Prognostic and immunotherapeutic implications of bilirubin metabolism-associated genes in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a major subtype of non-small-cell lung cancer and accompanies high mortality rates. While the role of bilirubin metabolism in cancer is recognized, its specific impact on LUAD and patient response to immunotherapy needs to be elucidated. This study aimed to develop a prognostic signature of bilirubin metabolism-associated genes (BMAGs) to predict outcomes and efficacy of immunotherapy in LUAD. We analysed gene expression data from The Cancer Genome Atlas (TCGA) to identify survival-related BMAGs and construct a prognostic model in LUAD. The prognostic efficacy of our model was corroborated by employing TCGA-LUAD and five Gene Expression Omnibus datasets, effectively stratifying patients into risk-defined cohorts with marked disparities in survival. The BMAG signature was indeed an independent prognostic determinant, outperforming established clinical parameters. The low-risk group exhibited a more favourable response to immunotherapy, highlighted by increased immune checkpoint expression and immune cell infiltration. Further, somatic mutation profiling differentiated the molecular landscapes of the risk categories. Our screening further identified potential drug candidates preferentially targeting the high-risk group. Our analysis of critical BMAGs showed the tumour-suppressive role of FBP1, highlighting its suppression in LUAD and its inhibitory effects on tumour proliferation, migration and invasion, in addition to its involvement in cell cycle and apoptosis regulation. These findings introduce a potent BMAG-based prognostic indicator and offer valuable insights for prognostication and tailored immunotherapy in LUAD.

Determination of the influence of water on the SO3 + CH3OH reaction in the gas phase and at the air-water interface.

Liu et al. (Proc. Natl. Acad. Sci. U. S. A, 2019, 116, 24966-24971) showed that at an altitude of 0 km, the reaction of SO3 with CH3OH to form CH3OSO3H reduces the amount of H2SO4 produced by the hydrolysis of SO3 in regions polluted with CH3OH. However, the influence of the water molecule has not been fully considered yet, which will limit the accuracy of calculating the loss of SO3 in regions polluted with CH3OH. Here, the influence of water molecules on the SO3 + CH3OH reaction in the gas phase and at the air-water interface was comprehensively explored by using high-level quantum chemical calculations and Born-Oppenheimer molecular dynamics (BOMD) simulations. Quantum chemical calculations show that both pathways for the formation of CH3OSO3H and H2SO4 with water molecules have greatly lowered energy barriers compared to the naked SO3 + CH3OH reaction. The effective rate coefficients reveal that H2O-catalyzed CH3OSO3H formation (a favorable route for CH3OSO3H formation) can be competitive with H2O-assisted H2SO4 formation (a favorable process for H2SO4 formation) at high altitudes up to 15 km. BOMD simulations found that H2O-induced formation of the CH3OSO3-⋯H3O+ ion pair and CH3OH-assisted formation of HSO4- and H3O+ ions were observed at the droplet surface. These interfacial routes followed a loop-structure or chain reaction mechanism and proceeded on a picosecond time scale. These results will contribute to better understanding of SO3 losses in the polluted areas of CH3OH.

Deformation Mechanism and Control of In-Situ Assembling Caisson Technology in Soft Soil Area under Field Measurement and Numerical Simulation.

With urban space becoming much more crowded, the construction of underground spaces continues to expand to deeper, and the requirements for the large depth and minor deformation in urban engineering construction are getting more urgent. A new kind of in-situ assembling caisson technology (called VSM) is a vertical shaft method (VSM), which excavates the stratum under water with a mechanical arm and assembles the prefabricated caisson segments at the same time. This paper takes the Shanghai Zhuyuan Bailonggang Sewage Connecting Pipe Project as an example, which is the first construction project in the soft soil area, such as Shanghai, and makes a technical analysis of the VSM by comparing the field measurement and numerical simulation. Ground settlements and layered deep displacements were monitored in the field measurement during the VSM construction. It shows that the maximum ground settlement caused by the VSM is 15.2 mm and the maximum horizontal displacement is 3.74 mm. The influence range of the shaft excavation on the ground settlement is about 30 m away from the shaft center. The results demonstrate that the VSM construction has great applicability in the soft soil area. A finite element simulation model of the VSM shaft is established and verified by field measurement. There is a certain error between the traditional theoretical calculation by analogy to the common retaining walls of the deep foundation pit and the measured results, while the simulation results are relatively consistent with field measurements. The reasons for the difference are well-analyzed. Finally, the effects of the VSM construction method on the engineering environment are analyzed, and the suggestions for deformation control in the future are put forward.

Quasispecies of SARS-CoV-2 revealed by single nucleotide polymorphisms (SNPs) analysis.

New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies' composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual's respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies' profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak.

Mutant Kras co-opts a proto-oncogenic enhancer network in inflammation-induced metaplastic progenitor cells to initiate pancreatic cancer.

Kras-activating mutations display the highest incidence in pancreatic ductal adenocarcinoma. Pancreatic inflammation accelerates mutant Kras-driven tumorigenesis in mice, suggesting high selectivity in the cells that oncogenic Kras transforms, although the mechanisms dictating this specificity are poorly understood. Here we show that pancreatic inflammation is coupled to the emergence of a transient progenitor cell population that is readily transformed in the presence of mutant KrasG12D. These progenitors harbor a proto-oncogenic transcriptional program driven by a transient enhancer network. KrasG12D mutations lock this enhancer network in place, providing a sustained Kras-dependent oncogenic program that drives tumors throughout progression. Enhancer co-option occurs through functional interactions between the Kras-activated transcription factors Junb and Fosl1 and pancreatic lineage transcription factors, potentially accounting for inter-tissue specificity of oncogene transformation. The pancreatic ductal adenocarcinoma cell of origin thus provides an oncogenic transcriptional program that fuels tumor progression beyond initiation, accounting for the intra-tissue selectivity of Kras transformation.

Prevalence of phase variable epigenetic invertons among host-associated bacteria.

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.

Genome-wide piggyBac transposon-based mutagenesis and quantitative insertion-site analysis in haploid Candida species.

Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.

Feasibility Investigation of Ipsilateral Reoperations by Thoracoscopy for Major Lung Resection.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) has become the preferred approach for minimizing harm from thoracic operations. There is no report, however, which has discussed the feasibility of VATS in ipsilateral reoperation of major lung resection. METHODS: The present study included patients who had undergone ipsilateral reoperation of major lung resection by VATS from October 2009 to May 2017. Referring clinical data were recruited for analysis. RESULTS: Fourteen patients were recruited in the present study, including nine patients who underwent lobectomy and five who underwent segmentectomy during the second operation. Different hila were found in 6 patients, and pleural adhesions appeared in 10 patients. The average intraoperative blood loss was 203.6 ± 121.7 mL, and the mean operating room time was 2.2 ± 0.5 hours. There were no intraoperative deaths, and only one patient required conversion to thoracotomy. The average drainage time was 5.9 ± 4.6, and the mean hospital stay was 6.7 ± 4.2 days. CONCLUSION: Though it is technically demanding to safely handle the changed hilum structure caused by the last operation, major lung resection by VATS is feasible for ipsilateral reoperation in appropriate candidates.

Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling.

RNA molecules are highly compartmentalized in eukaryotic cells, with their localizations intimately linked to their functions. Despite the importance of RNA targeting, our current knowledge of the spatial organization of the transcriptome has been limited by a lack of analytical tools. In this study, we develop a chemical biology approach to label RNAs in live cells with high spatial specificity. Our method, called CAP-seq, capitalizes on light-activated, proximity-dependent photo-oxidation of RNA nucleobases, which could be subsequently enriched via affinity purification and identified by high-throughput sequencing. Using this technique, we investigate the local transcriptomes that are proximal to various subcellular compartments, including the endoplasmic reticulum and mitochondria. We discover that messenger RNAs encoding for ribosomal proteins and oxidative phosphorylation pathway proteins are highly enriched at the outer mitochondrial membrane. Due to its specificity and ease of use, CAP-seq is a generally applicable technique to investigate the spatial transcriptome in many biological systems.

Expanding APEX2 Substrates for Proximity-Dependent Labeling of Nucleic Acids and Proteins in Living Cells.

The subcellular organization of biomolecules such as proteins and nucleic acids is intimately linked to their biological functions. APEX2, an engineered ascorbate peroxidase that enables proximity-dependent labeling of proteins in living cells, has emerged as a powerful tool for deciphering the molecular architecture of various subcellular structures. However, only phenolic compounds have thus far been employed as APEX2 substrates, and the resulting phenoxyl radicals preferentially react with electron-rich amino acid residues. This narrow scope of substrates could potentially limit the application of APEX2. In this study, we screened a panel of aromatic compounds and identified biotin-conjugated arylamines as novel probes with significantly higher reactivity towards nucleic acids. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we applied APEX2 labeling with biotin-aniline (Btn-An) in the mitochondrial matrix, capturing all 13 mitochondrial messenger RNAs and none of the cytoplasmic RNAs. APEX2-mediated Btn-An labeling of RNA is thus a promising method for mapping the subcellular transcriptome, which could shed light on its functions in cell physiology.

Management of calcified hilar lymph nodes during thoracoscopic lobectomies: avoidance of conversions.

BACKGROUND: Our objective in this paper is to introduce a new method for handling calcified hilar lymph nodes during lobectomies by video-assisted thoracoscopy that effectively avoids arterial injury and possible conversions. METHODS: The 12 cases in this study were initially evaluated as eligible and were subsequently scheduled for thoracoscopic lobectomies. However, calcification of the hilar lymph nodes was discovered during the operations, and its presence hampered the conventional process of pulmonary artery dissection. To avoid vessel injuries and subsequent massive bleeding, we developed two techniques specific to the position and exposure of the target vessels and nodes. The space between the bronchus and lymph nodes is exposed by sharp dissection ("scissor first") either before or after suturing the artery. These techniques are illustrated in detail. RESULTS: Seven male and five female patients participated in this study, with an average age of 72.5 years. We performed five right-upper lobectomies, three right-middle lobectomies, three right-lower lobectomies, and a left-lower lobectomy. Using this new technique, no conversions to thoracotomy occurred. The average operation time was 125 minutes, the mean blood loss was 275 mL, and no intra-operative massive bleeding occurred. Two patients experienced minor complications, one pulmonary infection and another postoperative subcutaneous emphysema. CONCLUSIONS: The proposed "scissor first" technique provides an effective solution for the thoracoscopic management of calcified hilar lymph nodes, and is a safe and effective method for avoiding arterial injury and conversion.

Author Correction: Candida albicans gains azole resistance by altering sphingolipid composition.

In the original version of this Article, Haoping Liu, who conceptualized, designed and supervised the project and acquired funding, was inadvertently omitted from the author list. Furthermore, the affiliation of Jiaxin Gao and Haoping Liu with 'Department of Biological Chemistry, University of California, Irvine, CA 92697, USA' was omitted. Finally, funding from NIH grant GM117111, and contributions from Dr. Li-lin Du of NIBS for providing pPB[ura4] and pDUAL-PBase and Allan Bradley of Sanger for hyPBase, were not acknowledged. These errors have now been corrected in both the PDF and HTML versions of the Article.

Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems.

Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

Candida albicans gains azole resistance by altering sphingolipid composition.

Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen's diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.

Three-dimensional printing of navigational template in localization of pulmonary nodule: A pilot study.

BACKGROUND: Small pulmonary nodules are a common problem, especially with the wide implementation of lung cancer-screening program. This poses a great challenge to thoracic surgeons because of the difficulty of nodule localization. We recently built an efficient, customized navigational template using 3-dimensional (3D) printing technology to facilitate the procedure of lung nodule localization. This study aims to investigate its feasibility in clinical application. METHODS: Patients with peripheral lung nodules (<2 cm) were enrolled. Preadmission computed tomography images were downloaded and reconstructed into a 3D model. A digital model of the navigational template was designed via computer-aided design software and then exported into 3D printer to produce physical template. The precision of the template-guided nodule localization and associated complications were evaluated. RESULTS: A total of 16 patients were enrolled, and 18 nodules were localized through template-guided localization. The success rate of lung nodule localization was 100%, and the median time of localization was 13 minutes (range 10-16 minutes). In our series, no significant complication occurred, except for 2 asymptomatic pneumothoraxes. The median deviation between the localizer and the center of the nodule was 10.0 mm, ranging from 5 to 20 mm. CONCLUSIONS: This novel navigational template created by 3D printing technology is feasible, and it has acceptable accuracy for the application in lung nodule localization. The use of this navigational template could facilitate the procedure of lung nodule localization and may potentially break the dependence of percutaneous localization on computed tomography scanning.