Long-read sequencing reveals the landscape of aberrant alternative splicing and novel therapeutic target in colorectal cancer.

BACKGROUND: Alternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC). METHODS: Here, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4-5 exclusion was introduced to inhibit neoplasm growth. RESULTS: A total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients' prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4-5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4-5 exclusion have the potential to restrain the CRC growth. CONCLUSIONS: This data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

Expert consensus on indocyanine green fluorescence imaging for thoracoscopic lung resection (The Version 2022).

The use of the white-light thoracoscopy is hampered by the low contrast between oncologic margins and surrounding normal parenchyma. As a result, many patients with in situ or micro-infiltrating adenocarcinoma have to undergo lobectomy due to a lack of tactile and visual feedback in the resection of solitary pulmonary nodules. Near-infrared (NIR) guided indocyanine green (ICG) fluorescence imaging technique has been widely investigated due to its unique capability in addressing the current challenges; however, there is no special consensus on the evidence and recommendations for its preoperative and intraoperative applications. This manuscript will describe the development process of a consensus on ICG fluorescence-guided thoracoscopic resection of pulmonary lesions and make recommendations that can be applied in a greater number of centers. Specifically, an expert panel of thoracic surgeons and radiographers was formed. Based on the quality of evidence and strength of recommendations, the consensus was developed in conjunction with the Chinese Guidelines on Video-assisted Thoracoscopy, and the National Comprehensive Cancer Network (NCCN) guidelines on the management of pulmonary lesions. Each of the statements was discussed and agreed upon with a unanimous consensus amongst the panel. A total of 6 consensus statements were developed. Fluorescence-guided thoracoscopy has unique advantages in the visualization of pulmonary nodules, and recognition and resection of the anterior plane of the pulmonary segment. The expert panel agrees that fluorescence-guided thoracoscopic surgery has the potential to become a routine operation for the treatment of pulmonary lesions.

Andrographolide attenuates epithelial-mesenchymal transition induced by TGF-ß1 in alveolar epithelial cells.

Andrographolide (Andro), a component from Chinese medicinal herb Andrographis paniculata, could alleviate pulmonary fibrosis in rodents. Yet, whether and how Andro mitigates epithelial-mesenchymal transition (EMT) induced by TGF-ß1 remain unknown. This study aimed to explore the effect of Andro on TGF-ß1-induced EMT in human alveolar epithelial cells (AECs) and the mechanisms involved. We illustrated that Andro inhibited TGF-ß1-induced EMT and EMT-related transcription factors in alveolar epithelial A549 cells. Andro also reduced TGF-ß1-induced cell migration and synthesis of pro-fibrotic factors (ie CCN-2, TGF-ß1), matrix metalloproteinases (ie MMP-2, MMP-9) and extracellular matrix (ECM) components (ie collagen 1), implying the inhibiting effect of Andro on TGF-ß1-induced EMT-like cell behaviours. Mechanistically, Andro treatment not only repressed TGF-ß1-induced Smad2/3 phosphorylation and Smad4 nuclear translocation, but also suppressed TGF-ß1-induced Erk1/2 phosphorylation and nuclear translocation in A549 cells. And treatment with ALK5 inhibitor (SB431542) or Erk1/2 inhibitors (SCH772984 and PD98059) remarkably reduced EMT evoked by TGF-ß1. In addition, Andro also reduced TGF-ß1-induced intracellular ROS generation and NOX4 expression, and elevated antioxidant superoxide dismutase 2 (SOD2) expression, demonstrating the inhibiting effect of Andro on TGF-ß1-induced oxidative stress, which is closely linked to EMT. Furthermore, Andro remarkably attenuated TGF-ß1-induced down-regulation of sirtuin1 (Sirt1) and forkhead box O3 (FOXO3), implying that Andro protects AECs from EMT partially by activating Sirt1/FOXO3-mediated anti-oxidative stress pathway. In conclusion, Andro represses TGF-ß1-induced EMT in AECs by suppressing Smad2/3 and Erk1/2 signalling pathways and is also closely linked to the activation of sirt1/FOXO3-mediated anti-oxidative stress pathway.

Modulation of gut microbiota to overcome resistance to immune checkpoint blockade in cancer immunotherapy.

Immune checkpoint blockade therapies that target CTLA-4 and PD-1/PD-L1 have ushered in a new era of cancer treatment. Nevertheless, a significant proportion of patients demonstrated primary or acquired resistance. Harnessing gut microbiota has been an emerging novel therapeutic strategy to overcome resistance. Here we summarized the current research status of gut microbiota in immune checkpoint blockade therapies, clinical trials, underlying mechanisms and challenges of microbiome research in checkpoint immunotherapy. Findings from preclinical models, standardized microbiome analysis and progress of multi-omic approaches may better disclose the interaction between gut microbiota and immune checkpoint inhibitors (ICIs) and traditional Chinese medicine can be a potential microbiome modulator to sensitize the response to ICIs.

Andrographolide ameliorates bleomycin-induced pulmonary fibrosis by suppressing cell proliferation and myofibroblast differentiation of fibroblasts via the TGF-ß1-mediated Smad-dependent and -independent pathways.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no effective medication. Andrographolide (Andro), extracted from Chinese herbal Andrographis paniculata, could attenuate bleomycin (BLM)-induced pulmonary fibrosis via inhibition of inflammation and oxidative stress, however, the anti-fibrotic mechanisms have not been clarified. Myofibroblasts are the primary cell types responsible for the accumulation of extracellular matrix (ECM) in fibrotic diseases, and targeting fibroblast proliferation and differentiation is an important therapeutic strategy for the treatment of IPF. Hence, this study aimed to investigate the effects of Andro on the fibroblast proliferation and differentiation in the in vivo and in vitro models. The results showed that Andro improved pulmonary function and inhibited BLM-induced fibroblast proliferation and differentiation and ECM deposition in the lungs. In vitro, Andro inhibited proliferation and induced apoptosis of TGF-ß1-stimulated NIH 3T3 fibroblasts and primary lung fibroblasts (PLFs). Andro also inhibited TGF-ß1-induced myofibroblast differentiation and ECM deposition in both cells. We also found that Andro suppressed TGF-ß1-induced Smad2/3 and Erk1/2 activation, suggesting that Smad2/3 and Erk1/2 inactivation mediates Andro-induced effects on TGF-ß1-induced fibroblast proliferation and differentiation. These results indicated that Andro has novel and potent anti-fibrotic effects in lung fibroblasts via inhibition of the proliferation and myofibroblast differentiation of fibroblasts and subsequent ECM deposition, which are modulated by TGF-ß1-mediated Smad-dependent and -independent pathways.

Diaphragm electromyography guidance for a lung transplant recipient with difficult weaning from mechanical ventilation: A case report.

RATIONALE: Many factors contribute to a complicated postoperative course following difficult weaning off a ventilator after lung transplantation. PATIENT CONCERNS: A female patient underwent a successful surgery but received a size-mismatched lung graft. The graft had been pruned before transplantation. She experienced delayed ventilator weaning 3 days after lung transplantation. DIAGNOSES: A postoperative X-ray revealed a normal mediastinal structure and diaphragm position. Diaphragmatic function was assessed by diaphragm electromyography (EMGdi) via esophageal and surface electrodes. EMGdi showed decreased left compound motor action potentials (CMAPs), prolonged left phrenic nerve conduction time (PNCT), failure to induce right CMAPs and PNCT under bilateral magnetic stimulation, and right phrenic nerve injury. INTERVENTIONS: She was treated with neural nutritional support and prescribed rehabilitation measures such as strengthening limb activities on the bed. OUTCOMES: The patient finally achieved satisfactory outcomes after an early diagnosis and medical interventions. LESSONS: Lung size mismatch before transplantation and phrenic nerve injury during surgery should be avoided wherever possible.

Suppression of Lipogenesis via Reactive Oxygen Species-AMPK Signaling for Treating Malignant and Proliferative Diseases.

AIMS: Systemic diseases often have common characteristics. The aim of this study was to investigate the feasibility of targeting common pathological metabolism to inhibit the progression of malignant and proliferative diseases. RESULTS: Gefitinib-resistant (G-R) nonsmall-cell lung cancer (NSCLC) and rheumatoid arthritis (RA) were studied as conditions representative of malignant and proliferative diseases, respectively. Strong lipogenic activity and high expression of sterol regulatory element-binding protein 1 (SREBP1) were found in both G-R NSCLC cells and synovial fibroblasts from RA patients (RASFs). Berberine (BBR), an effective suppressor of SREBP1 and lipogenesis regulated through reactive oxygen species (ROS)/AMPK pathway, selectively inhibited the growth of G-R NSCLC cells and RASFs but not that of normal cells. It effectively caused mitochondrial dysfunction, activated ROS/AMPK pathway, and finally suppressed cellular lipogenesis and cell proliferation. Addition of ROS blocker, AMPK inhibitor, and palmitic acid significantly reduced the effect of BBR. In an in vivo study, treatment of BBR led to significant inhibition of mouse tumor xenograft growth and remarkably slowed down the development of adjuvant-induced arthritis in rats. Innovation and Conclusion: Targeting ROS/AMPK/lipogenesis signaling pathway selectively inhibited the growth of G-R NSCLC cells and the progress of RASFs in vitro and in vivo, which provides a new avenue for treating malignancies and proliferative diseases. Antioxid. Redox Signal. 28, 339-357.

Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis.

Chloride intracellular channel 1 (CLIC1) is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM) database using structure-based virtual screening and molecular dynamics (MD) simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

[Propensity Score Matching Analysis of VATS Lobectomy and Sublobar Resection for Stage I Lung Adenocarcinoma].

BACKGROUND: National Comprehensive Cancer Network (NCCN) guidelines recommend video-assisted thoracoscopic surgery (VATS) anatomical lobectomy as the first choice for the treatment of resectable lung cancer. However, sublobar resection offers significantly better functional preservation compared with lobectomy for stage I lung cancer. At present, the inferiority of sublobar resection to lobectomy is still uncertain. Herein, we compared the prognoses of these two types of surgical treatment for stage I lung adenocarcinoma. METHODS: Retrospective research was conducted on 258 patients with stage I lung adenocarcinomas who underwent VATS lobectomy and sublobar resection at the First Affiliated Hospital of Guangzhou Medical University between January 2009 and December 2011. VATS lobectomy was performed on 222 patients, and VATS sublobe resection was performed on 36 patients. Propensity score matching analyses were conducted on the two groups. RESULTS: A total of 70 patients were matched in the two groups. No significant difference was observed between the lobectomy and sublobar resection groups after matching (P=0.137). The disease-free survival (DFS) of the two groups were 49.3 and 42.7 months, and their overall survival (OS) were 50.3 and 49.0 months (P=0.122). Further, stratified analysis showed no significant differences in DFS and OS between the two groups with stage Ia lung adenocarcinoma. Nevertheless, the DFS and OS of the two groups significantly differed in matched patients with stage Ib lung adenocarcinomas. CONCLUSIONS: Sublobar resection could achieve a similar prognosis to VATS lobectomy for stage Ia lung adenocarcinoma. Lobectomy should still be the first choice for the treatment of stage Ib lung adenocarcinoma.

Spontaneous ventilation anaesthesia: total intravenous anaesthesia with local anaesthesia or thoracic epidural anaesthesia for thoracoscopic bullectomy.

OBJECTIVES: At present, few data exist regarding the comparisons of perioperative outcomes and recurrence of spontaneous ventilation (SV) video-assisted thoracic surgery (VATS) bullectomy using total intravenous anaesthesia (TIVA) with local anaesthesia (LA) or thoracic epidural anaesthesia (TEA). We evaluated the feasibility and safety of TIVA with LA in the management of primary spontaneous pneumothorax (PSP). METHODS: We conducted a single-institution retrospective analysis of patients undergoing VATS bullectomy between July 2011 and May 2015; 240 patients were included for analysis. Preoperative, intraoperative and postoperative variables of patients undergoing VATS bullectomy using TIVA-TEA (n = 140) were compared with those using TIVA-LA (n = 100). RESULTS: Baseline demographics were similar between groups. No patients in either group required conversion to thoracotomy. Three patients (TIVA-TEA: 2; TIVA-LA: 1) required conversion to intubated general anaesthesia. Both groups had comparable surgical duration, estimated blood loss, peak EtCO2 and lowest intraoperative SpO2 level. Postoperatively, thoracic drainage volume, duration of chest tube drainage and hospitalization cost did not differ between groups. The incidence of postoperative complications between groups was not significant (2% for TIVA-TEA vs 2% for TIVA-LA, P = 1.00). Pneumothorax recurrence rate was 3% in TIVA-TEA cases (n = 4) and 2% in TIVA-LA cases (n = 2). CONCLUSIONS: SV-VATS bullectomy using TIVA with LA or TEA is technically feasible and safe. Both groups have comparable short-term outcomes and recurrence rates; TIVA-LA seems a valid alternative to TIVA-TEA for the surgical management of PSP under SV.

Compound Library Screening Identified Cardiac Glycoside Digitoxin as an Effective Growth Inhibitor of Gefitinib-Resistant Non-Small Cell Lung Cancer via Downregulation of α-Tubulin and Inhibition of Microtubule Formation.

Non-small-cell lung cancer (NSCLC) dominates over 85% of all lung cancer cases. Epidermal growth factor receptor (EGFR) activating mutation is a common situation in NSCLC. In the clinic, molecular-targeting with Gefitinib as a tyrosine kinase inhibitor (TKI) for EGFR downstream signaling is initially effective. However, drug resistance frequently happens due to additional mutation on EGFR, such as substitution from threonine to methionine at amino acid position 790 (T790M). In this study, we screened a traditional Chinese medicine (TCM) compound library consisting of 800 single compounds in TKI-resistance NSCLC H1975 cells, which contains substitutions from leucine to arginine at amino acid 858 (L858R) and T790M mutation on EGFR. Attractively, among these compounds there are 24 compounds CC50 of which was less than 2.5 µM were identified. We have further investigated the mechanism of the most effective one, Digitoxin. It showed a significantly cytotoxic effect in H1975 cells by causing G2 phase arrest, also remarkably activated 5' adenosine monophosphate-activated protein kinase (AMPK). Moreover, we first proved that Digitoxin suppressed microtubule formation through decreasing α-tubulin. Therefore, it confirmed that Digitoxin effectively depressed the growth of TKI-resistance NSCLC H1975 cells by inhibiting microtubule polymerization and inducing cell cycle arrest.

A practical molecular assay to predict survival in resected non-squamous, non-small-cell lung cancer: development and international validation studies.

BACKGROUND: The frequent recurrence of early-stage non-small-cell lung cancer (NSCLC) is generally attributable to metastatic disease undetected at complete resection. Management of such patients depends on prognostic staging to identify the individuals most likely to have occult disease. We aimed to develop and validate a practical, reliable assay that improves risk stratification compared with conventional staging. METHODS: A 14-gene expression assay that uses quantitative PCR, runs on formalin-fixed paraffin-embedded tissue samples, and differentiates patients with heterogeneous statistical prognoses was developed in a cohort of 361 patients with non-squamous NSCLC resected at the University of California, San Francisco. The assay was then independently validated by the Kaiser Permanente Division of Research in a masked cohort of 433 patients with stage I non-squamous NSCLC resected at Kaiser Permanente Northern California hospitals, and on a cohort of 1006 patients with stage I-III non-squamous NSCLC resected in several leading Chinese cancer centres that are part of the China Clinical Trials Consortium (CCTC). FINDINGS: Kaplan-Meier analysis of the Kaiser validation cohort showed 5 year overall survival of 71·4% (95% CI 60·5-80·0) in low-risk, 58·3% (48·9-66·6) in intermediate-risk, and 49·2% (42·2-55·8) in high-risk patients (p(trend)=0·0003). Similar analysis of the CCTC cohort indicated 5 year overall survivals of 74·1% (66·0-80·6) in low-risk, 57·4% (48·3-65·5) in intermediate-risk, and 44·6% (40·2-48·9) in high-risk patients (p(trend)<0·0001). Multivariate analysis in both cohorts indicated that no standard clinical risk factors could account for, or provide, the prognostic information derived from tumour gene expression. The assay improved prognostic accuracy beyond National Comprehensive Cancer Network criteria for stage I high-risk tumours (p<0·0001), and differentiated low-risk, intermediate-risk, and high-risk patients within all disease stages. INTERPRETATION: Our practical, quantitative-PCR-based assay reliably identified patients with early-stage non-squamous NSCLC at high risk for mortality after surgical resection. FUNDING: UCSF Thoracic Oncology Laboratory and Pinpoint Genomics.

[Microscopical study of original plant of Chinese drug "Dragon's Blood" Dracaena cochinchinensis and distribution and constituents detection of its resin].

OBJECTIVE: To study the anatomy of Dracaena cochinchinensis systematically, and find out the distribution and detect the constituents of its resin, in order to provide substantial foundation for the formation mechanism of its red resin. METHOD: The microscopic structures of D. cochinchinensis were systematically observed by using color micrographics, including stem with and without resin, roots, barks and leaves. The HPLC fingerprints of the stem with and without resin were compared. RESULT: Characteristics of the tangentical longitudinal section of stem with resin and surface view of leaves were elucidated. Besides xylem vessels and fibers of the stem, it was found that the red resin also exists in the cortex parenchyma cells of the stem and the medulla and xylem of the root. According to the HPLC fingerprint analysis result of the stems with and without resin, a number of flavones and stilbenoids were detected in the stem in which resin appeared after it wounded. CONCLUSION: No secretory tissue to secrete resin was found in D. cochinchinensis, further study is needed to elucidate the formation mechanism of its resin.