Mammalian Lung Research Articles

3D reconstruction of the bronchial tree of the Gray short-tailed opossum (Monodelphis domestica) in the postnatal period.

Recent didelphid marsupials resemble the assumed mammalian ancestor and are suitable to inform on the evolution of the mammalian lung. This study uses X-ray computed tomography (μCT) to three-dimensionally reconstruct the bronchial tree of the marsupial Gray short-tailed opossum (Monodelphis domestica) in order to reveal the timeline of morphogenesis during the postnatal period. The development of the bronchial tree was examined in 37 animals from embryonic day 13, during the postnatal period (neonate to 57 days) and in adults. The first appearance and the branching of lobar, segmental and sub-segmental bronchioles in the lungs were documented. Based on the reconstructions, the generation of end-branching airways, the median and maximum generation and the number of branches were calculated for each pulmonary lobe. At birth, the lung of M. domestica has a primitive appearance since it consists of a simple system of branching airways that end in a number of terminal air spaces, lobar bronchioles, and first segmental bronchioles are present. During the postnatal period, the volumes of the lung and bronchial tree steadily increase and development, differentiation, and expansion of the bronchial tree takes place. By 14 days, the fundamental bronchial tree consisting of lobar, segmental, and sub-segmental bronchioles has been established. A mature bronchial tree, including respiratory bronchioles and alveolar ducts is present by day 35. The asymmetry of the right (predominately four lobes) and the left lung (predominately two lobes), as present in M. domestica, can be considered as plesiomorphic for Mammalia. In marsupials, the process of branching morphogenesis, which takes place intrauterine in the placental fetus, is shifted to the postnatal period, but follows similar patterns as described in placentals. Lung maturation in general and the branching morphogenesis in particular seems to be highly conservative within mammalian evolution.

Conserved acidic second shell residue modulates the structure, stability and activity of non-seleno human peroxiredoxin 6

1-Cys peroxiredoxin6 (Prdx6) is unique and inducible bifunctional enzyme in the mammalian lungs and plays a role in the progression and inhibition of cancerous cells at different stages. The enzyme possesses two distinct active sites for phospholipase A2 and peroxidase activity. The conserved residues surrounding the peroxidase active site, also called as second shell residues are Glu50, Leu71, Ser72, His79 and Arg155. Since there is no study done about the active site stabilization of the transition state of Prdx6, there are a lot of questions unanswered regarding the Prdx6 peroxidase activity. In order to evaluate the role of second shell conserved residue Glu50, present in close vicinity to peroxidatic active site, we substituted this negatively charged residue with Alanine and Lysine. To explore the effect of mutation on the biophysical parameters, the mutant proteins were compared with Wild-Type by using biochemical, biophysical, and in silico methods. Comparative spectroscopic methods and enzyme activity demonstrate that the Glu50 plays a significant role in maintaining the structure, stability, and function of protein. From the results we conclude that Glu50 significantly controls the structure; stability and may be involved in the active site stabilization of transition state for proper position of diverse peroxides.

A mammalian lung's immune system minimizes tissue damage by initiating five major sequential phases of defense.

The mammalian lungs encounter several pathogens, but have a sophisticated multi-phase immune defense. Furthermore, several immune responses to suppress pulmonary pathogens can damage the airway epithelial cells, particularly the vital alveolar epithelial cells (pneumocytes). The lungs have a sequentially activated, but overlapping, five phase immune response to suppress most pathogens, while causing minimal damage to the airway epithelial cells. Each phase of the immune response may suppress the pathogens, but if the previous phase proves inadequate, a stronger phase of immune response is activated, but with an increased risk of airway epithelial cell damage. The first phase immune response involves the pulmonary surfactants, which have proteins and phospholipids with potentially sufficient antibacterial, antifungal and antiviral properties to suppress many pathogens. The second phase immune response involves the type III interferons, having pathogen responses with comparatively minimal risk of damage to airway epithelial cells. The third phase immune response involves type I interferons, which implement stronger immune responses against pathogens with an increased risk of damage to airway epithelial cells. The fourth phase immune response involves the type II interferon, interferon-γ, which activates stronger immune responses, but with considerable risk of airway epithelial cell damage. The fifth phase immune response involves antibodies, potentially activating the complement system. In summary, five major phases of immune responses for the lungs are sequentially initiated to create an overlapping immune response which can suppress most pathogens, while usuallycausing minimal damage to the airway epithelial cells, including the pneumocytes.

Water and heat exchanges in mammalian lungs

A secondary function of the respiratory system of the mammals is, during inspiration, to heat the air to body temperature and to saturate it with water before it reaches the alveoli. Relying on a mathematical model, we propose a comprehensive analysis of this function, considering all the terrestrial mammals (spanning six orders of magnitude of the body mass, M) and focusing on the sole contribution of the lungs to this air conditioning. The results highlight significant differences between the small and the large mammals, as well as between rest and effort, regarding the spatial distribution of heat and water exchanges in the lungs, and also in terms of regime of mass transfer taking place in the lumen of the airways. Interestingly, the results show that the mammalian lungs appear to be designed just right to fully condition the air at maximal effort (and clearly over-designed at rest, except for the smallest mammals): all generations of the bronchial region of the lungs are mobilized for this purpose, with calculated values of the local evaporation rate of water from the bronchial mucosa that can be very close to the maximal ability of the serous cells to replenish this mucosa with water. For mammals with a mass above a certain threshold (simeq 5 kg at rest and simeq 50 g at maximal effort), it appears that the maximal value of this evaporation rate scales as M^{-1/8} at rest and M^{-1/16} at maximal effort and that around 40% (at rest) or 50% (at maximal effort) of the water/heat extracted from the lungs during inspiration is returned to the bronchial mucosa during expiration, independently of the mass, due to a subtle coupling between different phenomena. This last result implies that, above these thresholds, the amounts of water and heat extracted from the lungs by the ventilation scale with the mass such as the ventilation rate does (i.e. as M^{3/4} at rest and M^{7/8} at maximal effort). Finally, it is worth to mention that these amounts appear to remain limited, but not negligible, when compared to relevant global quantities, even at maximal effort (4–6%).

Plasticity in airway smooth muscle differentiation during mouse lung development.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

Features of lung organometry in domestic animals of the Mammalian class (Mammalia)

The study of the animal body and its morphoarchitectonics is a priority area for the successful development of animal husbandry. The purpose of the study is to establish the macroscopic structure of the lungs and provide an organometric assessment of their morphological structures in domestic sexually mature animals. The study used 30 clinically healthy individuals of 6 animal species belonging to the class Mammalia – Mammals. According to the results of the morphometry analysis of absolute and relative lung values in domestic mammals and their right and left lobes, a pronounced asymmetry was established, the coefficient of which corresponded to: in rabbits – 1:1.30, in dogs – 1:1.33, in sheep – 1:1.37, in pigs – 1:1.34, in cattle – 1:1.37, in horses – 1:1.2. For morphological assessment of typical lung features characteristic of a particular animal species, a morphological scale of marker features of the organ is proposed. According to the results of the study, it is proposed to classify the lungs of domestic mammals, considering the structure and shape, into 4 types: 1st – expanded-shortened (lung development index (LDI) = 85– 100%); 2nd –intermediate (LDI = 101–120%); 3rd – moderately elongated (LDI = 121–130%); 4th – elongated (LDI = 131–140%). Based on a detailed analysis of organometric studies, a scale for assessing marker features of the lung is proposed – in rabbits (LDI = 90 ± 1.89%) assigned to the extended-shortened type, in cattle (LDI=117±2.21%) and sheep (LDI=114±2.08%) – intermediate type, in horses (LDI = 127 ± 2.74%) – moderately elongated type, in pigs (LDI = 136 ± 3.01%) and dogs (LDI = 137 ± 2.84%) – elongated type. The obtained results of morphological studies of the lungs of the representatives of the mammalian class are of practical importance in biology and veterinary medicine since they are marker signs of their morphofunctional state and criteria for pathomorphological diagnosis of respiratory diseases

Engineering and Modeling the Lung Mesenchyme.

The structure of the mammalian lung controls the flow of air through the airways and into the distal alveolar region where gas exchange occurs. Specialized cells in the lung mesenchyme produce the extracellular matrix (ECM) and growth factors required for lung structure. Historically, characterizing the mesenchymal cell subtypes was challenging due to their ambiguous morphology, overlapping expression of protein markers, and limited cell-surface molecules needed for isolation. The recent development of single-cell RNA sequencing (scRNA-seq) complemented with genetic mouse models demonstrated that the lung mesenchyme comprises transcriptionally and functionally heterogeneous cell-types. Bioengineering approaches that model tissue structure clarify the function and regulation of mesenchymal cell types. These experimental approaches demonstrate the unique abilities of fibroblasts in mechanosignaling, mechanical force generation, ECM production, and tissue regeneration. This chapter will review the cell biology of the lung mesenchyme and experimental approaches to study their function.

Lack of evidence for increased transcriptional noise in aged tissues.

Aging is often associated with a loss of cell type identity that results in an increase in transcriptional noise in aged tissues. If this phenomenon reflects a fundamental property of aging remains an open question. Transcriptional changes at the cellular level are best detected by single-cell RNA sequencing (scRNAseq). However, the diverse computational methods used for the quantification of age-related loss of cellular identity have prevented reaching meaningful conclusions by direct comparison of existing scRNAseq datasets. To address these issues we created Decibel, a Python toolkit that implements side-to-side four commonly used methods for the quantification of age-related transcriptional noise in scRNAseq data. Additionally, we developed Scallop, a novel computational method for the quantification of membership of single cells to their assigned cell type cluster. Cells with a greater Scallop membership score are transcriptionally more stable. Application of these computational tools to seven aging datasets showed large variability between tissues and datasets, suggesting that increased transcriptional noise is not a universal hallmark of aging. To understand the source of apparent loss of cell type identity associated with aging, we analyzed cell type-specific changes in transcriptional noise and the changes in cell type composition of the mammalian lung. No robust pattern of cell type-specific transcriptional noise alteration was found across aging lung datasets. In contrast, age-associated changes in cell type composition of the lung were consistently found, particularly of immune cells. These results suggest that claims of increased transcriptional noise of aged tissues should be reformulated.

MTORC1 signaling facilitates differential stem cell differentiation to shape the developing murine lung and is associated with mitochondrial capacity

Formation of branched organs requires sequential differentiation of stem cells. In this work, we find that the conducting airways derived from SOX2+ progenitors in the murine lungs fail to form without mTOR complex 1 (mTORC1) signaling and are replaced by lung cysts. Proximal-distal patterning through transitioning of distal SOX9+ progenitors to proximal SOX2+ cells is disrupted. Mitochondria number and ATP production are reduced. Compromised mitochondrial capacity results in a similar defect as that in mTORC1-deficient lungs. This suggests that mTORC1 promotes differentiation of SOX9+ progenitors to form the conducting airways by modulating mitochondrial capacity. Surprisingly, in all mutants, saccules are produced from lung cysts at the proper developmental time despite defective branching. SOX9+ progenitors also differentiate into alveolar epithelial type I and type II cells within saccules. These findings highlight selective utilization of energy and regulatory programs during stem cell differentiation to produce distinct structures of the mammalian lungs.

LungMAP Portal Ecosystem: Systems-level Exploration of the Lung.

An improved understanding of the human lung necessitates advanced systems models informed by an ever-increasing repertoire of molecular omics, cellular imaging, and pathological datasets. To centralize and standardize information across broad lung research efforts, we expanded the LungMAP.net website into a new gateway portal. This portal connects a broad spectrum of research networks, bulk and single-cell multiomics data, and a diverse collection of image data that span mammalian lung development and disease. The data are standardized across species and technologies using harmonized data and metadata models that leverage recent advances, including those from the Human Cell Atlas, diverse ontologies, and the LungMAP CellCards initiative. To cultivate future discoveries, we have aggregated a diverse collection of single-cell atlases for multiple species (human, rhesus, and mouse) to enable consistent queries across technologies, cohorts, age, disease, and drug treatment. These atlases are provided as independent and integrated queryable datasets, with an emphasis on dynamic visualization, figure generation, reanalysis, cell-type curation, and automated reference-based classification of user-provided single-cell genomics datasets (Azimuth). As this resource grows, we intend to increase the breadth of available interactive interfaces, supported data types, data portals and datasets from LungMAP, and external research efforts.

Decellularized Spinach Biomaterials Support Physiologically Relevant Mechanical Cyclic Strain and Prompt a Stretch-Induced Cellular Response.

Recently, decellularized plant biomaterials have been explored for their use as tissue engineered substitutes. Herein, we expanded upon the investigation of the mechanical properties of these materials to explore their elasticity as many anatomical areas of the body require biomechanical dynamism. We first constructed a device to secure the scaffold and induce a strain within the physiological range of the normal human adult lung during breathing (12-20 movements/min; 10-20% elongation). Results showed that decellularized spinach leaves can support cyclic strain for 24 h and displayed heterogeneous local strain values (7.76-15.88%) as well as a Poisson's ratio (0.12) similar to that of mammalian lungs (10.67-19.67%; 0.01), as opposed to an incompressible homogeneous standard polymer (such as PDMS (10.85-12.71%; 0.4)). Imaging and mechanical testing showed that the vegetal scaffold exhibited strain hardening but maintained its structural architecture and water retention capacity, suggesting an unaltered porosity. Interestingly, we also showed that cells seeded on the scaffold can also sense the mechanical strain as demonstrated by a nuclear reorientation perpendicular to strain direction (63.3° compared to 41.2° for nonstretched cells), a nuclear location of YAP and increased expression of YAP target genes, a high cytoplasmic calcium level, and an elevated expression level of collagen genes (COL1A1, COL3A1, COL4A1, and COL6A) with an increased collagen secretion at the protein level. Taken together, these data demonstrated that decellularized plant leaf tissues have an inherent elastic property similar to that found in the mammalian system to which cells can sense and respond.

Spontaneous phase coordination and fluid pumping in model ciliary carpets

Ciliated tissues, such as in the mammalian lungs, brains, and reproductive tracts, are specialized to pump fluid. They generate flows by the collective activity of hundreds of thousands of individual cilia that beat in a striking metachronal wave pattern. Despite progress in analyzing cilia coordination, a general theory that links coordination and fluid pumping in the limit of large arrays of cilia remains lacking. Here, we conduct in silico experiments with thousands of hydrodynamically interacting cilia, and we develop a continuum theory in the limit of infinitely many independently beating cilia by combining tools from active matter and classical Stokes flow. We find, in both simulations and theory, that isotropic and synchronized ciliary states are unstable. Traveling waves emerge regardless of initial conditions, but the characteristics of the wave and net flows depend on cilia and tissue properties. That is, metachronal phase coordination is a stable global attractor in large ciliary carpets, even under finite perturbations to cilia and tissue properties. These results support the notion that functional specificity of ciliated tissues is interlaced with the tissue architecture and cilia beat kinematics and open up the prospect of establishing structure to function maps from cilium-level beat to tissue-level coordination and fluid pumping.

Avian surfactant protein (SP)-A2 first arose in an early tetrapod before the divergence of amphibians and gradually lost the collagen domain

The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of specific proteins and lipids that serve a dual purpose-enabling air-breathing and protection against pathogens. In mammals, surfactant proteins A (SP-A) and D (SP -D) are involved in innate defence of the lung. Birds seem to lack the SP-D gene, but possess SP-A2, an additional SP-A-like gene. Here we investigated the evolution of the SP-A and SP-D genes using computational gene prediction, homology, simulation modelling and phylogeny with published avian and other vertebrate genomes. PCR was used to confirm the identity and expression of SP-A analogues in various tissue homogenates of zebra finch and turkey. In silico analysis confirmed the absence of SP-D-like genes in all 47 published avian genomes. Zebra finch and turkey SP-A1 and SP-A2 sequences, confirmed by PCR of lung homogenates, were compared with sequenced and in silico predicted vertebrate homologs to construct a phylogenetic tree. The collagen domain of avian SP-A1, especially that of zebra finch, was dramatically shorter than that of mammalian SP-A. Amphibian and reptilian genomes also contain avian-like SP-A2 protein sequences with a collagen domain. NCBI Gnomon-predicted avian and alligator SP-A2 proteins all lacked the collagen domain completely. Both avian SP-A1 and SP-A2 sequences form separate clades, which are most closely related to their closest relatives, the alligators. The C-terminal carbohydrate recognition domain (CRD) of zebra finch SP-A1 was structurally almost identical to that of rat SP-A. In fact, the CRD of SP-A is highly conserved among all the vertebrates. Birds retained a truncated version of mammalian type SP-A1 as well as a non-collagenous C-type lectin, designated SP-A2, while losing the large collagenous SP-D lectin, reflecting their evolutionary trajectory towards a unidirectional respiratory system. In the context of zoonotic infections, how these evolutionary changes affect avian pulmonary surface protection is not clear.

Detection of microplastics in domestic and fetal pigs’ lung tissue in natural environment: A preliminary study

Microplastics (MPs) are ubiquitous in the environment. However, it is unclear whether MPs are present in mammalian lungs through inhalation, and if so, could be possibly found in fetal tissues. In this study, we aim to determine the presence and characteristics of particles in domestic and fetal pig lung tissue in the natural environment. Specimens from the lungs of domestic pigs (n = 10) and fetal pigs that already died in matrix during vaginal birth from the non-contaminated area (n = 10) were obtained from farmers’ nearby sludge treatment plant. These specimens were compressed between two glass microscope slides, which were examined under polarized light microscopy. In addition, Agilent 8700 LDIR Chemical imaging system (LDIR) was used to determine the quantitative and qualitative characteristics of MPs. According to the polarized light microscope survey of domestic pig lungs, we observed an average of 12 particles/g, which was more than the 6 particles/g observed in fetal pig lungs, which ranged in size from 115.14 μm to 1370.43 μm. All the observed MP particles were fiber in shape. LDIR indicated an average of 180 particles/g of domestic pig lungs, ranging in size from 20.34 μm to 916.36 μm, which was twice as many MPs observed in fetal pig lungs. Furthermore, the compositions of MPs were different between them. LDIR indicated that polyamide (PA) was the most common polymer identified in domestic pig lungs (46.11%), while polycarbonate (PC) was the most common polymer in fetal pig lungs (32.99%). These findings confirmed the presence of MPs in the lung tissue of both domestic and fetal pigs in the natural environment, but the main characteristics differed. This fact indicated the increasing risk of MPs to human respiratory tract is increasing. Further research should be conducted to entirely estimate the specific exposure level on humans and offspring.

After the Storm: Regeneration, Repair, and Reestablishment of Homeostasis Between the Alveolar Epithelium and Innate Immune System Following Viral Lung Injury.

The mammalian lung has an enormous environmental-epithelial interface that is optimized to accomplish the principal function of the respiratory system, gas exchange. One consequence of evolving such a large surface area is that the lung epithelium is continuously exposed to toxins, irritants, and pathogens. Maintaining homeostasis in this environment requires a delicate balance of cellular signaling between the epithelium and innate immune system. Following injury, the epithelium can be either fully regenerated in form and function or repaired by forming dysplastic scar tissue. In this review, we describe the major mechanisms of damage, regeneration, and repair within the alveolar niche where gas exchange occurs. With a focus on viral infection, we summarize recent work that has established how epithelial proliferation is arrested during infection and how the innate immune system guides its reconstitution during recovery. The consequences of these processes going awry are also considered, with an emphasis on how this will impact postpandemic pulmonary biology and medicine.

Generation of Plvap-CreER and Car4-CreER for genetic targeting of distinct lung capillary populations

It has been reported recently that there are two distinct subpopulations of capillary endothelial cells in the mammalian lungs: gCap (general capillary) and aCap (aerocyte). They are identified by two unique markers, respectively: plasmalemmal vesicle-associated protein (PLVAP) and carbonic anhydrase IV (CAR4). Here, we report two novel knock-in mouse lines Plvap-CreER and Car4-CreER, which genetically target gCap and aCap, respectively. Induced by tamoxifen, the Plvap-CreER and Car4-CreER alleles mediate specific and efficient Cre-loxP recombinations in PLVAP+ gCap and CAR4+ aCap, respectively, in the lungs. These two mouse lines are useful genetic tools to investigate cell fates and functions of PLVAP+ and CAR4+ cells in lung homeostasis, injury and repair.

Predicting Species Boundaries and Assessing Undescribed Diversity in Pneumocystis, an Obligate Lung Symbiont.

Far more biodiversity exists in Fungi than has been described, or could be described in several lifetimes, given current rates of species discovery. Although this problem is widespread taxonomically, our knowledge of animal-associated fungi is especially lacking. Fungi in the genus Pneumocystis are obligate inhabitants of mammal lungs, and they have been detected in a phylogenetically diverse array of species representing many major mammal lineages. The hypothesis that Pneumocystis cospeciate with their mammalian hosts suggests that thousands of Pneumocystis species may exist, potentially equal to the number of mammal species. However, only six species have been described, and the true correspondence of Pneumocystis diversity to host species boundaries is unclear. Here, we use molecular species delimitation to estimate the boundaries of Pneumocystis species sampled from 55 mammal species representing eight orders. Our results suggest that Pneumocystis species often colonize several closely related mammals, especially those in the same genus. Using the newly estimated ratio of fungal to host diversity, we estimate ≈4600 to 6250 Pneumocystis species inhabit the 6495 currently recognized extant mammal species. Additionally, we review the literature and find that only 240 (~3.7%) mammal species have been screened for Pneumocystis, and many detected Pneumocystis lineages are not represented by any genetic data. Although crude, our findings challenge the dominant perspective of strict specificity of Pneumocystis to their mammal hosts and highlight an abundance of undescribed diversity.

Programmable Ligation-Transcription Circuit-Driven Cascade Amplification Machinery for Multiple Long Noncoding RNAs Detection in Lung Tissues

The measurement of long noncoding RNAs (lncRNAs) is essential to diagnosis and treatment of various diseases such as cancers. Herein, we develop a simple method to simultaneously detect multiple lncRNAs using programmable ligation-transcription circuit-driven cascade amplification and single-molecule counting. The presence of targets lncRNA HOTAIR and lncRNA MALAT1 activates the ligation-transcription circuits to produce two corresponding functional RNAs. The functional RNAs then cyclically initiate the digestion of signal probes by duplex-specific nuclease to liberate Cy5 and Cy3 molecules. After magnetic separation, the liberated Cy5 and Cy3 molecules are measured by single-molecule counting. In this assay, a single lncRNA can activate ligation-transcription circuit to generate abundant functional RNAs, endowing this assay with high sensitivity. Integration of single-molecule counting ensures the high sensitivity. This method shows extremely high sensitivity with a limit of detection (LOD) of 0.043 aM for HOX gene antisense intergenic RNA (lncRNA HOTAIR) and 0.126 aM for mammalian metastasis-related lung adenocarcinoma transcript 1 (lncRNA MALAT1). Importantly, this method enables simultaneous measurement of multiple endogenous lncRNAs at the single-cell level, and it may discriminate the expressions of various lncRNA in lung tumor tissues of nonsmall cell lung cancer (NSCLC) patients and their corresponding healthy adjacent tissues, offering a promising platform for clinical diagnosis and biomedical research.

Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans

BackgroundPulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in...