内耳前庭細胞由来因子とオルガノイド培養を組み合わせたES細胞から内耳前庭有毛細胞への分化誘導

Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs.博士（医学）・甲第877号・令和5年3月15

サリチル酸誘発耳鳴に対する牛車腎気丸の抑制効果の行動学的および免疫組織化学的な根拠

Many people are affected by tinnitus, a sensation of ringing in the ear despite the absence of external sound. Goshajinkigan (GJG) is one of the formulations of Japanese traditional herbal medicine and is prescribed for the palliative treatment of patients with tinnitus. Although GJG is clinically effective in these patients, its behavioral effects and the underlying neuroanatomical substrate have not been modeled in animals. We modeled tinnitus using salicylate-treated rats, demonstrated the effectiveness of GJG on tinnitus, and examined the underlying neuronal substrate with c-Fos expression. Intraperitoneal injection of sodium salicylate (400 mg/kg) into rats for three consecutive days significantly increased false positive scores, which were used to assess tinnitus behavior. When GJG was orally administered one hour after each salicylate injection, the increase in tinnitus behavior was suppressed. The analysis of c-Fos expression in auditory-related brain areas revealed that GJG significantly reduced the salicylate-induced increase in the number of c-Fos-expressing cells in the auditory cortices, inferior colliculus, and dorsal cochlear nucleus. These results suggest a suppressive effect of GJG on salicylate-induced tinnitus in animal models.博士（医学）・甲第851号・令和4年9月28日Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

Streptococcus suis in Humans, Thailand

Streptococcus suis in Humans, Thailan

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection

Effect of probiotics on gut microbiome in patients with administration of surgical antibiotic prophylaxis: A randomized controlled study

Surgical antibiotic prophylaxis (SAP) is recommended for the prevention of surgical site infections. However, there is a concern about adverse effects of SAP, such as antibiotic-associated diarrhea (AAD). To prevent AAD, administration of probiotics has been investigated. Although recent advances in next-generation sequencing makes it possible to analyze the gut microbiome, the effect of probiotics on the gut microbiome in the patients with SAP remains unknown. To test a hypothesis that SAP influences the gut microbiome and probiotics prevent the influence, a randomized controlled study was conducted with patients who underwent spinal surgery at Nagasaki University Hospital. After obtaining informed consent, the patients were automatically classified into the non-probiotics group and the probiotics group. In the probiotics group, the patients took 1 g of Enterococcus faecium 129 BIO 3B-R, 3 times a day on postoperative days (PODs) 1–5. The feces of all patients were sampled before administration of SAP and on PODs 5 and 10. We compared alpha and beta diversity and differential abundance analysis of the gut microbiome before and after SAP. During the study period, a total of 33 patients were evaluated, comprising 17 patients in the non-probiotics group and 16 in the probiotics group. There was no significant difference between the groups regarding patient characteristics. In alpha and beta diversity, there were no significant differences among all combinations. In differential abundance analysis at operational taxonomic unit level, Streptococcus gallolyticus and Roseburia were significantly increased in the non-probiotics group and significantly decreased in the probiotics group

Regulation of Transgene Expression in Tumor Cells by Exploiting Endogenous Intracellular Signals

Recently, we have proposed a novel strategy for a cell-specific gene therapy system based on responses to intracellular signals. In this system, an intracellular signal that is specifically and abnormally activated in the diseased cells is used for the activation of transgene expression. In this study, we used protein kinase C (PKC)α as a trigger to activate transgene expression. We prepared a PKCα-responsive polymer conjugate [PPC(S)] and a negative control conjugate [PPC(A)], in which the phosphorylation site serine (Ser) was replaced with alanine (Ala). The phosphorylation for polymer/DNA complexes was determined with a radiolabel assay using [γ-32P]ATP. PPC(S)/DNA complexes were phosphorylated by the addition of PKCα, but no phosphorylation of the PPC(A)/DNA complex was observed. Moreover, after microinjection of polymer/GFP-encoding DNA complexes into HepG2 cells at cation/anion (C/A) ratios of 0.5 to 2.0, significant expression of GFP was observed in all cases using PPC(S)/DNA complexes, but no GFP expression was observed in the negative control PPC(A)/DNA complex-microinjected cells at C/A ratios of 1.0 and 2.0. On the other hand, GFP expression from PPC(S)/DNA complexes was completely suppressed in cells pretreated with PKCα inhibitor (Ro31-7549). These results suggest that our gene regulation system can be used for tumor cell-specific expression of a transgene in response to PKCα activity

Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research

Evidence for Lateral Transfer of the Suilysin Gene Region of Streptococcus suis

Suilysin is a cholesterol-binding cytolysin encoded by sly in Streptococcus suis. DNA sequence determination of the sly locus in a strain lacking sly revealed the presence of another gene, designated orf102, in the place of sly. No transposable element or long-repeat sequence was found in the close vicinity. Except for six strains whose corresponding loci have been rearranged, all of the remaining 62 strains examined had either sly or orf102 at the same locus and their flanking regions were conserved. The genetic organizations having either sly or orf102 were found in the strains whose 16S rRNA sequences were identical. These results suggest that S. suis acquired sly or orf102 from a foreign source and that these genes subsequently spread among S. suis strains by homologous recombination

Rice Root Growth with Increasing in Plant Hormone and Allantoin by Inosine in Nutrient Solution

The presence of low concentration of inosine in solution in soil has increased the root growth as shown in our previous work. Thus the aim of this work was to clear the mechanism of the inosine-induced enhancement of root growth with emphasis on plant hormone and metabolites profile. In the first experiment the rice plant (Oryza sativa L. cv Nipponbare) was precultivated in nutrient solution for 3 weeks. Then seedlings with the same size were transferred to pots with the following treatment solutions (0 N + 0 inosine, 0 N + 20 mg L^[-1] inosine, 4 mg L^[-1] N + 0 inosine and 4 mg L^[-1] N + 20 mg L^[-1] inosine). Two weeks after the treatment started, xylem sap was collected for determining cytokinin and auxin concentration by ELISA, then fresh and dry weight of shoot and root, and root length were measured. In the second experiment, rice seedling were prepared as Experiment 1 then samples were collected after inosine treatment (0, 1, 2, 24, 72, and 96 hours) for the analysis of metabolites profiling by using GC-MS. Dry weight (shoot and root), and root length and number of lateral root were measured. As a result, inosine application increased root length and concentrations of trans-zeatin riboside and indole-3-acetic acid content in xylem sap significantly. The allantoin was the most up regulated compound in the metabolite profile. Thus, it is suggested that inosine application increased concentration of plant hormones and allantoin, possibly resulting in growth enhancement of plant root.XVI International Plant Nutrition Colloquium, Plant Nutrition for Sustainable Development and Global Health. 26-30 August 2009. Sacramento, California, USA