Major Susceptibility Gene Epistasis over Minor Gene Resistance to Spot Form Net Blotch in a Commercial Barley Cultivar.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant global disease of barley (Hordeum vulgare). Baudin, a barley cultivar that was until recently extensively grown in Western Australia, was reported as having minor seedling resistance. However, Baudin was highly susceptible to a local isolate, M3, suggesting that this isolate had gained virulence against a major susceptibility gene. M3 causes atypical lesions with pale centers early in the infection, with initial screens of a segregating population indicating that this was determined by a single locus in the Baudin genome. The susceptibility was semidominant in F1 progeny and the susceptibility gene, designated Spm1 (Susceptibility to P. teres f. maculata 1), mapped to a 190-kb section of the resistance gene-rich Mla region of chromosome 1H. Phenotyping with Ptm SP1, a non-M3 pathotype, identified a seedling resistance locus on 2H. Minor gene resistance is generally regarded as potentially durable, but our findings suggest the resistance to spot form net blotch in Baudin is nullified by strong susceptibility conferred by a separate locus on 1H. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Potentiators of Disease During Barley Infection by Pyrenophora teres f. teres in a Susceptible Interaction.

Pyrenophora teres f. teres is a necrotrophic fungal pathogen and causal agent of net form net blotch (NFNB), a significant disease of barley. RNA-seq data encompassing asymptomatic and subsequent necrotrophic phases of the pathogen was obtained for P. teres f. teres isolate W1-1 in NFNB-sensitive cultivar Baudin. Host genes notably regulated during infection included concerted induction of over half the repertoire of disease resistance genes, together with genes involved in oxidation-reduction processes, characteristic of a hypersensitive response. Several systemic acquired resistance response genes were suppressed and there was a complete absence of defense-related thionin gene expression. In P. teres f. teres, genes involved in hydrolase activities and cell-wall catabolic processes were induced during infection, while nitrate assimilation and response to oxidative stress processes were suppressed. Timecourse data allowed a number of predicted P. teres f. teres effector genes with differing expression profiles to be identified that may underlie barley sensitivity to NFNB. Candidate genes involved in the host-pathogen interaction provide a basis for functional characterization and control strategies based on fungicide or mutation targets, which will facilitate further research aimed at controlling NFNB disease.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Ultra-Processed Food Intake and Associations With Demographic Factors in Young New Zealand Children.

BACKGROUND: Children consume ultra-processed food (UPF) from a young age, but the proportional contribution of UPF to young children's total energy intakes has not been evaluated in developed countries. OBJECTIVES: To describe UPF intake and associations with demographic factors in young children from 12 to 60 months of age. DESIGN: Cohort study comprising a secondary analysis of data from a randomized controlled trial. Demographic data were collected by questionnaire. At 12, 24, and 60 months of age validated food frequency questionnaires estimated percentage of energy intake from UPF (%kcal UPF). PARTICIPANTS/SETTING: The 669 children were born in Dunedin, New Zealand, between May 2009 and December 2010. MAIN OUTCOME MEASURES: Mean percentage of energy intake from UPF at 12, 24, and 60 months of age, mean differences in %kcal UPF by demographic variables. STATISTICAL ANALYSES PERFORMED: Mixed effects regression models were used to estimate relationships between demographics and %kcal UPF. Multiple imputation methods were used to impute missing UPF data. RESULTS: UPF contributed mean (95% confidence interval) 45% (44%, 47%), 42% (41%, 44%), and 51% (50%, 52%) of energy intake to the diets of children at 12, 24, and 60 months of age, respectively. Energy intake from UPF was moderately correlated between 24 and 60 months (r = 0.36). No demographic factors were associated with mean %kcal UPF across time points, except for maternal obesity predicting higher UPF intake at 12 months. Bread, yoghurt, crackers, whole-wheat breakfast cereal, sausages, and muesli bars were among the 10 foods making the greatest contribution to mean %kcal UPF intakes at all time points. CONCLUSIONS: UPF contribute a substantial proportion of energy to the diets of young children. A range of foods with varying nutritional profiles contribute to these high intakes.

Reference Genome Assembly for Australian Ascochyta rabiei Isolate ArME14.

Ascochyta rabiei is the causal organism of ascochyta blight of chickpea and is present in chickpea crops worldwide. Here we report the release of a high-quality PacBio genome assembly for the Australian A. rabiei isolate ArME14. We compare the ArME14 genome assembly with an Illumina assembly for Indian A. rabiei isolate, ArD2. The ArME14 assembly has gapless sequences for nine chromosomes with telomere sequences at both ends and 13 large contig sequences that extend to one telomere. The total length of the ArME14 assembly was 40,927,385 bp, which was 6.26 Mb longer than the ArD2 assembly. Division of the genome by OcculterCut into GC-balanced and AT-dominant segments reveals 21% of the genome contains gene-sparse, AT-rich isochores. Transposable elements and repetitive DNA sequences in the ArME14 assembly made up 15% of the genome. A total of 11,257 protein-coding genes were predicted compared with 10,596 for ArD2. Many of the predicted genes missing from the ArD2 assembly were in genomic regions adjacent to AT-rich sequence. We compared the complement of predicted transcription factors and secreted proteins for the two A. rabiei genome assemblies and found that the isolates contain almost the same set of proteins. The small number of differences could represent real differences in the gene complement between isolates or possibly result from the different sequencing methods used. Prediction pipelines were applied for carbohydrate-active enzymes, secondary metabolite clusters and putative protein effectors. We predict that ArME14 contains between 450 and 650 CAZymes, 39 putative protein effectors and 26 secondary metabolite clusters.

Optimized plant compound with potent anti-biofilm activity across gram-negative species.

Many human diseases, including cystic fibrosis lung infections, are caused or exacerbated by bacterial biofilms. Specialized modes of motility, including swarming and twitching, allow gram-negative bacteria to spread across surfaces and form biofilms. Compounds that inhibit these motilities could slow the spread of biofilms, thereby allowing antibiotics to work better. We previously demonstrated that a set of plant-derived triterpenes, including oleanolic acid and ursolic acid, inhibit formation of Escherichia coli and Pseudomonas aeruginosa biofilms, and alter expression of genes involved in chemotaxis and motility. In the present study, we have prepared a series of analogs of oleanolic acid. The analogs were evaluated against clinical isolates of E. coli and P. aeruginosa in biofilm formation assays and swarming assays. From these analogs, compound 9 was selected as a lead compound for further development. Compound 9 inhibits E. coli biofilm formation at 4 µg/mL; it also inhibits swarming at ≤1 µg/mL across multiple clinical isolates of P. aeruginosa, E. coli, Burkholderia cepacia, and Salmonella enterica, and at <0.5 µg/mL against multiple agricultural strains. Compound 9 also potentiates the activity of the antibiotics tobramycin and colistin against swarming P. aeruginosa; this is notable, as tobramycin and colistin are inhaled antibiotics commonly used to treat P. aeruginosa lung infections in people with cystic fibrosis. qPCR experiments suggested that 9 alters expression of genes involved in regulating Type IV pili; western blots confirmed that expression of Type IV pili components PilA and PilY1 decreases in P. aeruginosa in the presence of 9.

Using compositional principal component analysis to describe children's gut microbiota in relation to diet and body composition.

BACKGROUND: Gut microbiota data obtained by DNA sequencing are complex and compositional because of large numbers of detectable taxa, and because microbiota characteristics are described in relative terms. Nutrition researchers use principal component analysis (PCA) to derive dietary patterns from food data. Although compositional PCA methods are not commonly used to describe patterns from complex microbiota data, this approach would be useful for identifying gut microbiota patterns associated with diet and body composition. OBJECTIVES: To use compositional PCA to describe the principal components (PCs) of gut microbiota in 5-y-old children and explore associations between microbiota components, diet, and BMI z-score. METHODS: A fecal sample was provided by 319 children aged 5 y. Their primary caregiver completed a validated 123-item quantitative FFQ. Body composition was determined using DXA, and a BMI z-score was calculated. Compositional PCA identified characterizing taxa and weightings for calculation of gut microbiota PC scores at the genus level, and was examined in relation to diet and body size. RESULTS: Three gut microbiota PCs were found. PC1 (negative loadings on uncultured Christensenellaceae and Ruminococcaceae) was related to lower BMI z-scores and longer duration of breastfeeding (per month) (ß = -0.14; 95% CI: -0.26, -0.02; and ß = 0.02; 95% CI: 0.003, 0.34, respectively). PC2 (positive loadings on Fusicatenibacter and Bifidobacterium; negative loadings on Bacteroides) was associated with a lower intake of nuts, seeds, and legumes (ß = -0.05 per gram; 95% CI: -0.09, -0.01). When adjusted for fiber intake, PC2 was also associated with higher BMI z-scores (ß = 0.12; 95% CI: 0.01, 0.24). PC3 (positive loadings on Faecalibacterium, Eubacterium, and Roseburia) was associated with higher intakes of fiber (ß = 0.02 per gram; 95% CI: 0.003, 0.04) and total nonstarch polysaccharides (ß = 0.02 per gram; 95% CI: 0.003, 0.04). CONCLUSIONS: Our results suggest that specific gut microbiota components determined using compositional PCA are associated with diet and BMI z-score.This trial was registered at clinicaltrials.gov as NCT00892983.

SQ1274, a novel microtubule inhibitor, inhibits ovarian and uterine cancer cell growth.

OBJECTIVE: Paclitaxel, a microtubule inhibitor, is subject to tumor resistance while treating high-grade serous ovarian and uterine cancer. This study aims to directly compare the effects of SQ1274, a novel microtubule inhibitor that binds to the colchicine-binding site on tubulin, and paclitaxel in high-grade serous ovarian and uterine cancer cell lines both in vitro and in vivo. METHODS: We assessed the sensitivity of ovarian (OVCAR8) and uterine (ARK1) cancer cell lines to SQ1274 and paclitaxel using XTT assays. We used western blot and quantitative real-time PCR to analyze changes in AXL RNA and protein expression by SQ1274 and paclitaxel. Differences in cell-cycle arrest and apoptosis were investigated using flow cytometry. Finally, we treated ovarian and uterine xenograft models with vehicle, paclitaxel, or SQ1274. RESULTS: First, we demonstrate that SQ1274 has a much lower IC50 than paclitaxel in both ARK1 (1.26 nM vs. 15.34 nM, respectively) and OVCAR8 (1.34 nM vs. 10.29 nM, respectively) cancer cell lines. Second, we show SQ1274 decreases both RNA and protein expression of AXL. Third, we show that SQ1274 causes increased cell-cycle arrest and apoptosis compared to paclitaxel. Finally, we report that SQ1274 more effectively inhibits tumor growth in vivo compared to paclitaxel. CONCLUSIONS: SQ1274 presents as a viable alternative to paclitaxel for treating ovarian and uterine cancer. This study supports the development of SQ1274 as a chemotherapeutic to treat ovarian and uterine cancer.

Bifidenone: Structure-Activity Relationship and Advanced Preclinical Candidate.

Bifidenone is a novel natural tubulin polymerization inhibitor that exhibits antiproliferative activity against a range of human cancer cell lines, making it an attractive candidate for development. A synthetic route was previously developed to alleviate supply constraints arising from its isolation in microgram quantities from a Gabonese tree. Using that previously published route, we present here 42 analogues that were synthesized to examine the structure-activity relationship of bifidenone derivatives. In addition to in vitro cytotoxicity data, data from murine xenograft and pharmacokinetic studies were used to evaluate the analogues. Compounds 45b and 46b were found to demonstrate promising efficacy in murine xenograft experiments, and 46b had significantly more potent in vitro antiproliferative activity against taxane-resistant cell lines compared to that of paclitaxel.

Transposable Element Genomic Fissuring in Pyrenophora teres Is Associated With Genome Expansion and Dynamics of Host-Pathogen Genetic Interactions.

Pyrenophora teres, P. teres f. teres (PTT) and P. teres f. maculata (PTM) cause significant diseases in barley, but little is known about the large-scale genomic differences that may distinguish the two forms. Comprehensive genome assemblies were constructed from long DNA reads, optical and genetic maps. As repeat masking in fungal genomes influences the final gene annotations, an accurate and reproducible pipeline was developed to ensure comparability between isolates. The genomes of the two forms are highly collinear, each composed of 12 chromosomes. Genome evolution in P. teres is characterized by genome fissuring through the insertion and expansion of transposable elements (TEs), a process that isolates blocks of genic sequence. The phenomenon is particularly pronounced in PTT, which has a larger, more repetitive genome than PTM and more recent transposon activity measured by the frequency and size of genome fissures. PTT has a longer cultivated host association and, notably, a greater range of host-pathogen genetic interactions compared to other Pyrenophora spp., a property which associates better with genome size than pathogen lifestyle. The two forms possess similar complements of TE families with Tc1/Mariner and LINE-like Tad-1 elements more abundant in PTT. Tad-1 was only detectable as vestigial fragments in PTM and, within the forms, differences in genome sizes and the presence and absence of several TE families indicated recent lineage invasions. Gene differences between P. teres forms are mainly associated with gene-sparse regions near or within TE-rich regions, with many genes possessing characteristics of fungal effectors. Instances of gene interruption by transposons resulting in pseudogenization were detected in PTT. In addition, both forms have a large complement of secondary metabolite gene clusters indicating significant capacity to produce an array of different molecules. This study provides genomic resources for functional genetics to help dissect factors underlying the host-pathogen interactions.

Anticipatory guidance to prevent infant sleep problems within a randomised controlled trial: infant, maternal and partner outcomes at 6 months of age.

OBJECTIVE: To evaluate the effectiveness of sleep education delivered antenatally and at 3 weeks postpartum to prevent infant sleep problems at 6 months of age. DESIGN: Sleep intervention within a randomised controlled trial for the Prevention of Overweight in Infancy (POI) study. PARTICIPANTS: 802 families were randomly allocated to one of four groups: usual care (control), sleep intervention (sleep), food, activity and breastfeeding intervention (FAB), and combined group receiving both interventions (combination). INTERVENTIONS: All groups received standard Well Child care. The sleep intervention groups (sleep and combination) received an antenatal group education session (all mothers and most partners) emphasising infant self-settling and safe sleeping, and a home visit at 3 weeks reinforcing the antenatal sleep education. FAB and combination groups received four contacts providing education and support on breast feeding, food and activity up to 4 months postpartum. OUTCOME MEASURES: Here we report secondary sleep outcomes from the POI study: the prevalence of parent-reported infant sleep problems and night waking, and differences in sleep duration. Additional outcomes reported include differences in infant self-settling, safe sleep practices, and maternal and partner reports of their own sleep, fatigue and depression symptoms. RESULTS: Linear or mixed linear regression models found no significant intervention effects on sleep outcomes, with 19.1% of mothers and 16.6% of partners reporting their infant's sleep a problem at 6 months. Actigraphy estimated the number of night wakings to be significantly reduced (8%) and the duration of daytime sleep increased (6 min) in those groups receiving the sleep intervention compared with those who did not. However, these small differences were not clinically significant and not observed in 24 hours infant sleep diary data. No other differences were observed. CONCLUSION: A strategy delivering infant sleep education antenatally and at 3 weeks postpartum was not effective in preventing the development of parent-reported infant sleep problems.

Isolation and Identification of the Novel Tubulin Polymerization Inhibitor Bifidenone.

The pursuit of structurally novel compounds has led to the isolation of a series of neolignans (2-6), for which the structures have been determined from microgram quantities using microcryoprobe NMR technology. Compounds 2-6 provided some unexpectedly clear structure-activity relationship data, with compound 2 demonstrating significantly more potency in the in vitro cytotoxicity assay than the other analogues. Further screening found that compound 2 induces apoptosis with activation of caspase 3/7. The NCI Compare algorithm suggested that compound 2 acts through the inhibition of tubulin/microtubule dynamics. Compound 2 was confirmed to be a tubulin polymerization inhibitor that binds directly to tubulin.

Eating frequency in relation to BMI in very young children: a longitudinal analysis.

OBJECTIVE: Eating less frequently is associated with increased obesity risk in older children but data are potentially confounded by reverse causation, where bigger children eat less often in an effort to control their weight. Longitudinal data, particularly in younger children, are scarce. We aimed to determine whether eating frequency (meals and snacks) at 2 years of age is associated with past, current or subsequent BMI. DESIGN: Cohort analysis of a randomised controlled trial. Eating frequency at 2 years of age was estimated using 48 h diaries that recorded when each child ate meals and snacks (parent-defined) in five-minute blocks. Body length/height and weight were measured at 1, 2 and 3·5 years of age. Linear regression assessed associations between the number of eating occasions and BMI Z-score, before and after adjustment for potential confounding variables. SETTING: Prevention of Overweight in Infancy (POI) study, Dunedin, New Zealand. SUBJECTS: Children (n 371) aged 1-3·5 years. RESULTS: On average, children ate 5·5 (sd 1·2) times/d at 2 years of age, with most children (88-89 %) eating 4-7 times/d. Eating frequency at 2 years was not associated with current (difference in BMI Z-score per additional eating occasion; 95 % CI: -0·02; -0·10, 0·05) or subsequent change (0·02; -0·03, 0·06) in BMI. Similarly, BMI at age 1 year did not predict eating frequency at 2 years of age (difference in eating frequency per additional BMI Z-score unit; 95 % CI: -0·03; -0·19, 0·13). CONCLUSIONS: Number of eating occasions per day was not associated with BMI in young children in the present study.

Three-year follow-up of a randomised controlled trial to reduce excessive weight gain in the first two years of life: protocol for the POI follow-up study.

BACKGROUND: The Prevention of Overweight in Infancy (POI) study was a four-arm randomised controlled trial (RCT) in 802 families which assessed whether additional education and support on sleep (Sleep group); food, physical activity and breastfeeding (FAB group); or both (Combination group), reduced excessive weight gain from birth to 2 years of age, compared to usual care (Control group). The study had high uptake at recruitment (58 %) and retention at 2 years (86 %). Although the FAB intervention produced no significant effect on BMI or weight status at 2 years, the odds of obesity were halved in those who received the sleep intervention, despite no apparent effect on sleep duration. We speculate that enhanced self-regulatory behaviours may exist in the Sleep group. Self-regulation was not measured in our initial intervention, but extensive measures have been included in this follow-up study. Thus, the overall aim of the POI follow-up is to determine the extent to which augmented parental support and education on infant sleep, feeding, diet, and physical activity in the first 2 years of life reduces BMI at 3.5 and 5 years of age, and to determine the role of self-regulation in any such relationship. METHODS/DESIGN: We will contact all 802 families and seek renewed consent to participate in the follow-up study. The families have received no POI intervention since the RCT finished at 2 years of age. Follow-up data collection will occur when the children are aged 3.5 and 5 years (i.e. up to 3 years post-intervention). Outcomes of interest include child anthropometry, body composition (DXA scan), diet (validated food frequency questionnaire), physical activity (accelerometry), sleep (questionnaire and accelerometry), and self-regulation (questionnaires and neuropsychological assessment). DISCUSSION: Our follow-up study has been designed primarily to enable us to determine whether the intriguing benefit of the sleep intervention suggested at 2 years of age remains as children approach school age. However, cohort analyses will also investigate how BMI, self-regulation, and sleep consolidation develop during the early years. This information will be valuable to researchers and policy makers progressing the field of early childhood obesity prevention. TRIAL REGISTRATION: ClinicalTrials.gov number NCT00892983 .

Impact of an early-life intervention on the nutrition behaviors of 2-y-old children: a randomized controlled trial.

BACKGROUND: Despite an extensive well-child health service, 30% of New Zealand's 2- to 4-y old children are overweight or obese. This suggests that additional intervention is necessary to establish healthy nutrition behaviors. OBJECTIVE: The aim of this study was to assess the effect of intervention from 0 to 18 mo of age on food and nutrient intake, eating behaviors, and parental feeding practices in 18- to 24-mo-old children. DESIGN: In total, 802 families with healthy infants were randomly allocated to 1 of 4 groups: Usual Care (UC); Food, Activity, and Breastfeeding (FAB); Sleep; or FAB and Sleep (Combination). All groups received standard "well-child" care. The FAB intervention comprised 7-8 additional contacts for education and support around breastfeeding, food, and activity. The Sleep intervention comprised 2 additional contacts for guidance about sleeping habits. Combination families received both interventions. A validated food-frequency questionnaire assessed food intake at 2 y. A questionnaire assessed eating behaviors and parental feeding practices at 18 and 24 mo. RESULTS: At 2 y, there were no statistically significant differences in food and nutrient intake or eating behaviors in the groups receiving the FAB intervention (FAB, Combination; 325 children) compared with the groups who did not (Sleep, UC; 341 children). With the use of a 5-point scale, small but statistically significant differences in parental feeding practices were observed in the groups receiving the FAB intervention: greater child control over eating (difference: 0.14; 95% CI: 0.02, 0.26) and less pressure to eat (difference: 0.18; 95% CI: 0.04, 0.32) at 18 mo, as well as greater encouragement of nutrient-dense foods at 24 mo (difference: 0.16; 95% CI: 0.03, 0.30). No statistically significant differences were observed between the groups who received the Sleep intervention (Sleep, Combination; 313 children) and those who did not, except higher meat intake in the former (11 g/d). CONCLUSION: Additional education and support for parents from birth did not improve nutrition behaviors in this population at 2 y of age. This trial was registered at clinicaltrials.gov as NCT00892983.

Lactation Consultant Support from Late Pregnancy with an Educational Intervention at 4 Months of Age Delays the Introduction of Complementary Foods in a Randomized Controlled Trial.

BACKGROUND: Although the WHO recommends that complementary feeding in infants should begin at 6 mo of age, it often begins before this in developed countries. OBJECTIVE: Our objective was to determine whether lactation consultant (LC) support, with educational resources given at 4-mo postpartum, can delay the introduction of complementary foods until around 6 mo of age. METHODS: A total of 802 mother-infant pairs were recruited from the single maternity hospital serving Dunedin, New Zealand (59% response rate) and randomly assigned to the following: 1) usual care (control group); 2) infant sleep education intervention (Sleep); 3) food, activity, and breastfeeding intervention (FAB); or 4) combination (both) intervention (Combo). Certified LCs delivered 3 intervention sessions (late pregnancy and 1-wk and 4-mo postpartum). The 4-mo contact used educational resources focused on developmental readiness for complementary foods. Age when complementary foods were introduced was obtained from repeated interviews (monthly from 3- to 27-wk postpartum). RESULTS: A total of 49.5% and 87.2% of infants received complementary foods before 5 and 6 mo of age, respectively. There was evidence of group differences in the number of infants introduced to complementary foods before 5 mo (P = 0.006), with those receiving support and resources (FAB and Combo groups combined; 55.6%) more likely to wait until at least 5 mo compared with controls (control and Sleep groups combined; 43.3%) (OR: 1.52; 95% CI: 1.08, 2.16). However, there was no evidence they were more likely to wait until 6 mo of age (P = 0.52). Higher maternal age, higher parity, and a less positive attitude toward breastfeeding were positively associated, and drinking alcohol during pregnancy was negatively associated, with later age of introduction of complementary foods. CONCLUSIONS: Providing an LC and educational resources at 4-mo postpartum to predominantly well-educated, mainly European, women can delay the introduction of complementary foods until 5 mo of age, but not until the WHO recommendation of 6 mo. This trial was registered at clinicaltrials.gov as NCT00892983.

Antibacterial chromene and chromane stilbenoids from Hymenocardia acida.

Six chromene stilbenoids and one chromane stilbenoid were isolated from the African tree Hymenocardia acida. Several were moderately active against methicillin-resistant Staphylococcus aureus clinical isolate MRSA-108, including hymenocardichromanic acid, which was active at 8 µg/ml. None had IC50 values <20 µM in antiproliferation assays against several human cancer cell lines.

Phenylpropanoids from Phragmipedium calurum and their antiproliferative activity.

Seven stilbenes and one alkylresorcinol were isolated from the orchid Phragmipedium calurum during a screen for anticancer compounds. They were evaluated for antiproliferative activity against multiple human cancer cell lines, and two displayed moderate activity against several cell lines.

Construction and screening of attenuated ΔphoP/Q Salmonella typhimurium vectored plague vaccine candidates.

Preclinical studies evaluating plague vaccine candidates have demonstrated that the F1 and V protein antigens of Yersinia pestis confer protection against challenge from virulent strains. Live-attenuated ΔphoP/Q Salmonella typhimurium recombinants were constructed expressing either F1, V antigens, F1 and V antigens, or a F1-V fusion from Asd (+) balanced-lethal plasmids. To improve antigen delivery, genes encoding plague antigens were modified in order to localize antigens to specific bacterial cellular compartments which include cytoplasmic, outer membrane, or secreted. Candidate vaccine strains were evaluated for growth characteristics, full-length lipopolysaccharide (LPS), plasmid stability, and antigen expression in vitro. Plague vaccine candidate strains with favorable in vitro profiles were evaluated in murine or rabbit preclinical oral immunogenicity studies. Attenuated S. typhimurium strains expressing cytoplasmically localized F1-V and V antigen antigens were more immunogenic than strains that secreted or localized plague antigens to the outer membrane. In particular, S. typhimurium M020 and M023, which express Asd(+)-plasmid derived soluble F1-V and soluble V antigen, respectively, at high levels in the bacterial cell cytoplasm were found to induce the highest levels of plague-specific serum antibodies. To further evaluate balanced-lethal plasmid retention capacity, ΔphoP/Q S. typhimurium PurB(+) and GlnA(+) balanced-lethal plasmid systems harboring F1-V were compared with M020 in vitro and in BALB/c mice in a immunogenicity study. Although there was no detectable difference in plague antigen expression in vitro, S. typhimurium M020 was the most immunogenic plague antigen vector strain evaluated, inducing high-titer serum IgG antibodies specific against F1, V and F1-V.

Isolation of apoptosis-inducing stilbenoids from four members of the Orchidaceae family.

High-throughput natural product research produced a suite of anticancer hits among several species of the Orchidaceae family (Oncidium microchilum, O. isthmi, and Myrmecophila humboldtii). A commercial Oncidium sp. was also examined as a convenient source of additional material. Isolation and structure elucidation led to the identification of fifteen stilbenoids including a new phenanthraquinone and two new dihydrostilbenes. NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7-mm MicroCryoProbe or a 5-µL CapNMR capillary microcoil. Several compounds inhibited proliferation of NCI-H460 and M14 cancer cell lines. All compounds were also examined for their ability to induce apoptosis. Apoptosis induction was determined by measuring caspase 3/7 activation and LDH release in a NCI-H460 cell line. Based on these results, a portion of the extract from a commercially available Oncidium sp. was chemically modified in an attempt to obtain additional phenanthraquinones.

Polyoxygenated cyclohexene derivatives from Monanthotaxis congoensis.

A phytochemical investigation of Monanthotaxis congoensis afforded eight polyoxygenated cyclohexenes as well as the known crotepoxide. Structures were determined using NMR, MS, and optical rotation analyses. One compound displayed moderate antiproliferative activity against NCI-H460 and M14 cancer cells.