Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

A TA/Cu2+ Nanoparticle Enhanced Carboxymethyl Chitosan-Based Hydrogel Dressing with Antioxidant Properties and Promoting Wound Healing.

Background: As the first line of immune defense and the largest organ of body, skin is vulnerable to damage caused by surgery, burns, collisions and other factors. Wound healing in the skin is a long and complex physiological process that is influenced by a number of different factors. Proper wound care can greatly improve the speed of wound healing and reduce the generation of scars. However, traditional wound dressings (bandages, gauze, etc.) often used in clinical practice have a single function, lack of active ingredients and are limited in use. Hydrogels with three-dimensional network structure are a potential biomedical material because of their physical and chemical environment similar to extracellular matrix. In particular, hydrogel dressings with low price, good biocompatibility, degradability, antibacterial and angiogenic activity are favored by the public. Methods: Here, a carboxymethyl chitosan-based hydrogel dressing (CMCS-TA/Cu2+) reinforced by copper ion crosslinked tannic acid (TA/Cu2+) nanoparticles was developed. This study investigated the physical and chemical characteristics, cytotoxicity, and angiogenesis of TA/Cu2+ nanoparticles and CMCS-TA/Cu2+ hydrogels. Furthermore, a full-thickness skin defect wound model was employed to assess the in vivo wound healing capacity of hydrogel dressings. Results: The introduction of TA/Cu2+ nanoparticles not only could increase the mechanical properties of the hydrogel but also continuously releases copper ions to promote cell migration (the cell migration could reach 92% at 48 h) and tubule formation, remove free radicals and promote wound healing (repair rate could reach 90% at 9 days). Conclusion: Experiments have proved that CMCS-TA/Cu2+ hydrogel has good cytocompatibility, antioxidant and wound healing ability, providing an advantageous solution for skin repair.

Mnox Nanoenzyme Armed CAR-NK Cells Enhance Solid Tumor Immunotherapy by Alleviating the Immunosuppressive Microenvironment.

Adoptively transferred cells usually suffer from exhaustion, limited expansion, and poor infiltration, partially attributing to the complicated immunosuppressive microenvironment of solid tumors. Therefore, it is necessary to explore more effective strategies to improve the poor tumor microenvironment (TME) to efficaciously deliver and support extrinsic effector cells in vivo. Herein, an intelligent biodegradable hollow manganese dioxide nanoparticle (MnOX) that possesses peroxidase activity to catalyze excess H2O2 in the TME to produce oxygen and relieve the hypoxia of solid tumors is developed. MnOX nanoenzymes modified with CD56 antibody could specifically bind CAR-NK (chimeric antigen receptor modified natural killer) cells. It is demonstrated that CAR-NK cells incorporated with MnOX nanoenzymes effectively infiltrate into tumor tissues with an improved TME, which results in superior antitumor activity in solid tumor-bearing mice. The antibody connection between MnOX nanoenzymes and CAR-NK endows the lowest efficient dosage of MnOX. This study features a smart synergistic immunotherapy approach for solid tumors using MnOX nanoenzyme-armed CAR-NK cells, which would provide a valuable tool for immunocyte therapy in solid tumors.

Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.

Early human lung immune cell development and its role in epithelial cell fate.

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

Risk Factors for Hypocoagulability After Cardiac Surgery: A Retrospective Study.

Hemostatic disturbances after cardiac surgery can lead to excessive postoperative bleeding. Thromboelastography (TEG) was employed to evaluate perioperative coagulative alterations in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB), investigating the correlation between factors concomitant with cardiac surgery and modifications in coagulation. Coagulation index as determined by TEG correlated significantly with postoperative bleeding at 24-72 h after cardiac surgery (P < .001). Among patients with a normal preoperative coagulation index, those with postoperative hypocoagulability showed significantly lower nadir temperature (P = .003), larger infused fluid volume (P = .003), and longer CPB duration (P = .033) than those with normal coagulation index. Multivariate logistic regression showed that nadir intraoperative temperature was an independent predictor of postoperative hypocoagulability (adjusted OR: 0.772, 95% CI: 0.624-0.954, P = .017). Multivariate linear regression demonstrated linear associations of nadir intraoperative temperature (P = .017) and infused fluid volume (P = .005) with change in coagulation index as a result of cardiac surgery. Patients are susceptible to hypocoagulability after cardiac surgery, which can lead to increased postoperative bleeding. Ensuring appropriate temperature and fluid volume during cardiac surgery involving CPB may reduce risk of postoperative hypocoagulability and bleeding.

OncoCTMiner: streamlining precision oncology trial matching via molecular profile analysis.

By establishing omics sequencing of patient tumors as a crucial element in cancer treatment, the extensive implementation of precision oncology necessitates effective and prompt execution of clinical studies for approving molecular-targeted therapies. However, the substantial volume of patient sequencing data, combined with strict clinical trial criteria, increasingly complicates the process of matching patients to precision oncology studies. To streamline enrollment in these studies, we developed OncoCTMiner, an automated pre-screening platform for molecular cancer clinical trials. Through manual tagging of eligibility criteria for 2227 oncology trials, we identified key bio-concepts such as cancer types, genes, alterations, drugs, biomarkers and therapies. Utilizing this manually annotated corpus along with open-source biomedical natural language processing tools, we trained multiple named entity recognition models specifically designed for precision oncology trials. These models analyzed 460 952 clinical trials, revealing 8.15 million precision medicine concepts, 9.32 million entity-criteria-trial triplets and a comprehensive precision oncology eligibility criteria database. Most significantly, we developed a patient-trial matching system based on cancer patients' clinical and genetic profiles, which can seamlessly integrate with the omics data analysis platform. This system expedites the pre-screening process for potentially suitable precision oncology trials, offering patients swifter access to promising treatment options. Database URL  https://oncoctminer.chosenmedinfo.com.

Nadir Hemoglobin Concentration After Spinal Tumor Surgery: Association With Risk of Composite Adverse Events.

STUDY DESIGN: Retrospective case-control study. OBJECTIVE: To explore the association of early postoperative nadir hemoglobin with risk of a composite outcome of anemia-related and other adverse events. METHODS: We retrospectively analyzed data from spinal tumor patients who received intraoperative blood transfusion between September 1, 2013 and December 31, 2020. Uni- and multivariate logistic regression was used to explore relationships of clinicodemographic and surgical factors with risk of composite in-hospital adverse events, including death. Subgroup analysis explored the relationship between early postoperative nadir hemoglobin and composite adverse events. RESULTS: Among the 345 patients, 331 (95.9%) experienced early postoperative anemia and 69 (20%) experienced postoperative composite adverse events. Multivariate logistic regression analysis showed that postoperative nadir Hb (OR = .818, 95% CI: .672-.995, P = .044), ASA ≥3 (OR = 2.007, 95% CI: 1.086-3.707, P = .026), intraoperative RBC infusion volume (OR = 1.133, 95% CI: 1.009-1.272, P = .035), abnormal hypertension (OR = 2.199, 95% CI: 1.085-4.457, P = .029) were correlated with composite adverse events. The lumbar spinal tumor was associated with composite adverse events with a decreased odds compared to thoracic spinal tumors (OR = .444, 95% CI: .226-.876, P = .019). Compared to patients with postoperative nadir hemoglobin ≥11.0 g/dL, those with nadir <9.0 g/dL were at significantly higher risk of postoperative composite adverse events (OR = 2.709, 95% CI: 1.087-6.754, P = .032). CONCLUSION: Nadir hemoglobin <9.0 g/dL after spinal tumor surgery is associated with greater risk of postoperative composite adverse events in patients who receive intraoperative blood transfusion.

Identification and validation of a novel HOX-related classifier signature for predicting prognosis and immune microenvironment in pediatric gliomas.

Background: Pediatric gliomas (PGs) are highly aggressive and predominantly occur in young children. In pediatric gliomas, abnormal expression of Homeobox (HOX) family genes (HFGs) has been observed and is associated with the development and progression of the disease. Studies have found that overexpression or underexpression of certain HOX genes is linked to the occurrence and prognosis of gliomas. This aberrant expression may contribute to the dysregulation of important pathological processes such as cell proliferation, differentiation, and metastasis. This study aimed to propose a novel HOX-related signature to predict patients' prognosis and immune infiltrate characteristics in PGs. Methods: The data of PGs obtained from publicly available databases were utilized to reveal the relationship among abnormal expression of HOX family genes (HFGs), prognosis, tumor immune infiltration, clinical features, and genomic features in PGs. The HFGs were utilized to identify heterogeneous subtypes using consensus clustering. Then random forest-supervised classification algorithm and nearest shrunken centroid algorithm were performed to develop a prognostic signature in the training set. Finally, the signature was validated in an internal testing set and an external independent cohort. Results: Firstly, we identified HFGs significantly differentially expressed in PGs compared to normal tissues. The individuals with PGs were then divided into two heterogeneous subtypes (HOX-SI and HOX-SII) based on HFGs expression profiles. HOX-SII showed higher total mutation counts, lower immune infiltration, and worse prognosis than HOX-SI. Then, we constructed a HOX-related gene signature (including HOXA6, HOXC4, HOXC5, HOXC6, and HOXA-AS3) based on the cluster for subtype prediction utilizing random forest supervised classification and nearest shrunken centroid algorithm. The signature was revealed to be an independent prognostic factor for patients with PGs by multivariable Cox regression analysis. Conclusion: Our study provides a novel method for the prognosis classification of PGs. The findings also suggest that the HOX-related signature is a new biomarker for the diagnosis and prognosis of patients with PGs, allowing for more accurate survival prediction.

Causal relationship between obesity and iron deficiency anemia: a two-sample Mendelian randomization study.

Background: Observational studies have suggested an association between obesity and iron deficiency anemia, but such studies are susceptible to reverse causation and residual confounding. Here we used Mendelian randomization to assess whether the association might be causal. Methods: Data on single-nucleotide polymorphisms that might be associated with various anthropometric indicators of obesity were extracted as instrumental variables from genome-wide association studies in the UK Biobank. Data on genetic variants in iron deficiency anemia were extracted from a genome-wide association study dataset within the Biobank. Heterogeneity in the data was assessed using inverse variance-weighted regression, Mendelian randomization Egger regression, and Cochran's Q statistic. Potential causality was assessed using inverse variance-weighted, Mendelian randomization Egger, weighted median, maximum likelihood and penalized weighted median methods. Outlier SNPs were identified using Mendelian randomization PRESSO analysis and "leave-one-out" analysis. Results: Inverse variance-weighted regression associated iron deficiency anemia with body mass index, waist circumference, trunk fat mass, body fat mass, trunk fat percentage, and body fat percentage (all odds ratios 1.003-1.004, P ≤ 0.001). Heterogeneity was minimal and no evidence of horizontal pleiotropy was found. Conclusion: Our Mendelian randomization analysis suggests that obesity can cause iron deficiency anemia.

RTK signalling promotes epithelial columnar cell shape and apical junction maintenance in human lung progenitor cells.

Multipotent epithelial progenitor cells can be expanded from human embryonic lungs as organoids and maintained in a self-renewing state using a defined medium. The organoid cells are columnar, resembling the cell morphology of the developing lung tip epithelium in vivo. Cell shape dynamics and fate are tightly coordinated during development. We therefore used the organoid system to identify signalling pathways that maintain the columnar shape of human lung tip progenitors. We found that EGF, FGF7 and FGF10 have distinct functions in lung tip progenitors. FGF7 activates MAPK/ERK and PI3K/AKT signalling, and is sufficient to promote columnar cell shape in primary tip progenitors. Inhibitor experiments show that MAPK/ERK and PI3K/AKT signalling are key downstream pathways, regulating cell proliferation, columnar cell shape and cell junctions. We identified integrin signalling as a key pathway downstream of MAPK/ERK in the tip progenitors; disrupting integrin alters polarity, cell adhesion and tight junction assembly. By contrast, stimulation with FGF10 or EGF alone is not sufficient to maintain organoid columnar cell shape. This study employs organoids to provide insight into the cellular mechanisms regulating human lung development.

Conversion therapy in liver transplantation for hepatocellular carcinoma: What's new in the era of molecular and immune therapy?

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common cancer globally, with limited therapies and unsatisfactory prognosis once in the advanced stages. With promising advances in locoregional and systematic treatments, fast development of targeted drugs, the success of immunotherapy, as well as the emergence of the therapeutic alliance, conversion therapy has recently become more well developed and an effective therapeutic strategy. This article aimed to review recent developments in conversion therapy in liver transplantation (LT) for HCC. DATA SOURCES: We searched for relevant publications on PubMed before September 2022, using the terms "HCC", "liver transplantation", "downstaging", "bridging treatment" and "conversion therapy." RESULTS: Conversion therapy was frequently represented as a combination of multiple treatment modalities to downstage HCC and make patients eligible for LT. Although combining various local and systematic treatments in conversion therapy is still controversial, growing evidence has suggested that multimodal combined treatment strategies downstage HCC in a shorter time, which ultimately increases the opportunities for LT. Moreover, the recent breakthrough of immunotherapy and targeted therapy for HCC also benefit patients with advanced-stage tumors. CONCLUSIONS: In the era of targeted therapy and immunotherapy, applying the thinking of transplant oncology to benefit HCC patients receiving LT is a new topic that has shed light on advanced-stage patients. With the expansion of conversion therapy concepts, further investigation and research is required to realize the full potential of conversion treatment strategies, including accurately selecting candidates, determining the timing of surgery, improving the conversion rate, and guaranteeing the safety and long-term efficacy of treatment.

Large volume acute normovolemic hemodilution in patients undergoing cardiac surgery with intermediate-high risk of transfusion: A randomized controlled trial.

STUDY OBJECTIVE: To investigate whether large volume acute normovolemic hemodilution (L-ANH), compared with moderate acute normovolemic hemodilution (M-ANH), can reduce perioperative allogeneic blood transfusion in patients with intermediate-high risk of transfusion during cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Prospective randomized controlled trial. SETTING: University hospital. PATIENTS: Patients with transfusion risk understanding scoring tool ("TRUST") ≥2 points undergoing cardiac surgery with CPB in the Second Affiliated Hospital of Zhejiang University from May 2020 to January 2021 were included. INTERVENTIONS: The patients were randomly assigned with a 1:1 ratio to M-ANH (5 to 8 mL/kg) or L-ANH (12 to 15 mL/kg). MEASUREMENTS: The primary outcome was perioperative red blood cell (RBC) transfusion units. The composite outcome included new-onset atrial fibrillation, pulmonary infection, cardiac surgery associated acute kidney injury (CSA-AKI) class ≥2, surgical incision infection, postoperative excessive bleeding, and resternotomy. MAIN RESULTS: Total 159 patients were screened and 110 (55 L-ANH and 55 M-ANH) were included for final analysis. Removed blood volume of L-ANH is significantly higher than M-ANH (886 ± 152 vs. 395 ± 86 mL, P < 0.001). Perioperative RBC transfusion was median 0 unit ([25th, 75th] percentiles: 0-4.4) in M-ANH group vs. 0 unit ([25th, 75th] percentiles: 0-2.0) in L-ANH group (P = 0.012) and L-ANH was associated with lower incidence of transfusion (23.6% vs. 41.8%, P = 0.042, rate difference: 0.182, 95% confidence interval [0.007-0.343]). The incidence of postoperative excessive bleeding was significantly lower in L-ANH vs. M-ANH (3.6% vs. 18.2%, P = 0.029, rate difference: 0.146, 95% confidence interval [0.027-0.270]) without significant difference for other second outcomes. The volume of ANH was inversely related to perioperative RBC transfusion units (Spearman r = -0.483, 95% confidence interval [-0.708 to -0.168], P = 0.003), and L-ANH in cardiac surgery was associated with a significantly reduced risk of perioperative RBC transfusion (odds ratio: 0.43, 95% confidence interval: 0.19-0.98, P = 0.044). CONCLUSIONS: Compared with M-ANH, L-ANH during cardiac surgery inclined to be associated with reduced perioperative RBC transfusion and the volume of RBC transfusion was inversely proportional to the volume of ANH. In addition, LANH during cardiac surgery was associated with a lower incidence of postoperative excessive bleeding.

miR-34a-5p facilitates osteogenic differentiation of bone marrow mesenchymal stem cells and modulates bone metabolism by targeting HDAC1 and promoting ER-α transcription.

OBJECTIVE: Metabolism is essential for bone development. The expressions of catabolic markers in chondrocytes show association with miR-34a-5p. This study discussed the mechanism by which miR-34a-5p regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) as well as bone metabolism. METHODS: Expressions of BMSC surface markers were determined via flow cytometry. Osteogenic differentiation of BMSCs was subsequently induced. miR-34a-5p mimic, oe-HDAC1, or ER-α activator Ferutinin was introduced in BMSCs. Alkaline phosphatase activity and calcification were detected. Expressions of miR-34a-5p, HDAC1, ER-α, and osteogenic markers were determined via RT-qPCR and Western blot. The binding relationship between miR-34a-5p and HDAC1 was verified by a dual-luciferase assay. Mice at the age of 6 months and 18 months were assigned to the young group and age group for in vivo experiments, and aged mice were treated with agomiR miR-34a-5p. Expressions of serum miR-34a-5p, HDAC1, ER-α, and bone metabolism markers in mice were determined. RESULTS: Osteogenic medium-induced BMSCs manifested increased expressions of miR-34a-5p and ER-α and decreased HDAC1 expression. miR-34a-5p overexpression promoted osteogenic differentiation of BMSCs. miR-34a-5p targeted HDAC1. HDAC1 overexpression partially counteracted the promotional action of miR-34a-5p overexpression on osteogenic differentiation of BMSCs. miR-34a-5p overexpression activated ER-α. ER-α activator Ferutinin partially nullified the regulatory function of miR-34a-5p/HDAC1 on osteogenic differentiation of BMSCs. In vivo experiments showed that miR-34a-5p overexpression enhanced the potential of bone metabolism in aged mice. CONCLUSION: miR-34a-5p overexpression promoted osteogenic differentiation of BMSCs and enhanced bone metabolism by promoting ER-α activation via targeting HDAC1.

A study of the relationship between cytokine levels and the response to anti-VEGF therapy in polypoid choroidal vasculopathy with different choroidal thicknesses.

Purpose: Polypoidal choroidal vasculopathy (PCV) is an irreversible retinal choroidal disease. Individuals with PCV exhibit diverse baseline characteristics, including systemic characteristics, ocular traits, metabolic factor levels, and different responses to intravitreal anti-VEGF therapy. This study aims to investigate the pathogenesis of PCV by analyzing the systemic characteristics, ocular traits, and cytokine levels at baseline within a cohort of patients who exhibit different responses to anti-VEGF treatment. Methods: We conducted a retrospective analysis involving 80 eyes diagnosed with PCV. Patients were categorized into two groups based on responses to suboptimal intravitreal ranibizumab injection therapy: those with suboptimal responses and optimal responses. Aqueous humor samples were collected from the experimental eyes, and cytokine expression levels were assessed using cytometric bead array analysis. All subjects were further stratified into two groups according to the median choroidal thickness. Subsequently, logistic regression analysis and the ROC curve were employed to examine the relationship between cytokine expression levels, choroidal thickness, and anti-VEGF response. Results: The results revealed that compared to the group of optimal anti-VEGF response, the choroid in the suboptimal response group exhibited a significantly greater thickness. Additionally, compared to the suboptimal anti-VEGF response group, the expression levels of VEGF and VCAM-1 were markedly lower observed in the optimal anti-VEGF response group, while TNF-α showed the opposite trend. Logistic regression analysis indicated that VEGF, VCAM-1, and TNF-α in the aqueous humor were independent risk factors for a suboptimal anti-VEGF response. After adjusting other risk factors, the risk of suboptimal anti-VEGF response decreased to 0.998-fold, 0.997-fold, and 1.294-fold. The AUC values for VEGF, VCAM-1, and TNF-α were determined to be 0.805, 0.846, and 0.897, respectively. Furthermore, the risk of VEGF, VCAM-1, and TNF-α were significantly associated with an increased risk of suboptimal anti-VEGF response after correction for risk factors in the thick choroid group. Conclusions: Our study demonstrated that PCV exhibits systemic and ocular characteristics variations based on different anti-VEGF responses. The levels of cytokines in aqueous humor were found to have a significant correlation with the anti-VEGF response in PCV. VEGF, VCAM-1, and TNF-α are potential targets for assessing treatment response in thick choroidal PCV.

A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates.

We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5-22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models.

Generation of multicellular tumor spheroids with micro-well array for anticancer drug combination screening based on a valuable biomarker of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. More than 30% of patients with diagnosed HCC have abnormally high expression of fibroblast growth factor receptor 4 (FGFR4). Currently, clinical trials for a variety of FGFR4-specific inhibitors have started. However, the effect of these inhibitors is not ideal, and it is necessary to find a drug combination to synergistically exert anti-tumor effects. We found strong correlations between FGFR4 and HCC clinicopathological characteristics in the present study. After grouping patients according to FGFR4 expression, the key gene signatures were inputted the drug-gene related databases, which predicted several potential drug candidates. More importantly, to achieve the reliable and high throughput drug cytotoxicity assessment, we developed an efficient and reproducible agarose hydrogel microwells to generate uniform-sized multicellular tumor spheroids, which provide better mimicry of conventional solid tumors that can precisely represent anticancer drug candidates' effects. Using high content screening, we quickly evaluated the enhanced anti-tumor effects of these combinations. Finally, we demonstrated that Parthenolide is a potential drug that can significantly enhance the clinical efficacy of FGFR4 receptor inhibitors. In general, we offered a new therapeutic way for FGFR4 positive HCC patients.

Mineralize It or Not: Comparative Proteomics and Elemental Analysis Reveal Ancestral Compositions of Iron Mineralized Molluscan Radulae.

The radula is a unique foraging organ to Mollusca, which is important for their evolution and taxonomic classification. Many radulae are mineralized with metals. Although the remarkable mechanical properties of mineralized radulae are well-studied, the formation of mineralization from nonmineralized radulae is poorly understood. Taking advantage of the recently sequenced octopus and chiton genomes, we were able to identify more species-specific radular proteins by proteomics. Comparing these proteomes with the known limpet radula proteome enabled us to gain insight into the molecular components of nonmineralized and mineralized radula, highlighting that iron mineralization in the chiton radula is possibly due to the evolution of ferritins and peroxiredoxins. Through an in vitro binding assay, ferritin is shown to be important to iron accumulation into the nonmineralized radula. Moreover, radular proteomes reflect their adaption to dietary habits to some extent. The octopus radula has many scaffold modification proteins to suit flexibility while the chiton radula has abundant sugar metabolism proteins (e.g., glycosyl hydrolases) to adapt to algae feeding. This study provides a foundation for the understanding of molluscan radula formation and evolution and may inspire the synthesis of iron nanomaterials.

Co-administration of MDR1 and BCRP or EGFR/PI3K inhibitors overcomes lenvatinib resistance in hepatocellular carcinoma.

Lenvatinib is the first-line treatment for hepatocellular carcinoma (HCC), the most common type of primary liver cancer; however, some patients become refractory to lenvatinib. The underlying mechanism of lenvatinib resistance (LR) in patients with advanced HCC remains unclear. We focused on exploring the potential mechanism of LR and novel treatments of lenvatinib-resistant HCC. In particular, we established a Huh7 LR cell line and performed in vitro, bioinformatic, and biochemical assays. Additionally, we used a Huh7-LR cell-derived xenograft mouse model to confirm the results in vivo. Following LR induction, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) transporters were markedly upregulated, and the epidermal growth factor receptor (EGFR), MEK/ERK, and PI3K/AKT pathways were activated. In vitro, the co-administration of elacridar, a dual MDR1 and BCRP inhibitor, with lenvatinib inhibited proliferation and induced apoptosis of LR cells. These effects might be due to inhibiting cancer stem-like cells (CSCs) properties, by decreasing colony formation and downregulating CD133, EpCAM, SOX-9, and c-Myc expression. Moreover, the co-administration of gefitinib, an EGFR inhibitor, with lenvatinib retarded proliferation and induced apoptosis of LR cells. These similar effects might be caused by the inhibition of EGFR-mediated MEK/ERK and PI3K/AKT pathway activation. In vivo, co-administration of lenvatinib with elacridar or gefitinib suppressed tumour growth and angiogenesis. Therefore, inhibiting MDR1 and BCRP transporters or targeting the EGFR/PI3K pathway might overcome LR in HCC. Notably, lenvatinib should be used to treat HCC after LR induction owing to its role in inhibiting tumour proliferation and angiogenesis. Our findings could help develop novel and effective treatment strategies for HCC.

SOX9 maintains human foetal lung tip progenitor state by enhancing WNT and RTK signalling.

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.