The aging lung: Physiology, disease, and immunity.

The population is aging at a rate never seen before in human history. As the number of elderly adults grows, it is imperative we expand our understanding of the underpinnings of aging biology. Human lungs are composed of a unique panoply of cell types that face ongoing chemical, mechanical, biological, immunological, and xenobiotic stress over a lifetime. Yet, we do not fully appreciate the mechanistic drivers of lung aging and why age increases the risk of parenchymal lung disease, fatal respiratory infection, and primary lung cancer. Here, we review the molecular and cellular aspects of lung aging, local stress response pathways, and how the aging process predisposes to the pathogenesis of pulmonary disease. We place these insights into context of the COVID-19 pandemic and discuss how innate and adaptive immunity within the lung is altered with age.

May the (Mechanical) Force Be with AT2.

Idiopathic pulmonary fibrosis is a fatal disease involving destruction of the lung alveolar structure. In this issue of Cell, Wu et al. (2020) show that impaired alveolar (AT2) stem cells produce mechanical tension that leads to spatially regulated fibrosis, initiating a new chapter in understanding what underlies the periphery to center progression of this lung disease.

Don't Stop Re-healin'! Cancer as an Ongoing Stem Cell Affair.

Tumors have long been suspected of hijacking stem cell mechanisms used for tissue maintenance and repair. Ge et al. now show that skin tumors exhibit merged chromatin profiles from distinct stem cell lineages. This "lineage infidelity" recreates a state akin to transient wound repair that persists to maintain uncontrolled growth.

Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6.

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.

Mammalian Autophagy: How Does It Work?

Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis.

Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal 3D cocultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell cocultures. Gain- and loss-of-function experiments showed that BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1 null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide tools to understand the mechanisms of respiratory diseases at the single-cell level.

Early-stage lung cancer is driven by a transitional cell state dependent on a KRAS-ITGA3-SRC axis.

Glycine-12 mutations in the GTPase KRAS (KRASG12) are an initiating event for development of lung adenocarcinoma (LUAD). KRASG12 mutations promote cell-intrinsic rewiring of alveolar type-II progenitor (AT2) cells, but to what extent such changes interplay with lung homeostasis and cell fate pathways is unclear. Here, we generated single-cell RNA-seq (scRNA-seq) profiles from AT2-mesenchyme organoid co-cultures, mice, and stage-IA LUAD patients, identifying conserved regulators of AT2 transcriptional dynamics and defining the impact of KRASG12D mutation with temporal resolution. In AT2WT organoids, we found a transient injury/plasticity state preceding AT2 self-renewal and AT1 differentiation. Early-stage AT2KRAS cells exhibited perturbed gene expression dynamics, most notably retention of the injury/plasticity state. The injury state in AT2KRAS cells of patients, mice, and organoids was distinguishable from AT2WT states via altered receptor expression, including co-expression of ITGA3 and SRC. The combination of clinically relevant KRASG12D and SRC inhibitors impaired AT2KRAS organoid growth. Together, our data show that an injury/plasticity state essential for lung repair is co-opted during AT2 self-renewal and LUAD initiation, suggesting that early-stage LUAD may be susceptible to interventions that target specifically the oncogenic nature of this cell state.

Diverse autophagosome membrane sources coalesce in recycling endosomes.

Autophagic protein degradation is mediated by autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.

Premalignant Progression in the Lung: Knowledge Gaps and Novel Opportunities for Interception of Non-Small Cell Lung Cancer. An Official American Thoracic Society Research Statement.

Rationale: Despite significant advances in precision treatments and immunotherapy, lung cancer is the most common cause of cancer death worldwide. To reduce incidence and improve survival rates, a deeper understanding of lung premalignancy and the multistep process of tumorigenesis is essential, allowing timely and effective intervention before cancer development. Objectives: To summarize existing information, identify knowledge gaps, formulate research questions, prioritize potential research topics, and propose strategies for future investigations into the premalignant progression in the lung. Methods: An international multidisciplinary team of basic, translational, and clinical scientists reviewed available data to develop and refine research questions pertaining to the transformation of premalignant lung lesions to advanced lung cancer. Results: This research statement identifies significant gaps in knowledge and proposes potential research questions aimed at expanding our understanding of the mechanisms underlying the progression of premalignant lung lesions to lung cancer in an effort to explore potential innovative modalities to intercept lung cancer at its nascent stages. Conclusions: The identified gaps in knowledge about the biological mechanisms of premalignant progression in the lung, together with ongoing challenges in screening, detection, and early intervention, highlight the critical need to prioritize research in this domain. Such focused investigations are essential to devise effective preventive strategies that may ultimately decrease lung cancer incidence and improve patient outcomes.

The Peptide Antibiotic Corramycin Adopts a ß-Hairpin-like Structure and Is Inactivated by the Kinase ComG.

The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.

Long-term Outcomes Following Colectomy and Liver Transplantation for Inflammatory Bowel Disease with Primary Sclerosing Cholangitis.

OBJECTIVE: To investigate the long-term outcomes of patients with combined primary sclerosing cholangitis/inflammatory bowel disease (PSC-IBD) undergoing both liver transplantation (LT) and total abdominal colectomy (TAC). SUMMARY BACKGROUND DATA: The fraction of patients with PSC-IBD that require both LT and TAC is small, thereby limiting significant conclusions regarding long-term outcomes. METHODS: Adult and pediatric patients from nine centers from the US IBD Surgery Collaborative who underwent staged LT and TAC for PSC-IBD were included. Long-term outcomes, including survival, were assessed. RESULTS: Among 127 patients, 66 underwent TAC-before-LT, with a median time from TAC to LT of 7.9 yrs, while 61 underwent LT-before-TAC, with a median time from LT to TAC of 4.4 years. Median patient survival post TAC was significantly worse in those undergoing LT-before-TAC (16.0 yrs vs. 42.6 yrs, P=0.007), while post LT survival was not impacted by the order of TAC and LT (21.6 yrs vs. 22.0 yrs, P=0.81). Patients undergoing TAC for medically refractory disease had a higher incidence of recurrent PSC (rPSC) (P=0.02) and biliary complications (0.09) compared to those undergoing TAC for oncologic indications. Definitive TAC reconstruction with either end ileostomy or ileal-pouch anal anastomosis (IPAA) did not impact post-LT or post-TAC outcomes. CONCLUSIONS: Long term survival in PSC-IBD was contingent upon progression to LT and was not impacted by the need for TAC. PSC-IBD patients undergoing TAC for medically refractory disease had a higher incidence of rPSC and biliary complications. The use of IPAA in PSC-IBD was a viable alternative to end ileostomy.

Single-Cell Untargeted Lipidomics Using Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection.

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level. The method is then coupled to a system that can provide automated sampling of live, single cells into capillaries under microscope observation. This workflow retains the spatial information and morphology of cells during sampling and highlights the heterogeneity in lipid profiles observed at the single-cell level. The workflow is applied to show changes in single-cell lipid profiles as a response to oxidative stress, coinciding with expanded lipid droplets. This demonstrates that the workflow is sufficiently sensitive to observing changes in lipid profiles in response to a biological stimulus. Understanding how lipids vary in single cells will inform future research into a multitude of biological processes as lipids play important roles in structural, biophysical, energy storage, and signaling functions.

SPCS1-Dependent E2-p7 processing determines HCV Assembly efficiency.

Recent studies identified signal peptidase complex subunit 1 (SPCS1) as a proviral host factor for Flaviviridae viruses, including HCV. One of the SPCS1's roles in flavivirus propagation was attributed to its regulation of signal peptidase complex (SPC)-mediated processing of flavivirus polyprotein, especially C-prM junction. However, whether SPCS1 also regulates any SPC-mediated processing sites within HCV polyprotein remains unclear. In this study, we determined that loss of SPCS1 specifically impairs the HCV E2-p7 processing by the SPC. We also determined that efficient separation of E2 and p7, regardless of its dependence on SPC-mediated processing, leads to SPCS1 dispensable for HCV assembly These results suggest that SPCS1 regulates HCV assembly by facilitating the SPC-mediated processing of E2-p7 precursor. Structural modeling suggests that intrinsically delayed processing of the E2-p7 is likely caused by the structural rigidity of p7 N-terminal transmembrane helix-1 (p7/TM1/helix-1), which has mostly maintained membrane-embedded conformations during molecular dynamics (MD) simulations. E2-p7-processing-impairing p7 mutations narrowed the p7/TM1/helix-1 bending angle against the membrane, resulting in closer membrane embedment of the p7/TM1/helix-1 and less access of E2-p7 junction substrate to the catalytic site of the SPC, located well above the membrane in the ER lumen. Based on these results we propose that the key mechanism of action of SPCS1 in HCV assembly is to facilitate the E2-p7 processing by enhancing the E2-p7 junction site presentation to the SPC active site. By providing evidence that SPCS1 facilitates HCV assembly by regulating SPC-mediated cleavage of E2-p7 junction, equivalent to the previously established role of this protein in C-prM junction processing in flavivirus, this study establishes the common role of SPCS1 in Flaviviridae family virus propagation as to exquisitely regulate the SPC-mediated processing of specific, suboptimal target sites.

Systematic Review of Neoadjuvant Immunotherapy for Mismatch Repair Deficient Locally Advanced Colon Cancer: An Emerging Strategy.

BACKGROUND: In April 2023, the National Comprehensive Cancer Network endorsed neoadjuvant immunotherapy for select patients with nonmetastatic mismatch repair deficient colon cancer. Approximately 15% of incident colon cancers are mismatch repair deficient, resulting in a distinct molecular subtype with high microsatellite instability that is responsive to immune checkpoint inhibition. OBJECTIVE: To describe the existing evidence supporting neoadjuvant immunotherapy for mismatch repair deficient, microsatellite unstable nonmetastatic colon cancer. DATA SOURCES: A medical librarian performed PubMed, Embase, and Web of Science searches most recently on April 24, 2023. The PubMed search was re-run on September 26, 2023, to identify any additional studies published between April 24 and September 26, 2023. STUDY SELECTION: Two authors screened titles and abstracts in the published studies. The inclusion criteria were 1) English language, 2) adults with primary cancer of the colon, 3) nonmetastatic disease, 4) neoadjuvant immunotherapy, and 5) reporting on 10 or more cases. INTERVENTION: Neoadjuvant immunotherapy. MAIN OUTCOME MEASURES: Safety (grade 3+ treatment-related adverse events) and efficacy (complete pathologic responses). RESULTS: From 7691 studies identified, 6370 were screened and 8 were included. Various agents, dosing regimens, and treatment durations were used, with durations of immunotherapy ranging from 1 to 16 cycles. Complete R0 resections were consistently achieved in 98% to 100% of resections. Of patients who received neoadjuvant immunotherapy and underwent resection, 50% to 91% had ypT0N0 pathology. The safety profiles were generally favorable, with grade 1 to 2 treatment-related adverse events (mostly immune-related) during immunotherapy reported in 22.2% to 70% of patients. Postoperative complications after neoadjuvant immunotherapy were reassuring, with no severe complications reported. LIMITATIONS: Small number of heterogeneous and uncontrolled studies precluding a meta-analysis. CONCLUSIONS: Neoadjuvant immune checkpoint inhibition is associated with high rates of pathologic complete responses in locally advanced colon cancer. The literature is limited, particularly for postoperative outcomes, and more studies are needed to understand the safety and positioning of these regimens in the neoadjuvant context.

Perceptions of the White Coat: Generational, Regional, and Gender Differences Among Surgeons.

INTRODUCTION: Recent studies have evaluated patient perception of physician attire; however, few studies have considered physician perceptions of workplace attire. This study aimed to assess current trends regarding attire preferences among surgeons. METHODS: A national, population-based survey was distributed via email and "X" (Twitter). Participants were asked to complete an online questionnaire regarding their perception of the white coat, preferred attire in clinical settings, and reasons for choice of attire. RESULTS: Of 481 participants, 172 (36%) were attendings, 164 (34%) were residents, 125 (26%) were medical students, and 20 (4%) were fellows. Those who practiced in the Midwest region were more likely to wear a white coat daily (35.1% versus 28.5% South, 23.5% Northeast, 20.0% West, P < 0.05). Late career surgeons (practicing >20 y) were more likely to wear a white coat in the hospital and wear it daily (56% versus 36% of middle-career surgeons, 34% early-career surgeons, and 26% in training, P < 0.05). Women surgeons more frequently wore a white coat in clinic (64% versus 54% men, P < 0.05), reported that wearing a white coat was influenced by their program's culture (61% versus 46% of men surgeons, P < 0.05), that they would stop wearing a white coat if other members of their department stopped (50% versus 35% of men, P < 0.05), and that they believe the white coat helps distinguish female doctors from nurses (61% versus 50% of men surgeons, P < 0.05). CONCLUSIONS: This study demonstrates generational, regional, and gender differences among surgeons in their perception of the white coat at a national level.

Author Correction: EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

We wish to correct two mutations in Supplementary Table 4 of this Letter. The NCI-H460 cell line was annotated as being mutant for TP53. NCI-H460 has been verified to be TP53 wild type by several sources1. The NCI-H2009 cell line was annotated as being mutant for PIK3CA. As annotated by COSMIC (ref. 24 of the original Letter) and CCLE (ref. 25 of the original Letter), the NCI-H2009 cell line has a mutation in PIK3C3, rather than PIK3CA. The cell line is wild type for PIK3CA. The Supplementary Information of this Amendment contains the corrected Supplementary Table 4. These errors do not affect our conclusions. The original Letter has not been corrected.

The role of advanced endoscopy in appendiceal polyp management and outcomes.

BACKGROUND: Appendiceal orifice lesions are often managed operatively with limited or oncologic resections. The aim is to report the management of appendiceal orifice mucosal neoplasms using advanced endoscopic interventions. METHODS: Patients with appendiceal orifice mucosal neoplasms who underwent advanced endoscopic resections between 2011 and 2021 with either endoscopic mucosal resection (EMR), endoscopic mucosal dissection (ESD), hybrid ESD, or combined endoscopic laparoscopic surgery (CELS) were included from a prospectively collected dataset. Patient and lesion details and procedure outcomes are reported. RESULTS: Out of 1005 lesions resected with advanced endoscopic techniques, 41 patients (4%) underwent appendiceal orifice mucosal neoplasm resection, including 39% by hybrid ESD, 34% by ESD, 15% by EMR, and 12% by CELS. The median age was 65, and 54% were male. The median lesion size was 20 mm. The dissection was completed piecemeal in 49% of patients. Post-procedure, one patient had a complication within 30 days and was admitted with post-polypectomy abdominal pain treated with observation for 2 days with no intervention. Pathology revealed 49% sessile-serrated lesions, 24% tubular adenomas, and 15% tubulovillous adenomas. Patients were followed up for a median of 8 (0-48) months. One patient with a sessile-serrated lesion experienced a recurrence after EMR which was re-resected with EMR. CONCLUSION: Advanced endoscopic interventions for appendiceal orifice mucosal neoplasms can be performed with a low rate of complications and early recurrence. While conventionally lesions at the appendiceal orifice are often treated with surgical resection, advanced endoscopic interventions are an alternative approach with promising results which allow for cecal preservation.

Vaginal prevention of Candida albicans: synergistic effect of lactobacilli and mannan oligosaccharides (MOS).

Vulvovaginal candidiasis (VVC) affects approximately 30-50% of women at least once during their lifetime, causing uncomfortable symptoms and limitations in their daily quality of life. Antifungal therapy is not very effective, does not prevent recurrencies and usually causes side effects. Therefore, alternative therapies are urgently needed. The goal of this work was to investigate the potential benefits of using mannan oligosaccharides (MOS) extracts together with a Lactobacillus sp. pool, composed by the most significant species present in the vaginal environment, to prevent infections by Candida albicans. Microbial growth of isolated strains of the main vaginal lactobacilli and Candida strains was assessed in the presence of MOS, to screen their impact upon growth. A pool of the lactobacilli was then tested against C. albicans in competition and prophylaxis studies; bacterial and yeast cell numbers were quantified in specific time points, and the above-mentioned studies were assessed in simulated vaginal fluid (SVF). Finally, adhesion to vaginal epithelial cells (HeLa) was also evaluated, once again resorting to simultaneous exposure (competition) or prophylaxis assays, aiming to measure the effect of MOS presence in pathogen adherence. Results demonstrated that MOS extracts have potential to prevent vaginal candidiasis in synergy with vaginal lactobacilli, with improved results than those obtained when using lactobacilli alone. KEY POINTS: Potential benefits of MOS extracts with vaginal lactobacilli to prevent C. albicans infections. MOS impacts on growth of vaginal lactobacilli pool and C. albicans in SVF. MOS extracts in synergy with L. crispatus inhibit C. albicans adhesion in HeLa cells.

Evaluation of the feasibility for replacing sheep blood with human blood in culture media used in microbiological diagnostics.

The present study aimed to suggest the replacement of animal blood with human blood in culture media, involving alternative methods and ethical considerations, such as animal welfare, in addition to potential laboratory cost reduction. Characteristics of growth and hemolysis development were compared in different culture media, using both sheep blood and human blood. Blood types from the ABO blood group system were tested, and commercially acquired sheep blood agar was used for comparison. Bacteria of the genus Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were tested. It was observed that growth in media with type A and O positive blood showed closer similarities to those performed in agar with sheep blood. Depending on the bacterial species, the results were either more positive or not, with faster-growing and less demanding bacteria showing better results than, for example, S. pneumoniae, which demonstrated difficulty in the growth process and hemolysis generation in human blood agar. The research suggests that in some situations, sheep blood could be replaced, especially when the goal is growth and isolation, but may not be as suitable when the objective is to analyze hemolysis or when the studied species is demanding.

Harvesting the Power of Green Synthesis: Gold Nanoparticles Tailored for Prostate Cancer Therapy.

Biosynthetic gold nanoparticles (bAuNPs) present a promising avenue for enhancing bio-compatibility and offering an economically and environmentally responsible alternative to traditional production methods, achieved through a reduction in the use of hazardous chemicals. While the potential of bAuNPs as anticancer agents has been explored, there is a limited body of research focusing on the crucial physicochemical conditions influencing bAuNP production. In this study, we aim to identify the optimal growth phase of Pseudomonas aeruginosa cultures that maximizes the redox potential and coordinates the formation of bAuNPs with increased efficiency. The investigation employs 2,6-dichlorophenolindophenol (DCIP) as a redox indicator. Simultaneously, we explore the impact of temperature, pH, and incubation duration on the biosynthesis of bAuNPs, with a specific emphasis on their potential application as antitumor agents. Characterization of the resulting bAuNPs is conducted using ATR-FT-IR, TEM, and UV-Vis spectroscopy. To gain insights into the anticancer potential of bAuNPs, an experimental model is employed, utilizing both non-neoplastic (HPEpiC) and neoplastic (PC3) epithelial cell lines. Notably, P. aeruginosa cultures at 9 h/OD600 = 1, combined with biosynthesis at pH 9.0 for 24 h at 58 °C, produce bAuNPs that exhibit smaller, more spherical, and less aggregated characteristics. Crucially, these nanoparticles demonstrate negligible effects on HPEpiC cells while significantly impacting PC3 cells, resulting in reduced viability, migration, and lower IL-6 levels. This research lays the groundwork for the development of more specialized, economical, and ecologically friendly treatment modalities.