Effector Binding Sequentially Alters KRAS Dimerization on the Membrane: New Insights Into RAS-Mediated RAF Activation.

RAS proteins are peripheral membrane GTPases that activate multiple downstream effectors for cell proliferation and differentiation. The formation of a signaling RAS-RAF complex at the plasma membrane is implicated in a quarter of all human cancers; however, the underlying mechanism remains unclear. In this work, nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses to determine the structure of a hetero-tetrameric complex comprising KRAS and the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of activated RAF1 are employed. The binding of the RBD or RBD-CRD differentially alters the dimerization modes of KRAS on both anionic and neutral membranes, validated by interface-specific mutagenesis. Notably, the RBD binding allosterically generated two distinct KRAS dimer interfaces in equilibrium, favored by KRAS free and in complex with the RBD-CRD, respectively. Additional interactions of the CRD with both KRAS protomers are mutually cooperative to stabilize a new dimer configuration of KRAS bound to the RBD-CRD. The RAF binding sequentially alters KRAS dimerization, providing new insights into RAF activation, including a configurational transition of the KRAS dimer to provide an interaction site for the CRD and release the autoinhibited RAF complex. These methods are applicable to many other signaling protein complexes on the membrane.

L-asparaginase induces IP3R-mediated ER Ca2+ release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells.

L-asparaginase is a standard therapeutic option for acute lymphoblastic leukemia (aLL), a hematologic cancer that claims the most lives of pediatric cancer patients. Previously, we demonstrated that L-asparaginase kills aLL cells via a lethal rise in [Ca2+]i due to IP3R-mediated ER Ca2+ release followed by calpain-1-Bid-caspase-3/12 activation (Blood, 133, 2222-2232). However, upstream targets of L-asparaginase that trigger IP3R-mediated ER Ca2+ release remain elusive. Here, we show that L-asparaginase targets µ-OR1 and PAR2 and induces IP3R-mediated ER Ca2+ release in aLL cells. In doing so, µ-OR1 plays a major role while PAR2 plays a minor role. Utilizing PAR2- and µ-OR1-knockdown cells, we demonstrate that L-asparaginase stimulation of µ-OR1 and PAR2 relays its signal via Gαi and Gαq, respectively. In PAR2-knockdown cells, stimulation of adenylate cyclase with forskolin or treatment with 8-CPT-cAMP reduces L-asparaginase-induced µ-OR1-mediated ER Ca2+ release, suggesting that activation of µ-OR1 negatively regulates AC and cAMP. In addition, the PKA inhibitor 14-22 amide (myr) alone evokes ER Ca2+ release, and subsequent L-asparaginase treatment does not induce further ER Ca2+ release, indicating the involvement of PKA inhibition in L-asparaginase-induced µ-OR1-mediated ER Ca2+ release, which can bypass the L-asparaginase-µ-OR1-AC-cAMP loop. This coincides with (a) the decreases in PKA-dependent inhibitory PLCß3 Ser1105 phosphorylation, which prompts PLCß3 activation and ER Ca2+ release, and (b) BAD Ser118 phosphorylation, which leads to caspase activation and apoptosis. Thus, our findings offer new insights into the Ca2+-mediated mechanisms behind L-asparaginase-induced aLL cell apoptosis and suggest that PKA may be targeted for therapeutic intervention for aLL.

Emission characteristics of volatile organic compounds from consumer spray products based on product type, spray method, and distance.

Consumer spray products (CSPs) are widely used in daily life, yet it is challenging to find products that fully disclose all components posing health risks. Existing studies primarily focus on product components or VOC quantities emitted during use. Therefore, this study aimed to measure the VOC concentrations emitted by CSPs at varying distances. 47 CSPs available in the Korean market were selected, spanning three spray groups: antiseptics/insecticides (11), aromatic deodorants (16), and coating/polishing agents (20). VOC in air samples were collected using Tenax TA tube at a distance of 1 and 3 m from the sprayed CSPs and then analyzed by thermal desorption-gas chromatography-mass spectrometry system. Discrepancies were found between labeled and actual product components. Aromatic deodorants exhibited the highest total VOCs (TVOCs), while antiseptic/insecticide sprays exhibited the lowest. In the antiseptic/insecticide group and coating/polishing agent group, benzene as a propellant had a maximum concentration (30.9 ± 25.6 ppb), and as trigger, its concentration was 33.7 ± 30.7 ppb. Quantitative analysis using advanced analytical instruments only explained 26.1 ± 20.4% of toluene-equivalent TVOCs, suggesting the presence of additional substances. Concentrations varied by distance due to substance volatility and usage. Maintaining a distance of at least 1 m from CSPs is recommended.

PKA inhibition is a central step in D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a hematologic cancer that mostly affects children. It accounts for over a quarter of ALL pediatric cancers, causing most of the cancer death among children. Previously, we demonstrated that D,L-methadone causes ALL cell apoptosis via µ-opioid receptor 1 (OPRM1)-triggered ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i. However, the precise mechanism by which D,L-methadone induces ER Ca2+ release remains to be defined. Here, we show that in ALL cells, D,L-methadone-induced ER Ca2+ release is blocked by inhibition of Gαi, but not Gßϒ, indicating that the process is dependent on Gαi. Activation of adenylyl cyclase (AC) with forskolin or treatment with 8-CPT-cAMP blocks D,L-methadone-induced ER Ca2+ release, indicating that the latter results from Gαi-dependent downregulation of AC and cAMP. The 14-22 amide (myr) PKA inhibitor alone elicits ER Ca2+ release, and subsequent treatment with D,L-methadone does not cause additional ER Ca2+ release, indicating that PKA inhibition is a key step in D,L-methadone-induced ER Ca2+ release and can bypass the D,L-methadone-OPRM1-AC-cAMP step. This is consistent with the decrease in PKA-dependent (i) inhibitory PLCß3 Ser1105 phosphorylation that leads to PLCß3 activation and ER Ca2+ release, and (ii) BAD Ser118 phosphorylation, which together ultimately result in caspase activation and apoptosis. Thus, our findings indicate that D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in ALL cells is mediated by Gαi-dependent downregulation of the AC-cAMP-PKA-PLCß3/BAD pathway. The fact that 14-22 amide (myr) alone effectively kills ALL cells suggests that PKA may be targeted for ALL therapy.

PKA inhibition kills L-asparaginase-resistant leukemic cells from relapsed acute lymphoblastic leukemia patients.

Despite the success in treating newly diagnosed pediatric acute lymphoblastic leukemia (aLL), the long-term cure rate for the 20% of children who relapse is poor, making relapsed aLL the primary cause of cancer death in children. By unbiased genome-wide retroviral RNAi screening and knockdown studies, we previously discovered opioid receptor mu 1 (OPRM1) as a new aLL cell resistance biomarker for the aLL chemotherapeutic drug, L-asparaginase, i.e., OPRM1 loss triggers L-asparaginase resistance. Indeed, aLL cell OPRM1 level is inversely proportional to L-asparaginase IC50: the lower the OPRM1 level, the higher the L-asparaginase IC50, indicating that aLL cells expressing reduced OPRM1 levels show resistance to L-asparaginase. In the current study, we utilized OPRM1-expressing and -knockdown aLL cells as well as relapsed patient aLL cells to identify candidate targeted therapy for L-asparaginase-resistant aLL. In OPRM1-expressing cells, L-asparaginase induces apoptosis via a cascade of events that include OPRM1-mediated decline in [cAMP]i, downregulation of PKA-mediated BAD S118 phosphorylation that can be reversed by 8-CPT-cAMP, cyt C release from the mitochondria, and subsequent caspase activation and PARP1 cleavage. The critical role of PKA inhibition due to a decrease in [cAMP]i in this apoptotic process is evident in the killing of OPRM1-knockdown and low OPRM1-expressing relapsed patient aLL cells by the PKA inhibitors, H89 and 14-22 amide. These findings demonstrate for the first time that PKA can be targeted to kill aLL cells resistant to L-asparaginase due to OPRM1 loss, and that H89 and 14-22 amide may be utilized to destroy L-asparaginase-resistant patient aLL cells.

PTK2 is a potential biomarker and therapeutic target for EGFR- or TLRs-induced lung cancer progression via the regulation of the cross-talk between EGFR- and TLRs-mediated signals.

Protein tyrosine kinase 2 (PTK2), epidermal growth factor receptor (EGFR), and toll-like receptor (TLRs) are amplified in non-small cell lung cancer (NSCLC). However, the functional and clinical associations between them have not been elucidated yet in NSCLC. By using microarray data of non-small cell lung cancer (NSCLC) tumor tissues and matched normal tissues of 42 NSCLC patients, the genetic and clinical associations between PTK2, EGFR, and TLRs were analyzed in NSCLC patients. To verify the functional association, we generated PTK2-knockout (PTK2-KO) lung cancer cells by using CRISPR-Cas9 gene editing method, and performed in vitro cancer progression assay, including 3D tumor spheroid assay, and in vivo xenografted NSG (NOD/SCID/IL-2Rγnull) mouse assay. Finally, therapeutic effects targeted to PTK2 in lung cancer in response to EGF and TLR agonists were verified by using its inhibitor (Defactinib). In summary, we identified that up-regulated PTK2 might be a reliable marker for EGFR- or TLRs-induced lung cancer progression in NSCLC patients via the regulation of the cross-talk between EGFR- and TLRs-mediated signaling. This study provides a theoretical basis for the therapeutic intervention of PTK2 targeting EGFR- or TLRs-induced lung cancer progression.

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.

Temporal trend of microenvironmental time-activity patterns of the Seoul population from 2004 to 2022 and its potential impact on exposure assessment.

BACKGROUND: Time-activity pattern (TAP) is an important parameter for determining personal exposure to environmental pollutants. Changes in TAPs could have significant implications for the alterations in outcomes of exposure assessments. OBJECTIVE: This study aimed to evaluate the Seoul population's long-term change in TAPs, along with variations by sociodemographic group. METHODS: In 2004, 2009, 2014, and 2019, the Time Use Survey of Statistics Korea collected the TAP information of 4036, 2610, 3337, and 2793 Seoul residents, respectively. In 2022, the TAP information of 4401 Seoul residents was collected for Korean Air Pollutant Exposure (KAPEX) research. The microenvironmental TAP changes in the Seoul population from 2004 to 2022 were assessed based on age, gender, work status, and day type. RESULTS: From 2004 to 2022, Seoul people increasingly spent more time in indoor residences (from 14.8 ± 5.1 h to 15.8 ± 4.5 h) and less time in other indoors (from 7.2 ± 4.5 h to 5.9 ± 4.2 h). Their transit time constantly decreased from 2004 (1.4 ± 1.8 h) to 2022 (1.2 ± 1.3 h), whereas the outdoor time fluctuated throughout the years. From 2004 to 2022, the time of the day spent by Seoul people in residential indoor shifted to later in the morning (2004: 8:30 am; 2022: 9:00 am) and earlier in the evening (2004: 9:30 pm; 2022: 7:00 pm); however, the opposite was true for other indoors (2004: from 8:30 am to 9:30 pm; 2022: from 9:00 am to 7:00 pm) and transits (2004: 7:30-9:30 am and 3:00-8:00 pm; 2022: 7:30-9:00 pm and 5:00-9:00). The time of the day spent in outdoors increased from 2004 to 2019, with a distinct peak observed in 2022 (12:00 pm-2:00 pm). The microenvironmental time trends of adolescents and late-adulthoods differed from those of the other age groups, while those of males differed from females. Also, the microenvironmental time trends of the employed differed from those of the unemployed, and those during weekdays differed from those during weekends. IMPACT STATEMENT: Microenvironmental TAP should be essentially considered to estimate the actual exposure to pollutants. This study demonstrates the Seoul population's long-term changes in TAP throughout the 18 years as the significant parameter in exposure assessment. Notably, the microenvironmental TAPs of Seoul people shifted, with variations across different sociodemographic groups. Previous studies in Korea did not consider the TAP shifts in exposure assessment; this study highlights the importance of aligning TAP data with concurrent environmental pollutant data and emphasizes the need for refined data collection in future exposure assessments.

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.

Development of nationally representative exposure factor database for children's products in Korea.

BACKGROUND: Children may be exposed to harmful chemicals from their products. Accurate exposure factors are critical for exposure assessment of children's products. Product usage pattern parameters are relatively limited compared with the chemical concentration, children's physiological and behavioral parameters. OBJECTIVE: The aim of this study was to determine nationally representative Korean exposure factors for the usage patterns of children's products by sex, age, and season. METHODS: Using proportional quota sampling, a survey of 10,000 households with children aged 0-12 years was conducted twice, once in summer and winter. The children's ages were divided into four groups: infant (0-2 years old), toddler (3-6), lower-grade elementary student (7-9), and higher-grade elementary student (10-12). Data on exposure factors such as use rate, use frequency, and use duration of 57 children's products were collected. RESULTS: The 57 products were classified into five categories: baby products (13), toys (12), daily products (10), sporting goods (8), and stationery (14). The use rates of products in the daily products and stationery category were >90% in both seasons. Two of the 57 products showed significant sex differences in all three exposure factors (p < 0.001). Twenty-five of the 44 non-baby products showed significant age differences for all three exposure factors. Twenty-three of the 57 products varied significantly with season for all three exposure factors. IMPACT: This study generated a nationally representative exposure factor database for the usage patterns of children's products in Korea. The exposure factors for 57 children's products were investigated through twice survey with quota sampling with each 10,000 children nationwide. Sex, age, and seasonal differences for children's products were identified. These accurate exposure factors by sex, age, and season can be used as input parameters for refined exposure assessment.

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.

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.

Resting-state prefrontal EEG biomarker in correlation with postoperative delirium in elderly patients.

Postoperative delirium (POD) is associated with adverse outcomes in elderly patients after surgery. Electroencephalography (EEG) can be used to develop a potential biomarker for degenerative cerebral dysfunctions, including mild cognitive impairment and dementia. This study aimed to explore the relationship between preoperative EEG and POD. We included 257 patients aged >70 years who underwent spinal surgery. We measured the median dominant frequency (MDF), which is a resting-state EEG biomarker involving intrinsic alpha oscillations that reflect an idle cortical state, from the prefrontal regions. Additionally, the mini-mental state examination and Montreal cognitive assessment (MoCA) were performed before surgery as well as 5 days after surgery. For long-term cognitive function follow up, the telephone interview for cognitive status™ (TICS) was performed 1 month and 1 year after surgery. Fifty-two (20.2%) patients were diagnosed with POD. A multivariable logistic regression analysis that included age, MoCA score, Charlson comorbidity index score, Mini Nutritional Assessment, and the MDF as variables revealed that the MDF had a significant odds ratio of 0.48 (95% confidence interval 0.27-0.85). Among the patients with POD, the postoperative neurocognitive disorders could last up to 1 year. Low MDF on preoperative EEG was associated with POD in elderly patients undergoing surgery. EEG could be a novel potential tool for identifying patients at a high risk of POD.

Comparison of sampling methods for the determination of volatile organic compounds in consumer aerosol sprays.

Many studies have evaluated the hazardous substances contained in various household chemical products. However, for aerosol spray products there is currently no international standard sampling method for use in a component analysis. The aim of this study was to develop an appropriate sampling method for the analysis of volatile organic compounds (VOCs) in consumer aerosol sprays. Two different sampling methods, spraying (into a vial) and perforating (and transferring the contents into a vial), were used to evaluate the levels of 16 VOC components in eight different aerosol spray products. All eight products contained trace amounts of hazardous VOCs, and a quantitative analysis showed that, for the same product, VOC concentrations were higher when spraying than when perforating. Using the spraying method, average toluene, ethylbenzene, p-xylene, o-xylene, and styrene concentrations were 1.80-, 2.10- 2.25-, 2.03-fold, and 1.28-fold higher, respectively, than when using the perforating method. The spraying method may provide more realistic estimates of the user's exposure to harmful substances and the associated health risks when using spray products. Of the two representative methods widely used to analyze harmful substances in consumer aerosol sprays, the spraying method is recommended over the perforating method for the analysis of VOCs.

Prognostic implication of downregulated exosomal miRNAs in patients with sepsis: a cross-sectional study with bioinformatics analysis.

BACKGROUND: Despite the understanding of sepsis-induced extracellular vesicles (EVs), such as exosomes, and their role in intercellular communication during sepsis, little is known about EV contents such as microRNA (miRNA), which modulate important cellular processes contributing to sepsis in body fluids. This study aimed to analyze the differential expression of exosomal miRNAs in plasma samples collected from sepsis patients and healthy controls, and to identify potential miRNA regulatory pathways contributing to sepsis pathogenesis. METHODS: Quantitative real-time PCR-based microarrays were used to profile plasma exosomal miRNA expression levels in 135 patients with sepsis and 11 healthy controls from an ongoing prospective registry of critically ill adult patients admitted to the intensive care unit. The identified exosomal miRNAs were tested in an external validation cohort (35 sepsis patients and 10 healthy controls). And then, functional enrichment analyses of gene ontology, KEGG pathway analysis, and protein-protein interaction network and cluster analyses were performed based on the potential target genes of the grouped miRNAs. Finally, to evaluate the performance of the identified exosomal miRNAs in predicting in-hospital and 90-day mortalities of sepsis patients, receiver operating characteristic curve (ROC) and Kaplan-Meier analyses were performed. RESULTS: Compared with healthy controls, plasma exosomes from sepsis patients showed significant changes in 25 miRNAs; eight miRNAs were upregulated and 17 downregulated. Additionally, the levels of hsa-let-7f-5p, miR-331-3p miR-301a-3p, and miR-335-5p were significantly lower in sepsis patients than in healthy controls (p < 0.0001). These four miRNAs were confirmed in an external validation cohort. In addition, the most common pathway for these four miRNAs were PI3K-Akt and mitogen-activated protein kinase (MAPK) signaling pathways based on the KEGG analysis. The area under the ROC of hsa-let-7f-5p, miR-331-3p, miR-301a-3p, and miR-335-5p level for in-hospital mortality was 0.913, 0.931, 0.929, and 0.957, respectively (p < 0.001), as confirmed in an external validation cohort. Also, the Kaplan-Meier analysis showed a significant difference in 90-day mortality between sepsis patients with high and low miR-335-5p, miR-301a-3p, hsa-let-7f-5p, and miR-331-3p levels (p < 0.001, log-rank test). CONCLUSION: Among the differentially-expressed miRNAs detected in microarrays, the top four downregulated exosomal miRNAs (hsa-let-7f-5p, miR-331-3p miR-301a-3p, and miR-335-5p) were identified as independent prognostic factors for in-hospital and 90-day mortalities among sepsis patients. Bioinformatics analysis demonstrated that these four microRNAs might provide a significant contribution to sepsis pathogenesis through PI3K-Akt and MAPK signaling pathway.

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.

ß-arrestin 2 negatively regulates lung cancer progression by inhibiting the TRAF6 signaling axis for NF-κB activation and autophagy induced by TLR3 and TLR4.

ß-arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain clinical insight on its function in lung cancer, microarray data from lung tumor tissues (LTTs) and matched lung normal tissues (mLNTs) of primary non-small cell lung cancer (NSCLC) patients (n = 37) were utilized. ARRB2 expression levels were markedly decreased in all 37 LTTs compared to those in matched LNTs of NSCLC patients. They were significantly co-related to enrichment gene sets associated with oncogenic and cancer genes. Importantly, Gene Set Enrichment Analysis (GSEA) between three LTTs with highly down-regulated ARRB2 and three LTTs with lowly down-regulated ARRB2 revealed significant enrichments related to toll-like receptor (TLR) signaling and autophagy genes in three LTTs with highly down-regulated ARRB2, suggesting that ARRB2 was negatively involved in TLR-mediated signals for autophagy induction in lung cancer. Biochemical studies for elucidating the molecular mechanism revealed that ARRB2 interacted with TNF receptor-associated factor 6 (TRAF6) and Beclin 1 (BECN1), thereby inhibiting the ubiquitination of TRAF6-TAB2 to activate NF-κB and TRAF6-BECN1 for autophagy stimulated by TLR3 and TLR4, suggesting that ARRB2 could inhibit the TRAF6-TAB2 signaling axis for NF-κB activation and TRAF6-BECN1 signaling axis for autophagy in response to TLR3 and TLR4. Notably, ARRB2-knockout (ARRB2KO) lung cancer cells exhibited marked enhancements of cancer migration, invasion, colony formation, and proliferation in response to TLR3 and TLR4 stimulation. Altogether, our current data suggest that ARRB2 can negatively regulate lung cancer progression by inhibiting TLR3- and TLR4-induced autophagy.