Chloroplast-actin filaments decide the direction of chloroplast avoidance movement under strong light in Arabidopsis thaliana.

Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.

A purified diet affects intestinal epithelial proliferation and barrier functions through gut microbial alterations.

The gut microbiota plays a crucial role in maintaining epithelial barrier function. Although multiple studies have demonstrated the significance of dietary factors on the gut microbiota and mucosal barrier function, the impact of a purified diet, which has long been used in various animal experiments, on intestinal homeostasis remains to be elucidated. Here, we compared the impact of two different types of diets, a crude diet and an AIN-93G-formula purified diet, on epithelial integrity and the gut microbiota. Purified diet-fed mice exhibited shorter villi and crypt lengths and slower epithelial turnover, particularly in the ileum. In addition, antimicrobial products, including REG3γ, were substantially decreased in purified diet-fed mice. Purified diet feeding also suppressed α1,2-fucosylation on the epithelial surface. Furthermore, the purified diet induced metabolic rewiring to fatty acid oxidation and ketogenesis. 16S ribosomal RNA gene sequencing of the ileal contents and mucus layer revealed distinct gut microbiota compositions between the purified and crude diet-fed mice. Purified diet feeding reduced the abundance of segmented filamentous bacteria (SFB), which potently upregulate REG3γ and fucosyltransferase 2 (Fut2) by stimulating group 3 innate lymphoid cells (ILC3s) to produce IL-22. These observations illustrate that the intake of a crude diet secures epithelial barrier function by facilitating SFB colonization, whereas a purified diet insufficiently establishes the epithelial barrier, at least partly owing to the loss of SFB. Our data suggest that the influence of purified diets on the epithelial barrier integrity should be considered in experiments using purified diets.

D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease.

BACKGROUND & AIMS: D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression. METHODS: The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model. RESULTS: The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K. pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E. coli and generation of antibiotic-resistant strains. CONCLUSIONS: D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Heart-derived collagen promotes maturation of engineered heart tissue.

Although the extracellular matrix (ECM) plays essential roles in heart tissue engineering, the optimal ECM components for heart tissue organization have not previously been elucidated. Here, we focused on the main ECM component, fibrillar collagen, and analyzed the effects of collagens on heart tissue engineering, by comparing the use of porcine heart-derived collagen and other organ-derived collagens in generating engineered heart tissue (EHT). We demonstrate that heart-derived collagen induces better contraction and relaxation of human induced pluripotent stem cell-derived EHT (hiPSC-EHT) and that hiPSC-EHT with heart-derived collagen exhibit more mature profiles than those with collagens from other organs. Further, we found that collagen fibril formation and gel stiffness influence the contraction, relaxation, and maturation of hiPSC-EHT, suggesting the importance of collagen types III and type V, which are relatively abundant in the heart. Thus, we demonstrate the effectiveness of organ-specific collagens in tissue engineering and drug discovery.

Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis.

Cell transplantation using mesenchymal stem cells (MSCs) has emerged as a promising approach to repairing and regenerating injured or impaired organs. However, the survival and retention of MSCs following transplantation remain a challenge. Therefore, we investigated the efficacy of co-transplantation of MSCs and decellularized extracellular matrix (dECM) hydrogels, which have high cytocompatibility and biocompatibility. The dECM solution was prepared by enzymatic digestion of an acellular porcine liver scaffold. It could be gelled and formed into porous fibrillar microstructures at physiological temperatures. MSCs expanded three-dimensionally in the hydrogel without cell death. Compared to the 2-dimensional cell culture, MSCs cultured in the hydrogel showed increased secretion of hepatocyte growth factor (HGF) and tumor necrosis factor-inducible gene 6 protein (TSG-6), both of which are major anti-inflammatory and anti-fibrotic paracrine factors of MSCs, under TNFα stimulation. In vivo experiments showed that the co-transplantation of MSCs with dECM hydrogel improved the survival rate of engrafted cells compared to those administered without the hydrogel. MSCs also demonstrated therapeutic effects in improving inflammation and fibrosis of pancreatic tissue in a dibutyltin dichloride (DBTC)-induced rat pancreatitis model. Combinational use of dECM hydrogel with MSCs is a new strategy to overcome the challenges of cell therapy using MSCs and can be used for treating chronic inflammatory diseases in clinical settings.

Sharing of Moonlighting Proteins Mediates the Symbiotic Relationship among Intestinal Commensals.

The human gastrointestinal tract is inhabited by trillions of symbiotic bacteria that form a complex ecological community and influence human physiology. Symbiotic nutrient sharing and nutrient competition are the most studied relationships in gut commensals, whereas the interactions underlying homeostasis and community maintenance are not fully understood. Here, we provide insights into a new symbiotic relationship wherein the sharing of secreted cytoplasmic proteins, called "moonlighting proteins," between two heterologous bacterial strains (Bifidobacterium longum and Bacteroides thetaiotaomicron) was observed to affect the adhesion of bacteria to mucins. B. longum and B. thetaiotaomicron were cocultured using a membrane-filter system, and in this system the cocultured B. thetaiotaomicron cells showed greater adhesion to mucins compared to that shown by monoculture cells. Proteomic analysis showed the presence of 13 B. longum-derived cytoplasmic proteins on the surface of B. thetaiotaomicron. Moreover, incubation of B. thetaiotaomicron with the recombinant proteins GroEL and elongation factor Tu (EF-Tu)-two well-known mucin-adhesive moonlighting proteins of B. longum-led to an increase in the adhesion of B. thetaiotaomicron to mucins, a result attributed to the localization of these proteins on the B. thetaiotaomicron cell surface. Furthermore, the recombinant EF-Tu and GroEL proteins were observed to bind to the cell surface of several other bacterial species; however, the binding was species dependent. The present findings indicate a symbiotic relationship mediated by the sharing of moonlighting proteins among specific strains of B. longum and B. thetaiotaomicron. IMPORTANCE The adhesion of intestinal bacteria to the mucus layer is an important colonization strategy in the gut environment. Generally, the bacterial adhesion process is a characteristic feature of the individual cell surface-associated adhesion factors secreted by a particular bacterium. In this study, coculture experiments between Bifidobacterium and Bacteroides show that the secreted moonlighting proteins adhere to the cell surface of coexisting bacteria and alter the adhesiveness of the bacteria to mucins. This finding indicates that the moonlighting proteins act as adhesion factors for not only homologous strains but also for coexisting heterologous strains. The presence of a coexisting bacterium in the environment can significantly alter the mucin-adhesive properties of another bacterium. The findings from this study contribute to a better understanding of the colonization properties of gut bacteria through the discovery of a new symbiotic relationship between them.

Escherichia coli-derived outer-membrane vesicles induce immune activation and progression of cirrhosis in mice and humans.

BACKGROUND AND AIMS: Decompensated cirrhosis with fibrosis progression causes portal hypertension followed by an oedematous intestinal tract. These conditions weaken the barrier function against bacteria in the intestinal tract, a condition called leaky gut, resulting in invasion by bacteria and bacterial components. Here, we investigated the role of outer-membrane vesicles (OMVs) of Escherichia coli, which is the representative pathogenic gut-derived bacteria in patients with cirrhosis in the pathogenesis of cirrhosis. METHODS: We investigated the involvement of OMVs in humans using human serum and ascites samples and also investigated the involvement of OMVs from E. coli in mice using mouse liver-derived cells and a mouse cirrhosis model. RESULTS: In vitro, OMVs induced inflammatory responses to macrophages and neutrophils, including the upregulation of C-type lectin domain family 4 member E (Clec4e), and induced the suppression of albumin production in hepatocytes but had a relatively little direct effect on hepatic stellate cells. In a mouse cirrhosis model, administration of OMVs led to increased liver inflammation, especially affecting the activation of macrophages, worsening fibrosis and decreasing albumin production. Albumin administration weakened these inflammatory changes. In addition, multiple antibodies against bacterial components were increased with a progressing Child-Pugh grade, and OMVs were detected in ascites of patients with decompensated cirrhosis. CONCLUSIONS: In conclusion, OMVs induce inflammation, fibrosis and suppression of albumin production, affecting the pathogenesis of cirrhosis. We believe that our study paves the way for the future prevention and treatment of cirrhosis.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana.

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

A Novel Alaska Pollock Gelatin Sealant Shows Higher Bonding Strength and Nerve Regeneration Comparable to That of Fibrin Sealant in a Cadaveric Model and a Rat Model.

BACKGROUND: A novel biocompatible sealant composed of Alaska pollock-derived gelatin (ApGltn) has recently shown good burst strength and biocompatibility in a porcine aorta. The purpose of this study was to investigate the bonding strength and biocompatibility of the ApGltn sealant in transected digital nerves of fresh frozen cadavers and in the sciatic nerves of a rat model. METHODS: Eighty human digital nerves of fresh frozen cadavers were transected for biomechanical traction testing. They were treated with four surgical interventions: (1) suture plus ApGltn sealant; (2) suture; (3) ApGltn sealant; and (4) fibrin sealant. Forty-three sciatic nerves of male Wistar rats were used for functional and histopathologic evaluation. They were treated with six surgical interventions: (1) suture plus ApGltn sealant; (2) suture; (3) ApGltn sealant; (4) fibrin sealant; (5) resection with a 5-mm gap (10 rats per group); and (6) sham operation (three rats). Macroscopic confirmation, muscle weight measurement, and histopathologic findings including G-ratio were examined 8 weeks after the procedure. RESULTS: The maximum failure load of the ApGltn sealant was significantly higher than that of a fibrin sealant (0.22 ± 0.05 N versus 0.06 ± 0.04 N). The maximum failure load of the ApGltn sealant was significantly lower that of suture plus ApGltn sealant (1.37 N) and suture (1.27 N). Functional evaluation and histologic examination showed that sciatic nerves repaired with ApGltn sealant showed similar nerve recovery compared to repair with the suture and fibrin sealant. CONCLUSION: The ApGltn sealant showed higher bonding strength and equal effect of nerve regeneration when compared with the fibrin sealant.

Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians.

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases1-3. Here we show that centenarians have a distinct gut microbiome that is enriched in microorganisms that are capable of generating unique secondary bile acids, including various isoforms of lithocholic acid (LCA): iso-, 3-oxo-, allo-, 3-oxoallo- and isoallolithocholic acid. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from the faecal microbiota of a centenarian, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5α-reductase (5AR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSDH) were responsible for the production of isoalloLCA. IsoalloLCA exerted potent antimicrobial effects against Gram-positive (but not Gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that the metabolism of specific bile acids may be involved in reducing the risk of infection with pathobionts, thereby potentially contributing to the maintenance of intestinal homeostasis.

The adeno-associated virus rh10 vector is an effective gene transfer system for chronic spinal cord injury.

Treatment options for chronic spinal cord injury (SCI) remain limited due to unfavourable changes in the microenvironment. Gene therapy can overcome these barriers through continuous delivery of therapeutic gene products to the target tissue. In particular, adeno-associated virus (AAV) vectors are potential candidates for use in chronic SCI, considering their safety and stable gene expression in vivo. Given that different AAV serotypes display different cellular tropisms, it is extremely important to select an optimal serotype for establishing a gene transfer system during the chronic phase of SCI. Therefore, we generated multiple AAV serotypes expressing ffLuc-cp156, a fusion protein of firefly luciferase and Venus, a variant of yellow fluorescent protein with fast and efficient maturation, as a reporter, and we performed intraparenchymal injection in a chronic SCI mouse model. Among the various serotypes tested, AAVrh10 displayed the highest photon count on bioluminescence imaging. Immunohistological analysis revealed that AAVrh10 showed favourable tropism for neurons, astrocytes, and oligodendrocytes. Additionally, with AAVrh10, the area expressing Venus was larger in the injury epicentre and extended to the surrounding tissue. Furthermore, the fluorescence intensity was significantly higher with AAVrh10 than with the other vectors. These results indicate that AAVrh10 may be an appropriate serotype for gene delivery to the chronically injured spinal cord. This promising tool may be applied for research and development related to the treatment of chronic SCI.

Large-Area Fluorescence and Electron Microscopic Correlative Imaging With Multibeam Scanning Electron Microscopy.

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

Evolution of root apical meristem structures in vascular plants: plasmodesmatal networks.

PREMISE OF THE STUDY: The apical meristem generates indeterminate apical growth of the stem and root of vascular plants. Our previous examination showed that shoot apical meristems (SAMs) can be classified into two types based on plasmodesmatal networks (PNs), which are important elements in symplasmic signaling pathways within the apical meristem. Here, we examined the PNs of root apical meristems (RAMs) in comparison with those of SAMs. METHODS: Root apical meristems of 18 families and 22 species of lycophytes and euphyllophytes were analyzed. Plasmodesmata (PD) in cell walls in median longitudinal sections of RAMs were enumerated using transmission electron micrographs, and the PD density per 1 µm2 of each cell wall was calculated. KEY RESULTS: Root apical meristems with prominent apical cells of monilophytes (euphyllophytes) and Selaginellaceae (lycophytes) had high PD densities, while RAMs with plural initial cells of gymnosperms and angiosperms (euphyllophytes), and of Lycopodiaceae and Isoetaceae (lycophytes) had low PD densities. This correlation between structures of apical meristems and PD densities is identical to that in SAMs already described. CONCLUSIONS: Irrespective of their diversified structures, the RAMs of vascular plants can be classified into two types with respect to PNs: the fern (monilophyte) type, which has a lineage-specific PN with only primary PD, and the seed-plant type, which has an interspecific PN with secondary PD in addition to primary PD. PNs may have played a key role in the evolution of apical meristems in vascular plants.

Enhancement of bactericidal activity against group B streptococci with reduced penicillin susceptibility by uptake of gentamicin into cells resulting from combination with ß-lactam antibiotics.

Combined effects of penicillin (PEN) and gentamicin (GM) against Streptococcus agalactiae, i.e. group B streptococci (GBS), are known to occur, but synergy has not been examined in strains with reduced PEN susceptibility, usually called PEN-resistant GBS (PRGBS). We therefore studied combined effects of ß-lactam antibiotics and GM in cultures of 3 PRGBS strains belonging to serotype Ia or III that were isolated from Japanese adults with invasive infections. Killing kinetics were determined at 2-h intervals from 0 to 6 h after exposure to ampicillin (AMP) or cefotaxime (CTX) combined with GM. Concentrations of GM in bacterial cells were measured by liquid chromatography-tandem mass spectrometry. Morphologic changes after exposure to agents were observed by transmission electron microscopy. Combining AMP or CTX with GM synergistically increased bactericidal activity against PRGBS beyond that of either ß-lactam alone. GM concentrations in bacterial cells increased 5- to 8-fold when GM was combined with AMP or CTX. Electron microscopically, bacterial cells showed aggregates of strands and ribosomal damage most likely reflecting enhanced GM uptake into bacterial cells. This uptake appeared to result from cell wall damage caused by ß-lactam antibiotics. This study suggests that combining ß-lactam antibiotics with GM might be useful against severe PRGBS infection.

Size-dependent toxicity of silica nano-particles to Chlorella kessleri.

SiO(2) nano-particles were found to exhibit size-dependent toxicity toward the alga, Chlorella kessleri. Small SiO(2) nano-particles exhibit stronger toxicity: 50% inhibitory concentrations (IC(50)) value for 5 nm = 0.8 +/- 0.6%, 26 nm = 7.1 +/- 2.8%, and 78 nm = 9.1 +/- 4.7%. Enlargement of the cell body was observed by flow cytometry, which is due to the presence of structures that obstructed cell division. Optical and transmission microscopes were used to observe coagulated cells with incomplete division. Although the physiological effect of SiO(2) nano-particles was not clear, SiO(2) nano-particles are toxic, at least for algae in aquatic media. Under the transmission electron microscope, several amorphous structures appeared in the cells that were exposed to 5-nm silica nano-particles.