PKCε promotes oncogenic functions of ATF2 in the nucleus while blocking its apoptotic function at mitochondria.

The transcription factor ATF2 elicits oncogenic activities in melanoma and tumor suppressor activities in nonmalignant skin cancer. Here, we identify that ATF2 tumor suppressor function is determined by its ability to localize at the mitochondria, where it alters membrane permeability following genotoxic stress. The ability of ATF2 to reach the mitochondria is determined by PKCε, which directs ATF2 nuclear localization. Genotoxic stress attenuates PKCε effect on ATF2; enables ATF2 nuclear export and localization at the mitochondria, where it perturbs the HK1-VDAC1 complex; increases mitochondrial permeability; and promotes apoptosis. Significantly, high levels of PKCε, as seen in melanoma cells, block ATF2 nuclear export and function at the mitochondria, thereby attenuating apoptosis following exposure to genotoxic stress. In melanoma tumor samples, high PKCε levels associate with poor prognosis. Overall, our findings provide the framework for understanding how subcellular localization enables ATF2 oncogenic or tumor suppressor functions.

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.

PM2.5 concentrations of outdoor tobacco smoke at different distances from the smoking source: Is there an optimal distance for a designated smoking area?

INTRODUCTION: Many countries have enacted indoor smoke-free policies, and some have established outdoor non-smoking areas. However, no clear standard for determining the optimal distance for these outdoor non-smoking zones remains. This study aimed to evaluate outdoor tobacco smoke (OTS) exposure up to a distance of 21 m and to identify factors influencing OTS levels. METHODS: To assess OTS levels, PM2.5 concentrations were measured at distances of 6, 12, 15, 18, and 21 m using real-time aerosol monitors. Between August and October 2022, a total of 164 measurements were undertaken. The background PM2.5 concentration was gauged for 5 minutes before smoking commenced and then re-measured 3 minutes during smoking. OTS levels were determined by calculating the difference between the average background PM2.5 and the average PM2.5 concentrations during smoking. A one-sample t-test was employed to ascertain if the OTS levels at each distance were significantly elevated compared to 0 µg/m3. Furthermore, a multiple linear regression analysis was conducted to determine the factors influencing OTS levels. RESULTS: The mean OTS levels recorded at all specified distances significantly surpassed 0 µg/m3. The regression analysis revealed that the OTS levels correlated significantly with distance and wind speed. Specifically, OTS levels diminished as distance expanded and wind speed reduced. CONCLUSIONS: OTS levels, even at 21 m, were significantly greater than 0 µg/m3. Our results provide robust evidence supporting the establishment of outdoor non-smoking zone up to 21 m. IMPLICATIONS: Outdoor tobacco smoke (OTS) level was determined by PM2.5 concentration. The OTS levels significantly exceeded 0 µg/m3 at every measured distance up to 21 m. In the regression model, OTS levels notably correlated with distance and wind speed. OTS levels diminished as distance expanded and wind speed reduced.

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.

Non-target screening of volatile organic compounds in spray-type consumer products and their potential health risks.

Widespread use of spray-type consumer products can raise significant concerns regarding their effects on indoor air quality and human health. In this study, we conducted non-target screening using gas chromatography-mass spectrometry (GC-MS) to analyze VOCs in 48 different spray-type consumer products. Using this approach, we tentatively identified a total of 254 VOCs from the spray-type products. Notably, more VOCs were detected in propellant-type products which are mostly solvent-based than in trigger-type ones which are mostly water-based. The VOCs identified encompass various chemical classes including alkanes, cycloalkanes, monoterpenoids, carboxylic acid derivatives, and carbonyl compounds, some of which arouse concerns due to their potential health effects. Alkanes and cycloalkanes are frequently detected in propellant-type products, whereas perfumed monoterpenoids are ubiquitous across all product categories. Among the identified VOCs, 12 compounds were classified into high-risk groups according to detection frequency and signal-to-noise (S/N) ratio, and their concentrations were confirmed using reference standards. Among the identified VOCs, D-limonene was the most frequently detected compound (freq. 21/48), with the highest concentration of 1.80 mg/g. The risk assessment was performed to evaluate the potential health risks associated with exposure to these VOCs. The non-carcinogenic and carcinogenic risks associated with the assessed VOC compounds were relatively low. However, it is important not to overlook the risk faced by occupational exposure to these VOCs, and the risk from simultaneous exposure to various VOCs contained in the products. This study serves as a valuable resource for the identification of unknown compounds in the consumer products, facilitating the evaluation of potential health risks to consumers.

Plasma and urinary extracellular vesicle microRNAs and their related pathways in diabetic kidney disease.

To explore extracellular vesicle microRNAs (EV miRNAs) and their target mRNAs in relation to diabetic kidney disease (DKD), we performed paired plasma and urinary EV small RNA sequencing (n = 18) in patients with type 2 diabetes and DKD (n = 5) and healthy subjects (n = 4) and metabolic network analyses using our own miRNA and public mRNA datasets. We found 13 common differentially expressed EV miRNAs in both fluids and 17 target mRNAs, including RRM2, NT5E, and UGDH. Because succinate dehydrogenase B was suggested to interact with proteins encoded by these three genes, we measured urinary succinate and adenosine in a validation study (n = 194). These two urinary metabolite concentrations were associated with DKD progression. In addition, renal expressions of NT5E and UGDH proteins were increased in db/db mice with DKD compared to control mice. In conclusion, we profiled DKD-related EV miRNAs in plasma and urine samples and found their relevant target pathways.

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.

Estimation of multi-route exposures to various chemicals during Children's clay toy use.

Clay toys have been used as play materials and educational tools for children. Clay toys exhibit adherent properties, and may facilitate chemical ingestion via dermal absorption and oral (hand-to-mouth, HTM) exposures. Inhalation exposure also be considered when contain volatile chemicals. The purpose of this study was to estimate the exposure dose for chemicals in clay toys via three exposure routes, and to evaluate the relationship between the exposure contribution of each route considering both the chemical properties and children's age. Chemical analysis was conducted for 9 semi-volatile organic compounds (SVOCs), 17 volatile organic compounds (VOCs), and 7 metal elements in clay toys (n = 66) purchased from Korean market. Exposure factors for usage pattern of clay toys were conducted based on a nationally representative survey in Korea. A total of 12,144 (60.7%) children responded positively to playing with clay toys. Exposure to SVOCs and VOCs in clay toys via HTM, inhalation, and dermal absorption were estimated. The exposure level was the highest in styrene with 5.2 × 10-3 mg/kg-bw/day (95th percentile population), which was approximately 13% of the acceptable daily dose for styrene. In 3-year-old children, dermal absorption route contributed the highest at 59.2-100%. Chemicals with higher octanol-water partition coefficient (Kow) had the greater the contribution of the dermal absorption route and the weaker the contribution of the HTM route. In infants (0-2 years), the contribution via HTM exposure was higher than that in the other age groups. The contribution of inhalation exposure differed depending on the volatility of the chemicals. Furthermore, the exposure route contribution significantly differed due to age-dependent behavioral changes in children. These results suggest that the exposure assessments for children could be considered with multiple exposure routes related to chemical properties.

Concentrations of blood and urinary arsenic species and their characteristics in general Korean population.

Arsenic (As) exposure has been extensively studied by investigating As species (e.g., inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA)) in urine, yet recent research suggests that blood could be a possible biomarker of As exposure. These investigations, however, were conducted on iAs-contaminated areas, and evidence on populations exposed to low levels of iAs is limited. This study aimed to describe the levels and distributions of As species in urine and blood, as well as to estimate methylation efficiency and related factors in the Korean population. Biological samples were obtained by the Korean Ministry of Food and Drug Safety. A total of 2025 urine samples and 598 blood samples were utilized in this study. Six As species were measured using ultra-high-performance liquid chromatography with inductively coupled plasma mass spectrometry (UPLC-ICP-MS): As(V), As(III), MMA, DMA, arsenobetaine (AsB), and arsenocholine (AsC). Multiple linear regression models were used to examine the relationship between As species (concentrations and proportions) and covariates. AsB was the most prevalent species in urine and blood. The relative composition of iAs, MMA, DMA, and AsC in urine and blood differed significantly. Consumption of blue-backed fish was linked to higher levels of AsB in urine and blood. Type of drinking water and multigrain rice consumption were associated with increased iAs concentration in urine. Except for iAs, every species had correlations in urine and blood in both univariate and multivariate analyses. Adolescents and smokers presented a lower methylation efficiency (higher %MMA and lower %DMA in urine) and females presented a higher methylation efficiency (lower %iAs, %MMA, and higher %DMA in urine). In conclusion, blood iAs concentration cannot represent urinary iAs; nonetheless, different compositions of urine and blood might reflect distinct information about iAs exposure. Further investigations on exposure factors and health are needed using low-exposure groups.

Characteristics of ultrafine particles emitted from 3D-pens and effect of partition on children's exposure during 3D-pen operation.

A three-dimensional (3D) printing pen is a popular writing instrument that uses a heated nozzle, and is similar to a 3D-printer. Processing thermoplastic filaments with a 3D-pen can emit ultrafine particles (UFPs). 3D-pen education sessions were held with "∏"-shaped partitions for the prevention of coronavirus disease (COVID-19). This study aimed to characterize UFP emissions from two types of 3D-pens and evaluate the influence of "∏"-shaped partitions on UFP exposure. Measurements of UFP emission rates and the size distribution of particles emitted from 3D-pens were conducted in a chamber (2.5 m3 ). The partition's influence on UFP exposure was evaluated with and without a "∏"-shaped partition on a desk. A scanning mobility particle sizer (SMPS) and an optical particle spectrometer (OPS) were used to measure the particle number concentration (PNC) and size distribution. For both 3D-pen A and B, the average emission rates were statistically significantly highest for acrylonitrile butadiene styrene (ABS) filament (8.4 × 106 [3.4] particles/min and 1.1 × 106 [1.8] particles/min), followed by polylactic acid (PLA) (2.8 × 105 [1.5] particles/min and 4.8 × 104 [1.8] particles/min) and polycaprolactone (PCL) filaments (1.4 × 104 [2.8] particles/min and 2.0 × 104 [2.8] particles/min). For all filaments, particles in the Aitken mode (30-100 nm) accounted for the highest proportion. In 3D-pen A, PNCs were higher with the partition than without it for ABS (1.2 × 106 [1.15] particles/cm3 vs. 1.4 × 105 [1.29] particles/cm3 ) and PLA (6.2 × 105 [1.38] particles/cm3 vs. 8.9 × 104 [1.12] particles/cm3 ), whereas for 3D-pen B, they were higher with the partition for ABS (9.6 × 105 [1.13] particles/cm3 vs. 4.9 × 105 [1.22] particles/cm3 ) only. With the partition installed, PNCs decreased to the background level after the operation ended, whereas it took 2-6 min without the partition. However, the mass concentrations of PLA and PCL with 3D-pen A were not statistically significantly different with respect to the partition status. The use of 3D-pens with a partition can lead to high UFP exposure. Therefore, guidelines are required for the safe use of 3D-pens and partitions.

Simultaneous assessment of organophosphate flame retardants, plasticizers, trace metals, and house dust mite allergens in settled house dust.

Settled house dust (SHD) is a reservoir for various contaminants, including endocrine-disrupting chemicals (EDCs), trace metals, and house dust mite allergens. This study aimed to characterize various chemical and biological contaminants in SHD and identify determinants governing the indoor contaminants. In total, 106 SHD samples were collected from 106 houses in Seoul and Gyeonggi Province, Korea, in 2021. Bedding dust samples were collected from 30 of these 106 houses. All participants completed a questionnaire comprised of housing and lifestyle-related factors. The samples were analyzed for 18 organophosphate flame retardants (OPFRs), 16 phthalates, five alternative plasticizers (APs), seven trace metals, and two house dust mite allergens (Dermatophagoides farinae type 1 [Der f1] and Dermatophagoides pteronyssinus type 1 [Der p1]). A multiple regression analysis was conducted to identify the determinants governing the concentrations and profiles of various contaminants. OPFRs, phthalates, APs, and trace metals were detected in all SHD samples, indicating ubiquitous contamination in indoor environments. Among the three EDC groups, APs were detected at the highest concentrations (geometric mean [GM] (geometric standard deviation, [GSD]): 1452 (1.6) µg/g in total), followed by phthalates (GM (GSD): 676 (1.4) µg/g in total) and OPFRs (GM (GSD): 10 (1.4) µg/g in total). Der f1 was detected in all bedding dust samples with significantly higher levels than Der p1 (GM (GSD): 0.1 (1.8) µg/g vs. 1.4 × 10-3 (2.3) µg/g). The concentrations of OPFRs, plasticizers, and trace metals in SHD were significantly associated with the type and number of electronic appliances and combustion activities. Der f1 was significantly associated with the number of occupants and water penetration. Ventilation, vacuum cleaning, and wet cleaning or dry mopping significantly reduced the levels of most contaminants in SHD. As residents are persistently exposed to a wide array of pollutants, comprehensive and adequate measures are required to prevent potential exposures.

DNA metabarcoding-based study on bacteria and fungi associated with house dust mites (Dermatophagoides spp.) in settled house dust.

House dust mites (HDMs) including Dermatophagoides spp. are an important cause of respiratory allergies. However, their relationship with microorganisms in house dust has not been fully elucidated. Here, we characterized bacteria and fungi associated with HDMs in house dust samples collected in 107 homes in Korea by using DNA barcode sequencing of bacterial 16S rRNA gene, fungal internal transcribed spacer 2 (ITS2) region, and arthropod cytochrome c oxidase I (COI) gene. Our inter-kingdom co-occurrence network analysis and/or indicator species analysis identified that HDMs were positively related with a xerophilic fungus Wallemia, mycoparasitic fungi such as Cystobasidium, and some human skin-related bacterial and fungal genera, and they were negatively related with the hygrophilous fungus Cephalotrichum. Overall, our study has succeeded in adding novel insights into HDM-related bacteria and fungi in the house dust ecosystem, and in confirming the historically recognized fact that HDMs are associated with xerophilic fungi such as Wallemia. Understanding the microbial ecology in house dust is thought to be important for elucidating the etiology of human diseases including allergies, and our study revealed baseline information of house dust ecology in relation to HDMs. The findings could be useful from a perspective of human health.

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.