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Sphagnum-dominated peatlands are major carbon pools and sinks, but these functions are threatened by climate change. There is, therefore, a need to better understand how microclimatic changes (soil temperature, soil moisture and water table depth) are affecting their functioning. Experimental studies on Sphagnum peatlands conducted under precisely controlled (e.g. mesocosm) conditions are relatively rare, especially those aiming to understand the system as a whole. Furthermore, mesocosm designs are generally described only briefly in the literature. In this article we provide a comprehensive account of a mesocosm experiment designed to study the response of Sphagnum peatlands to water table manipulation. We describe our experimental setup (3 water levels × 3 amplitudes of water table fluctuation × 5 replicates); and explain how we built the mesocosms, the issues we faced and the solutions we chose to solve them. We provide a detailed description of the devices we conceived to manipulate the water level, including software codes and electronic diagrams (as supplementary material), and explain how to address data loss in such an experimental design. We show that it is possible to build a reliable and powerful experimental setup at moderate cost using standard technology. The aim of this article is to provide a useful resource for researchers wishing to design similar experiments in the future. 

KEY WORDS: bog; ecohydrology; equipment; mesocosm experiment; water manipulation; water table depth

A mesocosm approach to study the response of Sphagnum peatlands to hydrological changes: setup, optimisation and performance

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro‐inflammatory cytokines such as interleukin‐2 (IL‐2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL‐2 signalling strength also directs Myc expression but in an analogue process that fine‐tunes Myc quantity in individual cells via post‐transcriptional control of Myc protein. Fine‐tuning Myc matters and is possible as Myc protein has a very short half‐life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin‐2 in T lymphocytes

The thickness dependence of tunnel magnetoresistance and resistance area product in Co40Fe40B20/MgO wedge/Co40Fe40B20 magnetic tunnel junctions(MTJs) has been studied for multiple Ar partial pressure (PAr) values during MgO sputtering. The extension of the simple equivalent circuit model [B. Oliver et al., J. Appl. Phys. 91, 4348 (2002)] has been suggested in order to include different transport mechanism contributions to the overall conductance of the MTJ as a function of the MgO barrier thickness. Parameters of the model, used for quantitative description of the conductivity of unpatterned MTJ stacks, have been analyzed as a function of PAr.

The study of conductance in magnetic tunnel junctions with a thin MgO barrier: The effect of Ar pressure on tunnel magnetoresistance and resistance area product

Spin-transfer ferromagnetic resonance (ST-FMR) in symmetric magnetic tunnel junctions (MTJs) with a varied thickness of the MgO tunnel barrier (0.75nm<tMgO<1.05nm) is studied using the spin-torque diode effect. The application of an rf current into nanosized MTJs generates a dc mixing voltage across the device when the frequency is in resonance with the resistance oscillations arising from the spin-transfer torque. Magnetization precession in the free and reference layers of the MTJs is analyzed by comparing ST-FMR signals with macrospin and micromagnetic simulations. From ST-FMR spectra at different dc bias voltage, the in-plane and perpendicular torkances are derived. The experiments and free electron model calculations show that the absolute torque values are independent of tunnel barrier thickness. The influence of coupling between the free and reference layer of the MTJs on the ST-FMR signals and the derived torkances are discussed.

Influence of MgO tunnel barrier thickness on spin-transfer ferromagnetic resonance and torque in magnetic tunnel junctions

A semiclassical description of diffusive spin transport in spin valves, which takes into account the transverse components of spin accumulation, is used to calculate the second harmonic voltage response to a low-frequency current. The description is applied to single as well as dual spin valves, with the magnetic moment of the sensing layer slightly tilted out of the equilibrium position by an in-plane external magnetic field. In the case of double spin valves, only the antiparallel configuration is considered since the spin torque in this configuration is enhanced, while in the parallel configuration it is significantly reduced. In both cases considered, the second harmonic voltage response and the relevant magnetoresistance are shown to be significantly dependent on the transverse spin penetration length.

Transverse spin penetration length in metallic spin valves

Stability conditions for a spin valve with a composite free layer consisting of two antiferromagnetically coupled films—known as synthetic antiferromagnet or ferrimagnet—is studied theoretically by means of the linearized coupled Landau-Lifshitz-Gilbert equations. The Lyapunov and Routh-Hurwitz methods have been used to examine stability of the free layer subject to external in-plane magnetic field and electric current flowing perpendicularly to the layers’ plane. A simple formula for the critical current density, valid for a variety of composite free layer structures, also has been derived. The analytical results are compared with those obtained from numerical simulations in which we took into account the spin transfer torque in the diffusive spin-dependent transport model. An excellent agreement between the analytical and numerical results has been achieved.

Current-induced instability of a composite free layer with antiferromagnetic interlayer coupling

We consider the magnetization dynamics induced by thermally induced spin transfer torques in thin Fe|MgO|Fe tunnel junctions. The magnetization dynamics is described by the Landau-Lifshitz-Gilbert equation, including the thermal torques as computed from first principles. We show that the angular skewness of the torques importantly modifies the dynamics, e.g., leading to different instability criteria.

Thermally induced dynamics in ultrathin magnetic tunnel junctions

The irreversible thermodynamics of a continuous medium with magnetic dipoles predicts that a temperature gradient in the presence of magnetization waves induces a magnetic induction field, which is the magnetic analog of the Seebeck effect. This thermal gradient modulates the precession and relaxation. The magnetic Seebeck effect implies that magnetization waves propagating in the direction of the temperature gradient and the external magnetic induction field are less attenuated, while magnetization waves propagating in the opposite direction are more attenuated.

Evidence for a Magnetic Seebeck Effect

We present a theoretical description of spin-transfer torque in a spin valve with perpendicularly magnetized polarizer. The polarizer consisting of several ultrathin layers is considered as a single interfacial magnetic scatterer between two nonmagnetic layers, and is included in the theory based on diffusive transport via appropriate boundary conditions. The model has been used to study systematically the spin-transfer torque and current-induced switching in a spin valve with both perpendicular and in-plane polarizers and with in-plane magnetized free layer. The wave-function matching ab initio calculations have been used to determine transport parameters of the perpendicular polarizer. Additionally, the effect of disorder on the spin-transfer torque has been examined.

Spin-transfer torque and current-induced switching in metallic spin valves with perpendicular polarizers

We present a model introducing the Landau–Lifshitz–Gilbert equation with a Slonczewski's Spin-Transfer-Torque (STT) component in order to take into account spin polarized current influence on the magnetization dynamics, which was developed as an Object Oriented MicroMagnetic Framework extension. We implement the following computations: magnetoresistance of vertical channels is calculated from the local spin arrangement, local current density is used to calculate the in-plane and perpendicular STT components as well as the Oersted field, which is caused by the vertical current flow. The model allows for an analysis of all listed components separately, therefore, the contribution of each physical phenomenon in dynamic behavior of Magnetic Tunnel Junction (MTJ) magnetization is discussed. The simulated switching voltage is compared with the experimental data measured in MTJ nanopillars.

Micromagnetic model for studies on Magnetic Tunnel Junction switching dynamics, including local current density

We report broadband ferromagnetic resonance measurements based on a coplanar waveguide (CPW) of ultrathin magnetic film structures that comprise in-plane/out-of-plane decoupled layers deposited on nonmagnetic buffer layers of various thickness and diverse sheet resistance values. We show that the excitation of the fundamental mode can be enhanced up to 10 times in the structures deposited on buffer layers with a low sheet resistance compared to what it is in the structures deposited on thin or weakly conducting buffer layers. The results are analyzed in terms of shielding of the electromagnetic field of CPW by the conducting buffer layers.

Coplanar waveguide based ferromagnetic resonance in ultrathin film magnetic nanostructures: Impact of conducting layers

Micromagnetic simulations of Current Induced Magnetization Switching (CIMS) loops in CoFeB/MgO/CoFeB exchange-biased Magnetic Tunnel Junctions (MTJ) are discussed. Our model uses the Landau–Lifshitz–Gilbert equation with the Slonczewski׳s Spin-Transfer-Torque (STT) component. The current density for STT is calculated from the applied bias voltage and tunnel magnetoresistance which depends on the local magnetization vectors arrangement. We take into account the change in the anti-parallel state resistance with increasing bias voltage. Using such model we investigate influence of the interlayer exchange coupling, between free and reference layers across the barrier, on the backhopping effect in anti-parallel to parallel switching. We compare our simulated CIMS loops with the experimental data obtained from MTJs with different MgO barrier thicknesses.

Backhopping in magnetic tunnel junctions: Micromagnetic approach and experiment

We present a detailed study of Ta/Ru-based buffers and their influence on features crucial from the point of view of applications of Magnetic Tunnel Junctions (MTJs) such as critical switching current and thermal stability. We study buffer/FeCoB/MgO/Ta/Ru and buffer/MgO/FeCoB/Ta/Ru layers, investigating the crystallographic texture, the roughness of the buffers, the magnetic domain pattern, the magnetic dead layer thickness, and the perpendicular magnetic anisotropy fields for each sample. Additionally, we examine the effect of the current induced magnetization switching for complete nanopillar MTJs with lateral dimensions of 270 × 180 nm. Buffer Ta 5/Ru 10/Ta 3 (thicknesses in nm), which has the thickest dead layer, exhibits a much larger thermal stability factor (63 compared to 32.5) while featuring a slightly lower critical current density value (1.25 MA/cm2 compared to 1.5 MA/cm2) than the buffer with the thinnest dead layer Ta 5/Ru 20/Ta 5. We can account for these results by considering the difference in damping which compensates for the difference in the switching barrier heights.

Buffer influence on magnetic dead layer, critical current, and thermal stability in magnetic tunnel junctions with perpendicular magnetic anisotropy

The existence of a heat-driven spin torque is demonstrated using Co/Cu/Co spin valves embedded in metallic nanowires. Heat currents flowing in one direction or its opposite were obtained by heating optically one end or the other of the nanowires. The spin torque associated with the heat-driven spin current pushes the magnetization out of equilibrium, resulting in a change of the magnetoresistance, which is detected using a charge current small enough not to cause heating or induced fields of any significance. The giant magnetoresistance response to this torque peaks with the magnetic susceptibility, whereas the spurious signal coming from the temperature dependence of the resistance produces merely a field independent baseline.

Linear response to a heat-driven spin torqu

We present the research on magnetic tunnel junction bottom electrodes CoFeB prepared on different buffer layers: 5Ta/30CuN/5Ta, 5Ta/15CuN/3Ta/15CuN/5Ta, 5Ta/10CuN/5Ta, 5Ta, 10Ta, and 5Ta/10Ru/5Ta (all thicknesses in nanometers). The buffer crystallographic microstructures were characterized with X-ray diffraction measurements while the roughness was investigated with atomic force microscopy measurements. We show the influence of crystallographic texture and surface roughness on the coercivity and anisotropy fields. Ferromagnetic resonance measurements were performed with a scalar network analyzer to determine the spin-wave dispersion relation of the precession frequency and the damping parameter. The results were confirmed with pulse inductive microwave magnetometry measurements.

Magnetic Properties and Magnetization Dynamics of Magnetic Tunnel Junction Bottom Electrode With Different Buffer Layers

Spin waves in articially layered magnetic structures were extensively studied two decades ago [1, 2]. Of particular interest were spin waves in exchange-coupled double layers, where two metallic magnetic lms were separated by a metallic nonmagnetic spacer [35]. The two magnetic lms in such structures are coupled via indirect exchange interaction of RKKY type, which oscillates between ferromagnetic and antiferromagnetic with increasing thickness of the spacing layer [6]. The exchange interaction was shown to signicantly modify the corresponding spin wave spectra [35, 7], which in turn were used to determine the interlayer exchange coupling parameter. Recent interest in spin waves is associated, among others, with spin currents inherently accompanying spin waves, and with such phenomena like spin transfer torque and spin pumping [8, 9]. In the latter case, a spin current is pumped through a ferromagnet/nonmagnet interface by precessing magnetic moment of the ferromagnetic layer [10, 11]. In this paper we consider the inuence of spin pumping on the spin wave spectra in antiferromagnetically coupled magnetic double layers with perpendicular surface anisotropy. For simplicity, we assume that the spin pumping and surface anisotropy occur only at the top interface of the studied structure, see Fig. 1. By generalizing the macroscopic approach by Vohl et al. [3], we calculate the spin-wave eigenfrequencies and the related spin-wave life times.

Effects of Spin Pumping on Spin Waves in Antiferromagnetically Exchange-Coupled Double Layers with Surface Anisotropy

Spin diode effect in a giant magnetoresistive strip is measured in a broad frequency range, including resonance and off-resonance frequencies. The off-resonance dc signal is relatively strong and also significantly dependent on the exchange coupling between magnetic films through the spacer layer. The measured dc signal is described theoretically by taking into account magnetic dynamics induced by Oersted field created by an ac current flowing through the system. 

The influence of interlayer exchange coupling in giant-magnetoresistive devices on spin diode effect in wide frequency range

The thermodynamics of irreversible processes in continuous media predicts the existence of a magnetic Nernst effect that results from a magnetic analog to the Seebeck effect in a ferromagnet and magnetophoresis occurring in a paramagnetic electrode in contact with the ferromagnet. Thus, a voltage that has DC and AC components is expected across a Pt electrode as a response to the inhomogeneous magnetic induction field generated by magnetostatic waves of an adjacent YIG slab subject to a temperature gradient. The voltage frequency and dependence on the orientation of the applied magnetic induction field are quite distinct from that of spin pumping.

Magnetic Nernst effect

A theoretical description of spin current injection from a nonmagnetic layer into a magnetic one is presented, with the main emphasis on the description and determination of the penetration depth of spin current component transverse to the magnetization. This penetration depth also determines the depth of spin transfer torque generation. Physically, the spin current may be driven by an external electric field or by a temperature gradient. To determine the penetration depth we used ab initio calculations of channel and mixing conductances as well as of mixing transmission. The results are then used to determine the second harmonic voltage response, which in turn can be used to determine the penetration depth experimentally.

Estimation of transverse spin penetration length using second-harmonic measurement: Proposal of an experimental method

The damping of spin waves transmitted through a two-port magnonic device implemented on a yttrium iron garnet thin film is shown to be proportional to the temperature gradient imposed on the device. The sign of the damping depends on the relative orientation of the magnetic field, the wave vector, and the temperature gradient. The observations are accounted for qualitatively and quantitatively by using an extension of the variational principle that leads to the Landau-Lifshitz equation. All parameters of the model can be obtained by independent measurements.

Thermal spin torques in magnetic insulators

When a current is passed through a non-magnetic metal with strong spin-orbit coupling, an orthogonal spin current is generated. This spin current can be used to switch the magnetization of an adjacent ferromagnetic layer or drive its magnetization into continuous precession. The interface, which is not necessarily sharp, and the crystallographic structure of the nonmagnetic metal can both affect the strength of current-induced spin-orbit torques. Here, we investigate the effects of interface intermixing and film microstructure on spin-orbit torques in perpendicularly magnetized Ta/Co40Fe40B20/MgO trilayers with different Ta layer thickness (5nm, 10nm, 15nm), greater than the spin diffusion length. Effective spin-orbit torques are determined from harmonic Hall voltage measurements performed at temperatures ranging from 20K to 300K. We account for the temperature dependence of damping-like and field-like torques by including an additional contribution from the Ta/CoFeB interface in the spin diffusion model. Using this approach, the temperature variations of the spin Hall angle in the Ta underlayer and at the Ta/CoFeB interface are determined separately. Our results indicate an almost temperature-independent spin Hall angle of θ ≈ −0 2. SH N in Ta and a strongly temperature-dependent θSH N for the intermixed Ta/CoFeB interface.

Influence of intermixing at the Ta/ CoFeB interface on spin Hall angle in Ta/CoFeB/MgO heterostructures

We report on long-range spin wave (SW) propagation in nanometer-thick yttrium iron garnet (YIG) film with an ultralow Gilbert damping. The knowledge of a wavenumber value → k is essential for designing SW devices. Although determining the wavenumber → k in experiments like Brillouin light scattering spectroscopy is straightforward, quantifying the wavenumber in all-electrical experiments has not been widely commented upon so far. We analyze magnetostatic spin wave (SW) propagation in YIG films in order to determine the SW wavenumber → k excited by the coplanar waveguide. We show that it is crucial to consider the influence of magnetic anisotropy fields present in YIG thin films for precise determination of SW wavenumber. With the proposed methods we find that experimentally derived values of → k are in perfect agreement with that obtained from electromagnetic simulation only if anisotropy fields are included. Keywords: spin waves, ferromagnetic resonance, low damping materials

Characterization of spin wave propagation in (111) YIG thin films with large anisotropy

The number and variety of factors underlying control of gene expression have been frequently underestimated. Non-coding RNAs generated through pervasive transcription have recently been implicated in shaping the transcriptional landscape in different organisms from bacteria to higher eukaryotes, adding a previously unexpected layer of complexity to the process of gene regulation. In this review, we highlight non-coding transcription-dependent regulatory mechanisms linked to chromatin organization and environmental changes, and particular emphasis is given to single-cell approaches, which have been crucial in dissecting cell-to-cell variability. These studies have revealed that non-coding transcription can underlie the extensive heterogeneity in patterns of gene expression within a cell population.

Role of chromatin, environmental changes and single cell heterogeneity in non-coding transcription and gene regulation

Gene loops have been described in different organisms from yeast to human and form through interaction between components of the transcription pre-initiation complex and Ssu72, a member of the 3' end cleavage and polyadenylation complex. A recent study by Tan-Wong et al. reports a new role for gene loops in promoting ORF transcription directionality from otherwise bidirectional promoters.

Gene loops and HDACs to promote transcription directionality

DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry– based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologousrecombination– based protein tagging on a multigene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.

A new strategy for gene targeting and functional proteomics using the DT40 cell line

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in 10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.

Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target

Mature tRNA 3′ ends in the yeast Saccharomyces cerevisiae are generated by two pathways: endonucleolytic and exonucleolytic. Although two exonucleases, Rex1 and Rrp6, have been shown to be responsible for the exonucleolytic trimming, the identity of the endonuclease has been inferred from other systems but not confirmed in vivo. Here, we show that the yeast tRNA 3′ endonuclease tRNase Z, Trz1, is catalyzing endonucleolytic tRNA 3′ processing. The majority of analyzed tRNAs utilize both pathways, with a preference for the endonucleolytic one. However, 3′-end processing of precursors with long 3′ trailers depends to a greater extent on Trz1. In addition to its function in the nucleus, Trz1 processes the 3′ ends of mitochondrial tRNAs, contributing to the general RNA metabolism in this organelle.

tRNA 3′ processing in yeast involves tRNase Z, Rex1, and Rrp6

The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5’-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3’>5’direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsensemediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main modelspecies: yeast, Drosophila, plants and mammals.

Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems

Many Saccharomyces cerevisiae genes encode antisense transcripts, some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense (AS) RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single-molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation. We show that PHO84 AS RNA acts as a bimodal switch, in which continuous, low-frequency antisense transcription represses sense expression within individual cells. Surprisingly, antisense RNAs do not accumulate at the PHO84 gene but are exported to the cytoplasm. Furthermore, rather than stabilizing PHO84 AS RNA, the loss of Rrp6 favors its elongation by reducing early transcription termination by Nrd1–Nab3–Sen1. These observations suggest that PHO84 silencing results from antisense transcription through the promoter rather than the static accumulation of antisense RNAs at the repressed gene.

Bimodal expression of PHO84 is modulated by early termination of antisense transcription

Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased ∼2.5-fold, covering ∼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of ∼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome.

DIS3 shapes the RNA polymerase II transcriptome in humans by degrading a variety of unwanted transcripts

Increasing evidence indicates that besides promoters, enhancers, and epigenetic modifications, nuclear organization is another parameter contributing to optimal control of gene expression. Although differences between species exist, the influence of gene positioning on expression seems to be a conserved feature from yeast to Drosophila and mammals. The nuclear periphery is one of the nuclear compartments implicated in gene regulation. It consists of the nuclear envelope (NE) and the nuclear pore complexes (NPC), which have distinct roles in the control of gene expression. The NPC has recently been shown to tether proteins involved in the sumoylation pathway. Here, we will focus on the importance of gene positioning and NPClinked sumoylation/desumoylation in transcription regulation. We will mainly discuss observations made in the yeast Saccharomyces cerevisiae model system and highlight potential parallels in metazoan species.

Sumoylation and transcription regulation at nuclear pores

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

Endocytic Adaptor Protein Tollip Inhibits Canonical Wnt Signaling.

Signaling of plasma membrane receptors can be regulated by endocytosis at different levels, including receptor internalization, endocytic sorting towards degradation or recycling, and using endosomes as mobile signaling platforms. Increasing number of reports underscore the importance of endocytic mechanisms for signaling of cytokine receptors. In this short review we present both consistent and conflicting data regarding endocytosis and its role in signaling of receptors from the tumor necrosis factor receptor superfamily (TNFRSF) and those for interleukins (ILRs) and interferons (IFNRs). These receptors can be internalized through various endocytic routes and most of them are able to activate downstream pathways from endosomal compartments. Moreover, some of the cytokine receptors clearly require endocytosis for proper signal transduction. Still, the data describing internalization mechanisms and fate of cytokine receptors are often fragmentary and barely address the relation between their endocytosis and signaling. In the light of growing knowledge regarding different mechanisms of endocytosis, extending it to the regulation of cytokine receptor signaling may improve our understanding of the complex and pleiotropic functions of these molecules.

Endocytic regulation of cytokine receptor signaling.

The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery.

Effects of membrane trafficking on signaling by receptor tyrosine kinases.

We used in vivo and in vitro strategies to study the mechanisms of multivesicular endosome biogenesis. We found that, whereas annexinA2 and ARP2/3 mediate F-actin nucleation and branching, respectively, the ERM protein moesin supports the formation of F-actin networks on early endosomes. We also found that moesin plays no role during endocytosis and recycling to the plasma membrane but is absolutely required, much like actin, for early-to-late-endosome transport and multivesicular endosome formation. Both actin network formation in vitro and early-to-late endosome transport in vivo also depend on the F-actin-binding protein cortactin. Our data thus show that moesin and cortactin are necessary for formation of F-actin networks that mediate endosome biogenesis or maturation and transport through the degradative pathway. We propose that the primary function of endosomal F-actin is to control the membrane remodeling that accompanies endosome biogenesis. We also speculate that this mechanism helps segregate tubular and multivesicular membranes along the recycling and degradation pathways, respectively.

Moesin and cortactin control actin-dependent multivesicular endosome biogenesis.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

Wnt directs the endosomal flux of LDL-derived cholesterol and lipid droplet homeostasis.

Efficient sorting of the material internalized by endocytosis is essential for key cellular functions and represents a, if not the, major trafficking pathway in mammalian cells. Incoming material - solutes, receptors and cargos, lipids and even pathogenic agents - are routed to various destinations within mammalian cells at two major sorting stations: the early and late endosome. The early endosome receives all manner of incoming material from the plasma membrane, as well as from the Golgi, and serves as an initial sorting nexus routing molecules back to the cell surface through recycling endosomes, to the trans-Golgi network by retrograde transport, or on to the late endosome/lysosome. The early endosome also regulates cell signaling, through the downregulation of internalized receptors, which are packaged into intralumenal vesicles that arise from inward invaginations of the limiting membrane. These multivesicular regions detach or mature from early endosomes and become free endocytic carrier vesicle/multivesicular body, which transports cargoes to late endosomes. The late endosome provides a central hub for incoming traffic from the endocytic, biosynthetic and autophagic pathways and outgoing traffic to the lysosomes, the Golgi complex or the plasma membrane. They also function as a key sensing/signaling platform that inform the cell about the nutrient situation. Herein we summarize the current understanding of the organization and functions of the endocytic pathway, differences across species, and the process of endosome maturation.

Endosome maturation, transport and functions.

In yeast and mammalian cells, endosomal sorting complexes required for transport (ESCRT) assist in sorting ubiquitinated proteins into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs) for degradation in the lysosome/vacuole. In mammalian cells, ESCRTs also drive other topologically identical membrane deformation processes, including cytokinesis, exosome release, and virus budding. Although the ESCRT-associated protein ALIX regulates these mammalian cell-specific functions, it was believed to be dispensable for receptor sorting into ILVs, unlike its yeast homolog Bro1. Despite these differences, recent evidence suggests ALIX and Bro1 share common properties in cargo sorting and ILV formation. We review these commonalities and discuss the role of ALIX in operating 'behind the mirror' during ILV back-fusion with the limiting membrane. We also propose models of how ALIX and some ESCRTs regulate the back-fusion process.

ALIX and the multivesicular endosome: ALIX in Wonderland.

ALIX plays a role in nucleocapsid release during viral infection, as does lysobisphosphatidic acid (LBPA). However, the mechanism remains unclear. Here we report that LBPA is recognized within an exposed site in ALIX Bro1 domain predicted by MODA, an algorithm for discovering membrane-docking areas in proteins. LBPA interactions revealed a strict requirement for a structural calcium tightly bound near the lipid interaction site. Unlike other calcium- and phospholipid-binding proteins, the all-helical triangle-shaped fold of the Bro1 domain confers selectivity for LBPA via a pair of hydrophobic residues in a flexible loop, which undergoes a conformational change upon membrane association. Both LBPA and calcium binding are necessary for endosome association and virus infection, as are ALIX ESCRT binding and dimerization capacity. We conclude that LBPA recruits ALIX onto late endosomes via the calcium-bound Bro1 domain, triggering a conformational change in ALIX to mediate the delivery of viral nucleocapsids to the cytosol during infection.

Viral infection controlled by a calcium-dependent lipid-binding module in ALIX.

Intracellular organelles, including endosomes, show differences not only in protein but also in lipid composition. It is becoming clear from the work of many laboratories that the mechanisms necessary to achieve such lipid segregation can operate at very different levels, including the membrane biophysical properties, the interactions with other lipids and proteins, and the turnover rates or distribution of metabolic enzymes. In turn, lipids can directly influence the organelle membrane properties by changing biophysical parameters and by recruiting partner effector proteins involved in protein sorting and membrane dynamics. In this review, we will discuss how lipids are sorted in endosomal membranes and how they impact on endosome functions.

Lipid sorting and multivesicular endosome biogenesis.

Nucleic acid templated reactions are enabled by the hybridization of probe-reagent conjugates resulting in high effective reagent concentration and fast chemical transformation. We have developed a reaction that harnesses cellular microRNA (miRNA) to yield the cleavage of a linker releasing fluorogenic rhodamine in a live vertebrate. The reaction is based on the catalytic photoreduction of an azide by a ruthenium complex. We showed that this system reports specific expression of miRNA in living tissues of a vertebrate.

Nucleic Acid Templated Chemical Reaction in a Live Vertebrate.

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.

Asymmetric division of neural precursor cells contributes to the generation of a variety of neuronal types. Asymmetric division is mediated by the asymmetric inheritance of fate determinants by the two daughter cells. In vertebrates, asymmetric fate determinants, such as Par3 and Mib, are only now starting to be identified. Here we show that, during mitosis of neural precursors in zebrafish, directional trafficking of Sara endosomes to one of the daughters can function as such a determinant. In asymmetric lineages, where one daughter cell becomes a neuron (n cell) whereas the other divides again to give rise to two neurons (p cell), we found that the daughter that inherits most of the Sara endosomes acquires the p fate. Sara endosomes carry an endocytosed pool of the Notch ligand DeltaD, which is thereby itself distributed asymmetrically. Sara and Notch are both essential for cell fate assignation within asymmetric lineages. Therefore, the Sara endosome system determines the fate decision between neuronal differentiation and mitosis in asymmetric lineages and thereby contributes to controlling the number of neural precursors and differentiated neurons during neurogenesis in a vertebrate.

Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord.

How a developing organ grows and patterns to its final shape is an important question in developmental biology. Studies of growth and patterning in the Drosophila wing imaginal disc have identified a key player, the morphogen Decapentaplegic (Dpp). These studies provided insights into our understanding of growth control and scaling: expansion of the Dpp gradient correlated with the growth of the tissue. A recent report on growth of a Drosophila organ other than the wing, the eye imaginal disc, prompts a reconsideration of our models of growth control. Despite striking differences between the two, the Dpp gradient scales with the target tissues of both organs and the growth of both the wing and the eye is controlled by Dpp. The goal of this review is to discuss whether a parsimonious model of scaling and growth control can explain the relationship between the Dpp gradient and growth in these two different developmental systems.

The wing and the eye: a parsimonious theory for scaling and growth control?

During morphogenesis, organs grow to stereotyped sizes, but growth control mechanisms are poorly understood. Here, we measured the signaling dynamics of the morphogen Dpp, one of several Drosophila factors controlling morphogenetic growth, in the developing eye. In this tissue, the Dpp expression domain advances from the posterior to the anterior tissue edge. In front of this moving morphogen source, signaling inputs including Dpp activate the target gene hairy in a gradient that scales with tissue size. Proliferation, in turn, occurs in a mitotic wave in front of the source, whereas behind it, cells arrest and differentiate. We found that cells divide when their signaling levels have increased by around 60%. This simple mechanism quantitatively explains the proliferation and differentiation waves in wild type and mutants. Furthermore, this mechanism may be a common feature of different growth factors, because a Dpp-independent growth input also follows this growth rule.

Growth control by a moving morphogen gradient during Drosophila eye development.

Tissue homeostasis is maintained by adult stem cells, which self-renew and give rise to differentiating cells. The generation of daughter cells with different fates is mediated by signalling molecules coming from an external niche or being asymmetrically dispatched between the two daughters upon stem cell mitosis. In the adult Drosophila midgut, the intestinal stem cell (ISC) divides to generate a new ISC and an enteroblast (EB) differentiating daughter. Notch signalling activity restricted to the EB regulates intestinal cell fate decision. Here, we show that ISCs divide asymmetrically, and Sara endosomes in ISCs are specifically dispatched to the presumptive EB. During ISC mitosis, Notch and Delta traffic through Sara endosomes, thereby contributing to Notch signalling bias, as revealed in Sara mutants: Sara itself contributes to the control of the ISC asymmetric division. Our data uncover an intrinsic endosomal mechanism during ISC mitosis, which participates in the maintenance of the adult intestinal lineage.

Sara endosomes and the asymmetric division of intestinal stem cells.

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis [1-6]. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate [1-6]. We have previously shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter [6]. Here we show that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. Our data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis.

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division.

The Notch signaling pathway plays important roles in many organisms and developmental contexts. The activities of the Notch receptor and of its ligand Delta are known to be regulated at several steps along the endocytic pathway. However, the precise molecular mechanism of Notch activation and the role played by endosomal sorting and trafficking remain elusive. We developed an antibody uptake assay to enable live imaging of endogenous internalized Notch and Delta in Drosophila tissues. In this chapter, we describe how to perform live antibody uptake assays in the Drosophila notum. In this tissue, Notch signaling plays a crucial role in the regulation of cell fate decisions in the lineage of sensory organ precursor (SOP) cells. We describe here how to do a correlative analysis of Notch/Delta live imaging in dividing SOPs and of the lineage of these particular SOPs. Combined with the wide range of genetic and chemical tools available in Drosophila research, these two methods will provide a better understanding of the role played by endocytic proteins and endosomal trafficking in Notch regulation, in terms of botch Notch trafficking and Notch signaling output.

Monitoring notch/delta endosomal trafficking and signaling in Drosophila.

Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GlcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase.

Biochemical membrane lipidomics during Drosophila development.

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.

Anthrax toxin receptor 2a controls mitotic spindle positioning.

Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O(6)-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag.

Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins.

Morphogen gradients regulate the patterning and growth of many tissues, hence a key question is how they are established and maintained during development. Theoretical descriptions have helped to explain how gradient shape is controlled by the rates of morphogen production, spreading and degradation. These effective rates have been measured using fluorescence recovery after photobleaching (FRAP) and photoactivation. To unravel which molecular events determine the effective rates, such tissue-level assays have been combined with genetic analysis, high-resolution assays, and models that take into account interactions with receptors, extracellular components and trafficking. Nevertheless, because of the natural and experimental data variability, and the underlying assumptions of transport models, it remains challenging to conclusively distinguish between cellular mechanisms.

Investigating the principles of morphogen gradient formation: from tissues to cells.

This paper reports on the operando study of degradation mechanisms in carbon-based electrochemical capacitors with Li2SO4 and LiNO3 electrolytes. Several electrochemical techniques, coupled with mass and Raman spectrometry have been implemented to recognize and describe the processes occurring at the carbon electrode/aqueous electrolyte interface. It has been shown that for the systems operating with mild, water-based electrolytes, the performance fade might have a different origin; dependently on the salt used for electrolyte preparation, carbon surface might be either oxidized or anion might be reduced. A detrimental effect of oxygen evolution at elevated voltages (up to 1.8 V) has also been observed. An in-depth study involving the implementation of the operando techniques also allowed the most optimal voltage for both systems to be determined, taking into account their long-term performance as well.

Comparative operando study of degradation mechanisms in carbon-based electrochemical capacitors with Li2SO4 and LiNO3 electrolytes

This paper reports on a primary study of the capacitor system that can successfully overcome the main drawback of aqueous electrolytes, i.e. low max. voltage and then low energy density of the device. Our idea employs hybridization of electrolytes, i.e. use of two separated electrolytes with different pH values, each for one electrode. In such a way, the capacitor voltage is extended to 2.1 V. 6 mol L−1 KOH plays the role of electrolyte for the negative electrode whereas 1 mol L−1 H2SO4 or 5 mol L−1 LiNO3 are electrolytic solutions for a positive one. This idea allows us to take advantage of very negative hydrogen evolution potential for KOH and high overpotential of oxygen evolution, especially for LiNO3 solutions. Accordingly, it was possible to maintain almost 100 F g−1 after 25 000 cycles at 1 A g−1, gaining the energy more than 10 Wh kg−1 along the average power of 1 kW kg−1.

Around the thermodynamic limitations of supercapacitors operating in aqueous electrolytes

Increasing operating voltage is a straightforward approach to increase the specific energy of aqueous electrolyte based electrical double-layer capacitors (EDLCs). A broader operating voltage window, however, comes at the expense of accelerated cell ageing processes. Two complementary in situ gas analysis techniques, i.e., internal cell pressure measurement and Online Electrochemical Mass Spectrometry (OEMS), were applied to study the influence of electrolyte salt concentration on the electrochemical degradation of the electrode and decomposition of electrolyte during cycling of symmetrical carbon based EDLCs. Higher concentrations of Li2SO4 aqueous electrolyte salt increase the coulombic efficiency of the cell and reduce the amount of volatile side reaction products (e.g., CO, CO2, and H2) arising from carbon corrosion and water electrolysis. The lower amount of degradation products when increasing the salt to solvent ratio is believed to result from a favorable stabilization of the increasingly coordinated electrolyte solvent-salt bonds, which in turn increases the energy barrier for H2O decomposition. Higher salt concentrations not only increase the electrochemical reversibility at high cell voltages but also increase cell charging capacities as a result of increased electrolyte conductivity, which all-in-all facilitates implementation of future high energy aqueous EDLCs.

Influence of aqueous electrolyte concentration on parasitic reactions in high-voltage electrochemical capacitors

This paper reports on the performance of the supercapacitor operating in aqueous acetic acid salts. Lithium, sodium and magnesium acetate aqueous solutions at various concentrations have been selected as electrolytes. Maximum operational voltage and the overall capacitor performance have been determined by several electrochemical techniques. Floating and galvanostatic charge/discharge tests proved the promising performance at high voltages (1.5 V); the capacitance values have been retained at more than 80% of initial value for all tested electrolytes. Additionally, due to the the ability to operate at high voltages, the maximum energy obtained in the system with 0.5 mol L−1CH3COONa is more than two times higher than with 6 mol L−1 KOH, i.e., conventional aqueous capacitor. Taking into account a mild character of the electrolytes used, a novel concept of eco-friendly energy storage device has been proposed.

Carbon-based electrochemical capacitors with acetate aqueous electrolytes

High-voltage aqueous electrolyte based supercapacitors (U > 1.23 V) attract significant attention for next-generation high power, low cost and environmentally friendly energy storage applications. Cell ageing is however markedly pronounced at elevated voltages and results in accelerated overall performance fade and increased safety concerns. Online electrochemical mass spectrometry, combined with cell pressure analysis, is for the first time shown to provide a powerful means for in situ investigation of degradation mechanisms in aqueous electrolyte/carbon based supercapacitors. The activated carbon electrodes possess high specific surface area and oxygen-based surface functionalities (mainly phenol, lactone and anhydride groups), which are oxidized already at a cell voltage of 0.6 V to provoke the evolution of minor amounts of CO and CO2. Noticeable water decomposition starts at a high voltage of 1.6 V with the evolution of H2 on the negative electrode and carbon corrosion on the positive electrode with the generation of predominantly CO. In this paper we also report that short-term cycling leads to partly reversible gas evolution/consumption side-reactions giving negligible capacitance. On the other hand, long-term cycling causes irreversible side-reactions, deteriorates the electrochemical performance, and increases the internal pressure of the cell. Repeated cycling (U < 2 V) is confirmed as a more harmful technique for the electrode integrity compared to the voltage holding in a floating test. In situ gas analysis is shown to provide valuable insights into the electrochemical cell ageing aspects, such as the nature and potential onsets of side-reactions, hence paving the way for fundamental understanding and mitigating the performance and safety loss of high-energy aqueous supercapacitors.

Ageing phenomena in high-voltage aqueous supercapacitors investigated by in situ gas analysis

This paper presents supercapacitors utilizing new redox-active electrolytes with bromine species. Two sources of Br specimen were investigated, i.e. dibromodihydroxybenzene dissolved in KOH and potassium bromide dissolved in KOH with hydroxybenzene additive. KOH-activated carbon, exhibiting a well-developed porosity, was incorporated as an electrode material. The tested systems revealed a capacitance enhancement explained by Br and partial BrO3 redox activity. The optimisation of the electrolyte concentration resulted in a capacitance value of 314 F g−1 achieved at 1.1 V voltage range. Good cyclability performance (11% capacitance loss) combined with a high capacitance value (244 F g−1) were obtained for the system operating in 0.2 mol L− 1 C6H4Br2O2 in 2 mol L 1 KOH electrolytic solution.


Redox-active electrolyte
Activated carbon

Enhancement of the carbon electrode capacitance by brominated hydroquinones

This work reports on a high-voltage, hybrid capacitor involving two separate redox reactions. Aqueous solutions of Mg(NO3)2 and KI have been used for negative and positive electrode, respectively. Adjusting pH=2 for electrode (+) with KI solution and modifying Mg(NO3)2 solution to pH=9 for negative side play a crucial role for a stable long-term operation of capacitor at enhanced voltage. A benefit from such a construction is a pseudocapacitive contribution from hydrogen sorption reaction on the negative electrode and high iodine/iodide activity on the positive electrode, enhancing the energy with no remarkable impact on the power profile. Proposed solution allows a high voltage (1.8 V) to be reached and thereby high power and energy performance (~20 W h/kg at 1 kW/kg) to be obtained. High long-term stability has been confirmed by floating and galvanostatic tests.


Electrochemical capacitors
High-voltage systems
Hybrid systems
Carbon electrodes
Aqueous systems
Neutral electrolytes

Hybrid aqueous capacitors with improved energy/power performance

The paper reports on electrochemical characterization of several supercapacitors operating in aqueous electrolytes and discusses a strategy for enhancing their performance. Various electrolytes such as alkaline (6 mol L−1 KOH), acidic (1 mol L−1H2SO4) and neutral (1 mol L−1 KI and 1 mol L−1 Na2S2O3) were investigated in terms of electrochemical stability and redox activity. A new concept of capacitor with two electrodes operating in different electrolytes is demonstrated. The positive electrode worked in KI solution whereas KOH solution served as an electrolytic medium for the negative electrode. As a result, improved capacitance values and energy density for a capacitor with a combined electrolyte have been obtained because of the beneficial redox phenomena on both electrodes.

Interfacial Redox Phenomena for Enhanced Aqueous Supercapacitors

Chitin – a naturally occurring biopolymer – was employed for the first time as a binder for carbon electrodes and studied in electrochemical capacitors. Chitin-bound electrodes have shown excellent performance in neutral aqueous electrolytes up to 5 A g−1 current load with a capacitance retention of ca. 80% of the initial value for mild regimes. This study reports on the electrochemical behaviour of commercially available activated carbon (Supra 30 NORIT) bound with chitin (10% wt.) in the form of pellets, operating in two different aqueous electrolytes, i.e. 1 M Li2SO4 and 1 M KI solutions. It has been found that, for the 1 M Li2SO4 solution, the carbon electrodes demonstrate a moderate capacitance value of 65 F g−1 at 1 A g−1 current density. In 1 M KI solution merging electrical double-layer capacitance and faradaic contribution of the iodide/iodine redox couple, at the same current load, the capacitance was 175 F g−1 and it significantly increased with cycling to 260 F g−1 in the case of the chitin binder, and 300 F g−1 for the PTFE-bound electrodes taking into account the total charge supplied during capacitor discharging. Moreover, for the 1 M Li2SO4 solution, the chitin-bound electrodes display slightly better charge propagation than the PTFE-bound ones, whereas for the 1 M KI solution, the energy of the capacitor has been improved by 1 W h kg−1. Considering the rather negative impact of the commonly used binding fluoropolymers on the environment, chitin may become a great alternative for the development of cheap and environmentally benign electrochemical capacitors, while preserving their mechanical and electrochemical performance. Additionally, fluorine-based (e.g. PVDF or PTFE) electrodes are more hydrophobic and thus electrolyte penetration into the bulk of electrodes is unfavoured. It is noteworthy that the formation of a chitin complex with electrochemically generated iodine, which has a tendency to leave the system, may enhance the reversibility of the iodide/iodine redox couple and improve both the capacitance value as well as the cycle life.

Towards sustainable power sources: chitin-bound carbon electrodes for electrochemical capacitors

The electrochemical performance of various carbon materials as supercapacitor electrodes with redox active electrolytes has been presented. In situ Raman investigation was carried out to show possible iodine species, polyiodides and carbon/iodine interactions during electrode polarization. Apart from the electrolyte also the kind of current collector plays an important role. Gold current collector is not adapted because of its reactivity with iodides whereas stainless steel is convenient. The conjugated iodide and vanadium species as the electrolyte were also investigated with Nafion separation. The best performance was obtained for AAC 1 and AAC 2 carbon material especially after adding 10% of carbon nanotubes.


Vanadium species

Electrochemical capacitors as attractive power sources

Presented paper describes and critically comments major recent strategies for improving electrochemical capacitor performance. Particularly, carbon based electrodes and aqueous electrolytes have been considered. A novel concept of redox active electrolytes as a source of pseudocapacitance effect as well as profits and cons of such system have been discussed. The electrochemical performance of capacitor operating in such electrolyte solution is reported. Furthermore, some advantageous features of bio-inspired system based on bromine-cerium solution acting as oscillator are also presented.


Electrochemical capacitor
Redox active electrolytes
Carbon materials

Strategies for enhancing the performance of carbon/carbon supercapacitors in aqueous electrolytes

Capacitance of typical double layer capacitors is generally limited due to electrochemically available surface area of carbon electrode. Hence, significant increase of capacitance can be done by introducing some faradaic reactions that may occur at electrode/electrolyte interface. This kind of effect is called pseudocapacitance. Different solutions based on electroactive species, such as iodides, bromides or di-hydroxybenzenes seem to be very promising ones in order to increase capacitance. However, generally only one electrode is responsible for high capacitance enhancement. In case of iodides, bromides, di-hydroxybenzenes operating range of potential for faradaic reactions is quite narrow (0.1-0.4V) being connected with positive electrode. Unfortunately electrode with smaller capacitance will affect a total capacitor performance. A few examples of pseudocapacitive phenomena originated from electroactive species present in electrolyte will be shown. They can serve as great source of pseudocapacitance and may significantly enhance total capacitance of the system.

Electrode/Electrolyte Interface with Various Redox Couples

This paper reports the electrochemical behaviour of supercapacitor carbon electrodes operating in different aqueous solutions modified by various redox-active species (hydroxybenzenes, bromine derivatives and iodide). Three dihydroxybenzenes with varying stereochemistry, i.e., –OH substitution, have been considered as electrolyte additives (0.38 mol L−1) in acidic, alkaline and neutral solutions. High capacitance values have been obtained, especially for the acidic and alkaline solutions containing 1,4-dihydroxybenzene (hydroquinone). Bromine derivatives of dihydroxybenzenes were also considered as the additive in alkaline solution for use as a supercapacitor electrolyte, and a significant increase in capacitance value was observed. The redox couple investigated next was an iodide/iodine system, where 2 mol L−1 NaI aqueous electrolyte was utilized. In this case, the most promising faradaic contribution during capacitor operation was achieved. In particular, stable capacitance values from 300–400 F g−1 have been confirmed by long-term galvanostatic cycling (over 100 000 cycles), cycling voltammetry and floating. The mechanism of pseudocapacitance phenomena was discussed and supported by electrochemical and physicochemical measurements, e.g., in situ Raman spectroscopy.

Redox-active electrolyte for supercapacitor application

Solid supported phospholipid bilayers (SPB) formed by fusion of small unilamellar vesicles on glass, quartz and mica surfaces constitute an attractive model for studying lipid membrane properties and functions. Therefore, it is crucial to understand the mechanisms of SPB formation under different experimental conditions. In situ atomic force microscopy imaging can reveal the details of this process.

Supported Phospholipid Bilayers

Fourteen Ganoderma lucidum strains from different geographic regions were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship between the analyzed strains was determined. All G. lucidum strains were also genetically characterized using the AFLP technique. G. lucidum strains included in the analysis displayed an AFLP profile similarity level in the range from 9.6 to 33.9%. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mitochondrial activity. The analysis allowed comparison of functional diversity of the fungal strains. The substrate utilization profiles for the isolates tested revealed a broad variability within the analyzed G. lucidum species and proved to be a good profiling technology for studying the diversity in fungi. Significant differences have been demonstrated in substrate richness values. Interestingly, the analysis of growth and biomass production also differentiated the strains based on the growth rate on the agar and sawdust substrate. In general, the mycelial growth on the sawdust substrate was more balanced and the fastest fungal growthwas observed forGRE3 and FCL192.

Genetic and Metabolic Intraspecific Biodiversity of Ganoderma lucidum

The effect of different agriculture waste polysaccharides as an only carbon source in culture medium of wood–degrading basidiomycete Cerrena unicolor C–139 were investigated. The maximal growth and laccase synthesis in shaken flask was observed in mineral salts broth containing potato starch as the carbon source and asparagine as the nitrogen source (11,000 nkat/L). When an optimized medium was stimulated by addition of 10 μM Cu2+ to the culture medium on days 3–5 the maximal activity 24,000 nkt/L was obtained. Next the influence of the stabilization of the medium pH after 48–h incubation on laccase activity in fermentor cultures had to be estimated. The obtained data show that use of an automatic pH control set at pH 5.0 increased laccase productivity significantly (by 12 times) as compared to that obtained in the fermentor culture with a non–stabilized pH–value. Under the new conditions, the highest enzyme activity of 290,000 nkat/L was reached after 13–day incubation.

Large Scale Production of Cerrena unicolor Laccase on Waste Agricultural Based Media

Transformations of molecular structures formed by perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) molecules on a rutile TiO2(110) surface are studied with low-temperature scanning tunnelling microscopy. We demonstrate that metastable molecular assemblies transform into differently ordered structures either due to additional energy provided by thermal annealing or when the influence of intermolecular forces is increased by the enlarged amount of deposited molecules. Proper adjustment of molecular coverage and substrate temperature during deposition allows for fabrication of desired assemblies. Differences between PTCDA/TiO2(110) and PTCDA/TiO2(011) systems obtained through identical experimental procedures are discussed.

Transformations of PTCDA structures on rutile TiO2 induced by thermal annealing and intermolecular forces

Functionalized materials consisting of inorganic substrates with organic adsorbates play an increasing role in emerging technologies like molecular electronics or hybrid photovoltaics. For such applications, the adsorption geometry of the molecules under operating conditions, e.g., ambient temperature, is crucial because it influences the electronic properties of the interface, which in turn determine the device performance. So far detailed experimental characterization of adsorbates at room temperature has mainly been done using a combination of complementary methods like photoelectron spectroscopy together with scanning tunneling microscopy. However, this approach is limited to ensembles of adsorbates. In this paper, we show that the characterization of individual molecules at room temperature, comprising the determination of the adsorption configuration and the electrostatic interaction with the surface, can be achieved experimentally by atomic force microscopy(AFM) and Kelvin probe force microscopy (KPFM). We demonstrate this by identifying two different adsorption configurations of isolated copper(ii) meso-tetra (4-carboxyphenyl) porphyrin (Cu-TCPP) on rutile TiO2 (110) in ultra-high vacuum. The local contact potential difference measured by KPFM indicates an interfacial dipole due to electron transfer from the Cu-TCPP to the TiO2. The experimental results are verified by state-of-the-art first principles calculations. We note that the improvement of the AFM resolution, achieved in this work, is crucial for such accurate calculations. Therefore, high resolution AFM at room temperature is promising for significantly promoting the understanding of molecular adsorption.

Characterization of individual molecular adsorption geometries by atomic force microscopy: Cu-TCPP on rutile TiO2

Adsorption of carboxylic-substituted Zn porphyrin molecules (COOH-ZnTPP) on the (011) surface of rutile TiO2 is examined by scanning tunneling microscopy at room temperature. The molecules are either flat, stabilized by the surface defects, or mobile with their movement limited to a reconstruction row of the substrate. Upon increasing the molecular coverage the molecules form 1D structures along the [1–10] direction, in which molecular boards are no longer parallel to the surface. This behavior is contrasted with the structures formed by Zn-TPP lacking the carboxylic group, which remain flat up to a monolayer coverage.

Role of a Carboxyl Group in the Adsorption of Zn Porphyrins on TiO2(011)-2×1 Surface

Molecular heterostructures are formed from meso-tetraphenyl porphyrins-Zn(ii) (ZnTPP) and Cu(ii)-phthalocyanines (CuPc) on the rutile TiO2(011) surface. We demonstrate that ZnTPP molecules form a quasi-ordered wetting layer with flat-lying molecules, which provides the support for growth of islands comprised of upright CuPc molecules. The incorporation of the ZnTPP layer and the growth of heterostructures increase the stability of the system and allow for room temperature scanning tunneling microscopy (STM) measurements, which is contrasted with unstable STM probing of only CuPc species on TiO2. We demonstrate that within the CuPc layer the molecules arrange in two phases and we identify molecular dimers as basic building blocks of the dominant structural phase.

Ordered heteromolecular overlayers formed by metal phthalocyanines and porphyrines on rutile titanium dioxide surface studied at room temperature

Sensitized mesoporous titania is of increasing interest for catalysis and photovoltaic devices such as dye-sensitized solar cells (DSCs). For photovoltaic applications, the catalytic properties of TiO2 can cause degradation of the dyes during device fabrication. This is especially the case if natural sensitizers are used. We addressed this issue by fabrication of carotenoic acid sensitized solar cells under inert and ambient assembly conditions. The DSCs were investigated by currentvoltage and quantum efficiency measurements. Further characterization of the cells was made using impedance spectroscopy. The conversion efficiency of the DSCs prepared under inert conditions improved by at least 25% and the devices showed an enhanced reproducibility. The improvement of the DSCs correlated with the conversion efficiency of the sensitizers under inert conditions. We conclude that the photocatalytic bleaching depends on the electron injection efficiency of the sensitizer. Hence carotenoic acids support their own degradation. However, the photocatalytic decomposition of the sensitizers can be avoided by fabrication of the DSCs under inert conditions.

Impact of Photocatalysis on Carotenoic Acid Dye-Sensitized Solar Cells

The ordering of zinc containing porphyrin molecules on surface of rutile TiO2(110)-(1×1) has been investigated using scanning tunneling microscopy (STM) in ultra-high vacuum at room temperature. It is demonstrated that a carboxylic group (COOH) has a profound impact on the immobilization of the molecules. At coverages below 0.1 monolayer only molecules equipped with the group COOH could be anchored to the surface and imaged with STM. At higher coverage both species, with and without the carboxyl substituent, assemble into ordered structures, forming complete monolayers. It is found, however, that the rhomboid unit cells of these structures exhibit differences in size.

Self-assembling of Zn porphyrins on a (110) face of rutile TiO2–The anchoring role of carboxyl groups

The adsorption of Cu–porphyrin derivatives terminated with peripheral carboxyphenyl side groups on hydroxylated TiO2(110) surfaces is investigated by combined scanning tunneling microscopy (STM), atomic force microscopy (AFM), and density functional theory (DFT). Two distinct contrasts of the molecules are revealed by STM and tunneling spectroscopy. Via single-molecule manipulations, the origin of these peculiar contrasts is found to arise from the presence or not of hydroxyl groups below the molecules. Hence, the electronic coupling of the molecule with the underlying TiO2 surface is locally modified altering the amount of charge transfer and thus their charge state at the molecular scale. Our results particularly underline the fundamental role of hydroxyls of TiO2 on the charge state of adsorbed organic molecules potentially used in dye-sensitized solar cells.

Hydroxyl-Induced Partial Charge States of Single Porphyrins on Titania Rutile

Titanium dioxide, or titania, sensitized with organic dyes is a very attractive platform for photovoltaic applications. In this context, the knowledge of properties of the titania–sensitizer junction is essential for designing efficient devices. Consequently, studies on the adsorption of organic dyes on titania surfaces and on the influence of the adsorption geometry on the energy level alignment between the substrate and an organic adsorbate are necessary. The method of choice for investigating the local environment of a single dye molecule is high-resolution scanning probe microscopy. Microscopic results combined with the outcome of common spectroscopic methods provide a better understanding of the mechanism taking place at the titania–sensitizer interface. In the following paper, we review the recent scanning probe microscopic research of a certain group of molecular assemblies on rutile titania surfaces as it pertains to dye-sensitized solar cell applications. We focus on experiments on adsorption of three types of prototypical dye molecules, i.e., perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA), phtalocyanines and porphyrins. Two interesting heteromolecular systems comprising molecules that are aligned with the given review are discussed as well.

Keywords: dye molecules; perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA); phtalocyanines; porphyrins; rutile; scanning probe microscopy; scanning tunneling microscopy (STM); titanium dioxide (TiO2)

Scanning probe microscopy studies on the adsorption of selected molecular dyes on titania

Zn(II)phthalocyanine molecules (ZnPc) were thermally deposited on a rutile TiO2(011) surface and on Zn(II)meso-tetraphenylporphyrin (ZnTPP) wetting layers at room temperature and after elevated temperature thermal processing. The molecular homo- and heterostructures were characterized by high-resolution scanning tunneling microscopy (STM) at room temperature and their geometrical arrangement and degree of ordering are compared with the previously studied copper phthalocyanine (CuPc) and ZnTPP heterostructures. It was found that the central metal atom may play some role in ordering and growth of phthalocyanine/ ZnTPP heterostructures, causing differences in stability of upright standing ZnPc versus CuPc molecular chains at given thermal annealing conditions.

Ordering of Zn-centered porphyrin and phthalocyanine on TiO2(011): STM studies

Functionalization of surfaces has become of high interest for a wealth of applications such as sensors, hybrid photovoltaics, catalysis, and molecular electronics. Thereby molecule-surface interactions are of crucial importance for the understanding of interface properties. An especially relevant point is the anchoring of molecules to surfaces. In this work, we analyze this process for a zinc-porphyrin equipped with carboxylic acid anchoring groups on rutile TiO2TiO2(110) using scanning probe microscopy. After evaporation, the porphyrins are not covalently bound to the surface. Upon annealing, the carboxylic acid anchors undergo deprotonation and bind to surface titanium atoms. The formation of covalent bonds is evident from the changed stability of the molecule on the surface as well as the adsorption configuration. Annealed porphyrins are rotated by 45° and adopt another adsorption site. The influence of binding on electronic coupling with the surface is investigated using photoelectron spectroscopy. The observed shifts of Zn 2p and N 1s levels to higher binding energies indicate charging of the porphyrin core, which is accompanied by a deformation of the macrocycle due to a strong interaction with the surface.

Thermally induced anchoring of a zinc-carboxyphenylporphyrin on rutile TiO2 (110)”

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Projekt współfinansowany przez Szwajcarię w ramach szwajcarskiego programu współpracy z nowymi krajami członkowskimi Unii Europejskiej

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