CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

Quantitative systems pharmacology modeling of HER2-positive metastatic breast cancer for translational efficacy evaluation and combination assessment across therapeutic modalities.

HER2-positive (HER2+) metastatic breast cancer (mBC) is highly aggressive and a major threat to human health. Despite the significant improvement in patients' prognosis given the drug development efforts during the past several decades, many clinical questions still remain to be addressed such as efficacy when combining different therapeutic modalities, best treatment sequences, interindividual variability as well as resistance and potential coping strategies. To better answer these questions, we developed a mechanistic quantitative systems pharmacology model of the pathophysiology of HER2+ mBC that was extensively calibrated and validated against multiscale data to quantitatively predict and characterize the signal transduction and preclinical tumor growth kinetics under different therapeutic interventions. Focusing on the second-line treatment for HER2+ mBC, e.g., antibody-drug conjugates (ADC), small molecule inhibitors/TKI and chemotherapy, the model accurately predicted the efficacy of various drug combinations and dosing regimens at the in vitro and in vivo levels. Sensitivity analyses and subsequent heterogeneous phenotype simulations revealed important insights into the design of new drug combinations to effectively overcome various resistance scenarios in HER2+ mBC treatments. In addition, the model predicted a better efficacy of the new TKI plus ADC combination which can potentially reduce drug dosage and toxicity, while it also shed light on the optimal treatment ordering of ADC versus TKI plus capecitabine regimens, and these findings were validated by new in vivo experiments. Our model is the first that mechanistically integrates multiple key drug modalities in HER2+ mBC research and it can serve as a high-throughput computational platform to guide future model-informed drug development and clinical translation.

Wand-Based Calibration of Unsynchronized Multiple Cameras for 3D Localization.

Three-dimensional (3D) localization plays an important role in visual sensor networks. However, the frame rate and flexibility of the existing vision-based localization systems are limited by using synchronized multiple cameras. For such a purpose, this paper focuses on developing an indoor 3D localization system based on unsynchronized multiple cameras. First of all, we propose a calibration method for unsynchronized perspective/fish-eye cameras based on timestamp matching and pixel fitting by using a wand under general motions. With the multi-camera calibration result, we then designed a localization method for the unsynchronized multi-camera system based on the extended Kalman filter (EKF). Finally, extensive experiments were conducted to demonstrate the effectiveness of the established 3D localization system. The obtained results provided valuable insights into the camera calibration and 3D localization of unsynchronized multiple cameras in visual sensor networks.

In vitro co-culture systems of hepatic and intestinal cells for cellular pharmacokinetic and pharmacodynamic studies of capecitabine against colorectal cancer.

BACKGROUND: As a prodrug of 5-fluorouracil (5-FU), orally administrated capecitabine (CAP) undergoes preliminary conversion into active metabolites in the liver and then releases 5-FU in the gut to exert the anti-tumor activity. Since metabolic changes of CAP play a key role in its activation, a single kind of intestinal or hepatic cell can never be used in vitro to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) nature. Hence, we aimed to establish a novel in vitro system to effectively assess the PK and PD of these kinds of prodrugs. METHODS: Co-culture cellular models were established by simultaneously using colorectal cancer (CRC) and hepatocarcinoma cell lines in one system. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis were used to evaluate cell viability and apoptosis, respectively. Apoptosis-related protein expression levels were measured using western blot analysis. A selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for cellular PK in co-culture models. RESULTS: CAP had little anti-proliferative effect on the five monolayer CRC cell lines (SW480, LoVo, HCT-8, HCT-116 and SW620) or the hepatocarcinoma cell line (HepG2). However, CAP exerted marked anti-tumor activities on each of the CRC cell lines in the co-culture models containing both CRC and hepatocarcinoma cell lines, although its effect on the five CRC cell lines varied. Moreover, after pre-incubation of CAP with HepG2 cells, the culture media containing the active metabolites of CAP also showed an anti-tumor effect on the five CRC cell lines, indicating the crucial role of hepatic cells in the activation of CAP. CONCLUSION: The simple and cost­effective co-culture models with both CRC and hepatocarcinoma cells could mimic the in vivo process of a prodrug dependent on metabolic conversion to active metabolites in the liver, providing a valuable strategy for evaluating the PK and PD characteristics of CAP-like prodrugs in vitro at the early stage of drug development.

A Novel Vectorized Curved Road Representation Based Aerial Guided Unmanned Vehicle Trajectory Planning.

Unmanned vehicles frequently encounter the challenge of navigating through complex mountainous terrains, which are characterized by numerous unknown continuous curves. Drones, with their wide field of view and ability to vertically displace, offer a potential solution to compensate for the limited field of view of ground vehicles. However, the conventional approach of path extraction solely provides pixel-level positional information. Consequently, when drones guide ground unmanned vehicles using visual cues, the road fitting accuracy is compromised, resulting in reduced speed. Addressing these limitations with existing methods has proven to be a formidable task. In this study, we propose an innovative approach for guiding the visual movement of unmanned ground vehicles using an air-ground collaborative vectorized curved road representation and trajectory planning method. Our method offers several advantages over traditional road fitting techniques. Firstly, it incorporates a road star points ordering method based on the K-Means clustering algorithm, which simplifies the complex process of road fitting. Additionally, we introduce a road vectorization model based on the piecewise GA-Bézier algorithm, enabling the identification of the optimal frame from the initial frame to the current frame in the video stream. This significantly improves the road fitting effect (EV) and reduces the model running time (T-model). Furthermore, we employ smooth trajectory planning along the "route-plane" to maximize speed at turning points, thereby minimizing travel time (T-travel). To validate the efficiency and accuracy of our proposed method, we conducted extensive simulation experiments and performed actual comparison experiments. The results demonstrate the superior performance of our approach in terms of both efficiency and accuracy.

Distribution and identification of Mycobacterium tuberculosis lineage in Kashgar prefecture.

OBJECTIVES: Kashgar prefecture is an important transportation and trade hub with a high incidence of tuberculosis. The following study analyzed the composition and differences in Mycobacterium tuberculosis (M.tb) lineage and specific tags to distinguish the lineage of the M.tb in Kashgar prefecture, thus providing a basis for the classification and diagnosis of tuberculosis in this area. METHODS: Whole-genome sequencing (WGS) of 161 M.tb clinical strains was performed. The phylogenetic tree was constructed using Maximum Likelihood (ML) based on single nucleotide polymorphisms (SNPs) and verified through principal component analysis (PCA). The composition structure of M.tb in different regions was analyzed by combining geographic information. RESULTS: M.tb clinical strains were composed of lineage 2 (73/161, 45.34%), lineage 3 (52/161, 32.30%) and lineage 4 (36/161, 22.36%). Moreover, the 3 lineages were subdivided into 11 sublineages, among which lineage 2 included lineage 2.2.2/Asia Ancestral 1 (9/73, 12.33%), lineage 2.2.1-Asia Ancestral 2 (9/73, 12.33%), lineage 2.2.1-Asia Ancestral 3 (18/73, 24.66%), and lineage 2.2.1-Modern Beijing (39/73, 53.42%). Lineage 3 included lineage 3.2 (14/52, 26.92%) and lineage 3.3 (38/52, 73.08%), while lineage 4 included lineage 4.1 (3/36, 8.33%), lineage 4.2 (2/36, 5.66%), lineage 4.4.2 (1/36, 2.78%), lineage 4.5 (28/36, 77.78%) and lineage 4.8 (2/36, 5.66%), all of which were consistent with the PCA results. One hundred thirty-six markers were proposed for discriminating known circulating strains. Reconstruction of a phylogenetic tree using the 136 SNPs resulted in a tree with the same number of delineated clades. Based on geographical location analysis, the composition of Lineage 2 in Kashgar prefecture (45.34%) was lower compared to other regions in China (54.35%-90.27%), while the composition of Lineage 3 (32.30%) was much higher than in other regions of China (0.92%-2.01%), but lower compared to the bordering Pakistan (70.40%). CONCLUSION: Three lineages were identified in M.tb clinical strains from Kashgar prefecture, with 136 branch-specific SNP. Kashgar borders with countries that have a high incidence of tuberculosis, such as Pakistan and India, which results in a large difference between the M.tb lineage and sublineage distribution in this region and other provinces of China.

Efficacy and safety of anti-PD-1-based therapy in combination with PARP inhibitors for patients with advanced solid tumors in a real-world setting.

BACKGROUND: Rationale exists for combining immune checkpoint inhibitors and PARP inhibitors (PARPi), and results of clinical trials in ovarian cancer are promising, but data in other cancers are limited. METHOD: Efficacy and safety of PARPi/anti-PD-1 in advanced solid tumors were retrospectively analyzed. The efficacy measures included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). RESULTS: This retrospective study included data from 40 patients. The ORR was 27.5% (95% CI, 13.0-42.0%), with a DCR of 85.0% (95% CI, 73.4-96.6%). Except four patients in first-line treatment (three with PR and one with SD), the ORR of ≥second-line treatment, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) was 22.2%, 23.1% and 28.6%, and the DCR was 83.3%, 84.6% and 71.4%, separately. The median PFS of all patients, ≥second-line treatment, NSCLC and SCLC was 4.6 m, 4.2 m, 4.5 m and 3.7 m. The median OS was 9.4 m, 11.4 m, 12.7 m and 5.4 m, respectively. Multivariable analysis revealed that BRCA1/2 mutation was positively correlated with ORR (P = 0.008), and LDH≥250U/L was negatively correlated with lowered DCR (P = 0.018), while lymphocyte number, ECOG and LDH significantly influenced both PFS and OS. We found that the possible resistant mechanisms were sarcomatous degeneration and secondary mutation, including BRCA2 truncation mutation, A2M, JAK1,T790M, KEAP1 and mTOR mutation. 37.5% patients had ≥grade 3 adverse events. CONCLUSION: PARPi/anti-PD-1 is an effective and tolerable method for patients with advanced solid tumors, and BRCA1/2 is a potential biomarker.

Predicting disease progression in advanced non-small cell lung cancer with circulating neutrophil-derived and platelet-derived microparticles.

BACKGROUND: Microparticles (MPs) are extracellular vesicles that are associated with cancer development and progression. Advanced non-small cell lung cancer (NSCLC) still shows disease progression after multiple lines of treatment. Therefore, the objective of this study was to explore the correlation between circulating MPs and disease progression in advanced NSCLC, and to find a new method for concise and rapid determination of disease progression. METHODS: Patients with advanced NSCLC admitted to hospital between October 2019 and October 2020 were included and divided into objective remission (OR) and progressive disease (PD) groups. The morphology of MPs was observed using transmission electron microscopy. The circulating total MPs, neutrophil MPs (NMPs), and platelet MPs (PMPs) before and after treatment were detected by flow cytometry, and a predictive model for disease progression in advanced NSCLC was developed. RESULTS: Eighty-six patients were included; 60 in the OR group and 26 in the PD group. There was no significant difference in total MPs, NMPs, or PMPs at baseline between the two groups. After treatment, total MPs, NMPs, and PMPs were significantly higher in the PD than those in the OR group. Multivariate regression analysis showed that post-treatment NMPs≥160 events/µL(OR,3.748;95%CI,1.147-12.253,p = 0.029), PMPs≥80 events/µL(OR,10.968;95%CI,2.973-40.462,p < 0.0001) and neutrophil/lymphocyte ratio (NLR) ≥3.3 (OR,4.929;95%CI,1.483-16.375,p = 0.009) were independently associated with progression of advanced NSCLC. Post-treatment NMPs and PMPs combined with NLR were used to build a predictive model for progression of advanced NSCLC. The area under the curve was 0.825 (95%CI,0.715-0.934, p < 0.0001), optimal cut-off value was 16, sensitivity was 80.8%, and specificity was 88.3%. CONCLUSION: NMPs and PMPs are associated with progression of advanced NSCLC. The predictive model for progression of advanced NSCLC, established combining NMPs, PMPs, and NLR, can screen out 80.8% of patients with PD. This is helpful for real-time accurate, concise and rapid assessment of disease progression and timely adjustment of drug therapy. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800020223 . Registered 20 December 2018, http://www.chictr.org.cn/index.aspx .

ASPP2 suppresses stem cell-like characteristics and chemoresistance by inhibiting the Src/FAK/Snail axis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of death in cancer patients worldwide. Understanding the molecular pathogenesis of HCC recurrence and chemoresistance is key to improving patients' prognosis. In this study, we report that downregulation of ASPP2, a member of the ankyrin-repeat-containing, SH3-domain-containing, and proline-rich-region-containing protein (ASPP) family, bestowed HCC cells with stem-like properties and resistance to chemotherapy, including the expansion of side population fractions, formation of hepatospheroids, expression of stem cell-associated genes, loss of chemosensitivity, and increased tumorigenicity in immunodeficient mice. An expression profiling assay revealed that ASPP2 specifically repressed focal adhesion kinase (FAK)/Src/extracellular signal regulated kinase (ERK) signaling. ASPP2 does this by physically interacting with C-terminal Src kinase (CSK) and stimulating its kinase activity, which eventually leads to activator protein 1 (AP1)-mediated downregulation of Snail expression. In addition, pharmacologic inhibition of Src attenuated the effects of ASPP2 deficiency. Our findings present functional and mechanistic insight into the critical role of ASPP2 in the inhibition of HCC stemness and drug resistance and may provide a new strategy for therapeutic combinations to treat HCC.

Biomarkers for systemic lupus erythematosus: A scoping review.

BACKGROUND: In recent years, newly discovered potential biomarkers have great research potential in the diagnosis, disease activity prediction, and treatment of systemic lupus erythematosus (SLE). OBJECTIVE: In this study, a scoping review of potential biomarkers for SLE over several years has identified the extent to which studies on biomarkers for SLE have been conducted, the specificity, sensitivity, and diagnostic value of potential biomarkers of SLE, the research potential of these biomarkers in disease diagnosis, and activity detection is discussed. METHODS: In PubMed and Google Scholar databases, "SLE," "biomarkers," "predictor," "autoimmune diseases," "lupus nephritis," "neuropsychiatric SLE," "diagnosis," "monitoring," and "disease activity" were used as keywords to systematically search for SLE molecular biomarkers published from 2020 to 2024. Analyze and summarize the literature that can guide the article. CONCLUSIONS: Recent findings suggest that some potential biomarkers may have clinical application prospects. However, to date, many of these biomarkers have not been subjected to repeated clinical validation. And no single biomarker has sufficient sensitivity and specificity for SLE. It is not scientific to choose only one or several biomarkers to judge the complex disease of SLE. It may be a good direction to carry out a meta-analysis of various biomarkers to find SLE biomarkers suitable for clinical use, or to evaluate SLE by combining multiple biomarkers through mathematical models. At the same time, advanced computational methods are needed to analyze large data sets and discover new biomarkers, and strive to find biomarkers that are sensitive and specific enough to SLE and can be used in clinical practice, rather than only staying in experimental research and data analysis.

Protocol for deep mining the correlation between acute pancreatitis and ferroptosis using the MIMIC-III database and STATA software.

The limitations associated with distinguishing serum Fe2+ and Fe3+ hinder the widespread application of ferroptosis, beyond laboratory settings. Here, we present a protocol for deep mining the correlation between acute pancreatitis and ferroptosis using the MIMIC-III database and STATA software. We describe steps for using Cox regression, decision curve analysis (DCA), and receiver operating characteristic (ROC) approaches to establish the relationship between them and determine the relevant factors. This protocol has potential application in establishing novel research models that integrate both fundamental and clinical methodologies. For complete details on the use and execution of this protocol, please refer to Yueling Deng et al.1.

Multivariate Analysis Compares and Evaluates Heat Tolerance of Potato Germplasm.

High temperature is the most important environmental factor limiting potato (Solanum tuberosum L.) yield. The tuber yield has been used to evaluate the heat tolerance of some potato cultivars, but potato yield was closely correlated with the maturation period. Therefore, it is necessary to employ different parameters to comprehensively analyze and evaluate potato tolerance to heat stress. This study aimed to investigate physiologic changes during growth and development, and develop accurate heat tolerance evaluation methods of potato cultivars under heat stress. About 93 cultivars (including foreign elite lines, local landraces and cultivars) were screened using an in vitro tuber-inducing system (continuous darkness and 8% sucrose in the culture medium) under heat stress (30 °C) and normal (22 °C) conditions for 30 days. The tuber yield and number decreased significantly under heat stress compared to the control. A total of 42 cultivars were initially selected depending on tuber formation, after in vitro screening, further testing of selected cultivars was conducted in ex vitro conditions. The screened cultivars were further exposed to heat stress (35 °C/28 °C, day/night) for 60 days. Heat stress led to an increase in the plant height growth rate, fourth internode growth rate, and membrane damage, and due to heat-induced damage to chloroplasts, decrease in chlorophyll biosynthesis and photosynthetic efficiency. Three principal components were extracted by principal component analysis. Correlation and regression analysis showed that heat tolerance is positively correlated with the plant height growth rate, fourth internode growth rate, the content of chlorophyll b, photosynthetic rate, stomatal conductance, transpiration rate, tuber number, and tuber yield, and negatively correlated with the cell membrane injury level. The nine traits are accurate and representative indicators for evaluating potato tolerance to heat stress and could determine a relatively high mean forecast accuracy of 100.0% for the comprehensive evaluation value. Through cluster analysis and screening, cultivar FA, D73, and C132 had the highest heat comprehensive evaluation value, which could be further selected as heat-resistant varieties. This study provides insights into the different physiological mechanisms and accurate evaluation methods of potato cultivars under heat stress, which could be valuable for further research and breeding.

Potent covalent irreversible inhibitor of KRAS G12C IBI351 in patients with advanced solid tumors: First-in-human phase I study.

BACKGROUND: IBI351 is an irreversible and covalent inhibitor of KRAS G12C. Despite FDA approval of two KRAS G12C inhibitors, there are still significant unmet clinical needs in Chinese patients and ongoing concerns about the optimal dosage. Herein, we presented the phase Ia/Ib study of IBI351 monotherapy in Chinese patients with advanced solid tumors harboring KRAS G12C mutation. METHODS: In phase Ia dose escalation, IBI351 at 250/450/700/900 mg once daily and 450/600/750 mg twice daily (BID) were evaluated. Potentially efficacious doses and optimal recommended phase 2 dose (RP2D) were further evaluated in patients with advanced non-small cell lung cancer (NSCLC) in phase Ia dose expansion and phase Ib. Safety, pharmacokinetics, and investigator-assessed tumor response were evaluated. RESULTS: As of June 13, 2023, 176 patients were enrolled. IBI351 was well tolerated with no dose-limiting toxicity reported across all evaluated doses. The RP2D was determined as 600 mg BID by considering safety, efficacy and pharmacokinetics. A total of 168 patients (95.5 %) had at least one treatment-related adverse event (TRAE), and 64 patients (36.4 %) had grade 3 or higher TRAEs, most commonly gamma-glutamyl transferase increased (10.2 %) and anemia (6.8 %). For patients with NSCLC, the confirmed objective response rate (ORR) was 45.5 % across all doses. At 600 mg BID, the confirmed ORR was 46.8 % and median progression-free survival was 9.6 months with a median follow-up of 6.9 months. CONCLUSIONS: IBI351 was well tolerated in patients with advanced solid tumors and showed promising antitumor activity in advanced NSCLC patients with KRAS G12C mutation.

Efficacy and Safety of KRASG12C Inhibitor IBI351 Monotherapy in Patients With Advanced NSCLC: Results From a Phase 2 Pivotal Study.

INTRODUCTION: KRAS glycine-to-cysteine substitution at codon 12 (G12C) mutation is a well-recognized and increasingly promising therapeutic target with huge unmet clinical needs in NSCLC patients. IBI351 is a potent covalent and irreversible inhibitor of KRAS G12C. Here, we present the efficacy and safety of IBI351 from an open-label, single-arm, phase 2 pivotal study. METHODS: Eligible patients with NSCLC with KRAS G12C who failed standard therapy were enrolled. IBI351 was orally administered at a dose of 600 mg twice daily. The primary endpoint was confirmed objective response rate assessed by an independent radiological review committee (IRRC) as per Response Evaluation Criteria in Solid Tumors v1.1. Other endpoints were safety, IRRC-confirmed disease control rate, duration of response, progression-free survival (PFS), and overall survival. RESULTS: As of December 13, 2023, 116 patients were enrolled (Eastern Cooperative Oncology Group Performance Status 1: 91.4%; brain metastasis: 30.2%; prior treatments with both anti-PD-1 or anti-PD-L1 inhibitors and platinum-based chemotherapy: 84.5%). As per the IRRC assessment, the confirmed objective response rate was 49.1% (95% confidence interval [CI]: 39.7-58.6), and the disease control rate was 90.5% (95% CI: 83.7-95.2). The median duration of response was not reached whereas disease progression or death events occurred in 22 patients (38.6%), and the median PFS was 9.7 months (95% CI: 5.6-11.0). overall survival data was immature. Treatment-related adverse events (TRAEs) occurred in 107 patients (92.2%) whereas 48 patients (41.4%) had equal to or higher than grade three TRAEs. Common TRAEs were anemia (44.8%), increased alanine aminotransferase (28.4%), increased aspartate aminotransferase (27.6%), asthenia (26.7%) and presence of protein in urine (25.0%). TRAEs leading to treatment discontinuation occurred in nine patients (7.8%). In biomarker evaluable patients (n = 95), all patients had positive KRAS G12C in tissue whereas 72 patients were blood-positive and 23 were blood-negative for KRAS G12C. Patients with KRAS G12C in both blood and tissue had higher tumor burden at baseline (p < 0.05) and worse PFS (p < 0.05). Tumor mutation profiling identified tumor protein p53 (45.3%), serine/threonine kinase 11 (STK11) (30.5%), and kelch-like ECH-associated protein 1 (21.1%) as the most common genes co-mutated with KRAS G12C. Among 13 genes with mutation frequency equal to or higher than 5%, mutations of six genes (STK11, kelch-like ECH-associated protein 1, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma, DNA polymerase epsilon, SMAD family member 4, and BMP/retinoic acid-inducible neural-specific protein 3) were significantly associated with worse PFS (p < 0.05). Mutation in STK11 was also found to have a significant association with higher tumor burden at baseline and lower response rate (p < 0.05). CONCLUSIONS: IBI351 monotherapy demonstrated promising and sustained efficacy with manageable safety, supporting its potential as a new treatment option for KRAS G12C-mutant NSCLC.

Demethylation of miR-9-3 and miR-193a genes suppresses proliferation and promotes apoptosis in non-small cell lung cancer cell lines.

BACKGROUND: MicroRNAs miR-9-3 and miR-193a have recently been found to be hypermethylated in a variety of non-small cell lung cancer (NSCLC) cells and primary human tumors. The objectives of this study were to investigate the role of demethylation of miR-9-3 and miR-193a genes in regulating proliferation and apoptosis in NSCLCs, and to decipher the potential mechanisms underlying the properties. METHODS: MTT and population doubling time by flow cytometry were used to assess cell proliferation. Enzyme-Linked Immunosorbent Assay and caspase-3 activity assay were employed to evaluate apoptosis. Real-time RT-PCR and Western blot were used to quantify gene expression at mRNA and protein levels, respectively. Methylation-specific PCR was utilized to assess methylation status. RESULTS: We found that demethylation agent 5-Aza-2'-deoxycytidine (5-AzaC) reduced cell numbers and prolonged population doubling time (PDT), and promoted doxorubicin-induced apoptosis in seven NSCLC cell lines with different methylation statuses on miR-9-3 and miR-193a promoter regions: NCI-H1993/NCI-H1915 (miR-9-3(+)/miR-193a(+)), NCI-H1975/NCI-H200 (miR-9-3(+)/miR-193a(-)), A427/NCI-H2073 (miR-9-3(-)/miR-193a(+)), and NCI-H1703 (miR-9-3(-)/miR-193a(-)). Treatment with 5-AzaC concomitantly upregulated expression of miR-9-3 and miR-193a, and downregulated their respective target genes NF-κB and Mcl-1. The effects of 5-AzaC were abolished by concomitant knockdown of miR-9-3 and miR-193a using the complex antisense technique, whereas forced ectopic expression of miR-9-3 and miR-193a mimicked the effects of 5-AzaC. We further observed that the strength of proliferation inhibition and apoptosis promotion elicited by 5-AzaC was in the order of NCI-H1993/NCI-H1915 > A427/NCI-H2073 > NCI-H1975/NCI-H200 > NCI-H1703. CONCLUSIONS: Methylation-silencing of miR-9-3 and miR-193a may be an important epigenetic mechanisms favoring NSCLC cell growth and survival for carcinogenesis and cancer progression, and demethylation to reactivate expression of miR-9-3 and miR-193a genes contributes, at least partially, to the anti-cancer properties of 5-AzaC and thereby may be worthy of future studies for the possibility of being a new therapeutic strategy for the treatment of human NSCLCs.

Multi-omics integrated circulating cell-free DNA genomic signatures enhanced the diagnostic performance of early-stage lung cancer and postoperative minimal residual disease.

BACKGROUND: Liquid biopsy is a promising non-invasive alternative for cancer screening and minimal residual disease (MRD) detection, although there are some concerns regarding its clinical applications. We aimed to develop an accurate detection platform based on liquid biopsy for both cancer screening and MRD detection in patients with lung cancer (LC), which is also applicable to clinical use. METHODS: We applied a modified whole-genome sequencing (WGS) -based High-performance Infrastructure For MultIomics (HIFI) method for LC screening and postoperative MRD detection by combining the hyper-co-methylated read approach and the circulating single-molecule amplification and resequencing technology (cSMART2.0). FINDINGS: For early screening of LC, the LC score model was constructed using the support vector machine, which showed sensitivity (51.8%) at high specificity (96.3%) and achieved an AUC of 0.912 in the validation set prospectively enrolled from multiple centers. The screening model achieved detection efficiency with an AUC of 0.906 in patients with lung adenocarcinoma and outperformed other clinical models in solid nodule cohort. When applied the HIFI model to real social population, a negative predictive value (NPV) of 99.92% was achieved in Chinese population. Additionally, the MRD detection rate improved significantly by combining results from WGS and cSMART2.0, with sensitivity of 73.7% at specificity of 97.3%. INTERPRETATION: In conclusion, the HIFI method is promising for diagnosis and postoperative monitoring of LC. FUNDING: This study was supported by CAMS Innovation Fund for Medical Sciences, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, Beijing Natural Science Foundation and Peking University People's Hospital.

Mutation of the Nrf2 gene in non-small cell lung cancer.

Nrf2 (NFE2L2) is a transcription factor belonging to the Cap'N'Collar subfamily of basic-leucine zipper (bZIP) family of transcription factors, which plays a significant role in adaptive responses to oxidative stress. To investigate the relationship of between the mutation of Nrf2 gene and non-small cell lung cancer (NSCLC), in this study, we sequenced the Nrf2 gene from a total of 103 patients with NSCLC and corresponding blood samples. It is found that there is a discordance of Nrf2 mutations between NSCLC and corresponding blood samples. These differences may indicate that the variants in the Nrf2 gene are associated with an increased risk for lung cancer. In addition, the factor of smoking status is observed to play important roles in triggering the occurrence of the disorder.

[Clinical characteristics and prognostic factors of malignancy-associated hypercalcemia in squamous cell carcinoma].

OBJECTIVE: To explore the clinical characteristics and prognostic factors of patients diagnosed with squamous cell carcinoma (SCC) and presented malignancy-associated hypercalcemia (MAH). METHODS: We retrospectively analyzed the clinical data of 36 patients with biopsy-proven SCC and presented MAH who were treated at the our department from January 2001 to December 2010. The survival were analyzed using the Kaplan-Meier method and Cox analysis. RESULTS: Among these 36 patients, the median blood calcium level was 2.94 mmol/L (2.77-4.87 mmol/L), and the median survival time was only 45 days (1-839 d). Log-rank test showed that central nervous system symptoms, bone metastasis, and hypercalcemia occurring over 160 days after cancer diagnosis were predictors for poor survival(p=0.003, P=0.049, P=0.005). In the COX proportional hazard model analysis, central nervous system symptoms and hypercalcemia occurring over 160 days after cancer diagnosis were independent prognostic factors for survival time (RR=5.721, P=0.000; RR=4.624, P=0.001). CONCLUSIONS: Patients with squamous cell carcinoma (SCC) and presented MAH have poor prognosis. Central nervous system symptoms and hypercalcemia occurring over 160 days after cancer diagnosis are independent predictors of the prognosis.

The gut microbiota modulates responses to anti-PD-1 and chemotherapy combination therapy and related adverse events in patients with advanced solid tumors.

Background: Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) have been widely used in treating different malignancies. Several studies have reported that the gut microbiota modulates the response and adverse events (AEs) to ICIs in melanoma, non-small cell lung cancer (NSCLC), renal cell cancer and hepatocellular carcinoma, but data on other cancer types and ICI combination therapy are limited. Methods: Stool samples were collected from patients with cancer who received anti-PD-1 and chemotherapy combination treatment and were analyzed by fecal metagenomic sequencing. The microbiota diversity and composition were compared between the responder (R) and non-responder (NR) groups and the AE vs. the non-AE (NAE) groups. In addition, associated functional genes and metabolic pathways were identified. Results: At baseline, the microbiota diversity of the groups was similar, but the genera Parabacteroides, Clostridia bacterium UC5.1_2F7, and Bifidobacterium dentium were enriched in the R group, whereas Bacteroides dorei and 11 species of Nocardia were enriched in the NR group. At 6 weeks, the beta diversity was significantly different between the R and NR groups. Further analysis found that 35 genera, such as Alipes, Parabacteroides, Phascolarctobacterium, Collinsella, Ruminiclostridium, Porphyromonas, and Butyricimonas and several genera of the Fibrobacteraceae family, were frequently distributed in the R group, whereas 17 genera, including Enterococcus, Lachnoclostridium, Hungatella, and Bilophila and several genera of the Pseudonocardiaceae and Beijerinckiaceae families, were more abundant in the NR group. A total of 66 and 52 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs (KOs) were significantly enriched in the R and NR groups, respectively. In addition, pathway analysis revealed functional differences in the gut microbacteria in the R group, including the enrichment of anabolic pathways and DNA damage repair (DDR) pathways. Dynamic comparisons of the bacterial composition at baseline, 6 weeks, and 12 weeks showed that the abundance of Weissella significantly increased in the R group at 6 weeks and the abundance of Fusobacterium and Anaerotruncus significantly increased in the NR group at 12 weeks. Linear discriminant analysis effect size analysis indicated that bacteria of Bacteroidetes, especially Bacteroides, were enriched in the NAE group, whereas flora of Firmcutes, such as Faecalibacterium prausnitzii, Bacteroides fragilis, and Ruminococcus lactaris, were enriched in the AE group. Conclusion: Beta diversity and differences in the gut microbiota modulated AEs and the response to anti-PD-1 blockade combined with chemotherapy, by regulating related anabolic and DDR pathways. Dynamic changes in the intestinal microbiome may predict the efficacy of PD-1 inhibitor-based therapy.

Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer.

Anti-PD-1 treatment has shown unprecedented clinical success in the treatment of non-small-cell lung cancer (NSCLC), but the underlying mechanisms remain incompletely understood. Here, we performed temporal single-cell RNA and paired T-cell receptor sequencing on 47 tumor biopsies from 36 patients with NSCLC following PD-1-based therapies. We observed increased levels of precursor exhausted T (Texp) cells in responsive tumors after treatment, characterized by low expression of coinhibitory molecules and high expression of GZMK. By contrast, nonresponsive tumors failed to accumulate Texp cells. Our data suggested that Texp cells were unlikely to be derived from the reinvigoration of terminally exhausted cells; instead, they were accumulated by (1) local expansion and (2) replenishment by peripheral T cells with both new and pre-existing clonotypes, a phenomenon we named clonal revival. Our study provides insights into mechanisms underlying PD-1-based therapies, implicating clonal revival and expansion of Texp cells as steps to improve NSCLC treatment.