Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Global gene expression in rice blast pathogen Magnaporthe oryzae treated with a natural rice soil isolate

The rhizospheric microbiome is comprised of many microbes, some of which reduce the virulence of their phytopathogenic neighbors; however, the mechanisms underlying these interactions are largely unknown. Rice soil isolate Pseudomonas chlororaphis EA105 strongly inhibits Magnaporthe oryzae’s in vitro growth by restricting fungal diameter as well as inhibiting the formation of the appressorium, required for penetration. We were interested in elucidating M. oryzae’s response to EA105 treatment, and utilized a microarray approach to obtain a global perspective of EA105 elicited changes in this pathogen. Based on this analysis, three genes of interest were knocked out in M. oryzae 70-15, and their sensitivity to EA105 treatment as well as their ability to infect rice was determined. Priming rice plants with EA105 prior to M. oryzae infection decreased lesion size, and the mutants were tested to see if this effect was retained. A null 70-15 mutant in a trichothecene biosynthesis gene showed less susceptibility to bacterial treatment, forming more appressoria than the parental type 70-15. A similar pattern was seen in a null mutant for a stress-inducible protein, MGG_03098. In addition, when this mutant was inoculated onto the leaves of EA105-primed rice plants, lesions were reduced to a greater extent than in 70-15, implicating the lack of this gene with an increased ISR response in rice. Understanding the global effect of biocontrol bacteria on phytopathogens is a key for developing successful and lasting solutions to crop loss caused by plant diseases and has the potential to greatly increase food supply.     

Dexamethasone effects on liver pyruvate kinase

Dexamethasone in the medium perfusin isolated rabbit livers caused a fast-acting and reversible effect on liver pyruvate kinase. The effect was to lower th assayable V activity (units/g tissue) without changing the concentration (nmol/g enzyme protein). In effect, glucocorticoid lowered the specific activity (units/nmol of enzyme) by direct action on liver. The effect on liver pyruvate kinase is mediated by a relatively stable alteration; 30 min after perfusate (with steroid) was replaced by perfusate (without steroid), the effect remained strongly evident.     

Nuclear protein matrix of seminal vesicle epithelium

Nuclear protein matrix was isolated from guinea pig seminal vesicle epithelium and liver. The two matrices were similar in fine structure as seen by transmission electron microscopy, in protein electropherograms, and in percent composition relative to protein, DNA, and RNA. Scanning electron microscopy was used to examine intact seminal vesicle nuclei, nuclei after treatment with Triton X-100 and DNAse I, and purified nuclear matrix. The matrix surface presented a 'porous' appearance by both scanning and transmission electron microscopy. The matrices of liver and seminal vesicle epithelium (SVE) and the intact nuclei of SVE were assayed for specific binding of free synthetic androgen, 17 alpha-methyltrienolone (R1881). Saturable specific binding was demonstrable for seminal vesicle matrix but not for liver matrix. Maximal binding of androgen occurred at a concentration of approximately 12 nM and was demonstrated to be 1.34 +/- 0.22 pmol of R1881 per mg of seminal vesicle matrix protein; the Kd was approximately 8 nM. The binding of labeled R1881 to matrix could be inhibited with low concentrations of unlabeled androgens, but not with estrogens or other steroids. Our data indicate that the binding of androgen to matrix could account for at least 21% of the binding to intact nuclei.     

O-GlcNAcylation of αB-crystallin regulates its stress-induced translocation and cytoprotection

Under normal conditions, the ubiquitously expressed αB-crystallin functions as a chaperone. αB-crystallin has been implicated in a variety of pathologies, consistent with a build-up of protein aggregates, such as neuromuscular disorders, myofibrillar myopathies, and cardiomyopathies. αB-crystallins’ cardioprotection is partially attributed to its translocation and binding to cytoskeletal elements in response to stress. The triggers for this translocation are not clearly understood. In the heart, αB-crystallin undergoes at least three significant post-translational modifications: phosphorylation at ser-45 and 59 and O-GlcNAcylation (O-linked attachment of the monosaccharide β-N-acetyl-glucosamine) at thr-170. Whether phosphorylation status drives translocation remains controversial. Therefore, we evaluated the role of αB-crystallins’ O-GlcNAcylation in its stress-induced translocation and cytoprotection in cardiomyocytes under stress. Immunoblotting and precipitation experiments with anti-O-GlcNAc antibody (CTD110.6) and glycoprotein staining (Pro-Q Emerald) both demonstrate robust stress-induced O-GlcNAcylation of αB-crystallin. A non-O-GlcNAcylatable αB-crystallin mutant (αB-T170A) showed diminished translocation in response to heat shock and robust phosphorylation at both ser-45 and ser-59. Cell survival assays show a loss of overexpression-associated cytoprotection with the non-glycosylatable mutant to multiple stresses. While ectopic expression of wild-type αB-crystallin strongly stabilized ZsProSensor, a fusion protein rapidly degraded by the proteasome, the non-O-GlcNAcylatable version did not. Therefore, we believe the O-GlcNAcylation of αB-crystallin is a dynamic and important regulator of both its localization and function.     

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating <Emphasis Type="Italic">Brevundimonas diminuta</Emphasis> in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100–200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 × 10³ colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5′ 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems.     

Transfection of bovine cell culture with bovine herpesvirus 4 DNA obtained by cell nuclear extraction

Bovine herpes virus 4 (BHV-4) is a gamma-herpesvirus not associated with clearly defined clinical entities in cattle. The BHV-4 genome consists of a linear dsDNA of approximately 145 Kbp which is only partially characterized and sequenced. We set up a rapid and practical method to isolate BHV-4 DNA from infected cell culture. Microfuged infected cells after exposure to high salt concentration and detergent allowed viral DNA to be purified. Electrophoretic analysis of the digested DNA showed a complete digestion, corresponding to a classical EcoRI banding pattern of strains Movar 33/63, LVR and DN 599. Moreover the biological integrity of viral DNA here obtained, was demonstrated by transfection experiments. BHV-4 DNA was capable of forming CPE after transfection into BAE-7372 cells. Transfected cells specifically reacted with a BHV-4 infected cow serum demonstrating the presence of viral particles. The possibility of obtaining infectious viral DNA using this method may facilitate the construction of recombinant viruses. Specifically, through the use of cotransfection experiments with deleted or mutated viral DNA sequences, the infectious clones isolated could provide the basis for an increased understanding of BHV-4 viral gene expression, replication and pathogenesis.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.