Nonciliated Cells Research Articles

MICROSCOPIC ARCHITECTURE OF THE RESPIRATORY AND CONDUCTING SYSTEM OF THE LUNG OF NILE MONITOR

Using semi-thin sections, the present investigation examined the microscopic characteristics of the lungs of the Nile monitor (Varanus niloticus). The lungs were composed of Intrapulmonary conducting airways and respiratory faveoli. Intrapulmonary airways originate from the terminal portion of the bronchus, which extends into the lung to create the bronchial tree. The bronchus was lined with pseudostratified ciliated epithelium composed of both ciliated and non-ciliated cells, and it was supported by plates of hyaline cartilage. The central lumen is surrounded by contractile fibers that contain smooth muscle cell bundles and are covered by ciliated and non-ciliated cells. The central lumen communicates with the faveoli. Separating adjacent faveoli are pulmonary trabeculae covered with various cell types, including type I pneumocytes, type II pneumocytes, and pulmonary macrophages. Some substantial pulmonary bronchi were also supported by small cartilage plate granules and lined with ciliated epithelium. Type I pneumocytes were flat cells, whereas type II pneumocytes had cuboidal cells with vacuolated cytoplasm. Surface irregularity and vacuolated cytoplasm were features of pulmonary macrophages. In addition, the connective tissue of the pulmonary septa contained immune cells, such as Mast and Eosinophils. In conclusion, the microstructure of the lung of the Nile monitor closely resembles that of other reptile species. However, the distinction between intrapulmonary cartilage palates and pulmonary septa raises the concept of species differentiation. In addition, the discovery of various types of pulmonary immune cells enhances the Nile monitor's ability to persist in a variety of environments by enhancing its pulmonary immunity.

Histological Study of Development Structure of Testis and Epididymis in Gazelle Gazelle Subgutturosa

Objective: The aim of this study for focused the light on the development structure of the testis of gazelle. Materials and Method: 10 healthy male gazelle were used to study the histology structure of testis (5 pre- Puberty and 5 post- Puberty). Results: The testis is surrounded by a thick, irregular connective tissue capsule. Each lobule of the testis is composed of seminiferous tubules and interstitial tissue, and trabecular and interlobular septa divide the lobules apart. Neighboring the seminiferous tubules are Sertoli cells and spermatogenic cells. From spermatogenic cells, spermatozoa are produced. The first spermatogonia are small, spherical cells with black, spherical nuclei that reside on the basement membrane. Spermatogonia were earliest stages of the spermatogenic cells. Larger cells with frequently distinct chromatin are called primary spermatocytes. Secondary spermatocytes are rare because of their quick second meiotic division and the haploid spermatids that follow. Spermatids are round, pale-nucleated cells that are grouped close to the lumen of the seminiferous tubule. Prostatogenic cells outnumber Sertoli cells in the body. Oval or triangular in shape, the nucleus has a light coloration. In the connective tissue separating adjacent tubules, Leydig cells are present. Lines the epididymis are pseudostratified epithelium. Extensions of the cytoplasm into the lumen are called stereocilia. In different proportions, there is circular smooth muscle, and a connective tissue lamina propria supports the epithelium. The rete testis is connected to the ductus epididymidis by the efferent ductules, which are convolutions of tubules. Numerous valved veins were visible in the tunica albuginea, which covered the initial ascending portion of the epididymal head. Cuboidal epithelium was lining extratesticular rete testis. Three different cell types; migrating, basal, and columnar had epithelial membranes lining the efferent ductules. Many columnar, non-ciliated cells with many cytoplasmic vacuoles and tiny granules lined the epithelium of the first segment of the efferent ductules. Conclusion: In post-puberty age, the structure of the gazelle's testis exhibited a pattern resembling that of other ruminants; the seminiferous tubules contain adult sperm and a broad lumen.

Human efferent ductules and epididymis display unique cell lineages with motile and primary cilia.

Previous research has illustrated the role of cilia as mechanical and sensory antennae in various organs within the mammalian male reproductive system across different developmental stages. Despite their significance in both organ development and homeostasis, primary cilia in the human male reproductive excurrent duct have been overlooked due to limited access to human specimens. This study aimed to characterize the unique cellular composition of human efferent and epididymal ducts, with a focus on their association with primary cilia. Human efferent ductules/epididymides from five donors aged 32-47 years, were obtained through our local organ transplant program. Cell lineage specificity and primary cilia features were examined by immunofluorescent staining and confocal microscopy in the efferent ductules and the distinct segments of the epididymis. The epithelium of the human efferent duct exhibited estrogen receptor-positive cells with primary cilia, FoxJ1-positive multiciliated cells with numerous motile cilia, and non-ciliated intraepithelial immune cells. Notably, intraluminal macrophages, identified by CD163/CD68 positivity, were observed to engage in sperm phagocytosis. In all three segments of the human epididymis, primary cilia were found on the surface of principal and basal cells. Our research indicates that the human efferent ductules create a distinct environment, characterized by the presence of two types of ciliated cells that are in contact with immune cells. The discovery of sensory primary cilia exposed on the surface of reabsorptive cells in the efferent ductules, as well as on basal and principal cells in the epididymis, lays the foundation for complementary functional studies. This research uncovers novel characteristics exclusive to human efferent ductules and epididymides, providing a basis for exploring innovative approaches to male contraception and infertility treatment.

Rhinovirus infection of airway epithelial cells uncovers the non-ciliated subset as a likely driver of genetic risk to childhood-onset asthma

Asthma is a complex disease caused by genetic and environmental factors. Studies show that wheezing during rhinovirus infection correlates with childhood asthma development. Over 150 non-coding risk variants for asthma have been identified, many affecting gene regulation in Tcells, but the effects of most risk variants remain unknown. We hypothesized that airway epithelial cells could also mediate genetic susceptibility to asthma given they are the first line of defense against respiratory viruses and allergens. We integrated genetic data with transcriptomics of airway epithelial cells subject to different stimuli. We demonstrate that rhinovirus infection significantly upregulates childhood-onset asthma-associated genes, particularly in non-ciliated cells. This enrichment is also observed with influenza infection but not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or cytokine activation. Overall, our results suggest that rhinovirus infection is an environmental factor that interacts with genetic risk factors through non-ciliated airway epithelial cells to drive childhood-onset asthma.

Morphology, Glycan Pattern, Heat Shock Proteins, and Sex Steroid Receptors Expression in the Tubal Fimbria Epithelium of the Baboon Papio hamadryas during the Menstrual Cycle.

The oviductal fimbria is the first extraovarian anatomical structure that the cumulus-oocyte complex (COC) encounters, and is sensitive to sex hormone changes. The morphology, glycan pattern, expression of heat shock proteins (HSPs), estradiol receptor (ER), and progesterone receptor (PR) were investigated in the oviductal fimbria epithelium of the baboon (Papio hamadryas) during the menstrual cycle. The morphology was investigated by light and scanning electron microscopy; the glycopattern was characterized using conventional and lectin histochemistry; HSPs (60, -70, -90), ER, and PR were localized immunohistochemically. Well-differentiated ciliated and nonciliated cells were present only during the preovulatory phase. The nonciliated cells contained small apical protrusions and thin microvilli. During the preovulatory phase (1) the luminal surface of the fimbria displayed acidic glycans, complex N-glycans containing fucose, and oligolactosamine residues; (2) nonciliated cells expressed HSP60 and HSP90 in the apical blebs, HSP70 in the nucleus and cytoplasm, as well as nuclear ERα and PR; (3) ciliated cells showed HSP70 in the nucleus, cytoplasm, and cilia that also expressed HSP90 and PR. These results are related to the function of the fimbria where the early COC-oviduct crosstalk occurs and may represent a benchmark for translational studies of other primates.

The multiple facets of the club cell in the pulmonary epithelium.

The non-ciliated bronchiolar cell, also referred to as "club cell", serves as a significant multifunctional component of the airway epithelium. While the club cell is a prominent epithelial type found in rodents, it is restricted to the bronchioles in humans. Despite these differences, the club cell's importance remains undisputed in both species due to its multifunctionality as a regulatory cell in lung inflammation and a stem cell in lung epithelial regeneration. The objective of this review is to examine different aspects of club cell morphology and physiology in the lung epithelium, under both normal and pathological conditions, to provide a comprehensive understanding of its importance in the respiratory system.

Uterotubal junction of the bovine (Bos taurus) versus the dromedary camel (Camelus dromedarius): Histology and histomorphometry.

This study aims to conduct a comparative analysis of the uterotubal junction in two distinct mammalian species, the bovine (Bos taurus) and the dromedary camel (Camelus dromedarius), focusing on histological and histomorphometric parameters. Uterotubal junction (UTJ) was dissected from 8 cows and 12 camels with dominant follicles, and processed for H&E staining for histology, and histomorphometry examination. The results showed that the camel uterotubal junction papillae (UTJP) existed only in camels and was completely absent in cattle. Histologically, the cow UTJ appears with a star-shaped lumen, and the mucosa is lined by a simple columnar epithelium with ciliated and non-ciliated cells. Superficial (SG) and deep glands (DG) were abundant in the submucosa. Camel UTJP is a conical structure, it has a pale yellowish colour, 0.5 ± 0.2 cm height, and 0.3 ± 0.1 cm width, and it has a distinct sphincter at the tip towards the uterine lumen. In the camel UTJP, the lumen is wider, and the mucosa showed large multiple folds lined with ciliated and non-ciliated cells in a pseudostratified columnar epithelium. The submucosa showed no mucosal glands. Lumen area, lumen epithelial height, luminal epithelial density, the thickness of the muscular layer, number of folds, fold height, epithelial height, fold area and epithelial perimeter were higher in camel UTJP (p < .001) than cow UTJ, while the total endosalpinx area and glandular epithelial density were lower in camel UTJP (p < .001) than in cow UTJ. In conclusion, this study elucidates potential variations in the uterotubal junction between bovines and dromedary camels, providing valuable insights into their reproductive adaptations. The epithelial lining, absence of glands and the thick layer of tunica muscularis might indicate that camel UTJP could play a mechanical role in selecting spermatozoa and assisting the hatching of blastocysts during their passage into the uterus.

Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

The abundance and localization of heat shock proteins (HSP)-60, -70, and -90 in the oviductal ampulla of hamadryas baboon (Papio hamadryas) during the menstrual cycle.

The presence of HSPs in female reproductive and their relationship with the steroid hormone fluctuation have been reported in several mammals but not in non-human primates. The present research dealt with the oviductal expression and localization of the more studied HSPs (60, 70, and 90) as well as the morphological changes in the Hamadryas baboon (Papio hamadryas) during the follicular, preovulatory, and luteal phases of the menstrual cycle. Therefore, western blots, histomorphological, and immunohistochemical analyses were carried out. The results of western blot analysis displayed the lowest HSP expression in the luteal phase. The histomorphology showed that the mucosal epithelium consisted of undifferentiated cuboidal cells in follicular and luteal phases and well-distinguishable columnar ciliated and non-ciliated cells during the preovulatory phase. Immunohistochemistry evidenced that the mucosal epithelium contained cytoplasmic and nuclear HSP60, 70, and 90 immunostaining in the follicular and luteal phases. During the preovulatory phase, the non-ciliated cells showed: (i) cytoplasmic HSP60; (ii) nuclear and cytoplasmic HSP90. Ciliated cells showed cytoplasmic and ciliary HSP70 and ciliary HSP90. The stromal cells and myocytes of muscular layer displayed a decreased cytoplasmic HSP60 in the preovulatory phase and nuclear and low cytoplasmic HSP70 throughout the menstrual cycle. Nuclear HSP90 decreased in ampulla stromal cells and the follicular phase myocytes. These findings indicate that the expression pattern of HSP60,70, and 90 is related to the morphofunctional features of the baboon oviductal ampulla during the menstrual cycle and could represent a referent point for further studies in the oviduct of Primates.

CC16 drives VLA-2-dependent SPLUNC1 expression.

CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. We identified 8 antimicrobial proteins significantly decreased by CC16-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.

The ultrastructural features of the infundibulum of the green iguana, Iguana iguana.

The purpose of this study is to describe, in detail, the ultrastructure of the infundibulum of the sexually mature and active female green iguana, Iguana iguana. The infundibulum of five iguanas was remarkably distinct from the uterus, and was also clearly demarcated into cranial (expanded v-shaped) and caudal (tubular) divisions. Tissue samples obtained from five portions (three from the cranial division and two from the caudal division) of the infundibulum were processed conventionally for light and electron microscopy. The epithelial lining of the most anterior, middle, and posterior, parts of the cranial division displayed nonciliated cells predominantly, and occasionally ciliated cells. The numerous secretory granules in nonciliated type 1 cell found in the fimbrial aspect of the infundibulum were homogenous and deeply electron-dense, but those in the other two regions were variants of this cell type because they contained variably electron-dense secretory granules. Two main types of nonciliated cells (type 2 and its variant, type 3, as well as type 4) occurred in the epithelial lining of the caudal division of the infundibulum, but they, clearly, showed no dense secretory granules. Whereas the nonciliated type 2 cell and its variant (type 3 cell) contained large glycogen deposits, the type 4 cell lacked these deposits but its apical part contained large lipid-like droplets and, remarkably, blebbed into the duct lumen. The nonciliated cells lining the mucosal tubular glands contained highly electron-dense secretory granules, which were similar to those found in the nonciliated type 1 cell in the epithelial lining of the fimbrial part of the cranial division of the infundibulum.

Transcriptional regulation by the NSL complex enables diversification of IFT functions in ciliated versus nonciliated cells.

Members of the NSL histone acetyltransferase complex are involved in multiorgan developmental syndromes. While the NSL complex is known for its importance in early development, its role in fully differentiated cells remains enigmatic. Using a kidney-specific model, we discovered that deletion of NSL complex members KANSL2 or KANSL3 in postmitotic podocytes led to catastrophic kidney dysfunction. Systematic comparison of two primary differentiated cell types reveals the NSL complex as a master regulator of intraciliary transport genes in both dividing and nondividing cells. NSL complex ablation led to loss of cilia and impaired sonic hedgehog pathway in ciliated fibroblasts. By contrast, nonciliated podocytes responded with altered microtubule dynamics and obliterated podocyte functions. Finally, overexpression of wild-type but not a double zinc finger (ZF-ZF) domain mutant of KANSL2 rescued the transcriptional defects, revealing a critical function of this domain in NSL complex assembly and function. Thus, the NSL complex exhibits bifurcation of functions to enable diversity of specialized outcomes in differentiated cells.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

An RFX transcription factor regulates ciliogenesis in the closest living relatives of animals

Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey.1,2,3 Regulated ciliogenesis was likely critical for early animal evolution,2,4,5,6 and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals7,8,9 but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.10,11 By comparing the genomes of animals with those of their closest living relatives, the choanoflagellates, we found that the genome of their last common ancestor encoded at least three RFX paralogs and a FoxJ1 homolog. Disruption of the RFX homolog cRFXa in the model choanoflagellate Salpingoeca rosetta resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. In cRFXa mutants, ciliogenesis genes and foxJ1 were significantly downregulated. Moreover, the promoters of S.rosetta ciliary genes are enriched for DNA motifs matching those bound by the cRFXa protein invitro. These findings suggest that an ancestral cRFXa homolog coordinated ciliogenesis in the progenitors of animals and choanoflagellates and that the selective deployment of the RFX regulatory module may have been necessary to differentiate ciliated from non-ciliated cell types during early animal evolution.

SUFU promotes GLI activity in a Hedgehog-independent manner in pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signaling pathway, through which the GLI family of transcription factors (TF) is stimulated, is commonly observed in cancer cells. One well-established mechanism of this increased activity is through the inactivation of Suppressor of Fused (SUFU), a negative regulator of the Hh pathway. Relief from negative regulation by SUFU facilitates GLI activity and induction of target gene expression. Here, we demonstrate a novel role for SUFU as a promoter of GLI activity in pancreatic ductal adenocarcinoma (PDAC). In non-ciliated PDAC cells unresponsive to Smoothened agonism, SUFU overexpression increases GLI transcriptional activity. Conversely, knockdown (KD) of SUFU reduces the activity of GLI in PDAC cells. Through array PCR analysis of GLI target genes, we identified BCL2 among the top candidates downregulated by SUFU KD. We demonstrate that SUFU KD results in reduced PDAC cell viability, and overexpression of BCL2 partially rescues the effect of reduced cell viability by SUFU KD. Further analysis using as a model GLI1, a major TF activator of the GLI family in PDAC cells, shows the interaction of SUFU and GLI1 in the nucleus through previously characterized domains. ChIP assay shows binding of both SUFU and GLI1 at the promoter of BCL2 in PDAC cells. Finally, we demonstrate that SUFU promotes GLI1 activity without affecting its protein stability. Through our findings, we propose a novel role of SUFU as a positive regulator of GLI1 in PDAC, adding a new mechanism of Hh/GLI signaling pathway regulation in cancer cells.

Genome Editing in Ferret Airway Epithelia Mediated by CRISPR/Nucleases Delivered with Amphiphilic Shuttle Peptides.

Gene editing strategies are attractive for treating genetic pulmonary diseases such as cystic fibrosis (CF). However, challenges have included the development of safe and effective vector systems for gene editing of airway epithelia and model systems to report their efficiency and durability. The domestic ferret (Mustela putorius furo) has a high degree of conservation in lung cellular anatomy with humans, and has served as an excellent model for many types of lung diseases, including CF. In this study, we evaluated the efficiency of amphiphilic shuttle peptide S10 for protein delivery and gene editing using SpCas9, and AsCas12a (Cpf1) ribonucleoproteins (RNPs). These approaches were evaluated in proliferating ferret airway basal cells, polarized airway epithelia in vitro, and lungs in vivo, by accessing the editing efficiency using reporter ferrets and measuring indels at the ferret CFTR locus. Our results demonstrate that shuttle peptides efficiently enable delivery of reporter proteins/peptides and gene editing SpCas9 or Cpf1 RNP complexes to ferret airway epithelial cells in vitro and in vivo. We measured S10 delivery efficiency of green fluorescent protein (GFP)-nuclear localization signal (NLS) protein or SpCas9 RNP into ferret airway basal cells and fully differentiated ciliated and nonciliated epithelial cells in vitro. In vitro and in vivo gene editing efficiencies were determined by Cas/LoxP-gRNA RNP-mediated conversion of a ROSA-TG Cre recombinase reporter using transgenic primary cells and ferrets. S10/Cas9 RNP was more effective, relative to S10/Cpf1 RNP at gene editing of the ROSA-TG locus. Intratracheal lung delivery of the S10 shuttle combined with GFP-NLS protein or D-Retro-Inverso (DRI)-NLS peptide demonstrated efficiencies of protein delivery that were ∼3-fold or 14-fold greater, respectively, than the efficiency of gene editing at the ROSA-TG locus using S10/Cas9/LoxP-gRNA. Cpf1 RNPs was less effective than SpCas9 at gene editing of LoxP locus. These data demonstrate the feasibility of shuttle peptide delivery of Cas RNPs to the ferret airways and the potential utility for developing ex vivo stem cell-based and in vivo gene editing therapies for genetic pulmonary diseases such as CF.

Sex differences in vanadium inhalation effects in non-ciliated bronchiolar cells.

The non-ciliated bronchiolar cell (NCBC) is responsible for the defense of the lung and responds to negative stimuli such as exposure to toxic pro-oxidant substances, which triggers the hyperproduction and hypersecretion of mucins and CC16 protein. The literature demonstrates that physiological and pathological responses in the lung can be influenced by the organism's sex. The objective of this report was to evaluate response differences to vanadium (V) inhalation in male and female CD-1 mice. Mice were exposed to V for four weeks. Hyperplasia of bronchiolar epithelium, small inflammatory foci and sloughing of the NCBC were observed, without changes between sexes and throughout the exposure time. Mucosecretory metaplasia was found in both males and females, however it was more drastic in males. The expression of CC16 increased in both sexes. This study demonstrated a different susceptibility between male and female mice exposed to V inhalation regarding mucosecretory metaplasia.

Epididymis of the Lizard &lt;i&gt;Eutropis carinata&lt;/i&gt;: A Light Microscopic and Ultrastructural Seasonal Study

The epididymis of the lizard Eutropis carinata can be divided into four regions viz., an initial segment (extra testicular) Efferent ductules (Eds) and three regions, the anterior region, a broad middle, and a narrow posterior, comparable to the caput, corpus, and cauda epididymidis of mammals. The anterior region of the epididymis is closely associated with a whitish initial segment (extra testicular), the Efferent ductules (Eds). The epididymal wall consists of an epithelial layer lining the lumen resting on a basement membrane surrounded by four to five layers of smooth circular muscles, a layer of connective tissue, and a layer of serosa. The ultrastructure study of the initial segment of epididymis the Eds during breeding season discloses four cell types in its epithelium viz., ciliated, non-ciliated, Apical-Mitochondria Rich Cells (AMRC), and basal cells. The anterior, middle, and posterior regions of the epididymis show five different cell types namely principal, basal, AMRC, narrow, and clear cells. Ciliated and non-ciliated cells are limited to the initial segment of the epididymis while, principal, narrow, and clear cells are found in the rest of the three regions of the epididymis. Basal and AMRC are found in all four regions. AMRCs are the most abundant cell type in the initial segment while principal cells are the major components of the epithelial lining of the remaining regions of the epididymis. During the non-breeding season, all the cell types are present but with regression and altered cytology of the cells without any sign of cellular activity in the different regions of the epididymis. During the breeding season, even the circulating testosterone levels are significantly higher compared to the non-breeding season. This is the first report describing different cell types in the initial segment and three different regions of the epididymis with ultrastructural seasonal variations in the Keeled Indian Mabuya, Eutropis carinata (Scincidae).

The Human Early Maternal–Embryonic Interactome

Background: Single cell transcriptomics offers an avenue for predicting, with improved accuracy, the gene networks that are involved in the establishment of the first direct cell–cell interactions between the blastocyst and the maternal luminal epithelium. We hypothesised that in silico modelling of the maternal–embryonic interface may provide a causal model of these interactions, leading to the identification of genes associated with a successful initiation of implantation. Methods: Bulk and single cell RNA-sequencing of endometrial epithelium and scRNAseq of day 6 and 7 trophectoderm (TE) were used to model the initial encounter between the blastocyst and the maternal uterine lining epithelium in silico. In silico modelling of the maternal–embryonic interface was performed using hypernetwork (HN) analysis of genes mediating endometrial–TE interactions and the wider endometrial epithelial transcriptome. A hypernetwork analysis identifies genes that co-ordinate the expression of many other genes to derive a higher order interaction likely to be causally linked to the function. Potential interactions of TE with non-ciliated luminal cells, ciliated cells, and glandular cells were examined. Results: Prominent epithelial activities include secretion, endocytosis, ion transport, adhesion, and immune modulation. Three highly correlated clusters of 25, 22 and 26 TE-interacting epithelial surface genes were identified, each with distinct properties. Genes in both ciliated and non-ciliated luminal epithelial cells and glandular cells exhibit significant functional associations. Ciliated cells are predicted to bind to TE via galectin–glycan interaction. Day 6 and day 7 embryonic–epithelial interactomes are largely similar. The removal of aneuploid TE-derived mRNA invoked only subtle differences. No direct interaction with the maternal gland epithelial cell surface is predicted. These functional differences validate the in silico segregation of phenotypes. Single cell analysis of the epithelium revealed significant change with the cycle phase, but differences in the cell phenotype between individual donors were also present. Conclusions: A hypernetwork analysis can identify epithelial gene clusters that show correlated change during the menstrual cycle and can be interfaced with TE genes to predict pathways and processes occurring during the initiation of embryo–epithelial interaction in the mid-secretory phase. The data are on a scale that is realistic for functional dissection using current ex vivo human implantation models. A focus on luminal epithelial cells may allow a resolution to the current bottleneck of endometrial receptivity testing based on tissue lysates, which is confounded by noise from multiple diverse cell populations.