Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos.

This study aimed to assess the cryoprotectant role of exopolysaccharide (EPS) ID1, produced by Antarctic Pseudomonas sp., in the vitrification of in vitro-produced (IVP) bovine embryos. IVP day 7 (D7) and day 8 (D8) expanded blastocysts derived from cow or calf oocytes were vitrified without supplementation (EPS0) or supplemented with 10 µg/mL (EPS10) or 100 µg/mL (EPS100) EPS ID1. The effect of EPS ID1 was assessed in post-warming re-expansion and hatching rates, differential cell count, apoptosis rate, and gene expression. EPS100 re-expansion rates were significantly higher than those observed for the EPS0 and EPS10 treatments, regardless of culture length or oocyte source. EPS100 hatching rate was similar to the one of the fresh blastocysts except for those D7 blastocysts derived from calf oocytes. No differences were observed among EPS ID1 treatments when the inner cell mass, trophectoderm, and total cell number were assessed. Although apoptosis rates were higher (p ≤ 0.05) in vitrified groups compared to fresh embryos, EPS100 blastocysts had a lower number (p ≤ 0.05) of apoptotic nuclei than the EPS0 or EPS10 groups. No differences in the expression of BCL2, AQP3, CX43, and SOD1 genes between treatments were observed. Vitrification without EPS ID1 supplementation produced blastocysts with significantly higher BAX gene expression, whereas treatment with 100 µg/mL EPS ID1 returned BAX levels to those observed in non-vitrified blastocysts. Our results suggest that 100 µg/mL EPS ID1 added to the vitrification media is beneficial for embryo cryopreservation because it results in higher re-expansion and hatching ability and it positively modulates apoptosis.

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos.

The cold-adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in-straw warming protocol of in vitro-produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7-expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 µg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non-vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3 h post-warming between vitrification treatments. At 24 h post-warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop-vitrified embryos and similar hatching rates to fresh non-vitrified or Cryotop-vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p < .05) and higher GPX1 (p < .05) and CDX2 (p < .1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans-EPS-vitrified embryos when compared to those from the non-supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality.

Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7T.

Shewanella vesiculosa M7T is a cold-adapted Antarctic bacterium that has a great capacity to secrete membrane vesicles (MVs), making it a potentially excellent model for studying the vesiculation process. S. vesiculosa M7T undergoes a blebbing mechanism to produce different types of MVs, including outer membrane vesicles and outer-inner membrane vesicles (O-IMVs). More recently, other mechanisms have been considered that could lead to the formation of O-IMVs derived from prophage-mediated explosive cell lysis in other bacteria, but it is not clear if they are of the same type. The bacterial growth phase could also have a great impact on the type of MVs, although there are few studies on the subject. In this study, we used high-resolution flow cytometry, transmission electron microscopy, and cryo-electron microscopy (Cryo-EM) analysis to determine the amount and types of MVs S. vesiculosa M7T secreted during different growth phases. We show that MV secretion increases during the transition from the late exponential to the stationary phase. Moreover, prophage-mediated explosive cell lysis is activated in S. vesiculosa M7T, increasing the heterogeneity of both single- and double-layer MVs. The sequenced DNA fragments from the MVs covered the entire genome, confirming this explosive cell lysis mechanism. A different structure and biogenesis mechanisms for the explosive cell lysis-derived double-layered MVs was observed, and we propose to name them explosive O-IMVs, distinguishing them from the blebbing O-IMVs; their separation is a first step to elucidate their different functions. In our study, we used for the first time sorting by flow cytometry and Cryo-EM analyses to isolate bacterial MVs based on their nucleic acid content. Further improvements and implementation of bacterial MV separation techniques is essential to develop more in-depth knowledge of MVs.

RNA release via membrane vesicles in Pseudomonas aeruginosa PAO1 is associated with the growth phase.

Pseudomonas aeruginosa PAO1 membrane vesicles (MVs) are known to play a role in cell-to-cell communication. Several studies have shown that the MV composition and physicochemical properties vary according to the bacterial growth stage, but the impact this might have on the externalization of RNA via MVs has not been addressed. Therefore, a study to characterize the RNA content from MVs retrieved at different growth phases was conducted. First, the transcriptome analyses revealed a higher abundance of around 300 RNA species in MVs when compared with the cells. The vesiculation rate along the growth curve was determined, showing that the release of MVs increased during the transition to the stationary phase, whereas it decreased in the late stationary phase. RNA-seq of MVs retrieved along the transition to the stationary phase demonstrated that the RNA cargo of vesicles did not vary. However, the amount of smaller RNAs (<200 nt) inside MVs retrieved in the late exponential phase was higher than in the stationary phase MVs. These results indicate that the externalization of RNA via MVs occurs during late exponential phase and implies the secretion of different types of MVs during growth.

Relationship Between Membrane Vesicles, Extracellular ATP and Biofilm Formation in Antarctic Gram-Negative Bacteria.

Biofilms offer a safe environment that favors bacterial survival; for this reason, most pathogenic and environmental bacteria live integrated in biofilm communities. The development of biofilms is complex and involves many factors, which need to be studied in order to understand bacterial behavior and control biofilm formation when necessary. We used a collection of cold-adapted Antarctic Gram-negative bacteria to study whether their ability to form biofilms is associated with a capacity to produce membrane vesicles and secrete extracellular ATP. In most of the studied strains, no correlation was found between biofilm formation and these two factors. Only Shewanella vesiculosa M7T secreted high levels of extracellular ATP, and its membrane vesicles caused a significant increase in the speed and amount of biofilm formation. In this strain, an important portion of the exogenous ATP was contained in membrane vesicles, where it was protected from apyrase treatment. These results confirm that ATP influences biofilm formation. Although the role of extracellular ATP in prokaryotes is still not well understood, the metabolic cost of its production suggests it has an important function, such as a role in biofilm formation. Thus, the liberation of extracellular ATP through membrane vesicles and its function deserve further study.

Cryoprotectant role of exopolysaccharide of Pseudomonas sp. ID1 in the vitrification of IVM cow oocytes.

Biological molecules isolated from organisms that live under subzero conditions could be used to protect oocytes from cryoinjuries suffered during cryopreservation. This study examined the cryoprotectant role of exopolysaccharides of Pseudomonas sp. ID1 (EPS ID1) in the vitrification of prepubertal and adult cow oocytes. IVM oocytes were vitrified and warmed in media supplemented with 0, 1, 10, 100 or 1000µgmL-1 EPS ID1. After warming, oocytes were fertilised and embryo development, spindle morphology and the expression of several genes in Day 8 blastocysts were assessed. Vitrification led to significantly lower proportion of prepubertal oocytes exhibiting a normal spindle configuration. In fresh control oocytes and most groups of vitrified adult oocytes, similar percentages of oocytes with a normal spindle configuration were observed. Percentages of Day 8 blastocysts were similar for prepubertal oocytes vitrified in the absence or presence of 1 or 10µgmL-1 EPS ID1 and for adult oocytes vitrified in the presence of 10µgmL-1 EPS ID1 compared with non-vitrified oocytes. EPS ID1 supplementation had no effect on solute carrier family 2 member 3 (SLC2A3), ubiquitin-conjugating enzyme E2A (UBE2A) and histone deacetylase 1 (HDAC1) expression in Day 8 blastocysts form adult oocytes. However, supplementation with 10 and 100µgmL-1 EPS ID1 led to increased expression of genes involved in epigenetic modifications (DNA methyltransferase 3 alpha (DNMT3A) and K (lysine) acetyltransferase 2A (KAT2A)) and apoptosis (BCL2 associated X apoptosis regulator (BAX) and BCL2-like 1 (BCL2L1)). The lowest BAX:BCL2L1 ratio was found in the 10µgmL-1 EPS ID1-supplemented group. The results suggest that 10µgmL-1 EPS ID1 added to vitrification and warming media may help protect bovine oocytes against cryodamage.

Author Correction: DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton.

In the version of this Letter originally published, the Methods incorrectly stated that all phytoplankton cultures were sampled in mid-exponential phase. The low-nitrogen cultures were sampled in early stationary phase and at the point at which Fv/Fm values decreased, to indicate that cultures were experiencing low-nitrogen conditions. All other phytoplankton cultures were sampled in exponential phase. Growth and Fv/Fm data are provided here on high- and low-nitrogen cultures (Figs 1, 2 and 3) to clarify and support this correction. The Methods also stated that cell counting was done using a Beckman Multisizer 3 Coulter Counter, but a CASY Model TT Cell Counter was used.

Cryo-transmission Electron Microscopy of Outer-inner Membrane Vesicles Naturally Secreted by Gram-negative Pathogenic Bacteria.

A protocol was developed to visualize and analyze the structure of membrane vesicles (MVs) from Gram-negative bacteria. It is now accepted that these micrometric spherical vesicles are commonly produced by cells from all three domains of life, so the protocol could be useful in the study of vesicles produced by eukaryotes and archaea as well as bacteria. The multiplicity of functions performed by MVs, related to cell communication, interaction with the immune system, pathogenesis, and nutrient acquisition, among others, has made MVs a hot topic of research. Due to their small size (25-300 nm), the observation of MVs requires electron microscopy and is usually performed by transmission electron microscopy (TEM) of negatively stained MVs. Other protocols applied for their visualization include scanning electron microscopy, TEM after fixation and embedding of vesicles, or even atomic force microscopy. In some of these techniques, vesicle structure is altered by drying, while others are time-consuming and most of them can generate artifacts. Cryo-TEM after plunge freezing allows the visualization of samples embedded in a thin film of vitreous ice, which preserves their native cellular structures and provides the highest available resolution for the imaging. This is achieved by very high cooling rates that turn the intrinsic water of cells into vitreous ice, avoiding crystal formation and phase segregation between water and solutes. In addition to other types of characterization, an accurate knowledge of MV structure, which can be obtained by this protocol, is essential for MV application in different fields.

DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton.

Dimethylsulfoniopropionate (DMSP) is a globally important organosulfur molecule and the major precursor for dimethyl sulfide. These compounds are important info-chemicals, key nutrients for marine microorganisms, and are involved in global sulfur cycling, atmospheric chemistry and cloud formation1-3. DMSP production was thought to be confined to eukaryotes, but heterotrophic bacteria can also produce DMSP through the pathway used by most phytoplankton 4 , and the DsyB enzyme catalysing the key step of this pathway in bacteria was recently identified 5 . However, eukaryotic phytoplankton probably produce most of Earth's DMSP, yet no DMSP biosynthesis genes have been identified in any such organisms. Here we identify functional dsyB homologues, termed DSYB, in many phytoplankton and corals. DSYB is a methylthiohydroxybutryate methyltransferase enzyme localized in the chloroplasts and mitochondria of the haptophyte Prymnesium parvum, and stable isotope tracking experiments support these organelles as sites of DMSP synthesis. DSYB transcription levels increased with DMSP concentrations in different phytoplankton and were indicative of intracellular DMSP. Identification of the eukaryotic DSYB sequences, along with bacterial dsyB, provides the first molecular tools to predict the relative contributions of eukaryotes and prokaryotes to global DMSP production. Furthermore, evolutionary analysis suggests that eukaryotic DSYB originated in bacteria and was passed to eukaryotes early in their evolution.

Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals.

Secretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM+ plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM+ B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA+ B cells, memory IgM+ B cells were related to some IgA+ clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM+ B cells and could help SIgA to anchor highly diverse commensal communities to mucus.

Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry.

Membrane vesicles (MVs) produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN) are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM) of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs) and the recently described double-bilayer vesicles (O-IMVs), other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications.

Linear Morphea Mimicking Lichen Striatus in Its Early Presentation.

Linear morphea and lichen striatus are distinct conditions that have been linked in only one previous case report. We describe two patients with facial lichen striatus preceding linear morphea at the same site. A possible pathogenic relationship is discussed.

Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria.

Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.

Cryo-electron tomography of plunge-frozen whole bacteria and vitreous sections to analyze the recently described bacterial cytoplasmic structure, the Stack.

Cryo-electron tomography (CET) of plunge-frozen whole bacteria and vitreous sections (CETOVIS) were used to revise and expand the structural knowledge of the "Stack", a recently described cytoplasmic structure in the Antarctic bacterium Pseudomonas deceptionensis M1(T). The advantages of both techniques can be complementarily combined to obtain more reliable insights into cells and their components with three-dimensional imaging at different resolutions. Cryo-electron microscopy (Cryo-EM) and CET of frozen-hydrated P. deceptionensis M1(T) cells confirmed that Stacks are found at different locations within the cell cytoplasm, in variable number, separately or grouped together, very close to the plasma membrane (PM) and oriented at different angles (from 35° to 90°) to the PM, thus establishing that they were not artifacts of the previous sample preparation methods. CET of plunge-frozen whole bacteria and vitreous sections verified that each Stack consisted of a pile of oval disc-like subunits, each disc being surrounded by a lipid bilayer membrane and separated from each other by a constant distance with a mean value of 5.2±1.3nm. FM4-64 staining and confocal microscopy corroborated the lipid nature of the membrane of the Stacked discs. Stacks did not appear to be invaginations of the PM because no continuity between both membranes was visible when whole bacteria were analyzed. We are still far from deciphering the function of these new structures, but a first experimental attempt links the Stacks with a given phase of the cell replication process.

New emulsifying and cryoprotective exopolysaccharide from Antarctic Pseudomonas sp. ID1.

Pseudomonas sp. ID1 is a cold-adapted bacterium isolated from a marine sediment sample collected from South Shetland Islands (Antarctica) that is noted for the highly mucous appearance of its colonies. In this work, we have characterized an exopolysaccharide (EPS) produced by this strain, which is mainly composed of glucose, galactose and fucose, and has a molecular mass higher than 2×10(6) Da. We have also studied its potential biotechnological applications as an emulsifier and cryoprotectant agent. The EPS emulsifying activity against different food and cosmetic oils was much higher than commercial gums such as xanthan gum and arabic gum, and surfarctants such as Span 20. It formed highly stable emulsions against the cosmetic oil cetiol V, exhibiting pseudoplastic flow behavior, low thixotrophy and yield stress. The EPS of Pseudomonas sp. ID1 conferred significant cryoprotection for the strain itself as well as for other bacteria, including Escherichia coli, suggesting a universal cryoprotectant role. The cryoprotective activity of the EPS showed a clear dose-response relation at -20 °C and -80 °C and was significantly higher than that observed for the membrane stabilizer fetal bovine serum (FBS). These properties make the EPS of Pseudomonas sp. ID1 a promising alternative to commercial polysaccharides as an emulsifier and cryoprotectant agent for food, pharmaceutical and cosmetic industries.

Antimicrobial activity of Antarctic bryozoans: an ecological perspective with potential for clinical applications.

The antimicrobial activity of Antarctic bryozoans and the ecological functions of the chemical compounds involved remain largely unknown. To determine the significant ecological and applied antimicrobial effects, 16 ether and 16 butanol extracts obtained from 13 different bryozoan species were tested against six Antarctic (including Psychrobacter luti, Shewanella livingstonensis and 4 new isolated strains) and two bacterial strains from culture collections (Escherichia coli and Bacillus cereus). Results from the bioassays reveal that all ether extracts exhibited antimicrobial activity against some bacteria. Only one butanol extract produced inhibition, indicating that antimicrobial compounds are mainly lipophilic. Ether extracts of the genus Camptoplites inhibited the majority of bacterial strains, thus indicating a broad-spectrum of antimicrobial activity. Moreover, most ether extracts presented activities against bacterial strains from culture collections, suggesting the potential use of these extracts as antimicrobial drugs against pathogenic bacteria.

The stack: a new bacterial structure analyzed in the Antarctic bacterium Pseudomonas deceptionensis M1(T) by transmission electron microscopy and tomography.

In recent years, improvements in transmission electron microscopy (TEM) techniques and the use of tomography have provided a more accurate view of the complexity of the ultrastructure of prokaryotic cells. Cryoimmobilization of specimens by rapid cooling followed by freeze substitution (FS) and sectioning, freeze fracture (FF) and observation of replica, or cryoelectron microscopy of vitreous sections (CEMOVIS) now allow visualization of biological samples close to their native state, enabling us to refine our knowledge of already known bacterial structures and to discover new ones. Application of these techniques to the new Antarctic cold-adapted bacterium Pseudomonasdeceptionensis M1(T) has demonstrated the existence of a previously undescribed cytoplasmic structure that does not correspond to known bacterial inclusion bodies or membranous formations. This structure, which we term a "stack", was mainly visualized in slow growing cultures of P. deceptionensis M1(T) and can be described as a set of stacked membranous discs usually arranged perpendicularly to the cell membrane, but not continuous with it, and found in variable number in different locations within the cell. Regardless of their position, stacks were mostly observed very close to DNA fibers. Stacks are not exclusive to P. deceptionensis M1(T) and were also visualized in slow-growing cultures of other bacteria. This new structure deserves further study using cryoelectron tomography to refine its configuration and to establish whether its function could be related to chromosome dynamics.

New type of outer membrane vesicle produced by the Gram-negative bacterium Shewanella vesiculosa M7T: implications for DNA content.

Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7(T) has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/µg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7(T) that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).

Pseudomonas deceptionensis sp. nov., a psychrotolerant bacterium from the Antarctic.

During the taxonomic investigation of cold-adapted bacteria from samples collected in the Antarctic area of the South Shetland Islands, one Gram-reaction-negative, psychrotolerant, aerobic bacterium, designated strain M1(T), was isolated from marine sediment collected on Deception Island. The organism was rod-shaped, catalase- and oxidase-positive and motile by means of a polar flagellum. This psychrotolerant strain grew at temperatures ranging from -4 °C to 34 °C. Phylogenetic studies based on 16S rRNA gene sequences confirmed that Antarctic isolate M1(T) was a member of the genus Pseudomonas and was located in the Pseudomonas fragi cluster. 16S rRNA gene sequence similarity values were >98 % between 13 type strains belonging to the Pseudomonas fluorescens lineage. However, phylogenetic analysis of rpoD gene sequences showed that strain M1(T) exhibited high sequence similarity only with respect to Pseudomonas psycrophila (97.42 %) and P. fragi (96.40 %) and DNA-DNA hybridization experiments between the Antarctic isolate M1(T) and the type strains of these two closely related species revealed relatedness values of 58 and 57 %, respectively. Several phenotypic characteristics, together with the results of polar lipid and cellular fatty acid analyses, were used to differentiate strain M1(T) from related pseudomonads. Based on the evidence of this polyphasic taxonomic study, strain M1(T) represents a novel species, for which the name Pseudomonas deceptionensis sp. nov. is proposed. The type strain is M1(T) ( = LMG 25555(T)  = CECT 7677(T)).

Hyaluronidase expression by an oncolytic adenovirus enhances its intratumoral spread and suppresses tumor growth.

Successful virotherapy requires efficient virus spread within tumors. We tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix (ECM), could enhance the intratumoral distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in mice-bearing tumor xenografts improves the antitumor activity of an oncolytic adenovirus. Next, we constructed a replication-competent adenovirus expressing a soluble form of the human sperm hyaluronidase (PH20) under the control of the major late promoter (MLP) (AdwtRGD-PH20). Intratumoral treatment of human melanoma xenografts with AdwtRGD-PH20 resulted in degradation of hyaluronan (HA), enhanced viral distribution, and induced tumor regression in all treated tumors. Finally, the PH20 cDNA was inserted in an oncolytic adenovirus that selectively kills pRb pathway-defective tumor cells. The antitumoral activity of the novel oncolytic adenovirus expressing PH20 (ICOVIR17) was compared to that of the parental virus ICOVIR15. ICOVIR17 showed more antitumor efficacy following intratumoral and systemic administration in mice with prestablished tumors, along with an improved spread of the virus within the tumor. Importantly, a single intravenous dose of ICOVIR17 induced tumor regression in 60% of treated tumors. These results indicate that ICOVIR17 is a promising candidate for clinical testing.