African lungfish genome sheds light on the vertebrate water-to-land transition.

Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.

Acto3D: an open-source user-friendly volume rendering software for high-resolution 3D fluorescence imaging in biology.

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research.

Gα13 controls pharyngeal endoderm convergence by regulating E-cadherin expression and RhoA activation.

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.

A pharynx-to-brain axis controls pharyngeal inflammation-induced anxiety.

Anxiety is a remarkably common condition among patients with pharyngitis, but the relationship between these disorders has received little research attention, and the underlying neural mechanisms remain unknown. Here, we show that the densely innervated pharynx transmits signals induced by pharyngeal inflammation to glossopharyngeal and vagal sensory neurons of the nodose/jugular/petrosal (NJP) superganglia in mice. Specifically, the NJP superganglia project to norepinephrinergic neurons in the nucleus of the solitary tract (NTSNE). These NTSNE neurons project to the ventral bed nucleus of the stria terminalis (vBNST) that induces anxiety-like behaviors in a murine model of pharyngeal inflammation. Inhibiting this pharynx→NJP→NTSNE→vBNST circuit can alleviate anxiety-like behaviors associated with pharyngeal inflammation. This study thus defines a pharynx-to-brain axis that mechanistically links pharyngeal inflammation and emotional response.

Fgf signalling is required for gill slit formation in the skate, Leucoraja erinacea.

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm-ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm-ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal-ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.

The pseudobranch of jawed vertebrates is a mandibular arch-derived gill.

The pseudobranch is a gill-like epithelial elaboration that sits behind the jaw of most fishes. This structure was classically regarded as a vestige of the ancestral gill arch-like condition of the gnathostome jaw. However, more recently, hypotheses of jaw evolution by transformation of a gill arch have been challenged, and the pseudobranch has alternatively been considered a specialised derivative of the second (hyoid) pharyngeal arch. Here, we demonstrate in the skate (Leucoraja erinacea) that the pseudobranch does, in fact, derive from the mandibular arch, and that it shares gene expression features and cell types with gills. We also show that the skate mandibular arch pseudobranch is supported by a spiracular cartilage that is patterned by a shh-expressing epithelial signalling centre. This closely parallels the condition seen in the gill arches, where cartilaginous appendages called branchial rays, which support the respiratory lamellae of the gills, are patterned by a shh-expressing gill arch epithelial ridge. Together with similar discoveries in zebrafish, our findings support serial homology of the pseudobranch and gills, and an ancestral origin of gill arch-like anatomical features from the gnathostome mandibular arch.

Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis.

Developmentally, the great vessels of the heart originate from the pharyngeal arch arteries (PAAs). During PAA vasculogenesis, PAA precursors undergo sequential cell fate decisions that are accompanied by proliferative expansion. However, how these two processes are synchronized remains poorly understood. Here, we find that the zebrafish chemokine receptor Cxcr4a is expressed in PAA precursors, and genetic ablation of either cxcr4a or the ligand gene cxcl12b causes PAA stenosis. Cxcr4a is required for the activation of the downstream PI3K/AKT cascade, which promotes not only PAA angioblast proliferation, but also differentiation. AKT has a well-known role in accelerating cell-cycle progression through the activation of cyclin-dependent kinases. Despite this, we demonstrate that AKT phosphorylates Etv2 and Scl, the key regulators of angioblast commitment, on conserved serine residues, thereby protecting them from ubiquitin-mediated proteasomal degradation. Altogether, our study reveals a central role for chemokine signaling in PAA vasculogenesis through orchestrating angioblast proliferation and differentiation.

Differential retinoic acid sensitivity of oral and pharyngeal teeth in medaka (Oryzias latipes) supports the importance of pouch-cleft contacts in pharyngeal tooth initiation.

BACKGROUND: Previous studies have claimed that pharyngeal teeth in medaka (Oryzias latipes) are induced independent of retinoic acid (RA) signaling, unlike in zebrafish (Danio rerio). In zebrafish, pharyngeal tooth formation depends on a proper physical contact between the embryonic endodermal pouch anterior to the site of tooth formation, and the adjacent ectodermal cleft, an RA-dependent process. Here, we test the hypothesis that a proper pouch-cleft contact is required for pharyngeal tooth formation in embryonic medaka, as it is in zebrafish. We used 4-[diethylamino]benzaldehyde (DEAB) to pharmacologically inhibit RA production, and thus pouch-cleft contacts, in experiments strictly controlled in time, and analyzed these using high-resolution imaging. RESULTS: Pharyngeal teeth in medaka were present only when the corresponding anterior pouch had reached the ectoderm (i.e., a physical pouch-cleft contact established), similar to the situation in zebrafish. Oral teeth were present even when the treatment started approximately 4 days before normal oral tooth appearance. CONCLUSIONS: RA dependency for pharyngeal tooth formation is not different between zebrafish and medaka. We propose that the differential response to DEAB of oral versus pharyngeal teeth in medaka could be ascribed to the distinct germ layer origin of the epithelia involved in tooth formation in these two regions.

SMAD4: A critical regulator of cardiac neural crest cell fate and vascular smooth muscle development.

BACKGROUND: During embryogenesis, cardiac neural crest-derived cells (NCs) migrate into the pharyngeal arches and give rise to the vascular smooth muscle cells (vSMCs) of the pharyngeal arch arteries (PAAs). vSMCs are critical for the remodeling of the PAAs into their final adult configuration, giving rise to the aortic arch and its arteries (AAAs). RESULTS: We investigated the role of SMAD4 in NC-to-vSMC differentiation using lineage-specific inducible mouse strains. We found that the expression of SMAD4 in the NC is indelible for regulating the survival of cardiac NCs. Although the ablation of SMAD4 at E9.5 in the NC lineage led to a near-complete absence of NCs in the pharyngeal arches, PAAs became invested with vSMCs derived from a compensatory source. Analysis of AAA development at E16.5 showed that the alternative vSMC source compensated for the lack of NC-derived vSMCs and rescued AAA morphogenesis. CONCLUSIONS: Our studies uncovered the requisite role of SMAD4 in the contribution of the NC to the pharyngeal arch mesenchyme. We found that in the absence of SMAD4+ NCs, vSMCs around the PAAs arose from a different progenitor source, rescuing AAA morphogenesis. These findings shed light on the remarkable plasticity of developmental mechanisms governing AAA development.

Genetic deletion of hspa8 leads to selective tissue malformations in zebrafish embryonic development.

The heat shock cognate 71 kDa protein HSPA8 (also known as HSC70), a constitutively expressed cognate member of the heat shock protein 70 family, plays an essential role in protein quality control and cell homeostasis maintenance. HSPA8 has been implicated in many diseases, including cancers and neurodegenerative diseases. Owing to massive cell death after knockdown of HSPA8 and nonviable Hspa8 knockout mice, the physiological role of HSPA8 in vertebrates and its underlying mechanisms of action have not yet been elucidated. To address this issue, we used CRISPR/Cas9 technology and genetically deleted hspa8 in zebrafish embryos. Genetic deletion of hspa8 resulted in malformations of the pharyngeal arches, pectoral fins, head and eyes at the later stages. We next focused on pharyngeal arch deficiency and found that pharyngeal arches in hspa8 mutant embryos exhibited induction of endoplasmic reticulum stress and activation of the unfolded protein response via the Perk/p-eIF2α/Atf4 signaling cascade. Inhibition of Perk/p-eIF2α/Atf4 signaling rescued the developmental deficiency of pharyngeal arches caused by depletion of Hspa8. Taken together, our results provide novel insights into the tissue-specific roles of Hspa8 in the regulation of vertebrate embryonic development.

Pharyngeal pouches provide a niche microenvironment for arch artery progenitor specification.

The paired pharyngeal arch arteries (PAAs) are transient blood vessels connecting the heart with the dorsal aorta during embryogenesis. Although PAA malformations often occur along with pharyngeal pouch defects, the functional interaction between these adjacent tissues remains largely unclear. Here, we report that pharyngeal pouches are essential for PAA progenitor specification in zebrafish embryos. We reveal that the segmentation of pharyngeal pouches coincides spatiotemporally with the emergence of PAA progenitor clusters. These pouches physically associate with pharyngeal mesoderm in discrete regions and provide a niche microenvironment for PAA progenitor commitment by expressing BMP proteins. Specifically, pouch-derived BMP2a and BMP5 are the primary niche cues responsible for activating the BMP/Smad pathway in pharyngeal mesoderm, thereby promoting progenitor specification. In addition, BMP2a and BMP5 play an inductive function in the expression of the cloche gene npas4l in PAA progenitors. cloche mutants exhibit a striking failure to specify PAA progenitors and display ectopic expression of head muscle markers in the pharyngeal mesoderm. Therefore, our results support a crucial role for pharyngeal pouches in establishing a progenitor niche for PAA morphogenesis via BMP2a/5 expression.

Identifying COVID-19 subtypes by single-sample gene set enrichemnt analysis and providing guidance for sensitive drug selection.

This study aimed at using single-sample gene set enrichment analysis scores to cluster naso/pharyngeal swab specimen samples from coronavirus disease 2019 (COVID-19) patients into two clusters. One cluster with higher fractions of immune cells and more active inflammatory-related pathways was called the Immunity-High (Immunity-H) group, and the other one was called the Immunity-Low group. We explored impacts of the method on COVID-19 treatment. First, given that the Immunity-H group was mainly enriched in inflammatory-related pathways and had higher fractions of inflammatory cells, the Immunity-H group may obtain more curative effects from anti-inflammatory treatment. Second, we searched some hot genes from the PubMed platform that had been studied by researchers and found these genes upregulated in the Immunity-H group, so we speculated the Immunity-H group and Immunity-Low group may have different curative effects from drugs targeting these genes. Finally, we screened out hub genes for the Immunity-H group and predicted potential drugs for these hub genes by a public data set (http://dgidb.genome.wustl.edu). These hub genes are significantly upregulated in the Immunity-H group and neutrophils so that the Immunity-H group may obtain different treatment results from potential drugs compared with the Immunity-Low group. Therefore, the cluster method may provide help in drug development and administration for COVID-19 patients.

Signatures of convergence in Neotropical cichlid fish.

Convergent evolution of similar phenotypes suggests some predictability in the evolutionary trajectories of organisms, due to strong and repeated selective pressures, and/or developmental constraints. In adaptive radiations, particularly in cichlid fish radiations, convergent phenotypes are commonly found within and across geographical settings. Cichlids show major repeated axes of morphological diversification. Recurrent changes in body patterns reveal adaption to alternative habitats, and modifications of the trophic apparatus respond to the exploitation of different food resources. Here we compare morphologically and genetically two Neotropical cichlid assemblages, the Mexican desert cichlid and the Nicaraguan Midas cichlid, with similar polymorphic body and trophic adaptations despite their independent evolution. We found a common morphological axis of differentiation in trophic structures in both cichlid radiations, but two different axes of differentiation in body shape, defining two alternative limnetic body patterns. Adaptation to limnetic habitats implied regulation of immune functions in the Midas cichlid, while morphogenesis and metabolic functions in the desert cichlid. Convergent phenotypic adaptions could be associated to divergent gene regulation.

Abnormalities in pharyngeal arch-derived structures in SATB2-associated syndrome.

SATB2-associated syndrome (SAS, glass syndrome, OMIM#612313) is a neurodevelopmental autosomal dominant disorder with frequent craniofacial abnormalities including palatal and dental anomalies. To assess the role of Satb2 in craniofacial development, we analyzed mutant mice at different stages of development. Here, we show that Satb2 is broadly expressed in early embryonic mouse development including the mesenchyme of the second and third arches. Satb2-/- mutant mice exhibit microglossia, a shortened lower jaw, smaller trigeminal ganglia, and larger thyroids. We correlate these findings with the detailed clinical phenotype of four individuals with SAS and remarkable craniofacial phenotypes with one requiring mandibular distraction in childhood. We conclude that the mouse and patient data presented support less well-described phenotypic aspects of SAS including mandibular morphology and thyroid anatomical/functional issues.

Case-control study of heart rate variability and sleep apnea in childhood sickle cell disease.

Obstructive sleep apnea (OSA) is common in sickle cell disease (SCD) despite the absence of overweight, suggesting a specific pathophysiology. We previously showed that otherwise healthy children with increased pharyngeal compliance, a main endotype of OSA, exhibited decreased sympathetic modulation. Our objective was to assess whether modifications of heart rate variability (HRV) and compliance are associated in SCD. Cases (children with SCD, African or Caribbean ethnicity) and controls (otherwise healthy children, same ethnicity), aged 4-18 years, were selected from our database of children referred for OSA and matched for sex, age, and obstructive apnea-hypopnoea index (OAHI) score. The children underwent polysomnography and acoustic pharyngometry (to compute compliance). HRV analyses were performed from 5 min ECG recordings in wakeful, NREM, and REM sleep states and from the whole night. Twenty-one pairs were analysed (median age 10.5 years, 24 girls). Children with SCD had lower BMI z-scores and more tonsil hypertrophy than control children. Children with SCD and OSA (OAHI ≥2/hour) were characterised by lower compliance than children with SCD without OSA. An inverse relationship between compliance and SD2 (HRV from whole night, inversely related to sympathetic modulation) was evidenced (negative relationship in SCD: R = -0.63, p = 0.002 vs. positive relationship in controls R = 0.59, p = 0.006). In conclusion, while the decrease in sympathetic modulation in control children may contribute to increasing pharyngeal compliance, its decrease seems protective in children with sickle cell disease, which underlines the specificity of OSAS pathophysiology in SCD that could be due to sickle cell disease related smooth muscle dystonia.

Topical pharyngeal anesthesia with articaine for gastroscopy: a double-blinded, randomized cross-over study in healthy volunteers.

OBJECTIVES: The benefits of topical pharyngeal anesthesia for gastroscopy remain under debate. Articaine, a local anesthetic with fast onset and offset of action as well as low systemic toxicity, could be a promising choice for topical anesthesia. The objective of this study was to assess whether topical pharyngeal anesthesia with articaine is beneficial in sedated gastroscopy. MATERIALS AND METHODS: This randomized double-blinded cross-over study included nine volunteers who underwent two gastroscopies under conscious sedation. One was performed with topical pharyngeal anesthesia with articaine and the other with placebo. Hemodynamic parameters including autonomic nervous system state were recorded prior to and during the endoscopic procedure. The endoscopist and the volunteer assessed the endoscopy after the examination. RESULTS: Topical pharyngeal anesthesia with articaine resulted in less discomfort during esophageal intubation and higher patient satisfaction with the procedure. Topical pharyngeal anesthesia with articaine did not increase satisfaction or facilitate the procedure as rated by the endoscopist. There were no clinically relevant differences in hemodynamic parameters. CONCLUSION: The use of articaine for topical pharyngeal anesthesia results in less intubation-related discomfort and better satisfaction.

Characterization of pharyngeal contractile integral using pharyngeal manometry in children.

OBJECTIVES: Pharyngeal contractile integral (PhCI) is the product of mean pharyngeal contractile amplitude, length, and duration, and provides a single metric for the vigor of entire pharyngeal contraction. A major limitation in children is lack of characterization of PhCI on high-resolution pharyngeal manometry. We aimed to determine and compare the values of PhCI in children with the abnormal and normal videofluoroscopic study of swallow (VFSS). METHODS: Children who underwent high-resolution pharyngeal and esophageal manometry (HRPM/HREM), as well as VFSS, were divided into two groups; "normal VFSS" and "abnormal VFSS" groups. PhCI was calculated from the pharyngo-esophageal manometry analysis software (MMS, v9.5, Laborie Medical Technologies), and compared in these two groups. RESULTS: Of 67 children, 9 had abnormal VFSS (mean age 64 ± 50 months; 66.7% males), while 58 had normal VFSS (mean age 123 ± 55 months; 47% males). The mean PhCI in abnormal and normal VFSS groups was 82.00 ± 51.90 and 147.28 ± 53.89 mmHg.s.cm, respectively (p = 0.001). Subjects with abnormal VFSS were significantly younger than those with normal VFSS (p = 0.003). However, after adjusting for the VFSS result, age was no longer related to PhCI (p = 0.364). In subgroup analysis of children presenting with dysphagia, the mean PhCI in abnormal (9 subjects) and normal (36 subjects) VFSS groups was 82.00 ± 51.90 and 141.86 ± 50.39 mmHg.s.cm, respectively (p = 0.003). CONCLUSIONS: PhCI was significantly lower in children with abnormal VFSS than in those with normal VFSS. We did not find a significant impact of age on PhCI in our pediatric populations.

Measuring immediate surgical success in children undergoing adenotonsillectomy.

Adenotonsillectomy is the most common indication for sleep-disordered breathing in children. Measuring pharyngeal closing pressures in anaesthetised children allows identification of severe obstructive sleep apnoea. This technique could help quantify immediate surgical impact and risk stratify postoperative treatment in these patients.

Discovery of novel 20S proteasome subunit ß5 PROTAC degraders as potential therapeutics for pharyngeal carcinoma and Bortezomib-resistant multiple myeloma.

Resistance to proteasome inhibitors like Bortezomib is a major challenge in the treatment of multiple myeloma (MM). Proteolysis targeting chimeras (PROTACs), an emerging therapeutic approach that induces selective degradation of target proteins, offer a promising solution to overcome drug resistance. In this study, we designed and synthesized novel small-molecule PROTACs that induce 20S proteasome subunit ß5 degradation as a strategy to overcome Bortezomib resistance. These 20S proteasome subunit ß5 PROTACs demonstrated considerable binding affinity to 20S proteasome subunit ß5 and cereblon (CRBN), effectively induced 20S proteasome subunit ß5 degradation, and exhibited potent antiproliferative activity against a panel of cancer cell lines. Notably, PROTACs 12f and 14 displayed robust antitumor effects against both the pharyngeal carcinoma cell line FaDu and the Bortezomib-resistant MM cell line KM3/BTZ in vitro and in vivo with excellent safety profiles. Taken together, our findings highlight the potential of PROTACs 12f and 14 as novel 20S proteasome subunit ß5-degrading agents for the treatment of pharyngeal carcinoma and overcoming Bortezomib resistance in MM.

Efficacy of endoscopic surveillance for pharyngeal mucosa during endoscopic resection for pharyngeal carcinoma: a multicenter prospective study.

INTRODUCTION: Since patients with pharyngeal squamous cell carcinoma (SCC) often have multiple pharyngeal lesions, evaluation of pharyngeal lesions before endoscopic resection (ER) is important. However, detailed endoscopic observation of the entire pharyngeal mucosa under conscious sedation is difficult. We examined the usefulness of endoscopic surveillance with narrow band imaging (NBI) and lugol staining for detection of pharyngeal sublesions during ER for pharyngeal SCC under general anesthesia (endoscopic surveillance during treatment; ESDT). METHODS: From January 2021 through June 2022, we examined 78 patients who were diagnosed with superficial pharyngeal SCC and underwent ER. They underwent the ESDT and for patients who were diagnosed with new lesions of pharyngeal SCC or high-grade dysplasia (HGD) that were not detected in the endoscopic examination before treatment, ER were performed simultaneously for new lesions and the main lesions. The primary endpoint of this study was the detection rate of new lesions of pharyngeal SCC or HGD in the ESDT. RESULTS: Fifteen of the 78 patients were diagnosed as having undetected new pharyngeal lesions in the ESDT and 10 (12.8%) (95% CI 6.9-22.2%) were histopathologically confirmed to have new lesions of pharyngeal SCC or HGD. Among the 13 lesions of SCC or HGD, 8 were found by NBI observation; however, 5 were undetectable using NBI but detectable by lugol staining. All of the 13 lesions had endoscopic findings of pink color sign on lugol staining. CONCLUSIONS: Endoscopic surveillance for pharyngeal sublesions during ER for pharyngeal SCC is feasible and useful.