Efficient recovery of attenuated canine distemper virus from cDNA.

To provide insights into the biology of the attenuated canine distemper virus (CDV) Onderstepoort (OP) strain (large plaque forming variant), design next-generation multivalent vaccines, or further investigate its promising potential as an oncolytic vector, we employed contemporary modifications to establish an efficient OP-CDV-based reverse genetics platform. Successful viral rescue was obtained however only upon recovery of a completely conserved charged residue (V13E) residing at the N-terminal region of the large protein (L). Although L-V13 and L-V13E did not display drastic differences in cellular localization and physical interaction with P, efficient polymerase complex (P+ L) activity was recorded only with L-V13E. Interestingly, grafting mNeonGreen to the viral N protein via a P2A ribosomal skipping sequence (OPneon) and its derivative V-protein-knockout variant (OPneon-Vko) exhibited delayed replication kinetics in cultured cells. Collectively, we established an efficient OP-CDV-based reverse genetics system that enables the design of various strategies potentially contributing to veterinary medicine and research.

Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering.

Morbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerization per se was not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCE MeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity.

Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity.IMPORTANCE Despite the availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that postexposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, the lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here we combined structure-guided mutagenesis with cellular, biochemical, and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provide evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher-order oligomers necessary to promote plasma membrane-budding activity.

Assessment of the expression of biomarkers of uremic inflammation in dogs with renal disease.

OBJECTIVE: To assess the expression of inflammatory cytokines and enzymes in venous whole blood of dogs with impaired renal function attributable to various causes. ANIMALS: 46 dogs with acute kidney injury (AKI), 8 dogs with chronic kidney disease (CKD), and 10 healthy dogs. PROCEDURES: Dogs with AKI and CKD were prospectively enrolled during 2010 if they met inclusion criteria. Demographic and laboratory characteristics were evaluated for each dog, and expression of inflammatory cytokines (interleukin [IL]-1α, IL-1ß, IL-8, tumor necrosis factor [TNF]-α, IL-10, and transforming growth factor [TGF]-ß) and enzymes (inducible nitric oxide synthase [iNOS] and 5-lipoxygenase [5-LO]) was measured in venous whole blood obtained at initial evaluation. RESULTS: Dogs with impaired renal function had markedly higher expression of the cytokines IL-1α, IL-1ß, and TGF-ß and the enzyme 5-LO, compared with expression in healthy dogs. Additionally, 17 of 46 AKI dogs (but none of the CKD dogs) had higher IL-8 mRNA expression and 3 of 8 CKD dogs (but only 2/46 AKI dogs) had higher TNF-α expression, compared with results for healthy dogs. No significant difference between renal disease groups was detected for inflammatory markers and laboratory variables, degree of azotemia, or cause of impaired renal function. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, expression of the cytokines IL-1α, IL-1ß, and TGF-ß and the enzyme 5-LO was clearly increased in dogs with renal disease, which suggested that these markers were part of an inflammatory response in animals with AKI or CKD.

Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome.

BACKGROUND: Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. METHODOLOGY AND PRINCIPAL FINDINGS: The mRNA abundance of cytokines (IL-1α, IL-1ß, IL-8, IL-10, TNF-α, TGF-ß) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1ß, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. CONCLUSION: The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.

Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM.

UNLABELLED: Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces ("front" and "back"). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. IMPORTANCE: A complete understanding of the measles virus and canine distemper virus (CDV) cell entry molecular framework is still lacking, thus impeding the rational design of antivirals. Both viruses share many biological features that partially rely on the use of analogous Ig-like host cell receptors, namely, SLAM and nectin 4, for entering immune and epithelial cells, respectively. Here, we provide evidence that the mode of binding between the membrane-distal V domain of SLAM and the attachment protein (H) of morbilliviruses is very likely conserved. Moreover, although structural information revealed two discrete conformational states of H, one of the structures displayed two H-SLAM binding interfaces ("front" and "back"). Our data not only spotlight the front H-binding site of SLAM as the main determinant of membrane fusion promotion but suggest that the triggering efficiency of the viral entry machinery may rely on a local conformational change within the front H-SLAM interactive site rather than the binding affinity.

Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

BACKGROUND: Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). RESULTS: All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. CONCLUSIONS: Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice model. The data indicates that MLVA complex A strains are particularly adept at establishing encephalitis possibly by virtue of their higher resistance to antibacterial defense mechanisms in microglia cells, the main target of L. monocytogenes.

Sequential conformational changes in the morbillivirus attachment protein initiate the membrane fusion process.

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 "spacer" section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced "head-stalk" rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal "spacer" domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This "head-to-spacer" interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk's bioactivity that may prematurely activate F. Receptor-contact disrupts the "head-to-spacer" interaction, which subsequently "unlocks" the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk "spacer" microdomain as a possible drug target for antiviral therapy.

SLAM- and nectin-4-independent noncytolytic spread of canine distemper virus in astrocytes.

UNLABELLED: Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered "nectin-4-blind" recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes. IMPORTANCE: While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS) infections is that persistence is based on noncytolytic cell-to-cell spread, which, in the case of CDV, was demonstrated to rely on functional membrane fusion machinery complexes. This inferred a mechanism where nucleocapsids are transmitted through macroscopically invisible microfusion events between infected and target cells. Here, we provide evidence that CDV induces such microfusions in a SLAM- and nectin-4-independent manner, thereby strongly suggesting the existence of a third receptor expressed in glial cells (referred to as GliaR). We propose that GliaR governs intercellular transfer of nucleocapsids and hence contributes to viral persistence in the brain and ensuing demyelinating lesions.

Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head.

Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F's interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways.

Neurotropic astrovirus in cattle with nonsuppurative encephalitis in Europe.

Encephalitis is a frequently diagnosed condition in cattle with neurological diseases. Many affected animals present with a nonsuppurative inflammatory reaction pattern in the brain. While this pattern supports a viral etiology, the causative pathogen remains unknown in a large proportion of cases. Using viral metagenomics, we identified an astrovirus (bovine astrovirus [BoAstV]-CH13) in the brain of a cow with nonsuppurative encephalitis. Additionally, BoAstV RNA was detected with reverse transcription-PCR and in situ hybridization in about one fourth (5/22 animals) of cattle with nonsuppurative encephalitis of unknown etiology. Viral RNA was found primarily in neurons and at the site of pathology. These findings support the notion that BoAstV infection is a common cause of encephalitis in cattle. Phylogenetically, BoAstV-CH13 was closely related to rare astrovirus isolates from encephalitis cases in animals and a human patient. Future research needs to be directed toward the pathogenic mechanisms, epidemiology, and potential cross-species transmission of these neurotropic astroviruses.

Molecular determinants defining the triggering range of prefusion F complexes of canine distemper virus.

UNLABELLED: The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this "pocket'" appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from small-molecule refolding blockers. IMPORTANCE: With the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering "stimulus" (attachment protein and receptor types) define a "triggering range," which governs the initiation of the membrane fusion process. A central "pocket" microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency.

Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene.

Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene (C5H8) hydrocarbons production. Continuous production of isoprene from CO2 and H2O was achieved in the light, occurring via the endogenous methylerythritol-phosphate (MEP) pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid (MVA) pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate (IPP) and dimethylallyl-diphosphate (DMAPP) precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.

Mechanism for active membrane fusion triggering by morbillivirus attachment protein.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

Truncated photosystem chlorophyll antenna size in the green microalga Chlamydomonas reinhardtii upon deletion of the TLA3-CpSRP43 gene.

The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.

In vitro cytoprotective effects of acetylsalicylic acid, carprofen, meloxicam, or robenacoxib against apoptosis induced by sodium nitroprusside in canine cruciate ligament cells.

OBJECTIVE: To determine whether incubation of cruciate ligament cells with acetylsalicylic acid, carprofen, meloxicam, or robenacoxib provides protection against apoptosis induced by sodium nitroprusside (SNP). SAMPLE: Explants of cranial (CCL) and caudal (CaCL) cruciate ligaments from eight 1-day-old Beagles. PROCEDURES: Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 µg of acetylsalicylic acid/mL; 0.1, 1, or 10 µg of carprofen/mL; 0.1, 1, or 10 µg of meloxicam/mL; or 0.1, 1, or 10 µg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E(2) concentrations were measured via an ELISA. RESULTS: Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 µg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE(2) production from SNP-damaged cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments.

Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis.

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes.

Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies. EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP. Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ∼13 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking. In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.

Comparative spatiotemporal analysis of the intrathecal immune response in natural listeric rhombencephalitis of cattle and small ruminants.

This study examined the spatiotemporal immune response in listeric rhombencephalitis of ruminants in situ. Our data support the view that astrocytes facilitate the containment of infectious lesions. Results on the natural disease recapitulate observations in experimental rodent models and suggest that the mounted adaptive lymphocytic response of ruminants is effective in eliminating Listeria monocytogenes (LM). However, our data indicate earlier participation of the adaptive immune response, a stronger B lymphocyte contribution and a more protracted macrophage infiltration in the natural disease than it has been deduced from experimental models. Therefore, such models should be complemented by studies in natural host systems. Various macrophage and microglia subsets are involved in listeric rhombencephalitis and their differential contribution may account for species differences in clinical course and outcome of infection as might species differences in the B-cell response. Future functional ex vivo and in vitro studies are necessary to further investigate the findings obtained in the present study.