Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.     

The Magnetohydrodynamic Stagnation Point Flow of a Nanofluid over a Stretching/Shrinking Sheet with Suction

The magnetohydrodynamic (MHD) stagnation point flow of a nanofluid over a permeable stretching/shrinking sheet is studied. Numerical results are obtained using boundary value problem solver bvp4c in MATLAB for several values of parameters. The numerical results show that dual solutions exist for the shrinking case, while for the stretching case, the solution is unique. A stability analysis is performed to determine the stability of the dual solutions. For the stable solution, the skin friction is higher in the presence of magnetic field and increases when the suction effect is increased. It is also found that increasing the Brownian motion parameter and the thermophoresis parameter reduces the heat transfer rate at the surface.     

Influence of type of explant,plant growth regulators,salt composition of basal medium,and light on callogenesis and regeneration in <Emphasis Type="Italic">Passiflora suberosa</Emphasis> L. (Passifloraceae)

Passiflora suberosa is used in popular medicine, improvement programs, and as an ornamental plant. The goal of this study was to establish efficient protocols for plant regeneration and callus induction from nodal, internodal and leaf segments excised from in vitro-grown plants. The different morphogenetic responses were modulated by the type and concentration of plant growth regulators, according to the basal medium and light conditions. Shoot formation occurred through three pathways: (1) development of preexisting meristems, (2) direct organogenesis, and (3) indirect organogenesis. Development of preexisting meristems was observed from nodal segments (1 shoot/explant) in response to α-naphthaleneacetic acid (NAA), picloram (PIC), and 2,4-dichlorophenoxyacetic acid (2,4-D), using two basal media (MS and MSM). Direct organogenesis in this species was obtained for the first time in this work, through shoot development from internodal segments in the presence of 6-benzyladenine (BA). The highest regeneration rates were achieved on MSM medium, regardless of the BA concentration. Indirect organogenesis was achieved from all explant types on media supplemented with BA, used alone or in combination with NAA. The highest regeneration efficiency was obtained from internodal segments cultured on MSM medium plus 44.4 μM BA. Compact, friable, or mucilaginous non-morphogenic calluses were induced by thidiazuron, PIC, 2,4-D, and NAA. High-yielding friable calluses obtained on MSM medium supplemented with 28.9 μM PIC are being used for the establishment of suspension cultures and further analysis of the production of bioactive compounds.     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

Effect of ivermectin on the cellular and humoral immune responses of rabbits

The objective of this paper is to determine the effect of ivermectin administration on cell mediated (CMI) and humoral immunity (HI) of rabbits. CMI against dinitrochlorobenzene (DNCB) and sheep red blood cells (SRBC) in rabbits was determined by delayed-type hypersensitivity and macrophage engulfment assay (MEA), respectively; whereas, HI to Pasteurella multocida B2 vaccine and SRBC was determined by indirect haemagglutination assay (IHA) and Jerne hemolytic plaque formation assay (JHPFA), respectively. The rabbits were divided into four major groups (A through D) each subdivided into four sub-groups (1 through 4). Rabbits of group A served as vehicle control while those of groups B, C and D were treated with ivermectin at the dose rates of 200 microg/kg, 400 microg/kg and 600 microg/kg b.w., respectively. Cellular immunity was determined in sub-groups 1 and 2 through DNCB and MEA, respectively while HI was determined in sub-groups 3 and 4 through IHA and JHPFA, respectively. The skin sensitivity to DNCB at 24 and 48 h and macrophage engulfment of SRBC were highest (P>0.05) in rabbits administered with 600 microg/kg b.w. The highest geometric mean titers (14.00+/-0.31) and number of plaque forming units (1860+/-0.75) were found in rabbits that received ivermectin at a dose of 600 microg/kg b.w. followed, in order by the groups that received 400 microg/kg, 200 microg/kg b.w. and controls. Leukocyte counts were significantly higher in ivermectin-treated groups (C and D) than group A (vehicle control) and B (ivermectin at the rate of 200 microg/kg). A graded dose immune response suggested an immunopotentiating effect of ivermectin at higher doses.     

Determination of antioxidant activity and phenolic content of extracts from in vivo plants and in vitro materials of Passiflora alata Curtis

Passiflora alata Curtis, commonly known as sweet passion fruit, is one of the commercially cultivated species of the genus Passiflora, whose fruits can be consumed in natura due to their sweet taste. It is also used worldwide as an ornamental and in folk medicine. The goal of this work was the evaluation of the antioxidant potential of extracts from in vivo plants, and in vitro-derived materials of P. alata. Leaves from in vivo plants were used for the optimization of parameters that affect the efficiency of extraction of antioxidant compounds (proportions of ethanol:water, maceration period, solvent:plant tissue ratio, and number of extraction stages), by employing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant activity and the extract yields were significantly influenced by the proportion of ethanol:water and maceration period. The optimized protocol was applied to obtain the extracts of in vitro-derived materials. Total phenolic content was determined using the Folin–Ciocalteu method. Higher antioxidant activities and phenolic contents were observed in extracts from leaves of in vivo-seed derived and from acclimatized plants when compared to in vitro plants, calluses and suspension cultures. Differences in the reaction kinetics of DPPH scavenging activity were also observed.     

Statistical Cluster Analysis of the British Thoracic Society Severe Refractory Asthma Registry: Clinical Outcomes and Phenotype Stability

Background

Severe refractory asthma is a heterogeneous disease. We sought to determine statistical clusters from the British Thoracic Society Severe refractory Asthma Registry and to examine cluster-specific outcomes and stability.

Methods

Factor analysis and statistical cluster modelling was undertaken to determine the number of clusters and their membership (N = 349). Cluster-specific outcomes were assessed after a median follow-up of 3 years. A classifier was programmed to determine cluster stability and was validated in an independent cohort of new patients recruited to the registry (n = 245).

Findings

Five clusters were identified. Cluster 1 (34%) were atopic with early onset disease, cluster 2 (21%) were obese with late onset disease, cluster 3 (15%) had the least severe disease, cluster 4 (15%) were the eosinophilic with late onset disease and cluster 5 (15%) had significant fixed airflow obstruction. At follow-up, the proportion of subjects treated with oral corticosteroids increased in all groups with an increase in body mass index. Exacerbation frequency decreased significantly in clusters 1, 2 and 4 and was associated with a significant fall in the peripheral blood eosinophil count in clusters 2 and 4. Stability of cluster membership at follow-up was 52% for the whole group with stability being best in cluster 2 (71%) and worst in cluster 4 (25%). In an independent validation cohort, the classifier identified the same 5 clusters with similar patient distribution and characteristics.

Interpretation

Statistical cluster analysis can identify distinct phenotypes with specific outcomes. Cluster membership can be determined using a classifier, but when treatment is optimised, cluster stability is poor.     

Alterations in architecture and metabolism induced by ultraviolet radiation-B in the carragenophyte <Emphasis Type="Italic">Chondracanthus teedei</Emphasis> (Rhodophyta,Gigartinales)

The in vivo effect of ultraviolet radiation-B (UVBR) in apical segments of Chondracanthus teedei was examined. Over a period of 7 days, the segments were cultivated and exposed to photosynthetically active radiation (PAR) at 80 μmol photons m⁻² s⁻¹ and PAR + UVBR at 1.6 W m⁻² for 3 h per day. The samples were processed for electron microscopy and histochemistry; also was analyzed growth rates, mitochondrial activity, protein levels, content of photosynthetic pigments and photosynthetic performance. UVBR elicited increased cell wall thickness and accumulation of plastoglobuli, changes in mitochondrial organization and destruction of chloroplast internal organization. Compared to controls, algae exposed to PAR + UVBR showed a growth rate reduction of 55%. The content of photosynthetic pigments, including chlorophyll a and phycobiliproteins, decreased after exposure to PAR + UVBR. This result agrees with the decreased photosynthetic performance observed after exposing algae to PAR + UVBR. Irradiation also elicited increased activity of the antioxidant enzyme glutathione peroxidase and decreased mitochondrial NADH dehydrogenase activity, which correlated with the decreased protein content in plants exposed to PAR + UVBR. Taken together, these findings strongly indicate that UVBR negatively affects the architecture and metabolism of the carragenophyte C. teedei.