Performance of mNGS in bronchoalveolar lavage fluid for the diagnosis of invasive pulmonary aspergillosis in non-neutropenic patients.

The diagnosis of invasive pulmonary aspergillosis (IPA) diseases in non-neutropenic patients remains challenging. It is essential to develop optimal non-invasive or minimally invasive detection methods for the rapid and reliable diagnosis of IPA. Metagenomic next-generation sequencing (mNGS) in bronchoalveolar lavage fluid (BALF) can be a valuable tool for identifying the microorganism. Our study aims to evaluate the performance of mNGS in BALF in suspected IPA patients and compare it with other detection tests, including serum/BALF galactomannan antigen (GM) and traditional microbiological tests (BALF fungal culture and smear and lung biopsy histopathology). Ninety-four patients with suspicion of IPA were finally enrolled in our study. Thirty-nine patients were diagnosed with IPA, and 55 patients were non-IPA. There was significance between the IPA and non-IPA groups, such as BALF GM (P < 0.001), history of glucocorticoid use (P = 0.004), and pulmonary comorbidities (P = 0.002), as well as no significance of the other demographic data including age, sex, BMI, history of cigarette, blood GM assay, T-SPOT.TB, and NEUT#/LYMPH#. The sensitivity of the BALF mNGS was 92.31%, which was higher than that of the traditional tests or the GM assays. The specificity of BALF mNGS was 92.73%, which was relatively similar to that of the traditional tests. The AUC of BALF mNGS was 0.925, which presented an excellent performance compared with other traditional tests or GM assays. Our study demonstrated the important role of BALF detection by the mNGS platform for pathogen identification in IPA patients with non-neutropenic states, which may provide an optimal way to diagnose suspected IPA disease.

Characteristics and comparison of rapidly growing and slowly growing nontuberculous mycobacterial pulmonary disease.

Background: Nontuberculous mycobacterial (NTM) pulmonary disease (PD) has rapidly increased globally. The characteristics and comparison of rapidly growing mycobacteria PD (RGM-PD) and slowly growing mycobacteria PD (SGM-PD) are still unclear. Methods: Our study enrolled 31 NTM-PD patients. Clinical data, including baseline, symptoms, underlying disease, laboratory tests, metagenomic next-generation sequencing (mNGS) results, radiological images, treatment, and outcome were recorded and analyzed. Results: Of the 31 patients with NTM-PD, 22 patients were female and 9 were male. It included 11 RGM-PD and 20 SGM-PD. There was no difference in age (P = 0.425) and body mass index (P = 0.152) between the two groups. The common respiratory diseases in prevalence included bronchiectasis and chronic obstructive pulmonary disease. Three patients had positive results of T-SPOT tuberculosis (TB), and none had positive Xpert-Mycobacterium tuberculosis/rifampin results. On admission, patients were symptomatic and included cough/sputum production, fever, weight loss, fatigue, and hemoptysis. In comparison to RGM-PD, patients with SGM-PD had a greater chance of experiencing fatigue (P = 0.012). No significance was found in serum biomarkers between RGM and SGM-PD, including CD4/CD8 ratio, white blood cells, neutrophils, lymphocytes, procalcitonin, ferritin, C-reactive protein, and erythrocyte sedimentation rate. No liver or kidney impairment was found. Patients with RGM-PD were more likely to have right lower lobe (RLL) impairment (P = 0.021) and a cavity characteristic (P = 0.012). All 31 cases had positive mNGS results. The duration of mNGS was shorter than conventional methods (3.4 ± 0.7 vs. 26.4 ± 20.9, P < 0.001). Conclusions: Patients with SGM-PD were more likely to experience fatigue. The cavity and RLL involvement were more frequent in the RGM-PD. mNGS increases the identification of NTM specimens and complements the capabilities of conventional methods.

Hub Genes and Immune Cell Infiltration in Hypoxia-Induced Pulmonary Hypertension: Bioinformatics Analysis and In Vivo Validation.

BACKGROUND: Hypoxia-induced pulmonary hypertension (HPH) represents a severe pulmonary disorder with high morbidity and mortality, which necessitates identifying the critical molecular mechanisms underlying HPH pathogenesis. METHODS: The mRNA expression microarray GSE15197 (containing 8 pulmonary tissues from HPH and 13 normal controls) was downloaded from Gene Expression Omnibus (GEO). Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) were executed by RStudio software. The Protein-Protein Interaction (PPI) network was visualized and established using Cytoscape, and the cytoHubba app from Cytoscape was used to pick out the hub modules. The infiltration of immune cells in HPH was analyzed using the CIBERSORTx. To confirm the potential hub genes, real-time quantitative reverse transcription PCR (qRT-PCR) was conducted using lung tissues of rat HPH models and controls. RESULTS: A total of 852 upregulated and 547 downregulated genes were identified. The top terms in biological processes were apoptosis, proliferation, and regulation of the MAPK cascade, including ERK1/2. Cytoplasm, cytosol, and membrane were enriched in cellular component groups. Molecular functions mainly focus on protein binding, protein serine/threonine kinase activity and identical protein binding. KEGG analysis identified pathways in cancer, regulation of actin cytoskeleton and rap1 signaling pathway. There was significantly different immune cell infiltration between HPH and normal control samples. High proportions of the memory subsets of B cells and CD4 cells, Macrophages M2 subtype, and resting Dendritic cells were found in HPH samples, while high proportions of naive CD4 cells and resting mast cells were found in normal control samples. The qRT-PCR results showed that among the ten identified hub modules, FBXL3, FBXL13 and XCL1 mRNA levels were upregulated, while NEDD4L, NPFFR2 and EDN3 were downregulated in HPH rats compared with control rats. CONCLUSION: Our study revealed the key genes and the involvement of immune cell infiltration in HPH, thus providing new insight into the pathogenesis of HPH and potential treatment targets for patients with HPH.

EphrinA3 is a key regulator of malignant behaviors and a potential prognostic factor in lung adenocarcinoma.

BACKGROUND: As a member of the Ephrin protein family that elicits short distance cell-cell signaling, EphrinA3 has been shown to promote or inhibit tumorigenesis depending on tumor types, but its roles and the underlying mechanisms in lung adenocarcinoma (LUAD) have not been reported. MATERIALS AND METHODS: The TCGA database and Kaplan-Meier Plotter database were used to analyze the differential expression of EphrinA3 between LUAD and para-carcinoma tissues, and its effect on overall survival of LUAD patients. CCK-8 assay, Edu assay, and flow cytometry were used to probe the effect of EphrinA3 on the proliferation of LUAD cells, and transwell assay was employed to examine its effect on migration and invasion. In addition, the effect of EphrinA3 on the growth of LUAD was further evaluated using a xenograft tumor model. RESULTS: EphrinA3 was expressed highly in LUAD, and its expression level was negatively correlated with the prognosis of LUAD patients. In addition, EphrinA3 promoted proliferation, migration, and invasion of LUAD cells, and accelerated tumor growth in a xenograft LUAD model. The reported EphrinA3 receptors, EphA1 and EphA10, were expressed in clinical LUAD tissues and co-localized with EphrinA3 in LUAD cells. Mechanistically, EphrinA3/Eph signaling activated AKT, ERK, and p38MAPK, induced epithelial-mesenchymal transition (EMT), and upregulated matrix metalloproteases-2 and -9 (MMP-2/-9). CONCLUSION: EphrinA3 expression was negatively correlated with prognosis of patients with LUAD. EphrinA3 promoted proliferation, migration, and invasion of LUAD cells. EphrinA3 enhanced the phosphorylation of ERK and AKT, and potentiates EMT and MMP expression in LUAD cells.

Transcriptional upregulation of MAPK15 by NF-κB signaling boosts the efficacy of combination therapy with cisplatin and TNF-α.

The efficacy of cisplatin in treating advanced non-small cell lung cancer is limited mainly because of insensitivity and/or acquired resistance. MAPK15, previously shown by us to enhance the sensitivity of the anti-cancer drug arsenic trioxide, could also enhance the sensitivity of other anti-cancer drugs. Here, we explore the potential role of MAPK15 in chemosensitivity to cisplatin in human lung cancer cells. Our results indicated that the expression level of MAPK15 was positively correlated with cisplatin sensitivity through affecting the DNA repair capacity of cisplatin-treated cells. The expression of MAPK15 was transcriptionally regulated by the TNF-α-activated NF-κB signaling pathway, and TNF-α synergized with cisplatin, in a MAPK15-dependent manner, to exert cytotoxicity in vitro and in vivo. Therefore, levels of TNF-α dictate the responsiveness/sensitivity of lung cancer cells to cisplatin by transcriptionally upregulating MAPK15 to enhance chemosensitivity, suggesting manipulation of MAPK15 as a strategy to improve the therapeutic efficacy of chemotherapeutic drugs.

Protein tyrosine phosphatase PTPL1 suppresses lung cancer through Src/ERK/YAP1 signaling.

BACKGROUND: To reveal the function of protein tyrosine phosphatase-L1 (PTPL1) in lung adenocarcinoma. METHODS: Lung cancer cell lines were transfected with short hairpin RNA against PTPL1 (shPTPL1 group) or negative control (shmock group). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to verify the transfection efficacy. Cell proliferation was analyzed by ethynyldeoxyuridine (EdU), Cell counting kit 8 (CCK8), and colony formation assay after PTPL1 or PTPL1 and yes-associated protein (YAP1) knockdown. The effect of PTPL1 on tumor growth was examined in a xenograft lung cancer model. RESULTS: PTPL1 was downregulated in various types of lung cancer cell lines. The EdU, CCK8, colony formation assays and investigation using a xenograft lung cancer model indicated that PTPL1 knockdown increased the proliferation of lung cancer cells. Mechanistically, PTPL1 knockdown induced the activation of the Proto-oncogene tyrosine-protein kinase SRC (Src)/Extracellular regulated MAP kinase (ERK) pathway and thereby promoted yes-associated protein (YAP1) nuclear translocation and activation. CONCLUSIONS: In our study, PTPL1 played a crucial suppressive role in the pathogenesis of lung cancer potentially through counteracting the Src/ERK/YAP1 pathway.

Discovery and optimization of 4-anilinoquinazoline derivatives spanning ATP binding site and allosteric site as effective EGFR-C797S inhibitors.

Epidermal growth factor receptor (EGFR) is an effective drug target for the treatment of non-small cell lung cancer (NSCLC). However, a tertiary point mutation (C797S) at the ATP binding pocket of the EGFR induces resistance to the third-generation EGFR inhibitors, due to the loss of covalent interaction with Cys797. Here, we designed a series of 4-anilinoquinazoline derivatives that simultaneously occupied the ATP binding pocket and the allosteric site. The newly-synthesized compounds displayed high potency against EGFR-C797S resistance mutation. Among them, compound 14d presented high anti-proliferative effect against BaF3-EGFRL858R/T790M/C797S (IC50 = 0.75 µM) and BaF3-EGFR19del/T790M/C797S (IC50 = 0.09 µM) cells. Moreover, 14d resulted in obvious inhibition activities against EGFR and its downstream signaling pathways in a dose-dependent manner in BaF3-EGFR19del/T790M/C797S cells. Finally, 14d significantly inhibited tumor growth in BaF3-EGFR19del/T790M/C797S xenograft model (30 mg/kg, TGI = 67.95%). These results demonstrated that 14d is a novel and effective EGFR-C797S inhibitor which spanning the ATP binding pocket and the allosteric site and effective both in vitro and in vivo.

Mxi1-0 Promotes Hypoxic Pulmonary Hypertension Via ERK/c-Myc-dependent Proliferation of Arterial Smooth Muscle Cells.

Background: Hypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality, and so far patients have failed to benefit from therapeutics clinically available. Max interacting protein 1-0 (Mxi1-0) is one of the functional isoforms of Mxi1. Although it also binds to Max, Mxi1-0, unlike other Mxi1 isoforms, cannot antagonize the oncoprotein c-Myc because of its unique proline rich domain (PRD). While Mxi1-0 was reported to promote cell proliferation via largely uncharacterized mechanisms, it is unknown whether and how it plays a role in the pathogenesis of HPH. Methods: GEO database was used to screen for genes involved in HPH development, and the candidate players were validated through examination of gene expression in clinical HPH specimens. The effect of candidate gene knockdown or overexpression on cultured pulmonary arterial cells, e.g., pulmonary arterial smooth muscle cells (PASMCs), was then investigated. The signal pathway(s) underlying the regulatory role of the candidate gene in HPH pathogenesis was probed, and the outcome of targeting the aforementioned signaling was evaluated using an HPH rat model. Results: Mxi1 was significantly upregulated in the PASMCs of HPH patients. As the main effector isoform responding to hypoxia, Mxi1-0 functions in HPH to promote PASMCs proliferation. Mechanistically, Mxi1-0 improved the expression of the proto-oncogene c-Myc via activation of the MEK/ERK pathway. Consistently, both a MEK inhibitor, PD98059, and a c-Myc inhibitor, 10058F4, could counteract Mxi1-0-induced PASMCs proliferation. In addition, targeting the MEK/ERK signaling significantly suppressed the development of HPH in rats. Conclusion: Mxi1-0 potentiates HPH pathogenesis through MEK/ERK/c-Myc-mediated proliferation of PASMCs, suggesting its applicability in targeted treatment and prognostic assessment of clinical HPH.

Effects on the Cell Barrier Function of L-Met and DL-HMTBA Is Related to Metabolic Characteristics and m6A Modification.

Methionine is a substrate for protein synthesis and participates in many other biological events via its metabolism. We have previously demonstrated significant differences in the metabolism of L-methionine (L-Met) and its precursor DL-2-hydroxy-4-methylthiobutyric acid (DL-HMTBA) in IPEC-J2 cells. When DL-HMTBA is added to the diet, intracellular methionine (Met) sources also contain the natural form of L-Met. Then, what is the effect on Met metabolism when these two Met sources exist simultaneously? Moreover, the effects of metabolic differences on cell function remain unclear. In this study, it was found that when the proportion of L-Met to DL-HMTBA was ≤ 40%:60%, Met transmethylation was promoted and when the proportion of L-Met to DL-HMTBA was ≤ 85%:15%, Met trans-sulfuration and regeneration were improved. In addition, DL-HMTBA improved the cell barrier function when the ratio of L-Met to DL-HMTBA was ≤ 40%:60%. This finding may be due to the decrease in the proportion of S-adenosylmethionine to S-adenosylhomocysteine and mRNA N 6-methyladenosine (m6A) levels, which increase the mRNA stability and protein expression of tight junction zona occludens-1. To sum up, the effects of L-Met and DL-HMTBA on Met metabolism, especially transmethylation, suggest that DL-HMTBA has the potential to influence the intestinal barrier function of animals through epigenetic processes.

Diagnostic efficiency and safety of rapid on-site evaluation combined with CT-guided transthoracic core needle biopsy in suspected lung cancer patients.

OBJECTIVE: The efficacy of rapid on-site evaluation (ROSE) combined with computed tomography-guided transthoracic core needle biopsy (CT-guided TCNB) is rarely investigated. This study aimed to evaluate the diagnostic efficiency and safety of ROSE combined with CT-guided TCNB for suspected lung cancer patients. MATERIALS AND METHODS: Clinical data from 285 patients who received CT-guided TCNB for suspected lung cancer in Huashan Hospital from 2015 to 2018 were retrospectively analysed. Of these 163 patients underwent CT-guided TCNB combined with ROSE (ROSE group), while the remaining 122 patients underwent without ROSE (non-ROSE group). The smears from TCNB were quickly processed with Diff-Quick staining and analysed by a skilled cytologist on-site. The consistency of ROSE with the final clinicopathological diagnosis and the diagnostic efficiency and safety of ROSE combined with CT-guided TCNB in suspected lung cancer patients were evaluated. RESULTS: ROSE was highly concordant with pathological diagnosis (κ = 0.791; P < 0.001), with an accuracy of 95.7%. Diagnostic accuracy was significantly higher in the ROSE compared with the non-ROSE group (96.3% vs 86.1%; P = 0.002), with overall incidences of complications of 36.8% and 23.8%, respectively. Minor pneumothorax without drainage was slightly greater in the ROSE compared with the non-ROSE group (14.1% vs 6.6%; P = 0.046). However, there was no significant difference in serious complications between the two groups. CONCLUSION: ROSE was highly consistent with the final clinicopathological diagnosis for suspected lung cancer. ROSE further improved the diagnostic efficiency of CT-guided TCNB with no increased incidence of serious complications.

Downregulation of NEDD4L by EGFR signaling promotes the development of lung adenocarcinoma.

Cumulative evidence indicates that the abnormal regulation of the NEDD4 family of E3-ubiquitin ligases participates in the tumorigenesis and development of cancer. However, their role in lung adenocarcinoma (LUAD) remains unclear. This study comprehensively analyzed the NEDD4 family in LUAD data sets from public databases and found only NEDD4L was associated with the overall survival of LUAD patients. Gene set enrichment analysis (GSEA) indicated that NEDD4L might be involved in the regulation of mTORC1 pathway. Both cytological and clinical assays showed that NEDD4L inhibited the activity of the mTOR signaling pathway. In vivo and in vitro experiments showed that NEDD4L could significantly inhibit the proliferation of LUAD cells. In addition, this study also found that the expression of NEDD4L was regulated by EGFR signaling. These findings firstly revealed that NEDD4L mediates an interplay between EGFR and mTOR pathways in LUAD, and suggest that NEDD4L held great potential as a novel biomarker and therapeutic target for LUAD.

Diagnostic Accuracy of Metagenomic Next-Generation Sequencing in Sputum-Scarce or Smear-Negative Cases with Suspected Pulmonary Tuberculosis.

OBJECTIVE: To investigate the diagnostic accuracy of metagenomic next-generation sequencing (mNGS) in bronchoalveolar lavage fluid (BALF) samples or lung biopsy specimens from which suspected pulmonary tuberculosis (PTB) patients have no sputum or negative smear. MATERIALS AND METHODS: Sputum-scarce or smear-negative cases with suspected PTB (n = 107) were analyzed from January 2018 to June 2020. We collected BALF or lung tissue biopsy samples with these cases of suspected TB during hospitalization. The diagnostic accuracy of mNGS for these samples was compared with those of conventional tests or the T-SPOT.TB assay. RESULTS: 46 cases of PTB patients and 61 cases of non-PTB patients were finally enrolled and analyzed. mNGS exhibited a sensitivity of 89.13%, which was higher than conventional tests (67.39%) but equivalent to those of the T-SPOT.TB assay alone (76.09%) or T-SPOT.TB assay in combination with conventional tests (91.30%). The specificity of mNGS was 98.36%, similar to conventional tests (95.08%) but significantly higher than those of the T-SPOT.TB assay alone (65.57%) or the T-SPOT.TB assay in combination with conventional tests (63.93%). There was no significant difference in the diagnostic accuracy of mNGS in BALF samples and lung biopsy tissue specimens. CONCLUSION: Our findings demonstrate that mNGS could offer improved detection of Mycobacterium tuberculosis in BALF or lung tissue biopsy samples in sputum-scarce or smear-negative cases with suspected PTB.

Distribution of COPD Comorbidities and Creation of Acute Exacerbation Risk Score: Results from SCICP.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) often coexists with multiple comorbidities which may have a significant impact on acute exacerbations of patients. At present, what kind of comorbidities affects acute exacerbations and how comorbidities lead to poor prognosis are still controversial. The purpose of our study is to determine the impact of comorbidities on COPD exacerbation and establish an acute exacerbation risk assessment system related to comorbidities. METHODS: A total of 742 COPD patients participated in the Shanghai COPD Investigation on Comorbidity Program (SCICP, ChiCTR2000030911). Finally, the baseline information of 415 participants and one-year follow-up data were involved in the analysis. We collected hemogram indices, pulmonary function tests and acute exacerbation of COPD with regular medical follow-up. Q-type cluster analysis was used to determine the clusters of participants. Receiver operating characteristic (ROC) analysis was constructed to assess the ability of indicators in predicting acute exacerbations. RESULTS: Almost 65% of the population we investigated had at least one comorbidity. The distribution and incidence of comorbidities differed between exacerbation group and non-exacerbation group. Three comorbidity clusters were identified: (1) respiratory, metabolic, immune and psychologic disease (non-severe cases); (2) cardiovascular and neoplastic disease (severe cases); (3) less comorbidity. Different sub-phenotypes of COPD patients showed significant distinction in health status. Anxiety (OR=5.936, P=0.001), angina (OR=10.155, P=0.025) and hypertension (OR=3.142, P=0.001) were found to be independent risk factors of exacerbation in a year. The novel risk score containing BODEx and four diseases showed great prognostic value of COPD exacerbation in developing sample. CONCLUSION: Our study detailed the major interaction between comorbidities and exacerbation in COPD. Noteworthily, a novel risk score using comprehensive index - BODEx - and comorbidity parameters can identify patients at high risk of acute exacerbation.

Peroxisome proliferator-activated receptor γ improves pemetrexed therapeutic efficacy in non-squamous non-small cell lung cancer.

OBJECTIVE: The folic acid analog pemetrexed (PMX) is recommended for the first-line chemotherapy for advanced non-squamous non-small cell lung cancer (NSCLC). However, the mechanisms underlying PMX cytotoxicity in NSCLC remain to be fully explored. METHODS: PMX effect was evaluated in a urethane-induced lung adenocarcinoma mouse model. The interaction between PMX and intracellular proteins, particularly peroxisome proliferator-activated receptor γ (PPARγ), was investigated. The role of PPARγ in mediating pemetrexed cytotoxicity was investigated using NSCLC cell lines, mouse models and clinical specimens. RESULTS: This study found that PPARγ expression was correlated with prolonged progression-free survival in NSCLC patients. PPARγ downregulated hypoxanthine-guanine phosphoribosyl transferase (HGPRT), a key enzyme for nucleotide salvage synthesis, thereby sensitizing cells to PMX inhibition on nucleotide de novo synthesis. PMX was also a candidate partial agonist of PPARγ, and PMX-activated PPARγ bound to NF-κB and transcriptionally suppressed the NF-κB target gene, c-Myc. PMX inhibited tumor growth by activating PPARγ in a urethane-induced lung cancer model characterized by elevated NF-κB activity. CONCLUSION: PPARγ improves pemetrexed therapeutic efficacy in non-squamous NSCLC. The cytotoxicity effect of PMX can be synergized by activating PPARγ and thereby inhibiting NF-κB pathway.

PTPL1 suppresses lung cancer cell migration via inhibiting TGF-ß1-induced activation of p38 MAPK and Smad 2/3 pathways and EMT.

Epithelial-mesenchymal transition (EMT) enables dissemination of neoplastic cells and onset of distal metastasis of primary tumors. However, the regulatory mechanisms of EMT by microenvironmental factors such as transforming growth factor-ß (TGF-ß) remain largely unresolved. Protein tyrosine phosphatase L1 (PTPL1) is a non-receptor protein tyrosine phosphatase that plays a suppressive role in tumorigenesis of diverse tissues. In this study we investigated the role of PTPL1/PTPN13 in metastasis of lung cancer and the signaling pathways regulated by PTPL1 in terms of EMT of non-small cell lung cancer (NSCLC) cells. We showed that the expression of PTPL1 was significantly downregulated in cancerous tissues of 23 patients with NSCLC compared with adjacent normal tissues. PTPL1 expression was positively correlated with overall survival of NSCLC patients. Then we treated A549 cells in vitro with TGF-ß1 (10 ng/mL) and assessed EMT. We found that knockdown of PTPL1 enhanced the migration and invasion capabilities of A549 cells, through enhancing TGF-ß1-induced EMT. In nude mice bearing A549 cell xenografts, knockdown of PTPL1 significantly promoted homing of cells and formation of tumor loci in the lungs. We further revealed that PTPL1 suppressed TGF-ß-induced EMT by counteracting the activation of canonical Smad2/3 and non-canonical p38 MAPK signaling pathways. Using immunoprecipitation assay we demonstrated that PTPL1 could bind to p38 MAPK, suggesting that p38 MAPK might be a direct substrate of PTPL1. In conclusion, these results unravel novel mechanisms underlying the regulation of TGF-ß signaling pathway, and have implications for prognostic assessment and targeted therapy of metastatic lung cancer.

Ubiquitin carboxyl-terminal hydrolase isozyme L1/UCHL1 suppresses epithelial-mesenchymal transition and is under-expressed in cadmium-transformed human bronchial epithelial cells.

Cadmium (Cd), a highly toxic heavy metal, is widespreadly distributed in the environment. Chronic exposure to Cd is associated with the development of several diseases including cancers. Over the decade, many researches have been carried on various models to examine the acute effects of Cd; yet, limited knowledge is known about the long-term Cd exposure, especially in the human lung cells. Previously, we showed that chronic Cd-exposed human bronchial epithelial BEAS-2B cells exhibited transformed cell properties, such as anchorage-independent growth, augmented cell migration, and epithelial-mesenchymal transition (EMT). To study these Cd-transformed cells more comprehensively, here, we further characterized their subproteomes. Overall, a total of 63 differentially expressed proteins between Cd-transformed and passage-matched control cells among the five subcellular fractions (cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal) were identified by mass spectrometric analysis and database searching. Interestingly, we found that the thiol protease ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is one of the severely downregulated proteins in the Cd-transformed cells. Notably, the EMT phenotype of Cd-transformed cells can be suppressed by forced ectopic expression of UCHL1, suggesting UCHL1 as a crucial modulator in the maintenance of the proper differentiation status in lung epithelial cells. Since EMT is considered as a critical step during malignant cell transformation, finding novel cellular targets that can antagonize this transition may lead to more efficient strategies to inhibit cancer development. Our data report for the first time that UCHL1 may play a function in the suppression of EMT in Cd-transformed human lung epithelial cells, indicating that UCHL1 might be a new therapeutic target for chronic Cd-induced carcinogenesis. Graphical abstract.

Pharmacokinetic comparative study of GAS with different concentration of tetramethylpyrazine and ferulic acid on liver-yang hyperactivity migraine model by blood-brain microdialysis method.

Gastrodia elata and Ligusticum chuanxiong are used to treat primary headaches for many years. Gastrodin (GAS) and tetramethylpyrazine (TMP)/ferulic acid (FA) are the main active ingredients of Gastrodia elata and Ligusticum chuanxiong, respectively. Previous studies demonstrated the pharmacokinetics of GAS, TMP, and FA in the blood and brain interstitial fluids (BIF) in healthy animals but not in animal model with liver-yang hyperactivity migraine. Hence, this study examined the pharmacokinetics of GAS after its oral administration in the presence of different concentrations of TMP and FA in animals with liver-yang migraine hyperactivity. In the control group, GAS was administrated without TMP and FA. Pharmacokinetic parameters were determined using the blood-brain microdialysis in combination with the high-performance liquid chromatography method. Results revealed that the maximum drug concentrations (Cmax) in the serum, area under curve (AUC), and mean residence time (MRT) of GAS decreased in normal animals, whereas Cmax increased significantly in model animals. These findings indicate that varying concentrations of TMP and FA play an important role in the pharmacokinetics of GAS in both normal and migraine model animals, validating the utility of the ancient formulation.

Expert consensus for diagnosis and treatment using medical thoracoscopy in China.

Medical thoracoscopy is a commonly used endoscopic technique for the diagnosis and treatment of respiratory diseases. As an invasive technique, it is mainly used for pleural effusions and pleural diseases that cannot be diagnosed by non-invasive methods. It is also of great application in the diagnosis and treatment of certain other diseases. Any technical operation requires special skills. There must be a learning process for mastering these skills. Although internal thoracoscopic surgery is simple, especially for respiratory specialists who have undergone training for thoracentesis or closed drainage, there are discrepancies in thoracoscopic diagnosis and treatment in hospitals in China; furthermore, the surgical methods are not uniform, and some even lead to serious complications. Therefore, the thoracoscopic diagnostic and treatment technology in China needs to be standardized. The Respiratory Professional Committee of the Integrated Medical Branch of the Chinese Medical Doctor Association invited relevant Chinese experts to formulate this standard after several rounds of discussion.

Design, synthesis and biological evaluation of potent EGFR kinase inhibitors against 19D/T790M/C797S mutation.

The efficacy of EGFR inhibitors is frequently affected by acquired resistance. EGFR19D/T790M/C797S mutation is one of the primary reasons for the emergence of resistance after treatment with the third-generation EGFR inhibitors such as AZD9291, CO1686 and Olmutinib. To overcome the resistance mutation 19D/T790M/C797S, we designed and prepared a series of indole derivatives with the terminal hydroxyl of alkyl chain to increase extra interaction with the Asp855 in the conservative DFG site. Activity evaluation, structure-activity relationship and docking analysis were also carried out. Among them, compound 12e displayed significant inhibitory activity against EGFR19D/T790M/C797S (IC50 = 15.3 nM) and good selectivity over EGFR WT (IC50 > 1000 nM), L858R/T790M (IC50, 156.6 nM) and L858R/T790M/C797S (IC50, 218.3 nM) respectively. Furthermore, 12e exhibited good growth inhibition activity, induced G1 phase cell cycle arrest and apoptosis in BaF3/EGFR19D/T790M/C797S cells by suppressing EGFR phosphorylation signaling pathway. In all, our study might provide a novel structural design method and lay the solid foundation for the development of the 4th generation EGFR19D/T790M/C797S inhibitors.