Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Roles of adjuvants in aphicidal activity of enzymes from Beauveria bassiana (Ascomycota: Hypocreales) SFB-205 supernatant

Enzymes from Hypocrealean entomopathogenic fungi often encounter unfavorable abiotic and biotic factors during pathogenesis. The present work describes the roles of adjuvants, such as corn oil and polyoxyethylene-(3)-isotridecyl ether (TDE-3), in promoting aphicidal activity of enzyme precipitate from Beauveria bassiana SFB-205 supernatant. Supernatant enzymes including chitinase and proteases were lyophilized by attagel-mediated protein precipitation to produce attagel-mediated enzyme powder (AMEP). Corn oil-based AMEP + TDE-3 suspension showed 96.3% control efficacy against cotton aphids, Aphis gossypii Glover (Hemiptera: Aphididae), in glasshouse conditions at 2 days post-application, whereas water-based AMEP + TDE-3 suspension or TDE-3 alone showed < 20% efficacy. Corn oil-based AMEP + TDE-3 suspension was superior in degrading chitinase-specific substrate (p-nitrophenyl-β-d-acetylglucosaminide) under a drying condition compared to water-based AMEP + TDE-3 suspension. TDE-3 made supernatant, a source material of AMEP, degraded more cotton aphid proteins than supernatant alone in SDS-polyacrylamide gel electrophoresis; supernatant was used to clearly show the degradation without interfering with other proteins such as AMEP. These results suggest that 1) corn oil can slow down the evaporation of the diluted suspension drop and provide more time for enzymes from AMEP to degrade more cuticles at the time of application and 2) TDE-3 can disrupt chitin–protein matrixes within procuticle and facilitate enzymes from AMEP in degrading proteins which increases the exposure of chitin fibers to the attack of chitinases. This approach can provide another strategy in developing biopesticides using entomopathogenic fungi.     

A high throughput method to screen companion bacterium for 2-keto-l-gulonic acid biosynthesis by co-culturing Ketogulonicigenium vulgare

Bacillus megaterium is widely used as companion bacterium in the two-step biosynthesis of 2-keto-l-gulonic acid (2-KLG) by Ketogulonicigenium vulgare. To screen efficiently target companion strains from large numbers of random mutants, a screen method based on spectrophotometry and 24-well microtiter plates was developed and validated on an integrated library of 450 transposon random insertional mutants and two sporulation-defective mutants. The co-culture processes were classified into three groups (low, intermediate and high performance) by K-mean clustering analysis. In addition, mutant m71 was successfully screened out from the library. The substrate conversion ratio of m71 and K. vulgare co-culture process after 72 h was decreased by about 38% compared with that of the wild-type co-culture process in 750 ml flasks. These results indicated that the proposed high throughput method is feasible for screening target companions for the co-culture process of 2-KLG biosynthesis.     

Immunotherapy with Dendritic Cells Modified with Tumor-Associated Antigen Gene Demonstrates Enhanced Antitumor Effect Against Lung Cancer

BACKGROUND: Immunotherapy using dendritic cell (DC) vaccine has the potential to overcome the bottleneck of cancer therapy. METHODS: We engineered Lewis lung cancer cells (LLCs) and bone marrow–derived DCs to express tumor-associated antigen (TAA) ovalbumin (OVA) via lentiviral vector plasmid encoding OVA gene. We then tested the antitumor effect of modified DCs both in vitro and in vivo. RESULTS: The results demonstrated that in vitro modified DCs could dramatically enhance T-cell proliferation (P < .01) and killing of LLCs than control groups (P < .05). Moreover, modified DCs could reduce tumor size and prolong the survival of LLC tumor-bearing mice than control groups (P < .01 and P < .01, respectively). Mechanistically, modified DCs demonstrated enhanced homing to T-cell–rich compartments and triggered more naive T cells to become cytotoxic T lymphocytes, which exhibited significant infiltration into the tumors. Interestingly, modified DCs also markedly reduced tumor cells harboring stem cell markers in mice (P < .05), suggesting the potential role on cancer stem-like cells. CONCLUSION: These findings suggested that DCs bioengineered with TAA could enhance antitumor effect and therefore represent a novel anticancer strategy that is worth further exploration.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects

The oral toxicity of 5 Photorhabdus spp. strains collected in different regions of Korea was determined in the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, and Culex pipiens pallens. When diet or water containing culture media containing 1 of the 5 different strains was ingested by immature insects, the first instar larvae of both G. mellonella and L. caesar and young larvae of C. pipiens pallens died within 3–5 days after treatment. However, mortality of P. interpunctella neonate larvae was slightly slower and reached 94.4%–100% within 7 days after treatment. The mortality rate of a control group given a diet containing water, the medium without cultured bacteria, or Escherichia coli culture medium was not affected. The mortality rates were 100%, 45.3%, 2.8%, and 0% for Galleria, Lucilia, Plodia, and Culex, respectively, in another control group given a culture medium of Photorhabdus luminescens ssp. laumondii (TT01). In addition, culture media containing Photorhabdus strains significantly inhibited molting of third instar Plodia larvae by as much as 88% 7 days after treatment, whereas molting inhibition was reduced by 0%, 4%, and 20% following treatments with water, E. coli, or TT01 culture media, respectively. Our results suggest that the oral administration of Photorhabdus bacterial medium was highly effective for controlling various immature insects.     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Metal uptake by medicinal plant species grown in soils contaminated by a smelter

The hypothesis tested in this study was if medicinal plants could be grown as alternative crops in heavy metal polluted soils without contamination of the final marketable produce. Furthermore, medicinal crops may offer a phytoremediation option for mildly heavy metal polluted agricultural soils. The effect of metal-enriched soils was evaluated in five medicinal species (Bidens tripartita L., Leonurus cardiaca L., Marrubium vulgare L., Melissa officinalis L. and Origanum heracleoticum L.). Soils were sampled in the vicinities of the Non-Ferrous Metals Combine (Pb–Zn smelter) near Plovdiv, Bulgaria, from plots at 0.5 km (soil 1), 3 km (soil 2), 6 km (soil 3) and 9 km (control soil) from the smelter. Cadmium, Pb and Zn concentration in soil 1 were above the critical total (HNO₃-extractable) concentrations for these elements in soils. Generally, heavy metals in soil 1 decreased dry mater yields of the five species relative to the control. However, the essential oil content of M. vulgare, M. officinalis and O. heracleoticum was within the usual range for respective species and was not affected by the treatments. The overall metal uptake was in the order: B. tripartita > M. vulgare > O. heracleoticum > L. cardiaca > M. officinalis for Cd, L. cardiaca = M. vulgare > B. tripartita = M. officinalis = O. heracleoticum for Pb, L. cardiaca = M. vulgare > O. heracleoticum > B. tripartita = M. officinalis for Cu and B. tripartita > L. cardiaca = M. vulgare > M. officinalis = O. heracleoticum for Mn and Zn. Overall, metal concentration in plant parts was in the order: roots > leaves > flowers > stems for Cd, Pb and Cu, leaves > roots > flowers > stems for Mn and Zn. The concentration of Cd, Pb, Cu and Zn in plant tissue correlated to the exchangeable (EXCH) and the carbonate (CARB) bound fractions of metals in soil. Heavy metals caused disruptions of the plasma membrane of some root cortical cells and alterations in chloroplasts thylakoids in plants grown in soil 1. Metal content in teas prepared from the species was negligible, the essential oils were free of metals. Generally, the transfer factor (TF) was less than 1, indicating the tested species did not have a significant phytoextraction potential. This study demonstrated the three essential oil species M. vulgare, M. officinalis and O. heracleoticum can be grown as alternative high-value crops in metal polluted agricultural soils around the smelter and provide metal-free marketable produce.     

Metal uptake by medicinal plant species grown in soils contaminated by a smelter

The hypothesis tested in this study was if medicinal plants could be grown as alternative crops in heavy metal polluted soils without contamination of the final marketable produce. Furthermore, medicinal crops may offer a phytoremediation option for mildly heavy metal polluted agricultural soils. The effect of metal-enriched soils was evaluated in five medicinal species (Bidens tripartita L., Leonurus cardiaca L., Marrubium vulgare L., Melissa officinalis L. and Origanum heracleoticum L.). Soils were sampled in the vicinities of the Non-Ferrous Metals Combine (Pb–Zn smelter) near Plovdiv, Bulgaria, from plots at 0.5 km (soil 1), 3 km (soil 2), 6 km (soil 3) and 9 km (control soil) from the smelter. Cadmium, Pb and Zn concentration in soil 1 were above the critical total (HNO₃-extractable) concentrations for these elements in soils. Generally, heavy metals in soil 1 decreased dry mater yields of the five species relative to the control. However, the essential oil content of M. vulgare, M. officinalis and O. heracleoticum was within the usual range for respective species and was not affected by the treatments. The overall metal uptake was in the order: B. tripartita > M. vulgare > O. heracleoticum > L. cardiaca > M. officinalis for Cd, L. cardiaca = M. vulgare > B. tripartita = M. officinalis = O. heracleoticum for Pb, L. cardiaca = M. vulgare > O. heracleoticum > B. tripartita = M. officinalis for Cu and B. tripartita > L. cardiaca = M. vulgare > M. officinalis = O. heracleoticum for Mn and Zn. Overall, metal concentration in plant parts was in the order: roots > leaves > flowers > stems for Cd, Pb and Cu, leaves > roots > flowers > stems for Mn and Zn. The concentration of Cd, Pb, Cu and Zn in plant tissue correlated to the exchangeable (EXCH) and the carbonate (CARB) bound fractions of metals in soil. Heavy metals caused disruptions of the plasma membrane of some root cortical cells and alterations in chloroplasts thylakoids in plants grown in soil 1. Metal content in teas prepared from the species was negligible, the essential oils were free of metals. Generally, the transfer factor (TF) was less than 1, indicating the tested species did not have a significant phytoextraction potential. This study demonstrated the three essential oil species M. vulgare, M. officinalis and O. heracleoticum can be grown as alternative high-value crops in metal polluted agricultural soils around the smelter and provide metal-free marketable produce.     

Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-l-gulonic acid production in Ketogulonigenium vulgare–Bacillus cereus consortium

The expression levels of sorbose/sorbosone dehydrogenase genes (sdh and sndh) and the synthesis genes (pqqABCDEN) of the adjoint cofactor pyrroloquinoline quinone (PQQ) were genetically manipulated in Ketogulonigenium vulgare to increase the production of 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, in the consortium of K. vulgare and Bacillus cereus. We found that overexpression of sdh–sndh alone in K. vulgare could not significantly enhance the production of 2-KLG, revealing the cofactor PQQ was required for the biosynthesis of 2-KLG. Various expression levels of PQQ were achieved by differential expression of pqqA, pqqABCDE and pqqABCDEN, respectively. The combinatorial expression of sdh/sndh and pqqABCDEN in K. vulgare enabled a 20% increase in the production of 2-KLG (79.1±0.6 g l⁻¹) than that of the parental K. vulgare (65.9±0.4 g l⁻¹) in shaking flasks. Our results demonstrated the balanced co-expression of both the key enzymes and the related cofactors was an efficient strategy to increase chemicals' biosynthesis.     

Efficacy of entomopathogenic hypocrealean fungi against Periplaneta americana

The American cockroach Periplaneta americana, one of the worlds' most important urban insect pests was tested with entomopathogenic fungi. Most promising Metarhizium anisopliae, Metarhizium robertsii and Beauveria bassiana killed nymphs (≥ 81.7% mortality, 25 days after treatment), and these fungi developed on all dead insects. Other fungi tested were less virulent (Metarhizium frigidum and Purpureocillium lilacinum) or avirulent (Isaria cateniobliqua, Isaria farinosa, Simplicillium lanosoniveum, Sporothrix insectorum and Tolypocladium cylindrosporum). Intrageneric and intraspecific variability of fungal activity was detected. Adults were highly susceptible, and oothecae proved to be more resistant than nymphs and adults to infection with M. anisopliae IP 46. Findings of the study underscore the potential of fungi as biocontrol agents against this pest.     

Rhodomyrtone: A new candidate as natural antibacterial drug from Rhodomyrtus tomentosa

Rhodomyrtone [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl-1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1H-xanthene-1,3(2H)-di-one] from Rhodomyrtus tomentosa (Aiton) Hassk. displayed significant antibacterial activities against Gram-positive bacteria including Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Streptococcus gordonii, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus salivarius. Especially noteworthy was the activity against MRSA with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) ranging from 0.39 to 0.78 μg/ml. As shown for S. pyogenes, no surviving cells were detected within 5 and 6 h after treatment with the compound at 8MBC and 4MBC concentrations, respectively. Rhodomyrtone displays no bacteriolytic activity, as determined by measurement of the optical density at 620 nm. A rhodomyrtone killing test with S. mutans using phase contrast microscopy showed that this compound caused a few morphological changes as the treated cells were slightly changed in color and bigger than the control when they were killed. Taken together, the results support the view that rhodomyrtone has a strong bactericidal activity on Gram-positive bacteria, including major pathogens.     

Tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP): Rejuvenation of an obsolete antibiotic to further action

BackgroundIncreasing resistance in bacteria towards antibiotics has made it imperative to research on their revitalization to combat infectious diseases. This study dealt with synthesis of a nano-form of the antibiotic tetracycline, its characterization and potency of killing different multi-drug resistant diarrhea-causing bacteria.MethodsNano-formulation was done by loading tetracycline within biocompatible calcium phosphate nanoparticle. The synthesized tetracycline-loaded calcium phosphate nanoparticle (Tet-CPNP) was characterized by the techniques like TEM, DLS, EDS, FTIR, spectrofluorimetry and dialysis. Bactericidal activity of nano-particulate tetracycline was investigated by agar plating, spectrophotometry, phase contrast-fluorescence-atomic force microscopy and flow cytometry techniques.ResultsThe Tet-CPNPs were 8 ± 5 nm in size and nearly spherical in shape, efficiency of tetracycline loading in CPNP was about 20% and the release of antibiotic from Tet-CPNPs was sustainable during 7 days. Minimum inhibitory concentration (MIC) of Tet-CPNP on multiple antibiotic (including tetracycline) resistant bacteria like Escherichia coli, Salmonella kentuckey and Shigella flexneri was in the range of 20–40 μg/ml, whereas MIC of free tetracycline was in the range of 150–180 μg/ml. NP-mediated cell filamentation and cell membrane disintegration caused cell killing. Moreover, death of Shigella-infected Zebra fish larvae was stalled by Tet-CPNP treatment. CPNP itself had no toxic effect on bacteria as well as on Zebra fish.ConclusionOur nano-formulation of tetracycline might reclaim a nearly obsolete antibiotic to further potential function.General significanceSuch a study on revival of an old, cheap, broad-spectrum antibiotic to further action is highly beneficial to developing countries with limited health care budgets.     

Biodegradability and mechanical properties of polycaprolactone composites encapsulating phosphate-solubilizing bacterium Bacillus sp. PG01

This study examined the feasibility of using polycaprolactone (PCL) and its composites (with starch and/or clay) in encapsulating cells of phosphate-solubilizing bacteria (PSB) for the development of biodegradable and “controlled-release” bacterial fertilizer. The PSB used in this work was an indigenous Bacillus sp. PG01 isolate. The results show that the PG01 strain was able to degrade all the cell-loaded capsules made of PCL and PCL composites, resulting in a continual cell release. Morphology observation indicates that severe disruption of the capsule structure occurred after incubation for 15–20 days. The biodegradability of the capsules decreased in the order of PCL/starch (20 wt%) > PCL/starch (20 wt%)/cay (7 wt%) > PCL alone > PCL/clay (7 wt%). Similar trends were also observed for the decrease in tensile strength and elongation at break, suggesting strong connections between biodegradability and the mechanical properties. Addition of starch appeared to enhance the biodegradability of the capsules, whereas the clay-blended composites were less biodegradable. The amount and rate of cell release from cell-encapsulated PCL-based capsules were positively dependent on the biodegradability and on the decrease in the mechanical strength. Nevertheless, the pattern of cell release was quite similar for all types of capsules. The outcome of this work seems to suggest that by proper manipulation of composite compositions, the controlled release of the bacterial fertilizer (i.e., Bacillus sp. PG01 cells) might be achievable.     

STK15 rs2273535 polymorphism and cancer risk: A meta-analysis of 74,896 subjects

Background: It has been suggested that the serine/threonine kinase 15 (STK15) T91A rs2273535 polymorphism is associated with susceptibility to cancer. However, the results are conflicting. We performed this meta-analysis to derive a more precise estimation of the relationship. Methods: PubMed was searched to select studies. Case–control studies containing available genotype frequencies of the STK15 rs2273535 polymorphism were chosen, and the odds ratio (OR) with its 95% confidence interval (CI) was utilized to assess the strength of association. Results: 52 studies – including 34,057 cases and 40,839 controls – were identified. A significant effect of the STK15 rs2273535 polymorphism on cancer risk was found (AA vs. TT: OR = 1.13, 95%CI = 1.01–1.26, P_{heterogeneity} < 0.001; AA vs. TA/TT: OR = 1.12, 95%CI = 1.02–1.22, P_{heterogeneity} < 0.001; TA/AA vs. TT: OR = 1.06, 95%CI = 1.01–1.12, P_{heterogeneity} < 0.001). Stratified analysis by cancer type revealed that the STK rs2273535 polymorphism may contribute to the risk of breast cancer (AA vs. TT: OR = 1.21, 95%CI = 1.01–1.44, P_{heterogeneity} = 0.002), colorectal cancer (AA vs. TA/TT: OR = 1.24, 95%CI = 1.05–1.47, P_{heterogeneity} = 0.124), and esophageal cancer (AA vs. TA/TT: OR = 1.19, 95%CI = 1.02–1.39, P_{heterogeneity} = 0.148). Further subgroup analysis by ethnicity indicated that there was a statistically increased cancer risk in Asians (AA vs. TA/TT: OR = 1.20, 95%CI = 1.05–1.37, P_{heterogeneity} = 0.004). Conclusion: This meta-analysis suggests that the STK15 rs2273535 polymorphism is a candidate gene polymorphism for cancer susceptibility, especially in Asian populations.     

The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters

The dynamics of Dinophysis acuminata and its associated diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX1) as well as pectenotoxins (PTXs), were investigated within plankton and shellfish in Northport Bay, NY, USA, over a four year period (2008–2011). Over the course of the study, Dinophysis bloom densities ranged from ~10⁴ to 10⁶ cells L⁻¹ and exceeded 10⁶ L⁻¹ in 2011 when levels of total OA, total DTX1, and PTX in the water column were 188, 86, and 2900 pg mL⁻¹, respectively, with the majority of the DSP toxins present as esters. These cell densities exceed – by two orders of magnitude – those previously reported within thousands of samples collected from NY waters from 1971 to 1986. The bloom species was positively identified as D. acuminata via scanning electron microscopy and genetic sequencing (cox1 gene). The cox1 gene sequence from the D. acuminata populations in Northport Bay was 100% identical to D. acuminata from Narragansett Bay, RI, USA and formed a strongly supported phylogenetic cluster (posterior probability = 1) that included D. acuminata and Dinophysis ovum from systems along the North Atlantic Ocean. Shellfish collected from Northport Bay during the 2011 bloom had DSP toxin levels (1245 ng g⁻¹ total OA congeners) far exceeding the USFDA action level (160 ng g⁻¹ total OA of shellfish tissue) representing the first such occurrence on the East Coast of the U.S. D. acuminata blooms co-occurred with paralytic shellfish poisoning (PSP) causing blooms of Alexandrium fundyense during late spring each year of the study. D. acuminata cell abundances were significantly correlated with levels of total phytoplankton biomass and Mesodinium spp., suggesting food web interactions may influence the dynamics of these blooms. Given that little is known regarding the combined effects of DSP and PSP toxins on human health and the concurrent accumulation and depuration of these toxins in shellfish, these blooms represent a novel managerial challenge.     

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.     

Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease,the redbay ambrosia beetle,Xyleborus glabratus (Coleoptera: Curculionidae)

The redbay ambrosia beetle (RAB), Xyleborus glabratus, is a wood-boring insect that vectors the fungal pathogen, Raffaelea lauricola, which causes laurel wilt, a lethal disease of avocado. The objective of this study was to determine the susceptibility of RAB to infection and subsequent death by exposure to three commercial strains of entomopathogenic fungi [two strains of Isaria fumosorosea (Ifr 3581 and PFR), and strain GHA of Beauveria bassiana]. RAB females were dipped in fungal spore solutions and their median survivorship times (MST) determined. Contact with any of the biopesticides resulted in death of all RAB females. MSTs of RAB females ranged from 3 days (B. bassiana) to 5 days (I. fumosorosea PFR). B. bassiana killed RAB females faster, followed by Ifr 3581 and PFR. RAB females dipped in B. bassiana suspensions had the highest number of viable spores attached to their bodies, followed by Ifr 3581. Beetles dipped in PFR suspension had significantly less viable spores attached to their bodies. No significant differences were observed in the mortality of beetles exposed to entomopathogenic fungi by dipping in a fungal suspension or walking on treated avocado bolts. Beetles bored into the logs and constructed galleries, but they were found dead inside the galleries a few days after exposure to the entomopathogens. Entomopathogenic fungal infection in dead beetles was confirmed through molecular techniques. This is the first study to demonstrate that entomopathogenic fungi are potential biological control agents against RAB.     

Characterization,N-terminal sequencing and classification of Tolworthcin 524: A bacteriocin produced by Bacillus thuringiensis subsp. tolworthi

Bacteriocins synthesized by entomopathogenic Bacillus thuringiensis are gaining attention owing to their inhibitory effects against a wide variety of pathogenic bacteria. In the present study, we purified and characterized Tolworthcin 524, a bacteriocin synthesized by B. thuringiensis subsp. tolworthi, and compared it with other bacteriocins synthesized by B. thuringiensis. Tolworthcin 524 was separated and purified from the secretome of B. thuringiensis by fast protein liquid chromatography with a gel filtration column to obtain yields of 17% and a specific activity of ∼3600 U/mg protein. The purified product showed two peptides of ∼9 and 6 kDa with antimicrobial activity in a gel-screening assay. The purified product was analyzed by two-dimensional electrophoresis and the resolved peptides of ∼9 and 6 kDa with isoelectric points of ∼8 were sequenced. Partial sequences (METPVVQPR and DWTCWSCLVCAACS) were obtained suggesting that the ∼9 and 6 kDa correspond to the prebacteriocin and mature Tolworthcin 524, respectively. Sequences showed high identity with Thurincin H and Thuricin 17 and had a conserved motif with other bacteriocins of B. thuringiensis. Based on sequence data, Tolworthcin 524 was classified in subclass II.2 (Thuricin-like peptides) of the Bacillus bacteriocin classification scheme. The larger peptide did not harbor a sequence suggestive of a signal peptide neither did it contain the double-glycine (GG) motif characteristic of the secretion leader recognized by the ABC transport system. Implications of these properties in Tolworthcin 524 secretion are discussed.