Quantification of image texture in X-ray phase-contrast-enhanced projection images of in vivo mouse lungs observed at varied inflation pressures

To date, there are very limited noninvasive, regional assays of in vivo lung microstructure near the alveolar level. It has been suggested that x-ray phase-contrast enhanced imaging reveals information about the air volume of the lung; however, the image texture information in these images remains underutilized. Projection images of in vivo mouse lungs were acquired via a tabletop, propagation-based, X-ray phase-contrast imaging system. Anesthetized mice were mechanically ventilated in an upright position. Consistent with previously published studies, a distinct image texture was observed uniquely within lung regions. Lung regions were automatically identified using supervised machine learning applied to summary measures of the image texture data. It was found that an unsupervised clustering within predefined lung regions colocates with expected differences in anatomy along the cranial-caudal axis in upright mice. It was also found that specifically selected inflation pressures-here, a purposeful surrogate of distinct states of mechanical expansion-can be predicted from the lung image texture alone, that the prediction model itself varies from apex to base and that prediction is accurate regardless of overlap with nonpulmonary structures such as the ribs, mediastinum, and heart. Cross-validation analysis indicated low inter-animal variation in the image texture classifications. Together, these results suggest that the image texture observed in a single X-ray phase-contrast-enhanced projection image could be used across a range of pressure states to study regional variations in regional lung function

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies

Temporal Relationship between Primary and Motile Ciliogenesis in Airway Epithelial Cells

Cilia are traditionally classified as motile or primary. Motile cilia are restricted to specific populations of well-differentiated epithelial cells, including those in the airway, brain ventricles, and oviducts. Primary cilia are nonmotile, solitary structures that are present in many cell types, and often have sensory functions such as in the retina and renal tubules. Primary cilia were also implicated in the regulation of fundamental processes in development. Rare depictions of primary cilia in embryonic airways led us to hypothesize that primary cilia in airway cells are temporally related to motile ciliogenesis. We identified primary cilia in undifferentiated, cultured airway epithelial cells from mice and humans and in developing lungs. The solitary cilia in the airways express proteins considered unique to primary cilia, including polycystin-1 and polycystin-2. A temporal analysis of airway epithelial cell differentiation showed that cells with primary cilia acquire markers of motile ciliogenesis, suggesting that motile ciliated cells originate from primary ciliated cells. Whereas motile ciliogenesis requires Foxj1, primary ciliogenesis does not, and the expression of Foxj1 was associated with a loss of primary cilia, just before the appearance of motile cilia. Primary cilia were not found in well-differentiated airway epithelial cells. However, after injury, they appear in the luminal layer of epithelium and in basal cells. The transient nature of primary cilia, together with the temporal and spatial patterns of expression in the development and repair of airway epithelium, suggests a critical role of primary cilia in determining outcomes during airway epithelial cell differentiation

Development of Fully Degradable Phosphonium-Functionalized Amphiphilic Diblock Copolymers for Nucleic Acids Delivery

To expand the range of functional polymer materials to include fully hydrolytically degradable systems that bear bioinspired phosphorus-containing linkages both along the backbone and as cationic side chain moieties for packaging and delivery of nucleic acids, phosphonium-functionalized polyphosphoester-<i>block</i>-poly­(l-lactide) copolymers of various compositions were synthesized, fully characterized, and their self-assembly into nanoparticles were studied. First, an alkyne-functionalized polyphosphoester-<i>block</i>-poly­(l-lactide) copolymer was synthesized via a one pot sequential ring opening polymerization of an alkyne-functionalized phospholane monomer, followed by the addition of l-lactide to grow the second block. Second, the alkynyl side groups of the polyphosphoester block were functionalized via photoinitiated thiol–yne radical addition of a phosphonium-functionalized free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation of well-defined core–shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-phosphate (P⁺/P^–) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various copolymers at a P⁺/P^– ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might provide biocompatible nanocarriers for therapeutic nucleic acid delivery

IL13 activates autophagy to regulate secretion in airway epithelial cells

<p>Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene <i>Atg16l1</i>-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene <i>ATG5</i> (autophagy-related 5) or <i>ATG14</i> (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.</p

The translational landscape of SARS-CoV-2-infected cells reveals suppression of innate immune genes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development

The translational landscape of SARS-CoV-2-infected cells reveals suppression of innate immune genes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development

Synthesis, Characterization, and In Vivo

[Image: see text] The use of nebulizable, nanoparticle-based antimicrobial delivery systems can improve efficacy and reduce toxicity for treatment of multi-drug-resistant bacteria in the chronically infected lungs of cystic fibrosis patients. Nanoparticle vehicles are particularly useful for applying broad-spectrum silver-based antimicrobials, for instance, to improve the residence time of small-molecule silver carbene complexes (SCCs) within the lung. Therefore, we have synthesized multifunctional, shell cross-linked knedel-like polymeric nanoparticles (SCK NPs) and capitalized on the ability to independently load the shell and core with silver-based antimicrobial agents. We formulated three silver-loaded variants of SCK NPs: shell-loaded with silver cations, core-loaded with SCC10, and combined loading of shell silver cations and core SCC10. All three formulations provided a sustained delivery of silver over the course of at least 2–4 days. The two SCK NP formulations with SCC10 loaded in the core each exhibited excellent antimicrobial activity and efficacy in vivo in a mouse model of Pseudomonas aeruginosa pneumonia. SCK NPs with shell silver cation-load only, while efficacious in vitro, failed to demonstrate efficacy in vivo. However, a single dose of core SCC10-loaded SCK NPs (0.74 ± 0.16 mg Ag) provided a 28% survival advantage over sham treatment, and administration of two doses (0.88 mg Ag) improved survival to 60%. In contrast, a total of 14.5 mg of Ag(+) delivered over 5 doses at 12 h intervals was necessary to achieve a 60% survival advantage with a free-drug (SCC1) formulation. Thus, SCK NPs show promise for clinical impact by greatly reducing antimicrobial dosage and dosing frequency, which could minimize toxicity and improve patient adherence