Efficacy of Cricoid Pressure Application during Esophagogastroduodenoscopy in Patients with Poor Gastric Wall Extension.

INTRODUCTION: Esophagogastroduodenoscopy (EGD) requires adequate air infusion. However, cases of poor gastrointestinal wall extension due to frequent eructation have been reported. Sufficient gastrointestinal wall extension can be achieved by applying cricoid pressure during EGD. Herein, we evaluated the frequency of cases with poor gastrointestinal wall extension and the efficacy and safety of applying cricoid pressure during EGD. METHODS: This interventional study included patients who underwent EGD between January 2020 and December 2020 at the JA Akita Koseiren Yuri Kumiai General Hospital. Cases wherein folds of the greater curvature of the upper gastric body were not sufficiently extended during EGD were considered to have poor gastrointestinal wall extension. In such cases, air infusion was performed while applying cricoid pressure. This procedure was considered effective when gastric wall extension was achieved. RESULTS: A total of 2,000 patients were enrolled and underwent upper gastrointestinal endoscopy; however, five were excluded because of upper gastrointestinal tract stenosis. Observation of gastric wall extension of the greater curvature in the upper gastric body with normal air insufflation was difficult in 113 (5.7%) cases. Applying cricoid pressure was effective in 93 (82.3%) patients with poor gastric wall extension. Sufficient gastric wall extension was achieved within an average of 12.8 s in cases where cricoid pressure application was effective. No adverse events were associated with cricoid pressure application. CONCLUSIONS: Cricoid pressure application for patients with poor gastric wall extension during EGD is useful for ensuring a sufficient field of view during observation of the gastric body.

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by ß-1,6-branched ß-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and ß-1,3-glucans under turgor pressure in vivo.

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

This study reports that a high concentration of the endo-ß-1,3-glucanase ENG (200 µg ml-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular ß-glucosidase BGL2 (1,000 µg ml-1) did not; however, in combination, low concentrations of ENG (25 µg ml-1) and BGL2 (260 µg ml-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ß-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of ß-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ß-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ß-1,6 branched ß-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ß-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that ß-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ß-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ß-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ß-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ß-glucans during stipe elongation growth.

Accumulation and cross-linkage of ß-1,3/1,6-glucan lead to loss of basal stipe cell wall extensibility in mushroom Coprinopsis cinerea.

The mature basal stipe of mushroom Coprinopsis cinerea loses wall extensibility. We found that an endo-ß-1,3-glucanase ENG from C. cinerea could restore mature basal stipe wall extensibility via pretreatment such that the ENG-pretreated basal stipe walls could be induced to extend by chitinase ChiIII. ENG pretreatment released glucose, laminaribiose, and 3-O-D-gentiobiose-D-glucose from the basal stipe walls, consistent with ENG-digested products of ß-1,6-branched ß-1,3-glucan. Different effects of endo-ß-1,3-glucanase ENG and exo-ß-1,3-glucanase EXG pretreatment on the structure, amount and ratio (ß-1,3-glucoside bonds to ß-1,6-glucoside bonds) of products from the basal stipe and the apical stipe cell walls, respectively, and on the cell wall extensibility and the cell wall ultra-architecture of the basal stipes were analyzed. All results demonstrate that the more accumulation and cross-linkage of ß-1,6-branched ß-1,3-glucan with wall maturation lead to loss of wall extensibility of the basal stipe regions compared to the apical stipe cell walls.

BrEXLB1, a Brassica rapa Expansin-Like B1 Gene is Associated with Root Development, Drought Stress Response, and Seed Germination.

Expansins are structural proteins prevalent in cell walls, participate in cell growth and stress responses by interacting with internal and external signals perceived by the genetic networks of plants. Herein, we investigated the Brassica rapa expansin-like B1 (BrEXLB1) interaction with phytohormones (IAA, ABA, Ethephon, CK, GA3, SA, and JA), genes (Bra001852, Bra001958, and Bra003006), biotic (Turnip mosaic Virus (TuMV), Pectobacterium carotovorum, clubroot disease), and abiotic stress (salt, oxidative, osmotic, and drought) conditions by either cDNA microarray or qRT-PCR assays. In addition, we also unraveled the potential role of BrEXLB1 in root growth, drought stress response, and seed germination in transgenic Arabidopsis and B. rapa lines. The qRT-PCR results displayed that BrEXLB1 expression was differentially influenced by hormones, and biotic and abiotic stress conditions; upregulated by IAA, ABA, SA, ethylene, drought, salt, osmotic, and oxidative conditions; and downregulated by clubroot disease, P. carotovorum, and TuMV infections. Among the tissues, prominent expression was observed in roots indicating the possible role in root growth. The root phenotyping followed by confocal imaging of root tips in Arabidopsis lines showed that BrEXLB1 overexpression increases the size of the root elongation zone and induce primary root growth. Conversely, it reduced the seed germination rate. Further analyses with transgenic B. rapa lines overexpressing BrEXLB1 sense (OX) and antisense transcripts (OX-AS) confirmed that BrEXLB1 overexpression is positively associated with drought tolerance and photosynthesis during vegetative growth phases of B. rapa plants. Moreover, the altered expression of BrEXLB1 in transgenic lines differentially influenced the expression of predicted BrEXLB1 interacting genes like Bra001852 and Bra003006. Collectively, this study revealed that BrEXLB1 is associated with root development, drought tolerance, photosynthesis, and seed germination.

A novel endo-ß-1,6-glucanase from the mushroom Coprinopsis cinerea and its application in studying of cross-linking of ß-1,6-glucan and the wall extensibility in stipe cell walls.

We previously reported that chitinases reconstituted heat-inactivated stipe cell wall extension in a steady and continuous extension profile by cleaving chitins cross-linked to various polysaccahrides, whereas, endo-ß-1,3-glucanases reconstituted heat-inactivated stipe wall extension in a profile of an initially fast extension and subsequent termination of extension due to its degradation of ß-1,3-glucan but not other polysaccharides such as ß-1,6-glucans cross-linked to chitins. Thus, a novel endo-ß-1,6-glucanase, GH30A, from Coprinopsis cinerea was cloned and characterized to study cross-linking of ß-1,6-glucan and wall extensibility in stipe walls. GH30A had higher activity and better thermophilicity than reported ß-1,6-glucanases. GH30A hydrolyzed pustulan having ß-1,6-linkages but not other polysaccharides without ß-1,6-linkages; GH30A did not cleave gentiobiose and single ß-1,6-linkage branches in laminarin from Laminaria digitata but cut consecutive ß-1,6-linkage branches in laminarin from Eisenia bicyclis. GH30A reconstituted heated-inactivated stipe cell wall extension with release of glucose and gentiobiose, indicating that ß-1,6-glucans were present and cross-linked to chitins in stipe walls, and cleaving ß-1,6-glucans cross-linked to chitins by GH30A led to wall loosening for extension. However, GH30A individually or in combination with endo-ß-1,3-glucanase reconstituted-stipe wall extension profile was similar to individual endo-ß-1,3-glucanase's, exploring that chitins were also cross-linked to other polysaccharides besides ß-1,3-glucans and ß-1,6-glucans.

Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa.

Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains.