TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41.

Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms.

Transcription factor TFEB cell-autonomously modulates susceptibility to intestinal epithelial cell injury in vivo.

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn's disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1.

RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy.

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process.

Autophagy and checkpoints for intracellular pathogen defense.

PURPOSE OF REVIEW: Autophagy plays a crucial role in intracellular defense against various pathogens. Xenophagy is a form of selective autophagy that targets intracellular pathogens for degradation. In addition, several related, yet distinct, intracellular defense responses depend on autophagy-related genes. This review gives an overview of these processes, pathogen strategies to subvert them, and their crosstalk with various cell death programs. RECENT FINDINGS: The recruitment of autophagy-related proteins plays a key role in multiple intracellular defense programs, specifically xenophagy, microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis, and the interferon gamma-mediated elimination of pathogens, such as Toxoplasma gondii and murine norovirus. Recent progress has revealed methods employed by pathogens to resist these intracellular defense mechanisms and/or persist in spite of them. The intracellular pathogen load can tip the balance between cell survival and cell death. Further, it was recently observed that LC3-associated phagocytosis is indispensable for the efficient clearance of dying cells. SUMMARY: Autophagy-dependent and autophagy-related gene-dependent pathways are essential in intracellular defense against a broad range of pathogens.

A graphene-based physiometer array for the analysis of single biological cells.

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer - a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene - in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.

Evolution of physical and electronic structures of bilayer graphene upon chemical functionalization.

The chemical behavior of bilayer graphene under strong covalent and noncovalent functionalization is relatively unknown compared to monolayer graphene, which has been far more widely studied. Bilayer graphene is significantly less chemically reactive than monolayer graphene, making it more challenging to study its chemistry in detail. However, bilayer graphene is increasingly attractive for electronic applications rather than monolayer graphene because of its electric-field-controllable band gap, and there is a need for a greater understanding of its chemical functionalization. In this paper, we study the covalent and noncovalent functionalization of bilayer graphene using an electrochemical process with aryl diazonium salts in the high conversion regime (D/G ratio >1), and we use Raman spectroscopic mapping and conductive atomic force microscopy (cAFM) to study the resulting changes in the physical and electronic structures. Covalent functionalization at high chemical conversion induces distinct changes in the Raman spectrum of bilayer graphene including the broadening and shift in position of the split G peak. Also, the D peak becomes active with four components. We report for the first time that the broadening of the 2D22 and 2D21 components is a distinct indicator of covalent functionalization, whereas the decrease in intensity of the 2D11 and 2D12 peaks corresponds to doping. Conductive AFM imaging shows physisorbed species from noncovalent functionalization can be removed by mechanical and electrical influence from the AFM tip, and that changes in conductivity due to functionalization are inhomogeneous. These results allow one to distinguish covalent from noncovalent chemistry as a guide for further studies of the chemistry of bilayer graphene.

Excess thermopower and the theory of thermopower waves.

Self-propagating exothermic chemical reactions can generate electrical pulses when guided along a conductive conduit such as a carbon nanotube. However, these thermopower waves are not described by an existing theory to explain the origin of power generation or why its magnitude exceeds the predictions of the Seebeck effect. In this work, we present a quantitative theory that describes the electrical dynamics of thermopower waves, showing that they produce an excess thermopower additive to the Seebeck prediction. Using synchronized, high-speed thermal, voltage, and wave velocity measurements, we link the additional power to the chemical potential gradient created by chemical reaction (up to 100 mV for picramide and sodium azide on carbon nanotubes). This theory accounts for the waves' unipolar voltage, their ability to propagate on good thermal conductors, and their high power, which is up to 120% larger than conventional thermopower from a fiber of all-semiconducting SWNTs. These results underscore the potential to exceed conventional figures of merit for thermoelectricity and allow us to bound the maximum power and efficiency attainable for such systems.

Metallized DNA nanolithography for encoding and transferring spatial information for graphene patterning.

The vision for graphene and other two-dimensional electronics is the direct production of nanoelectronic circuits and barrier materials from a single precursor sheet. DNA origami and single-stranded tiles are powerful methods to encode complex shapes within a DNA sequence, but their translation to patterning other nanomaterials has been limited. Here we develop a metallized DNA nanolithography that allows transfer of spatial information to pattern two-dimensional nanomaterials capable of plasma etching. Width, orientation and curvature can be programmed by specific sequence design and transferred, as we demonstrate for graphene. Spatial resolution is limited by distortion of the DNA template upon Au metallization and subsequent etching. The metallized DNA mask allows for plasmonic enhanced Raman spectroscopy of the underlying graphene, providing information on defects, doping and lattice symmetry. This DNA nanolithography enables wafer-scale patterning of two-dimensional electronic materials to create diverse circuit elements, including nanorings, three- and four-membered nanojunctions, and extended nanoribbons.

Disorder imposed limits of mono- and bilayer graphene electronic modification using covalent chemistry.

A central question in graphene chemistry is to what extent chemical modification can control an electronically accessible band gap in monolayer and bilayer graphene (MLG and BLG). Density functional theory predicts gaps in covalently functionalized graphene as high as 2 eV, while this approach neglects the fact that lattice symmetry breaking occurs over only a prescribed radius of nanometer dimension, which we label the S-region. Therefore, high chemical conversion is central to observing this band gap in transport. We use an electrochemical approach involving phenyl-diazonium salts to systematically probe electronic modification in MLG and BLG with increasing functionalization for the first time, obtaining the highest conversion values to date. We find that both MLG and BLG retain their relatively high conductivity after functionalization even at high conversion, as mobility losses are offset by increases in carrier concentration. For MLG, we find that band gap opening as measured during transport is linearly increased with respect to the I(D)/I(G) ratio but remains below 0.1 meV in magnitude for SiO(2) supported graphene. The largest transport band gap obtained in a suspended, highly functionalized (I(D)/I(G) = 4.5) graphene is about 1 meV, lower than our theoretical predictions considering the quantum interference effect between two neighboring S-regions and attributed to its population with midgap states. On the other hand, heavily functionalized BLG (I(D)/I(G) = 1.8) still retains its signature dual-gated band gap opening due to electric-field symmetry breaking. We find a notable asymmetric deflection of the charge neutrality point (CNP) under positive bias which increases the apparent on/off current ratio by 50%, suggesting that synergy between symmetry breaking, disorder, and quantum interference may allow the observation of new transistor phenomena. These important observations set definitive limits on the extent to which chemical modification can control graphene electronically.

Charge transfer at junctions of a single layer of graphene and a metallic single walled carbon nanotube.

Junctions between a single walled carbon nanotube (SWNT) and a monolayer of graphene are fabricated and studied for the first time. A single layer graphene (SLG) sheet grown by chemical vapor deposition (CVD) is transferred onto a SiO2/Si wafer with aligned CVD-grown SWNTs. Raman spectroscopy is used to identify metallic-SWNT/SLG junctions, and a method for spectroscopic deconvolution of the overlapping G peaks of the SWNT and the SLG is reported, making use of the polarization dependence of the SWNT. A comparison of the Raman peak positions and intensities of the individual SWNT and graphene to those of the SWNT-graphene junction indicates an electron transfer of 1.12 × 10¹³ cm⁻² from the SWNT to the graphene. This direction of charge transfer is in agreement with the work functions of the SWNT and graphene. The compression of the SWNT by the graphene increases the broadening of the radial breathing mode (RBM) peak from 3.6 ± 0.3 to 4.6 ± 0.5 cm⁻¹ and of the G peak from 13 ± 1 to 18 ± 1 cm⁻¹, in reasonable agreement with molecular dynamics simulations. However, the RBM and G peak position shifts are primarily due to charge transfer with minimal contributions from strain. With this method, the ability to dope graphene with nanometer resolution is demonstrated.

Covalent electron transfer chemistry of graphene with diazonium salts.

Graphene is an atomically thin, two-dimensional allotrope of carbon with exceptionally high carrier mobilities, thermal conductivity, and mechanical strength. From a chemist's perspective, graphene can be regarded as a large polycyclic aromatic molecule and as a surface without a bulk contribution. Consequently, chemistries typically performed on organic molecules and surfaces have been used as starting points for the chemical functionalization of graphene. The motivations for chemical modification of graphene include changing its doping level, opening an electronic band gap, charge storage, chemical and biological sensing, making new composite materials, and the scale-up of solution-processable graphene. In this Account, we focus on graphene functionalization via electron transfer chemistries, in particular via reactions with aryl diazonium salts. Because electron transfer chemistries depend on the Fermi energy of graphene and the density of states of the reagents, the resulting reaction rate depends on the number of graphene layers, edge states, defects, atomic structure, and the electrostatic environment. We limit our Account to focus on pristine graphene over graphene oxide, because free electrons in the latter are already bound to oxygen-containing functionalities and the resulting chemistries are dominated by localized reactivity and defects. We describe the reaction mechanism of diazonium functionalization of graphene and show that the reaction conditions determine the relative degrees of chemisorption and physisorption, which allows for controlled modulation of the electronic properties of graphene. Finally we discuss different applications for graphene modified by this chemistry, including as an additive in polymer matrices, as biosensors when coupled with cells and biomolecules, and as catalysts when combined with nanoparticles.

Understanding and controlling the substrate effect on graphene electron-transfer chemistry via reactivity imprint lithography.

Graphene has exceptional electronic, optical, mechanical and thermal properties, which provide it with great potential for use in electronic, optoelectronic and sensing applications. The chemical functionalization of graphene has been investigated with a view to controlling its electronic properties and interactions with other materials. Covalent modification of graphene by organic diazonium salts has been used to achieve these goals, but because graphene comprises only a single atomic layer, it is strongly influenced by the underlying substrate. Here, we show a stark difference in the rate of electron-transfer reactions with organic diazonium salts for monolayer graphene supported on a variety of substrates. Reactions proceed rapidly for graphene supported on SiO(2) and Al(2)O(3) (sapphire), but negligibly on alkyl-terminated and hexagonal boron nitride (hBN) surfaces, as shown by Raman spectroscopy. We also develop a model of reactivity based on substrate-induced electron-hole puddles in graphene, and achieve spatial patterning of chemical reactions in graphene by patterning the substrate.

Understanding surfactant/graphene interactions using a graphene field effect transistor: relating molecular structure to hysteresis and carrier mobility.

Manipulation of transport hysteresis on graphene transistors and understanding electron transfer between graphene and polar/ionic adsorbates are important for the development of graphene-based sensor devices and nonvolatile memory electronics. We have investigated the effects of commonly used surfactants for graphene dispersion in aqueous solution on transport characteristics of graphene transistors. The adsorbates are found to transfer electrons to graphene, scatter carrier transport, and induce additional electron-hole puddles when the graphene is on an SiO(2) substrate. We relate the change in transport characteristics to specific chemical properties of a series of anionic, cationic, and neutral surfactants using a modification of a self-consistent transport theory developed for graphene. To understand the effects of surfactant adsorbates trapped on either side of the graphene, suspended devices were fabricated. Strong hysteresis is observed only when both sides of the graphene were exposed to the surfactants, attributable to their function as charge traps. This work is the first to demonstrate the control of hysteresis, allowing us to eliminate it for sensor and device applications or to enhance it to potentially enable nonvolatile memory applications.

Anomalous thickness-dependence of photocurrent explained for state-of-the-art planar nano-heterojunction organic solar cells.

Due to their simple geometry and design, planar heterojunction (PHJ) solar cells have advantages both as potential photovoltaics with more efficient charge extraction than their bulk heterojunction (BHJ) counterparts, and as idealized interfaces to study basic device operation. The main reason for creating BHJs was the limited exciton diffusion length in the active materials of the PHJ: if an exciton is generated at a distance greater than its diffusion length from the hetero-interface of the PHJ, it would be very unlikely to be able to contribute to the photocurrent. Based on this argument one expects a maximum in the photocurrent of PHJs for a thickness of the active layer equal to the exciton diffusion length (~10 nm). However, in two recently developed PHJs that have appeared in the literature, a maximum photocurrent is observed for 60-65 nm of poly(3-hexylthiophene) (P3HT). In this work, we explore this anomaly by combining both an optical T-matrix and a kinetic Monte Carlo simulation that tracks the exciton behavior in the PHJs. The two systems considered are a P3HT/single walled carbon nanotube (SWNT) device, and a P3HT/phenyl-C61-butyric acid methyl ester (PCBM) device. The model demonstrates how a bulk exciton sink can explain the shifted maximum in the P3HT/SWNT case, whereas in the P3HT/PCBM case the maximum is mainly determined by PCBM molecules interdiffusing in the P3HT upon annealing. Based upon the results of this model it will be possible to more intelligently design nanostructured photovoltaics and optimize them toward higher efficiencies.

Bi- and trilayer graphene solutions.

Bilayer and trilayer graphene with controlled stacking is emerging as one of the most promising candidates for post-silicon nanoelectronics. However, it is not yet possible to produce large quantities of bilayer or trilayer graphene with controlled stacking, as is required for many applications. Here, we demonstrate a solution-phase technique for the production of large-area, bilayer or trilayer graphene from graphite, with controlled stacking. The ionic compounds iodine chloride (ICl) or iodine bromide (IBr) intercalate the graphite starting material at every second or third layer, creating second- or third-stage controlled graphite intercolation compounds, respectively. The resulting solution dispersions are specifically enriched with bilayer or trilayer graphene, respectively. Because the process requires only mild sonication, it produces graphene flakes with areas as large as 50 µm(2). Moreover, the electronic properties of the flakes are superior to those achieved with other solution-based methods; for example, unannealed samples have resistivities as low as ∼1 kΩ and hole mobilities as high as ∼400 cm(2) V(-1) s(-1). The solution-based process is expected to allow high-throughput production, functionalization, and the transfer of samples to arbitrary substrates.

Evidence for high-efficiency exciton dissociation at polymer/single-walled carbon nanotube interfaces in planar nano-heterojunction photovoltaics.

There is significant interest in combining carbon nanotubes with semiconducting polymers for photovoltaic applications because of potential advantages from smaller exciton transport lengths and enhanced charge separation. However, to date, bulk heterojunction (BHJ) devices have demonstrated relatively poor efficiencies, and little is understood about the polymer/nanotube junction. To investigate this interface, we fabricate a planar nano-heterojunction comprising well-isolated millimeter-long single-walled carbon nanotubes underneath a poly(3-hexylthiophene) (P3HT) layer. The resulting junctions display photovoltaic efficiencies per nanotube ranging from 3% to 3.82%, which exceed those of polymer/nanotube BHJs by a factor of 50-100. The increase is attributed to the absence of aggregate formation in this planar device geometry. It is shown that the polymer/nanotube interface itself is responsible for exciton dissociation. Typical open-circuit voltages are near 0.5 V with fill factors of 0.25-0.3, which are largely invariant with the number of nanotubes per device and P3HT thickness. A maximum efficiency is obtained for a 60 nm-thick P3HT layer, which is predicted by a Monte Carlo simulation that takes into account exciton generation, transport, recombination, and dissociation. This platform is promising for further understanding the potential role of polymer/nanotube interfaces for photovoltaic applications.

Exciton antennas and concentrators from core-shell and corrugated carbon nanotube filaments of homogeneous composition.

There has been renewed interest in solar concentrators and optical antennas for improvements in photovoltaic energy harvesting and new optoelectronic devices. In this work, we dielectrophoretically assemble single-walled carbon nanotubes (SWNTs) of homogeneous composition into aligned filaments that can exchange excitation energy, concentrating it to the centre of core-shell structures with radial gradients in the optical bandgap. We find an unusually sharp, reversible decay in photoemission that occurs as such filaments are cycled from ambient temperature to only 357 K, attributed to the strongly temperature-dependent second-order Auger process. Core-shell structures consisting of annular shells of mostly (6,5) SWNTs (E(g)=1.21 eV) and cores with bandgaps smaller than those of the shell (E(g)=1.17 eV (7,5)-0.98 eV (8,7)) demonstrate the concentration concept: broadband absorption in the ultraviolet-near-infrared wavelength regime provides quasi-singular photoemission at the (8,7) SWNTs. This approach demonstrates the potential of specifically designed collections of nanotubes to manipulate and concentrate excitons in unique ways.