3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.     

Shifts in the rhizobiome during consecutive in planta enrichment for phosphate-solubilizing bacteria differentially affect maize P status

Phosphorus (P) is despite its omnipresence in soils often unavailable for plants. Rhizobacteria able to solubilize P are therefore crucial to avoid P deficiency. Selection for phosphate-solubilizing bacteria (PSB) is frequently done in vitro; however, rhizosphere competence is herein overlooked. Therefore, we developed an in planta enrichment concept enabling simultaneous microbial selection for P-solubilization and rhizosphere competence. We used an ecologically relevant combination of iron- and aluminium phosphate to select for PSB in maize (Zea mays L.). In each consecutive enrichment, plant roots were inoculated with rhizobacterial suspensions from plants that had grown in substrate with insoluble P. To assess the plants’ P statuses, non-destructive multispectral imaging was used for quantifying anthocyanins, a proxy for maize’s P status. After the third consecutive enrichment, plants supplied with insoluble P and inoculated with rhizobacterial suspensions showed a P status similar to plants supplied with soluble P. A parallel metabarcoding approach uncovered that the improved P status in the third enrichment coincided with a shift in the rhizobiome towards bacteria with plant growth-promoting and P-solubilizing capacities. Finally, further consecutive enrichment led to a functional relapse hallmarked by plants with a low P status and a second shift in the rhizobiome at the level of Azospirillaceae and Rhizobiaceae.     

A thermoelectrically regulated multipurpose cuvette for simultaneous time-dependent measurements

An improved design of a thermostatically controlled reaction cuvette for time-dependent biochemical measurements is described. The design is such that a multiple choice of single and simultaneous spectroscopic and electrode analyses can be performed in a sample of about 1.8 ml. This choice is quite flexible due to the use of exchangeable tapered plugs suited with either optical quartz-rod windows for absorption or fluorescence measurements or selective electrodes for changes of O₂, H₂, H⁺, etc. Temperature is accurately controlled by a thermoelectric (Peltier) module. An overhead constant-stirring device includes solute addition and gasflow ports. A bottom window allows actinic illumination for photobiological and photochemical experiments. Some examples of application in combination with commercial or laboratorymade instruments are presented.     

A new family of soft corals (Anthozoa,Octocorallia, Alcyonacea) from the aphotic tropical eastern Pacific waters revealed by integrative taxonomy

The new family Aquaumbridae is described based on distinct morphological characters and supported by a molecular phylogenetic analysis. Aquaumbridae is similar to Nidaliidae, Nepththeidae and Alcyoniidae in having arborescent colonies and lacking axis structure or stolons, but differs from them in having very different sclerite composition and having conspicuous transparent jelly-like lobes. Phylogenetic analysis of two mitochondrial genes, ND2 and mtMutS, strongly supports its placement in a separated clade. Herein we describe Aquaumbra klapferi sp. nov., gen. nov. in the new family. The organisms were obtained from the seamounts, ridges and canyons out of the insular shelf of Isla del Coco, Costa Rica, down to 400 m depth. The new species represents the first discovery of a soft coral in an eastern Pacific oceanic island, and provides hints of the biodiversity of the largely unexplored deep waters of the tropical eastern Pacific.     

Lysine degradation in Candida. Characterization and probable role of L-norleucine-leucine, 4-aminobutyrate and delta-aminovalerate:2-oxoglutarate aminotransferases

Three enzymes partially purified that catalyze respectively the transamination of L-norleucine, 4-aminobutyrate and delta-aminovalerate with alpha-ketoglutarate as aminoacceptor were characterized and isolated from L-lysine adapted cell of Candida guilliermondii var. membranaefaciens. The transaminases have a maximum activity in the pH range of 7.8-8.5 and at 55 degrees C, 45 degrees C and 40 degrees C respectively. alpha-Ketoglutarate and to a lesser extent pyridoxal-5'-phosphate were effective protecting agents against rise in temperature. The enzymes exhibit absorption maximum at 280 nm, 330 nm and 410 nm. The fact that L-norleucine-leucine aminotransferase, 4-aminobutyrate aminotransferase and delta-aminovalerate aminotransferase are strongly induced by growing the yeast Candida on L-lysine suggests new hypothetic pathways for the catabolism of L-lysine where the main substrate of each aminotransferase could be an intermediary metabolite.     

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x10⁵/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture – total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 10⁶ cells that is reduced by >90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.