BOD1L mediates chromatin binding and non-canonical function of H3K4 methyltransferase SETD1A.

The H3K4 methyltransferase SETD1A plays an essential role in both development and cancer. However, essential components involved in SETD1A chromatin binding remain unclear. Here, we discovered that BOD1L exhibits the highest correlated SETD1A co-dependency in human cancer cell lines. BOD1L knockout reduces leukemia cells in vitro and in vivo, and mimics the transcriptional profiles observed in SETD1A knockout cells. The loss of BOD1L immediately reduced SETD1A distribution at transcriptional start sites (TSS), induced transcriptional elongation defect, and increased the RNA polymerase II content at TSS; however, it did not reduce H3K4me3. The Shg1 domain of BOD1L has a DNA binding ability, and a tryptophan residue (W104) in the domain recruits SETD1A to chromatin through the association with SETD1A FLOS domain. In addition, the BOD1L-SETD1A complex associates with transcriptional regulators, including E2Fs. These results reveal that BOD1L mediates chromatin and SETD1A, and regulates the non-canonical function of SETD1A in transcription.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

SETD1A function in leukemia is mediated through interaction with mitotic regulators BuGZ/BUB3.

The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.

Improving Proteomic Identification Using Narrow Isolation Windows with Zeno SWATH Data-Independent Acquisition.

Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized the SWATH-DIA method using a narrow isolation window placement approach, improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios ofEscherichia coli, yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.

Association of immune-related adverse events with durvalumab efficacy after chemoradiotherapy in patients with unresectable Stage III non-small cell lung cancer.

BACKGROUND: Immune-related adverse events (irAEs) have been found to predict PD-L1 inhibitor efficacy in metastatic NSCLC. However, the relation of irAEs to clinical outcome for nonmetastatic NSCLC has remained unknown. METHODS: In this multicenter prospective study of Stage III NSCLC treated with PACIFIC regimen, the relation of irAEs to PFS was evaluated by 8-week landmark analysis to minimise lead-time bias as well as by multivariable analysis adjusted for baseline factors. irAEs were categorised as mild or nonmild according to whether they were treated with systemic steroid. RESULTS: Median PFS was 16.0 months, not reached, and 9.7 months for patients without (85 cases) or with mild (21 cases) or nonmild (21 cases) irAEs, respectively. Multivariable analysis indicated that nonmild irAEs were associated with poor PFS, with HRs of 3.86 (95% CI, 1.31-11.38) compared with no irAEs and 11.58 (95% CI, 2.11-63.63) compared with mild irAEs. This pattern was consistent after irAE grade, the number of durvalumab doses and immune profiles (PD-L1 score, CD8+ tumour-infiltrating lymphocyte density, and tumour mutation burden) were taken into consideration. CONCLUSIONS: The development of mild irAEs might predict a better survival outcome, whereas immunosuppressive steroid-treated irAEs were associated with a worse outcome, regardless of baseline clinical and immune profiles.

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis.

BACKGROUND: Lipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model. METHODS: We measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice. RESULTS: Serum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration. CONCLUSIONS: Our findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Intestinal overgrowth of Candida albicans exacerbates bleomycin-induced pulmonary fibrosis in mice with dysbiosis.

Increasing evidence indicates an interaction between the intestinal microbiota and diseases in distal organs. However, the relationship between pulmonary fibrosis and the intestinal microbiota, especially intestinal fungal microbiota, is poorly understood. Thus, this study aimed to determine the effects of changes in the intestinal fungal microbiota on the pathogenesis of pulmonary fibrosis. Mice with intestinal overgrowth of Candida albicans, which was established by oral administration of antibiotics plus C. albicans, showed accelerated bleomycin-induced pulmonary fibrosis relative to the control mice (i.e. without C. albicans treatment). In addition, the mice with intestinal overgrowth of C. albicans showed enhanced Th17-type immunity, and treatment with IL-17A-neutralizing antibody alleviated pulmonary fibrosis in these mice but not in the control mice. This result indicates that IL-17A is involved in the pathogenesis of C. albicans-exacerbated pulmonary fibrosis. Even before bleomycin treatment, the expression of Rorc, the master regulator of Th17, was already upregulated in the pulmonary lymphocytes of the mice with intestinal overgrowth of C. albicans. Subsequent administration of bleomycin triggered these Th17-skewed lymphocytes to produce IL-17A, which enhanced endothelial-mesenchymal transition. These results suggest that intestinal overgrowth of C. albicans exacerbates pulmonary fibrosis via IL-17A-mediated endothelial-mesenchymal transition. Thus, it might be a potential therapeutic target in pulmonary fibrosis. This study may serve as a basis for using intestinal fungal microbiota as novel therapeutic targets in pulmonary fibrosis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

D-dimer cut-off value for predicting venous thromboembolism at the initial diagnosis in Japanese patients with advanced lung cancer.

OBJECTIVE: Cancer is a well-known risk factor for venous thromboembolism. The D-dimer level is used to predict venous thromboembolism; however, reports on an appropriate D-dimer cut-off value in Japanese patients with advanced lung cancer are lacking. Therefore, this study aimed to calculate the D-dimer cut-off value for venous thromboembolism at the time of lung cancer diagnosis. METHODS: The Rising-venous thromboembolism/NEJ037 study was a multicenter, prospective observational study. Patients with lung cancer who were contraindicated for radical resection or radiation were enrolled and followed up for 2 years. In the present study (jRCT no. 061180025), a receiver operating characteristic curve for D-dimer levels was created using the dataset of the Rising-venous thromboembolism/NEJ037 study. RESULTS: The Rising-venous thromboembolism/NEJ037 study included a total of 1008 patients, of whom 976, whose D-dimer levels had been measured at the time of cancer diagnosis, were included in the present study. At the time of lung cancer diagnosis, 62 (6.3%) and 914 (93.7%) patients presented with and without venous thromboembolism, respectively. The D-dimer values ranged from 0.1 to 180.1 µg/ml and from 0.1 to 257.2 µg/ml in patients with and without venous thromboembolism, respectively. The receiver operating characteristic curve was discriminative with a cut-off value of 3.3 µg/ml and an area under the curve of 0.794 (sensitivity, 0.742; specificity, 0.782; 95% confidence interval, 0.725-0.863). CONCLUSIONS: This is the first study to calculate the D-dimer cut-off value in Japanese patients with advanced lung cancer. Patients with D-dimer levels ≥3.3 µg/ml at the time of initial diagnosis may have coexisting venous thromboembolism.

Minimum 10-Year Survivorship of Uncemented Acetabular Reconstruction With Bulk Femoral Head Autografting in Crowe Type IV Developmental Hip Dysplasia.

BACKGROUND: In total hip arthroplasty (THA) for severe dislocations such as Crowe type IV developmental dysplasia of the hip (DDH), sufficient bone volume for stable fixation of the acetabular component can be achieved by placing a reinforcing bone graft prepared from the resected femoral head into the deficient acetabulum. The purpose of the current study was to examine the long-term survivorship of uncemented THA in conjunction with a bulk femoral head autograft in patients who have Crowe type IV DDH. METHODS: A total of 35 patients (42 hips) who have Crowe type IV DDH and underwent THA using uncemented cup fixation with bulk femoral head autografting were followed up for a mean period of 15.0 years (range, 10.0 to 20.0) postoperatively. Anteroposterior pelvic radiographs were used for measurements such as the horizontal coverage of the grafted bone and the center-edge angle. Kaplan-Meier survivorship analyses were performed with revision of the acetabular component as the endpoint. RESULTS: The Kaplan-Meier analysis indicated 15-year survival rates of 90.4%. The mean horizontal coverage of grafted bone was 46.1% (range, 23.7 to 66.0), and there were 16 cases with horizontal coverage of ≥ 50%. There was no difference in the appearance of a thin (< 1 mm) radiolucency line around the cup between cases with < 50% versus ≥ 50% of the horizontal coverage of grafted bone (4 versus 2 hips; P = .446). Trabecular bridging and remodeling were seen in all cases after mean periods of 4.1 and 9.0 months postoperatively, respectively. Trabecular reorientation was seen in 41 of 42 hips (97.6%) at a mean follow-up of 19.9 months. CONCLUSIONS: Acetabular reconstruction with femoral bulk bone grafting for Crowe type IV DDH resulted in high survival rates and was a good method to restore bone stock and obtain long-term fixation.

Hypoxia-inducible factor 1α modulates interstitial pneumonia-mediated lung cancer progression.

BACKGROUND: The prognosis of patients with lung cancer accompanied by interstitial pneumonia is poorer than that of patients with lung cancer but without interstitial pneumonia. Moreover, the available therapeutic interventions for lung cancer patients with interstitial pneumonia are limited. Therefore, a new treatment strategy for these patients is required. The aim of the present study was to investigate the pathophysiological relationship between interstitial pneumonia and lung cancer and explore potential therapeutic agents. METHODS: A novel hybrid murine model of lung cancer with interstitial pneumonia was established via bleomycin-induced pulmonary fibrosis followed by orthotopic lung cancer cell transplantation into the lungs. Changes in tumor progression, lung fibrosis, RNA expression, cytokine levels, and tumor microenvironment in the lung cancer with interstitial pneumonia model were investigated, and therapeutic agents were examined. Additionally, clinical data and samples from patients with lung cancer accompanied by interstitial pneumonia were analyzed to explore the potential clinical significance of the findings. RESULTS: In the lung cancer with interstitial pneumonia model, accelerated tumor growth was observed based on an altered tumor microenvironment. RNA sequencing analysis revealed upregulation of the hypoxia-inducible factor 1 signaling pathway. These findings were consistent with those obtained for human samples. Moreover, we explored whether ascorbic acid could be an alternative treatment for lung cancer with interstitial pneumonia to avoid the disadvantages of hypoxia-inducible factor 1 inhibitors. Ascorbic acid successfully downregulated the hypoxia-inducible factor 1 signaling pathway and inhibited tumor progression and lung fibrosis. CONCLUSIONS: The hypoxia-inducible factor 1 pathway is critical in lung cancer with interstitial pneumonia and could be a therapeutic target for mitigating interstitial pneumonia-mediated lung cancer progression.

Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial-mesenchymal transition in lung endothelial cells.

BACKGROUND: Nestin, an intermediate filament protein, participates in various pathophysiological processes, including wound healing, angiogenesis, endothelial-mesenchymal transition (EndoMT), and fibrosis. However, the pathophysiological roles of lung nestin-expressing cells remain unclear due to conflicting reports. The objective of this study is to elucidate the characteristics and functions of lung nestin-expressing cells. METHODS: We conducted a series of in vitro and in vivo experiments using endothelial cell line MS1 and nestin-GFP mice. This animal model allows for nestin-expressing cell detection without the use of anti-nestin antibodies. RESULTS: Lung nestin-expressing cells occurred in approximately 0.2% of CD45- cells and was co-expressed with epithelial, endothelial, and mesenchymal cell-surface markers. Importantly, virtually all nestin-expressing cells co-expressed CD31. When compared to lung nestin-nonexpressing endothelial cells, nestin-expressing endothelial cells showed robust angiogenesis with frequent co-expression of PDGFRß and VEGFR2. During TGFß-mediated EndoMT, the elevation of Nes mRNA expression preceded that of Col1a1 mRNA, and nestin gene silencing using nestin siRNA resulted in further upregulation of Col1a1 mRNA expression. Furthermore, Notch3 expression was regulated by nestin in vitro and in vivo; nestin siRNA resulted in reduced Notch3 expression accompanied with enhanced EndoMT. Contrary to previous reports, neither Nes mRNA expression nor nestin-expressing cells were increased during pulmonary fibrosis. CONCLUSIONS: Our study showed that (1) lung nestin-expressing cells are an endothelial lineage but are distinct from nestin-nonexpressing endothelial cells; (2) nestin regulates Notch3 and they act collaboratively to regulate angiogenesis, collagen production, and EndoMT; and (3) nestin plays novel roles in lung angiogenesis and fibrosis. Video Abstract.

Predictive value of serum high-mobility group box 1 levels for checkpoint inhibitor pneumonitis.

BACKGROUND AND OBJECTIVE: Checkpoint inhibitor pneumonitis (CIP), caused by the anti-programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) antibody, can be a fatal adverse event in cancer patients. However, no predictive biomarkers for CIP have been identified. Because high-mobility group box 1 (HMGB1) can aggravate lung injury and potentially increase the immune response, it was investigated as a predictive blood marker. METHODS: Blood samples, prospectively stored before anti-PD-1/PD-L1 monotherapy between December 2015 and October 2020, were obtained at two university hospitals from 87 and 43 non-small cell lung cancer (NSCLC) patients (discovery and validation cohorts, respectively). We retrospectively evaluated the association of serum HMGB1 levels with the incidence of CIP developed within 3 months of initiating anti-PD-1/PD-L1 therapy. RESULTS: CIP was observed in 9 (10.3%) and 6 (14.0%) patients in the discovery and validation cohorts, respectively. In each cohort, serum HMGB1 levels were significantly and reproducibly higher in patients with CIP. In the discovery cohort, an HMGB1 cut-off level of 11.24 ng/ml was identified by receiver operating characteristic analysis. CIP incidence in the HMGB1high subgroup was significantly higher than that in the HMGB1low subgroup in the discovery (41.2% vs. 2.9%) and validation cohorts (36.4% vs. 6.3%). In an exploratory pooled analysis, three patients died of grade 5 CIP; a 19.29 ng/ml HMGB1 cut-off level detected grade 5 CIP with 100% sensitivity and 96.85% specificity. CONCLUSION: Our results suggest that HMGB1 may be a potential blood marker to predict the development and severity of CIP in NSCLC patients.

Clinical characteristics of extrapulmonary nontuberculous mycobacteria infections in comparison with pulmonary infections: A single-center, retrospective study in Japan.

INTRODUCTION: The prevalence of nontuberculous mycobacteria (NTM) infections is increasing worldwide. Although NTM can affect extrapulmonary organs, studies on the clinical characteristics of extrapulmonary NTM are rare. METHODS: We retrospectively analyzed patients who were newly diagnosed with NTM infections at Hiroshima University Hospital between 2001 and 2021 to investigate species distribution, infected sites, and risk factors of extrapulmonary NTM compared to pulmonary NTM. RESULTS: Of the 261 NTM infections, 9.6% and 90.4% had extrapulmonary and pulmonary NTM, respectively. The mean ages of patients with extrapulmonary and pulmonary NTM were 53.4 and 69.3 years, 64.0% and 42.8% were male, 36.0% and 9.3% received corticosteroids, 20.0% and 0% had acquired immune deficiency syndrome (AIDS), and 56.0% and 16.1% had any immunosuppressive conditions, respectively. Younger age, corticosteroid use, and AIDS were associated with extrapulmonary NTM. In pulmonary NTM, Mycobacterium avium complex (MAC) accounted for 86.4% of NTM species, followed by M. abscessus complex (4.2%), whereas in extrapulmonary NTM, M. abscessus complex, MAC, M. chelonae, and M. fortuitum accounted for 36.0%, 28.0%, 12.0%, and 8.0%, respectively. Compared to pulmonary NTM, extrapulmonary NTM were significantly more likely to be rapid-growing mycobacteria (RGM) (56.0% vs. 5.5%). The most common sites of infection were the skin and soft tissues (44.0%), followed by the blood (20.0%), tenosynovium, and lymph nodes (12.0%). CONCLUSION: Younger age and immunosuppressive conditions are associated with extrapulmonary NTM, with a higher prevalence of RGM in extrapulmonary NTM than in pulmonary NTM. These results provide a better understanding of extrapulmonary NTM.

Identification of risk factors for venous thromboembolism and validation of the Khorana score in patients with advanced lung cancer: based on the multicenter, prospective Rising-VTE/NEJ037 study data.

BACKGROUND: Management of cancer-associated venous thromboembolism (VTE) is essential in cancer treatment selection and prognosis. However, currently, no method exists for assessing VTE risk associated with advanced lung cancer. Therefore, we assessed VTE risk, including driver gene mutation, in advanced lung cancer and performed a Khorana score validation. METHODS: The Rising-VTE/NEJ037 study was a multicenter prospective observational study that included patients with advanced lung cancer. In the Rising-VTE/NEJ037 study, the Khorana score was calculated for enrolled patients with available data on all Khorana score components. The modified Khorana score was based on the body mass index of ≥ 25 kg/m2, according to the Japanese obesity standard. A multivariate logistic regression analysis, including patient background characteristics, was performed to evaluate the presence of VTE 2 years after the lung cancer diagnosis. RESULTS: This study included 1008 patients with lung cancer, of whom 100 (9.9%) developed VTE. From the receiver operating characteristic curve analysis, VTE risk could not be determined because both the Khorana score (0.518) and modified Khorana score (0.516) showed very low areas under the curve. The risk factors for VTE in the multivariate analysis included female sex, adenocarcinoma, performance status, N factor, lymphocyte count, platelet count, prothrombin fragment 1 + 2 and diastolic blood pressure. CONCLUSION: The Khorana score, which is widely used in cancer-VTE risk assessment, was less useful for Japanese patients with advanced lung cancer. Prothrombin fragment 1 + 2, a serum marker involved in coagulation, was more suitable for risk identification. CLINICAL TRIAL INFORMATION: jRCTs061180025.

Exhaled Nitric Oxide and Olfactory Dysfunction in Patients with Asthma: Association with Chronic Rhinosinusitis.

Objectives: Olfactory dysfunction is a clinical sign that is important to detect with coexistent upper airway comorbidities in patients with asthma. This study aimed to investigate the etiology of olfactory dysfunction in patients with asthma and the relationship between fractional exhaled nitric oxide (FeNO) levels. Materials and Methods: This study included 47 asthma patients who were evaluated for olfactory dysfunction at Hiroshima University Hospital between 2012 and 2020. The etiologies of olfactory dysfunction were evaluated, and they were classified according to the FeNO levels of patients with asthma. Results: Olfactory dysfunction was observed in 30 patients with asthma, with chronic rhinosinusitis (77%) being the most prevalent etiology. Eosinophilic chronic rhinosinusitis (ECRS) was the most prevalent etiology of olfactory dysfunction in asthma patients with high FeNO levels (≥25 ppb), while non-eosinophilic chronic rhinosinusitis (NCRS) was the most prevalent etiology in asthma patients with low FeNO levels (<25 ppb). Additionally, the prevalence of ECRS was significantly higher in asthma patients with olfactory dysfunction and high FeNO levels (74%) than in those with either high FeNO levels or olfactory dysfunction and those with low FeNO levels and no olfactory dysfunction (12% and 9%, respectively). Conclusions: We found that ECRS was the predominant cause of olfactory dysfunction in patients with high FeNO levels, while NCRS was more common in those with low FeNO levels. The present study showed that both ECRS and NCRS are common etiologies of olfactory dysfunction in patients with asthma. Additionally, this study supports the link between upper and lower airway inflammation in patients with asthma complicated with olfactory dysfunction.

Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Accelerated decline in lung function in adults with a history of remitted childhood asthma.

BACKGROUND: A significant number of children with asthma show remission in adulthood. Although these adults are often diagnosed with COPD in later life, the effect of clinically remitted childhood asthma on the decline in lung function during adulthood is uncertain. We examined whether clinical remission of childhood asthma was associated with an accelerated decline in lung function in apparently nonasthmatic adults. METHODS: 3584 participants (mean (range) age 48.1 (35-65) years) who did not have adulthood asthma and other lung diseases and had normal lung function at the baseline visit were included. They were categorised as those with remitted childhood asthma (n=121) and healthy controls (n=3463) according to their self-reported childhood asthma history. Spirometry was performed at baseline and follow-up visits. RESULTS: The mean follow-up was 5.3 years. Multivariate regression analysis showed that remitted childhood asthma and smoking were independently associated with a rapid decline in forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC). Smoking was an independent predictor of a rapid decline in FEV1/FVC. The annual decline in FEV1 and FVC was significantly greater in participants with remitted childhood asthma than in healthy controls, and the differences remained significant after adjusting for the propensity score. CONCLUSIONS: A history of clinically remitted childhood asthma is an independent risk factor for accelerated decline in lung function in adults. Remitted childhood asthma and smoking may additively accelerate the development of obstructive lung disease.

Objective discrimination of fur type based on electrophoresis optimized for fur-hair proteins.

The accurate identification of animal species used for fur is important for conserving endangered animals, stopping illegal fur distribution, and addressing consumer concerns. Animal species used for fur are currently differentiated by observing species-specific morphological fur-hair features through a microscope. Although this method is simple, the results may differ among inspectors owing to its subjective nature. To develop an objective approach for differentiating animal species based on fur, we utilized the electrophoretic patterns of fur-hair proteins. First, we optimized protein extraction methods to produce clear electrophoretic patterns from fur-hair proteins. Then, we obtained 324 electrophoretic patterns from 54 fur samples belonging to 24 different animals; 216 of the 324 patterns were used for the construction of a discrimination model using two-way orthogonal partial least squares discriminant analysis. The model correctly discriminated between all the remaining 108 patterns without any false negatives or positives. Moreover, this model could discriminate between fur samples from closely related species that are difficult to distinguish using conventional microscopic identification because of the visual similarity of the fur hairs.

Potential use of EGFR-targeted molecular therapies for tumor suppressor CYLD-negative and poor prognosis oral squamous cell carcinoma with chemoresistance.

BACKGROUND: Tumor suppressor CYLD dysfunction by loss of its expression, triggers malignant transformation, especially drug resistance and tumor invasion/metastasis. Although loss of CYLD expression is significantly associated with poor prognosis in a large variety of tumors, no clinically-effective treatment for CYLD-negative cancer patients is available. METHODS: We focused on oral squamous cell carcinoma (OSCC), and sought to develop novel therapeutic agents for CYLD-negative cancer patients with poor prognosis. CYLD-knockdown OSCC cells by using CYLD-specific siRNA, were used to elucidate and determine the efficacy of novel drug candidates by evaluating cell viability and epithelial-mesenchymal transition (EMT)-like change. Therapeutic effects of candidate drug on cell line-derived xenograft (CDX) model and usefulness of CYLD as a novel biomarker using patient-derived xenograft (PDX) model were further investigated. RESULTS: CYLD-knockdown OSCC cells were resistant for all currently-available cytotoxic chemotherapeutic agents for OSCC, such as, cisplatin, 5-FU, carboplatin, docetaxel, and paclitaxel. By using comprehensive proteome analysis approach, we identified epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, played key roles in CYLD-knockdown OSCC cells. Indeed, cell survival rate in the cisplatin-resistant CYLD-knockdown OSCC cells was markedly inhibited by treatment with clinically available EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib. In addition, gefitinib was significantly effective for not only cell survival, but also EMT-like changes through inhibiting transforming growth factor-ß (TGF-ß) signaling in CYLD-knockdown OSCC cells. Thereby, overall survival of CYLD-knockdown CDX models was significantly prolonged by gefitinib treatment. Moreover, we found that CYLD expression was significantly associated with gefitinib response by using PDX models. CONCLUSIONS: Our results first revealed that EGFR-targeted molecular therapies, such as EGFR-TKIs, could have potential to be novel therapeutic agents for the CYLD-negative OSCC patients with poor prognosis.

Effect of Insulin Receptor-Knockdown on the Expression Levels of Blood-Brain Barrier Functional Proteins in Human Brain Microvascular Endothelial Cells.

PURPOSE: The insulin receptor (INSR) mediates insulin signaling to modulate cellular functions. Although INSR is expressed at the blood-brain barrier (BBB), its role in the modulation of BBB function is poorly understood. Therefore, in this study, we aimed to analyze the effect of INSR knockdown on the expression levels of functional proteins at the BBB. METHODS: We established the INSR-knockdown cell line (shINSR) using human cerebral microvascular endothelial cells (hCMEC/D3). The cellular proteome was analyzed using quantitative proteomics. RESULTS: INSR mRNA and protein expressions were decreased in shINSR cells. The suppression of INSR-mediated signaling in shINSR cells was evaluated. The proteins involved in glycolysis and glycogenolysis were suppressed in shINSR cells. As amyloid-ß peptide-related proteins, the expressions of presenilin-1 was increased, and those of the insulin-degrading enzyme and neprilysin were decreased. The expressions of BBB transporters, including the ABCB1/MDR1, ABCG2/BCRP, and SLCO2A1/OATP2A1 were significantly decreased by more than 50% in shINSR cells. The efflux activity of ABCB1/MDR1 was also suppressed. The expressions of the low-density lipoprotein receptor-related protein 1 were significantly increased, and those of the transferrin receptor were significantly decreased in shINSR cells. The expression of claudin-5 was also suppressed in shINSR cells. CONCLUSIONS: The present study suggests that INSR-mediated signaling is involved in the regulation of functional protein expression at the BBB and contributes to the maintenance of BBB function. Changes in the expressions of amyloid-ß peptide-related proteins may contribute to the development of cerebral amyloid angiopathy via the suppression of INSR-mediated signaling.