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We leverage machine learning approaches to adapt nanopore sequencing basecallers for nucleotide modification detection. We first apply the incremental learning (IL) technique to improve the basecalling of modification-rich sequences, which are usually of high biological interest. With sequence backbones resolved, we further run anomaly detection (AD) on individual nucleotides to determine their modification status. By this means, our pipeline promises the single-molecule, single-nucleotide, and sequence context-free detection of modifications. We benchmark the pipeline using control oligos, further apply it in the basecalling of densely-modified yeast tRNAs and E.coli genomic DNAs, the cross-species detection of N6-methyladenosine (m6A) in mammalian mRNAs, and the simultaneous detection of N1-methyladenosine (m1A) and m6A in human mRNAs. Our IL-AD workflow is available at: https://github.com/wangziyuan66/IL-AD . Here the authors adapt nanopore sequencing basecallers to detect RNA modifications. The authors first apply incremental learning to resolve modification-disturbed basecalling, then use anomaly detection to assess nucleotide modification status.

Accurately basecalling sequence backbones in the presence of nucleotide modifications remains a substantial challenge in nanopore sequencing bioinformatics. It has been extensively demonstrated that state-of-the-art basecallers are less compatible with modification-induced sequencing signals. A precise basecalling, on the other hand, serves as the prerequisite for virtually all the downstream analyses. Here, we report that basecallers exposed to diverse training modifications gain the generalizability to analyze novel modifications. With synthesized oligos as the model system, we precisely basecall various out-of-sample RNA modifications. From the representation learning perspective, we attribute this generalizability to basecaller representation space expanded by diverse training modifications. Taken together, we conclude increasing the training data diversity as a paradigm for building modification-tolerant nanopore sequencing basecallers. Accurately basecalling sequence backbones in the presence of nucleotide modifications remains a significant challenge in nanopore sequencing bioinformatics. Here, authors report that basecallers exposed to diverse training modifications gain the generalisability to analyse novel modifications.

Background Recently, increased tumor incidence and cancer-related mortality have been reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), the hallmark feature of OSA, contributes to the metastasis of tumors. However, the molecular mechanisms by which tumor metastasis is accelerated by OSA-like IH remain to be elucidated. Methods C57BL/6 J male mice were subjected to intravenous injection of B16F10 melanoma cells before receiving IH treatment. Then, the animals were randomly distributed into three groups ( n  = 8 each): normoxia (N) group, IH group, and antioxidant tempol group (IHT, exposed to IH after treatment with tempol). After the mice were sacrificed, the number and weight of lung metastatic colonies were assessed. The lung tissues with tumor metastasis were analyzed for markers of oxidative stress and inflammation and for HIF-1α using western blotting and real-time PCR (qRT-PCR). The level of reactive oxygen species (ROS) in B16F10 cell was also assessed after N, IH and IH with tempol treatments. Results Compared with normoxia, IH significantly increased the number and weight of mouse lung metastatic colonies. Treatment of B16F10 cells with IH significantly enhanced ROS generation. Lung tissues with tumor metastasis provided evidence of increased oxidative stress, as assessed by p22 phox and SOD mRNA levels and the NRF2 protein level, as well as increased inflammation, as assessed by TNF-α and IL-6 mRNA levels and the NF-κB P65 protein level. HIF-1α protein levels were increased in response to IH treatment. Tempol, an important antioxidant, ameliorated IH-induced melanoma lung metastasis in mice and reduced oxidative stress and inflammation responses. Conclusions These results support the hypothesis that oxidative stress and inflammation responses play an important role in the pathogenesis of OSA-like IH-induced melanoma lung metastasis in mice. Antioxidant intervention provides a novel strategy for the prevention and treatment of cancer in OSA populations.

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(β)—two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection—but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID-19.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

Tissue-resident immune cells such as innate lymphoid cells (ILC) are known to reside in the parenchymal compartments of tissues and modulate local immune protection. Here we use intravascular cell labeling, parabiosis and multiplex 3D imaging to identify a population of group 3 ILCs in mice that are present within the intravascular space of lung blood vessels (vILC3). vILC3s are distributed broadly in alveolar capillary beds from which inhaled pathogens enter the lung parenchyma. By contrast, conventional ILC3s in tissue parenchyma are enriched in lymphoid clusters in proximity to large veins. In a mouse model of pneumonia, Pseudomonas aeruginosa infection results in rapid vILC3 expansion and production of chemokines including CCL4. Blocking CCL4 in vivo attenuates neutrophil recruitment to the lung at the early stage of infection, resulting in prolonged inflammation and delayed bacterial clearance. Our findings thus define the intravascular space as a site of ILC residence in mice, and reveal a unique immune cell population that interfaces with tissue alarmins and the circulating immune system for timely host defense. Innate lymphoid cells (ILC) are instrumental to immediate, local response to pathogens. Here the authors use parabiosis and multiplex 3D imaging to identify a mouse type 3 ILC population that resides in the intravascular space of lung, produces CCL4 for neutrophil recruitment, and protects from Pseudomonas aeruginosa infection in mice.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don’t proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63 CreERT2 and KRT5-CreERT2 -labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63 CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63 CreERT2 -labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3 -/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.