Pathogenesis of Reinke's Edema of the Vocal Fold.

OBJECTIVES: The most frequent etiologic factor of Reinke's edema (RE) is considered to be smoking. However, the mechanism for the onset and development of the disease remains unclear. Hypoxia-inducible factor-1α (HIF-1α) is an oxygen-dependent transcriptional activator which plays crucial roles in angiogenesis in hypoxic microenvironments. HIF-1α induces the expression of vascular endothelial growth factor (VEGF) which involves angiogenesis and enhances vascular permeability. This study investigated the roles of HIF-1α in the pathogenesis of RE. METHODS: Surgical specimens of RE from patients who underwent endolaryngeal microsurgery were used. Normal vocal folds were used as a control group. Expression of HIF-1α and VEGF was analyzed by immunohistochemistry. Three-dimensional fine structures of the vessels in RE were investigated using correlative light and electron microscopy (CLEM) technique. RESULTS: HIF-1α and VEGF were broadly expressed in the stromal, inflammatory, and endothelial cells in the lamina propria of the vocal fold of RE. The expression of HIF-1α and VEGF of RE were significantly higher than in the lamina propria of the normal vocal fold mucosa. CLEM showed vascularization and telangiectasia and there were many dilated capillaries with thin endothelium not covered with pericytes indicating the vessels were fragile. CONCLUSION: Transcription factor HIF-1α and induced VEGF likely play roles in the pathogenesis of RE. And increased vascular permeability with fragile vessels in angiogenesis is likely to be an etiology of RE. Transcription factor HIF-1α and induced VEGF are potential therapeutic targets for RE. LEVEL OF EVIDENCE: NA Laryngoscope, 134:1785-1791, 2024.

Glycolytic Metabolism of the Tissue Stem Cells in the Maculae Flavae of the Human Vocal Fold.

OBJECTIVES: Metabolic programs in the stem cells are essential for maintaining homeostasis and protecting against stem cell aging. There is growing evidence that the tissue stem cells reside in the anterior and posterior maculae flavae of the human vocal fold mucosa. Our previous studies observed that the glycolysis of the cell in the human maculae flavae seems to rely more on anaerobic glycolysis for energy supply in comparison with oxidative phosphorylation. However, previous studies showed only the metabolic enzymes of glycolysis and functional morphology of the mitochondria, therefore, it has not yet been determined whether anaerobic glycolysis actually took place. The purpose of this study is to investigate the glycolytic metabolites of the cells in the maculae flavae of the human vocal fold in vitro. METHODS: Four normal human vocal folds were used. After extraction of the anterior maculae flavae, cells in the maculae flavae were cultured and proliferated. Glucose transporter-1 was assessed using immunocytochemistry and metabolites of glycolysis (lactate and NADPH) were measured. RESULTS: The cells in the maculae flavae expressed glucose transporter-1 in the cytoplasm and the cell membranes. In addition, the cultured cells produced lactate (metabolites of anaerobic glycolysis) and NADPH (metabolites of the pentose phosphate pathway). CONCLUSIONS: The cells in the maculae flavae of the human vocal folds were found to undergo anaerobic glycolysis via the pentose phosphate pathway. This suggests that the cells in the maculae flavae of the human vocal fold have a metabolism that favors the maintenance of stemness and undifferentiated states.

Enhanced Co-culture System Using Escherichia coli and Pichia pastoris (Komagataella phaffii) for Improved Microbial Production of Valuable Plant Alkaloids.

Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.

Comparative Histoanatomy of the Epiglottis and Pre-epiglottic Space of the Chimpanzee Larynx.

OBJECTIVES: Understanding of the evolution of the larynx clarifies the physiology and functional histoanatomy of the present-day human organ. Comparative histoanatomy of the epiglottis and pre-epiglottic space of the chimpanzee larynx (a hominid, phyletic closest relative of humans) was investigated. METHODS: Two normal adult chimpanzee larynges obtained from natural deaths were investigated. The whole organ serial section technique was employed. RESULTS: The histoanatomical structures of the chimpanzees' epiglottis and pre-epiglottic space were considerably similar to those of human adults. The chimpanzees' epiglottic cartilage was relatively thin and composed of elastic cartilage. These histologic findings of epiglottis indicate that the chimpanzee's epiglottis is flexible and plays the role of retroflection. The chimpanzees' larynges had a pre-epiglottic space composed of adipose tissue and loose connective tissue. Epiglottic cartilage was connected to the thyroid cartilage anteroinferiorly with an intervening thyroepiglottic ligament and to the hyoid bone anteriorly with an intervening hyoepiglottic ligament. These histoanatomical structures of the epiglottis and pre-epiglottic space reflect the fact that chimpanzees have a descended larynx and acquire the pharyngeal space of the vocal tract. CONCLUSION: The results of this study are consistent with the hypothesis that, in the process of evolution, the histoanatomical structures of the epiglottis and pre-epiglottic space change and allow the larynx to descend and lengthen the pharyngeal space of the vocal tract which facilitates speech production in humans. Moreover, the distribution of the human pre-epiglottic space likely allows the epiglottis to more effectively play the role of retroflection during swallowing in order to prevent aspiration.

Cerebellar Nuclei Receiving Orofacial Proprioceptive Signals through the Mossy Fiber Pathway from the Supratrigeminal Nucleus in Rats.

Proprioception from muscle spindles is necessary for motor function executed by the cerebellum. In particular, cerebellar nuclear neurons that receive proprioceptive signals and send projections to the lower brainstem or spinal cord play key roles in motor control. However, little is known about which cerebellar nuclear regions receive orofacial proprioception. Here, we investigated projections to the cerebellar nuclei from the supratrigeminal nucleus (Su5), which conveys the orofacial proprioception arising from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 resulted in a large number of labeled axon terminals bilaterally in the dorsolateral hump (IntDL) of the cerebellar interposed nucleus (Int) and the dorsolateral protuberance (MedDL) of the cerebellar medial nucleus. In addition, a moderate number of axon terminals were ipsilaterally labeled in the vestibular group Y nucleus (group Y). We electrophysiologically detected JCMS proprioceptive signals in the IntDL and MedDL. Retrograde tracing analysis confirmed bilateral projections from the Su5 to the IntDL and MedDL. Furthermore, anterograde tracer injections into the external cuneate nucleus (ECu), which receives other proprioceptive input from forelimb/neck muscles, resulted in only a limited number of ipsilaterally labeled terminals, mainly in the dorsomedial crest of the Int and the group Y. Taken together, the Su5 and ECu axons almost separately terminated in the cerebellar nuclei (except for partial overlap in the group Y). These data suggest that orofacial proprioception is differently processed in the cerebellar circuits in comparison to other body-part proprioception, thus contributing to the executive function of orofacial motor control.

Prognostic values of systemic inflammation and nutrition-based prognostic indices in oropharyngeal carcinoma.

Objective: Pretreatment systemic inflammation and nutrition-based prognostic indices (SINBPI) have demonstrated significance. This study investigated the prognostic value of pretreatment SINBPI for patients with oropharyngeal cancer and identified unfavorable prognostic markers. Methods: We retrospectively reviewed the data of 124 patients with oropharyngeal squamous cell carcinoma (OPSCC) who received definitive treatment between January 2010 and December 2018. The prognostic utility of the neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), prognostic nutritional index, and high-sensitivity modified Glasgow prognostic score (HS-mGPS) was assessed for disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) using univariate and multivariate analyses. Results: Multivariate analyses revealed that human papillomavirus (HPV) status and HS-mGPS were significantly associated with DFS, DSS, and OS. Patients with a HS-mGPS of 2 had a significantly higher rate of treatment-related deaths than those with a HS-mGPS of 0 or 1. The combination of the HS-mGPS and PLR had more accurate predictive ability in DFS and OS compared with the HS-mGPS alone, and the combination of the HS-mGPS and LMR had more accurate predictive ability in DSS and OS. Conclusion: Our results indicated that the HS-mGPS was a useful prognostic marker for patients with OPSCC, and combined markers consisting of the HS-mGPS and PLR or LMR may provide more accurate prognostic predictions.Level of Evidence: 3.

California poppy (Eschscholzia californica), the Papaveraceae golden girl model organism for evodevo and specialized metabolism.

California poppy or golden poppy (Eschscholzia californica) is the iconic state flower of California, with native ranges from Northern California to Southwestern Mexico. It grows well as an ornamental plant in Mediterranean climates, but it might be invasive in many parts of the world. California poppy was also highly prized by Native Americans for its medicinal value, mainly due to its various specialized metabolites, especially benzylisoquinoline alkaloids (BIAs). As a member of the Ranunculales, the sister lineage of core eudicots it occupies an interesting phylogenetic position. California poppy has a short-lived life cycle but can be maintained as a perennial. It has a comparatively simple floral and vegetative morphology. Several genetic resources, including options for genetic manipulation and a draft genome sequence have been established already with many more to come. Efficient cell and tissue culture protocols are established to study secondary metabolite biosynthesis and its regulation. Here, we review the use of California poppy as a model organism for plant genetics, with particular emphasis on the evolution of development and BIA biosynthesis. In the future, California poppy may serve as a model organism to combine two formerly separated lines of research: the regulation of morphogenesis and the regulation of secondary metabolism. This can provide insights into how these two integral aspects of plant biology interact with each other.

Vascularity in the macula flava of human vocal fold as a stem cell niche.

OBJECTIVES: There is growing evidence that the maculae flavae of the human vocal fold are a stem cell niche, which is a microenvironment nurturing tissue stem cells. This study investigated the microenvironment, especially vascularity, in the maculae flavae of the human vocal fold. METHODS: Three normal human adult, three normal newborn vocal folds obtained from autopsy cases and three surgical specimens of glottic carcinoma were investigated using light and electron microscopy. For scanning electron microscopy, a chemical digestion method (modified sodium hydroxide maceration method) was used to observe the inner 3-dimensional structure of the macula flava. RESULTS: Capillaries ran around the anterior and posterior maculae flavae in adults and newborns. However, there was no vascularity in the maculae flavae of the vocal fold. The inner 3-dimensional electron microscopic structure of the macula flava showed there were no blood vessels in the maculae flavae of the vocal fold. Glottic carcinoma (squamous cell carcinoma) surrounded and was in contact with the macula flava, however, the carcinoma did not invade the macula flava indicating there was no vascular supply into the macula flava from the surrounding tissue. CONCLUSIONS: There was no vascularity in the anterior and posterior maculae flavae in the human adult and newborn vocal folds. The present study is consistent with the hypothesis that the hypoxic microenvironment in the maculae flavae of the adult and newborn vocal fold as a stem cell niche is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.

The Cerebellar Cortex Receives Orofacial Proprioceptive Signals from the Supratrigeminal Nucleus via the Mossy Fiber Pathway in Rats.

Proprioceptive sensory information from muscle spindles is essential for the regulation of motor functions. However, little is known about the motor control regions in the cerebellar cortex that receive proprioceptive signals from muscle spindles distributed throughout the body, including the orofacial muscles. Therefore, in this study, we investigated the pattern of projections in the rat cerebellar cortex derived from the supratrigeminal nucleus (Su5), which conveys orofacial proprioceptive information from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 revealed that many bilateral axon terminals (rosettes) were distributed in the granular layer of the cerebellar cortex (including the simple lobule B, crus II and flocculus) in a various sized, multiple patchy pattern. We could also detect JCMS proprioceptive signals in these cerebellar cortical regions, revealing for the first time that they receive muscle proprioceptive inputs in rats. Retrograde tracer injections confirmed that the Su5 directly sends outputs to the cerebellar cortical areas. Furthermore, we injected an anterograde tracer into the external cuneate nucleus (ECu), which receives proprioceptive signals from the forelimb and neck muscle spindles, to distinguish between the Su5- and ECu-derived projections in the cerebellar cortex. The labeled terminals from the ECu were distributed predominantly in the vermis of the cerebellar cortex. Almost no overlap was seen in the terminal distributions of the Su5 and ECu projections. Our findings demonstrate that the rat cerebellar cortex receives orofacial proprioceptive input that is processed differently from the proprioceptive signals from the other regions of the body.

Distribution of Label-Retaining Cells and their Properties in the Newborn Vocal Fold Mucosa.

OBJECTIVES: There is growing evidence that the cells in the maculae flavae (MFe) are candidates for tissue stem cells of the vocal fold mucosa and the MFe are a stem cell niche. Distribution of label-retaining cells and their properties in the postnatal vocal fold mucosa were investigated. METHODS: Oral administration of bromodeoxyuridine (BrdU) was given to pregnant Sprague-Dawley rats and the label-retaining cells in the postnatal vocal fold mucosa were observed by immunohistochemistry. Immunoreactivity to antibodies directed to Ki-67 was studied to investigate the cell cycle. RESULTS: At day 1 after birth, BrdU positive cells were identified in the MFe (60.1 ± 1.7%), epithelium (58.7 ± 10.6%) and lamina propria (52.4 ± 7.8%) of the vocal fold mucosa. At day 56 after birth, the number of BrdU positive cells in the epithelium (4.8 ± 2.2%) and lamina propria (32.3 ± 16.5%) were significantly lower compared to day 1 after birth (P < 0.05). However, the number of BrdU positive cells remaining in the MFe was still high (56.2 ± 2.5%). The label-retaining cells were distributed throughout the MFe. Few Ki-67 positive cells were identified in the MFe indicating they were resting cells. CONCLUSIONS: The results of this study are consistent with the hypothesis that the cells in the postnatal MFe are candidates for tissue stem cells. At birth, these cells are already present in the MFe of the newborn vocal fold and they are likely ready to start the growth and development of the vocal fold mucosa.

Three-dimensional fine structures of the maculae flavae of the human vocal fold using correlative light and electron microscopy.

Objectives: To analyze various aspects of complex tissue, there is increasing demand to study each sample at different length scales in biology. Correlative light and electron microscopy (CLEM) is the latest technique to correlate two different types of information on the exact same histological area of interest: histology (from light microscopy) and ultrastructure (from electron microscopy). The three-dimensional fine structures of the maculae flavae (MFe) of the human vocal fold were investigated using CLEM. Methods: Five normal human adult vocal folds as specimens embedded in paraffin, sectioned, and mounted on glass slides with/without a chemical digestion method (modified sodium hydroxide maceration method) were investigated. Observations using CLEM were performed. Results: The fine structures of cells and extracellular matrices in the MFe and their peripheral regions were able to be observed on the exact same histological area of interest with the light microscope and field emission-scanning electron microscope. Cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells were intermingled in the MFe of the human vocal fold. The extracellular matrices surrounding each three types of cell in the MFe differed, suggesting the cells were different in functional property. Conclusion: CLEM is a useful technique to observe the three-dimensional fine structures of the human vocal fold mucosa. The results of the present study are consistent with the hypothesis that the cells in the MFe of the human vocal fold have heterogeneity and each three types of cell have different properties.

Comparative Histoanatomy of the Epiglottis and Preepiglottic Space of the Lemur Larynx.

OBJECTIVES: An adequate understanding of the evolution of the larynx presumably clarify the physiology and functional histoanatomy of the present-day human organ. This study investigated the comparative histoanatomy of the epiglottis and preepiglottic space of the lemur larynx (a non-human primate without vocal tract). METHODS: Two normal adult ring-tailed lemur larynges obtained from natural death were investigated. The whole organ serial section technique was employed. RESULTS: The histologic structures of the lemur epiglottis and preepiglottic space differed considerably from that of human adults. The lemur epiglottis was proportionally thick and vertical. It was connected to the thyroid cartilage anteroinferiorly without an intervening thyroepiglottic ligament and to the hyoid bone anteriorly with intervening large bundles of collagen fibers, indicating that the lemur epiglottis does not play the role of retroflection. The lemur larynges did not have a preepiglottic space. These findings also reflect the fact that lemurs do not have a descended larynx and do not acquire the pharyngeal space of the vocal tract. The lemur epiglottis was composed of fibrocartilage, adipose tissue and a small amount of elastic cartilage covered with mucosa (lamina propria and pseudostratified ciliated columnar epithelium). The histologic findings also indicate that the lemur epiglottis is not flexible and does not play the role of retroflection. CONCLUSIONS: The results of this study are consistent with the hypothesis that, in the process of evolution, the histologic structures of epiglottis and preepiglottic space likely change to make the larynx descend and lengthen the pharyngeal space of the vocal tract for speech production in humans. Moreover, the distribution of the human preepiglottic space likely allows the epiglottis to more effectively play the role of retroflection during swallowing in order to prevent aspiration.

Glycolysis of Tissue Stem Cells in the Macula Flava of Newborn Vocal Fold.

OBJECTIVES: There is growing evidence that the cells in the maculae flavae are tissue stem cells and the maculae flavae are a stem cell niche of the human vocal fold mucosa. This study investigated the metabolic activity, especially glycolysis, of the tissue stem cells in the maculae flavae of the human newborn vocal fold. METHODS: Three normal human newborn vocal folds obtained from autopsy cases were investigated using immunohistochemistry. RESULTS: Among the three phenotypes of cells (cobblestone-like polygonal cells, vocal fold stellate cell-like cells and fibroblast-like spindle cells) in the newborn maculae flavae, a small number of cobblestone-like polygonal cells strongly expressed glucose transporter-1. All three phenotypes of cells in the newborn maculae flavae expressed glycolytic enzymes (hexokinase II, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase A). The cells did not express phosphofructokinase-1 (rate-limiting enzyme of regular glucose metabolism pathway) but sparsely express glucose-6-phosphate dehydrogenase (rate-limiting enzyme) indicating the cells relied more on the pentose phosphate pathway. The cells' expression of lactate dehydrogenase A suggests the maculae flavae of the newborn vocal fold is likely to be an anaerobic microenvironment where cells perform anaerobic glycolysis. CONCLUSIONS: The present study is consistent with the hypothesis that the tissue stem cells in the maculae flavae of the newborn vocal fold seem to rely more on anaerobic glycolysis, especially by the pentose phosphate pathway, for energy supply. Already at birth, the metabolism of the tissue stem cells in the maculae flavae of the newborn vocal fold is likely to prevent the formation of toxic reactive oxygen species and is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.

Transport engineering using tobacco transporter NtJAT1 enhances alkaloid production in Escherichia coli.

Transporters have been used in the production of plant metabolites in microorganisms. This study introduced a tobacco multidrug and toxic compound extrusion transporter, NtJAT1, into alkaloid-producing Escherichia coli cells. NtJAT1 expression enhanced alkaloid production secretion into the medium by 14 folds. Our findings further demonstrate the usefulness of the transport-engineering approach.

Dysphagia Precipitated by Cervical Lordosis in the Aged.

A lordotic curve of the cervical spine in the aged is a physiological curvature of the cervical spine. The swallowing disorder precipitated by lordosis of the cervical spine in the aged, the swallowing function and intervention were reported on. An 86-year-old woman complained of swallowing difficulty for a few months. A lordotic curve (physiological curvature) of the cervical spine was observed. In the neutral neck position, the cervical lordotic angle was 29° using the Cobb C2-C7 method. When the lordotic curve of the cervical spine was intentionally straightened, the lordotic angle became 0°, the laryngeal elevation changed from 0.5 times the height of a cervical vertebra to 1.75 times, the laryngeal elevation delay time changed from 0.5 seconds to 0.23, the percent laryngeal elevation changed from 50% to 72% and pharyngeal transit time changed from 0.83 seconds to 0.75. Lordosis of the cervical spine inhibited laryngeal elevation and influenced the swallowing function. In cases in which lordotic curvature of the cervical spine adversely influences the swallowing function in the aged, rehabilitation to manage the lordosis of the cervical spine is useful. For the purpose of effective intervention, not only diagnostic but also therapeutic videofluorographic examination of swallowing is very important.

Metabolic Roles of Colony-Forming Tissue Stem Cells in the Maculae Flavae of Newborn Vocal Fold In Vivo.

OBJECTIVES: Tissue stem cells in the maculae flavae (a stem cell niche) of the human vocal fold form colonies in vivo like stem cells in vitro. However, the roles of colony-forming aggregated cells in the maculae flavae in vivo have not yet been determined. This study investigated the glycolysis, of the colony-forming aggregated cells in the maculae flavae of the human newborn vocal fold in vivo. METHODS: Three normal newborn vocal folds were investigated under light microscopy with immunohistochemistry and transmission electron microscopy. RESULTS: Colony-forming aggregated cells in the newborn maculae flavae strongly expressed glucose transporter-1 and glycolytic enzymes (hexokinase II, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase A). The colony-forming aggregated cells did not express phosphofructokinase-1 (rate-limiting enzyme of regular glucose metabolism pathway) but expressed glucose-6-phosphate dehydrogenase (rate-limiting enzyme) indicating the cells relied more on the pentose phosphate pathway. The colony-forming aggregated cells' strong expression of lactate dehydrogenase A indicated that they rely more on anaerobic glycolysis in an anaerobic microenvironment. Mitochondrial cristae of the colony-forming aggregated cells in the newborn maculae flavae were sparse. Consequently, the microstructural features of the mitochondria suggested that their metabolic activity and oxidative phosphorylation were low. CONCLUSIONS: The colony-forming aggregated cells in the maculae flavae of the newborn vocal fold seemed to rely more on anaerobic glycolysis using the pentose phosphate pathway for energy supply in vivo. Microstructural features of the mitochondria and the glycolytic enzyme expression of the colony-forming aggregated cells suggested that the oxidative phosphorylation activity was low. Already at birth, in the anaerobic microenvironment of the macular flavae in vivo, there is likely a complex cross-talk regarding metabolism between the colony-forming aggregated cells along the adhesion machinery and chemical signaling pathways which reduces toxic oxygen species and is favorable to maintaining the stemness and undifferentiated states of the tissue stem cells.

Efferent and afferent connections of supratrigeminal neurons conveying orofacial muscle proprioception in rats.

The supratrigeminal nucleus (Su5) is a key structure for controlling jaw movements; it receives proprioceptive sensation from jaw-closing muscle spindles (JCMSs) and sends projections to the trigeminal motor nucleus (Mo5). However, the central projections and regulation of JCMS proprioceptive sensation are not yet fully understood. Therefore, we aimed to reveal the efferent and afferent connections of the Su5 using neuronal tract tracings. Anterograde tracer injections into the Su5 revealed that the Su5 sends contralateral projections (or bilateral projections with a contralateral predominance) to the Su5, basilar pontine nuclei, pontine reticular nucleus, deep mesencephalic nucleus, superior colliculus, caudo-ventromedial edge of the ventral posteromedial thalamic nucleus, parafascicular thalamic nucleus, zona incerta, and lateral hypothalamus, and ipsilateral projections (or bilateral projections with an ipsilateral predominance) to the intertrigeminal region, trigeminal oral subnucleus, dorsal medullary reticular formation, and hypoglossal nucleus as well as the Mo5. Retrograde tracer injections into the Su5 demonstrated that the Su5 receives bilateral projections with a contralateral predominance (or contralateral projections) from the primary and secondary somatosensory cortices, granular insular cortex, and Su5, and ipsilateral projections (or bilateral projections with an ipsilateral predominance) from the dorsal peduncular cortex, bed nuclei of stria terminalis, central amygdaloid nucleus, lateral hypothalamus, parasubthalamic nucleus, trigeminal mesencephalic nucleus, parabrachial nucleus, juxtatrigeminal region, trigeminal oral and caudal subnuclei, and dorsal medullary reticular formation. These findings suggest that the Su5, which receives JCMS proprioception, has efferent and afferent connections with multiple brain regions that are involved in emotional and autonomic functions as well as orofacial motor functions.

Laryngeal spaces in the newborn larynx.

OBJECTIVES: Distributions of laryngeal spaces are important in understanding their functional significance. However, laryngeal spaces in the newborn larynx are enigmatic. METHODS: Five normal human newborn larynges were investigated using whole organ serial section technique. RESULTS: The chief newborn laryngeal spaces were the preepiglottic space, the paraglottic space and the cricoid area. They consisted of an areolar tissue area composed of brown adipose tissue (immature adipose tissue) and loose elastic and collagen fibers. The newborn preepiglottic space was immature and occupied a small area anterior to the epiglottis. The epiglottis lied on a somewhat horizontal axis and was partially obscured behind the hyoid bone. The hyoid bone overlapped the thyroid cartilage, partially obscuring the superior thyroid notch. The thyroid cartilage was present close to the hyoid bone and the thyrohyoid membrane ran between the superior surface of the thyroid lamina, and not the anteroinferior, but the posteroinferior surface of the hyoid bone. The preepiglottic space was located anterior, lateral and posterolateral to the thyroepiglottic ligament. However, the preepiglottic space was located anterior to the epiglottic cartilage. As seen in adults, the paraglottic space existed medial to the lamina of each bilateral thyroid cartilage. Each bilateral cricoid area was located along not only the superomedial but also the medial portion of the cricoid arch on both sides. CONCLUSIONS: As the child grows, the preepiglottic space, occupying a small area anterior to the epiglottis, likely grows as the larynx descends and acquires the human adult vocal tract. From the functional histoanatomical point of view, the newborn laryngeal spaces appear to be favored for respiration and maintenance of a protective sphincter for the lower airway over phonation.

Transcription Factors in Alkaloid Engineering.

Plants produce a large variety of low-molecular-weight and specialized secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used in the pharmaceutical industry. Although alkaloid chemistry has been intensively investigated, characterization of alkaloid biosynthesis, including biosynthetic enzyme genes and their regulation, especially the transcription factors involved, has been relatively delayed, since only a limited number of plant species produce these specific types of alkaloids in a tissue/cell-specific or developmental-specific manner. Recent advances in molecular biology technologies, such as RNA sequencing, co-expression analysis of transcripts and metabolites, and functional characterization of genes using recombinant technology and cutting-edge technology for metabolite identification, have enabled a more detailed characterization of alkaloid pathways. Thus, transcriptional regulation of alkaloid biosynthesis by transcription factors, such as basic helix-loop-helix (bHLH), APETALA2/ethylene-responsive factor (AP2/ERF), and WRKY, is well elucidated. In addition, jasmonate signaling, an important cue in alkaloid biosynthesis, and its cascade, interaction of transcription factors, and post-transcriptional regulation are also characterized and show cell/tissue-specific or developmental regulation. Furthermore, current sequencing technology provides more information on the genome structure of alkaloid-producing plants with large and complex genomes, for genome-wide characterization. Based on the latest information, we discuss the application of transcription factors in alkaloid engineering.

Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production.

BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli-E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.