Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Sensitivity of heat-stressed yeasts to essential oils of plants.

Eight strains of yeasts (Candida lipolytica, Debaryomyces hansenii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata) were examined for changes in sensitivity to eight essential oils of plants (allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme) after being sublethally heat stressed. With the exception of garlic oil for all test yeasts, onion oil for S. cerevisiae, and oregano oil for R. rubra, the essential oils at concentrations of up to 200 ppm in recovery media did not interfere with colony formation by unheated cells. However, some oils, at concentrations as low as 25 ppm in recovery media, reduced populations of sublethally heat-stressed cells compared to populations recovered in media containing no test oils. This demonstrates that the yeasts were either metabolically or structurally damaged as a result of being exposed to elevated temperatures and that essential oils prohibited repair of injury. The size (diameter) of colonies produced on oil-supplemented recovery agar by heat-stressed cells was reduced compared to that observed on unsupplemented agar. Pigment production by heated R. rubra was inhibited by oils of oregano, savory, and thyme, but enhanced by garlic and onion oils. The influence of essential oils on survival of yeasts in thermally processed foods and in the enumeration of stressed cells in these foods should not be minimized.     

Monitoring hairy-root growth by image analysis

Hairy roots, incited byAgrobacterium rhizogenes, form a useful system for analysing the expression and phenotypic effects of foreign genes in plant root tissue. Image analysis offers a non-invasive method of describing their growth in culture. Images of pea (coarse) andBrassica (fine) hairy roots were captured, processed and analysed without difficulty using a commercially available image analysis system. The value of this method in monitoring intermediate changes in growth pattern was illustrated by following the changes in five putatively chlorsulfuron-resistantBrassicc hairy-root lines cultured with and without a selective level of chlorsulfuron. Areas of hairy-root research where this technique will be particularly useful are discussed.     

The effect of temperature on unsaturated fatty acid loss in Tetrahymena pyriformis.

Cultures of Tetrahymena pyriformis W incorporate exogenous 3-[14C]-cilienic acid and gamma-[1(-14)C] linolenic acid, terminal products of unsaturated fatty acid synthesis, into glycerophosphatides without randomization of the radiolabel. There was no difference in the rate of loss of each of the two acids at 15 or 28.5 degrees C. Differential turnover of these fatty acids, therefore, does not appear to be the cause of the shift in fatty acid pattern observed with temperature reduction.     

Lymphotoxin Alpha-Deficient Mice Clear Persistent Rotavirus Infection after Local Generation of Mucosal IgA

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα^−/−) mice that lack Peyer''s patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα^−/− and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα^−/− mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα^−/− mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.     

Endovascular Treatment with Stent-Retriever Devices for Acute Ischemic Stroke: A Meta-Analysis of Randomized Controlled Trials

Importance

Acute ischemic stroke is a leading cause of death and disability worldwide. Several recent clinical trials have shown that endovascular treatment improves clinical outcomes among patients with acute ischemic stroke.

Objective

To provide an overall and precise estimate of the efficacy of endovascular treatment predominantly using second-generation mechanical thrombectomy devices (stent-retriever devices) compared to medical management on clinical and functional outcomes among patients with acute ischemic stroke.

Data Sources

MEDLINE, EMBASE, Cochrane Collaboration Central Register of Controlled Clinical Trials, Web of Science, and NIH ClinicalTrials.gov were searched through November 2015.

Study Selection

Searches returned 3,045 articles. After removal of duplicates, two authors independently screened titles and abstracts to assess eligibility of 2,495 potentially relevant publications. From these, 38 full-text publications were more closely assessed. Finally, 5 randomized controlled trials of endovascular treatment with predominant use of retrievable stents were selected.

Data Extraction and Synthesis

Three authors independently extracted information on participant and trial characteristics and clinical events using a standardized protocol. Random effects models were used to pool endovascular treatment effects across outcomes.

Main Outcomes and Measures

The primary outcome was better functional outcome as measured on the modified Rankin Scale at 90 days of follow-up. Secondary outcomes included all-cause mortality and symptomatic intra-cerebral hemorrhage.

Results

Five trials representing 1,287 patients were included. Overall, patients randomized to endovascular therapy experienced 2.22 times greater odds of better functional outcome compared to those randomized to medical management (95% CI, 1.66 to 2.98; P < 0.0001). Endovascular therapy was not associated with mortality [OR (95% CI), 0.78 (0.54, 1.12); P = 0.1056] or symptomatic intracerebral hemorrhage [OR (95% CI), 1.19 (0.69, 2.05); P = 0.5348]. Meta-regression analysis suggested that shorter times from stroke onset to groin puncture and from stroke onset to reperfusion result in better functional outcomes in ischemic stroke patients (P = 0.0077 and P = 0.0089). There were no significant differences in the beneficial effects of endovascular treatment on functional outcomes across categories of gender, age, stroke severity, ischemic changes on computed tomography, or intravenous tissue plasminogen activator administration.

Conclusions and Relevance

This meta-analysis demonstrated superior functional outcomes in subjects receiving endovascular treatment compared to medical management. Further, this analysis showed that acute ischemic stroke patients may receive enhanced functional benefit from earlier endovascular treatment.     

Fault detection and diagnosis in an industrial fed-batch cell culture process

A flexible process monitoring method was applied to industrial pilot plant cell culture data for the purpose of fault detection and diagnosis. Data from 23 batches, 20 normal operating conditions (NOC) and three abnormal, were available. A principal component analysis (PCA) model was constructed from 19 NOC batches, and the remaining NOC batch was used for model validation. Subsequently, the model was used to successfully detect (both offline and online) abnormal process conditions and to diagnose the root causes. This research demonstrates that data from a relatively small number of batches (approximately 20) can still be used to monitor for a wide range of process faults.