Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L.

BACKGROUND: We have recently identified a novel virus detected in alfalfa seed material. The virus was tentatively named alfalfa-associated potyvirus 1, as its genomic fragments bore similarities with potyvirids. In this study, we continued investigating this novel species, expanding information on its genomic features and biological characteristics. METHODS: This research used a wide range of methodology to achieve end results: high throughput sequencing, bioinformatics tools, reverse transcription-polymerase chain reactions, differential diagnostics using indicator plants, virus purification, transmission electron microscopy, and others. RESULTS: In this study, we obtained a complete genome sequence of the virus and classified it as a tentative species in the new genus, most closely related to the members of the genus Ipomovirus in the family Potyviridae. This assumption is based on the genome sequence and structure, phylogenetic relationships, and transmission electron microscopy investigations. We also demonstrated its mechanical transmission to the indicator plant Nicotiana benthamiana and to the natural host Medicago sativa, both of which developed characteristic symptoms therefore suggesting a pathogenic nature of the disease. CONCLUSIONS: Consistent with symptomatology, the virus was renamed to alfalfa vein mottling virus. A name Alvemovirus was proposed for the new genus in the family Potyviridae, of which alfalfa vein mottling virus is a tentative member.

Composition of the alfalfa pathobiome in commercial fields.

Through the recent advances of modern high-throughput sequencing technologies, the "one microbe, one disease" dogma is being gradually replaced with the principle of the "pathobiome". Pathobiome is a comprehensive biotic environment that not only includes a diverse community of all disease-causing organisms within the plant but also defines their mutual interactions and resultant effect on plant health. To date, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa (Medicago sativa L.), the most extensively cultivated forage legume in the world, is non-existent. Here, we approached this subject by characterizing the biodiversity of the alfalfa pathobiome using high-throughput sequencing technology. Our metagenomic study revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. Profiling the alfalfa pathobiome is a starting point to assess known and identify new and emerging stress challenges in the context of plant disease management. In addition, it allows us to address the complexity of microbial interactions within the plant host and their impact on the development and evolution of pathogenesis.

Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells.

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 µM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.

Characterization of the seed virome of alfalfa (Medicago sativa L).

BACKGROUND: Seed transmission of plant viruses can be important due to the role it plays in their dissemination to new areas and subsequent epidemics. Seed transmission largely depends on the ability of a virus to replicate in reproductive tissues and survive during the seed maturation process. It occurs through the infected embryo or mechanically through the contaminated seed coat. Alfalfa (Medicago sativa L.) is an important legume forage crop worldwide, and except for a few individual seedborne viruses infecting the crop, its seed virome is poorly known. The goal of this research was to perform initial seed screenings on alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System in order to identify pathogenic viruses and understand their potential for dissemination. METHODS: For the detection of viruses, we used high throughput sequencing combined with bioinformatic tools and reverse transcription-polymerase chain reactions. RESULTS: Our results suggest that, in addition to common viruses, alfalfa seeds are infected by other potentially pathogenic viral species that could be vertically transmitted to offspring. CONCLUSIONS: To the best of our knowledge, this is the first study of the alfalfa seed virome carried out by HTS technology. This initial screening of alfalfa germplasm accessions maintained by the NPGS showed that the crop's mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. The information gathered will be used to update germplasm distribution policies and to make decisions on the safety of distributing germplasm based on viral presence.

Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA.

Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa (Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae.

Influence of SHS Precursor Composition on the Properties of Yttria Powders and Optical Ceramics.

This study looked at optimizing the composition of precursors for yttria nanopowder glycine-nitrate self-propagating high-temperature synthesis (SHS). Based on thermodynamic studies, six different precursor compositions were selected, including with excesses of either oxidant or fuel. The powders from the precursors of all selected compositions were highly dispersed and had specific surface areas ranging from 22 to 57 m2/g. They were consolidated by hot pressing (HP) with lithium-fluoride sintering additive and subsequent hot isostatic pressing (HIP). The 1 mm thick HPed ceramics had transmittance in the range of 74.5% to 80.1% @ 1µm, which was limited by optical inhomogeneity due to incomplete evaporation of the sintering additive. Two-stage HIP significantly improves optical homogeneity of the ceramics. It was shown that an excess of oxidizer in the precursor decreases the powders' agglomeration degree, which forms large pore clusters in the ceramics.

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA.

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681-684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence "wildtype" spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

Dysregulated metabolic pathways in age-related macular degeneration.

Age-related macular degeneration is a major cause of vision impairment in the Western world among people of 55 years and older. Recently we have shown that autophagy is dysfunctional in the retinal pigment epithelium (RPE) of the AMD donor eyes (AMD RPE). We also showed increased reactive oxygen (ROS) production, increased cytoplasmic glycogen accumulation, mitochondrial dysfunction and disintegration, and enlarged and annular LAMP-1-positive organelles in AMD RPE. However, the underlying mechanisms inducing these abnormalities remain to be elucidated. Here, by performing a comprehensive study, we show increased PAPR2 expression, deceased NAD+, and SIRT1, increased PGC-1α acetylation (inactive form), lower AMPK activity, and overactive mTOR pathway in AMD RPE as compared to normal RPE. Metabolomics and lipidomics revealed dysregulated metabolites in AMD RPE as compared to normal RPE, including glycerophospholipid metabolism, involved in autophagy, lipid, and protein metabolisms, glutathione, guanosine, and L-glutamic acid, which are implicated in protection against oxidative stress and neurotoxicity, further supporting our observations. Our data show dysregulated metabolic pathways as important contributors to AMD pathophysiology, and facilitate the development of new treatment strategies for this debilitating disease of the visual system.

Stable Intronic Sequences and Exon Skipping Events in the Human RPE65 Gene: Analysis of Expression in Retinal Pigment Epithelium Cells and Cell Culture Models.

Currently, there is much interest in intronic sequence-containing long non-coding RNAs and the role of intronic transcription in regulation of cellular metabolism and fate. Several stable intronic sequence RNAs (sisRNAs) were recently implicated in regulation of parental genes. To investigate transcription from introns of the RPE65 gene, we analyzed RNA-seq and Nanopore sequencing data from different cell models of human retinal pigment epithelium (RPE) and native bovine RPE. We discovered putative stable poly-adenylated transcripts with sequences corresponding to intronic regions of the RPE65 gene in the cytoplasm of RPE cells. These stable intronic sequences could be important for RPE65 transcription, splicing or translation. We also analyzed alternative splicing events in RPE65. Frequent exon skipping events involving exons 2, 3, and 7 were detected. The rate of these events was much higher in human RPE cell cultures compared with native RPE , consistent with lack of translation of RPE65 mRNA in cell cultures.

Volatile Evolution of Long Non-Coding RNA Repertoire in Retinal Pigment Epithelium: Insights from Comparison of Bovine and Human RNA Expression Profiles.

Currently, several long non-coding RNAs (lncRNAs) (TUG1, MALAT1, MEG3 and others) have been discovered to regulate normal visual function and may potentially contribute to dysfunction of the retina. We decided to extend these analyses of lncRNA genes to the retinal pigment epithelium (RPE) to determine whether there is conservation of RPE-expressed lncRNA between human and bovine genomes. We reconstructed bovine RPE lncRNAs based on genome-guided assembly. Next, we predicted homologous human transcripts based on whole genome alignment. We found a small set of conserved lncRNAs that could be involved in signature RPE functions that are conserved across mammals. However, the fraction of conserved lncRNAs in the overall pool of lncRNA found in RPE appeared to be very small (less than 5%), perhaps reflecting a fast and flexible adaptation of the mammalian eye to various environmental conditions.

Proinflammatory cytokine interferon-γ increases the expression of BANCR, a long non-coding RNA, in retinal pigment epithelial cells.

The inflammatory response may contribute to retinal pigment epithelial (RPE) dysfunction associated with the pathogenesis of age-related macular degeneration (AMD). We investigated whether the inflammatory response affects the expression of long coding RNAs (lncRNAs) in human RPE-derived ARPE-19 cells. This class of regulatory RNA molecules recently came to prominence due to their involvement in many pathophysiological processes. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1ß and TNF-α altered the expression several lncRNAs including BANCR in these cells. The cytokine responsible for increasing BANCR expression in ARPE-19 cells was found to be IFN-γ. BANCR expression induced by IFN-γ was suppressed when STAT1 phosphorylation was blocked by JAK inhibitor 1. Thus, proinflammatory cytokines could modulate the expression of lncRNAs in RPE cells and IFN-γ could upregulate the expression of BANCR by activating JAK-STAT1 signaling pathway.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae.

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties.

Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.

PURPOSE: The RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter. METHODS: ARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting. RESULTS: ARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation. CONCLUSIONS: The ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated.

Antimicrobial Effects of Silver Nanoparticles Stabilized in Solution by Sodium Alginate.

BACKGROUND/PURPOSE: To investigate the effect of nanosilver particles in solution stabilized in a matrix of sodium alginate on the growth and development of pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Enterobacter cloacae, the antibiotic-resistant strain of Pseudomonas aeruginosa, the yeast-like fungus Candida albicans, and the luminescent bacteria Photobacterium leiognathi Sh1. METHODS: Isolates of pathogenic bacteria obtained from bronchoalveolar and peritoneal lavage samples from Wistar rats with experimental pneumonia and peritonitis were tested for their susceptibility to silver nanoparticles in solution with an alginate stabilizer. The antifungal activity of silver nanoparticles in sodium alginate was studied for C. albicans (strain CCM885) using the Sabouraud agar method. The biocidal impact of silver nanoparticles in solution with a sodium alginate matrix on the luminescent bacteria P. leiognathi Sh1 was investigated using a BLM 8801 luminometer. RESULTS: It was observed that a 0.02-0.05% nanosilver solution with an alginate stabilizer limits the growth and development of pathogenic bacteria within the first 24 hours of exposure. If the concentration of nanosilver solution is 0.0005-0.05%, it inhibits the viability of the fungus C. albicans. A nanosilver solution at a concentration of 0.05-0.2 µg/mL represses bioluminescence in the bacteria P. leiognathi Sh1. From these results, it appears that the biocidal effect of nanosilver is related either to the presence of ions that are formed during dissolution, or to the availability of nanoparticles that interrupt the membrane permeability of bacterial cells. CONCLUSION: Silver nanoparticles stabilized in a solution of sodium alginate possess significant in vitro antimicrobial activity, which is manifested by inhibition of the bioluminescence of P. leiognathi Sh1, and inhibition of the growth and development of the pathogenic bacteria S. aureus, E. faecalis, E. coli, P. vulgaris, E. cloacae, the antibiotic-resistant strain of P. aeruginosa, and the fungus C. albicans.

Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana.

In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development.

Complete Genome Sequence of the Alfalfa latent virus.

The first complete genome sequence of the Alfalfa latent carlavirus (ALV) was obtained by primer walking and Illumina RNA sequencing. The virus differs substantially from the Czech ALV isolate and the Pea streak virus isolate from Wisconsin. The absence of a clear nucleic acid-binding protein indicates ALV divergence from other carlaviruses.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Gene Expression Profiling in Viable but Nonculturable (VBNC) Cells of Pseudomonas syringae pv. syringae.

Pseudomonas syringae infects diverse crop plants and comprises at least 50 different pathovar strains with different host ranges. More information on the physiological and molecular effects of the host inhibitory environment on the pathogen is needed to develop resistant cultivars. Recently, we reported an in vitro model system that mimics the redox pulse associated with the oxidative burst in plant cells inoculated with Pseudomonas syringae pv. syringae. Using this system, we demonstrated that oxidation of acetosyringone, a major extracellular phenolic compound induced in some plants in response to bacteria, rendered Pseudomonas syringae pv. syringae to a "viable but nonculturable" (VBNC) state. Here we performed a large scale transcriptome profiling of P. s. pv. syringae in the VBNC state induced by acetosyringone treatment and identified bacterial genes and pathways presumably associated with this condition. The findings offer insight into what events occur when bacterial pathogens are first encountered and host defense responses are triggered. The acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance. We believe that this is the first work on global gene expression profiling of VBNC cells in plant pathogenic bacteria.

Joint Intratracheal Surfactant-Antibacterial Therapy in Experimental Pseudomonas-Induced Pneumonia.

BACKGROUND: The application of an exogenous pulmonary surfactant as a carrier for intratracheally administered antimicrobials represents a promising therapeutic modality that is still on its way to clinical practice. Owing to its ability to decrease surface tension, exogenous surfactant may enhance delivery of antibiotics into foci of pulmonary infection, thus increasing efficiency and safety of topical antimicrobial therapy in bacterial lung diseases. OBJECTIVES: To assess potential interactions between exogenous surfactant and amikacin in vitro, and to study the effects of their joint intratracheal instillation in rats with acute pneumonia caused by Pseudomonas aeruginosa. METHODS: The antibacterial and surface-active properties of amikacin (Amicil, Kievmedpreparat, Ukraine), porcine pulmonary surfactant (Suzacrin, Docpharm, Ukraine), and their combination were studied in vitro using standard microbiologic procedures and modified Pattle method (estimation of bubble diameter). Similar methods were utilized to study bacterial contamination of lungs and blood, and to assess the surface activity of bronchoalveolar wash (BAW) in 119 Wistar rats, including infected (intratracheal introduction of P. aeruginosa ATCC 27853) and noninfected animals. Histopathologic findings, differential leukocyte counts, and oxygenation parameters were recorded. RESULTS: Antibacterial and surface-active properties of the surfactant and amikacin remained unimpaired in vitro. In rats anti-pseudomonal and anti-inflammatory effects of the surfactant-amikacin mixture were more pronounced (p<0.05) than effects of pure amikacin as evidenced by recorded rates of bacterial growth and granulocytic response. The combined therapy considerably restricted tissue damage and mitigated reduction of BAW surface activity. CONCLUSION: The advantages of the joint surfactant-amikacin therapy of Pseudomonas-induced pneumonia may suggest further clinical trials.