DNA binding reveals hidden interdomain allostery of a MazE antitoxin from Mycobacterium tuberculosis.

Type II toxin-antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA-DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.

Membrane-Driven Dimerization of the Peripheral Membrane Protein KRAS: Implications for Downstream Signaling.

Transient homo-dimerization of the RAS GTPase at the plasma membrane has been shown to promote the mitogen-activated protein kinase (MAPK) signaling pathway essential for cell proliferation and oncogenesis. To date, numerous crystallographic studies have focused on the well-defined GTPase domains of RAS isoforms, which lack the disordered C-terminal membrane anchor, thus providing limited structural insight into membrane-bound RAS molecules. Recently, lipid-bilayer nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses have revealed several distinct structures of the membrane-anchored homodimers of KRAS, an isoform that is most frequently mutated in human cancers. The KRAS dimerization interface is highly plastic and altered by biologically relevant conditions, including oncogenic mutations, the nucleotide states of the protein, and the lipid composition. Notably, PRE-derived structures of KRAS homodimers on the membrane substantially differ in terms of the relative orientation of the protomers at an "α-α" dimer interface comprising two α4-α5 regions. This interface plasticity along with the altered orientations of KRAS on the membrane impact the accessibility of KRAS to downstream effectors and regulatory proteins. Further, nanodisc platforms used to drive KRAS dimerization can be used to screen potential anticancer drugs that target membrane-bound RAS dimers and probe their structural mechanism of action.

Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling.

Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/ß' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/ß interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.

Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3.

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.

Cdk5 regulates IP3R1-mediated Ca2+ dynamics and Ca2+-mediated cell proliferation.

Loss of cyclin-dependent kinase 5 (Cdk5) in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) increases ER-mitochondria tethering and ER Ca2+ transfer to the mitochondria, subsequently increasing mitochondrial Ca2+ concentration ([Ca2+]mt). This suggests a role for Cdk5 in regulating intracellular Ca2+ dynamics, but how Cdk5 is involved in this process remains to be explored. Using ex vivo primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5-/- mouse embryos, we show here that loss of Cdk5 causes an increase in cytosolic Ca2+concentration ([Ca2+]cyt), which is not due to reduced internal Ca2+ store capacity or increased Ca2+ influx from the extracellular milieu. Instead, by stimulation with ATP that mediates release of Ca2+ from internal stores, we determined that the rise in [Ca2+]cyt in Cdk5-/- MEFs is due to increased inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from internal stores. Cdk5 interacts with the IP3R1 Ca2+ channel and phosphorylates it at Ser421. Such phosphorylation controls IP3R1-mediated Ca2+ release as loss of Cdk5, and thus, loss of IP3R1 Ser421 phosphorylation triggers an increase in IP3R1-mediated Ca2+ release in Cdk5-/- MEFs, resulting in elevated [Ca2+]cyt. Elevated [Ca2+]cyt in these cells further induces the production of reactive oxygen species (ROS), which upregulates the levels of Nrf2 and its targets, Prx1 and Prx2. Cdk5-/- MEFs, which have elevated [Ca2+]cyt, proliferate at a faster rate compared to wt, and Cdk5-/- embryos have increased body weight and size compared to their wt littermates. Taken together, we show that altered IP3R1-mediated Ca2+ dynamics due to Cdk5 loss correspond to accelerated cell proliferation that correlates with increased body weight and size in Cdk5-/- embryos.

Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane.

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.

The Self-Association of the KRAS4b Protein is Altered by Lipid-Bilayer Composition and Electrostatics.

KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self-association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self-assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self-association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5-α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge-reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane.

Synthetic Topological Nodal Phase in Bilayer Resonant Gratings.

The notion of synthetic dimensions in artificial photonic systems has received considerable attention as it provides novel methods for exploring hypothetical topological phenomena as well as potential device applications. Here, we present nanophotonic manifestation of a two-dimensional topological nodal phase in bilayer resonant grating structures. Using the mathematical analogy between a topological semimetal and vertically asymmetric photonic lattices, we show that the interlayer shift simulates an extra momentum dimension for creating a two-dimensional topological nodal phase. We present a theoretical model and rigorous numerical analyses showing the two nodal points that produce a complex gapless band structure and localized edge states in the topologically nontrivial region. Therefore, our results provide a practical scheme for producing high-dimensional topological effects in simple low-dimensional photonic structures.

Analysis of measurement changes in pelvic incidence according to pelvic rotation using a three-dimensional model.

BACKGROUND: Pelvic incidence (PI) is used as a key parameter in surgical correction of adult spinal deformity (ASD). However, reflecting the exact center or inclination of the three-dimensional anatomical structures on the two-dimensional (2D) sagittal radiographs is limited, resulting in measurement errors. Therefore, we evaluated whether there is a change in PI measurement according to the actual rotation of the pelvis, and conducted a study on a more accurate method for PI measurement using 2D sagittal radiographs. METHODS: From 2014 to 2015, the data of 30 patients who visited our outpatient clinic were analyzed retrospectively. CT scans including those of the lower lumbar spine, pelvis, and both femurs in the DICOM format were imported to Mimics Research 17.0 (Materialise NV, Belgium), SolidWorks (Dassault systems, France), and AutoCAD 2014 (AUTODESK, US). The changes in PI according to vertical and horizontal pelvic rotations were evaluated. RESULTS: The average PIs according to the horizontal pelvic rotations measured on AutoCAD with 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, and 40° were 48.8°, 48.7°, 48.3°, 47.8°, 46.9°, 45.6°, 44.0°, 42.2°, and 39.9°, respectively. The PI with an acceptable error of 6° on radiographs was 35° in the horizontal pelvic rotation. The average PIs according to the vertical pelvic rotations measured on AutoCAD with 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, and 40° were 48.8°, 49.0°, 49.5°, 50.2°, 51.3°, 52.7°, 54.4°, 56.6°, and 59.4°, respectively. The PI with an acceptable error of 6° on radiographs was 30° in the vertical pelvic rotation. CONCLUSIONS: This study revealed that the PI value could differ from the actual anatomical value due to the horizontal and vertical rotation of the pelvis while acquiring the radiograph. Regarding whole-spine lateral radiographs, errors in PI measurement may occur due to pelvic rotation or nonvertical projection of X-rays. In the standing pelvic lateral radiographs, ensuring superposition of the femoral heads at the center and obtaining the straight sacral endplate by referring to CT or magnetic resonance imaging would be a more accurate measurement method to define PI.

HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway.

l-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3 Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase-induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

Crystal structure of proteolyzed VapBC and DNA-bound VapBC from Salmonella enterica Typhimurium LT2 and VapC as a putative Ca2+ -dependent ribonuclease.

Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency and antibiotic treatment. There are at least 14 TA systems in Salmonella enterica serovar Typhimurium LT2, a pathogenic bacterium, and none of the structures of these TA systems have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in proteolyzed and DNA-bound forms at 2.0 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a pilT N-terminal domain (PIN-domain) that shows ribonuclease activity, and the VapB antitoxin has an AbrB-type DNA binding domain. In addition, the structure revealed details of interaction mode between VapBC and the cognate promoter DNA, including the inhibition of VapC by VapB and linear conformation of bound DNA in the VapBC complex. The complexation of VapBC with the linear DNA is not consistent with known structures of VapBC homologs in complex with bent DNA. We also identified VapC from S. Typhimurium LT2 as a putative Ca2+ -dependent ribonuclease, which differs from previous data showing that VapC homologs have Mg2+ or Mn2+ -dependent ribonuclease activities. The present studies could provide structural understanding of the physiology of VapBC systems and foundation for the development of new antibiotic drugs against Salmonella infection.

Phase-coherent transport in trigonal gallium nitride nanowires.

Gallium nitride nanowires (GaN NWs) with triangular cross-section exhibit universal conductance fluctuations (UCF) originating from the quantum interference of electron wave functions in the NWs. The amplitude of UCF is inversely proportional to the applied bias current. The bias dependence of UCF, combined with temperature dependence of the resistance suggests that phase coherent transport dominates over normal transport in GaN NWs. A unique temperature dependence of phase-coherent length and fluctuation amplitude is associated with inelastic electron-electron scattering in NWs. The phase-coherence length extracted from the UCF is as large as 400 nm at 1.8 K, and gradually decreases as temperature increases up to 60 K.

Impact of BMI on Complications of Gastric Endoscopic Submucosal Dissection.

BACKGROUND: Gastric endoscopic submucosal dissection (ESD) has a high rate of complications. However, it is unclear whether BMI affects ESD complications. We aimed to investigate the impact of BMI on ESD complications. METHODS: A total of 7,263 patients who underwent gastric ESD were classified into 3 groups according to the Asia-Pacific classification of BMI: normal (BMI <23 kg/m2, n = 2,466), overweight (BMI 23-24.9 kg/m2, n = 2,117), and obese (BMI ≥25 kg/m2, n = 2,680). Adjusted logistic regression analyses were conducted to assess the association between BMI and ESD complications. RESULTS: Compared to the normal group, a lower incidence of perforation and a higher incidence of pneumonia and leukocytosis were found in the overweight and obese groups, and intra-ESD desaturation and hypertension were more frequent in the obese group. After adjustment for confounders, the risk of perforation significantly decreased in the overweight (odds ratio [OR] = 0.24, 95% confidence interval [CI]: 0.17-0.33) and obese (OR = 0.12, 95% CI: 0.08-0.18) groups compared to that in the normal group. Meanwhile, the risk of pneumonia significantly increased in the overweight (OR = 11.04, 95% CI: 6.31-19.31) and obese (OR = 10.71, 95% CI: 6.14-18.66) groups compared to the normal group. During sedation, the obese group had a significantly increased risk of desaturation (OR = 2.81, 95% CI: 1.18-6.69) and hypertension (OR = 1.35, 95% CI: 1.11-1.63) compared to the normal group. CONCLUSIONS: High BMI was significantly associated with ESD complications. More caution is needed in cases of obese patients undergoing ESD.

Comparison of high flow nasal oxygen and conventional nasal cannula during gastrointestinal endoscopic sedation in the prone position: a randomized trial.

PURPOSE: Deep sedation for endoscopic retrograde cholangiopancreatography (ERCP) can be challenging in elderly patients in the prone position. This study investigated the effect of a high flow nasal oxygen (HFNO) delivery system on oxygenation in this procedure compared with that of conventional nasal cannula oxygen administration. METHODS: A prospective randomized trial was conducted using HFNO and conventional nasal cannula in patients undergoing ERCP in the prone position. For each patient, the lowest oxygen saturation (SpO2), the incidence of hypoxemia defined as an SpO2 below 90%, and interruptions due to airway interventions were recorded during the procedure. RESULTS: The lowest mean (standard deviation) SpO2 recorded during the procedure was higher in the HFNO group than in the conventional control group [99.8 (0.6)% vs 95.1 (7.3)%; mean difference, 4.7%; 95% confidence interval, 2.3% to 7.1%; P Group x Time < 0.001]. While the lowest SpO2 during the procedure was lower than the baseline SpO2 in the control group, the lowest SpO2 during the procedure was higher than the baseline SpO2 in the HFNO group. Hypoxemia occurred only in the control group (n = 7; 19%; P = 0.01). Procedural interruptions, including discontinuation of sedation, patient stimulation, and jaw thrusting, occurred only in the control group (n = 9 [25%], n = 10 [28%], and n = 10 [28%] cases, respectively; P = 0.001 for each). CONCLUSION: In contrast to conventional nasal cannula, high flow nasal oxygen provided adequate oxygenation without causing procedural interruptions during ERCP, suggesting that HFNO may be used as a standard oxygen delivery method during these procedures. TRIAL REGISTRATION: www.ClinicalTrials.gov (NCT03872674); registered 11 March 2019.

RéSUMé: OBJECTIF: La sédation profonde pour cholangiopancréatographie rétrograde endoscopique (CPRE) peut être difficile à réaliser chez des patients âgés en position ventrale. Cette étude a exploré l'effet d'un système d'oxygénothérapie nasale à haut débit (ONHD) sur l'oxygénation pendant cette intervention par rapport à l'administration conventionnelle d'oxygène via une lunette nasale. MéTHODE: Une étude randomisée prospective a été réalisée en utilisant une ONHD ou une lunette nasale conventionnelle chez des patients subissant une CPRE en position ventrale. Pour chaque patient, la saturation en oxygène (SpO2) la plus basse, l'incidence d'hypoxémie définie en tant qu'une SpO2 inférieure à 90 %, et les interruptions provoquées par des interventions au niveau des voies aériennes ont été enregistrées au cours de l'intervention. RéSULTATS: La SpO2 moyenne (écart type) la plus basse enregistrée pendant l'intervention était plus élevée dans le groupe ONHD que dans le groupe témoin conventionnel [99,8 (0,6) % vs 95,1 (7,3) %; différence moyenne, 4,7%; intervalle de confiance 95 %, 2,3 % à 7,1 %; P Groupe x Temps < 0,001]. Alors que la SpO2 la plus basse pendant l'intervention était plus basse que la SpO2 de base dans le groupe témoin, la SpO2 la plus basse pendant l'intervention était plus élevée que la SpO2 de base dans le groupe ONHD. L'hypoxémie n'est survenue que dans le groupe témoin (n = 7; 19 %; P = 0,01). Il n'y a eu d'interruptions de l'intervention, y compris la cessation de la sédation, la stimulation du patient et le déplacement de la mâchoire inférieure vers l'avant, que dans le groupe témoin (n = 9 [25 %], n = 10 [28 %], et n = 10 [28 %] cas, respectivement; P = 0,001 pour chaque intervention). CONCLUSION: Comparativement à une lunette nasale conventionnelle, l'oxygénothérapie nasale à haut débit a procuré une oxygénation adéquate sans provoquer d'interruptions de l'intervention pendant une CPRE, suggérant que cette modalité pourrait être utilisée comme méthode standard d'oxygénothérapie pendant de telles interventions. ENREGISTREMENT DE L'éTUDE: www.ClinicalTrials.gov (NCT03872674); enregistrée le 11 mars 2019.

Antimicrobial Activity and Action Mechanisms of Arg-Rich Short Analog Peptides Designed from the C-Terminal Loop Region of American Oyster Defensin (AOD).

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures-including nine amino acids and a disulfide bond derived from the C-terminus of AOD-and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms-including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans-without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Fast Magneto-Ionic Switching of Interface Anisotropy Using Yttria-Stabilized Zirconia Gate Oxide.

Voltage control of interfacial magnetism has been greatly highlighted in spintronics research for many years, as it might enable ultralow power technologies. Among a few suggested approaches, magneto-ionic control of magnetism has demonstrated large modulation of magnetic anisotropy. Moreover, the recent demonstration of magneto-ionic devices using hydrogen ions presented relatively fast magnetization toggle switching, tsw ∼ 100 ms, at room temperature. However, the operation speed may need to be significantly improved to be used for modern electronic devices. Here, we demonstrate that the speed of proton-induced magnetization toggle switching largely depends on proton-conducting oxides. We achieve ∼1 ms reliable (>103 cycles) switching using yttria-stabilized zirconia (YSZ), which is ∼100 times faster than the state-of-the-art magneto-ionic devices reported to date at room temperature. Our results suggest that further engineering of the proton-conducting materials could bring substantial improvement that may enable new low-power computing scheme based on magneto-ionics.

NMR in integrated biophysical drug discovery for RAS: past, present, and future.

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.

Crystal structure of the YoeBSa1-YefMSa1 complex from Staphylococcus aureus.

Toxin-antitoxin (TA) systems are ubiquitously found in bacteria and are related to cell maintenance and survival under environmental stresses such as heat shock, nutrient starvation, and antibiotic treatment. Here, we report for the first time the crystal structure of the Staphylococcus aureus TA complex YoeBSa1-YefMSa1 at a resolution of 1.7 Å. This structure reveals a heterotetramer with a 2:2 stoichiometry between YoeBSa1 and YefMSa1. The N-terminal regions of the YefMSa1 antitoxin form a homodimer characteristic of a hydrophobic core, and the C-terminal extended region of each YefMSa1 protomer makes contact with each YoeBSa1 monomer. The binding stoichiometry of YoeBSa1 and YefMSa1 is different from that of YoeB and YefM of E. coli (YoeBEc and YefMEc), which is the only structural homologue among YoeB-YefM families; however, the structures of individual YoeBSa1 and YefMSa1 subunits in the complex are highly similar to the corresponding structures in E. coli. In addition, docking simulation with a minimal RNA substrate provides structural insight into the guanosine specificity of YoeBSa1 for cleavage in the active site, which is distinct from the specificity of YoeBEc for adenosine rather than guanosine. Given the previous finding that YoeBSa1 exhibits fatal toxicity without inducing persister cells, the structure of the YoeBSa1-YefMSa1 complex will contribute to the design of a new category of anti-staphylococcal agents that disrupt the YoeBSa1-YefMSa1 complex and increase YoeBSa1 toxicity.

Structural and functional study of SaAcP, an acylphosphatase from Staphylococcus aureus.

Acylphosphatase is the smallest enzyme that is widely distributed in many diverse organisms ranging from archaebacteria to higher-eukaryotes including the humans. The enzyme hydrolyzes the carboxyl-phosphate bonds of the acyl phosphates which are important intermediates in glycolysis, membrane pumps, tricarboxylic acid cycle, and urea biosynthesis. Despite its biological importance in critical cellular functions, very limited structural investigations have been conducted on bacterial acylphosphatases. Here, we first unveiled the crystal structure of SaAcP, an acylphosphatase from gram-positive S. aureus at the atomic level. Structural insights on the active site together with mutation study provided greater understanding of the catalytic mechanism of SaAcP as a bacterial acylphosphatase and as a putative apyrase. Furthermore, through NMR titration experiment of SaAcP in its solution state, the dynamics and the alterations of residues affected by the phosphate ion were validated. Our findings elucidate the structure-function relationship of acylphosphatases in gram-positive bacteria and will provide a valuable basis for researchers in the field related to bacterial acylphosphatases.

Effect of different general anaesthetics on ventricular repolarisation in robot-assisted laparoscopic prostatectomy.

BACKGROUND: Ventricular repolarisation is affected differently by the types of anaesthetics used. This study aimed to compare the effect of different types of anaesthetics on ventricular repolarisation during robot-assisted laparoscopic radical prostatectomy (RALP). METHODS: Sixty-nine patients were randomly assigned in a 1:1:1 ratio to the Sevoflurane (sevoflurane/remifentanil), Desflurane (desflurane/remifentanil) or total intravenous anaesthesia (TIVA [propofol/remifentanil]) groups; however, only 67 patients completed the study. The primary outcome was heart rate-corrected QT (QTc) interval collected at nine time points during RALP. Bazett's (QTcB) and Fridericia's (QTcF) formulae were used for QT interval correction. The secondary outcomes were Tpeak-Tend (Tp-e) interval and Tp-e/QT ratio that were collected at the same time points. RESULTS: The QTcB and QTcF intervals were significantly prolonged during surgery in all groups; however, these values showed significant intergroup differences with time. After assuming the Trendelenburg position, the QTcB and QTcF intervals were significantly longer in the Desflurane group than in the other two groups, and this prolongation continued until the end of surgery. Intra-operatively, the QTcB and QTcF intervals exceeded 450 ms in six and five patients, respectively, in the Desflurane group, but in none in the TIVA group. Moreover, the incidence of intra-operative QTc interval prolongation >20 ms and >60 ms was significantly higher in the Desflurane group than in the TIVA group. There were no significant differences in Tp-e intervals and Tp-e/QT ratio among the three groups during surgery. CONCLUSIONS: To minimise QTc interval prolongation during RALP, TIVA with propofol/remifentanil is recommended for general anaesthesia.