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development 1–6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas 7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

SUMMARYCurrent approaches to stage chronic liver diseases have limited utility to directly predict liver cancer risk. Here, we employed single nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and chronically injured pre-malignant livers using two distinct mouse models. Analysis of 40,748 hepatic nuclei unraveled a previously uncharacterized disease-associated hepatocyte transcriptional state (daHep). These cells were absent in healthy livers, but were increasingly prevalent as chronic liver disease progressed towards hepatocarcinogenesis. Gene expression deconvolution of 1,439 human liver transcriptomes from publicly available datasets revealed that daHep frequencies highly correlate with current histopathological liver disease staging systems. Importantly, we show that high daHep levels precede carcinogenesis in mice and humans and predict a higher risk of hepatocellular carcinoma (HCC) development. This novel transcriptional signature with diagnostic and, more importantly, prognostic significance has the potential to change the way chronic liver disease patients are staged, surveilled and risk-stratified.

Roots are fundamental organs for plant development and response to their environment: they anchor the plant to its growth substrate, uptake nutrients and water vital to plant growth, and can sense and respond to a variety of biotic and abiotic stresses. The architecture of root systems and their growth are known to be strongly affected by the environmental conditions found in the soil. However, the acquisition of cell identities at the root meristem is still mainly viewed as ontogenetically driven, where a small number of stem cells generate all the cell types through stereotyped divisions followed by differentiation, along a simple developmental trajectory. The extent to which environmental cues precisely shape and affect these developmental trajectories remains an open question. We used single-cell RNA-seq, combined with spatial mapping, to deeply explore the trajectories of cell states at the tip of Arabidopsis roots, known to contain multiple developing lineages. Surprisingly, we found that most lineage trajectories exhibit a stereotyped bifid topology with two developmental trajectories rather than one. The formation of one of the trajectories is driven by a strong and specific activation of genes involved in the responses to various environmental stimuli, that affects only of a subset of the cells in multiple cell types simultaneously, revealing another layer of patterning of cell identities in the root that is independent of cell ontogeny. We demonstrate the robustness of this environmentally responsive transcriptional state by showing that it is present in a mutant where cell type identities are greatly perturbed, as well as in different Arabidopsis ecotypes. We also show that the root can adapt the proportion of cells that acquire this particular state in response to environmental signals such as nutrient availability. The discovery of this cell state reveals new layers of cell identity that may underpin the adaptive potential of plant development.

Current approaches to stage chronic liver diseases have limited utility to predict liver cancer risk. Here, we employed single nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte transcriptional state (daHep). These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled and risk-stratified.

Background Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. Results Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. Conclusions These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.

Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development. This work was supported by the following grants: R.L.—National Health and Medical Research Council (NHMRC) Project Grant 1130168, NHMRC Investigator Grant 1178460, Silvia and Charles Viertel Senior Medical Research Fellowship, Howard Hughes Medical Institute International Research Scholarship, and Australian Research Council (ARC) LE170100225; S.F.—NHMRC Ideas Grant 1184421; I.V.—ARC Future Fellowship FT170100359, UNSW Scientia Fellowship, and NHMRC Project Grant RG170137; S.B.—NHMRC-ARC Dementia Research Development Fellowship 1111206; C.P.—Raine Foundation Priming Grant RPG66-21; J.M.P.—Silvia and Charles Viertel Senior Medical Research Fellowship, ARC Future Fellowship FT180100674. This work was supported by a Cancer Research Trust grant ‘‘Enabling advanced single cell cancer genomics in WA’’ and Cancer Council WA enabling grant. Genomic data were generated at the ACRF Centre for Advanced Cancer Genomics and Genomics WA. Human brain tissue was received from the UMB Brain and Tissue Bank at the University of Maryland, part of the NIH NeuroBioBank. The glioblastoma sample was procured and provided by the AGOG biobank, funded by CINSW grant SRP-08-10. L.M. was a fellow of The Lorenzo and Pamela Galli Medical Research Trust. We thank Ankur Sharma and Greg Neely for valuable feedback. The graphical abstract and elements of Figure 1A were created with BioRender. Sí

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage., This work was supported by the Australian Research Council (ARC) Centre of Excellence programme in Plant Energy Biology (grant no. CE140100008). R.L. was supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship, ARC Future Fellowship (no. FT120100862) and Howard Hughes Medical Institute International Research Scholarship. A.d.M. was funded by an EMBO long-term fellowship (no. ALTF 144-2014). J.L.G.-S. was supported by the Spanish government (grant no. BFU2016- 74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (no. MDM-2016-0687). B.V. was supported by the Biomedical Research Council of the Agency for Science, Technology and Research of Singapore. F.G. was supported by an ARC Future Fellowship (no. FT160100267). C.W.R. was supported by an NSF grant (no. IOS-1354898). J.R.E. is an investigator of the Howard Hughes Medical Institute. Genomic data was generated at the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system.

In order to combat human-caused climate change, the emission of greenhouse gases from the transportation sector must be addressed. However, to make a meaningful impact on emissions in a market with such a large number of distributed point sources poses a daunting challenge. Battery electric vehicles are a viable commercial option, but consumer demand for range and convenience have prevented their widespread adoption. Hydrogen fuel cell vehicles (FCV’s) offer the high performance of battery EV’s, but with the driving range and refueling times that most consumers demand. Presently, California leads the nation in the introduction of hydrogen filling stations and sales/leases of FCV’s by major automobile manufacturers. The California Energy Commission has been tasked with implementing 100 hydrogen fueling stations within the state with a near term goal of 50 hydrogen fueling stations open to the public by the end of 2017. Hydrogen is highly flammable, stored at high pressures, and quickly propagates if released into the air. The introduction of odorants to provide an automatic mechanism for human detection of leaks is not possible because these chemicals will damage the fuel cell stack. Yet hydrogen must not accumulate to concentrations greater than the lower flammability limit (LFL) of 4 vol%. Because the risk of a hazardous event involving hydrogen can be mitigated through use of hydrogen safety sensors, these devices are recognized as a critical component in the safety design for any hydrogen system whether production, distribution as in filling stations, or as part of the consumer product itself. The number of available sensor technologies is quite great and most mature hydrogen sensor technologies can be categorized into a relatively small number of platforms. The US DOE has published target specifications for hydrogen safety sensors for stationary and automotive systems. In order to understand the capabilities and limitations of the existing sensor platforms, the DOE created the Hydrogen Safety Sensor Testing Laboratory at the NREL. The findings from extensive sensor testing show that there are gaps in performance in all presently available sensor platforms and that none of the commercially available hydrogen sensors meet DOE requirements for hydrogen safety sensors and the needs of real-world applications. The principle issues are that (1) no one technology appears to meet all of the DOE requirements and (2) there is a clear absence of technology offering superior performance in the 1-4vol% H2concentration range which does not suffer from drift that may lead to false positives (a costly nuisance) or false negatives (potential for loss of life and property). The frequency for the occurrences of false responses can be mitigated by frequent testing and calibration, which adds operational expenses. To address sensor limitations, the US DOE began sensor research programs at Los Alamos and Lawrence Livermore National Laboratories in 2008 to develop a new safety sensor platform to precisely fill the critical performance gaps identified in the existing commercial platforms. In this work, zirconia mixed potential-based electrochemical hydrogen safety sensors were field-tested at hydrogen filling stations in the Los Angeles area over a two-year period. Two versions of the sensor – one with a tin-doped indium oxide working electrode and one with a strontium doped lanthanum chromite working electrode – were tested in four rounds of field trials experiments conducted at hydrogen stations located in Burbank and Chino CA. The sensor systems detected numerous hydrogen releases over two years of testing. These events were localized in time and always easily distinguished from the sensor baseline behavior. No evidence of sensor baseline drift was detected during extended baseline stability testing at the Chino station. No false alarms or false positives were encountered during the testing program at either station. We found excellent correlation of recorded H2 release events with customers filling their FCVs or related to normal station maintenance activities. Multiple fill events were recorded in filling station logs and sensor resolution was sufficient to identify fuel cell vehicles queued to refill. Multiple refueling events could be identified in the sensor data even when these actions took place in less than 15-minute intervals. Moreover, sensor sensitivity was sufficient to detect hydrogen permeating from dispenser components at the Burbank station during the refueling activities. There was confirmed exposure of the sensor enclosure to significant and sometimes severe weather events, with no discernable, deleterious effects to the sensor. The installation of two field trials units at the Burbank station together with the addition of a weather station permitted correlations to be made vis-à-vis hydrogen detection and prevailing wind direction at the outdoor station.

DNA methylation functions in genome regulation and is implicated in neuronal maturation. Early post-natal accumulation of atypical non-CG methylation (mCH) occurs in neurons of mice and humans, but its precise function remains unknown. Here we investigate mCH deposition in neurons derived from mouse ES-cellsin vitroand in cultured primary mouse neurons. We find that both acquire comparable levels of mCH over a similar period asin vivo. In vitromCH deposition occurs concurrently with a transient increase inDnmt3aexpression, is preceded by expression of the post-mitotic neuronal markerRbfox3(NeuN) and is enriched at the nuclear lamina. Despite these similarities, whole genome bisulfite sequencing reveals that mCH patterning in mESC-derived neurons partially differs fromin vivo. mESC-derived neurons therefore represent a valuable model system for analyzing the mechanisms and functional consequences of correct and aberrantly deposited CG and non-CG methylation in neuronal maturation.

Background Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. Results Here, we compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37 °C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries. Conclusions Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single-cell experiments to make an informed choice.

Gene expression measurements, similarly to DNA methylation and proteomic measurements, are influenced by the cellular composition of the sample analysed. Deconvolution of bulk transcriptome data aims to estimate the cellular composition of a sample from its gene expression data, which in turn can be used to correct for composition differences across samples. Although a multitude of deconvolution methods have been developed, it is unclear whether their performance is consistent across tissues with different complexities of cellular composition. For example, the human brain is unique in its transcriptomic diversity, and in the complexity of its cellularity, yet a comprehensive assessment of the accuracy of transcriptome deconvolution methods on human brain data is currently lacking. Here we carry out the first comprehensive comparative evaluation of the accuracy of deconvolution methods for human brain transcriptome data, and assess the tissue-specificity of our key observations by comparison with transcriptome data from human pancreas. We evaluate 22 transcriptome deconvolution approaches, covering all main classes: 3 partial deconvolution methods, each applied with 6 different categories of cell-type signature data, 2 enrichment methods and 2 complete deconvolution methods. We test the accuracy of cell type estimates using in silico mixtures of single-cell RNA-seq data, mixtures of neuronal and glial RNA, as well as nearly 2,000 human brain samples. Our results bring several important insights into the performance of transcriptome deconvolution: (a) We find that cell-type signature data has a stronger impact on brain deconvolution accuracy than the choice of method. In contrast, cell-type signature only mildly influences deconvolution of pancreas transcriptome data, highlighting the importance of tissue-specific benchmarking. (b) We demonstrate that biological factors influencing brain cell-type signature data (e.g. brain region, in vitro cell culturing), have stronger effects on the deconvolution outcome than technical factors (e.g. RNA sequencing platform). (c) We find that partial deconvolution methods outperform complete deconvolution methods on human brain data. (d) We demonstrate that the impact of cellular composition differences on differential expression analyses is tissue-specific, and more pronounced for brain than for pancreas. To facilitate wider implementation of correction for cellular composition, we develop a novel brain cell-type signature, MultiBrain, which integrates single-cell, immuno-panned, and single-nucleus datasets. We demonstrate that it achieves improved deconvolution accuracy over existing reference signatures. Deconvolution of transcriptome data from autism cases and controls using MultiBrain identified cell-type composition changes replicable across studies, and highlighted novel genes dysregulated in autism.

Hydrogen refueling stations (HRSs) that dispense hydrogen to fuel cell vehicles need to ensure the quality of hydrogen to avoid contamination of the vehicle’s expensive fuel cell stacks. Currently, stations verify their fuel quality only periodically to ensure that they meet the strict fuel quality standards specified by either International Organization for Standards (ISO) or Society for Automotive Engineers (SAE). The development of hydrogen contaminant detectors (HCDs) that can provide low cost continuous monitoring at the HRS can be an invaluable asset in protecting fuel cell vehicles from any fuel contamination in-between infrequent expensive analysis of hydrogen fuel quality. An HCD capable of detecting < 200 ppb of CO in hydrogen is presented in this paper. The HCD is based on an electrochemical hydrogen pumping cell whose ultra-low loaded working electrode is poisoned by the contaminant, thus reducing its hydrogen oxidation reaction rate. The hydrogen pumping cell consists of a Nafion® membrane, a sputtered Pt working electrode, a Pt/Ru counter/pseudo-reference electrode and an internal water wicking system that provides humidification to the membrane and electrodes. When this HCD is operated in a pulsed voltammetry mode, it can provide stable CO response for thousands of hours in a HRS.

Forced expression of proneural transcription factors has been shown to direct neuronal conversion of fibroblasts. Because neurons are postmitotic, conversion efficiencies are an important parameter for this process. We present a minimalist approach combining two-factor neuronal programming with small molecule-based inhibition of glycogen synthase kinase-3β and SMAD signaling, which converts postnatal human fibroblasts into functional neuron-like cells with yields up to >200% and neuronal purities up to >80%.

Alterations in the microtubule (MT)–associated protein, tau, have emerged as a pivotal phenomenon in several neurodegenerative disorders, including frontotemporal dementia and Alzheimer's disease. Although compelling lines of evidence from various experimental models suggest that hyperphosphorylation and conformational changes of tau can cause its aggregation into filaments, the actual tau species and effective mechanisms that conspire to trigger the degeneration of human neurons remain obscure. Herein, we explored whether human embryonic stem cell–derived neural stem cells can be exploited to study consequences of an overexpression of 2N4R tau (two normal N-terminal and four MT-binding domains; n-tau) versus pseudohyperphosphorylated tau (p-tau) directly in human neurons. Given the involvement of tau in MT integrity and cellular homeostasis, we focused on the effects of both tau variants on subcellular transport and neuronal survival. By using inducible lentiviral overexpression, we show that p-tau, but not n-tau, readily leads to an MC-1–positive protein conformation and impaired mitochondrial transport. Although these alterations do not induce cell death under standard culture conditions, p-tau–expressing neurons cultured under non–redox-protected conditions undergo degeneration with formation of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Our data support a causative link between the phosphorylation and conformational state of tau, microtubuli-based transport, and the vulnerability of human neurons to oxidative stress. They further depict human embryonic stem cell–derived neurons as a useful experimental model for studying tau-associated cellular alterations in an authentic human system.

Hydrogen sensors are recognized as an important to critical component in the safety design for any hydrogen system. In this role, sensors can perform several important functions including indication of unintended hydrogen releases, activation of mitigation strategies to preclude the development of dangerous situations, activation of alarm systems and communications to first responders, and may even be called upon to autonomously initiate a system shutdown. The hydrogen sensors may be used is such a manner that they operate separate from the system being monitored, thereby providing an independent safety component that is not affected by the system itself. The importance of hydrogen sensors has been recognized by the DOE Safety and Codes Standards sub-program within the Fuel Cell Technologies Office and has for the past several years supported hydrogen safety sensor research and development. Since 2008, Los Alamos National Laboratory (LANL) and Lawrence Livermore National Laboratory (LLNL) have been developing durable, high temperature electrochemical sensors based on zirconia oxygen ion-conducting solid electrolytes1 because, among the various modalities in hydrogen sensing, electrochemical solid-state sensors can be produced at low cost as evidenced by the widely used oxygen exhaust gas sensor. Also, solid-state electrochemical technology is well suited for meeting the most stringent requirements including operation over a wide temperature range, fast response times, and reliability under variations in humidity2-4. In 2013, several packaged prototype mixed-potential hydrogen safety sensors together with control electronics (designed and built by Custom Sensor Solutions, Inc. Oro Valley AZ) were tested in validation and verification experiments at the National Renewable Energy Laboratory (NREL) Hydrogen Sensor Test Laboratory5. This preliminary testing not only provided an independent evaluation and feedback for the performance of the sensor6, but also identified design requirements for the rest of sensing system that would be required to move forward with deployment in a field trials testing environment. Recently, we have started to test electrochemical, mixed-potential hydrogen sensor technology at a California commercial fuel cell vehicle hydrogen filling station. In the first field trials experiment, data were collected over a month time period during two modes of station operation: a) station hydrogen supplied by a hydrogen tube trailer and b) hydrogen generated on-site from a methane reformer. The sensor unit – comprised of a heater control board and commercial wireless transmitter inside of a NEMA-8 enclosure – was located inside the dispensing island at the City of Burbank Hydrogen Filling Station (Burbank CA). The inside of the dispensing island enclosure location was selected because it was in an area with least expectation of measuring a hydrogen exposure and because co-location with an existing commercial safety sensor was possible. The commercial sensor was one component of a larger safety system used to signal the authorities in case a customer ran into difficulties during refueling. Over the course of the testing, the mixed potential sensor was stable and showed no evidence of baseline drift and did not appear to be affected by over a month of unusually frequent and active weather systems that produced severe at times. The stable operation and lack of influence to temperature and humidity changes agreed well with earlier testing results from NREL. During the first field trials experiment, the mixed-potential sensor reported numerous hydrogen releases with some as high as 14% of LFL and these events correlated well to activities when customers resupplied their fuel cell vehicles. During periods that the station was supplied using hydrogen from the reformer, elevated levels of hydrogen (200-400 ppm) showed oscillations with regular periodicity over a 280hr experiment well within the 600hr reformer duty cycle. Releases up to 20% of LFL were reported from the mixed potential-based sensor system during this phase of station operation and these releases were attributed to the reformer and onsite compressor usage. In this presentation, we will present the first field trails results and discuss expansion of this work to include logging multiple mixed-potential sensors at distributed locations within the filling. References 1. Sekhar, PK et al., Sens Actuators B 148(2010) 469-77. 2. Korotcenkov G, Han SD, Stetter JR, Rev 109(2009), 1402-33. 3. Lange U, Hirsch T, Mirsky VM, Wolfbeis OS, Electrochem Acta 65(2011) 3707-12. 4. Hubert T, Boon-Brett L, Black G, Banach U, Sens Actuators B 157(2011) 329-52. 5. http://www.nrel.gov/hydrogen/facilities_hsl.htm 6. Sekhar, PK et al., Int. J. of Hydrogen Energy 39(2014) 4657-4663. Acknowledgements The authors would like to thank Charles (Will) James Jr. and the DOE Hydrogen Fuel Cell Technology Office and Hydrogen Safety Codes and Standards Sub-program provided funding for this work.