The nature and origin of nucleus‐like intracellular inclusions in Paleoproterozoic eukaryote microfossils

The well‐known debate on the nature and origin of intracellular inclusions (ICIs) in silicified microfossils from the early Neoproterozoic Bitter Springs Formation has recently been revived by reports of possible fossilized nuclei in phosphatized animal embryo‐like fossils from the Ediacaran Doushantuo Formation of South China. The revisitation of this discussion prompted a critical and comprehensive investigation of ICIs in some of the oldest indisputable eukaryote microfossils—the ornamented acritarchs Dictyosphaera delicata and Shuiyousphaeridium macroreticulatum from the Paleoproterozoic Ruyang Group of North China—using a suite of characterization approaches: scanning electron microscopy (SEM), transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB‐SEM). Although the Ruyang acritarchs must have had nuclei when alive, our data suggest that their ICIs represent neither fossilized nuclei nor taphonomically condensed cytoplasm. We instead propose that these ICIs likely represent biologically contracted and consolidated eukaryotic protoplasts (the combination of the nucleus, surrounding cytoplasm, and plasma membrane). As opposed to degradational contraction of prokaryotic cells within a mucoidal sheath—a model proposed to explain the Bitter Springs ICIs—our model implies that protoplast condensation in the Ruyang acritarchs was an in vivo biologically programmed response to adverse conditions in preparation for encystment. While the discovery of bona fide nuclei in Paleoproterozoic acritarchs would be a substantial landmark in our understanding of eukaryote evolution, the various processes (such as degradational and biological condensation of protoplasts) capable of producing nuclei‐mimicking structures require that interpretation of ICIs as fossilized nuclei be based on comprehensive investigations.     

There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator

Background

Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult configuration; this process is called 'inversion.' There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out?

Results

In this study, we investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri.

Conclusions

Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator.     

FINE STRUCTURE OF MISTLETOE POLLEN. IV. EURASIAN AND AUSTRALIAN VISCUM L. (VISCACEAE)

Fifteen Eurasian and Australian species of Viscum L. were examined by light, scanning, and transmission electron microscopy. Pollen characters divide the species into two groups, each correlated with differences in habit and inflorescence structure: Group I (12 spp.) characterized by psilate or slightly sculptured exines and a non-uniform ektexine pattern and Group II (3 spp.) possessing highly sculptured (echinate, rodlet) surfaces and uniform ektexine patterns. Within each of the groups, pollen characters divide the species into several subgroups. Among Group I, species V. nepalense, V. heyneanum and V. ovalifolium are particularly close. The Group I species, V. trilobatum, is placed in its own subgroup primarily because of its uniform ektexine pattern—a unique feature among Asian and Australian Viscum. Of the three Group II species, V. album and V. alniformosanae are palynologically almost indistinguishable. Pollen of the Group II V. cruciatum, though exomorphologically similar to V. album, is closest ultrastructurally to the Indian V. trilobatum. Overall, the most common and probably basic pollen characters among the Eurasian and Australian species include: subprolate, rounded convex almost spherical shapes; tricolporate apertures, and non-uniform sculpturing and ektexine patterns. Oblate-spheroidal or prolate-spheroidal shapes, prominent sculpturing, and a uniform ektexine pattern are derived characters largely restricted to the Eurasian and Asian Group II species (V. album, V. alniformosanae, V. cruciatum).     

Ultrastructural observations of the early and late stages of gorgonian coral (Junceella juncea) oocytes

The developmental oogenesis of gorgonian coral was investigated at the histological level. The objective of this study was to examine and improve the understanding of Junceella juncea oogenesis using ultrastructural methods, such as histological sectioning and transmission electron microscopy. At least three types of yolk materials were observed in this study: yolk body, lipid granules and cortical alveoli. Some of the complex yolk materials were encompassed by concentric or arched layers of smooth and rough endoplasmic reticulum and the Golgi complex in early stage oocytes. Different types of vesicles were found in both early and late stage oocytes and some granules could be seen inside the empty vesicles. This may be a possible method for elaborating complex yolk materials. Homogeneous yolks from different types of inclusions were abundant and the autosynthesis of yolk may be a major mechanism in J. juncea oocytes. This is the first report of the ultrastructural observation of oogenesis in gorgonian coral species using transmission electron microscopy. Our study obtained relatively detailed information at the ultrastructural level, and it provides an overview of the oocyte ultrastucture of the gorgonian coral J. juncea.     

Ultrastructural and molecular characterization of cyanobacterial symbionts in Dictyocoryne profunda (polycystine radiolaria)

Cyanobacterial symbionts were detected in the extracytoplasm of the polycystine radiolarian Dictyocoryne profunda Ehrenberg. The bacterial symbionts were observed as numerous spherical bodies ~0.5?C1.0???m in diameter under transmission electron microscopy. They were present in a very restricted location close to the periphery of the host radiolarian shell, adjacent to the central capsular wall. Several cells of them may have been in the process of cell division or just divided. The symbionts had thylakoid-like structures, which ran around the cell periphery in two or three concentric layers. Based on the small subunit ribosomal DNA (16S rDNA) phylogenetic analyses, the intracellular symbiotic bacteria grouped with cyanobacteria belonging to the genus Synechococcus. Three sequences, one from each of three specimens of D. profunda, collected in March/October 2009 and March 2010 from the East China Sea, were the same and branched within Synechococcus clade II, that is characterized by strains with low amounts of phycourobilin (PUB).     

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.     

Structural similarity between actin bundles from characean algal cells and sea urchin oocytes

The structure of actin bundles from internodal cells of Chara australis, an algal plant, was studied by electron microscopy of negatively stained specimens and optical diffraction. Gently prepared bundles revealed paracrystalline structures resembling the Mg²⁺-induced paracrystals of rabbit skeletal muscle actin (Hanson, 1968). In addition, the algal actin bundles sometimes had transverse striations at intervals of about 130 Å, as has been observed in actin bundles from sea urchin eggs (DeRosier et al., 1977; Spudich & Amos, 1979) and sea urchin coelomocytes (De Rosier & Edds, 1980; Otto & Bryan, 1981). This finding suggests that a common mechanism might be working in a variety of cells to organize actin filaments into functional bundles.     

Intracellular Biosynthesis and Removal of Copper Nanoparticles by Dead Biomass of Yeast Isolated from the Wastewater of a Mine in the Brazilian Amazonia

In this study was developed a natural process using a biological system for the biosynthesis of nanoparticles (NPs) and possible removal of copper from wastewater by dead biomass of the yeast Rhodotorula mucilaginosa. Dead and live biomass of Rhodotorula mucilaginosa was used to analyze the equilibrium and kinetics of copper biosorption by the yeast in function of the initial metal concentration, contact time, pH, temperature, agitation and inoculum volume. Dead biomass exhibited the highest biosorption capacity of copper, 26.2 mg g⁻¹, which was achieved within 60 min of contact, at pH 5.0, temperature of 30°C, and agitation speed of 150 rpm. The equilibrium data were best described by the Langmuir isotherm and Kinetic analysis indicated a pseudo-second-order model. The average size, morphology and location of NPs biosynthesized by the yeast were determined by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM). The shape of the intracellularly synthesized NPs was mainly spherical, with an average size of 10.5 nm. The X-ray photoelectron spectroscopy (XPS) analysis of the copper NPs confirmed the formation of metallic copper. The dead biomass of Rhodotorula mucilaginosa may be considered an efficiently bioprocess, being fast and low-cost to production of copper nanoparticles and also a probably nano-adsorbent of this metal ion in wastewater in bioremediation process.     

A peculiar mode of formation of the surface lining layer in the lungs of Salamandra salamandra

The pneumocytes of the larva of Salamandra salamandra contain numerous lamellar bodies and their precursors: electron-dense bodies at various stages of development. Both lamellar bodies and electron-dense bodies occur inside the fluid-filled lung. The former are spherical or bell-shaped and possess concentrically arranged smooth membranes, 8 nm thick; the latter have paracrystalline cores composed of alternately oriented clear and dark striations (3.6–3.9 nm and 2.6–3.6 nm, respectively). On all sides such cores separate membranes, which assume a concentric orientation. No tubular myelin was observed in any phase of the transformation of lamellar bodies and electron-dense bodies into the surface lining layer. Fixation of the lungs of adult individuals with tannic acid-containing fixative visualized the surface lining layer, but not tubular myelin.     

Mycocrystallization of gold ions by the fungus Cylindrocladium floridanum

The size and morphology determines the thermodynamic, physical and electronic properties of metal nanoparticles. The extracellular synthesis of gold nanoparticles by fungus, Cylindrocladium floridanum, which acts as a source of reducing and stabilizing agent has been described. The synthesized nanoparticles were characterized using techniques such as UV–Vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy, energy dispersive X-ray analysis (EDAX), and high-resolution transmission electron microscopy (HR-TEM). Based on the evidence of HR-TEM, the synthesized particles were found to be spherical with an average size of 19.05 nm. Powder XRD pattern proved the formation of (111)-oriented face-centered cubic crystals of metallic gold. This microbial approach by fungus for the green synthesis of spherical gold nanoparticles has many advantages such as economic viability, scaling up and environment friendliness.     

Synthesis and characterization of silver nanoparticles using Cynodon dactylon leaves and assessment of their antibacterial activity

Many methods of synthesizing silver nanoparticles (Ag-NPs) by reducing Ag⁺ ions using aqueous/organic extracts of various plants have been reported in the past, but the methods are rather slow. In this investigation, silver nanoparticles were quickly synthesized from aqueous silver nitrate through a simple method using leaf extract of a plant—Cynodon dactylon which served as reducing agent, while sunlight acted as a catalyst. The formation of Ag-NPs was indicated by gradual change in colour and pH and confirmed by ultraviolet–visible spectroscopy. The Ag-NPs showed a surface plasmon resonance at 451 nm. Based on the decrease in pH, a possible mechanism of the synthesis of Ag-NPs involving hydroxyl (OH^?) ions of polyphenols of the leaf extract is postulated. Ag-NPs having (111) and (200) crystal lattices were confirmed by X-ray diffraction. Scanning electron microscopy revealed the spherical nature of the Ag-NPs, while transmission electron microscopy showed that the nanoparticles were polydispersed with a size range of 8–10 nm. The synthesized Ag-NPs also demonstrated their antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhimurium.     

Phytosynthesis of silver nanoparticles using Mangifera indica flower extract as bioreductant and their broad-spectrum antibacterial activity

The present study focused on the evaluation of antibacterial property of silver nanoparticles (AgNPs) synthesized using mango flower extract. The morphology of the synthesized AgNPs was observed under transmission electron microscopy and the particles have shown spherical shape in the range of 10–20 nm. X-ray powder diffraction analysis confirmed the crystalline nature of the AgNPs. The atomic percentage of the Ag element in the nanoparticles was about 7.58% which is greater than the other elements present in the sample. The AgNPs showed extensive lethal effect on both Gram-positive (Staphylococcus sp.) and Gram-negative (Klebsiella sp., Pantoea agglomerans, and Rahnella sp.) bacteria. The extensive lethal effect of AgNPs against clinically important pathogens demonstrated that the mango flower mediated AgNPs could be applied as potential antibacterial agent to control the bacterial population in the respective industries.     

Nanoscale Diblock Copolymer Micelles: Characterizations and Estimation of the Effective Diffusion Coefficients of Biomolecules Release through Cylindrical Diffusion Model

Biomolecules have been widely investigated as potential therapeutics for various diseases. However their use is limited due to rapid degradation and poor cellular uptake in vitro and in vivo. To address this issue, we synthesized a new nano-carrier system comprising of cholic acid-polyethylenimine (CA-PEI) copolymer micelles, via carbodiimide-mediated coupling for the efficient delivery of small interfering ribonucleic acid (siRNA) and bovine serum albumin (BSA) as model protein. The mean particle size of siRNA- or BSA-loaded CA-PEI micelles ranged from 100–150 nm, with zeta potentials of +3-+11 mV, respectively. Atomic force, transmission electron and field emission scanning electron microscopy demonstrated that the micelles exhibited excellent spherical morphology. No significant morphology or size changes were observed in the CA-PEI micelles after siRNA and BSA loading. CA-PEI micelles exhibited sustained release profile, the effective diffusion coefficients were successfully estimated using a mathematically-derived cylindrical diffusion model and the release data of siRNA and BSA closely fitted into this model. High siRNA and BSA binding and loading efficiencies (95% and 70%, respectively) were observed for CA-PEI micelles. Stability studies demonstrated that siRNA and BSA integrity was maintained after loading and release. The CA-PEI micelles were non cytotoxic to V79 and DLD-1 cells, as shown by alamarBlue and LIVE/DEAD cell viability assays. RT-PCR study revealed that siRNA-loaded CA-PEI micelles suppressed the mRNA for ABCB1 gene. These results revealed the promising potential of CA-PEI micelles as a stable, safe, and versatile nano-carrier for siRNA and the model protein delivery.     

Effects of the bacterial algicide IRI-160AA on cellular morphology of harmful dinoflagellates

The algicide, IRI-160AA, induces mortality in dinoflagellates but not other species of algae, suggesting that a shared characteristic or feature renders this class of phytoplankton vulnerable to the algicide. In contrast to other eukaryotic species, the genome of dinoflagellates is stabilized by high concentrations of divalent cations and transition metals and contains large amounts of DNA with unusual base modifications. These distinctions set dinoflagellates apart from other phytoplankton and suggest that the nucleus may be a dinoflagellate-specific target for IRI-160AA. In this study, morphological and ultrastructural changes in three dinoflagellate species, Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum, were evaluated after short-term exposure to IRI-160AA using super resolution structured illumination microscopy (SR-SIM) and transmission electron microscopy (TEM). Exposure to the algicide resulted in cytoplasmic membrane blebbing, differing chloroplast morphologies, nuclear expansion, and chromosome expulsion and/or destabilization. TEM analysis showed that chromosomes of algicide-treated K. veneficum appeared electron dense with fibrous protrusions. In algicide-treated P. minimum and G. instriatum, chromosome decompaction occurred, while for P. minimum, nuclear expulsion was also observed for several cells. Results of this investigation demonstrate that exposure to the algicide destabilizes dinoflagellate chromosomes, although it was not clear if the nucleus was the primary target of the algicide or if the observed effects on chromosomal structure were due to downstream impacts. In all cases, changes in cellular morphology and ultrastructure were observed within two hours, suggesting that the algicide may be an effective and rapid approach to mitigate dinoflagellate blooms.     

Invasion of Spores of the Arbuscular Mycorrhizal Fungus Gigaspora decipiens by Burkholderia spp.

Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic Burkholderia spp., including B. vietnamiensis, B. cepacia, and B. pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens. Spore lysis assays revealed that all Burkholderia spp. tested were able to colonize the interior of G. decipiens spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B. vietnamiensis. Twelve percent of all spores were invaded by B. vietnamiensis, with an average of 1.5 × 10⁶ CFU recovered from individual infected spores. Of those spores inoculated with B. pseudomallei, 7% were invaded, with an average of 5.5 × 10⁵ CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G. decipiens and the attachment of bacteria. Burkholderia spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of Burkholderia spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.     

Antheridial dehiscence in ferns

We investigated the mechanism of antheridial dehiscence in ferns for the first time using fluorescence microscopy as well as scanning and transmission electron microscopy. The mechanism leading to antheridial dehiscence in Polystichum setiferum, Asplenium trichomanes and A. onopteris was found to depend on the different cellulose contents of the inner and outer walls of the ring cells detected with calcofluor white stain and the Thiéry test. The extremely low cellulose content of the ring cell walls facing spermatozoids made them less mechanically resilient than external wall cells. When the ring cells absorbed water they expanded only into the antheridial cavity, pushing the gametes against the cap cell, which detached from the ring cell below and enabled spermatozoid release. The newly released spermatozoids were spherical bodies covered in cellulose fibrils. The significance of cellulose fibrils could be to isolate the gametes from each other, to reinforce the electron transparent material and to protect the gamete from pressure created by the ring cells during release.     

Seed Epidermis Development and Histochemistry in Solanum melongena L. and S. violaceum Ort.

Structure, development and histochemistry of the seed epidermiswere studied inSolanum melongena L. andS. violaceum Ort. usinglight and scanning electron microscopy. The epidermal cellsat the endosperm mother cell stage of ovule development hadthickened outer periclinal walls, consisting of two layers,a thin inner layer, and a thick outer layer. The latter whichstained positively for pectic substances became further thickenedduring the course of seed development; more so inS. melongena.The inner layer of the outer periclinal wall also was thickenedby depositions of cellulose but remained comparatively thin.The development of the inner periclinal and anticlinal wallstook place by the uneven deposition of concentric layers. Thesesecondary wall thickenings which appeared as pyramids in transversesection stained for cellulose, lignin and pectin. Further unevensecondary thickenings near the outer part of the anticlinalwalls resulted in the formation of projections which were hair-or ribbon-like in appearance. InS. melongena, these projectionsprogressed only a short distance from the anticlinal wall. InS.violaceum, on the other hand, they grew much longer formingstriations on the inside of the outer periclinal wall. InS.melongena, partial removal of the outer periclinal wall by enzymeetching exposed to surface view a beaded appearance of the cellboundaries. Complete erosion of the outer periclinal wall revealedthe hair-like projections of the underlying anticlinal walls.InS. violaceum, enzyme treatment exposed the striations whichformed bridge-like structures over the curves in the anticlinalwalls. Solanum melongena ; Solanum violaceum; seed epidermis; seed structure; seed development; cell wall histochemistry; cell wall projections; cell wall striations     

Ultrastructure of the Acidophilic Aerobic Photosynthetic Bacterium Acidiphilium rubrum

The ultrastructure of cells of Acidiphilium rubrum, which is an acidophilic aerobic photosynthetic bacterium containing zinc-complexed bacteriochlorophyll a, was studied by electron microscopy with the rapid substitution technique. Thin-section electron microscopy indicated that any type of internal photosynthetic membranes was not present in this organism despite a relatively high content of the photopigment. The majority of cells had poly-β-hydroxybutyrate granules and electron-dense spherical bodies identified as being polyphosphate granules. When the organism was grown chemotrophically with 0.1% FeSO₄, it produced another group of electron-dense granules that were associated with the inner part of the cytoplasmic membrane. An energy-dispersive X-ray analysis showed that these membrane-bound, electron-dense granules contained iron. Received: 24 November 1999 / Accepted: 5 January 2000     

Microbial community associated with <Emphasis Type="Italic">Thioploca</Emphasis> sp. sheaths in the area of the posolsk bank methane seep,southern baikal

Bacterial mats formed by a colorless sulfur bacterium Thioploca sp. in the area of the Posolsk Bank cold methane seep (southern Baikal) were studied using electron microscopy and phylogenetic analysis. Morphologically the bacteria were identified as Thioploca ingrica. Confocal microscopy of DAPI-stained samples revealed numerous rod-shaped, filamentous, and spiral microorganisms in the sheaths, as well as inside and between the trichomes. Transmission electron microscopy revealed nonvacuolated bacteria and small cells without cell envelopes within the sheath. Bacteria with pronounced intracytoplasmic membranes characteristic of type I methanotrophs were observed at the outer side of the sheath. Based on analysis of the 16S rRNA gene sequences, the following phyla were identified in the sheath community: Bacteroidetes, Nitrospira, Chloroflexi, Planctomycetes, Verrucomicrobia, γ-, and δ-Proteobacteria, Euryarchaeota, Crenarchaeota, and Thaumarchaeota, as well as anammox bacteria. A hypothetical scheme of matter flows in the Lake Baikal bacterial mats was proposed based on the data on metabolism of the cultured homologues.