Polarity-dependent expression and localization of secretory glucoamylase mRNA in filamentous fungal cells.

In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we analyzed mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae, by the MS2 system, in which mRNA can be visualized in living cells. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion, in polarity-dependent expression and localization manners. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that although glaA mRNA localized to stress granules (SGs) and processing bodies (PBs) under high temperature, glaA mRNA was not seen under ER stress, suggesting that there are different regulatory mechanisms of glaA mRNA by SG and PB under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi.

Stress response abnormalities transgenerationally inherited via miR-23 downregulation are restored by a methyl modulator during the lactation period.

Low birthweight rats due to fetal undernutrition sustain higher corticosterone levels when exposed to stress. This is due to the upregulated expression of the pituitary-specific Gas5, a long noncoding RNA (lncRNA) that acts as a glucocorticoid receptor decoy and then competitively inhibiting the binding of glucocorticoids to DNA. However, the mechanism of Gas5 lncRNA upregulation remains unclear. Therefore, using the fetal undernourished model, we identified the factors that regulated Gas5 lncRNA expression and examined their effect on subsequent generations. We found that the expression levels of miR-23 was significantly lower in low birth-weight rats compared with controls. The expression of miR-23 was significantly lower and the expression levels of Gas5 lncRNA were significantly higher in the pituitary gland of low birth-weight offspring of the F2 and F3 generations compared with controls. The methyl modulator intervention in lactating F0 maternal rats restored miR-23 and Gas5 lncRNA expressions not only in F1, F2 and F3 offspring. Moreover, the intervention reduced circulating corticosterone levels and gene expressions in the pituitary gland after restraint stress exposure. In conclusion, miR-23-mediated alterations of the stress response are inherited and restored by methyl modulator intervention during lactation.

Control of One-Handed Helicity in Polyacetylenes: Impact of an Extremely Small Amount of Chiral Substituents.

Controlling the one-handed helicity in synthetic polymers is crucial for developing helical polymer-based advanced chiral materials. We now report that an extremely small amount of chiral biphenylylacetylene (BPA) monomers (ca. 0.3-0.5 mol %) allows complete control of the one-handed helicity throughout the polymer chains mostly composed of achiral BPAs. Chiral substituents introduced at the 2-position of the biphenyl units of BPA positioned in the vicinity of the polymer backbones contribute to a significant amplification of the helical bias, as interpreted by theoretical modeling and simulation. The helical structures, such as the helical pitch and absolute helical handedness (right- or left-handed helix) of the one-handed helical copolymers, were unambiguously determined by high-resolution atomic force microscopy combined with X-ray diffraction. The exceptionally strong helix-biasing power of the chiral BPA provides a highly durable and practically useful chiral material for the separation of enantiomers in chromatography by copolymerization of an achiral functional BPA with a small amount of the chiral BPA (0.5 mol %) due to the robust helical scaffold of the one-handed helical copolymer.

Prediction of the invasion depth of superficial nonampullary duodenal adenocarcinoma.

OBJECTIVES: Distinguishing between intramucosal cancer and submucosal invasive cancer is vital for optimal treatment selection for patients with superficial nonampullary duodenal adenocarcinoma (SNADAC); however, standard diagnostic systems for diagnosing invasion depth are as yet undetermined. METHODS: Of 205 patients with SNADAC who underwent treatment at our institution between 2006 and 2022, 188 had intramucosal cancer and 17 had submucosal invasive cancer. The clinical, endoscopic, and pathological features used in the preoperative diagnosis of invasion depth and the diagnostic performance of endoscopic ultrasonography (EUS) were retrospectively analyzed in 85 patients. RESULTS: The oral side of the papilla tumor location, protruded or mixed macroscopic type, and moderately-to-poorly differentiated adenocarcinoma based on biopsy specimens were significantly more frequent in submucosal invasive cancer than in intramucosal cancer (88% vs. 48%; 94% vs. 42%; 47% vs. 0%, respectively). From the relationship between the endoscopic features and the submucosal invasive cancer incidence, submucosal invasion risk was stratified as: (i) low-risk (risk, 2%), all lesions located on the anal side of the papilla and superficial macroscopic type on the oral side of the papilla; and (ii) high-risk (risk, 23%), protruded or mixed macroscopic type on the oral side of the papilla. Based on the biopsy specimens, all eight patients with moderately-to-poorly differentiated adenocarcinoma had submucosal invasive cancer. Furthermore, EUS was not associated with invasion depth's diagnostic accuracy improvements. CONCLUSION: Optimal treatment indications for SNADAC can be selected based on the risk factors of submucosal invasion by tumor location, macroscopic type, and biopsy diagnosis.

Dehalococcoides mccartyi strain NIT01 grows more stably in vessels made of pure titanium rather than the stainless alloy SUS304.

Advances in many isolation studies have revealed that pure Dehalococcoides grow stably, although the large-scale pure cultivation of Dehalococcoides has yet to be established. In this study, 7 L-culturing of Dehalococcoides mccartyi strain NIT01 was first performed using vessels made of glass and stainless alloy SUS304. All batches cultured in the glass vessel successfully dechlorinated >95% of 1 mM trichloroethene (TCE) to ethene (ETH), whereas only 5 out of 13 batches cultured in the SUS304 vessel did the same. The difference in dechlorination efficiency suggested the possible inhibition of dechlorination by SUS304. Also, the strain NIT01 showed long delays in dechlorination with pieces of SUS316, steel, and a repeatedly used SUS304, but not with titanium. The repeatedly used SUS304 cracked and increased the Fe2+ concentration to ≥76 µM. Dechlorination by this strain was also inhibited with ≥1000 µM Fe2+ and ≥23 µM Cr3+ but not with ≤100 µM Ni2+ , suggesting that Cr3+ eluted from solid stainless alloys inhibited the dechlorination. Culturing in a titanium vessel instead of a stainless alloy showed the complete dechlorination of 1 mM TCE within 12-28 days with a growth yield of 2.7 × 107 cells/µmol-released Cl- , even after repeating use of the vessels six times.

Intracellular Drug Delivery Process of Am80-Encapsulated Lipid Nanoparticles Aiming for Alveolar Regeneration.

Chronic obstructive pulmonary disease (COPD) results in obstructive ventilatory impairment caused by emphysema, and current treatment is limited to symptomatic therapy or lung transplantation. Therefore, the development of new treatments to repair alveolar destruction is especially urgent. Our previous study revealed that 1.0 mg/kg of synthetic retinoid Am80 had a repair effect on collapsed alveoli in a mouse model of elastase-induced emphysema. From these results, however, the clinical dose calculated in accordance with FDA guidance is estimated to be 5.0 mg/60 kg, and it is desirable to further reduce the dose to allow the formulation of a powder inhaler for clinical application. To efficiently deliver Am80 to the retinoic acid receptor in the cell nucleus, which is the site of action, we focused on SS-cleavable proton-activated lipid-like material O-Phentyl-P4C2COATSOME®SS-OP, hereinafter referred to as "SS-OP"). In this study, we investigated the cellular uptake and intracellular drug delivery process of Am80-encapsulated SS-OP nanoparticles to elucidate the mechanism of Am80 by nanoparticulation. Am80-encapsulated SS-OP nanoparticles were taken up into the cells via ApoE, and then Am80 was efficiently delivered into the nucleus via RARα. These results indicated the usefulness of SS-OP nanoparticles as drug delivery system carriers of Am80 for COPD treatment.

Postoperative analgesia following robot-assisted thoracic surgery for mediastinal disease: retrospective comparative study of general anesthesia alone, combined with epidural analgesia, and with ultrasound-guided thoracic paraspinal block.

Background: Recently, robot-assisted thoracic surgery has been increasingly performed for mediastinal disease. However, appropriate postoperative analgesic methods have not been evaluated. Methods: We retrospectively studied patients who underwent robot-assisted thoracic surgery for mediastinal disease at a single university hospital between January 2019 and December 2021. Patients were performed either general anesthesia alone, general anesthesia combined with thoracic epidural anesthesia, or general anesthesia combined with ultrasound-guided thoracic block. Patients were divided into three groups [non-block (NB), thoracic epidural analgesia (TEA), and thoracic paraspinal block (TB)] according to postoperative analgesic methods, and they compared with terms of postoperative pain scores by using numerical rating scale (NRS) at 0, 3, 6, 12, 18, 24, and 48 h. Additionally, rescue supplemental analgesic within 24 h, side effects of anesthesia such as respiratory depression, hypotension, postoperative nausea and vomiting, pruritus and urinary retention, time to ambulation after surgery, and hospital stay after surgery were also compared among the three groups. Results: Data from 169 patients (Group NB: 25, Group TEA: 102, and Group TB: 42) were progressed to the analysis. Postoperative pain scale at 6 and 12 h was significantly lower in Group TEA than NB (1.2±1.6 vs. 2.4±1.8, P<0.01; and 1.2±1.5 vs. 2.2±1.7, P=0.018, respectively). There were no differences in pain scores between Groups TB and TEA at any point. The incidence of patients using rescue analgesics within 24 h was significantly different between groups [Group NB: 15/25 (60%), Group TEA: 30/102 (29.4%), Group TB: 25/42 (59.5%), P=0.01]. For postoperative side effects, only the number of patients complaining of postoperative nausea and vomiting for 24 h after surgery differed significantly between groups [Group NB: 7/25 (28%), Group TEA: 19/102 (18.6%), Group TB: 1/42 (2.4%), P=0.01]. Conclusions: TEA provided better analgesia after robot-assisted thoracic surgery for mediastinal disease than NB as indicated by lower pain scores and fewer rescue analgesic requirements. However, the frequency of postoperative nausea and vomiting was lowest in Group TB of all the groups. Thus, TBs might also provide adequate postoperative analgesia following robot-assisted thoracic surgery for mediastinal disease.

Emergent endoscopic submucosal dissection for a polypoid fibroadipose tumor accidentally disgorged from the mouth: an organ-preserving minimally invasive treatment.

Video 1The yellow protruded lesion at the larynx is different from the main lesion.

Bromisoval-induced bromism with status epilepticus mimicking Wernicke's encephalopathy: report of two cases.

BACKGROUND: Bromine compounds are used in several drugs, including over-the-counter drugs. They sometimes cause intoxication known as bromism. Although the acute neurological symptoms and sequelae of bromism vary, few reports have mentioned acute encephalopathy. CASE PRESENTATION: We report two cases of bromisoval-induced bromism with status epilepticus. Presence of pseudohyperchloremia and history of over-the-counter medication use guided the diagnosis. In the acute phase, our patients showed bilateral medial thalamic lesions on magnetic resonance imaging. The imaging findings were similar to those of Wernicke's encephalopathy. Although these findings improved in the chronic phase, neuropsychiatric sequelae, such as confabulation and amnesia, occurred. CONCLUSION: Bromism can cause acute encephalopathy, and it is important to differentiate it from Wernicke-Korsakoff syndrome.

Am80-Encapsulated Lipid Nanoparticles, Developed with the Aim of Achieving Alveolar Regeneration, Have an Improvement Effect on Pulmonary Emphysema.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis and emphysema, and current drug treatments target its symptoms. Thus, the development of a therapeutic drug to repair alveolar destruction is urgently needed. Our previous research revealed that the synthetic retinoic acid Am80 (1.0 mg/kg) showed a repairing effect on collapsed alveoli in a mouse model of elastase-induced emphysema. However, a further reduction in the dose is desirable to facilitate the development of a powder inhalation formulation for clinical application. We, therefore, focused on SS-OP to deliver Am80 efficiently. As a result, 0.01 mg/kg of Am80-encapsulated SS-OP nanoparticles repaired collapsed alveoli and improved the respiratory function in the mouse model of elastase induced emphysema. The results suggested that, with the use of SS-OP, the Am80 dose could be reduced. This could contribute to the development of a powder inhalation system as a curative medicine for COPD.

Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.

Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes.

The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of the DNA scaffold, such as the modulation of the local pH near enzymes. In this study, two types of enzymes with optimal activity at pH 6 and 8 equally displayed significant catalytic enhancements on the DNA scaffold surface. By using a ratiometric pH indicator, a lower local pH shift of 0.8 was observed near the DNA scaffold surface. The postulated local pH change near the DNA scaffold surface is unlikely to play a general role in enhancing the activity of the scaffolded enzymes.

Characterization of a TNFR2-Selective Agonistic TNF-α Mutant and Its Derivatives as an Optimal Regulatory T Cell Expander.

Regulatory T cells (Tregs) are a subpopulation of lymphocytes that play a role in suppressing and regulating immune responses. Recently, it was suggested that controlling the functions and activities of Tregs might be applicable to the treatment of human diseases such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease. TNF receptor type 2 (TNFR2) is a target molecule that modulates Treg functions. In this study, we investigated the role of TNFR2 signaling in the differentiation and activation of mouse Tregs. We previously reported the generation of a TNFR2-selective agonist TNF mutant, termed R2agoTNF, by using our unique cytokine modification method based on phage display. R2agoTNF activates cell signaling via mouse TNFR2. In this study, we evaluated the efficacy of R2agoTNF for the proliferation and activation of Tregs in mice. R2agoTNF expanded and activated mouse CD4+CD25+ Tregs ex vivo. The structural optimization of R2agoTNF by internal cross-linking or IgG-Fc fusion selectively and effectively enhanced Treg expansion in vivo. Furthermore, the IgG-Fc fusion protein suppressed skin-contact hypersensitivity reactions in mice. TNFR2 agonists are expected to be new Treg expanders.

Correlative Localization Analysis Between mRNA and Enhanced Green Fluorescence Protein-Fused Protein by a Single-Molecule Fluorescence in situ Hybridization Using an egfp Probe in Aspergillus oryzae.

Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.

Synthesis of DFGH-Ring Derivatives of Physalins via One-Pot Construction of GH-Ring and Evaluation of Their NF-κB-Inhibitory Activity.

We designed and synthesized a series of derivatives containing the right-side DFGH-ring structure of physalin-type natural products, decorated with a hydrophobic substituent. The synthetic scheme utilizes a highly efficient, one-pot protocol for simultaneous construction of the GH-ring system, promoted by HF/pyridine. Among the compounds synthesized, 5d inhibited TNF-α-stimulated NF-κB activation with similar potency to physalin B.

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae.

Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using ß-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics.

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.