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.

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.

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.

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.

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.

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.

FFAR2 antagonizes TLR2- and TLR3-induced lung cancer progression via the inhibition of AMPK-TAK1 signaling axis for the activation of NF-κB.

BACKGROUND: Free fatty acid receptors (FFARs) and toll-like receptors (TLRs) recognize microbial metabolites and conserved microbial products, respectively, and are functionally implicated in inflammation and cancer. However, whether the crosstalk between FFARs and TLRs affects lung cancer progression has never been addressed. METHODS: We analyzed the association between FFARs and TLRs using The Cancer Genome Atlas (TCGA) lung cancer data and our cohort of non-small cell lung cancer (NSCLC) patient data (n = 42), and gene set enrichment analysis (GSEA) was performed. For the functional analysis, we generated FFAR2-knockout (FFAR2KO) A549 and FFAR2KO H1299 human lung cancer cells and performed biochemical mechanistic studies and cancer progression assays, including migration, invasion, and colony-formation assays, in response to TLR stimulation. RESULTS: The clinical TCGA data showed a significant down-regulation of FFAR2, but not FFAR1, FFAR3, and FFAR4, in lung cancer, and a negative correlation with TLR2 and TLR3. Notably, GSEA showed significant enrichment in gene sets related to the cancer module, the innate signaling pathway, and the cytokine-chemokine signaling pathway in FFAR2DownTLR2UpTLR3Up lung tumor tissues (LTTs) vs. FFAR2upTLR2DownTLR3Down LTTs. Functionally, treatment with propionate (an agonist of FFAR2) significantly inhibited human A549 or H1299 lung cancer migration, invasion, and colony formation induced by TLR2 or TLR3 through the attenuation of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB. Moreover, FFAR2KO A549 and FFAR2KO H1299 human lung cancer cells showed marked increases in cell migration, invasion, and colony formation in response to TLR2 or TLR3 stimulation, accompanied by elevations in NF-κB activation, cAMP levels, and the production of C-C motif chemokine ligand (CCL)2, interleukin (IL)-6, and matrix metalloproteinase (MMP) 2 cytokines. CONCLUSION: Our results suggest that FFAR2 signaling antagonized TLR2- and TLR3-induced lung cancer progression via the suppression of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB, and its agonist might be a potential therapeutic agent for the treatment of lung cancer.

Requirement for ER-mitochondria Ca2+ transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (aLL) is a malignant cancer in the blood and bone marrow characterized by rapid expansion of lymphoblasts. It is a common pediatric cancer and the principal basis of cancer death in children. Previously, we reported that L-asparaginase, a key component of acute lymphoblastic leukemia chemotherapy, causes IP3R-mediated ER Ca2+ release, which contributes to a fatal rise in [Ca2+]cyt, eliciting aLL cell apoptosis via upregulation of the Ca2+-regulated caspase pathway (Blood, 133, 2222-2232). However, the cellular events leading to the rise in [Ca2+]cyt following L-asparaginase-induced ER Ca2+ release remain obscure. Here, we show that in acute lymphoblastic leukemia cells, L-asparaginase causes mitochondrial permeability transition pore (mPTP) formation that is dependent on IP3R-mediated ER Ca2+ release. This is substantiated by the lack of L-asparaginase-induced ER Ca2+ release and loss of mitochondrial permeability transition pore formation in cells depleted of HAP1, a key component of the functional IP3R/HAP1/Htt ER Ca2+ channel. L-asparaginase induces ER Ca2+ transfer into mitochondria, which evokes an increase in reactive oxygen species (ROS) level. L-asparaginase-induced rise in mitochondrial Ca2+ and reactive oxygen species production cause mitochondrial permeability transition pore formation that then leads to an increase in [Ca2+]cyt. Such rise in [Ca2+]cyt is inhibited by Ruthenium red (RuR), an inhibitor of the mitochondrial calcium uniporter (MCU) that is required for mitochondrial Ca2+ uptake, and cyclosporine A (CsA), an mitochondrial permeability transition pore inhibitor. Blocking ER-mitochondria Ca2+ transfer, mitochondrial ROS production, and/or mitochondrial permeability transition pore formation inhibit L-asparaginase-induced apoptosis. Taken together, these findings fill in the gaps in our understanding of the Ca2+-mediated mechanisms behind L-asparaginase-induced apoptosis in acute lymphoblastic leukemia cells.

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.

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.