Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

Maintenance of appropriate size scaling of the C. elegans pharynx by YAP-1.

Even slight imbalance between the growth rate of different organs can accumulate to a large deviation from their appropriate size during development. Here, we use live imaging of the pharynx of C. elegans to ask if and how organ size scaling nevertheless remains uniform among individuals. Growth trajectories of hundreds of individuals reveal that pharynxes grow by a near constant volume per larval stage that is independent of their initial size, such that undersized pharynxes catch-up in size during development. Tissue-specific depletion of RAGA-1, an activator of mTOR and growth, shows that maintaining correct pharynx-to-body size proportions involves a bi-directional coupling between pharynx size and body growth. In simulations, this coupling cannot be explained by limitation of food uptake alone, and genetic experiments reveal an involvement of the mechanotransducing transcriptional co-regulator yap-1. Our data suggests that mechanotransduction coordinates pharynx growth with other tissues, ensuring body plan uniformity among individuals.

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRT-PCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis.

Integration of single-cell transcriptomes and chromatin landscapes reveals regulatory programs driving pharyngeal organ development.

Maldevelopment of the pharyngeal endoderm, an embryonic tissue critical for patterning of the pharyngeal region and ensuing organogenesis, ultimately contributes to several classes of human developmental syndromes and disorders. Such syndromes are characterized by a spectrum of phenotypes that currently cannot be fully explained by known mutations or genetic variants due to gaps in characterization of critical drivers of normal and dysfunctional development. Despite the disease-relevance of pharyngeal endoderm, we still lack a comprehensive and integrative view of the molecular basis and gene regulatory networks driving pharyngeal endoderm development. To close this gap, we apply transcriptomic and chromatin accessibility single-cell sequencing technologies to generate a multi-omic developmental resource spanning pharyngeal endoderm patterning to the emergence of organ-specific epithelia in the developing mouse embryo. We identify cell-type specific gene regulation, distill GRN models that define developing organ domains, and characterize the role of an immunodeficiency-associated forkhead box transcription factor.

Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta.

Using Filters to Estimate Regional Lung Deposition with Dry Powder Inhalers.

PURPOSE: To develop an in vitro method to rapidly evaluate regional lung doses delivered by pharmaceutical inhalers. Currently, cascade impactor measurements are used, but these are resource intensive and require significant post processing of in vitro data to arrive at regional deposition estimates. METHODS: We present a specialized filter apparatus that mimics tracheobronchial (TB) deposition of pharmaceutical aerosols emitted by commercially available dry powder inhalers (DPIs). The filter housing includes an electrostatic neutralizer to eliminate artificial electrostatic filtration effects. Regional deposition (tracheobronchial and alveolar) for four DPIs (Onbrez Breezhaler, Flovent Diskus, Pulmicort Turbuhaler, and Asmanex Twisthaler) was estimated using cascade impactor measurements and an in silico regional deposition model. These estimates were compared to direct measurements of regional deposition as provided by the TB filter mimic and an absolute filter placed downstream of the TB filter housing, representing the alveolar dose. RESULTS: The two methods were shown to provide similar estimates of extrathoracic, tracheobronchial, and alveolar deposition, as well as total recovery of active pharmaceutical ingredients. CONCLUSIONS: Because of its design, the TB filter apparatus makes it possible to estimate regional deposition with inhalers directly using variable inhalation profiles without any additional equipment or changes to the experimental configuration. This method may be useful to expedite development of both innovative and generic drug products as it provides regional respiratory tract deposition estimates using fewer resources than exisiting methods.

ceRNA Network Regulation of TGF-ß, WNT, FOXO, Hedgehog Pathways in the Pharynx of Ciona robusta.

The transforming growth factor-ß (TGF-ß) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-ß signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-ß, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3'UTR elements analyses of Tgf-ß, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3'UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta.

Morphology of GNAT3-immunoreactive chemosensory cells in the nasal cavity and pharynx of the rat.

Solitary chemosensory cells and chemosensory cell clusters are distributed in the pharynx and larynx. In the present study, the morphology and reflexogenic function of solitary chemosensory cells and chemosensory cell clusters in the nasal cavity and pharynx were examined using immunofluorescence for GNAT3 and electrophysiology. In the nasal cavity, GNAT3-immunoreactive solitary chemosensory cells were widely distributed in the nasal mucosa, particularly in the cranial region near the nostrils. Solitary chemosensory cells were also observed in the nasopharynx. Solitary chemosensory cells in the nasopharyngeal cavity were barrel like or slender in shape with long lateral processes within the epithelial layer to attach surrounding ciliated epithelial cells. Chemosensory cell clusters containing GNAT3-immunoreactive cells were also detected in the pharynx. GNAT3-immunoreactive cells gathered with SNAP25-immunoreactive cells in chemosensory clusters. GNAT3-immunoreactive chemosensory cells were in close contact with a few SP- or CGRP-immunoreactive nerve endings. In the pharynx, GNAT3-immunoreactive chemosensory cells were also attached to P2X3-immunoreactive nerve endings. Physiologically, the perfusion of 10 mM quinine hydrochloride (QHCl) solution induced ventilatory depression. The QHCl-induced reflex was diminished by bilateral section of the glossopharyngeal nerve, suggesting autonomic reflex were evoked by chemosensory cells in pharynx but not in nasal mucosa. The present results indicate that complex shape of nasopharyngeal solitary chemosensory cells may contribute to intercellular communication, and pharyngeal chemosensory cells may play a role in respiratory depression.

NLR-1/CASPR Anchors F-Actin to Promote Gap Junction Formation.

Gap junctions are present in most tissues and play essential roles in various biological processes. However, we know surprisingly little about the molecular mechanisms underlying gap junction formation. Here, we uncover the essential role of a conserved EGF- and laminin-G-domain-containing protein nlr-1/CASPR in the regulation of gap junction formation in multiple tissues across different developmental stages in C. elegans. NLR-1 is located in the gap junction perinexus, a region adjacent to but not overlapping with gap junctions, and forms puncta before the clusters of gap junction channels appear on the membrane. We show that NLR-1 can directly bind to actin to recruit F-actin networks at the gap junction formation plaque, and the formation of F-actin patches plays a critical role in the assembly of gap junction channels. Our findings demonstrate that nlr-1/CASPR acts as an early stage signal for gap junction formation through anchoring of F-actin networks.

Association of mRNA Expression Levels of LRP1 and Actin-Binding Proteins with the Development of Laryngeal and Laryngopharyngeal Squamous Cell Carcinoma.

We analyzed the association of the level of mRNA expression of the main endocytosis receptor LRP1 and actin-binding proteins (ezrin, profilin-1, cofilin-1, and adenylyl cyclase-associated protein 1) with the development and metastasis of laryngeal and laryngopharyngeal squamous cell carcinoma. The mRNA expression was evaluated in paired tissue samples using quantitative reverse transcription real-time PCR (RT-qPCR) and SYBR Green reagents. The study included 38 patients with stage T1-4N0-1M0 laryngeal and laryngopharyngeal squamous cell carcinoma and 10 patients with chronic hyperplastic laryngitis or grade II-III epithelial dysplasia. The expression of LRP1 in patients with laryngeal and laryngopharyngeal squamous cell carcinoma depended on the stage of the tumor process. Against the background of low expression of LRP1 mRNA, the relationship between cofilin 1 and profilin 1 expression became stronger (r=0.08; p=0.05) and a correlation between cofilin 1 and esrin expression (r=0.7; p=0.05) appeared. Studies on a larger patient cohort are required to make a definite conclusion on the role of LRP1 in the development of laryngeal and laryngopharyngeal squamous cell carcinoma.

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-ß and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 µm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

Age-Associated Role of Lingual and Pharyngeal Glands in Local Immunity in Oral Cavity.

The macroscopic and histological methods were employed to examine the autopsy specimens of salivary lingual glands obtained from 299 patients of both sexes and various age ranging from newborn to longevity. The age-associated alterations of minor lingual and pharyngeal glands were revealed, and the topographical relations between the glands and lymphoid cells were described. The characteristic sparsity of the glands in infancy is caused by nutritional uniformity at this period, when diminished production of secretory IgA results in frequent inflammatory processes in oral and pharyngeal cavities. With age, the glandular orifices widen, and their number increases thereby augmenting local immunity in the oral cavity and in oral aspect of the pharynx. Starting from elderly and senile age, the involutive alterations were observed, which were accompanied by diminished production of secretory immunoglobulin A and related degradation of local and humoral immunity.

Reliable Postmortem Molecular Diagnosis of Anaphylaxis: Co-localization of Mast Cell Degranulation and Immunoglobulin E in Allergic Throat Tissues.

Anaphylaxis is a serious reaction that may cause death in half an hour without diagnostic characteristic in autopsies. Mast cell (MC) degranulation combined with immunoglobulin E (IgE) plays the key roles in anaphylaxis. Unavailability of serum and instability of measured serum in postmortem diagnoses sometimes limit the opinion of medical experts. Allergic tissues are more accessible than serum, and there is a little research on degranulated mast cells and IgE in different human tissues, whereas we hardly know whether the expression will keep stable over the increasing postmortem interval (PMI). In this research, we examined the mast cell counts and degranulation rates and gE contents in human throat, lung, and intestine tissues and preliminarily investigated the correlation of these markers with PMI in anaphylaxis-associated death. Allergic samples showed a significant increase in mast cell degranulation accompanied by an increase in IgE levels than the control group, but the expression was not significantly correlated with increasing PMI only in throat tissues. Elevated mast cell degranulation combined with increased IgE levels may be a reliable biomarker for forensic diagnosis of human tissues due to IgE-mediated allergic sudden death.

Histochemical detection of free thiols in glandular cells and tissues of different marine Polychaeta.

Either through differentiated glands or specialised individual cells, the coating epithelia of soft-bodied marine invertebrates are responsible for the secretion of a broad span of peptidic substances, from protective mucins to biocides. These secretions are characterised by the presence of cysteine-rich proteins and peptides, rendering a distinct histochemical signature of secretory epithelia. Through a histochemical procedure for fluorescence microscopy in paraffin sections, we performed a comparative assessment of the distribution of thiol-rich compounds in multiple epithelia of different species of intertidal Polychaeta, which revealed distinctive patterns of distribution that closely relate to ecology, morphoanatomy and physiology. The presence of free thiols was notorious in mucocytes and enzyme-plus toxin-secreting cells. Consequently, strong signals were recorded in the mucocytes of the parapodia of Nereis splendida, the epidermis and pharynx epithelium of Mysta picta and the venom glands of Glycera alba. The findings show an investment in mucus secretion in foragers such as Nereis and Mysta, especially the latter, which is not a native burrower, as a protective response and as lubricant for locomotion. Additionally, nereidids are believed to secret integumentary toxins for defence. On the other hand, Glycera is an ambush predatorial burrower whose behaviour entirely revolves around the delivery of venom making use of its four jaws. The results showed that the detection of thiol-rich compounds in histological sections can be a tool to identify potential toxin secretion and delivery structures, with important consequences for the bioprospecting of novel bioreactives from marine invertebrates for the purpose of drug discovery.

Transcriptome Comparison Reveals Key Components of Nuptial Plumage Coloration in Crested Ibis.

Nuptial plumage coloration is critical in the mating choice of the crested ibis. This species has a characteristic nuptial plumage that develops from the application of a black sticky substance, secreted by a patch of skin in the throat and neck region. We aimed to identify the genes regulating its coloring, by comparing skin transcriptomes between ibises during the breeding and nonbreeding seasons. In breeding season skins, key eumelanin synthesis genes, TYR, DCT, and TYRP1 were upregulated. Tyrosine metabolism, which is closely related to melanin synthesis, was also upregulated, as were transporter proteins belonging to multiple SLC families, which might act during melanosome transportation to keratinocytes. These results indicate that eumelanin is likely an important component of the black substance. In addition, we observed upregulation in lipid metabolism in breeding season skins. We suggest that the lipids contribute to an oil base, which imbues the black substance with water insolubility and enhances its adhesion to feather surfaces. In nonbreeding season skins, we observed upregulation in cell adhesion molecules, which play critical roles in cell interactions. A number of molecules involved in innervation and angiogenesis were upregulated, indicating an ongoing expansion of nerves and blood vessels in sampled skins. Feather ß keratin, a basic component of avian feather filament, was also upregulated. These results are consistent with feather regeneration in the black skin of nonbreeding season ibises. Our results provide the first molecular evidence indicating that eumelanin is the key component of ibis coloration.

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor.

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

Comparative Transcriptomics Reveals Clues for Differences in Pathogenicity between Hysterothylacium aduncum, Anisakissimplex sensu stricto and Anisakis pegreffii.

Ascaridoid nematodes are widespread in marine fishes. Despite their major socioeconomic importance, mechanisms associated to the fish-borne zoonotic disease anisakiasis are still obscure. RNA-Seq and de-novo assembly were herein applied to RNA extracted from larvae and dissected pharynx of Hysterothylacium aduncum (HA), a non-pathogenic nematode. Assembled transcripts in HA were annotated and compared to the transcriptomes of the zoonotic species Anisakis simplex sensu stricto (AS) and Anisakis pegreffii (AP). Approximately 60,000,000 single-end reads were generated for HA, AS and AP. Transcripts in HA encoded for 30,254 putative peptides while AS and AP encoded for 20,574 and 20,840 putative peptides, respectively. Differential gene expression analyses yielded 471, 612 and 526 transcripts up regulated in the pharynx of HA, AS and AP. The transcriptomes of larvae and pharynx of HA were enriched in transcripts encoding collagen, peptidases, ribosomal proteins and in heat-shock motifs. Transcripts encoding proteolytic enzymes, anesthetics, inhibitors of primary hemostasis and virulence factors, anticoagulants and immunomodulatory peptides were up-regulated in AS and AP pharynx. This study represents the first transcriptomic characterization of a marine parasitic nematode commonly recovered in fish and probably of negligible concern for public health.

The ventral peptidergic system of the adult ascidian Ciona robusta (Ciona intestinalis Type A) insights from a transgenic animal model.

Ascidians are the sister group of vertebrates and occupy a critical position in explorations of the evolution of the endocrine and nervous systems of chordates. Here, we describe the complete ventral peptidergic system in adult transgenic Ciona robusta (Ciona intestinalis Type A) which expresses the Kaede reporter gene driven by the prohormone convertase 2 (PC2) gene promoter. Numerous PC2 promoter-driven fluorescent (Kaede-positive) non-neural cells were distributed in the blood sinus located at the anterior end of the pharynx, suggesting the acquisition of a peptidergic circulatory system in Ciona. Kaede-positive ciliated columnar cells, rounded cells, and tall ciliated cells were observed in the alimentary organs, including the endostyle, pharynx, esophagus, stomach, and intestine, suggesting that digestive functions are regulated by multiple peptidergic systems. In the heart, Kaede-positive neurons were located in the ring-shaped plexus at both ends of the myocardium. Nerve fiber-like tracts ran along the raphe and appeared to be connected with the plexuses. Such unique structures suggest a role for the peptidergic system in cardiac function. Collectively, the present anatomic analysis revealed the major framework of the ventral peptidergic system of adult Ciona, which could facilitate investigations of peptidergic regulation of the pharynx, endostyle, alimentary tissues, and heart.

Percutaneous Endoscopic Gastrostomy versus Nasogastric Tube Feeding: Oropharyngeal Dysphagia Increases Risk for Pneumonia Requiring Hospital Admission.

BACKGROUND: Aspiration pneumonia is the most common cause of death in patients with percutaneous endoscopic gastrostomy (PEG) and nasogastric tube (NGT) feeding. This study aimed to compare PEG versus NGT feeding regarding the risk of pneumonia, according to the severity of pooling secretions in the pharyngolaryngeal region. METHODS: Patients were stratified by endoscopic observation of the pooling secretions in the pharyngolaryngeal region: control group (<25% pooling secretions filling the pyriform sinus), pharyngeal group (25-100% pooling secretions filling the pyriform sinus), and laryngeal group (pooling secretions entering the laryngeal vestibule). Demographic data, swallowing level scale score, and pneumonia requiring hospital admission were recorded. RESULTS: Patients with NGT (n = 97) had a significantly higher incidence of pneumonia (episodes/person-years) than those patients with PEG (n = 130) in the pharyngeal group (3.6 ± 1.0 vs. 2.3 ± 2.1, P < 0.001) and the laryngeal group (3.8 ± 0.5 vs. 2.3 ± 2.2 vs, P < 0.001). The risk of pneumonia was significantly higher in patients with NGT than in patients with PEG (adjusted hazard ratio = 2.85, 95% CI: 1.46-4.98, P < 0.001). Cumulative proportion of pneumonia was significantly higher in patients with NGT than with PEG for patients when combining the two groups (pharyngeal + laryngeal groups) (P = 0.035). CONCLUSION: PEG is a better choice than NGT feeding due to the decrease in risk of pneumonia requiring hospital admission, particularly in patients with abnormal amounts of pooling secretions accumulation in the pyriform sinus or leak into the laryngeal vestibule.