Activation Kinetics Research Articles

Stress-induced pseudokinase TRB3 augments IL1β signaling by interacting with Flightless homolog 1

Interleukin-1β is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1β-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1β-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1β signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1β receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1β signaling to augment the proinflammatory response in beta cells.

Metal–Organic Frameworks Encaged Ru Single Atoms for Rapid Acetylene Harvest and Activation in Hydrochlorination

Ruthenium (Ru)-based catalysts have been candidates in hydrochlorination for vinyl chloride monomer (VCM) production, yet they are limited by efficient acetylene (C2H2) utilization. The strong adsorption performance of HCl can deactivate Ru active sites which resulted in weak C2H2 adsorption and slow activation kinetics. Herein, we designed a channel that employed metal-organic framework (MOF)-encaged Ru single atoms to achieve rapid adsorption and activation of C2H2. Low-Ru (∼0.5 wt %) single-atom catalysts (named Ru-NC@MIL) were assembled by hydrogen-bonding nanotraps (the H-C≡C-Hδ+···Oδ- interactions between C2H2 and carboxylate groups/furan rings). Results confirmed that C2H2 could easily enter the encapsulation channels in an optimal mode perpendicular to the channel with a potential energy of 42.3 kJ/mol. The harvested C2H2 molecules can be quickly passed to Ru-N4 active sites for activation by stretching the length of carbon-carbon triple bonds (C≡C) to 1.212 Å. Such a strategy guaranteed >99% C2H2 conversion efficiency and >99% VCM selectivity. Moreover, a stable long-term (>150 h) catalysis with high efficiency (∼0.85 kgvcm/h/kgcat.) and a low deactivation constant (0.001 h-1) was also achieved. This work provides an innovative strategy for precise C2H2 adsorption and activation and guidance for designing multi-functional Ru-based catalysts.

Computational Modeling Analysis of Kinetics of Fumarate Reductase Activity and ROS Production during Reverse Electron Transfer in Mitochondrial Respiratory Complex II.

Reverse electron transfer in mitochondrial complex II (CII) plays an important role in hypoxia/anoxia, in particular, in ischemia, when the blood supply to an organ is disrupted and oxygen is not available. A computational model of CII was developed in this work to facilitate the quantitative analysis of the kinetics of quinol-fumarate reduction as well as ROS production during reverse electron transfer in CII. The model consists of 20 ordinary differential equations and 7 moiety conservation equations. The parameter values were determined at which the kinetics of electron transfer in CII in both forward and reverse directions would be explained simultaneously. The possibility of the existence of the "tunnel diode" behavior in the reverse electron transfer in CII, where the driving force is QH2, was tested. It was found that any high concentrations of QH2 and fumarate are insufficient for the appearance of a tunnel effect. The results of computer modeling show that the maximum rate of succinate production cannot provide a high concentration of succinate in ischemia. Furthermore, computational modeling results predict a very low rate of ROS production, about 50 pmol/min/mg mitochondrial protein, which is considerably less than 1000 pmol/min/mg protein observed in CII in forward direction.

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR’s nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

Physical, optical and thermoluminescence properties of lithium aluminum borate glass co-doped with Dy2O3

The present study aimed to evaluate the physical, optical, and thermoluminescence (TL) properties of Li2O–Al2O3–B2O3 as a function of adding Dy2O3 dopant concentrations varying from 0.0 to 0.8 mol%. The glasses were produced through the melt-quenching technique. The optimal concentration of Dy3+ ions for the 4F9/2 → 6H11/2 and 4F9/2 → 6H15/2 transition was found to be at 0.8 mol%. The glassy nature of the quenched samples was confirmed by the XRD pattern. All samples have been subjected to measurements of various physical properties, including density, molar volume, Polaron radius, inter-nuclear distance, ion concentration, and field strength. The most optimal sample exhibited exceptional linearity within the dose range of 0.5–5.0 Gy during gamma irradiation. The un-doped and Dy3+ doped glasses exhibited a linear correlation coefficient of 0.446 and 0.995, respectively, while their tissue-equivalent effective atomic numbers were 7.4 and 10.5. Over the course of a month, the glasses exhibited a fading rate of only 15%. After being exposed to 3.0 Gy of gamma rays, the samples for minimum detectable dose and reproducibility were examined. The kinetic parameters of the glasses under investigation, such as activation energies and kinetics orders, were determined using the peak shape method. According to the findings, the sample that contained 0.8 mol% of Dy2O3 exhibited exceptional thermoluminescence characteristics, indicating its potential as a dosimeter for measuring exposure to ionizing radiation.

Tunable B cell response through allosteric modulation of Bruton’s tyrosine kinase

Abstract The B cell receptor (BCR) is essential for both the initial burst of B cell proliferation in response to antigens, and for the differentiation of plasma cells and memory B cells during germinal center reactions. However, the BCR’s precise role in germinal center selection has been difficult to assess: mechanistic investigations into its modulation during the decision of B cell fate suffer from experimental limitations due to the fundamental role of the BCR in B cell biology. Bruton’s tyrosine kinase (BTK), a member of the TEC family, is a key component of BCR signaling. Using a computational screening method based on small molecule docking outside of the BTK active site, we have identified putative allosteric modulators of BTK. In vitro primary B cell Nur77 upregulation, calcium flux, proliferation and plasma cell differentiation confirmed that these molecular probes alter B cell activation strength, kinetics, and fate only when the BCR is engaged. In vivo immunization using an antigen model reveled that BTK agonism strongly favors B cell differentiation into switched plasma cells. BTK agonism, either at the time of the immunization or at the peak of germinal center formation, enhanced humoral immunity, proving for the first time that transient, augmented BCR signaling during germinal center reaction has a direct impact on B cell differentiation ability. In addition to being powerful tools for basic B cell biology investigation, allosteric agonists of BTK could be developed into antigen-licensed B cell adjuvants for vaccines in at risk populations, as well as into cancer immunotherapy drugs which hyperactivate B cells in tumor microenvironments during checkpoint inhibitor blockade therapies.

Notch2 amplification of B cell division and differentiation fates

Abstract The efficiency with which B cells respond to activation signals varies depending on the subset. Whereas marginal zone (MZ) B cells exhibit rapid activation kinetics following stimulation, follicular (FO) B cells adopt delayed effector programs. The development and maintenance of MZ B cells uniquely requires signaling through transmembrane receptor family member Notch2, but whether this signal underlies distinctive response patterns among B cell subsets is not known. To examine the functional consequences of Notch2 signals on B cells with no Notch requirement, we cultured FO B cells on Notch2-ligand expressing OP9 stromal cells and subsequently stimulated them. Notch2-experienced FO B cells displayed a marked enhancement of several aspects of proliferation, including two-fold more rounds of division. Further, sensitivity to BCR cross-linking was enhanced by this signal, enabling cell cycle entry earlier and in response to lower concentrations of anti-BCR. Additionally, Notch2 signals modified responses to TLR stimuli Poly(I:C) and LPS by amplifying the time to first division and the propensity to become a plasma cell. Intriguingly, TLR3 responsiveness appeared to be an entirely Notch2-dependant mechanism. Employing a published mathematical model for dividing lymphocytes, we found Notch2 amplified the division destiny of both graded and quantal patterns of B cell responses. We next examined the timing and magnitude of Myc induction, a mitotic regulator and known Notch target. Remarkably, Myc levels prior to the first division appeared to be a direct predictor of division destiny. Together these observations highlight a novel role for Notch2 in modifying two distinct response patterns and their respective outcomes for B cells. Supported by grants from NIH (T32-AI00763,R01 AI139123,T R01 AI154932)

Reciprocity between IL-4 and opioid immune regulation: IL-4 primes human CD4+ T cells for opioid receptor driven MAP kinase signaling

Abstract Opioid receptor (OR) signaling induces transcriptomic and functional changes in human T cell responses at both a molecular and cellular level, yet the signaling pathways initiated by OR engagement in human T cells are poorly characterized. ORs are G-protein coupled receptors with three subtypes: MuOR, KappaOR, and DeltaOR. Using the human lymphoma Jurkat T cell line and negatively selected human peripheral blood CD4 +T cells, the effects of opioid exposure on the activation kinetics of the MAP kinases ERK 1/2, JNK, and p38, scored as their level of phosphorylation, was followed by immunoblot. Opioid-driven phosphorylation of the MAPKs was dependent on IL-4 pretreatment of Jurkat cells, but not blood-derived CD4+ T cells. After Jurkat cells were primed with a 3-day exposure to IL-4, a series of opioid agonists selective for each OR subtype induced the phosphorylation of all three MAPKs (ERK, JNK, p38) within 2 min. Phosphorylation of the MAPKs was independent of OR subtype and opioid agonist. Notably, the kinetics were different than that observed in neurons. When primary human CD4 +T cells were treated with opioids, only p38 MAPK, not ERK or JNK, was activated with similar kinetics and OR subtype independence as that seen in Jurkat cells. This difference between primary cells and Jurkat cells suggests an additional level of regulation of opioid signaling in primary cells not found in Jurkat cells. The requirement for IL-4 pretreatment suggests that opioids may play a role in Th2 biology. Preliminary data indicate that morphine modestly increases the expression of IL-4 mRNA. Overall, these results reveal that p38 functions as a primary effector of opioid receptor signal transduction in human T cells. Supported by grants from NIH / NIDA (R01 DA 043253, )

MHC Ib-restricted T Cells Generate the Dominant Memory T Cell Response in Early Stages of Mycobacterium tuberculosisInfection

Abstract Unconventional T cells, which recognize monomorphic MHC Ib molecules, such as Qa1, Qa2, M3, CD1, and MR1 have been implicated in the primary response to Mycobacterium tuberculosis(Mtb) infection. However, whether these T cells play a role and which MHC Ib molecules participate in the memory response against Mtbinfection remains unknown. To determine the optimal immunization strategy, we vaccinated mice that lack MHC Ia (K b−/−D b−/−) with an attenuated MtbH37Rv strain via intravenous (IV) or subcutaneous (SC) injection. We found both routes of immunization induced a comparable MHC Ib-restricted CD8 +T cell response after virulent MtbH37Rv re-challenge. Since bacteria were cleared through the SC but not the IV route, we selected SC as our primary route of immunization. MHC Ib-restricted CD8 +T cells in vaccinated mice produced multiple proinflammatory cytokines, expanded faster after Mtbchallenge than those in unvaccinated mice, and recognized several Mtbprotein antigens. Importantly, the memory response of MHC Ib-restricted CD8 +T cells was more dominant than that of MHC Ia-restricted CD8 +T cells at early stages of infection. We also observed an increased percentage and number of Qa1, Qa2 and M3-restricted CD8 +T cells in vaccinated mice in the early stages of infection, suggesting that these MHC Ib molecules can elicit MHC Ib-restricted memory CD8 +T cell response. While CD1d-restricted NKT cells did not differ significantly between vaccinated and non-vaccinated mice, the activation kinetics of MR1-restricted T cells was accelerated in vaccinated mice. Overall, we show that MHC Ib-restricted T cells memory response can play an important role at the early stages of Mtbinfection thus should be included in subunit vaccine formulation. Supported by grants from NIH (R01AI141083)

Rational Construction of 2D/3D Co3O4/NH2‐MIL‐53(Al) Heterostructures to Facilitate Photocatalytic Hydrogen Evolution

Based on the principle of strengthening photogenerated carrier separation and interfacial charge transfer, the heterojunction structure is constructed to improve the performance of photocatalytic hydrogen evolution. For this reason, a 3D gear‐like NH2‐MIL‐53(Al) material is synthesized by the template‐free hydrothermal method, and then the 2D irregular loose flake material Co3O4 derived from metal–organic framework is loaded. The hydrogen evolution rate of the composite photocatalyst Co3O4/NH2‐MIL‐53 (Al) is much greater than that of two single catalysts after progressively improving the experimental conditions. First of all, this is attributed to the heterojunction structure formed by the appropriate arrangement of energy bands. Second, the rationality of the heterojunction structure is confirmed by the change of binding energy of atoms in in situ irradiation X‐ray photoelectron spectroscopy and the calculation results of density functional theory. Further testing of the materials’ photoelectrochemical properties reveals that the composite photocatalyst Co3O4/NH2‐MIL‐53 (Al) has improved light adsorption, a lower overpotential, and a smaller Tafel slope. These results show that the hybrid material Co3O4/NH2‐MIL‐53 (Al) improves the charge separation activity and hydrogen evolution reaction kinetics, thus enhancing the photocatalytic performance.

The Effect of Drying Temperature and Thickness on the Drying Kinetic, Antioxidant Activity, Phenolic Compounds, and Color Values of Apple Slices

Dried fruit slices are important, healthy, and popular snacks and gain importance day by day due to their high nutritional content. In this context, this study mainly focused on the production of healthy apple chips snacks and the determination of degradation kinetics of antioxidant activity, total phenolic compounds, and color values of apple chip snacks during convective hot air drying at three different temperatures (45, 55, and 65°C) and sample thicknesses (1.5 and 5 ± 0.5 mm). The drying kinetics, desorption isotherms, activation energy, and half-life of the apple chip snack were also calculated. The Page and GAB models are the best models for the determination of the drying (>0.992) and desorption (>0.9979) behavior of apple snacks with the highest R2 values. The drying of all samples took place in the falling rate period. The Deff values increased depending on the increasing air temperature and slice thickness. The antioxidant activity, total phenolic compounds, and total color change of the 5 mm thick samples were degraded following the first-order reaction kinetics. The higher antioxidant activity, phenolic compounds, L∗ values, and lower half-life values were observed in conditions where the thickness (1.5 mm) and temperature (45°C) are low. The activation energy values calculated for the total phenolic compounds are higher than those calculated for the antioxidant activity. As a result, it can be concluded that apple chip snacks with high nutritional value can be produced by choosing low temperatures and slice thickness.

Acid-sensing (proton-gated) ion channels (ASICs) in GtoPdb v.2023.1

Acid-sensing ion channels (ASICs, nomenclature as agreed by NC-IUPHAR [48, 2, 3]) are members of a Na+ channel superfamily that includes the epithelial Na+ channel (ENaC), the FMRF-amide activated channel (FaNaC) of invertebrates, the degenerins (DEG) of Caenorhabitis elegans, channels in Drosophila melanogaster and 'orphan' channels that include BLINaC [70] and INaC [72] that have also been named BASICs, for bile acid-activated ion channels [90]. ASIC subunits contain 2 TM domains and assemble as homo- or hetero-trimers [45, 41, 7, 94, 93, 77] to form proton-gated, voltage-insensitive, Na+ permeable, channels that are activated by levels of acidosis occurring in both physiological and pathophysiological conditions with ASIC3 also playing a role in mechanosensation (reviewed in [44, 89, 48, 69, 23]). Splice variants of ASIC1 [termed ASIC1a (ASIC, ASICα, BNaC2α) [84], ASIC1b (ASICβ, BNaC2β) [19] and ASIC1b2 (ASICβ2) [79]; note that ASIC1a is also permeable to Ca2+], ASIC2 [termed ASIC2a (MDEG1, BNaC1α, BNC1α) [66, 85, 40] and ASIC2b (MDEG2, BNaC1β) [56]] differ in the first third of the protein. Unlike ASIC2a (listed in table), heterologous expression of ASIC2b alone does not support H+-gated currents. A third member, ASIC3 (DRASIC, TNaC1) [83] is one of the most pH-sensitive isoforms (along with ASIC1a) and has the fastest activation and desensitisation kinetics, however can also carry small sustained currents. ASIC4 (SPASIC) evolved as a proton-sensitive channel but seems to have lost this function in mammals [58]. Mammalian ASIC4 does not support a proton-gated channel in heterologous expression systems but is reported to downregulate the expression of ASIC1a and ASIC3 [1, 43, 34, 54]. ASICs channels are primarily expressed in central (ASIC1a, -2a, 2b and -4) and peripheral neurons including nociceptors (ASIC1-3) where they participate in neuronal sensitivity to acidosis. Humans express, in contrast to rodents, ASIC3 also in the brain [27]. ASICs have also been detected in taste receptor cells (ASIC1-3)), photoreceptors and retinal cells (ASIC1-3), cochlear hair cells (ASIC1b), testis (hASIC3), pituitary gland (ASIC4), lung epithelial cells (ASIC1a and -3), urothelial cells, adipose cells (ASIC3), vascular smooth muscle cells (ASIC1-3), immune cells (ASIC1,-3 and -4) and bone (ASIC1-3) (ASIC distribution is reviewed in [55, 28, 42]). A neurotransmitter-like function of protons has been suggested, involving postsynaptically located ASICs of the CNS in functions such as learning and fear perception [35, 50, 97], responses to focal ischemia [91] and to axonal degeneration in autoimmune inflammation in a mouse model of multiple sclerosis [39], as well as seizures [98] and pain [89, 29, 30, 13, 32]. Heterologously expressed heteromultimers form ion channels with differences in kinetics, ion selectivity, pH- sensitivity and sensitivity to blockers that resemble some of the native proton activated currents recorded from neurones [56, 5, 38, 11]. In general, the known small molecule inhibitors of ASICs are non-selective or partially selective, whereas the venom peptide inhibitors have substantially higher selectivity and potency. Several clinically used drugs are known to inhibit ASICs, however they are generally more potent at other targets (e.g. amiloride at ENaCs, ibuprofen at COX enzymes) [68, 63]. The information in the tables below are for the effects of inhibitors on homomeric channels, for information of known effects on heteromeric channels see the comments below.

A Review on Cassava Residues as Adsorbents for Removal of Organic and Inorganic Contaminants in Water and Wastewater

An increase in water demand for drinking, agriculture, and industries necessitates the treatment of water and wastewater. Among various conventional treatment techniques available, adsorption is found to be one of the most economical and feasible methods. Adsorbents from plant biomass are effective for the removal of organic and inorganic pollutants. Cassava has gained attention among researchers during the past decades due to its plentiful availability and resilient characteristics. Even though cassava contains cyanogenic glucosides as toxins, it is used in industries for development of various products. Cassava stem, rhizome, peel, and bagasse are industrial residues that are generated in abundance. The present review focusses on factors affecting adsorption using cassava residues, adsorbent preparation and activation methods, equilibrium, mass transfer, kinetics, and thermodynamic studies of adsorption.

Flower-Like Amorphous MoO3- x Stabilized Ru Single Atoms for Efficient Overall Water/Seawater Splitting.

Benefitting from the maximum atom utilization efficiency, special size quantum effects and tailored active sites, single-atom catalysts (SACs) have been promising candidates for bifunctional catalysts toward water splitting. Besides, due to the unique structure and properties, some amorphous materials have been found to possess better performance than their crystalline counterparts in electrocatalytic water splitting. Herein, by combining the advantages of ruthenium (Ru) single atoms and amorphous substrates, amorphous molybdenum-based oxide stabilized single-atomic-site Ru (Ru SAs-MoO3- x /NF) catalysts are conceived as a self-supported electrode. By virtue of the large surface area, enhanced intrinsic activity and fast reaction kinetics, the as-prepared Ru SAs-MoO3- x /NF electrode effectively drives both oxygen evolution reaction (209mV @ 10mA cm-2 ) and hydrogen evolution reaction (36mV @ 10mA cm-2 ) in alkaline media. Impressively, the assembled electrolyzer merely requires an ultralow cell voltage of 1.487V to deliver the current density of 10mA cm-2 . Furthermore, such an electrode also exhibits a great application potential in alkaline seawater electrolysis, achieving a current density of 100mA cm-2 at a low cell voltage of 1.759V. In addition, Ru SAs-MoO3- x /NF only has very small current density decay in the long-term constant current water splitting test.

Engineering kinetics of TLR7/8 agonist release from bottlebrush prodrugs enables tumor-focused immune stimulation.

Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7 (TLR7) and/or TLR8 on innate immune cells. Nevertheless, intravenous administration of IMDs causes severe immune-related toxicities, and attempts to improve their tissue-selective exposure while minimizing acute systemic inflammation have proven difficult. Here, using a library of R848 "bottlebrush prodrugs" (BPDs) that differ only by their R848 release kinetics, we explore how the timing of R848 exposure affects immune stimulation in vitro and in vivo. These studies led to the discovery of R848-BPDs that exhibit optimal activation kinetics to achieve potent stimulation of myeloid cells in tumors and substantial reductions in tumor growth following systemic administration in mouse syngeneic tumor models without any observable systemic toxicity. These results suggest that release kinetics can be tuned at the molecular level to provide safe yet effective systemically administered immunostimulant prodrugs for next-generation cancer immunotherapies.

The rate of platelet activation determines thrombus size and structure at arterial shear

BackgroundThe response of platelets to activating stimuli and pharmaceutical agents varies greatly within the normal population. Current platelet function tests are used to measure end-point levels of platelet activation without taking the speed at which platelets activate into account, potentially missing vital metrics to characterize platelet reactivity. ObjectivesTo identify variability, to agonists and among individuals, in platelet activation kinetics and assess the impact of this on thrombus formation. MethodsWe have developed a bespoke real-time flow cytometry assay and analysis package to measure the rate of platelet activation over time using 2 parameters of platelet activation, fibrinogen binding and P-selectin exposure. ResultsThe rate of platelet activation varied considerably within the normal population but did not correlate with maximal platelet activation, demonstrating that platelet activation rate is a separate and novel metric to describe platelet reactivity. The relative rate of platelet response between agonists was strongly correlated, suggesting that a central control mechanism regulates the rate of platelet response to all agonists. ConclusionFor the first time, we have shown that platelet response rate corresponds to thrombus size and structure, wherein faster responders form larger, more densely packed thrombi at arterial, but crucially not venous, shear. We have demonstrated that the rate of platelet activation is an important metric in stratifying individual platelet responses and will provide a novel focus for the design and development of antiplatelet therapy, targeting high-shear thrombosis without exacerbating bleeding at low shear.

Autonomous and Programmable Reorganization of DNA‐Based Polymers Using Redox Chemistry**

We demonstrate here a strategy that allows the programmable and autonomous reorganization of self-assembled DNA polymers using redox chemistry. We have rationally designed different DNA monomers (tiles) that can co-assemble into tubular structures. The tiles can be orthogonally activated/deactivated with disulfide-linked DNA fuel strands that are degraded over time upon reduction because of the presence of a reducing agent in the system. The concentration of the disulfide fuels determines the activation kinetics of each DNA tile, which controls the degree of order/disorder in the formed co-polymer. The disulfide-reduction pathway can be employed together with enzymatic fuel-degradation pathways providing an additional level of control in the re-organization of DNA structures. Taking advantage of the different pH-sensitivities of disulfide-thiol and enzymatic reactions, we show that we can control the order in DNA-based co-polymers as a function of pH.

Digital optimization of teeth setup in an edentulous patient with partial glossectomy: A case report.

The purpose of this clinical report was to describe the use of a piezographic impression associated with computer-aided design and computer-aided manufacturing (CAD-CAM) for teeth setup and of digital tools for neuro-musculo-kinetic analyses. An edentulous patient with hemiglossectomy and heavily resorbed mandible consulted for complete denture rehabilitation to improve their masticatory function and speech. Master casts, wax rims, and piezographic impression were scanned for digital prosthetic work. Two digital try-ins were performed to respect the neutral zone: try-in 1 with posterior crossbite and try-in 2 without crossbite. Muscle activity and mandibular kinetics were performed for each try-in following the MAC2 protocol (six criteria): muscular tone, contraction synchrony, contraction efficiency, interocclusal rest distance, amplitude of mandibular movement, and velocity. Try-in 2 showed better data than try-in 1 in all criteria: muscle tone (respectively 71% vs. 59%), contraction synchrony (79% vs. 75%), contraction efficiency (85% vs. 77%), an increase in range of motion of 3.3mm, and a better velocity (0.35±0.12s vs. 0.57±0.14s, p=0.008). The piezographic impression, in combination with CAD-CAM, allowed the comparison of two prosthetic designs and the selection of the try-in with the best neuro-musculo-kinetic results.

Abstract 3876: Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors

Abstract Combination treatment of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) produces remarkable response rates and is expected to prevent ERK activation in BRAF-mutant melanoma. However, sporadic ERK reactivation occurs in melanoma models, and efficacy is severely limited by the emergence of drug resistance. The underlying molecular mechanisms that drive ERK reactivation and adaptation to BRAFi/MEKi remain elusive. Here, using single-cell imaging and optogenetic approaches, we show that the strength and duration of receptor tyrosine kinase (RTK) activation determines ERK reactivation and the development of BRAFi/MEKi-resistant cells. Single-cell data reveal that despite global RTK activation by the same external stimuli, only small subsets of melanoma cells induce effective RTK and ERK activation to develop BRAFi/MEKi resistance. Furthermore, by directly controlling RTK activity, we demonstrate that the kinetics of RTK activation determine ERK signaling dynamics and the percentage of drug-resistant cells. While these drug-resistant cells are rare during early stages of drug treatment, they eventually become the dominant population through sustained RTK-mediated ERK activation that drives cell proliferation. Thus, RTK activation in established drug-resistant cells causes effective ERK activation and cell proliferation. Importantly, limiting RTK signaling activation reverses the BRAFi/MEKi-resistant state. We show that targeting SHP2, a mediator of RTK signaling, effectively suppresses RTK-mediated ERK activation and cell proliferation in BRAFi/MEKi-resistant cells. Our results provide mechanistic insights into the contribution of heterogeneity in RTK activation kinetics to ERK reactivation and BRAFi/MEKi resistance, also highlighting SHP2 activity as a promising target to overcome drug resistance in BRAF-mutant melanoma. Citation Format: Sungsoo Kim, Richard Carvajal, Minah Kim, Hee Won Yang. Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3876.

Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state.

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.