Typical Ultrastructure Research Articles

Morphological and anatomical study of the fruits of four species of Stachyurus Siebold et Zucc. (Stachyuraceae)

The genus Stachyurus Siebold et Zucc. has always been considered as the only genus in the Stachyuraceae, but the systematic position of this family is complicated, since it has been included in various orders of flowering plants by vegetative and generative features. A thorough study of the fruits of representatives of the genus Stachyurus has not yet been undertaken. This work presents the results of a morphological and anatomical study of the fruits of four species of the genus Stachyurus. Morphological and anatomical studies of the fruits of representatives of the genus Stachyurus Siebold et Zucc. from the monotypic family Stachyuraceae are carried out. The fruits are considered as many-seeded 4-false-loculed paracarpous berries. A peculiarity of the fruit anatomy of the studied species Stachyurus is the lack of differentiation of the mesocarp. It is composed of layers of similar parenchymatous isodiametric cells with a row of vascular bundles in the center. The exocarp and endocarp are monolayers composed of parenchymatous rectangular cells elongated in a tangential direction. Separate mechanical tissues and various inclusions (resins, gums, etc.) were not found. Stachyurus species differ in the features of fruit surface micromorphology: type of surface ultrastructure, presence of pore-like areas in the cuticle, presence and shape of epicuticular wax. The identified carpological features of Stachyurus along with other diagnostic features can be taken into account in further systematic and phylogenetic studies of Stachyuraceae.

Complex Brain Morphology Discovered in the Shark Parasite Nybelinia surmenicola (Cestoda: Trypanorhyncha).

The ultrastructure of the nervous system has been studied in sexually mature Nybelinia surmenicola (Cestoda: Trypanorhyncha) from the intestine of a shark Lamna ditropis. The central nervous system (CNS) reveals a complex organization within cestodes and corresponds to the trypanorhynch pattern of brain architecture. The brain of N. surmenicola is differentiated into nine clearly defined lobes and semicircular, median, and X-shaped cruciate commissures. A specific feature is the presence of a powerful extracellular capsule that surrounds the brain lobes with the cortical glial cells. Moreover, the architecture of the anterior lobes clearly distinguishes the species of Tentacularioidea. The neurons of the anterior lobes form compact groups looking like frontal horns. There are approximately 120 neurons in the anterior lobes and a preliminary estimate of more than 300 perikarya in the brain. Several ultrastructural types of neurons have been identified, differing in the size and shape of the soma, the density of the cytoplasm, and the ultrastructure of synaptic vesicles. Numerous synapses involving clear and electron-dense vesicles have been observed in neuropils. Two types of glial cells have been found in the brain that participate in neuronal metabolism and wrap around the giant axons, brain lobes, neuropil compartments, and the main nerve cords. Such a powerful extracellular fibrillar brain capsule has not been observed in the brain of other studied cestodes and has been demonstrated in this study for the first time. The differentiation of the brain lobes reveals the important role of the rhyncheal system in the evolution of cestodes and correlates with their behavior. The anterior nerves arising from the anterior lobes innervate the radial muscles stabilizing the position of the tentacle sheaths and movements of the attachment organs. The nervous system anatomy and the brain architecture may reflect the morphofunctional aspects of the tapeworm evolution.

A new cheirolepidiaceous microsporangiate cone Classostrobus archangelskyi with in situ pollen from the Lower Cretaceous of Figueira da Foz Formation, central-western mainland Portugal

A new cheirolepidiaceous conifer microsporangiate cone Classostrobus archangelskyi is described from the Carregueira opencast clay pit complex near the village of Juncal, in the Lusitanian Basin of central-western mainland Portugal. The plant-bearing horizon belongs to the Famalicão Member of the Figueira da Foz Formation, considered to be of Lower Cretaceous (upper Aptian–lower Albian) age. The new microsporangiate cone is characterised by the presence of distinctive and unusual pollen assigned to the genus Classopollis. The microsporangiate cone is ovoid and small, comprising only a few microsporophylls, each with a stalk and deltoid head bearing 4–5 pollen sacs. The new Portuguese species is compared to other microsporangiate cones attributed to the genus Classostrobus. The Classopollis pollen grains found in situ are characterised by a microechinate sculpture and typical ultrastructure. The pollen grains were studied using SEM, TEM and transmitted light microscopy. The studied pollen shares strong similarities with Classopollis martinotii, Classopollis torosus, but particularly with Classopollis noelii. The new microsporangiate cone Classostrobus archangelskyi co-occurs with frenelopsid leafy shoots of Frenelopsis antunesii, and their common association is suggested.

Environmental Biofilms from an Urban Community in Salvador, Brazil, Shelter Previously Uncharacterized Saprophytic Leptospira.

Biofilms are complex microecosystems with valuable ecological roles that can shelter a variety of microorganisms. Spirochetes from the genus Leptospira have been observed to form biofilms in vitro, in rural environments, and in the kidneys of reservoir rats. The genus Leptospira is composed of pathogenic and non-pathogenic species, and the description of new species is ongoing due to the advent of whole genome sequencing. Leptospires have increasingly been isolated from water and soil samples. To investigate the presence of Leptospira in environmental biofilms, we collected three distinct samples of biofilms formed in an urban setting with poor sanitation: Pau da Lima, in Salvador, Bahia, Brazil. All biofilm samples were negative for the presence of pathogenic leptospires via conventional PCR, but cultures containing saprophytic Leptospira were identified. Whole genomes were generated and analyzed for twenty isolates obtained from these biofilms. For species identification, we used digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis. The obtained isolates were classified into seven presumptive species from the saprophytic S1 clade. ANI and dDDH analysis suggest that three of those seven species were new. Classical phenotypic tests confirmed the novel isolated bacteria as saprophytic Leptospira. The isolates presented typical morphology and ultrastructure according to scanning electron microscopy and formed biofilms under in vitro conditions. Our data indicate that a diversity of saprophytic Leptospira species survive in the Brazilian poorly sanitized urban environment, in a biofilm lifestyle. We believe our results contribute to a better understanding of Leptospira biology and ecology, considering biofilms as natural environmental reservoirs for leptospires.

POLLEN MORPHOLOGY OF SOME PLANTS FROM THE GYZMEIDAN MUD VOLCANO (REPUBLIC OF AZERBAIJAN)

The aim of this study was to assess the influence of unfavorable conditions of the mud volcano on the morphological features of pollen. Using light, scanning and transmission electron microscopes, the morphology of pollen of 20 species from 14 families of dicotyledonous plants growing on the Gyzmeidan mud volcano (Shamakhi) in the Republic of Azerbaijan has been studied. According to the aperture patterns of the pollen grains in the studied species, there are 5 pollen types: 3-colpate (Brassicaceae, Convolvulaceae, Fabaceae (Hedysarum sericeum), Lamiaceae, Linaceae), 3-colporate (Asteraceae, Cistaceae, Clusiaceae, Euphorbiaceae, Fabaceae, Rosaceae), stepanocolporate (Polygalaceae), heteroaperturate (Boraginaceae), pantoporate (Ranunculaceae).No significant deviations of the main morphological features of pollen (shape, outline, size of pollen grains, type and number of apertures, exine sculpture, thickness and ultrastructure of the sporoderm) were found. Most of the studied species have a low percentage of deformed, underdeveloped, morphologically sterile, and atypical pollen grains. The number of deformed pollen grains differs between taxa. It can vary from single grains (most of the studied samples) to 56% in Potentilla pedata. However, its plants from the mud volcano produce less deformed pollen than the control ones (88%). This is most likely a feature of the taxon, rather than the influence of growing conditions.

Ontogenetical development of branchial chambers of Litopenaeus vannamei (Boone, 1931) and theirinvolvement in osmoregulation: ionocytes and Na+/K+-ATPase.

Branchial chambers constitute the main osmoregulatory site in almost all decapod crustaceans. However, few studies have been devoted to elucidate the cellular function of specific cells in every osmoregulatory structure of the branchial chambers. In decapod crustaceans, it is well-known that the osmoregulatory function is localized in specific structures that progressively specialize from early developmental stages while specific molecular mechanisms occur. In this study, we found that although the structures developed progressively during the larval and postlarval stages, before reaching juvenile or adult morphology, the osmoregulatory capabilities of Litopenaeus vannamei were gradually established only during the development of branchiostegites and epipodites, but not gills. The cellular structures of the branchial chambers observed during the larval phase do not present the typical ultrastructure of ionocytes, neither Na+/K+-ATPase expression, likely indicating that pleura, branchiostegites, or bud gills do not participate in osmoregulation. During early postlarval stages, the lack of Na+/K+-ATPase immunoreactivity of the ionocytes from the branchiostegites and epipodites suggests that they are immature ionocytes (ionocytes type I). It could be inferred from IIF and TEM results that epipodites and branchiostegites are involved in iono-osmoregulation from PL15, while gills and pleura do not participate in this function.

Ультраструктура сперматозоидов у подростков с варикоцеле.

Introduction. In modern reproductive medicine, increasingly attention is paid to pathospermia caused by the formation of oxidative stress, one of the causes of which is varicocele, leading to the formation of hyperthermia and testicular ischemia. The excess of reactive oxygen species, free radicals in the ejaculate during oxidative stress can provoke damage to spermatozoa at the subcellular level, leading not only to DNA fragmentation, but also damage to the acrosome, mitochondria, and increased apoptosis. The purpose of the study: to assess the ultrastructure of spermatozoa in adolescents, to identify changes in spermatozoa characteristic of varicocele. Material and methods. Electron microscopic examination of spermatozoa was performed on adolescents aged 17 years; 100 adolescents were diagnosed with left-sided varicocele; 30 teenagers without varicocele made up the comparison group. Results and discussion. In adolescence, the typical ultrastructure of spermatozoa is most often detected. In adolescents with varicocele, damage to chromatin, acrosome, mitochonria was more often detected, but no statistically significant differences were found compared to adolescents without varicocele. Conclusions. There were no specific changes in the structure of spermatozoa characteristic of varicocele.

Nanostructure and Nanomechanics of Prorocentrum donghaiense and Their Changes Under Nitrogen Limitation by Atomic Force Microscopy

In this paper, atomic force microscopy was employed to study the harmful algal bloom species Prorocentrum donghaiense for the first time. Cells were immobilized in pores of polycarbonate membrane to keep moist and to acquire images of P. donghaiense at different scales. Typical ultrastructures, such as knob-like spines and valve pores, were observed on cell surfaces. These structures had similar characteristics to those observed on scanning electron microscopy images. Moreover, the height and spacing of typical nanostructure, and nanomechanical parameters such as adhesion and elasticity, were also quantified by AFM. Additionally, the changes in cell surface nanostructure and nanomechanical characteristics under nitrogen limitation were further studied. Compared with the cells under normal culture conditions, the cell surface roughness and adhesion decreased, and the elastic modulus increased for cells under nitrogen limitation. Potential changes in the ability of P. donghaiense cells to perform normal physiological functions are reflected by changes in cell surface parameters, including cell surface roughness, cell surface adhesion, and cell surface elasticity. The results of this study reveal how P. donghaiense responds to changes in the external environment under approximately physiological conditions from the perspective of changes in cell surface nanostructures and nanomechanical characteristics and provide a new understanding of its cell biology.

Confocal Laser and Electron Microscopic Investigation of Gap Junctions in Anaplastic Astrocytomas.

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localisation and ultrastructure of gap junctions in samples of malignant brain tumour (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumour fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated and embedded in epoxy resin by a plane-parallel method. Ultra-thin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localised around or at the intersection of small processes containing GFAP. Electron microscopy of the tumour samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumour cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100μm2. Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.

Maternal selenium deficiency suppresses proliferation, induces autophagy dysfunction and apoptosis in the placenta of mice.

Selenium deficiency is thought to be associated with the occurrence of gestational complications. However, the underlying mechanism of selenium deficiency impairs placental function remains unclear. In this study, female mice were separately supplemented with a Se-deficient (0.02 mg/kg Se) or control diet (0.2 mg/kg Se) for 12 weeks before mating and throughout gestation. Maternal liver and placentas were collected at embryonic day 15.5 and analyzed for Se content. Oxidative stress status, proliferation capability, autophagy, and apoptosis of the placenta were determined. We found that maternal selenium deficiency decreased placental Se concentration and some antioxidant selenoproteins expressions. The concentrations of catalase and glutathione in selenium-deficient placentas were reduced, along with an increase in hydrogen peroxide (H2O2) content. Selenium deficiency inhibited the expression of proliferating cell nuclear antigen. Autophagosomes, autophagolysosomes, and upregulation of autophagy-related protein microtubule-associated protein 1 light chain 3 alpha II (LC3B), Beclin1, PTEN-induced putative kinase 1 (PINK1), and Parkin were found in the selenium-deficient trophoblasts. Autophagic substrate p62/sequestosome 1 was surprisingly increased, indicating autophagy flux dysfunction. Selenium deficiency increased expressions of B cell leukemia/lymphoma 2 associated X protein (Bax), cleaved caspase-9/-3, and decreased the B cell leukemia/lymphoma 2 (Bcl2) level. Moreover, typical apoptotic ultrastructure and apoptosis-positive cells were observed in the selenium-deficient placenta. Our results suggested that maternal selenium deficiency impaired placental proliferation, induced autophagy dysfunction and apoptosis via increasing oxidative stress, and the Akt/mechanistic target of rapamycin (mTOR) pathway involved in this process. This study revealed a novel mechanism by which maternal selenium deficiency caused impairment of the placenta.

Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. N/A.

Paediatric knee anterolateral capsule does not contain a distinct ligament: analysis of histology, immunohistochemistry and gene expression

ObjectivesThe presence of a discrete ligament within the knee anterolateral capsule (ALC) is controversial. Tendons and ligaments have typical collagens, ultrastructure, transcription factors and proteins. However, these characteristics have not been investigated in paediatric ALC. The purpose of this study was to characterise the paediatric ALC in terms of tissue ultrastructure and cellular expression of ligament markers scleraxis (SCX)—a basic helix-loop-helix transcription factor—and the downstream transmembrane glycoprotein tenomodulin (TNMD), as compared with the paediatric lateral collateral ligament (LCL) and paediatric quadriceps tendon (QT). We hypothesised that, in comparison to the LCL and QT, the ALC would possess poor collagen orientation and reduced SCX and TNMD expression.Methods15 paediatric ALCs (age 6.3±3.3 years), 5 paediatric LCLs (age 3.4±1.3 years) and 5 paediatric QTs (age 2.0±1.2 years) from fresh cadaveric knees were used in this study. Fresh-frozen samples from each region were cryosectioned and then stained with H&E to evaluate collagen alignment and cell morphology. Expression of SCX and TNMD was determined by gene expression analysis and immunohistochemistry.ResultsThe histological sections of the paediatric LCL and QT showed well-organised, dense collagenous tissue fibres with elongated fibroblasts, while the ALC showed more random collagen orientation without clear cellular directionality. The aspect ratio of cells in the ALC was significantly lower than that of the LCL and QT (p<0.0001 and p<0.0001, respectively). The normalised distribution curve of the inclination angles of the nuclei in the ALC was more broadly distributed than that of the LCL or QT, indicating random cell alignment in the ALC. SCX immunostaining was apparent in the paediatric LCL within regions of aligned fibres, while the comparatively disorganised structure of the ALC was negative for SCX. The paediatric LCL also stained positive for TNMD, while the ALC was only sparsely positive for this tendon/ligament cell-surface molecule. Relative gene expression of SCX and TNMD were higher in the LCL and QT than in the ALC.ConclusionIn this study, a distinct ligament could not be discerned in the ALC based on histology, immunohistochemistry and gene expression analysis.Level of evidenceControlled laboratory study.

Extracellular lipids of Camelina sativa: Characterization of cutin and suberin reveals typical polyester monomers and unusual dicarboxylic fatty acids

Camelina sativa is relatively drought tolerant and requires less fertilizer than other oilseed crops. Various lipid- and phenolic-based extracellular barriers of plants help to protect them against biotic and abiotic stresses. These barriers, which consist of solvent-insoluble polymeric frameworks and solvent-extractable waxes, include the cuticle of aerial plant surfaces and suberized cell walls found, for example, in periderms and seed coats. Cutin, the polymeric matrix of the cuticle, and the aliphatic domain of suberin are fatty acid- and glycerol-based polyesters. These polyesters were investigated by base-catalyzed transesterification of C. sativa aerial and underground delipidated tissues followed by gas chromatographic analysis of the released monomer mixtures. Seed coat and root suberin had similar compositions, with 18-hydroxyoctadecenoic and 1,18-octadecenedioic fatty acids being the dominant species. Root suberin presented a typical lamellar ultrastructure, but seed coats showed almost imperceptible, faint dark bands. Leaf and stem lipid polyesters were composed of fatty acids (FA), 1,ω-dicarboxylic fatty acids (DCA), ω-hydroxy fatty acids (HFA) and hydroxycinnamic acids (HCA). Dihydroxypalmitic acid (DHP) and caffeic acid were the major constituents of leaf cutin, whereas stem cutin presented similar molar proportions in several monomers across the four classes. Unlike the leaf cuticle, the C. sativa stem cuticle presented lamellar structure by transmission electron microscopy. Flower cutin was dominated by DHP, did not contain aromatics, and presented substantial amounts (>30%) of hydroxylated 1,ω-dicarboxylic acids. We found striking differences between the lipid polyester monomer compositions of aerial tissues of C. sativa and that of its close relatives Arabidopsis thaliana and Brassica napus.

Study on the ability of type I collagen bioactive nanomaterial scaffold to repair spontaneous cartilage injury

Objective: Natural bone tissue can be considered as a complex composed of type I collagen and light-based apatite with low crystallinity. Choosing type I collagen and light-based apatite as bone repair materials can realise the biomimetic of natural bone. Chitosan is easy to mix with collagen, and the interaction of the two at the molecular level can make the complex of the two more stable than any single component. However, due to the poor mechanical properties of collagen and chitosan, the high swelling degree of collagen, the greater brittleness of light-based apatite, and the incompatibility with organics, the high swelling degree and the rapid degradation of this material limit the use of similar materials in bone tissue engineering. In this paper, the ultrastructure of type I collagen scaffolds is analysed; goat knee cartilage defect models are established using green goats as experimental animals. Fifteen 12-month-old goats are randomly divided into three groups, each with 10 knees, and group A is blank, the control group, group B is a simple collagen I collagen double-layer scaffold group, and group C is a type I collagen double-layer scaffold combined with MSCs transplantation group, which is treated according to the experimental group; materials are taken at 6 weeks and 24 weeks after the operation gross observation and histological scores were used to evaluate the repair effect of each group. Successfully established goat knee cartilage defect model and type I double-layer collagen scaffold composite autologous bone marrow mesenchymal stem cells (BMSCs) implantation technology. At 24 weeks postoperative MRI, there was no effusion in the articular cavity and no synovial hyperplasia. The repair tissue in the experimental group had no difference in signal from normal cartilage; the repair tissue was observed to be flat with normal cartilage and similar in colour to normal cartilage. Histology confirmed the repaired tissue as hyaline cartilage. The Waitangi score in the experimental group was 2.65 ± 1.36, which was significantly better than that in the stent group alone (7.04 ± 2.14) and the blank control group (10.87 ± 3.51). The blank control group and the stent group had poor repair effect.

Differentiation of Human Parthenogenetic Embryonic Stem Cells into Functional Hepatocyte-like Cells

ABSTRACT Stem cell and tissue engineering-based therapies for acute liver failure (ALF) have been limited by the lack of an optimal cell source. We aimed to determine the suitability of human parthenogenetic embryonic stem cells (hPESCs) for the development of strategies to treat ALF. We studied the ability of human parthenogenetic embryonic stem cells (hPESCs) with high whole-genome SNP homozygosity, which were obtained by natural activation during in vitro fertilization (IVF), to differentiate into functional hepatocyte-like cells in vitro by monolayer plane orientation. hPESCs were induced on a single-layer flat plate for 21 d in complete medium with the inducers activin A, FGF-4, BMP-2, HGF, OSM, DEX, and B27. Polygonal cell morphology and binuclear cells were observed after 21 d of induction by using an inverted microscope. RT-qPCR results showed that the levels of hepatocyte-specific genes such as AFP, ALB, HNF4a, CYP3A4, SLCO1B3, and ABCC2 significantly increased after induction. Immunocytochemical assay showed CK18 and Hepa expression in the induced cells. Indocyanine green (ICG) staining showed that the cells had the ability to absorb and metabolize dyes. Detection of marker proteins and urea in cell culture supernatants showed that the cells obtained after 21 d of induction had synthetic and secretory functions. The typical ultrastructure of liver cells was observed using TEM after 21 d of induction. The results indicate that naturally activated hPESCs can be induced to differentiate into hepatocellular cells by monolayer planar induction.

The brain structure of selected trypanorhynch tapeworms.

The brain architecture in four species of tapeworms from the order Trypanorhyncha has been studied. In all species, the brain consists of paired anterior and lateral lobes, and an unpaired central lobe. The anterior lobes connect by dorsal and ventral semicircular commissures; the central and lateral lobes connect by a median and an X-shaped crisscross commissure. In the center of the brain, five well-developed compact neuropils are present. The brain occupies a medial position in the scolex pars bothrialis. The ventral excretory vessels are situated outside the lateral lobes of the brain; the dorsal excretory vessels are located inside the brain and dorsal to the median commissure. The brain gives rize four anterior proboscis nerves and four posterior bulbar nerves with myelinated giant axons (GAs). The cell bodies of the GAs are located within the X-commissure and in the bulbar nerves. Highly developed serotonergic neuropils are present in the anterior and lateral lobes; numerous 5-HT neurons are found in the brain lobes including the central unpaired lobe. The X-cross commissure consists of the α-tub-immunoreactive and 5-HT-IR neurites. Eight ultrastructural types of neurons were found in the brain of the three species investigated. In addition, different types of synapses were present in the neuropils. Glial cells ensheath the brain lobes, the neuropils, the GAs, and the bulbar nerves. Glia cell processes form complex branching patterns of thin cytoplasmic sheets sandwiched between adjacent neural processes and filling the space between neurons. Multilayer myelin-like envelopes and a mesaxon-like structure have been found in Trypanorhyncha nervous system. We compared the brain architecture of Trypanorhyncha with that of an early basal cestode taxon, that is, Diphyllobothriidea, and present a hypothesis about the homology of the anterior brain lobes in order Trypanorhyncha; and the lateral lobes and median commissure are homologous brain structures within Eucestoda.

Caproicibacter fermentans gen. nov., sp. nov., a new caproate-producing bacterium and emended description of the genus Caproiciproducens.

A strictly anaerobic bacterial strain designated EA1T was isolated from an enrichment culture inoculated with biogas reactor content. Cells of strain EA1T are spore-forming rods (1-3×0.4-0.8 µm) and stain Gram-negative, albeit they possess a Gram-positive type of cell-wall ultrastructure. Growth of strain EA1T was observed at 30 and 37 °C and within a pH range of pH 5-9. The major components recovered in the fatty acid fraction were C14:0, C16:0, C16:0 DMA (dimethyl acetal) and C16:1 ω7c. Strain EA1T fermented several mono- and disaccharides. Metabolic end products from fructose were acetate, butyrate, caproate and lactate. Furthermore, ethanol, CO2 and H2 were identified as products. The genome consists of a chromosome (3.9 Mbp) with 3797 predicted protein-encoding genes and a G+C content of 51.25 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EA1T represents a novel taxon within the family Oscillospiraceae. The most closely related type strains of EA1T, based on 16S rRNA gene sequence identity, are Caproiciproducens galactitolivorans BS-1T (94.9 %), [Clostridium] leptum DSM 753T (93.8 %), [Clostridium] sporosphaeroides DSM 1294T (91.7 %) and Ruminococcus bromii ATCC 27255T (91.0 %). Further phenotypic characteristics of strain EA1T differentiate it from related, validly described bacterial species. Strain EA1T represents a novel genus and novel species within the family Oscillospiraceae. The proposed name is Caproicibacter fermentans gen. nov., sp. nov. The type strain is EA1T (DSM 107079T=JCM 33110T).

A Widely Distributed Thraustochytrid Parasite of Diatoms Isolated from the Arctic Represents a gen. and sp. nov.

A unicellular, heterotrophic, eukaryotic parasite was isolated from nearshore Arctic marine sediment in association with the diatom Pleurosigma sp. The parasite possessed ectoplasmic threads that could penetrate diatom frustules. Healthy and reproducing Pleurosigma cultures would begin to collapse within a week following the introduction of this parasite. The parasite (2-10μm diameter) could reproduce epibiotically with biflagellate zoospores, as well as binary division inside and outside the diatom host. While the parasite grew, diatom intracellular content disappeared. Evaluation of electron micrographs from co-cultures revealed the presence of hollow tubular processes and amorphic cells that could transcend the diatom frustule, generally at the girdle band, as well as typical thraustochytrid ultrastructure, such as the presence of bothrosomes. After nucleotide extraction, amplification, and cloning, database queries of DNA revealed closest molecular affinity to environmental thraustochytrid clone sequences. Testing of phylogenetic hypotheses consistently grouped this unknown parasite within the Thraustochytriidae on a distinct branch within the environmental sequence clade Lab19. Reclassification of Arctic high-throughput sequencing data, with appended reference datasets that included this diatom parasite, indicated that the majority of thraustochytrid sequences, previously binned as unclassifiable stramenopiles, are allied to this new isolate. Based on the combined information acquired from electron microscopy, life history, and phylogenetic testing, this unknown isolate is described as a novel species and genus.

Elucidating the biomineralization of low-temperature hydrothermal precipitates with varying Fe, Si contents: Indication from ultrastructure and microbiological analyses

Fe, Si-rich low-temperature hydrothermal precipitates, typical hydrothermal products of low-temperature diffuse flow, have been found widely on the seafloor. It is believed that microorganisms play an important role in the formation of Fe, Si-rich low-temperature precipitates, yet, microbial biomineralization within these precipitates has not been well understood. In this study, we characterized different hydrothermal precipitates collected from the Lau Basin of the western Pacific Ocean, using an integrated mineralogical, geochemical, ultrastructural and microbiological approach. Three types of Fe, Si-rich precipitates were identified: Fe-rich, Fe–Si-rich and Si-rich, consisting of amorphous or poor crystalline minerals, from the two-line ferrihydrite to opal-A. Geochemical indices suggest that the precipitates are typical low-temperature hydrothermal origin, precipitated from mixing, at various extent, of hydrothermal fluid and seawater. All three types of Fe, Si-rich precipitates show abundant and distinctive biogenic ultrastructures, indicating different biomineralization experiences. The Fe-rich precipitates dominantly exhibited the typical Fe-enriched ultrastructures produced by putative iron-oxidizing bacteria (FeOB), suggesting the prevalence of Fe-oxidation catalyzed by FeOB in its formation process. Similar FeOB-catalyzed biomineralization process also happened in Fe–Si-rich precipitates with the abundant occurrence of FeOB-induced ultrastructures. By contrast, the Si-enriched architectures, encompassing mycelium-like networks, became absolutely prevailing within Si-rich precipitates. Based on their indicative morphologies, mycelium-like networks were ascribed as silicified actinobacteria or fungi, suggesting that silicification induced by actinobacteria or fungi dominated the biomineralization process. Microbiological analysis revealed the existence of a series of diverse, heterogeneous microbial populations within different Fe, Si-rich precipitates. However, no putative FeOB lineages were detected in the Fe-rich and Fe–Si-rich precipitates. Taken together, our results indicate that different biomineralization processes induced by different microbial lineages contributed to the formation of the three different types of Fe, Si-rich precipitates and the distinct environmental conditions existed for heterogeneous microorganisms within these different Fe, Si-rich precipitates.

TMIC-27. GLUTAMATERGIC NEURON-GLIOMA SYNAPSES DRIVE BRAIN TUMOUR PROGRESSION

Abstract A network of communicating tumour cells established by tumour microtubes (TMs) is supposed to mediate relevant aspects of progression and resistance of incurable gliomas. Moreover, neuronal activity has been shown to foster malignant behavior of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here, we report an unexpected direct communication channel between neurons and glioma cells in multiple disease models as well as in astrocytomas and glioblastomas (GBs) of adult patients: functional bona fide chemical synapses formed between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses (NGS) show a typical synaptic ultrastructure, are located on TM networks, and produce depolarizing postsynaptic currents mediated by glutamate receptors of the AMPA subtype. AMPA-type glutamate receptors (AMPAR) are expressed by a molecularly and morphologically distinct subpopulation of network-integrated glioma cells. Increased neuronal activity under epileptic conditions ex vivo or neuronal optogenetic stimulation in vivo enhanced, while general anesthesia diminished synchronized calcium transients in TM-connected glioma networks. Accordingly, anesthesia reduced invasiveness of TM-positive tumour cells in mice. Genetic perturbation of AMPAR or chronic AMPAR inhibition by perampanel decreased glioma invasion and proliferation in mice and deletion of GluRII in Drosophila glioma increased survival. These findings reveal a hitherto unappreciated direct synaptic communication between neurons and glioma cells that appears relevant for brain tumour biology, implying new avenues for glioma treatment.