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Recombinant protein expression and purification are crucial in modern life sciences research. A fluorescent immunosensor termed Quenchbody (Q-body) was developed for real-time monitoring of FLAG-fused protein expression. Detection results showed that the limit of detection of the 3 × FLAG peptide detected by the TAMRA-labeled anti-FLAG Q-body was as low as 3.1 nM, with a half-maximal effective concentration of 21.4 nM. Furthermore, the anti-FLAG Q-body was used for detecting different proteins fused with a FLAG-tag at the N- or C-terminal. Subsequently, the constructed Q-body was used to monitor the real-time fermentation process of single-strand DNA-binding protein in Escherichia coli. Unlike previously reported Q-bodies that widely used Fab or scFv, the present study used a full-length anti-FLAG IgG for the first time. Owing to its excellent detection speed and sensitivity, the FLAG Q-body immunosensor has the potential to quantify and monitor target recombinant proteins in multiple biological processes in real-time.
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Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Imunoensaio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes de FusãoRESUMO
Mechanical memory meant the mechanical properties of the matrix could influence the cell fate even after the matrix was changed and has been justified in many kinds of cells. To utilize the phenomenon to improve periodontal tissue engineering, we studied whether mechanical memory existed in human periodontal ligament stem cells and testified if ILK plays a role in this process. The substrate of different stiffness was fabricated by gelatin methacrylate hydrogel. Two groups of hPDLSCs with stiff (St) and soft (So) matrix, respectively, were cultivated. Then, half of the cells exchanged their matrix stiffness in the fourth passage and therefore So, St, So-St, and St-So were formed. Morphology of hPDLSCs and intracellular location of YAP was observed via fluorescence staining, osteogenic differentiation of hPDLSCs was assessed by real-time PCR, ALP staining, and Western blot. Then, all these were reassessed after the ILK gene had been knocked down. The results showed that morphology and YAP location of hPDLSCs were different between matrix changed and unchanged groups; osteogenic genes expression, ALP staining, and Western blot also varied. After the ILK gene had been knocked down, the YAP location and osteogenic activity of hPDLSCs were significantly influenced. Thus, it could be concluded that mechanical memory exists in hPDLSCs; ILK is involved in this process.
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Osteogênese , Ligamento Periodontal , Humanos , Osteogênese/genética , Células Cultivadas , Células-Tronco , Diferenciação Celular , Proliferação de CélulasRESUMO
BACKGROUND: Brachydactyly type B is an autosomal dominant disorder that is characterized by hypoplasia of the distal phalanges and nails and can be divided into brachydactyly type B1 (BDB1) and brachydactyly type B2 (BDB2). BDB1 is the most severe form of brachydactyly and is caused by truncating variants in the receptor tyrosine kinase-like orphan receptor 2 (ROR2) gene. CASE PRESENTATION: Here, we report a five-generation Chinese family with brachydactyly with or without syndactyly. The proband and her mother underwent digital separation in syndactyly, and the genetic analyses of the proband and her parents were provided. The novel heterozygous frameshift variant c.1320dupG, p.(Arg441Alafs*18) in the ROR2 gene was identified in the affected individuals by whole-exome sequencing and Sanger sequencing. The c.1320dupG variant in ROR2 is predicted to produce a truncated protein that lacks tyrosine kinase and serine/threonine- and proline-rich structures and remarkably alters the tertiary structures of the mutant ROR2 protein. CONCLUSION: The c.1320dupG, p.(Arg441Alafs*18) variant in the ROR2 gene has not been reported in any databases thus far and therefore is novel. Our study extends the gene variant spectrum of brachydactyly and may provide information for the genetic counselling of family members.
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Braquidactilia , Sindactilia , Braquidactilia/diagnóstico , Braquidactilia/genética , Ossos do Carpo/anormalidades , Feminino , Deformidades Congênitas do Pé , Deformidades Congênitas da Mão , Humanos , Linhagem , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Estribo/anormalidades , Sinostose , Ossos do Tarso/anormalidadesRESUMO
BACKGROUND AND AIMS: The decreasing Helicobacter pylori eradication rate and the increasing antibiotic resistance trend are of great concern. Therefore, new and effective therapies are needed for H. pylori infection. We conducted a systematic review and meta-analysis to assess the efficacy and safety of semisynthetic tetracycline regimens in H. pylori treatment. METHODS: PubMed, EMBASE, and the Cochrane library were searched. The outcome indicators were the eradication rate, risk ratio (RR, ie, the risk of the semisynthetic tetracycline regimen relative to the control), and 95% confidence interval (95% CI). Controls were patients undergoing any other treatment without semisynthetic tetracycline. RESULTS: Twenty-three studies with 5240 participants were included. The eradication rates of triple regimens with semisynthetic tetracyclines in most studies were less than 70% in both the intention-to-treat (ITT) and the per-protocol (PP) analyses. The pooled eradication rates of quadruple therapies with doxycycline and controls were 95% and 84% in the PP analyses, respectively. The pooled RR associated with efficacy in the quadruple therapy with doxycycline group compared with the control group was 1.12 (95% CI: 1.04-1.20) in the PP analysis. The pooled RR of side effects in the quadruple therapy with doxycycline group compared with the control group was 1.01 (95% CI: 0.65-1.55). CONCLUSION: Seven-day and ten-day quadruple therapy with doxycycline might be an optional first-line therapy. The safety of regimens containing semisynthetic tetracyclines was relatively satisfactory. However, the triple regimen is not recommended.
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Infecções por Helicobacter , Helicobacter pylori , Amoxicilina/uso terapêutico , Antibacterianos/efeitos adversos , Doxiciclina/efeitos adversos , Quimioterapia Combinada , Infecções por Helicobacter/tratamento farmacológico , Humanos , Minociclina/efeitos adversos , Resultado do TratamentoRESUMO
Acne is a common inflammatory skin disease, especially in adolescents. Certain Cutibacterium acnes subtypes are associated with acne, although more than one subtype of C. acnes strains may simultaneously reside on the surface of the skin of an individual. To better understand the relationship between the genomic characteristics of C. acnes subtypes and acnes, we collected 50 C. acnes strains from the facial skin of 10 people (5 healthy individuals, 5 patients with acne) in Liaoning, China and performed whole genome sequencing of all strains. We demonstrated that the six potential pathogenic C. acnes strains were all Type II subtype, and discovered 90 unique genes of the six strains related to acne using pan-genome analysis. The distribution of 2 of the 90 genes was identified by PCR in bacterial cultures collected from the facial skin of 171 individuals (55 healthy individuals, 52 patients with mild acne and 64 patients with moderate to severe acne). Both the genes were significantly associated with acne (Chi square test, P < 0.01). We conclude that Type II strains are associated with acne in Chinese patients.
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Acne Vulgar/microbiologia , Infecções por Actinomycetales/microbiologia , Propionibacterium/classificação , China , Genoma Bacteriano , Genômica/métodos , Humanos , Tipagem de Sequências Multilocus , Filogenia , Propionibacterium/genética , Sequenciamento Completo do GenomaRESUMO
The Mg/S battery is attractive because of its high theoretical energy density and the abundance of Mg and S on the earth. However, its development is hindered by the lack of understanding to the underlying electrochemical reaction mechanism of its charge-discharge processes. Here, using a unique in situ X-ray absorption spectroscopic tool, we systematically study the reaction pathways of the Mg/S cells in Mg(HMDS)2-AlCl3 electrolyte. We find that the capacity degradation is mainly due to the formation of irreversible discharge products, such as MgS and Mg3S8, through a direct electrochemical deposition or a chemical disproportionation of intermediate polysulfide. In light of the fundamental understanding, we propose to use TiS2 as a catalyst to activate the irreversible reaction of low-order MgS x and MgS, which results in an increased discharging capacity up to 900 mAh·g-1 and a longer cycling life.
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Previous studies have revealed that bone marrow-mesenchymal stem cells (BM-MSCs) from systemic lupus erythematosus (SLE) patients exhibited early signs of senescence, which may participate in the development of SLE. However, the molecular mechanisms about this phenomenon have not been fully elucidated. In the current study, we aimed to investigate whether Janus kinase (JAK)-signaling transducers and activators of transcription (STAT) signaling mediated the senescence of BM-MSCs from SLE patients. Twelve female SLE patients and healthy subjects were enrolled in the study. All BM-MSCs were isolated by density gradient centrifugation. Western blot analysis was used to test the expression of JAK-STAT signaling molecules. We observed the activity of ß-gal of cells, the changes of cytoskeletal structure by F-actin staining, and the distribution of cell cycle by flow cytometry. BM-MSCs from SLE patients showed prominent features of senescence, and abnormal activation of JAK-STAT signaling transduction, high level of phosphorylated JAK2, and STAT3. After stimulation of IFN-γ in normal MSCs, JAK-STAT signaling was activated. The cell volume and the number of senescence-associated ß-galactosidase (SA-ß-gal) positive in SLE BM-MSCs were increased. The organization of cytoskeleton was nearly disordered. The rate of cell proliferation was decreased. AG490, the inhibitor of JAK2, and knockdown of STAT3 in BM-MSCs, could significantly reverse the senescence. In summary, our study indicated that JAK-STAT signaling pathway may play a critical role in the senescence of SLE BM-MSCs.
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Medula Óssea/patologia , Senescência Celular , Janus Quinase 2/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Células-Tronco Mesenquimais/patologia , Transdução de Sinais , Adolescente , Adulto , Medula Óssea/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Adulto JovemRESUMO
PURPOSE: In this study, we investigated the expression and role of PSMB4 in human epithelial ovarian cancer(EOC). METHODS: Western blot was used to evaluate the expression of PSMB4 in EOC tissues, and immunohistochemical analysis was performed on 115 cases of ovarian cancers. Then, we used Fisher exact test to analyze the correlation between PSMB4 and clinicopathological parameters. Starvation and re-feeding assay was used to assess cell cycle. CCK-8 assay and plate colony formation assay showed the influence of PSMB4 on proliferation of EOC cells. RESULTS: The expression of PSMB4 in EOC tissues was higher than normal ovary tissues and was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of PSMB4 was related to poor prognosis of EOC patients. Starvation and re-feeding assay suggested that PSMB4 played a critical role in EOC cell proliferation. CCK-8 assay and plate colony formation assay showed that EOC cells treated with PSMB4-siRNA reduced cell proliferation of EOC cells. Additionally, PSMB4 knockdown decreased NF-κB activity. PSMB4 also regulated the expression of NF-κB mediated proteins, including cyclin D1, and cyclin E which involved in cell proliferation. CONCLUSIONS: Our findings implied that PSMB4 is involved in the progression of EOC and could serve as potential therapeutical target of EOC. These data suggested that PSMB4 may promote cell proliferation via the NF-κB-target gene in EOC.
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Biomarcadores Tumorais/metabolismo , Proliferação de Células/fisiologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Adulto , Biomarcadores Tumorais/genética , Carcinoma Epitelial do Ovário , Ciclina D1/genética , Ciclina D1/metabolismo , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , MicroRNAs/genética , Pessoa de Meia-Idade , NF-kappa B/genética , Neoplasias Epiteliais e Glandulares/patologia , Prognóstico , Complexo de Endopeptidases do Proteassoma/genética , RNA Interferente PequenoRESUMO
BACKGROUND: Cancers are complex diseases with heterogeneous genetic causes and clinical outcomes. It is critical to classify patients into subtypes and associate the subtypes with clinical outcomes for better prognosis and treatment. Large-scale studies have comprehensively identified somatic mutations across multiple tumor types, providing rich datasets for classifying patients based on genomic mutations. One challenge associated with this task is that mutations are rarely shared across patients. Network-based stratification (NBS) approaches have been proposed to overcome this challenge and used to classify tumors based on exome-level mutations. In routine research and clinical applications, however, usually only a small panel of pre-selected genes is screened for mutations. It is unknown whether such small panels are effective in classifying patients into clinically meaningful subtypes. RESULTS: In this study, we applied NBS to 13 major cancer types with exome-level mutation data and compared the classification based on the full exome data with those focusing only on small sets of genes. Specifically, we investigated three panels, FoundationOne (240 genes), PanCan (127 genes) and TruSeq (48 genes). We showed that small panels not only are effective in clustering tumors but also often outperform full exome data for most cancer types. We further associated subtypes with clinical data and identified 5 tumor types (CRC-Colorectal carcinoma, HNSC-Head and neck squamous cell carcinoma, KIRC-Kidney renal clear cell carcinoma, LUAD-Lung adenocarcinoma and UCEC-Uterine corpus endometrial carcinoma) whose subtypes are significantly associated with overall survival, all based on small panels. CONCLUSION: Our analyses indicate that effective patient subtyping can be carried out using mutations detected in smaller gene panels, probably due to the enrichment of clinically important genes in such panels.
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Biologia Computacional/métodos , Mutação , Neoplasias/genética , Neoplasias/patologia , Algoritmos , Bases de Dados Genéticas , Exoma , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genes Supressores de Tumor , Humanos , Oncogenes , Análise de SobrevidaRESUMO
With the aim of broadening the scope of Janus-type polymers with new functionalities, Janus-type miktoarm star copolymers comprising helical poly(phenyl isocyanide) (PPI) and a vinyl polymer were designed and synthesized via a combination of Pd(II)-initiated isocyanide polymerization and atom transfer radical polymerization (ATRP). A functional ß-cyclodextrin bearing 7 Pd(II) complexes at one side and 14 bromine groups at the other side ((Pd(II))7-CD-(Br)14) was prepared and used as an initiator for the one-pot polymerization of phenyl isocyanide and the ATRP of vinyl monomers in a living and controlled manner. A variety of Janus-type copolymers with different structures and tunable compositions were facilely obtained by using this method. Thus, Janus-type copolymers composed of helical PPIs and tetraphenylethylene-modified vinyl polymers exhibited a significant circularly polarized luminescence performance in both soluble and aggregated states. Meanwhile, Janus-type copolymers containing PPIs and hydrophilic vinyl polymers presented amphiphilicity and self-assembled into diverse morphologies.
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[This corrects the article DOI: 10.3389/fonc.2021.738801.].
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Optically active helical polycarbenes were constructed through the living and controlled helix-sense-selective polymerization (HSSP) of methyl salicylate modified diazoacetate monomer catalysed via π-allylPdCl with chiral phosphine ligands. The obtained helical polycarbenes could undergo postpolymerization modification to afford functional polycarbenes efficiently.
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Digoxin is a cardiac glycosylated steroid-like drug with a positive inotropic effect and has been widely used in treating congestive heart failure, atrial fibrillation, atrial flutter, and other heart diseases. Digoxin is also a dangerous drug, which can cause drug poisoning at a low blood drug concentration (2.73-3.9 nmol/L, i.e., 2.14-3.05 ng/mL). Therefore, the timely detection of a patient's blood drug concentration plays a significant role in controlling blood drug concentration, reducing the occurrence of drug poisoning events, and maximizing the role of drug therapy. In this study, a DNA vector for the expression of the antidigoxin antibody Fab fragment was constructed. With the vector, Fab was expressed in E. coli and purified, and 1.2 mg of antibodies was obtained from 100 mL of culture. An immunofluorescent sensor based on the mechanism of photoinduced electron transfer was constructed by labeling additional cysteines in the heavy chain variable region and light chain variable region of the antibody Fab fragment with fluorescent dyes. The assay for digoxin with the immunosensor could be finished within 5 min with a limit of detection of 0.023 ng/mL, a detectable range of 0.023 ng/mL to 100 µg/mL, and an EC50 of 0.256 ng/mL. A new approach for the rapid detection of digoxin was developed and will contribulte to therapeutic drug monitoring.
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Conventional ascorbic acid (AA) detection methods such as chromatography, capillary electrophoresis, colorimetry, electrochemical detection, and enzymatic analysis require expensive equipment and complicated operation. Simple, rapid, and accurate AA detection is essential to inspect food quality, diagnose diseases, and assess immunity in humans. In this study, the first near-infrared fluorescence sensor DBHM with aggregation-induced emission was developed to detect AA under the involvement of Cu2+. The DBHM + Cu2+ sensor showed high sensitivity to AA with a limit of detection of 2.37 µM. The AA detection mechanism was investigated by optical studies, 1H NMR titration, high-resolution mass spectrometry, and infrared spectroscopy. AA was detected qualitatively and quantitatively by the DBHM + Cu2+ sensor in beverages, fruits, and Vitamin C tablets using a dual-mode (fluorescence and smartphone app) sensing platform. The new sensing system also showed low toxicity and excellent bioimaging in HeLa cells, C. elegans, and mice. This sensor could advance AA detection technology in the food industry and has potential bioimaging applications.
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Corantes Fluorescentes , Pontos Quânticos , Camundongos , Humanos , Animais , Corantes Fluorescentes/toxicidade , Corantes Fluorescentes/química , Ácido Ascórbico/análise , Células HeLa , Caenorhabditis elegans , Pontos Quânticos/química , Limite de Detecção , Espectrometria de Fluorescência/métodosRESUMO
The hallmark of orthodontic tooth movement (OTM) is time-consuming during clinical treatments. The acceleration of OTM through modulating proliferation and apoptosis of periodontal ligament cells (PDLCs) possesses the potential application in clinical treatments. Here, we established an in vitro model with a graded increase in substrate stiffness to investigate the underlying mechanism of proliferation and apoptosis of PDLCs. The role of the integrin-linked kinase (ILK) in response to substrate stiffness was investigated by the depletion model of PDLCs. We found that the proliferation and apoptosis of PDLCs show a stiffness-dependent property with stiffer substrates favoring increased bias at the transcript level. Depleting integrin-linked kinase diluted the correlation between PDLCs behaviors and substrate stiffness. Our results suggest that ILK plays a significant role in modulating PDLC proliferation and apoptosis and can serve as a potential target for accelerating OTM.
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Apoptose , Ligamento Periodontal , Proliferação de Células , Proteínas Serina-Treonina Quinases/genética , HumanosRESUMO
Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. Combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.
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Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.
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Células Endoteliais , Fibrose Pulmonar Idiopática , Camundongos , Animais , Humanos , Células Endoteliais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , DNA Complementar/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Bleomicina/toxicidade , Fatores de Transcrição Forkhead/metabolismo , Fibroblastos/metabolismoRESUMO
Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.