Transient Kinetics Research Articles

Quantitative Insights into Processivity of an Hsp100 Protein Disaggregase on Folded Proteins.

The Hsp100 family of protein disaggregases play important roles in maintaining protein homeostasis in cells. E. coli ClpB is an Hsp100 protein that solubilizes protein aggregates. ClpB is proposed to couple the energy from ATP binding and hydrolysis to processively unfold and translocate protein substrates through its axial channel in the hexameric ring structure. However, many of the details of this reaction remain obscure. We have recently developed a transient state kinetics approach to study ClpB catalyzed protein unfolding and translocation. In the work reported here we have used the approach to examine how ATP is coupled to the protein unfolding reaction. Here we show that at saturating [ATP], ClpB induces the cooperative unfolding of a complete TitinI27 domain of 98 amino acids, which is represented by our measured kinetic step-size m ∼100 amino acids. This unfolding event is followed by rapid and undetected translocation up to the next folded domain. At sub-saturating [ATP], ClpB induces cooperative unfolding of a complete TitinI27 domain but translocation becomes partially rate-limiting, which leads to an apparent reduced kinetic step-size as small as ∼ 50 amino acids. Further, we show that ClpB exhibits an unfolding processivity of P = (0.74 ± 0.06) independent of [ATP]. These findings advance our understanding of the ATP coupling to enzyme catalyzed protein unfolding by E. coli ClpB and present a strategy that is broadly applicable to a variety of Hsp100 family members and AAA+ superfamily members.

A High Throughput Platform to Minimize Voltage and Fill Factor Losses

AbstractOrganic photovoltaics (OPV) now can exceed 20% power conversion efficiency in single junction solar cells. To close the remaining gap to competing technologies, both fill factor and open‐circuit voltage must be optimized. The Langevin reduction factor is a well‐known concept that measures the degree to which charge extraction is favored over charge recombination. It is therefore ideally suited as an optimization target in high‐throughput workflows; however, its evaluation so far requires expert interaction. Here, an integrated high‐throughput workflow is presented, able to obtain the Langevin reduction factor within a few seconds with high accuracy without human intervention and thus suited for autonomous experiments. This is achieved by combining evidence from UV–vis spectra, current–voltage curves, and a novel implementation of microsecond transient absorption kinetics allowing, for the first time, the intrinsic determination of charge absorption cross‐sections, which is crucial to reporting stationary charge densities. The method is demonstrated by varying the donor:acceptor ratio of the high performance OPV blend PM6:Y12. The high reproducibility of the method allows to find a strictly exponential relationship between the PM6 exciton energy and the Langevin reduction factor.

Transient Kinetic QXAFS Approach for Understanding the RDE-MEA Gap in Fuel Cell (Oxygen Reduction Reaction) Performances of Pt-Based Electrocatalysts.

There is a large gap between the performances indicated by rotating disk electrode (RDE) results in acidic media and the actual performances obtained in membrane-electrode assemblies (MEAs) composed of the same electrocatalysts. It is unclear whether the intrinsic kinetic reactivity of the available surface Pt sites of Pt-based cathode electrocatalysts is similar or different at RDE and in MEA. To address this, we used an operando element-selective time-resolved Pt LIII-edge quick X-ray absorption fine structure (QXAFS) technique to determine transient response profiles and rate constants, k d(WL), k d(CNPt-O), and k d(CNPt-Pt), corresponding to changes in the oxidation states [white line (WL) intensity] and local structures (coordination numbers of Pt-O and Pt-Pt bonds) at Pt sites for nine representative Pt-based cathode electrocatalysts under transient voltage operations, aiming to understand the oxygen reduction reaction (ORR) performance gap between RDE and MEA. For the first time, the transient kinetics and reactivity of electrocatalyst themselves in MEA, characterized by the operando QXAFS analysis technique, were systematically compared with the electrochemical activity [mass activity (I mass) and surface specific activity (I specific)] of the electrocatalysts in MEA and at RDE. The operando time-resolved QXAFS analysis revealed that the ORR activities of available surface Pt sites at RDEs of the electrocatalysts, including notably structured electrocatalysts (concave octahedral PtNi x /C and Pt nanowire/C), were kinetically reflected at good levels of k d(WL) and k d(CNPt-O) in MEA performances, despite large RDE-MEA gaps observed in the electrochemically determined I mass and I specific. As the I mass and I specific of MEA increased, the relaxation time k d(CNPt-Pt) -1, which indicates long-term durability, decreased, reflecting a dilemma in the development of remarkable Pt-based electrocatalysts, while the k d(CNPt-Pt) -1 was almost independent of ECSA. The differences and similarities in the kinetic reactivity and durability of the Pt surface between RDE and MEA were examined using operando QXAFS transient kinetics and electrochemical performance measurements to elucidate the underlying factors contributing to the performance gap between RDE and MEA. The insights gained aim to support the development of next-generation polymer electrolyte fuel cells with enhanced performance and durability by leveraging the operando time-resolved QXAFS technique under the transient kinetic-response operation.

Transient Kinetic Investigation of Chain Growth and the Accumulation of Surface Carbon During CO Hydrogenation on Cobalt Catalysts

AbstractCO hydrogenation is a promising approach for the storage of renewable energy in the form of hydrocarbons via the Fischer–Tropsch synthesis (FTS). Since transient operation of FTS reactors might be necessary and even be beneficial, transient kinetics for a rational catalyst and reactor design are essential. In order to advance the development of such transient kinetics, the periodic transient kinetics (PTK) method was applied to the CO hydrogenation on a Co/TiO2 catalyst under FT‐like conditions. It was revealed that there are two carbon species of different reactivity, Cα and Cβ, present on the catalyst surface during the reaction. Cα forms fast, within a few seconds, and is highly reactive. Whereas Cβ forms slowly, is accumulating on the surface over a longer time, and imposes an inhibiting effect. The results indicate an important role of the Cβ species to chain growth and the formation of C2+ products. Finally, the transient experimental results were evaluated based on a material balance and the amounts of Cα and Cβ present on the catalyst surface during the reaction were determined.

Quantifying the reversibility of ATP hydrolysis in rabbit skeletal myosin subfragment 1 using a luciferase assay

BackgroundIt is known that the adenosine triphosphate (ATP) hydrolysis in a number of ATPases is reversible. Radioactive methods are standard in measuring ATPase reversibility. We used myosin as a well-studied model enzyme to develop a luciferase-based assay to quantify the reversibility of ATP hydrolysis. In myosin, ATP bound to the active site is hydrolyzed into adenosine diphosphate (ADP) and inorganic phosphate (Pi) and recombined back into ATP multiple times before products of hydrolysis are released. The reversibility of ATP hydrolysis by skeletal myosin was previously confirmed using radioactively labeled isotopes. Transient kinetics studies indicated that the ATP hydrolysis step is temperature-sensitive, with a dissociation equilibrium constant of 1.6 at 3 °C. Consequently, the association equilibrium constant at this temperature is 0.63. The goal of our work was two-fold, (a) to develop a luciferase-based assay to measure the equilibrium constant of enzymatic ATP hydrolysis, eliminating the need for radioactively labeled Pi, and (b) refine the value of the association equilibrium constant of the myosin ATPase hydrolysis step.MethodsIn this assay, a reaction mixture containing myosin and saturating levels of ADP and Pi was incubated to reach equilibrium, then the reaction was terminated, and the amount of ATP produced by myosin was quantified using the luciferase assay. The equilibrium constant of ATP hydrolysis was defined as the ratio of ATP to ADP bound to myosin.ResultsWe obtained a value of 0.78 ± 0.14 for the association equilibrium constant of ATP hydrolysis at 0 °C. 50 μM ADP bound to myosin is turned into 21.9 ± 3.0 μM ATP. Our result is in excellent agreement with the literature data, supporting the viability of the new methodology.DiscussionThis methodology allows a more accessible and safe option to measure ATP production and reversibility of ATPase enzymes. Standard laboratory equipment is utilized in the assay, and the number of steps in the developed assay is reduced significantly compared to the radioactive method.

Developing a soft micropatterned substrate to enhance maturation of human induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSCCM immaturity by introducing a physiologically relevant micropatterned substrate for long-term culture and maturation. An innovative microfabrication methodology combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue. Platinum electrodes were integrated into the cell culture chamber enable pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+ transient, sarcomere orientation, and nucleus aspect ratio were analyzed in a 2D hiPSCCM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSCCMs for three weeks on a micropatterned PDMS substrate (2.5–5 µm deep, 20 µm center-to-center spacing of grooves, 2–5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, contractility, and cytosolic Ca2+ transient. The study provides insights into substrate stiffness effects on hiPSCCM contractility and Ca2+ transient at immature and mature states. Maximum contractility and fastest Ca2+transient kinetics occur in mature hiPSCCMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. MS proteomic analysis further revealed that hiPSCCMs cultured on soft micropatterned substrates exhibit advanced maturation, marked by significant upregulation of key structural, electrophysiological, and metabolic proteins. This new substrate offers a promising platform for disease modeling and therapeutic interventions. Statement of SignificanceHuman induced pluripotent stem cell derived cardiomyocytes (hiPSCCMs) have been transformative to disease-in-a-dish modeling, drug discovery and testing, and autologous regeneration for human hearts and their role will continue to expand dramatically. However, one of the major limitations of hiPSCCMs is that without intervention, the cells are immature and represent those in the fetal heart. We developed protocols for the fabrication of the PDMS matrices that includes variations in its stiffness and micropatterning. Growing our hiPSCCMs on matrices of comparable stiffness to a healthy heart (5 kPa) and grooves of 20 μm, generate heart cells typical of the healthy adult human heart.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s−1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by Escherichia coli ClpB.

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s-1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

Ultrafast transient absorption spectra and kinetics of human blue cone visual pigment at room temperature

The ultrafast photochemical reaction mechanism, transient spectra, and transition kinetics of the human blue cone visual pigment have been recorded at room temperature. Ultrafast time-resolved absorption spectroscopy revealed the progressive formation and decay of several metastable photo-intermediates, corresponding to the Batho to Meta-II photo-intermediates previously observed with bovine rhodopsin and human green cone opsin, on the picosecond to millisecond timescales following pulsed excitation. The experimental data reveal several interesting similarities and differences between the photobleaching sequences of bovine rhodopsin, human green cone opsin, and human blue cone opsin. While Meta-II formation kinetics are comparable between bovine rhodopsin and blue cone opsin, the transition kinetics of earlier photo-intermediates and qualitative characteristics of the Meta-I to Meta-II transition are more similar for blue cone opsin and green cone opsin. Additionally, the blue cone photo-intermediate spectra exhibit a high degree of overlap with uniquely small spectral shifts. The observed variation in Meta-II formation kinetics between rod and cone visual pigments is explained based on key structural differences.

Unravelling the multiple effects of H2O on the NH3-SCR over Mn2Cu1Al1Ox-LDO by transient kinetics and in situ DRIFTS

Understanding detrimental effects of water in the low-temperature NH3-SCR is very important for advancing the design of novel catalytic materials. Herein, the effects of 5 vol% H2O on the performance of NH3-SCR over Mn2Cu1Al1Ox-LDO were investigated through steady-state and transient kinetic analysis of step-gas switching experiments, in-situ DRIFTS and 15NO-SSITKA-DRIFTS operando. Water exhibited a significant negative activity effect at T<180 °C. Slightly positive activity (200–300 °C) and N2-selectvity (120–300 °C) effects were observed due to suppression of NH3 oxidation and NH4NO3 decomposition to N2O. The decline in activity at 140 °C by ∼ 45 % in the presence of water was found to be related to the decrease of the site activity (k) and not of surface coverage (θ) of active species formed in the NH3-SCR reaction paths of N2/N2O formation. An increase in the concentration of inactive NOx-s might also contribute to the inhibitive effect of water.

Operando spectroscopic studies on redox mechanism for CO2 hydrogenation to CO on In2O3 catalysts

The catalytic activities of In2O3 catalysts with different surface area for both cyclic unsteady-state (transient) and steady-state reverse water–gas shift (RWGS) reactions were systemically investigated. The initial CO formation rates during CO2-oxidation of the H2-reduced In2O3 catalysts were close to the CO formation rates in steady-state RWGS conditions at 325 °C, suggesting a redox mechanism for the steady-state RWGS reaction over the In2O3 catalysts. Transient kinetics for the In3+/In+ redox and products (H2O, CO) formation during the cyclic H2-reduction and CO2-oxidation of In2O3 under periodic feeding of H2 ↔ CO2 were studied by time-resolved operando UV–vis and In K-edge X-ray absorption experiments at 325 °C. During the H2-reduction, the surface In3+-O species was reduced to produce H2O and In+-□ (□: oxygen vacancy). Subsequent re-oxidation of the In+-□ by CO2 gave CO and the In3+-O species. The transient CO formation rates were close to the consumption rates of In+-□ under CO2, providing a quantitative evidence on the redox mechanism for unsteady-state RWGS reaction over In2O3. These results indicate that the unsteady-state and steady-state RWGS reactions are primary driven by the In3+/In+ redox mechanism. Kinetic results show that the reoxidation step is the rate-limiting step in the steady-state RWGS reaction.

Populus trichocarpa EXPA6 Facilitates Radial and Longitudinal Transport of Na+ under Salt Stress.

Expansins are cell wall (CW) proteins that mediate the CW loosening and regulate salt tolerance in a positive or negative way. However, the role of Populus trichocarpa expansin A6 (PtEXPA6) in salt tolerance and the relevance to cell wall loosening is still unclear in poplars. PtEXPA6 gene was transferred into the hybrid species, Populus alba × P. tremula var. glandulosa (84K) and Populus tremula × P. alba INRA '717-1B4' (717-1B4). Under salt stress, the stem growth, gas exchange, chlorophyll fluorescence, activity and transcription of antioxidant enzymes, Na+ content, and Na+ flux of root xylem and petiole vascular bundle were investigated in wild-type and transgenic poplars. The correlation analysis and principal component analysis (PCA) were used to analyze the correlations among the characteristics and principal components. Our results show that the transcription of PtEXPA6 was downregulated upon a prolonged duration of salt stress (48 h) after a transient increase induced by NaCl (100 mM). The PtEXPA6-transgenic poplars of 84K and 717-1B4 showed a greater reduction (42-65%) in stem height and diameter growth after 15 days of NaCl treatment compared with wild-type (WT) poplars (11-41%). The Na+ accumulation in roots, stems, and leaves was 14-83% higher in the transgenic lines than in the WT. The Na+ buildup in the transgenic poplars affects photosynthesis; the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); and the transcription of PODa2, SOD [Cu-Zn], and CAT1. Transient flux kinetics showed that the Na+ efflux of root xylem and leaf petiole vascular bundle were 1.9-3.5-fold greater in the PtEXPA6-transgenic poplars than in the WT poplars. PtEXPA6 overexpression increased root contractility and extensibility by 33% and 32%, indicating that PtEXPA6 increased the CW loosening in the transgenic poplars of 84K and 717-1B4. Noteworthily, the PtEXPA6-promoted CW loosening was shown to facilitate Na+ efflux of root xylem and petiole vascular bundle in the transgenic poplars. We conclude that the overexpression of PtEXPA6 leads to CW loosening that facilitates the radial translocation of Na+ into the root xylem and the subsequent Na+ translocation from roots to leaves, resulting in an excessive Na+ accumulation and consequently, reducing salt tolerance in transgenic poplars. Therefore, the downregulation of PtEXPA6 in NaCl-treated Populus trichocarpa favors the maintenance of ionic and reactive oxygen species (ROS) homeostasis under long-term salt stress.

Selective Oxidation of Methanol to Methyl Formate on Gold: The Role of Low-Coordinated Sites Revealed by Isothermal Pulsed Molecular Beam Experiments and AIMD Simulations.

To elucidate the role of low-coordinated sites in the partial methanol oxidation to methyl formate (MeFo), the isothermal reactivity of flat Au(111) and stepped Au(332) in pulsed molecular beam experiments was compared for a broad range of reaction conditions. Low-coordinated step sites were found to enhance MeFo selectivity, especially at low coverage conditions, as found at higher temperatures. The analysis of the transient kinetics provides evidence for the essential role of Au x O y phases for MeFo formation and the complex interplay of different oxygen species for the observed selectivity. Ab initio molecular dynamic simulations yielded microscopic insights in the formation of Au x O y phases on flat and stepped gold surfaces emphasizing the role of low-coordinated sites in their formation. Moreover, associated surface restructuring provides atomic-scale insights which align with the experimentally observed transient kinetics in MeFo formation.

NTPs compete in the active site of RNA polymerases I and II

Eukaryotes express at least three RNA polymerases (Pols) carry out transcription, while bacteria and archaea use only one. Using transient state kinetics, we have extensively examined and compared the kinetics of both single and multi-nucleotide additions catalyzed by the three Pols. In single nucleotide addition experiments we have observed unexpected extension products beyond one incorporation, which can be attributed to misincorporation, the presence of nearly undetectable amounts of contaminating NTPs, or a mixture of the two. Here we report the development and validation of an analysis strategy to account for the presence of unexpected extension products, when they occur. Using this approach, we uncovered evidence showing that non-cognate nucleotide, thermodynamically, competes with cognate nucleotide for the active site within the elongation complex of Pol I, ΔA12 Pol I, and Pol II. This observation is unexpected because base pairing interactions provide favorable energetics for selectivity and competitive binding indicates that the affinities of cognate and non-cognate nucleotides are within an order of magnitude. Thus, we show that application of our approach will allow for the extraction of additional information that reports on the energetics of nucleotide entry and selectivity.

Effect of Viscous Media on the Quantum Yield of Bioluminescence in a Reaction Catalyzed by Bacterial Luciferase

Based on previously obtained data on the transient kinetics of the bioluminescent reaction catalyzed by P. leiognathi luciferase in media with polyols and sugars, the relative quantum yield of bioluminescence per substrate molecule was determined using mathematical modeling. It was found that in some media the relative quantum yield per aldehyde molecule increases compared to the value in the buffer: by 18% in the presence of glycerol and by 33% in the presence of sucrose. The conformation of the side chain of αHis44, which is the functionally important and conservative in all bacterial luciferases, was analyzed using molecular dynamics methods. It has been established that in the presence of all cosolvents there is an increase in the probability of formation the optimal for catalysis orientation of this amino acid, which may contribute to the observed increase in the quantum yield of bioluminescence in viscous media.

Phosphorylation of cardiac sodium channel at Ser571 anticipates manifestations of the aging myopathy.

Diastolic dysfunction and delayed ventricular repolarization are typically observed in the elderly, but whether these defects are intimately associated with the progressive manifestation of the aging myopathy remains to be determined. In this regard, aging in experimental animals is coupled with increased late Na+ current (INa,L) in cardiomyocytes, raising the possibility that INa,L conditions the modality of electrical recovery and myocardial relaxation of the aged heart. For this purpose, aging male and female wild-type (WT) C57Bl/6 mice were studied together with genetically engineered mice with phosphomimetic (gain of function, GoF) or ablated (loss of function, LoF) mutations of the sodium channel Nav1.5 at Ser571 associated with, respectively, increased and stabilized INa,L. At ∼18 mo of age, WT mice developed prolonged duration of the QT interval of the electrocardiogram and impaired diastolic left ventricular (LV) filling, defects that were reversed by INa,L inhibition. Prolonged repolarization and impaired LV filling occurred prematurely in adult (∼5 mo) GoF mutant mice, whereas these alterations were largely attenuated in aging LoF mutant animals. Ca2+ transient decay and kinetics of myocyte shortening/relengthening were delayed in aged (∼24 mo) WT myocytes, with respect to adult cells. In contrast, delayed Ca2+ transients and contractile dynamics occurred at adult stage in GoF myocytes and further deteriorated in old age. Conversely, myocyte mechanics were minimally affected in aging LoF cells. Collectively, these results document that Nav1.5 phosphorylation at Ser571 and the late Na+ current modulate the modality of myocyte relaxation, constituting the mechanism linking delayed ventricular repolarization and diastolic dysfunction.NEW & NOTEWORTHY We have investigated the impact of the late Na current (INa,L) on cardiac and myocyte function with aging by using genetically engineered animals with enhanced or stabilized INa,L, due to phosphomimetic or phosphoablated mutations of Nav1.5. Our findings support the notion that phosphorylation of Nav1.5 at Ser571 prolongs myocardial repolarization and impairs diastolic function, contributing to the manifestations of the aging myopathy.

Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2

ASCT2 (alanine serine cysteine transporter 2), a member of the SLC1 (solute carrier 1) family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anti-cancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor Lc-BPE with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that, despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The pre-incubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

Modulating hot carrier cooling and extraction with A-site organic cations in perovskites.

Hot carrier solar cells could offer a solution to achieve high efficiency solar cells. Due to the hot-phonon bottleneck in perovskites, the hot carrier lifetime could reach hundreds of ps. Such that exploring perovskites could be a good way to promote hot carrier technology. With the incorporation of large organic cations, the hot carrier lifetime can be improved. By using ultrafast transient spectroscopy, the hot carrier relaxation and extraction kinetics are measured. From the transient kinetics, 2-phenyl-acetamidine cation based perovskites exhibit the highest initial carrier temperature, longest carrier relaxation, and slowest hot carrier relaxation. Such superior behavior could be attributed to reduced electron-phonon coupling induced by lattice strain, which is a result of the large organic cation and also a possible surface electronic state change. Our discovery exhibits the potential to use large organic cations for the use of hot carrier perovskite solar cells.

Silica-based scintillators: basic properties of radioluminescence kinetics.

Radioluminescent silica-based fiber dosimeters offer great advantages for designing miniaturized realtime sensors for high dose-rate dosimetry. Rise and fall kinetics of their response must be properly understood to better assess their performances in terms of measurement speed and repeatability. A standard model of radioluminescence (RL) has already been quantitatively validated for doped silica glasses, but beyond conclusive comparisons with specific experiments, a comprehensive understanding of the processes and parameters determining transient and equilibrium kinetics of RL is still lacking. We analyze in detail the kinetics inherent in the standard RL model. Several asymptotical regimes in the RL growth are demonstrated in the case of a pristine sample (succesive quadratic, linear and power-law time dependencies before the plateau is reached). We show how this situation is modified when a pre-irradiation partly fills traps beforehand. RL growth is then greatly accelerated because of the pre-formation of recombination centers (RCs) from dopant ions, but not due to pre-filling of trapping levels. In all cases, the RL intensity eventually tends to a constant level equal to the pair generation rate, long before all carrier densities themselves reach equilibrium. This occurs late under irradiation, when deep traps get to saturation. The fraction of dopants converted into RCs is then 'frozen' at a lower level the smaller the density of deep traps. Controlling RL kinetics through the engineering of material traps is not an option. Pre-irradiation appears to be the simplest way to obtain accelerated and repeatable kinetics.

A framework of computer vision-enhanced microfluidic approach for automated assessment of the transient sickling kinetics in sickle red blood cells.

The occurrence of vaso-occlusive crisis greatly depends on the competition between the sickling delay time and the transit time of individual sickle cells, i.e., red blood cells (RBCs) from sickle cell disease (SCD) patients, while they are traversing the circulatory system. Many drugs for treating SCD work by inhibiting the polymerization of sickle hemoglobin (HbS), effectively delaying the sickling process in sickle cells (SS RBCs). Most previous studies on screening anti-sickling drugs, such as voxelotor, rely on in vitro testing of sickling characteristics, often conducted under prolonged deoxygenation for up to 1 hour. However, since the microcirculation of RBCs typically takes less than 1 minute, the results of these studies may be less accurate and less relevant for in vitro-in vivo correlation. In our current study, we introduce a computer vision-enhanced microfluidic framework designed to automatically capture the transient sickling kinetics of SS RBCs within a 1-min timeframe. Our study has successfully detected differences in the transient sickling kinetics between vehicle control and voxelotor-treated SS RBCs. This approach has the potential for broader applications in screening anti-sickling therapies.