Identifying genes in Fusarium verticillioides through forward and reverse genetics.

The increasing availability of sequenced genomes for plant pathogenic fungi has revolutionized molecular plant pathology in recent years. However, the genetic regulatory networks underlying many important components of pathogenesis remain poorly defined. Although the protocols outlined in this chapter can be utilized to identify genes regulating a wide range of biological processes in many filamentous fungi, we focus on describing how to identify genes through forward and reverse genetics, using the plant pathogenic fungus Fusarium verticillioides as a model for the protocol. Specifically, this chapter explains how to create a collection of insertional mutants via Restriction Enzyme Mediated Integration (REMI) and how to screen mutants with a high-throughput method to visualize defects in amylolysis. Next, techniques are described to define the genomic lesions in REMI mutants with genome-walker PCR in order to identify candidate genes. Finally, protocols are presented describing a reverse-genetic approach to disrupt candidate genes in the wild-type strain with a split-marker strategy to confirm the phenotype observed in the REMI mutant.

First Report of Rust Caused by Puccinia emaculata on Switchgrass in Arkansas.

In May 2007, switchgrass (Panicum virgatum L.) cv. Alamo and a breeding line, OSU-NSL 2001-1, were planted at the Arkansas Agricultural Research and Extension Center, Fayetteville. In August 2008, a high incidence of dark brown-to-black rectangular foliar lesions delineated by major veins was observed throughout plots of both lines. Lesions covered 25% to nearly 100% of total leaf tissue. Similar symptoms were also observed on unknown switchgrass cultivars in Benton County in northwest Arkansas and in St. Francis County in east-central Arkansas, suggesting that the disease was widely distributed throughout the state. The pathogen produced epiphyllous and adaxial masses of dark brown-to-black telia from erumpent fissures on leaf surfaces. Dark brown teliospores were observed under magnification and were two-celled, oblong to ellipsoid, and 33 ± 3.5 µm long with an apical cell width of 17.5 ± 2.7 µm and basal cell width of 16.2 ± 2.8 µm (reported as mean ± standard deviation, n = 25). Pedicles were colorless to light brown and measured 25.4 ± 9.2 µm (n = 25). In June 2009, at the Fayetteville Research and Extension Center, several second-year stands of switchgrass developed amphigenous and adaxial foliar lesions containing urediniospores. The uredia were globose and finely echinulate, measuring 23.1 ± 2.2 µm (n = 25) with brown cell walls. Teliospore and urediniospore morphology from all collections was consistent with Puccinia emaculata Schw. (2). Genomic DNA was extracted from a representative infected leaf of cv. Alamo, collected in Fayetteville, AR in June 2009, and amplified by PCR with primer sets PRITS1F (3) and ITS4B (1), which amplified an 803-bp fragment of rDNA encoding the first internal transcribed spacer (ITS1), 5.8S subunit, and second internal transcribed spacer (ITS2). The fragment was cloned into pGEM T Easy (Promega Corp, Madison, WI) and sequenced. A BLAST search of GenBank revealed that the fragment was most similar to the rDNA of P. emaculata (GenBank Accession No. EU915294.1; 755 of 758 bases matching; 99% identity) previously reported as a pathogen on switchgrass in Tennessee (3). The incidence and severity of rust on the widely planted switchgrass cv. Alamo is considerable cause for concern as efforts are made to increase acreage and production. Climatic conditions in St. Francis County are generally consistent with locations in Tennessee where switchgrass rust was previously reported (3). However, northwest Arkansas represents the eastern edge of the southwestern United States, suggesting that P. emaculata may affect switchgrass in geographically diverse areas of the United States. To our knowledge, this study represents the first report of rust on switchgrass in Arkansas. Managing this disease will be an important consideration for large-scale switchgrass cultivation in the state. References: (1) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (2) P. Ramachar and G. Cummins. Mycopathol. Mycol. Appl. 25:7, 1965. (3) J. Zale et al. Plant. Dis. 92:1710, 2008.

Entry overload, emergency department overcrowding, and ambulance bypass.

OBJECTIVES: To describe an experience of emergency department (ED) overcrowding and ambulance bypass. METHODS: A prospective observational study at Royal Perth Hospital, a major teaching hospital. Episodes of ambulance bypass and their characteristics were recorded. RESULTS: From 1 July 1999 to 30 June 2001, there were 141 episodes of ambulance bypass (mean duration 187 min, range 35-995). Monday was the most common day with 39 (28%) episodes. Entry block alone was the most common reason bypass was activated (n=38, 30.4%). The mean number of patients in ED at these times was 40 (occupancy 174%), including nine in the corridor, seven awaiting admission, and 14 waiting to be seen. Episodes attributable to entry block were typically preceded by a presentation rate of >/=10 patients per hour for >/=2 hours (OR 6.2, 95% CI 4.3 to 8.5). Mid-afternoon to early evening was the most common time for activation. Ambulance bypass is increasing in frequency and duration. CONCLUSIONS: Entry overload resulting in entry block results from overwhelming numbers of patients presenting to the ED in a short space of time. Entry block impairs access to emergency care. Unless something is done in the near future, the general public may no longer be able to rely on EDs for quality and timely emergency care. A "whole of system" approach is necessary to tackle the problem.

A humanized monoclonal antibody against respiratory syncytial virus (palivizumab) inhibits RSV-induced neurogenic-mediated inflammation in rat airways.

Respiratory syncytial virus (RSV) is the most important respiratory pathogen in infancy and early childhood and may predispose to subsequent lower respiratory tract illness. Recent data indicate that RSV up-regulates the substance P receptor, making the airways abnormally susceptible to the proinflammatory effects of this peptide released from sensory nerves. The present study was designed to determine whether the administration of RSV antibodies prevents the potentiation of neurogenic inflammation in rat airways. Five days after inoculation, sensory nerve-mediated extravasation of Evans blue-labeled albumin was significantly greater in the airways of RSV-infected rats than in pathogen-free controls. Polyclonal immune globulin enriched for RSV-neutralizing antibodies (RSVIG) reduced neurogenic extravasation when injected 24 h before intranasal inoculation of the virus but not when injected before endotracheal inoculation. A humanized MAb against RSV fusion protein (palivizumab) was twice as potent as RSVIG when given before intranasal inoculation and also caused significant inhibition after endotracheal inoculation. Furthermore, palivizumab inhibited neurogenic inflammation in RSV-infected rats when given 72 h after virus inoculation. These data suggest that palivizumab protects the respiratory tract from RSV-induced inflammation when given before or in the early phase of the viral infection. The administration of palivizumab to high-risk infants may limit the severity of the acute airway inflammation and may protect against subsequent lower respiratory tract illness.

Respiratory syncytial virus immune globulin treatment of lower respiratory tract infection in pediatric patients undergoing bone marrow transplantation - a compassionate use experience.

Respiratory syncytial virus (RSV) pneumonia in BMT recipients carries a mortality rate of approximately 50-70% despite ribavirin (Virazole) treatment. In both immunocompetent and immunocompromised animal models, RSV neutralizing antibodies rapidly reduce pulmonary virus load after a single dose. RSV-IGIV (RespiGam) is an IgG immune globulin with high concentrations of RSV neutralizing antibody (>19 200 MU/ml). From June 1991 to February 1996, a compassionate-use protocol using RSV-IGIV for treatment of RSV infections was conducted. Eleven children at multiple centers, mean age 3.3 years (4 months to 9 years), were undergoing BMT and met the protocol criteria. They received a single 1500 mg/kg dose of RSV-IGIV infused over 12 h at a median of 5 days (1-37 days) after RSV symptom onset. Ten of these patients received prior or concurrent aerosolized ribavirin. Serum RSV neutralizing titers were measured in five patients and showed a 3- to 30-fold increase 24 h after RSV-IGIV infusion. Adverse events were mild. One of 11 (9.1%) patients died from their RSV illness (91% RSV survival). In comparison to previously published reports, RSV-IGIV treatment of RSV pneumonia in BMT patients may increase survival above that in such patients treated with ribavirin alone. Bone Marrow Transplantation (2000) 25, 161-165.

The prognostic significance of serum viral load, codon 215 reverse transcriptase mutation and CD4+ T cells on progression of HIV disease in a double-blind study of thymopentin.

OBJECTIVE: To determine in asymptomatic HIV-infected subjects the prognostic value of virion reverse transcriptase (RT) codon 215 mutation, serum HIV RNA level, CD4+ T-cell count and immune complex dissociated (ICD) p24 level. The retrospective evaluation of thymopentin treatment effect on subjects in high risk groups for progression was a secondary objective. PARTICIPANTS: Zidovudine (ZDV)-experienced asymptomatic HIV-infected subjects (n = 352) who had been enrolled in a 48-week placebo-controlled double-blind trial of thymopentin treatment were studied. METHODS: Post hoc analyses were conducted to determine which subjects at study entry were at greater risk for progression to AIDS-related complex (ARC), AIDS or death, and to determine the effect of treatment on these subjects. Four potential prognostic variables (virion RT codon 215 mutation, circulating HIV virion RNA copies, CD4+ T-cell count, and ICD p24) were evaluated by dichotomizing subjects for each variable based on the median of the observed values. CD4+ T-cell count was evaluated prospectively, whereas frozen samples were evaluated under blinded conditions for the other variables after the study was completed. RESULTS: The presence of the codon 215 mutation [P = 0.044; relative hazard (RH), 2.6], > or = 20,000 HIV RNA copies/ml (P = 0.002; RH, 5.5), and < 350 CD4+ cells 10(6)/l (P = 0.042; RH, 2.2) were prognostic factors, and > or = 30 pg/ml ICD p24 level (P = 0.52; RH, 1.4) was not a prognostic factor in predicting progression. Subjects were prestratified by previous ZDV use (< or = 6 or > 6 months). Across both strata thymopentin delayed treatment progression to ARC, AIDS, or death (P = 0.015; RH, 3.0). This effect was magnified in the ZDV-experienced subjects at greater risk, where thymopentin delayed progression compared to placebo in the presence of the codon 215 mutation (P = 0.007; RH, 10.1), > or = 20,000 RNA copies/ml (P = 0.012; RH, 8.9), and CD4+ T-cell count < 350 x 10(6)/l (P = 0.005; RH, 10.4). CONCLUSIONS: Codon 215 mutation, serum HIV RNA and CD4 T-cell count are independent predictors of progression in ZDV-experienced asymptomatic subjects. Furthermore, thymopentin delays HIV disease progression in the presence of a key ZDV resistance mutation as well as high viral load and low CD4+ T-cell counts.

Safety and efficacy of thymopentin in zidovudine (AZT)-treated asymptomatic HIV-infected subjects with 200-500 CD4 cells/mm3: a double-blind placebo-controlled trial.

Thymopentin, 50 mg subcutaneously (s.c.) 3 times per week, was evaluated in a double-blind, randomized, placebo-controlled trial of zidovudine (AZT)-treated asymptomatic human immunodeficiency virus (HIV)-infected subjects with 200-500 CD4 cells/mm3 at entry. The 352 subjects were prestratified by prior AZT use into stratum I (235 subjects, > 6 months AZT at entry) and stratum II (117 subjects, < or = 6 months AZT at entry). Clinical end points, CD4 cell counts, serum p24, serum immune complex dissociated (ICD) p24, and safety variables were evaluated through 48 weeks, using an intent-to-treat analysis. The two strata were analyzed individually because they yielded different clinical outcomes, with a statistically significant treatment-by-stratum interaction. In stratum I (mean, 16 months AZT at entry) two AIDS or death events occurred in thymopentin and 10 in placebo recipients (p = 0.024; relative risk (RR) estimate, 4.9 [95% confidence limit (CI), 1.1 to 22.2]). There were three AIDS-related complex (ARC), AIDS, or death events in thymopentin and 18 in placebo recipients [p = 0.001; RR estimate, 5.9 (95% CI, 1.7 to 20.0)]. In stratum II (mean, 3 months AZT at entry), four AIDS or death events occurred in thymopentin and none in placebo recipients (p = 0.11), and four ARC, AIDS, or death events occurred in thymopentin and two in placebo recipients (p = 0.79). The treatment groups did not differ significantly with respect to changes in CD4 counts or p24 antigen levels or with respect to clinical adverse experiences or laboratory abnormalities. Thus, AZT-experienced placebo-treated subjects had relatively high progression rates to AIDS or death and to ARC, AIDS, or death, and these rates were reduced by thymopentin treatment. In contrast, placebo-treated subjects with little prior AZT experience had low progression rates; these were not significantly changed by thymopentin treatment. There was no increase in the incidence of adverse reactions with thymopentin.

Maintenance of CD4+ cells by thymopentin in asymptomatic HIV-infected subjects: results of a double-blind, placebo-controlled study.

OBJECTIVE: To assess the efficacy and safety of thymopentin in HIV-infected patients who had not yet developed AIDS. DESIGN: Patients were stratified into asymptomatic or symptomatic groups and randomized to receive either thymopentin (50 mg) or placebo, subcutaneously, double-blind for 24 or 52 weeks, three times a week. SETTING: Patients were enrolled at three sites (two hospital clinics and one private practice). PATIENTS: Of 91 HIV-seropositive patients (52 asymptomatic and 39 symptomatic) from whom HIV could be isolated from peripheral blood, 45 were enrolled for 24 weeks and 46 for 52 weeks of double-blind evaluation. MAIN OUTCOME MEASURES: Virological, immunological and clinical evaluations were performed before and during treatment. RESULTS: Thymopentin-treated asymptomatic patients had more CD4+ cells, as demonstrated by a greater area under the percentage CD4+ cells curve (P = 0.03) and a shorter median time to a 20% increase in percentage of CD4+ cells (P = 0.04) in the first 24 weeks, with similar trends in the 52-week study. By 24 weeks no asymptomatic thymopentin-treated and two placebo-treated patients (9.1%, Kaplan-Meier estimate) had progressed to constitutional symptoms (P = 0.12; two-tailed Wilcoxon-Gehan test), with only one further progression in a placebo-treated patient in the subset followed for 52 weeks. Symptomatic patients receiving thymopentin or placebo were similar in both CD4+ cell levels and disease progression (two progressions to AIDS in each group). No serious adverse effects attributable to thymopentin were observed. CONCLUSIONS: These results, if confirmed, indicate that thymopentin, by maintaining CD4+ cells, could slow or arrest immune decline and consequent disease progression at the asymptomatic stage of HIV infection.

Admissions for suspected snake bite to the Perth adult teaching hospitals, 1979 to 1988.

OBJECTIVE: To describe the epidemiology of snake bite in Perth, and the likelihood of envenomation. DESIGN: Information from case notes was retrospectively analysed. SETTING: Emergency medicine, teaching hospitals. PATIENTS: All patients admitted to the three adult teaching hospitals in Perth for suspected snake bite from 1979 to 1988. MAIN OUTCOME MEASURE: Systemic envenomation, was accepted as present if there were definite symptoms, signs or laboratory evidence (vomiting, abdominal pain, ptosis, convulsions, difficulty with breathing or swallowing, coagulopathy, haemolysis, rhabdomyolysis or renal failure). RESULTS: Ninety-nine patients were definitely bitten, with 53 envenomed, including three snake handlers. Thirty others may have been envenomed. Nearly half (44%) of the 82 patients with witnessed snake bite were envenomed. The dugite (Pseudonaja affinis) caused most cases of envenomation, most often producing coagulopathy only. The remainder were probably due to bites by the tiger snake (Notechis after occidentalis) and gwardar (Pseudonaja nuchalis), with one by a sea snake. The Commonwealth Serum Laboratories Snake Venom Detection Kit (VDK) enabled identification of the genus in 36% of definite cases of snake bite, and in 51% of cases of envenomation. It may occasionally produce false-positive results. The VDK is of greatest value in establishing the genus of snake in envenomed patients. CONCLUSIONS: It is suggested that a mixture of brown and tiger snake antivenom be used to treat patients envenomed by an unidentified snake in the Perth metropolitan area. This does not apply to patients bitten elsewhere in Western Australia or transferred to Perth from country regions where other snakes are more prevalent.