Temporal Control Research Articles

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways

A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Autocatalytic Reaction Networks: A Pathway to Spatial Temporal Mastery in Dynamic Materials

AbstractAutocatalytic reaction present a significant opportunity for the precise spatial and temporal control of dynamic materials, mimicking the characteristics of living matter within autonomous chemical systems. Herein, we have crafted an autocatalytic chemical reaction network (CRN) designed to be incorporated into a dynamic system, allowing for efficient control of both sol(I)‐gel and gel‐sol(II) transitions through autocatalytic fronts. The CRN incorporates two autocatalytic reactions. The first reaction promotes the formation of disulfide crosslinks while increasing the local pH through base product generation, catalyzing further disulfide bond formation and initiating a polymerization front that transforms the liquid phase into a gel. A subsequent, slower reaction triggered at the gel/air interface, resulted in the breakage of disulfide crosslinks, transforming the gel back into a liquid state through accelerating fronts. The dynamics of these autocatalytic fronts are accurately predicted by a reaction‐diffusion model, providing a theoretical framework for system preprogramming. Moreover, our results show that the reversible sol‐gel transition can be reliably repeated multiple times. This approach not only enhances our understanding of autocatalytic CRNs but also pioneers a new approach for developing dynamic materials with life‐like properties, significantly impacting material science and bioengineering.

The Rho effector ARHGAP18 coordinates a Hippo pathway feedback loop through YAP and Merlin to regulate the cytoskeleton and epithelial cell polarity.

The organization of the cell's cytoskeletal filaments is coordinated through a complex symphony of signaling cascades originating from internal and external cues. Two major actin regulatory pathways are signal transduction through Rho family GTPases and growth and proliferation signaling through the Hippo pathway. These two pathways act to define the actin cytoskeleton, controlling foundational cellular attributes such as morphology and polarity. In this study, we use human epithelial cells to investigate the interplay between the Hippo and Rho Family signaling pathways, which have predominantly been characterized as independent actin regulatory mechanisms. We identify that the RhoA effector, ARHGAP18, forms a complex with the Hippo pathway transcription factor YAP to address a long-standing enigma in the field. Using super resolution STORM microscopy, we characterize the changes in the actin cytoskeleton, on the single filament level, that arise from CRISPR/Cas9 knockout of ARHGAP18. We report that the loss of ARHGAP18 results in alterations of the cell that derive from both aberrant RhoA signaling and inappropriate nuclear localization of YAP. These findings indicate that the Hippo and Rho family GTPase signaling cascades are coordinated in their temporal and spatial control of the actin cytoskeleton.

Transmembrane Delivery of an Aryl Azopyrazole Photo‐switchable Ion Transporter Relay

Stimuli‐responsive synthetic ionophores allow for spatial and temporal control over ion transport, with promise for applications in targeted therapy. Relay transporters have emerged as a new class of ion transporters ‐ these are anchored carriers that sit in both leaflets of the bilayer and mediate transport across the membrane by passing ions between them. The relays are themselves membrane impermeable, and so must be incorporated into the membrane during vesicle preparation. Here we show that relay transporters can be delivered to both sides of the membrane of vesicles using a synthetic flippase. By incorporating an aryl azopyrazole photo‐switch into the movable arm of the relay transporters the ion transport activity can be very efficiently and reversibly switched between off and on states. This control is achieved by extension and contraction of their movable arms via photo‐isomerization of the central aryl azopyrazole moiety, hence modulating the ability of the relays to pass ions across the membrane.

NIR photothermal activation in epoxy/anhydride systems for advanced polymerization

Photopolymerization is an important area of investigation due to its wide industrial applications; however, it generally employs quite energetic UV or near UV light to initiate the formation of the polymer. Even though it is very effective and offers several advantages, numerous drawbacks are also encountered such as low light penetration, harmfulness of wavelengths in the UV range and the use of photoinitiators that can be hazardous to health. In this context, many researchers have focused their interest in improving the spatial localization and the temporal control of the polymerization under safer irradiation wavelengths (NIR). In this work, we have successfully produced polyester thermosets by the reaction of epoxy resins with anhydride in the presence of a thermolatent N-Heterocyclic carbene generator using the NIR photothermal effect of a dye (heater) able to convert low-energetic NIR light into heat.

Secure control for uncertain nonlinear cyber–physical systems: A prescribed-time scheme based on temporal scaling

In this paper, the prescribed-time secure control is explored for uncertain nonlinear cyber–physical systems through temporal scaling transformation and gain control technique, where the nonlinearity is more generic than common linear growth conditions due to the presence of large uncertainties and low-order states. Given that security risks exist in open networks, the impact of deception attacks is considered, whereas, this prevents the gain control technique from being applied directly to control design. To lift the restriction, a novel auxiliary system is firstly constructed to cleverly turn the secure control to the boundedness control of the auxiliary system. Next, a crucial bridge, temporal scaling transformation, is employed to transform the control issue in the finite time into infinite time, which opens up more possibilities for solving studied issues. Then, by the gain control technique, a Zeno-free secure control strategy with dynamically adjustable triggering mechanism is devised, which successfully ensures the convergence of system states within the prescribed time meanwhile counteracting deception attacks. Further, an improved nonlinearity, i.e. containing both low-order and high-order states, is discussed. Particularly, the proposed control strategies not only avoid real-time control updates, but also the gains therein handle system nonlinearities efficiently. Eventually, the validity of major results is verified via simulation examples.

Poly(lactic-co-glycolic) acid nanoparticles with thermoresponsive shell for sustained release of dexamethasone

Polymeric nanocarriers based on poly(lactide-co-glycolide) (PLGA) enable drug encapsulation while achieving increased solubility of hydrophobic drugs, enhanced plasmatic half-life, and protecting the drug from early degradation/metabolization. Nevertheless, it is still difficult to effectively regulate the drug release from nanocarriers. Covering the surface of PLGA nanoparticles (NPs) with a thermoresponsive shell may be the key factor for building drug delivery systems with temporal and spatial control. In this respect, PLGA NPs with a mean size of 212 nm, containing dexamethasone (Dex) (Dex/PLGA NPs) have been produced by the dialysis technique without the use of a surfactant. Then, the nanoparticles were covered with poly(N-isopropylacrylamide-co-4-vinyl pyridine) (PVP) by ionic interactions between the amine groups of the PVP and carboxylic groups of PLGA. FT-IR and XPS spectroscopy were used to identify the new functional groups introduced on the PLGA nanoparticles' surface. The assembly of PVP on the PLGA NPs was analyzed by monitoring the particle size with temperature, zeta potential, AFM, and TEM analysis. The in vitro drug delivery investigation showed that temperature has significant control over the release rate of Dex from PVP-coated PLGA NPs. The thermosensitive polymer is hydrated at temperatures lower than LCST (20 °C) and forms a gel around the nanoparticles slowing down the release rate of Dex. Above the LCST, PVP becomes hydrophobic, collapses, inducing a substantially slower and sustained release of Dex, with 84 % released at 200 h.

Large-Scale, High-Density MicroLED Array-Based Optogenetic Device for Neural Stimulation and Recording.

Optogenetics has emerged as a pivotal tool in neuroscience, enabling precise control of neural activity through light stimulation. However, the current microLED arrays lack sufficient density and scalability. This study proposes an innovative optogenetic device capable of integrating hundreds of microLEDs and electrocorticography (ECOG) electrodes. Individual or multiple microLEDs in the device can be selectively controlled with a custom controller. The light intensity of microLEDs decreases with increasing brain tissue penetration while maintaining a low temperature rise during pulse stimulations. In addition, interference from microLED pulses on ECOG electrode recordings could be alleviated with local mean subtraction data processing. The optogenetic device enables high-quality neural signal recording and triggers a significant enhancement in neural activity following light stimulation. Integration of microLED arrays and ECOG electrodes in the optogenetic device represents a promising advancement in neuroscientific research, providing improved spatial and temporal recording and control over neural activity.

Forced MyD88 signaling in microglia impacts the production and survival of regenerated retinal neurons.

Inflammation and microglia appear to be key factors influencing the outcome of retinal regeneration following acute retinal damage. Despite such findings, direct connection of microglia-specific inflammatory factors as drivers of regenerative responses in the retina are still not defined, and intracellular pathways activated to stimulate such signals from microglia are currently unknown. We became interested in MyD88 regulation in microglia because transcriptomic datasets suggest myd88 could be regulated temporally in zebrafish microglia responding to damage in the central nervous system. MyD88 is an intracellular molecular adaptor that initiates signaling cascades downstream of several innate immune receptors, and probably most well-known for inducing gene expression of pro-inflammatory factors. Using zebrafish, which spontaneously regenerate retinal neurons after acute retinal damage, we studied the effects of overactivation of MyD88 signaling in microglia and macrophages on the Müller glia-mediated regenerative response. Our results indicate that increased MyD88 signaling in microglia/macrophages impacts the initial response of Müller glia entering a regenerative response after acute, neurotoxin-induced retinal damage to inner retinal neurons. In addition, increased MyD88 signaling in microglia/macrophages resulted in reduced survival of inner retinal neurons in regenerated retinas. This work supports the idea that temporal control of inflammatory signaling is a key component in the production of MG-derived progenitors yet further indicates that such control is important for differentiation and survival of regenerated neurons.

A versatile invivo platform for reversible control of transgene expression in adult tissues.

Temporal control of transgenes has advanced biomedical interventions, including invivo reprogramming, often utilizing the doxycycline (Dox)-mediated Tet-ON system. Here, we developed the Dox-mediated Tet-ON or complementary Tet-OFF counterpart to thoroughly investigate spatial and temporal transgene regulation in adult tissues, revealing inherent limitations and unexpected capabilities of each system. In stark contrast with the Tet-ON system, which was effective only in particular tissues and cell types, primarily epithelial cells, the Tet-OFF system proved capable of gene induction across diverse cell types. Despite the drawback of the Tet-OFF system in inducibility and tunability identified in our study, we demonstrated that use of tetracycline (Tc) effectively addresses these issues, possibly through its pharmacologic properties. Our data suggest that the Tc-mediated Tet-OFF system not only enables more versatile control of transgene expression but also offers a more biocompatible alternative for invivo applications such as tissue regeneration and organismal rejuvenation.

Temporal control of Staphylococcus aureus intracellular pH by sodium and potassium.

Adaptation to environmental change during both colonization and infection is essential to the pathogenesis of Staphylococcus aureus. Like other bacterial pathogens that require potassium to fulfill nutritional and chemiosmotic requirements, S. aureus has been shown to utilize potassium transport to modulate virulence gene expression, antimicrobial resistance, and osmotic tolerance. Recent studies examining the role for potassium uptake in mediating S. aureus physiology have also described its contribution in mediating carbon flux within central metabolism and generation of a proton motive force. Here, we utilize a pH-sensitive green fluorescent protein to examine the temporal regulation of S. aureus intracellular pH by potassium and sodium under various environmental conditions, including extracellular pH and antibiotic stress. Our results distinguish unique conditions and transport mechanisms that utilize these ions to modulate cytoplasmic pH homeostasis, and they identify these processes as a novel mechanism of intrinsic ampicillin resistance in S. aureus.

Loss of neuropeptide signalling alters temporal expression of mouse suprachiasmatic neuronal state and excitability.

Individual neurons of the hypothalamic suprachiasmatic nuclei (SCN) contain an intracellular molecular clock that drives these neurons to exhibit day-night variation in excitability. The neuropeptide vasoactive intestinal polypeptide (VIP) and its cognate receptor, VPAC2, are synthesized by SCN neurons and this intercellular VIP-VPAC2 receptor signal facilitates coordination of SCN neuronal activity and timekeeping. How the loss of VPAC2 receptor signalling affects the electrophysiological properties and states of SCN neurons as well as their responses to excitatory inputs is unclear. Here we used patch-clamp electrophysiology and made recordings of SCN neurons in brain slices prepared from transgenic animals that do not express VPAC2 receptors (Vipr2-/- mice) as well as animals that do (Vipr2+/+ mice). We report that while Vipr2+/+ neurons exhibit coordinated day-night variation in their electrical state, Vipr2-/- neurons lack this and instead manifest a range of states during both day and night. Further, at the population level, Vipr2+/+ neurons vary the membrane threshold potential at which they start to fire action potentials from day to night, while Vipr2-/- neurons do not. We provide evidence that Vipr2-/- neurons lack a component of voltage-gated sodium currents that contribute to SCN neuronal excitability. Moreover, we determine that this aberrant temporal control of neuronal state and excitability alters neuronal responses to a neurochemical mimic of the light-input pathway to the SCN. These results highlight the critical role VIP-VPAC2 receptor signalling plays in the temporal expression of individual neuronal states as well as appropriate ensemble activity and input gating of the SCN neural network.

N-Cadherin — A Potential Target for Psychopharmacology

Glycoprotein N-cadherin (Neuronal cadherin) belongs to the family of calcium-dependent cell adhesion molecules, representing a key element that carries out intercellular contacts in brain neurons. However, it is involved not only in the mechanical connection of neurons, but also influences the specifics of the further development and functional state of the neuron. This is due to the active interaction of N-cadherin with many proteins at the pre- and post-synapse, initiating a cascade of reactions that provide such processes as long-term potentiation (underlying learning and memory), morphogenesis, neuronal recognition, activation of receptors (NMDA and AMPA types), regulation of cytoskeleton formation. This polyfunctionality is necessary for specific neurons to connect to each other in a certain way, and such adhesion leads to the coordination of cell behavior through intercellular signaling and spatio–temporal control of differential gene expression. Mutations in the genes responsible for the expression of N-cadherin lead to various disorders of the functional activity of the synapse and the processes of spatial orientation and memory. Thus, involvement in important neuroplastic processes regulating cognitive functions and behavior determines interest in studying the effect of drugs on N-cadherin. In particular, N-cadherin deserves closer consideration by pharmacologists as a potential target in the mechanism of action of various psychoactive substances.

Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells

Bone Morphogenetic Protein (BMP) signaling is essential for craniofacial development, though little is known about the mechanisms that govern BMP secretion. We show that depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme. We show endogenous transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day 9.5 to 13.5 mice. Disruption of a potassium channel called Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Kcnj2 knockout mice have cleft palate and reduced BMP signaling. Our data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and intracellular calcium for mammalian craniofacial morphogenesis.

Cyclic Acetals as Expanding Monomers to Reduce Shrinkage.

Polarity-reversal catalysts (PRCs) for hydrogen-atom transfer reactions have been known in radical chemistry for more than 60 years but are rarely described and utilized in the field of photopolymerization up to now. Herein, we present the use of thiols in a unique dual function as thiol-ene click reagents and as polarity-reversal catalyst (PRC) for the radical-mediated redox rearrangements of benzylidene acetals. During the rearrangement reaction, cyclic benzylidene acetals are transformed into benzoate esters leading to a significant volumetric expansion to reduce thermoset shrinkage. We were able to show that this expansion on a molecular level reduces shrinkage and polymerization stress but does not significantly affect the (thermo-)mechanical properties of the cross-linked networks. One of the key advantages of this process lies in its simplicity. No additives like sensitizers or combinations of different initiators (radical and cationic) are needed. Furthermore, the same light source can be used for both the polymerization reaction and expansion through rearrangement. Additionally, the applied photoinitiator enables spatial and temporal control of the polymerization; thus, the developed system can be an excellent platform for additive manufacturing processes.

The control costs of human brain dynamics

Abstract The human brain is a complex system with high metabolic demands and extensive connectivity that requires control to balance energy consumption and functional efficiency over time. How this control is manifested on a whole-brain scale is largely unexplored, particularly what the associated costs are. Using network control theory, here we introduce a novel concept, time-averaged control energy (TCE), to quantify the cost of controlling human brain dynamics at rest, as measured from functional and diffusion MRI. Importantly, TCE spatially correlates with oxygen metabolism measures from positron emission tomography, providing insight into the bioenergetic footing of resting state control. Examining the temporal dimension of control costs, we find that brain state transitions along a hierarchical axis from sensory to association areas are more efficient in terms of control costs and more frequent within hierarchical groups than between. This inverse correlation between temporal control costs and state visits suggests a mechanism for maintaining functional diversity while minimizing energy expenditure. By unpacking the temporal dimension of control costs, we contribute to the neuroscientific understanding of how the brain governs its functionality while managing energy expenses.

Cyclische Acetale als expandierende Monomer zur Verringerung von Schrumpfung

AbstractUmpolungskatalysatoren (PRCs) für Wasserstoff‐Übertragungsreaktionen sind innerhalb der Radikalchemie seit mehr als 60 Jahren bekannt, werden allerdings im Bereich der Photopolymerisation bisher kaum beschrieben und genutzt. In diesem Artikel demonstrieren wir die Verwendung von Thiolen in einer einzigartigen Doppelfunktion als Thiol‐En‐Click‐Reagenzien und Umpolungskatalysatoren (PRC) für radikalvermittelte Redox‐Umlagerungsreaktionen von Benzylidenacetalen. Während der Umlagerungsreaktionen wandeln sich Benzylidenacetale in Benzoesäureester um, wodurch es zu einer signifikanten Volumenssteigerung kommt, die der Schrumpfung des Duromeren entgegenwirkt. Wir konnten zeigen, dass diese Expansion auf molekularer Ebene Schrumpfungs‐ und Polymerisationsspannungen reduziert ohne die (thermo‐) mechanischen Eigenschaften des Polymernetzwerkes wesentlich zu beeinflussen. Der Hauptvorteil dieser Herangehensweise liegt dabei in der Schlichtheit, da hier keine Additive wie Sensibilisatoren oder unterschiedliche Initiatorsysteme (radikalisch und kationisch) notwendig sind. Des Weiteren kann dieselbe Lichtquelle sowohl für die Polymerisation als auch für die Expansion im Zuge der Umlagerungsreaktion genutzt werden. Zusätzlich erlaubt der verwendete Photoinitiator eine Kontrolle der Polymerisation über Raum und Zeit, wodurch sich dieses System als hervorragende Grundlage zur additiven Fertigung anbietet.

Transient Photoactivation of Anionophores by Using Redshifted Fast-Relaxing Azobenzenes.

Photo-regulated transmembrane ionophores enable spatial and temporal control over activity, offering promise as targeted therapeutics. Key to such applications is control using bio-compatible visible light. Herein, we report red-shifted azobenzene-derived synthetic anionophores that use amber or red light to trigger (E)-(Z) photoisomerisation and activation of transmembrane chloride transport. We demonstrate that by tuning the thermal half-life of the more active, but thermodynamically unstable, Z isomer to relax on the timescale of minutes, transient activation of ion transport can be achieved by activating solely with visible light and deactivating by thermal relaxation.

Intermittent Flow Control Schemes for Heat Stress Mitigation in Lactating Sows on a Floor Cooling Pad

The Purdue hog cooling pad has previously been demonstrated to mitigate heat stress in lactating sows by conductively transferring heat from a sow to cool water running through an integral heat exchanger. Coolant effectiveness, which describes how much heat is removed per volume of water flushed through the cooling pad, is used to compare the operation under varying conditions. Past studies have indicated that the intermittent flow of cooling water achieves a greater coolant effectiveness than continuous flow operational schemes. An electronic control system was implemented with the current cooling pad design to allow for the automated control of a solenoid valve to create the intermittent flow conditions. All testing was performed using 18 ± 1 °C inlet water. Potential control schemes were categorized into two groups, temporal and temperature threshold. The temporal schemes opened the solenoid for 30 s, enough time to flush the entire contents of the cooling coils, before closing for 3, 6, or 9 min. The temperature threshold control schemes utilized feedback from thermal probes embedded beneath the surface of the cooling pad to open the solenoid for 30 s, when a maximum surface temperature was detected. Trigger temperatures of 28.0, 29.5, or 31.0 °C were used. The temperature threshold control schemes achieved greater heat transfer rates (348, 383, 268 W) compared to the temporal control schemes (324, 128, 84 W). The cooling effectiveness for all control schemes ranged from 46.6 to 64.7 kJ/L. The tested intermittent flow control schemes in this study achieved greater cooling effectiveness than continuous flow systems from previous studies (time: 51 kJ/L; temperature: 61 kJ/L; steady: 5.8 kJ/L), although the temporal control schemes exhibited lower heat transfer rates (time: 180 W; temperature: 330 W; steady: 305 W).

Spatiotemporal control of transgene expression using an infrared laser in the crustacean Daphnia magna

The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna.