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Enzyme activity is often regulated by conformational changes coupled to binding of an effector at an allosteric site, a feature especially important for enzymes involved in signaling cascades. Hadzipasic et al. studied the origins of allosteric regulation of Aurora A, a kinase involved in progression of the eukaryotic cell cycle. Aurora A is allosterically regulated through the binding of an effector protein named TPX2, which also targets the kinase to spindle microtubules. By reconstructing ancestor kinase sequences, they found that TPX2 bound to an early Aurora A but had very weak activation that was gradually strengthened by evolution of an allosteric network within the kinase. An evolutionary advantage from localizing the active protein at the mitotic spindle may have driven the development of this regulatory mechanism. Science , this issue p. 912 Ancestor reconstruction reveals a path in the evolution of an allosteric regulatory mechanism in Aurora kinase. A myriad of cellular events are regulated by allostery; therefore, evolution of this process is of fundamental interest. Here, we use ancestral sequence reconstruction to resurrect ancestors of two colocalizing proteins, Aurora A kinase and its allosteric activator TPX2 (targeting protein for Xklp2), to experimentally characterize the evolutionary path of allosteric activation. Autophosphorylation of the activation loop is the most ancient activation mechanism; it is fully developed in the oldest kinase ancestor and has remained stable over 1 billion years of evolution. As the microtubule-associated protein TPX2 appeared, efficient kinase binding to TPX2 evolved, likely owing to increased fitness by virtue of colocalization. Subsequently, TPX2-mediated allosteric kinase regulation gradually evolved. Surprisingly, evolution of this regulation is encoded in the kinase and did not arise by a dominating mechanism of coevolution.

Hepatocellular carcinoma frequently recurs after surgery, necessitating personalized clinical approaches based on tumor avatar models. However, location-dependent oxygen concentrations resulting from the dual hepatic vascular supply drive the inherent heterogeneity of the tumor microenvironment, which presents challenges in developing an avatar model. In this study, tissue samples from 12 patients with hepatocellular carcinoma are cultured directly on a chip and separated based on preference of oxygen concentration. Establishing a dual gradient system with drug perfusion perpendicular to the oxygen gradient enables the simultaneous separation of cells and evaluation of drug responsiveness. The results are further cross-validated by implanting the chips into mice at various oxygen levels using a patient-derived xenograft model. Hepatocellular carcinoma cells exposed to hypoxia exhibit invasive and recurrent characteristics that mirror clinical outcomes. This chip provides valuable insights into treatment prognosis by identifying the dominant hepatocellular carcinoma type in each patient, potentially guiding personalized therapeutic interventions. Hepatocellular carcinoma is the most common type of primary liver cancer. Here the authors show an oxygen gradient chip that separates aggressive hepatocellular carcinoma cells from a heterogeneous tumor mass, mirroring the conditions of the portal vein, hepatic artery, and liver.

Pesticides and veterinary pharmaceuticals are used for effective crop production and prevention of livestock diseases; these chemicals are released into the environment via various pathways. Although the chemicals are typically present in trace amounts post-release, they could disturb aquatic ecosystems and public health through resistance development toward drugs or diseases, e.g., reproductive disorders. Thus, the residues of pesticides and veterinary pharmaceuticals in the environment must be managed and monitored. To that end, we developed a simultaneous analysis method for 41 target chemicals in environmental water samples using ultra-high-performance liquid chromatography (UHPLC)–quadrupole-orbitrap high-resolution mass spectrometry (HRMS) coupled with an on-line solid-phase extraction system. Calibration curves for determining linearity were constructed for 10–750 ng∙L−1, and the coefficient of determination for each chemical exceeded 0.99. The method’s detection and quantitation limits were 0.32–1.72 ng∙L−1 and 1.02–5.47 ng∙L−1, respectively. The on-line solid-phase extraction system exhibited excellent method reproducibility and reduced experimental error. As the proposed method is applicable to the monitoring of pesticides and veterinary pharmaceuticals in surface water and groundwater samples acquired near agricultural areas, it allows for the management of chemicals released into the environment.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

Enzymes govern biological processes as they are biocatalysts that increase the rate of chemical reactions within cells, which would not spontaneously occur. Kinases catalyze the transfer of phosphate groups from high-energy molecules to their substrates. Since these processes are crucial for many biological signaling processes, dysregulation of kinases is correlated with many human diseases. Thus, inhibition of kinases has been investigated as a means of disease treatment for many years, but mainly via orthosteric sites. However, orthosteric inhibition of kinases often presented non-specificity due to the conserved active site architecture of kinases. Furthermore, long-term clinical use of orthosteric inhibitors resulted in emergence of resistance mutations, incapacitating the treatments. Hence, allosteric inhibition has recently been highlighted as an alternative but effective way treating diseases. Recent break-through research, which combined the use of both orthosteric and allosteric drugs inhibiting a single target (double-drugging), has presented a means of effectively overcoming the resistance mutations in clinics. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric inhibitors is less understood. In this work, we quantitatively investigated the modulation of kinases, Aurora A kinase and Abelson kinase, with rational selection of the combination between orthosteric and allosteric modulators. We find that conformational equilibria shift is the main principle for the cooperativities during double-drugging. We quantify the combined inhibition effect double-drugging provides, such as how much the required concentration for orthosteric inhibition decreases in the presence of the allosteric inhibitor. Furthermore, our structural characterization of these double-drugged complexes provides insights into the atomistic mechanism underlying double-drugging’s cooperativities. Our results present a framework for future design and evaluation of double-drugging strategies. Not only rationally designing drugs, but designing new enzymes has also garnered much interest in the past decades. Current approaches in enzyme design commonly involve multiple rounds of directed evolution mimicking the natural evolution. During this process, we expected that a more evolvable starting-point would provide wider genomic possibilities traveling the sequence (fitness) landscape, potentially achieving better end-points. However, biophysical properties affecting the evolution have not been fully elucidated despite decades of investigation by evolutionary biochemists. Guided by scientific intuition, we hypothesized that ancestral proteins are more evolvable than modern proteins due to their less exposure to evolutionary pressures. Indeed, our preliminary data suggest that ancestral adenylate kinases may be more evolvable than their extant forms, indicated by much diverse ancestral population after selection. Collectively, we employed various methods, including coupled-enzyme assays, discontinuous assays, isothermal titration calorimetry, X-ray crystallography, Förster resonance energy transfer, and molecular biology techniques, to better understand how kinases work. We believe our data provide valuable knowledge into design and evaluation of future double-drugging candidates as well as new enzymes.

A myriad of cellular events are regulated by allostery; therefore, evolution of this process is of fundamental interest. Here, we use ancestral sequence reconstruction to resurrect ancestors of two colocalizing proteins, Aurora A kinase and its allosteric activator TPX2 (targeting protein for Xklp2), to experimentally characterize the evolutionary path of allosteric activation. Autophosphorylation of the activation loop is the most ancient activation mechanism; it is fully developed in the oldest kinase ancestor and has remained stable over 1 billion years of evolution. As the microtubule-associated protein TPX2 appeared, efficient kinase binding to TPX2 evolved, likely owing to increased fitness by virtue of colocalization. Subsequently, TPX2-mediated allosteric kinase regulation gradually evolved. Surprisingly, evolution of this regulation is encoded in the kinase and did not arise by a dominating mechanism of coevolution.

A myriad of cellular events are regulated by allostery; therefore, evolution of this process is of fundamental interest. Here, we use ancestral sequence reconstruction to resurrect ancestors of two colocalizing proteins, Aurora A kinase and its allosteric activator TPX2 (targeting protein for Xklp2), to experimentally characterize the evolutionary path of allosteric activation. Autophosphorylation of the activation loop is the most ancient activation mechanism; it is fully developed in the oldest kinase ancestor and has remained stable over 1 billion years of evolution. As the microtubule-associated protein TPX2 appeared, efficient kinase binding to TPX2 evolved, likely owing to increased fitness by virtue of colocalization. Subsequently, TPX2-mediated allosteric kinase regulation gradually evolved. Surprisingly, evolution of this regulation is encoded in the kinase and did not arise by a dominating mechanism of coevolution.

Antibiotics used for the treatment of humans and livestock are released into the environment, whereby they pose a grave threat to biota (including humans) as they can cause the emergence of various strains of resistant bacteria. An improved understanding of antibiotics in the environment is thus vital for appropriate management and mitigation. Herein, surface water and groundwater samples containing antibiotics were analyzed in an urban–rural complex watershed (Cheongmi Stream) comprising intensive livestock farms by collecting samples across different time points and locations. The spatiotemporal trends of the residual antibiotics were analyzed, and ecological and antibiotic resistance-based risk assessments were performed considering their concentrations. The results showed that the concentrations and detection frequencies of the residual antibiotics in the surface water were affected by various factors such as agricultural activities and point sources, and were higher than those found in groundwater; however, frequent detection of antibiotics in groundwater showed that residual antibiotics were influenced by factors such as usage pattern and sewage runoff. Furthermore, few antibiotics posed ecological risks. The risk assessment methods adopted in this study can be applied elsewhere, and the results can be considered in the environmental management of residual antibiotics in the Cheongmi Stream watershed.

Hepatocellular carcinoma frequently recurs after surgery, necessitating personalized clinical approaches based on tumor avatar models. However, location-dependent oxygen concentrations resulting from the dual hepatic vascular supply drive the inherent heterogeneity of the tumor microenvironment, which presents challenges in developing an avatar model. In this study, tissue samples from 12 patients with hepatocellular carcinoma are cultured directly on a chip and separated based on preference of oxygen concentration. Establishing a dual gradient system with drug perfusion perpendicular to the oxygen gradient enables the simultaneous separation of cells and evaluation of drug responsiveness. The results are further cross-validated by implanting the chips into mice at various oxygen levels using a patient-derived xenograft model. Hepatocellular carcinoma cells exposed to hypoxia exhibit invasive and recurrent characteristics that mirror clinical outcomes. This chip provides valuable insights into treatment prognosis by identifying the dominant hepatocellular carcinoma type in each patient, potentially guiding personalized therapeutic interventions.

Previous general aviation (GA) accident studies showed that decision errors were more associated with fatal GA accidents than other kinds of human errors, and weather related accidents, especially continued visual flight rules (VFR) flight into instrument meteorological conditions (IMC), remained the major cause of fatal GA accidents. Thus, finding the underlying causes of GA pilots' decision errors and continued VFR flight into adverse weather conditions are needed to reduce weather related GA accidents as well as fatal GA accidents. Causal factors and hypotheses of weather related GA accidents show that knowledge, experience, motivation, and weather information frequently have been referred as causal factors of weather-related GA accidents. Among causal hypotheses, situation assessment and risk assessment hypotheses have been cited frequently as the causes of weather related GA accidents. The purpose of this study is to evaluate the effects of weather recognition training on GA pilots' situation assessment and tactical decision making under gradually aggravating weather conditions. To meet this purpose, WeatherWise and an X-Plane 9 flight simulation program has been used. WeatherWise is a computer based weather training program developed by Wiggins et al. (2000) to improve GA pilot weather-related decision making, and was approved by the Federal Aviation Administration (FAA) for free public use. Pilot situation assessment is a pilot's understanding of a current flight state, and was evaluated in terms of weather assessment and risk assessment. Weather assessment is the pilot's ability to recognize or estimate the changes in visibility, ceiling, and weather condition. Risk assessment is the understanding of the risks associated with flying in adverse weather conditions, and was measured in terms of risk perception and risk tolerance using the Hazardous Event Scale, personal weather minimums, and the Aviation Safety Attitude Scale. Pilot situation assessment was measured by a post experiment questionnaire. Pilot tactical decision making is in flight judgment, and was evaluated in terms of decision accuracy and decision confidence. Decision accuracy was evaluated by measuring the distance that a pilot has flown from an optimal divert point to an actual divert point, and the distance a pilot has flown into adverse weather conditions. Decision confidence is the pilot's confidence level in making diverting decisions when the pilot encounters adverse weather, and was measured by subjective rating method. Findings of the study indicated that the WeatherWise training group exhibited significantly higher weather assessment as measured by ceiling estimation ability and decision accuracy as measured by flown distance into adverse weather condition than the control group, but no significant differences were found in their risk assessment and decision confidence. Although the effects of weather training on the risk assessment were not significantly different between the two groups, participants in the WeatherWise training group was more conservative toward flying into adverse weather condition than the control group. It was hypothesized to find a positive relationship between pilots' situation assessments and their tactical decision-making because situation assessment forms a basis for decision making; however, positive relationship was found only between pilots' ceiling estimation and flown distances into adverse weather in this study. Thus, it can be concluded that the weather training was effective at least in part to pilot situation assessment and tactical decision making. In addition, considering the weather training was just one-time 30 minute training, long-term effects of weather training should be conducted to find further relationship between pilot situation assessment and tactical decision making. The results of this study can be expanded not only to GA pilots but also to commercial airline pilots and military pilots for various reasons. First, all pilots are expected to acquire weather recognition skills and knowledge to ensure a safe flight regardless of their flight types because the nature of weather condition changes is dynamic and hard to predict during the flight. Second, although those aircrafts are well equipped with navigation aid systems and weather display radar, they do not provide real–time weather information, and they sometimes malfunction. In conclusion, it is expected that this study will be helpful for GA pilots to understand the effects of weather recognition training on weather decision making, and eventually help them assess a situation correctly and make a timely in-flight decision. It is believed that this study will help to establish a sound foundation for weather training program and has the potential to reduce weather-related GA accidents by implementing weather training during flight training.