Visualization of Bacillus subtilis spore structure and germination using quick-freeze deep-etch electron microscopy.

Bacterial spores, known for their complex and resilient structures, have been the focus of visualization using various methodologies. In this study, we applied quick-freeze and replica electron microscopy techniques, allowing observation of Bacillus subtilis spores in high-contrast and three-dimensional detail. This method facilitated visualization of the spore structure with enhanced resolution and provided new insights into the spores and their germination processes. We identified and described five distinct structures: (i) hair-like structures on the spore surface, (ii) spike formation on the surface of lysozyme-treated spores, (iii) the fractured appearance of the spore cortex during germination, (iv) potential connections between small vesicles and the core membrane and (v) the evolving surface structure of nascent vegetative cells during germination.

A comparison of teleost rodlet cells with apicomplexan cells.

Rodlet cells are unique pear-shaped cells found primarily in the epithelium of the teleost fishes. The rodlet cell was first identified by Thèlohan in 1892 who named it Rhabdospora thelohani as it was believed to be a protozoan parasite of the phylum Apicomplexa. The rodlet cell as parasite paradigm persisted for several decades afterwards but has since faded in the last 20 years or so. The rodlet cell is now generally believed to be an immune cell, functioning as an early responder to parasite intrusion. This short review makes a detailed comparison of apicomplexan structure and behavior with that of the rodlet cell to further strengthen the argument against a parasitic nature for the fish cell. It is then proposed that apical microvilli of the rodlet cell serve as a mechanical trigger for rodlet discharge as possible defense against larger ectoparasites.

Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

Infection of endemic chub Squalius tenellus with the intestinal tapeworm Caryophyllaeus brachycollis (Cestoda): histopathology and ultrastructural surveys.

The endemic chub Squalius tenellus (Heckel, 1843) was introduced more than 100 years ago to Lake Blidinje (Bosnia-Herzegovina). Only 1 species of enteric helminth was found in a sample of 35 chubs, the tapeworm Caryophyllaeus brachycollis (Janiszewska, 1953). The paper includes histopathological investigation with identification of innate immune cells involved in host reaction and molecular data allowed correct designation of the cestode species. Of 35 specimens of chub examined, 21 (60%) harboured individuals of C. brachycollis and a total of 1619 tapeworms were counted, the intensity of infection ranged from 1 to 390 worms per fish (46.2 ± 15.3, mean ± s.e.). Histopathological and ultrastructural investigations showed strict contact between the worm's body and the epithelia and increase in the number of mucous cells, rodlet cells among the epithelial cells. Within the tunica propria-submucosa, beneath the site of scolex attachment, numerous neutrophils and mast cells were noticed. This is the first study of the occurrence of C. brachycollis in chub from Lake Blidinje and on the response of the innate immune cells of S. tenellus to this tapeworm. Interestingly, in 3 very heavily infected chubs, perforation of the intestinal wall was documented; this is uncommon among cestodes which use fish as a definitive host.

Teleost innate immunity, an intricate game between immune cells and parasites of fish organs: who wins, who loses.

Fish, comprising over 27,000 species, represent the oldest vertebrate group and possess both innate and adaptive immune systems. The susceptibility of most wild fish to parasitic infections and related diseases is well-established. Among all vertebrates, the digestive tract creates a remarkably favorable and nutrient-rich environment, which, in turn, renders it susceptible to microparasites and macroparasites. Consequently, metazoan parasites emerge as important disease agents, impacting both wild and farmed fish and resulting in substantial economic losses. Given their status as pathogenic organisms, these parasites warrant considerable attention. Helminths, a general term encompassing worms, constitute one of the most important groups of metazoan parasites in fish. This group includes various species of platyhelminthes (digeneans, cestodes), nematodes, and acanthocephalans. In addition, myxozoans, microscopic metazoan endoparasites, are found in water-dwelling invertebrates and vertebrate hosts. It is worth noting that several innate immune cells within the fish alimentary canal and certain visceral organs (e.g., liver, spleen, and gonads) play active roles in the immune response against parasites. These immune cells include macrophages, neutrophils, rodlet cells, and mast cells also known as eosinophilic granular cells. At the site of intestinal infection, helminths often impact mucous cells number and alter mucus composition. This paper presents an overview of the state of the art on the occurrence and characteristics of innate immune cells in the digestive tract and other visceral organs in different fish-parasite systems. The data, coming especially from studies employed immunohistochemical, histopathological, and ultrastructural analyses, provide evidence supporting the involvement of teleost innate immune cells in modulating inflammatory responses to metazoan and protozoan parasitic infections.

Characterization of the structure and self-assembly of two distinct class IB hydrophobins.

Hydrophobins are small proteins secreted by fungi that accumulate at interfaces, modify surface hydrophobicity, and self-assemble into large amyloid-like structures. These unusual properties make hydrophobins an attractive target for commercial applications as emulsifiers and surface modifying agents. Hydrophobins have diverse sequences and tertiary structures, complicating attempts to characterize how they function. Here we describe the atomic resolution structure of the unusual hydrophobin SLH4 (86 aa, 8.4 kDa) and compare its function to another hydrophobin, SC16 (99 aa, 10.2 kDa). Despite containing only one charged residue, SLH4 has a similar structure to SC16 yet has strikingly different rodlet morphology, propensity to self-assemble, and preferred assembly conditions. Secondary structure analysis of both SC16 and SLH4 suggest that during rodlet formation residues in the first intercysteine loop undergo conformational changes. This work outlines a representative structure for class IB hydrophobins and illustrates how hydrophobin surface properties govern self-assembly, which provides context to rationally select hydrophobins for applications as surface modifiers. KEY POINTS: • The atomic-resolution structure of the hydrophobin SLH4 was determined using nuclear magnetic resonance spectroscopy. • The structure of SLH4 outlines a representative structure for class IB hydrophobins. • The assembly characteristics of SLH4 and SC16 are distinct, outlining how surface properties of hydrophobins influence their function.

The immunity function of rodlet cells in the intestine of Binni fish (Mesopotamichthys sharpeyi).

Objective: Rodlet cells produce secretions of glycoproteins in nature. This study investigated the microscopic morphology, histochemical and immunohistochemical reactions, and distribution of the rodlet cells in the gut of Binni fish (Mesopotamichthys sharpeyi). Materials and Methods: Thirty samples were obtained from the cranial, middle, and caudal portions of Binni intestine immediately after being euthanized, fixed in Bouin's solution for 18 h at 24°C, and had undergone routine histological processing, different conventional histochemical stains, and immunostaining with TNF-α and S100 protein antibody. Results: The intestine of Binni fish showed different stages of rodlet cells classified into three distinctive forms: vesicular, granular, and mature cells. Rodlet cells are poorly stained with hematoxylin and eosin. Their secretory granules have a weak positive reaction with periodic acid-Schiff (PAS) and Alcian blue (AB), and react positively to combined AB and PAS. Rodlet cells were stained lightly with Safranin O, observed pink in color by Giemsa stain, and showed reactivity to Masson's and Mallory trichrome stains. Rodlet cells were immunostained positively against TNF-α and S100 antibodies, indicating that they have an immune function. Conclusions: Rodlet cells, with their neutral glycoprotein secretions, play a crucial role in the immunity of Binni fish intestine.

Histological and Chemical Analysis of Heavy Metals in Kidney and Gills of Boops boops: Melanomacrophages Centers and Rodlet Cells as Environmental Biomarkers.

Industrialization has resulted in a massive increase in garbage output, which is frequently discharged or stored in waterways like rivers and seas. Due to their toxicity, durability, bioaccumulation, and biomagnification, heavy metals (such as mercury, cadmium, and lead) have been identified as strong biological poisons. Their presence in the aquatic environment has the potential to affect water quality parameters and aquatic life in general. Teleosts' histopathology provides a sensitive indicator of pollutant-induced stress, because their organs have a central role in the transformation of different active chemical compounds in the aquatic environment. In particular, the gills, kidneys, and liver are placed at the center of toxicological studies. The purpose of this study is to examine the morphological changes caused by heavy metals in the kidney and gills of Boops boops, with a focus on melanomacrophages centers (MMCs) and rodlet cells (RCs) as environmental biomarkers, using histological and histochemical stainings (hematoxylin/eosin, Van Gieson trichrome, Periodic Acid Schiff reaction, and Alcian Blue/PAS 2.5), and immunoperoxidase methods. Our findings show an increase of MMCs and RCs linked to higher exposure to heavy metals, confirming the role of these aggregates and cells as reliable biomarkers of potential aquatic environmental changes reflected in fish fauna. The cytological study of RCs and MMCs could be important in gaining a better understanding of the complicated immune systems of teleosts.

Rodlet cells in kidney of goldfish (Carassius auratus, Linnaeus 1758): A light and confocal microscopy study.

Rodlet cells (RCs) have always been an enigma for scientists. RCs have been given a variety of activities over the years, including ion transport, osmoregulation, and sensory function. These cells, presumably as members of the granulocyte line, are present only in teleosts and play a role in the innate immune response. RCs are migratory cells found in a variety of organs, including skin, vascular, digestive, uropoietic, reproductive, and respiratory systems, and present distinct physical properties that make them easily recognizable in tissues and organs. The development of RCs can be divided into four stages: granular, transitional, mature, and ruptured, having different morphological characteristics. Our study aims to characterize the different stages of these cells by histomorphological and histochemical techniques. Furthermore, we characterized these cells at all stages with peroxidase and fluorescence immunohistochemical techniques using different antibodies: S100, tubulin, α-SMA, piscidin, and for the first time TLR-2. From our results, the immunoreactivity of these cells to the antibodies performed may confirm that RCs play a role in fish defense mechanisms, helping to expand the state of the art on immunology and immune cells of teleosts.

Effect of waterborne exposure to perfluorooctanoic acid on nephron and renal hemopoietic tissue of common carp Cyprinus carpio.

Per- and poly-fluoroalkyl substances (PFAS) are synthetic contaminants of global concern for environmental and public health. Perfluorooctanoic acid (PFOA) is an important PFAS, and considerable attention has been paid to its hepatotoxicity and reproductive and developmental impact, while potential nephrotoxic effects are largely ignored, especially in fish. This study documents the structural and ultrastructural effects on kidney of common carp Cyprinus carpio exposed to waterborne PFOA at an environmentally relevant concentration of 200 ng L-1 and at 2 mg L-1. Dilation of the glomeruli capillary bed, increased vesiculation in the proximal tubular segment, compromised mitochondria, apical blebbing, and sloughing of collecting duct cells occurred in exposed fish, primarily at 2 mg L-1. Perfluorooctanoic acid exposure resulted in higher numbers of rodlet cells (RC), putative immune cells exclusive to fish, mainly in the renal interstitium, than seen in controls, increased association with cells of myeloid lineage and modifications to ultrastructure. No differences in other cells of innate immunity were observed. Despite the absence of severe histological lesions, PFOA was shown to affect both nephron and hemopoietic interstitium at high concentration, raising concern of the impact on renal and immune function in fish. The response of RCs to PFOA concentration of 200 ng L-1 suggests a potential role as a biomarker of PFOA exposure.

Adsorption Kinetics and Self-Assembled Structures of Aspergillus oryzae Hydrophobin RolA on Hydrophobic and Charged Solid Surfaces.

Hydrophobins are small secreted amphipathic proteins ubiquitous among filamentous fungi. Hydrophobin RolA produced by Aspergillus oryzae attaches to solid surfaces, recruits polyesterase CutL1, and thus promotes hydrolysis of polyesters. Because the N-terminal region of RolA is involved in the interaction with CutL1, the orientation of RolA on the solid surface is important. However, the kinetic properties of RolA adsorption to solid surfaces with various chemical properties remain unclear, and RolA structures assembled after the attachment to surfaces are unknown. Using a quartz crystal microbalance (QCM), we analyzed the kinetic properties of RolA adsorption to the surfaces of QCM electrodes that had been chemically modified to become hydrophobic or charged. We also observed the assembled RolA structures on the surfaces by atomic force microscopy and performed molecular dynamics (MD) simulations of RolA adsorption to self-assembled monolayer (SAM)-modified surfaces. The RolA-surface interaction was considerably affected by the zeta potential of RolA, which was affected by pH. The interactions of RolA with the surface seemed to be involved in the self-assembly of RolA. Three types of self-assembled structures of RolA were observed: spherical, rod-like, and mesh-like. The kinetics of RolA adsorption and the structures formed depended on the amount of RolA adsorbed, chemical properties of the electrode surface, and the pH of the buffer. Adsorption of RolA to solid surfaces seemed to depend mainly on its hydrophobic interaction with the surfaces; this was supported by MD simulations, which suggested that hydrophobic Cys-Cys loops of RolA attached to all SAM-modified surfaces at all pH values. IMPORTANCE The adsorption kinetics of hydrophobins to solid surfaces and self-assembled structures formed by hydrophobin molecules have been studied mostly independently. In this report, we combined the kinetic analysis of hydrophobin RolA adsorption onto solid surfaces and observation of RolA self-assembly on these surfaces. Since RolA, whose isoelectric point is close to pH 4.0, showed higher affinity to the solid surfaces at pH 4.0 than at pH 7.0 or 10.0, the affinity of RolA to these surfaces depends mainly on hydrophobic interactions. Our combined analyses suggest that not only the adsorbed amount of RolA but also the chemical properties of the solid surfaces and the zeta potential of RolA affect the self-assembled RolA structures formed on these surfaces.

Rodlet cells, fish immune cells and a sentinel of parasitic harm in teleost organs.

Rodlet cells (RCs) are the enigmatic and distinctive pear-shaped cells had found in many tissues of marine and freshwater teleosts. They have a distinctive fibrous capsule or the cell cortex that envelopes conspicuous inclusions called rodlets, basally situated nucleus, and poorly developed mitochondria. The contraction of the cell cortex results in the expulsion of the cell contents through an apical opening. One hundred and thirty years since rodlet cells were first reported, many questions remain about their origin and a function. This review will present new evidence regarding the relationship between RCs and metazoan parasites, and a protozoan infecting organs of different fish species, and update the state of knowledge about the origin, structure and the function of these intriguing fish cells.

Microanalysis of the Intestinal Bulb of Grass Carp (Ctenopharyngodon Idella): Histological, Histochemical, Immunohistochemical, and Scanning Electron Microscopical Studies.

Cyprinid fishes have one of the simplest types of gastrointestinal tract among vertebrates. Those fish species do not possess a true stomach that is replaced by a simple dilatation at the anterior part of the intestine called the intestinal bulb. Twenty adult specimens of grass carp were used in the present study to identify the cellular components as well as the immunohistochemical and surface architectural characteristics of the intestinal bulb. The mucosa of the intestinal bulb shows numerous, deep longitudinal folds arranged in zigzagging-like patterns. The epithelium is composed mainly of absorptive columnar cells covered by microvilli and mucous goblet cells. Spindle-shaped enteroendocrine cells and some migratory immune cells such as intraepithelial lymphocytes and rodlet cells could be identified between the absorptive cells. The epithelium also contains many secretory granules and large numbers of vacuoles containing digestive enzymes mostly in the basal part. The immunohistochemistry revealed that CD20-positive B-lymphocytes are immunolocalized mainly in the connective tissue core lamina propria of the mucosal folds. However, CD3-immunopositive T-lymphocytes are highly concentrated in the lamina propria. In addition, intraepithelial T-lymphocytes expressed immunopositivity to CD3. The current study presented many types of immune cells and suggests their essential immunological role for the intestinal blub.

Characterization of the mbsA Gene Encoding a Putative APSES Transcription Factor in Aspergillus fumigatus.

The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ΔmbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ΔmbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ΔmbsA conidium. Comparative transcriptomic analyses revealed that the ΔmbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ΔmbsA mutant produced higher amount of GT, ΔmbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.

Functional analysis of the catalytic triad of the hAT-family transposase TcBuster.

TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid. Using a drug-resistant colony assay for transposon integration, we found that the D223N, D289N, and E589Q mutants of TcBuster transposase were inactive in human cells. We used a modified chromatin immunoprecipitation assay to determine that each mutant maintained binding to TcBuster transposon inverted repeat elements. Although the catalytic mutants retained their transposon binding properties, mutants displayed altered expression and localization in human cells. None of the catalytic mutants formed characteristic TcBuster transposase rodlet structures, and the D223N and D289N mutants were not able to be detected by immunofluorescence microscopy. Immunoblot analysis demonstrated that the E589Q mutant is less abundant than wild-type TcBuster transposase. Cells transfected with either TcBuster or TcBuster-E589Q transposase were imaged by structured illumination microscopy to quantify differences in the length of the transposase rodlets. The average length of the TcBuster transposase rodlets (N = 39) was 3.284 µm while the E589Q rodlets (N = 33) averaged 1.157 µm (p < 0.0001; t-test). The catalytic triad mutations decreased overall protein levels and disrupted transposase rodlet formation while nuclear localization and DNA binding to the inverted repeat elements were maintained. Our results may have broader implications for the overproduction inhibition phenomenon observed for DNA transposons.

Migratory Activities and Stemness Properties of Rodlet Cells.

The current study aimed to characterize different stages of rodlet cells using light microscopy, immunohistochemistry, and transmission electron microscopy. Granular rodlet cells have a distinct granular cytoplasm. Transitional rodlet cells had distinct capsules, and immature granules. Mature rodlet cells were pear-shaped and had elongated granules. Ruptured rodlet cells had a granular cytoplasm. The affinity of rodlet cells for different histochemical techniques was detected. Immunohistochemical analysis of rodlet cells for stem cell markers such as CD117, CD34, proliferation marker, proliferating cell nuclear antigen (PCNA), endopeptidase activity; matrix metalloproteinase-9 (MPP-9) and the angiogenic factor; vascular endothelial growth factor (VEGF) was investigated. All stages of rodlet cells were expressed CD117. However, the ruptured stage was CD117-negative. The granular, transitional, and mature stages had strong CD34 immunoaffinity, while the ruptured rodlet cells were CD34-negative. The most potent immunoreactivity for PCNA was the granular rodlet cells. The transitional cells exhibited less immunoreactivity, while mature rodlet cells had no immunoaffinity for PCNA. All stages of rodlet cells had high enzyme activity as indicated by Acridine orange and exhibited strong MPP-9 immunoaffinity. VEGF is mostly expressed by granular, transitional, and mature rodlet cells. In conclusion, rodlet cells relatively had stemness properties, endopeptidase activity, express a proliferation marker, and angiogenic factors. We suggest a potential role of rodlet cells in immune defense.

Functional Role of the Rodlet Cell and Macrophage in Neural Protection of the Olfactory Neuroepithelium in a Teleostean: Gobiid (Pseudapocryptes lanceolatus [Bloch and Schneider, 1801]): An Ultrastructural Study.

BACKGROUND: Neural protection of the olfactory epithelium against pathogenic invasion is still hardly addressed in fish chemosensory research. AIMS AND OBJECTIVES: The ultrastructural detail on the rodlet cell and macrophage has been studied within the olfactory neuroepithelium of Pseudapocryptes lanceolatus to correlate their role in the neural protection of the chemosensory system. MATERIALS AND METHODS: The cellular structures were examined under light microscope (LM: Primo Star; Carl Zeiss Microscopy, GmbH, Germany) and transmission electron microscope (Morgagni 268D). RESULTS: Three distinct stages of the rodlet cell (viz., immature, mature, and degenerative) and macrophages have been characterized at the various depths of the olfactory neuroepithelium in P. lanceolatus. The cytoarchitecture of degenerative rodlet cell indicates holocrine mode of secretion against pathogenic invasion into the nasal cavity. Macrophages possess prominent pseudopodia, extending toward invading pathogens. The interaction between macrophage and invading pathogens implicates the role of macrophage as a scavenger to eliminate the pathogens by phagocytosis from the neuroepithelial system. CONCLUSION: This study denotes a significant difference in the mode of action of rodlet cell and macrophages, but they are commonly involved in cell-mediated nonspecific immune response against the invading pathogens.

Development of molecular and histological methods to evaluate stress oxidative biomarkers in sea bass (Dicentrarchus labrax).

In aquaculture, fish species may experience stressful episodes caused by poor farming conditions. The exponential increase of global aquaculture has raised the number of research studies aimed at demonstrating the sensitivity of aquatic animals in confined environments. The development of a real-time PCR and immunohistochemistry methods were investigated to evaluate the presence, localization, and quantity of biomarkers of oxidative stress in European sea bass (Dicentrarchus labrax). In particular, stress tests such as manipulation and temperature changes were conducted through molecular methods to identify the expression level of heat shock protein 70 (HSP70) in stressed animals compared with a control group. The immunohistochemical technique was also applied to locate and study the trends-levels of nitrotyrosine (NT), heat shock protein 70 (HSP70), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in different tissues from stressed animals and control group. The presence of the rodlet cell (RCs) was evaluated by histology in both a control and stressed group. Our results show that the real-time PCR method developed is specific for the evaluated target gene and that manipulation and temperature increase are strong stressors for animals. Relative quantification data revealed a gene expression increase of HSP70 in the stressed group of animals compared to the control group. The antibodies used for the immunohistochemical staining were efficient, and it was possible to appreciate the increase of immunoprecipitates in European sea bass either manipulated or stressed by temperature increase. The present study can be a starting point to allow the quantification of HSP70 and the identification of other stress biomarkers in D. labrax.

Microanalysis of Fish Ovarian Follicular Atresia: A Possible Synergic Action of Somatic and Immune Cells.

The present study describes in detail the morphological characteristics of the process of ovarian follicular atresia in Redbelly tilapia (Coptodon zillii) during the nonbreeding season using light and electron microscopy and immunohistochemistry. The follicular regression process was initiated with shrinkage and disintegration of the nuclear membrane of oocytes resulting in dispersing of chromatin within the ooplasm, followed by marked hyperplasia and hypertrophy of follicular and granulosa cells, which exhibited a strong phagocytic activity to engulf the liquefied yolk particles. Rodlet cells and granulocytes were recorded on the follicular wall and invaded the regressed follicles. Rodlet cells expressed a strong immunoreactivity to matrix metalloperoxidase (MMP-9) and α-smooth muscle actin, while neutrophils expressed a strong reactivity to Myeloperoxidase-3 (MPO). In the advanced stage of follicular atresia, the yolk was almost phagocytized and resorbed and the regressed follicle lost its integrity and appeared to be formed of a cellular mass of phagocytic cells. Transmission electron microscopy revealed the presence of neutrophils, eosinophils, and dendritic cells within the atretic follicle in between these phagocytic cells. Moreover, numerous lysosomes, granules, and phagosomes were observed within the cytoplasm of both phagocytic cells and granulocytes. Telocytes were also demonstrated within the highly thickened richly vascularized theca layer during the late stages of follicular atresia. Immunohistochemical staining for caspase-3 established the participation of apoptosis in the advanced stages of follicular regression. Immune cells, rodlet cells, and telocytes in combination with follicular cells play an essential role in follicular atresia. In conclusion, the present study provides a new evidence on the role of both somatic and immune cells in the phenomenon of ovarian follicular atresia in Redbelly tilapia (Coptodon zillii) during the nonbreeding season.

Formation of Amphipathic Amyloid Monolayers from Fungal Hydrophobin Proteins.

The fungal hydrophobins are small proteins that are able to self-assemble spontaneously into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic nanomaterials. One subgroup of this family, the class I hydrophobins, forms monolayers that are composed of extremely robust amyloid-like fibrils, called rodlets. Here, we describe the protocols for the production and purification of recombinant hydrophobins and oxidative refolding to a biologically active, soluble, monomeric form. We describe methods to trigger the self-assembly into the fibrillar rodlet state and techniques to characterize the physicochemical properties of the polymeric forms.