The red alga Tsunamia transpacifica (Stylonematophyceae) from plastic drift shows adaptation to its uncommon habitat in ultrastructure and soluble low molecular weight carbohydrate composition

The recently described red alga Tsunamia transpacifica (Stylonematophyceae) was previously isolated from plastic drift found at the pacific coast, but the natural habitat remains unknown. Here, we investigate ultrastructural details and the low molecular weight soluble carbohydrate composition to get further insight into the adaptation to this uncommon habitat. By means of high pressure freeze fixation, followed by freeze substitution, we could detect an up to 2-µm-thick cell wall surrounded by a distinct layer of extracellular polymeric substances (EPS), likely responsible for the adhering capacities of Tsunamia. The central position of the nucleus and multilobed parietal chloroplast, already observed by light microscopy, could be confirmed. The ultrastructure revealed large electron-dense bodies (EB) in the central cytoplasm, likely resembling degradation products of the chloroplast. Interestingly, these structures contained phosphorous and cobalt, and iron was found in smaller rounded electron-dense bodies by electron energy loss spectroscopy (EELS). Accumulation of these elements suggests a high biosorption activity of Tsunamia. Liquid chromatography-mass spectrometry (LC–MS) data showed the presence of two heterosides (floridoside and digeneaside) together with the polyol sorbitol, which are known as organic osmolytes and compatible solutes. Taken together, these are the first observations on ultrastructural details, element storage and accumulation of protective compounds are contributing to our understanding of the ultrastructural and osmotic solute basis for the ability of Tsunamia to thrive on plastic surfaces.

Evaluation of the Microstructure and the Electrochemical Properties of Ce0.8(1−x)Gd0.2(1−x)CuxO[1.9(1−x)+x] Electrolytes for IT-SOFCs

The influence of copper addition (0.5–2 mol%) on the crystal structure, densification microstructure, and electrochemical properties of Ce0.8Gd0.2O1.9 synthesized in a one-step sol–gel combustion synthesis route has been studied. It has been found that Cu is very active as sintering aids, with a significative reduction of GDC firing temperature. A reduction of 500 °C with a small amount of copper (0.5 mol%) was observed achieving dense bodies with considerable ionic conductivities. Rietveld refined was used to investigate the crystal structure while relative density and microstructural examination were performed in the sintering temperature range of 1000–1200 °C after dilatometer analysis. High dense bodies were fabricated at the lowest sintering temperature, which promotes the formation of Ce0.8(1−x)Gd0.2(1−x)CuxO[1.9(1−x)+x] solid solution and the absence of secondary phase Cu-rich or the segregation or copper at the grain boundary. As compared to the pure GDC an improvement of total conductivity was achieved with a maximum for the highest copper content of 2.23·10−3–9.19·10−2 S cm−1 in the temperature range of 200–800 °C.

Ultrastructure of Spermatozoa of Elaphe schrenckii (Reptilia, Squamata)

Elaphe schrenckii (Serpentes, Colubridae), a kind of large nonvenomous snakes and great significance to maintain the stability of ecosystem in China. We provide detailed descriptions of the sperm microstructure and ultrastructure of E. schrenckii, experimented by light microscope and transmission electron microscope. The spermatozoon of E. schrenckii is filiform and consists of head and tail regions. The cross-section of acrosomal vesicle is always rounded and divided into medulla inside and cortex outside. The ultrastructure of acrosome complex observed the unilateral ridge, the single perforatorium, the perforatorium base plate, the epinuclear lucent zone, the subacrosomal space and the nuclear fossa at the end of nucleus connect the neck region. The neck region is short with the stratified laminar structure and observed the distal centriole and the proximal centriole are perpendicular and both consisted of nine triplets. Midpiece is long and observed the extracellular microtubules, the multilaminar membranec, the mitochondria with the dense bodies discontinuity distribting, the fibrous sheath, and the axoneme. The principal piece is after the annulus with no mitochondrias and the end piece with no mitochondrias neither the fibrous sheath. Our study contrasted the spermatozoa ultrastructure of 8 species belong to 5 families and 6 genera and added the sperm measurement compare, summarized that three Colubridae snakes are more similar than others momentarily but some specific characteristics in E. schrenckii and proved that the ultrastructure of sperm related to phylogeny in some ways.

P4734Electron microscopy reveals evidence of perinuclear clustering of mitochondria in cardiac biopsy proven allograft rejection

Background Despite improved efficacy of immunosuppression therapy, allograft rejection continues to be a significant risk, especially early after transplantation. Endomyocardial biopsy (EMB) is the standard tool with a recognized role in the surveillance of posttransplant cardiac rejection and is based on optical microscopy analysis. However, this method presents technical limitations. Purpose In this work we focus on the analysis of new ultrastructural findings in cardiac biopsy specimens. Methods This study include heart transplanted patients from a single center who were referred for EMB as a scheduled routine screening. Participants were divided into 2 groups: patients transplanted without allograft rejection (n=5), and patients with biopsy-proven allograft rejection (n=5). Rejection episodes were assessed according to the International Society for Heart and Lung Transplantation (ISHLT) consensus report. Results We detected by electronic microscopy a significative increase in the number of mitochondria (p<0.0001) and dense bodies in the rejection group (p<0.05). But the most significative finding was the presence of local accumulations of mitochondria close to the nuclear envelope, pressing and shaping the morphology of this membrane in all rejection samples. We found these perinuclear clustering of mitochondria in a 68±27% of the total cardiac nucleus observed from rejection samples. We not observed this phenomenon in non-rejection samples, thus reflecting an excellent sensitivity and specificity. Perinuclear clustering of mitochondria Conclusion We observed by electron microscopy a specific phenomenon, perinuclear clustering of mitochondria, in endomyocardial biopsies from patients with cardiac rejection that affects to the architecture of the nuclear membrane. This ultrastructural approach might complement and improve the diagnosis of rejection. Acknowledgement/Funding National Institute of Health [PI16/01627, PI17/01925, PI17/01232], “Consorcio Centro de Investigaciόn Biomédica en Red, M.P.”, and FEDER

Fatal Elephant Endotheliotropic Herpesvirus Infection of Two Young Asian Elephants

Elephant endotheliotropic herpesvirus (EEHV) can cause a devastating haemorrhagic disease in young Asian elephants worldwide. Here, we report the death of two young Asian elephants after suffering from acute haemorrhagic disease due to EEHV-1A infection. We detected widespread distribution of EEHV-1A in various organs and tissues of the infected elephants. Enveloped viral particles accumulated within and around cytoplasmic electron-dense bodies in hepatic endothelial cells were detected. Attempts to isolate the virus on different cell cultures showed limited virus replication; however, late viral protein expression was detected in infected cells. We further showed that glycoprotein B (gB) of EEHV-1A possesses a conserved cleavage site Arg-X-Lys/Arg-Arg that is targeted by the cellular protease furin, similar to other members of the Herpesviridae. We have determined the complete 180 kb genome sequence of EEHV-1A isolated from the liver by next-generation sequencing and de novo assembly. As virus isolation in vitro has been unsuccessful and limited information is available regarding the function of viral proteins, we have attempted to take the initial steps in the development of suitable cell culture system and virus characterization. In addition, the complete genome sequence of an EEHV-1A in Europe will facilitate future studies on the epidemiology and diagnosis of EEHV infection in elephants.

Production Strategies for Pentamer-Positive Subviral Dense Bodies as a Safe Human Cytomegalovirus Vaccine

Infections with the human cytomegalovirus (HCMV) are associated with severe clinical manifestations in children following prenatal transmission and after viral reactivation in immunosuppressed individuals. The development of an HCMV vaccine has long been requested but there is still no licensed product available. Subviral dense bodies (DB) are immunogenic in pre-clinical models and are thus a promising HCMV vaccine candidate. Recently, we established a virus based on the laboratory strain Towne that synthesizes large numbers of DB containing the pentameric protein complex gH/gL/UL128-131 (Towne-UL130repΔGFP). The work presented here focuses on providing strategies for the production of a safe vaccine based on that strain. A GMP-compliant protocol for DB production was established. Furthermore, the DB producer strain Towne-UL130rep was attenuated by deleting the UL25 open reading frame. Additional genetic modifications aim to abrogate its capacity to replicate in vivo by conditionally expressing pUL51 using the Shield-1/FKBP destabilization system. We further show that the terminase inhibitor letermovir can be used to reduce infectious virus contamination of a DB vaccine by more than two orders of magnitude. Taken together, strategies are provided here that allow for the production of a safe and immunogenic DB vaccine for clinical testing.

Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response

ABSTRACTThe development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.IMPORTANCEInfections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.