Determination of the effect of JO146, a CtHtrA inhibitor, on the chlamydial developmental cycle and persistence

The role of Chlamydia trachomatis HtrA (CtHtrA) for the growth and pathogenesis of this organism is presented. Inhibition of CtHtrA led to loss of infectious progeny in Chlamydia, particularly during heat stress or recovery from penicillin persistence. Isolation of CtHtrA inhibitor resistant mutants identified positive selection for mutants in fatty acid related pathways. Importantly, HtrA inhibition was effective against clinical isolates of C. trachomatis. Thus the findings combined indicate that CtHtrA is an important chlamydial growth factor that has the potential to be targeted for the development of anti-chlamydial drugs in the future.

LibVM: An architecture for shared library sandboxing

Many software applications extend their functionality by dynamically loading libraries into their allocated address space. However, shared libraries are also often of unknown provenance and quality and may contain accidental bugs or, in some cases, deliberately malicious code. Most sandboxing techniques which address these issues require recompilation of the libraries using custom tool chains, require significant modifications to the libraries, do not retain the benefits of single address-space programming, do not completely isolate guest code, or incur substantial performance overheads. In this paper we present LibVM, a sandboxing architecture for isolating libraries within a host application without requiring any modifications to the shared libraries themselves, while still retaining the benefits of a single address space and also introducing a system call inter-positioning layer that allows complete arbitration over a shared library’s functionality. We show how to utilize contemporary hardware virtualization support towards this end with reasonable performance overheads and, in the absence of such hardware support, our model can also be implemented using a software-based mechanism. We ensure that our implementation conforms as closely as possible to existing shared library manipulation functions, minimizing the amount of effort needed to apply such isolation to existing programs. Our experimental results show that it is easy to gain immediate benefits in scenarios where the goal is to guard the host application against unintentional programming errors when using shared libraries, as well as in more complex scenarios, where a shared library is suspected of being actively hostile. In both cases, no changes are required to the shared libraries themselves.

Role of polarization in probing anomalous gauge interactions of the Higgs boson

We explore the use of polarized e(+)/e(-) beams and/or the information on final state decay lepton polarizations in probing the interaction of the Higgs boson with a pair of vector bosons. A model independent analysis of the process e(+)e(-) -> f (f) over barH, where f is any light fermion, is carried out through the construction of observables having identical properties under the discrete symmetry transformations as different individual anomalous interactions. This allows us to probe an individual anomalous term independent of the others. We find that initial state beam polarization can significantly improve the sensitivity to CP-odd couplings of the Z boson with the Higgs boson (ZZH). Moreover, an ability to isolate events with a particular tau helicity, with even 40% efficiency, can improve sensitivities to certain ZZH couplings by as much as a factor of 3. In addition, the contamination from the ZZH vertex contributions present in the measurement of the trilinear Higgs-W (WWH) couplings can be reduced to a great extent by employing polarized beams. The effects of initial state radiation and beamstrahlung, which can be relevant for higher values of the beam energy are also included in the analysis.

Analysis of a Genomic DNA Expression Library of Mycobacterium tuberculosis Using Tuberculosis Patient Sera: Evidence for Modulation of Host Immune Response

DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments, The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins, Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, others whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients, This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression.

Development of temperature-tolerant strains of Thiobacillus ferrooxidans to improve bioleaching kinetics

An isolate of Thiobacillus ferrooxidans derived from gold mine water samples was repeatedly subcultured at increasing temperatures (from 30 degrees to 42 degrees C) in 9K medium. The temperature-adapted strain was found to be more efficient in the bioleaching of pyrite mineral than the wild type. When temperature-tolerant strains were cultured repeatedly in 9K medium at 30 degrees C, the temperature tolerance was completely lost, These results indicate that the temperature tolerance was stress-dependent and not a permanent trait of the adapted strain, The potential utility of such temperature-tolerant strains of Thiobacillus ferrooxidans in sulphide mineral dissolution is demonstrated.

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

Presence of Nicotinamide Adenine Dinucleotide–Independent Haemophilus paragallinarum in Mexico

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent—growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3 wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NADindependent H. paragallinarum of serovar B has been reported. Abbreviations: NAD ¼ nicotinamide adenine dinucleotide; NAM ¼ nicotinamide; PCR ¼ polymerase chain reaction; TM ¼ complete growth medium without chicken serum or nicotinamide adenine dinucleotide; TM/SN ¼ complete growth medium that contains both chicken serum and nicotinamide adenine dinucleotide

Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardmentwith the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov.

[Pasteurella] trehalosi is an important pathogen of sheep, being primarily associated with serious systemic infections in lambs but also having an association with pneumonia. The aim of the present investigation was to characterize a broad collection of strains tentatively identified as [P.] trehalosi in order to reclassify and rename this taxon to support improvements in our understanding of the pathogenesis and epidemiology of this important organism. The type strain for [P.] trehalosi, strain NCTC 10370T, was included along with 42 field isolates from sheep (21), cattle (14), goats (1), roe deer (3) and unknown sources (3). An extended phenotypic characterization was performed on all 43 strains. Amplified fragment length polymorphism (AFLP) was also performed on the isolates. Two of the field isolates were subjected to 16S rRNA gene sequencing. These sequences, along with five existing sequences for [P.] trehalosi strains and 12 sequences for other taxa in the family Pasteurellaceae, were subjected to a phylogenetic analysis. All the isolates and the reference strains were identified as [P.] trehalosi. A total of 17 out of 22 ovine isolates produced acid from all glycosides, while only four out of 14 bovine isolates produced acid from all glycosides. All 22 ovine isolates were haemolytic and CAMP-positive, while no other isolate was haemolytic and only two bovine isolates were CAMP-positive. Nineteen AFLP types were found within the [P.] trehalosi isolates. All [P.] trehalosi isolates shared at least 70% similarity in AFLP patterns. The largest AFLP type included the type strain and 7 ovine field isolates. Phylogenetic analysis indicated that the seven strains studied (two field isolates and the five serovar reference strains) are closely related, with 98.6% or higher 16S rRNA gene sequence similarity. As both genotypic and phenotypic testing support the separate and distinct nature of these organisms, we propose the transfer of [P.] trehalosi to a new genus, Bibersteinia, as Bibersteinia trehalosi comb. nov. The type strain is NCTC 10370T (=ATCC 29703T). Bibersteinia trehalosi can be distinguished from the existing genera of the family by the observation of only nine characteristics; catalase, porphyrin, urease, indole, phosphatase, acid from dulcitol, (+)-D-galactose, (+)-D mannose and (+)-D-trehalose.

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

Comparative growth and pathogenicity of geographical isolates of Sclerotinia sclerotiorum on lentil genotypes

Isolates of Sclerotinia sclerotiorum were collected from infected lentil plants from 2 agro-ecological zones of Syria and used to study their comparative growth on culture media and pathogenicity on different lentil genotypes. The growth studies were carried out on Potato Dextrose Agar (PDA) growth media under laboratory conditions. Mycelial radial growth and sclerotial production were the parameters used to compare the isolates. Pathogenicity studies were carried out with selected isolates on 10 lentil genotypes, infected as detached shoots and as whole potted-plants in the plastic house. The isolates showed considerable variation in cultural characteristics through mycelial growth, mycelial pigmentation and sclerotial production in the media plates. There were significant differences in the growth and sclerotial production of most of the isolates, but no apparent correlation between mycelial growth and sclerotial production among the isolates. Genotype by isolate interactions was significant for the isolates tested for pathogenicity. These interactions, however, appeared to be caused by differences in virulence of the isolates and did not suggest the occurrence of distinct pathogenic races of the pathogen isolates.

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed.

Phytogeny of the Quambalariaceae fam. nov., including important Eucalyptus pathogens in South Africa and Australia

The genus Quambalaria consists of plant-pathogenic fungi causing disease on leaves and shoots of species of Eucalyptus and its close relative, Corymbia. The phylogenetic relationship of Quambalaria spp., previously classified in genera such as Sporothrix and Ramularia, has never been addressed. It has, however, been suggested that they belong to the basidiomycete orders Exobasidiales or Ustilaginales. The aim of this study was thus to consider the ordinal relationships of Q. eucalypti and Q. pitereka using ribosomal LSU sequences. Sequence data from the ITS nrDNA were used to determine the phylogenetic relationship of the two Quambalaria species together with Fugomyces (= Cerinosterus) cyanescens. In addition to sequence data, the ultrastructure of the septal pores of the species in question was compared. From the LSU sequence data it was concluded that Quambalaria spp. and F. cyanescens form a monophyletic clade in the Microstromatales, an order of the Ustilaginomycetes. Sequences from the ITS region confirmed that Q. pitereka and Q. eucalypti are distinct species. The ex-type isolate of F. cyanescens, together with another isolate from Eucalyptus in Australia, constitute a third species of Quambalaria, Q. cyanescens (de Hoog & G.A. de Vries) Z.W. de Beer, Begerow & R. Bauer comb. nov. Transmission electron-microscopic studies of the septal pores confirm that all three Quambalaria spp. have dolipores with swollen lips, which differ from other members of the Microstromatales (i.e. the Microstromataceae and Volvocisporiaceae) that have simple pores with more or less rounded pore lips. Based on their unique ultrastructural features and the monophyly of the three Quambalaria spp. in the Microstromatales, a new family, Quambalariaceae Z.W. de Beer, Begerow & R. Bauer fam. nov., is described.

Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n=25) and Brachyspira pilosicoli (n=17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC >4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC >32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.

Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

The leader protease (L-pro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968-2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups - Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (<5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or onvergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the L-pro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the L-pro (P<0.05; 0.046*) and at aa 171 in the capsid protein VP1 (P<0.01; 0.003**).