Aureococcus anophagefferens, recur in several northeastern US coastal bays. Direct bloom control could alleviate the ecological and economic damage associated with bloom outbreak. This paper explored the effectiveness and safety of natural chemical biocide hydrogen peroxide (H2O2) for brown tide bloom control. Culture studies showed that H2O2 at 1.6 mg L−1 effectively eradicated high density A. anophagefferens within 24-hr, but caused no significant growth inhibition in the diatoms, prymnesiophytes, green algae and dinoflagellates of >2–3 μm cell sizes among 12 phytoplankton species tested over 1-week observation. When applied to brown tide bloom prone natural seawater in a microcosm study, this treatment effectively removed the developing brown tide bloom, while the rest of phytoplankton assemblage , particularly the diatoms and green algae, experienced only transient suppression then recovered with total chlorophyll a exceeding that in the controls within 72-hr; cyanobacteria was not eradicated but was still reduced about 50% at 72-hr, as compared to the controls. The action of H2O2 against phytoplankton as a function of cell size and cell wall structure, and a realistic scenario of H2O2 application were discussed.Brown tide algal blooms, caused by the excessive growth of  The toxin(s) secretion, thought to be associated with A. anophagefferens exopolymer on cell surface, affects shellfish and zooplankton feeding behavior The brown tide algal blooms in northeastern US coastal bays are caused by the excessive growth of Pelagophyte 2O2) is naturally produced in sunlit surface seawater from photochemical reaction of dissolved organic matter, oxygen and trace metals 2O2 mol−1 photons at <340 nm light 2O2 was reported to potentially accumulate at 30–59 nM hr−1 for several hours in 0.2 μm filtered seawater in Narragansett Bay, Rhode Island 2O2 concentrations averaged 122±38 nM 2O2 ranged from 21 to 123 nM along a meridional transect in the eastern Atlantic Ocean. Rain water also contains hydrogen peroxide; up to 57 µM, approximately 2 mg L−1, were observed in wet deposition in Bermuda Atlantic Time Series Stations Hydrogen peroxide . The results provided an initial assessment on the effectiveness and safety of hydrogen peroxide in brown tide bloom control.This study was carried out to examine the effectiveness of hydrogen peroxide in brown tide bloom control, and the safety of its application for the other and possibly co-occurring phytoplankton species. We first established a dose that effectively removed laboratory cultures of brown tide alga Aureococcus anophagefferens (Aa), five diatoms Phaeodactylum tricornutum (Pt), Minutocellus polymorphus (Mpo), Thalassiosira pseudonana (Tp), Skeletonema costatum (Sc), and Thalassiosira weissflogii (Tw), two green algae Micromonas pusilla (Mp) and Dunaliella tertiolecta (Dt), two dinoflagellates Amphidinium carterae (Ac) and Prorocentrum micans (Pm), and two Prymnesiophytes Isochrysis galbana and Emiliania huxleyi  and included the brown tide bloom alga  huxleyi . These sTraceSELECT®, 30% w:w, 9.8 M, kept in refrigerator and in dark) was first diluted with sterile culture medium to 10 mM (∼0.03%), then immediately added to the mid- or late-exponential phase cultures. Upon gentle mixing, the cultures  were returned to the growth chamber for incubation. The nominal H2O2 addition was 0, 0.8, 1.6, 3.2 and 6.4 mg L−1 for A. anophagefferens, and 0, 1.6 and 6.4 mg L−1 for other algal species. The nominal concentrations of hydrogen peroxide were confirmed with 10–15% accuracy using spectrophotometric method of Lu et al. 2O2 reacted with p-nitrophenylboronic acid stoichiometrically at pH 9 producing p-nitrophenol of 19400 cm−1 M−1 absorptivity. Cultures without H2O2 addition were used as controls. Cultures were in triplicates for A. anophagefferens, and duplicates for other species.Hydrogen peroxide , 3 hr, and then daily up to 1 week after H2O2 addition. The in vivo fluorescence (IVF) was monitored using Turner Designs' Trilogy Fluorometer . Cell density was measured by Coulter Counter Multisizer 3 . Total chlorophyll a (chl a) was measured with GF/F filtration, 90% acetone extraction, and UV-Vis spectrophotometer (Agilent 8453) quantification using Jeffery and Humphrey's trichromatic equation following EPA method 446.0.Parameters reflecting algal growth were monitored, typically at 0 hr  were used to compare temporal variation of the cultures. Exponential growth or log-linearity was determined with regression analysis. Regression was also used to determine the correlation between percent inhibition and both cell size and H2O2 dose.Throughout the study Minitab 16 Software Package was used in statistical analysis with probability Aureococcus-Chlorella-Nannochloris-Synechococcus complex, other small flagellates of 3–7 μm size , prymnesiophytes such as Chrysochromulia sp., diatoms of small or large sizes , and small numbers of larger species (>15 μm) e.g. eugalenophyte Eugalena sp, and raphidophyte Heterosigma carteraeNatural seawater was collected from Barnegat Bay, NJ, which is prone to summer brown tide, by pumping surface seawater into 20-L carboy (pre-cleaned with detergent and acid) from a boat on July 28, 2010.  Collected seawater was kept in a cooler and stored in cold-room (4°C) for processing next day. The upper Barnegat Bay summer phytoplankton assemblage typically consists of small coccoidal picophytoplankton complex (<5 μm) 2O2 action, a fraction of the collected natural seawater was passed through 5 µm PTFE membrane filters (Millipore) under low vacuum pressure (<5 psi). Unfiltered and 5 µm filtered seawater microcosms were set up by distributing 450 mL seawater into 500 mL sterilized polycarbonate bottles, and subsequently inoculating 35,000 cell mL−1 cultured A. anophagefferens (to mimic a medium density brown tide bloom) 2O2, freshly prepared in sterile Aquil at 10 mM, was added at 1.6 and 6.4 mg L−1. The controls (microcosms without H2O2 addition) and 1.6 mg L−1 H2O2 treatments were in triplicates; microcosms with 6.4 mg L−1 H2O2 addition were not replicated. The microcosms were incubated in the growth chamber as in the culture study.In order to test cell size dependency, if any, of HA. anophagefferens cell density, and phytoplankton community composition were analyzed at 0 hr (immediately before H2O2 addition), and then daily up to 72 hrs of H2O2 addition. Total phytoplankton growth was analyzed via in vivo chlorophyll fluorescence and total chl a (same as in culture study). Changes in phytoplankton community composition were tracked via accessory pigment analysis: 19′-butanoyloxyfucoxanthin (19′-bf) for brown tide alga A. anophagefferensA. anophagefferens cell density was further estimated from a standard curve that correlated the number of cultured A. anophagefferens cells used in the pigment analysis and the resulting chromatographic peak area of 19′-bf.Total phytoplankton growth, A. anophagefferens, Amphidinium carterae, Synechococcus sp. and Dunaliella tertiolecta for 19′-bf, peridinin, zeaxanthin and chl b respectively.Accessory pigment analysis followed Van Heukelem and Thomas' HPLC method −1 effectively inhibited A. anophagefferens growth as was observed with in vivo chlorophyll a fluorescence, total chl a, and cell density , total chl a 41.5±1.2 μg L−1, and cell density 1.98±0.4 million cells mL−1. The control cultures experienced 4.5-fold log-linear increase in in vivo fluorescence during 1-week incubation , but the cultures with 0.8 mg L−1 H2O2 addition had a decrease in in vivo fluorescence nearly 50% at 24-hr (p = 0.001) which then rebounded at a rate comparable to that of the control cultures (p = 0.975). More importantly, the cultures with 1.6, 3.2 and 6.4 mg L−1 H2O2 addition showed 98%, 99.7% and 99.7% (p = 0.000) decrease in in vivo fluorescence at 24-hr and re-growth was not observed over 1 week (−1 H2O2) decreased more than 90% (p≤0.002) at 24-hr, and were below detection limit at 1-week  from nearly 2 to 0.5, 0.34 and 0.16 million cells mL−1at 24-hr, 48-hr and 1-week respectively; and in the cultures with 3.2 and 6.4 mg L−1 H2O2 addition, it decreased >90% to below 0.19±0.02 million cells mL−1at and after 24-hr , K is the growth response of the control (K = 1), C is H2O2 concentration (mg L−1), EC50 is medium effective concentration. The resulting EC50s for A. anophagefferens were 1.39±0.06 mg L−1 at 3-hr, and 0.91±0.00 mg L−1 at 24-hr and remained at this level during 48-hr –1-week.Medium effective concentration, ECA. anophagefferens growth response suggested that H2O2 at 1.6 mg L−1 or higher concentrations effectively removed high density A. anophagefferens within 24-hr exposure , and re-growth was not observable during 1-week incubation.Overall, A. anophagefferens removal was accomplished by 1.6 mg L−1 H2O2 treatment, comparisons on the survival and re-growth of A. anophagefferens with 11 other marine phytoplankton species  were carried out, by exposing the growing or grown cultures of similar in vivo chlorophyll fluorescence  to 1.6 mg L−1 H2O2.Because effective  Micromonas pusilla (Mp)  was eradicated by 1.6 mg L−1 H2O2 , had an overall 17% lower growth rate as compared to its control cultures .The growth of the rest 9 species, i.e. the diatoms , Sc, Tw , the oth, Tw  the percent inhibition increased from non-inhibitive in Sc and Tw to more than 90% inhibition in Pt  where % inhibition is the average % inhibition at 24-hr, 48-hr and 72-hr upon 6.4 mg L−1 H2O2 addition as compared to the control, cell diameter is the mean cell size measured by Coulter counter (μm), and H2O2/chl a is the weight ratio, mg H2O2/mg chl a, at time zero. Since dinoflagellate Pm (45 μm) is substantially larger than the rest of the species (1.4–11.05 μm), a correlation of 11 species excluding Pm was also established, % inhibition  = 0.913–0.0952 x cell diameter +0.00224 x H2O2/chl a , to reflect species sensitivity as a function of cell size and H2O2 dose. In this regression, the cell diameter alone was found to explain 40% of the variations.Exposure of these species to Hsitivity . It was on in Pt . An empiA. anophagefferens cell density was estimated at 27,900±5,379 cell mL−1 (n = 2) in the original Barnegat Bay water samples, and at 54,150±3,188 cell mL−1 in the testing microcosms before H2O2 addition . In the control microcosms (i.e. no H2O2 addition), A. anophagefferens cell density increased 60% at 24-hr as compared to time zero (p<0.05), and remained nearly constant at 24-hr, 48-hr and 72-hr , suggesting a developing brown tide bloom  at 24-hr, and its cell density remained low during the entire 72-hr study period, in both unfiltered and 5-μm filtered seawater microcosms , but recovered, at 72-hr, to a level comparable to  or even higher  than those in the control microcosms (p<0.01) (While nd 48-hr . Specificrocosms . The tra(p<0.01) .−1 H2O2 treatment was estimated from algal marker pigments, fucoxanthin for diatoms and other chrysophytes, chl b for green algae, peridinin for dinoflagellates and zeaxanthin for cyanobacteria (−1 H2O2) by 55% at 24-hr (p = 0.006) and 44% at 48-hr (p = 0.000), but subsequently recovered and exceeded the controls by 104% at 72-hr (p = 0.003); chl b was reduced approximately 55% at 24-hr and 48-hr (p<0.01) then recovered to a level not significantly different from the controls (p = 0.267) (p<0.01) then exceeded the controls by 66% at 72-hr (p = 0.000); chl b was exceeded by the control by 54–67% at 24-hr and 48-hr (p<0.01) and then to a less degree of 17% at 72-hr (p = 0.018) (p<0.05) in unfiltered and 5 μm filtered seawater microcosms, respectively (The response of the rest of the phytoplankton assemblage to 1.6 mg Lbacteria . In the = 0.267) . In the = 0.018) . Peridinectively . Altoget2O2 was also evident, as the phytoplankton groups in 5 μm filtered seawater microcosms (dominated by picophytoplankton) experienced slightly greater percent inhibition and/or less robust recovery as compared to that in unfiltered seawater microcosms  (Cell size dependent action of Hlankton) .−1 H2O2. As expected, 6.4 mg L−1 H2O2 completely removed 19′-bf pigment during the 72-hr study period, suggesting A. anophagefferens eradication. Chl a, fucoxanthin, chl b, peridinin were still exceeded by the controls at 72-hr by 64–71%, 93–97%, 5–28% and 49% respectively, and zeaxanthin went below detection limit during 24–72 hr (data not shown).A limited numbers of microcosms were exposed to 6.4 mg L2O2 (e.g. 1.6 mg L−1) effectively removed more than 90% of brown tide alga Aureococcus anophagefferens even at peak bloom density, without devastating most of the other potentially coexisting phytoplankton species and the natural phytoplankton community. Mechanisms of the differential susceptibility among phytoplankton and a realistic application scenario are discussed below.This study presented strong evidence, from culture and microcosm studies, that low dose of HA. anophagefferens demonstrated sensitivity similar to those of cyanobacteria. For example the EC50 were 0.3–0.4 mg L−1 for Microcystis aeruginosa and Trichormus variabilis and were between 1.1 and 1.7 mg L−1 for Synechococcus nidulans and two other cyanobacteria species on photosynthetic yield over 3-hr H2O2 exposure 50 of 1.39 mg L−1 on in vivo fluorescence based growth inhibition of A. anophagefferens over 3-hr exposure. Cyanobacteria has been shown to be more sensitive than most eukaryotic phytoplankton A. anophagefferens to H2O2 may be attributed to both its small cell size and the fact that it lacks cell wall. A. anophagefferens has only a diffuse layer of polysaccharide external to plasma membrane 2O2 can easily penetrate into the cell, and damage the cell by producing hydroxyl radicals which inhibit the photosynthetic electron transfer and the photosynthetic activity 2O2 sensitivity is likely caused by both the surface area to volume ratio being a function of cell size, and the non-selective reactivity of H2O2 and hydroxyl radical (·OH) product. The ·OH, a product of H2O2 interacting with ferrous ion via Fenton reactions 2+ may be formed via extracellular Fe(III) reduction 2O2. It reacts with a wide range of organic compounds  very rapidly with a second order reaction rate constant in the range of 107–109 M−1S−12O2 and ·OH. As a result, the damage would be more extensive.The cell size dependent H2O2 resistance. For example, the silica-mucilage cell wall of the diatoms Sc and Tw their H2O2 resistance , as compared to the athecate dinoflagellate Ac of similar cell size   Eh (CCMP 374) is non-coccolith forming strain ; and both are more sensitive to H2O2  than the diatoms of similar cell size e.g. Mpo  and Tp    and 2. Tibition)  and 2. TLikewise, Jeong et al. 2O2 penetrates into a cell, reactive oxygen species scavenging systems would decompose and inactivate the excessive amounts of H2O22O2 and HO· scavenging. This, along with the capability of the cell to repair and replace the damaged biomolecules and cellular components, permits the culture to re-grow, as was observed in both the culture and the microcosm studies at relatively low H2O2 exposure.Hydrogen peroxide may be inactivated and detoxified by organisms' reactive oxygen species scavenging systems. Once H2 with a depth of 1 m  is to be treated ex situ within 5 days, it would require a treatment capacity of 1300 million gallon per day (mgd), nearly 4 times that of Passaic Valley Sewage Commissioner, New Jersey, which is among the largest wastewater treatment facilities in Eastern US. Further, if the water is to be retained for 1 day before disposal, it would need 1300 million gallon reactor, representing an enormous size.Ex situ treatment of coastal algal bloom could be very costly given the enormous large size of water to be treated. For example if a bay water of 5×5 km−1 H2O2, one challenge is to disperse the concentrated H2O2 stock solution homogenously into seawater with minimal localized high concentration. In our study the stock solution (30% H2O2) was diluted 1000-fold before adding to the cultures. This dilution scheme requires an addition of 4.8-mm water containing 0.03% H2O2 to bloom water of 1-m depth (or 4.8 µm of 30% H2O2); and the wave action or a mixing device makes the additional 200-fold dilution to arrive at 1.6 mg L−1. The rate of chemical dispersal facilitated by wave action should be examined ahead of time, if possible, using hydrologic tracer chemicals such as sulfur hexafluoride (SF6) −1 H2O2 using 10% H2O2 stock solution −1In situ treatment should be more feasible, although challenges exist. To treat brown tide bloom at 1.6 mg L+/Fe3+ and UV radiation could be important, as they would promote the conversion of H2O2 into OH radical through Fenton reaction  + was well controlled at 6.6 × 10−19 M by using 100 µM EDTA and 8.3 µM total iron, and no Fe2+ was externally added + and total dissolved iron (FeT) vary over a range of 1–2 orders of magnitude. For example, Peconic Estuary  had FeT at 10–100 nM + at 1.1×10−20–4.7×10−19 M averaging 1.3x10−19 M, where salinity ≥26 parts per thousand +/Fe3+ were not measured in our microcosm sample taken from Barnegat Bay, NJ; but they could be similar to those of Peconic Estuary, based on reports of FeT in river water that discharges to these estuaries, e.g. 926 µg L−1 in Metedeconk River in Aug. 2003 + concentrations in two types of media, suggesting limited variation in Fe3+ concentration and limited influence in shaping the outcome of bloom control in coastal water. Natural radiation  likely enhance the treatment efficacy in the field. Barrington et al. examined laboratory-, mesocosm- and full-scale trials of H2O2 treatment for cyanobacterial bloom control in waste stabilization pond; they found significant synergistic effect of H2O2 addition with environmental factors (e.g. radiation) in the field trials A second concern in in situ treatment is whether the efficacy of the treatment observed in the culture and microcosm studies is transferable in field applications, and further, what environmental factors may lead to variable success of bloom control. Since the OH radical is believed to be the active chemical species that attacks and kills the cells during H2O2 treatment, environmental factors that affect H2O2 decay and OH radical production likely also influence the efficacy of the treatment. Among other factors, Fe22O2 ≤0.7 mg L−1 in receiving water is not a significant threat to organisms, environmental and public health  −1 H2O2 and transient exposure  should exert little harm to zooplankton −1 H2O2 also evidenced relatively low environmental risk of this treatment to aquatic organisms, as the eukaryotic phytoplankton , zooplankton and macrofauna remained largely unaffected by the treatment 2O2 treatment to marine phytoplankton in that a) phytoplankton cultures of ≥2–3 µm cell size were largely unaffected, and b) in natural seawater microcosms the diatom and the green algae were only transiently suppressed and total chlorophyll a recovered and exceeded that of the control by 72-hr of H2O2 treatment Therefore, although some eukaryotic phytoplankton could be eliminated by the H2O2 treatment at 1.6 mg L−1 , there would be very low risk for the majority eukaryotic phytoplankton, zooplankton, fish, larval fish, and other larger aquatic animals.In terms of environmental risk of hydrogen peroxide in natural waters, Schmidt et al. reviewed and summarized that fish and fish eggs are relatively tolerant of hydrogen peroxide, other vertebrates and mammals are much more tolerant than fish, and that microorganisms and zooplankton in aquatic ecosystem are generally less tolerant than fish or other vertebrates 2O2 treatment, as H2O2 is also used as a therapeutic agent in controlling aquaculture bacterial infection 2O2 treatment −1 or higher. For example, 11 strains of 4 species of oral Streptococci had MIC and MBC of 3.2–14 mg L−1 and 3.5–28.2 mg L−1, respectively  −1 were observed for cultures of Bacillus subtilis, Bacillus stearothermophilus, Escherichia coli, Staphylococcus aureus Enterobacter cloacae, Serratia marcescens and Acinetobactev calcoaceticus (E. coli had MIC of 2505 mg L−1) 2O2 to be a poor bactericide but bacteriostatic effect was achieved at and above 0.15 mM (5.1 mg L−1) −1 for four marine bacteria Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and V. vulnificus in Mueler Hinton Broth and were 0.6–2.4 mg L−1 in 1.5% NaCl solution in situ abundance) showed that natural background levels of H2O2  could cause bacterial production reduction to 25% of its control over 24-hr, and induce 1–3.4% lysogenic (due to virus) bacterial mortality −1 H2O2 and were variably suppressed (dependent on the date and time of observation) at the exposure of as low as 0.0034 mg L−1E. coli very resistant. Nevertheless, bacteria are expected to inoculate from surrounding waters and quickly reproduce and repopulate the treated water once H2O2 is sufficiently dissipated after treatment. Thus, long lasting impacts are not expected.It is logical to think that aquatic bacterial community could be most sensitive to H2O2 treatment, as was seen in the field work of Matthij et al. where 2 mg L−1 H2O2 eradicated cyanobacterial bloom of Planktothrix agardhii in the freshwater lake 2O2 also varied substantially among the species. For example, EC50 for photosynthetic yield (3-hr H2O2 exposure) were 0.3–0.4 mg L−1 for Microcystis aeruginosa and Trichormus variabilis but were 4-fold higher at 1.1–1.7 mg L−1 for Synechococcus nidulans and two other cyanobacteria species −1 H2O2) for off-flavor producing cyanobacteria Oscillatoria cf. chalybea but it was 10-fold higher  for Anabaena sp LP691 Raphidiopsis sp. after 48-hr exposure to 200 µM H2O2 at 90 µmol m−2 s−1 but no significant bleach was observed in Anabaena sp under the same condition 2O2 tolerance among cyanobacteria, Schrader et al. concluded that H2O2-based algaecide “may be less disruptive to the aquatic ecosystem by not eliminating all of the cyanophytes so quickly as to create low dissolved oxygen levels which could stress and kill fish” 2O2 addition (as compared to the control without H2O2 addition). Furthermore, the most sensitive cyanobacteria species, though eliminated by H2O2 treatment, likely get re-inoculated from surrounding water via natural water exchange, while the more resistant ones would persist and repopulate the treated water.Cyanobacteria community could be most severely affected by H−1 was degraded to 0.7 mg L−1 after one day and to below detection limit (∼ 0.1 mg L−1) after two days 2O2, if applied at 1.6 mg L−1 to natural seawater, to be reduced to the natural background level within a couple of days due to decomposition and simultaneous dilution. The proposed concentration is indeed quite low as compared to a) the proposed receiving water quality criterion of 0.7 mg L−12O2 in North Carolina −1) at Bermuda Atlantic Time Series Stations 2O2 potentially improves water quality by oxygenating the water column, facilitating dissolved organic matter degradation 2O2−1 H2O2 may be effective in brown tide bloom control.Hydrogen peroxide is rapidly decomposed into water and oxygen −1 H2O2 effectively removed brown tide even at peak bloom intensity, and the other phytoplankton of >2–3 µm cell size remained largely unaffected by the treatment. Thus hydrogen peroxide is proposed as a natural and environmentally friendly agent potentially useful for brown tide control.Harmful algal bloom has become an increasingly important issue to the economic development and environmental sustainability, such that it demands both direct control methods and long term prevention measures. This study evidenced, from culture and microcosm studies, that 1.6 mg L


Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient  between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado. The study of population and genetic diversity is a complex subject that involves the analysis of DNA sequences, gene adaptability, inter-individual variation and speciation, and an understanding of the interactions among organisms that compose communities. Furthering knowledge about genes, individuals, species and communities provides an ever greater understanding of biodiversity and, consequently, allows the development of the most adequate strategies for environmental preservation. Molecular markers provide an important tool for assessing the genetic variability and structure of natural populations, and for studying biodiversity in general (The random amplification of polymorphic DNA (RAPD) provides markers that can be used to identify and discriminate genotypes, in addition to providing a means for assessing phenotypic expression and phylogenetic relations in the germplasm under study . SimilarJacaranda decurrens is a medicinal plant endemic to the Cerrado. One of its components, ursolic acid, is considered to have antitumoral activity   . The areulo (SP)  and are J. decurrens DNA were initially evaluated with 110 primers for RAPD (Operon Life Technology and Biosynthesis Incorporated), 30 of which were selected for further investigation. The reactions were optimized by the amplification protocol of Samples of J. decurrens : 3.0 μL of Tp 10X buffer, 3.0 μL of 2.5 mM dNTPs, 2.4 μL of 25.0 mM MgCl2, 5.0 μL of primer (10 ng/μL), 0.3 μL of Taq DNA polymerase (5 U/μL) and 4.0 μL of sample DNA (5 ng/μL). The amplifications were done in a thermocycler using the following conditions: 2 cycles at 94 °C for 2 min, 1 cycle at 37 °C for 1 min, 1 cycle at 72 °C for 2 min, and 33 cycles at 94 °C for 10 s, 40 °C for 20 s  and 72 °C for 2 min.Of 30 RAPD primers selected, 10 showed a high degree of polymorphism and better band intensity . After oEcoRI and MseI at 37 °C for 3 h in a model PTC-100 thermocycler (MJ Research Inc). The resulting fragments were ligated to specific adapting sequences complementary to the extremities cleaved off by the two enzymes in a reaction done at 23 °C for 3 h.AFLP was done using a protocol adapted from Eco-A/MseI-A and the PCR products of this pre-amplification were used as a template for the selective amplification in which primers with three selective nucleotides were added to the 3' extremity of the primers. Four combinations that were helpful in detecting polymorphisms were used, namely, Eco-ATC/MseI-ATC, Eco-ATT/MseI-ATG, Eco-ATG/MseI-AGT and Eco-AGT/MseI-ATT.The ligated fragments (50 μL) were pre-amplified with primers containing a selective base, EcoRI-primer with one selective base (25 ng/μL), 1 μL MseI + (primer with one selective base) (25 ng/μL), 0.8 μL of 2.5 mM dNTPs, 2 μL of 10 X buffer B (Promega), 1.2 μL 25 mM MgCl2, 0.6 μL Taq DNA polymerase (5 U/μL) (Promega) and 2 μL of the ligated DNA. The reaction conditions were as follows: 1) 94 °C for 2 min, 2) 94 °C for 1 min, 3) 56 °C for 1 min, 4) 72 °C for 1 min, and 5) 72 °C for 5 min; steps 2 to 4 were repeated 26 times. The amplification products were subsequently diluted four-fold and stored at -20 °C.The components of the pre-amplification reaction were: 1 μL of EcoRI + (primer three selective base) (25 ng/μL), 1.2 μL MseI + (primer with three selective bases) (25 ng/μL), 0.4 μL of 2.5 mM dNTPs, 2 μL 10 X buffer B (Promega), 1.2 μL of 25 mM MgCl2, 0.2 μL of Taq DNA polymerase (5 U/μL) (Promega) and 1.5 μL of pre-amplified DNA. The reaction conditions were as follows: 1) 94 °C for 2 min, 2) 94 °C for 30 s, 3) 65 °C for 30 s, 4) 72 °C for 1 min, 5) 94 °C for 30 s, 6) 56 °C for 30 s, 7) 72 °C for 1 min and 8) 72 °C for 2 min; steps 2 to 4 were repeated 12 times and steps 5 to 7, 23 times. The amplification products were stored at -20 °C.For selective amplification by AFLP-PCR the reaction mixture contained: 1 μL of The AFLP products were separated by electrophoresis in denaturing 6% polyacrylamide gels in TBE 1X buffer for 2 h at 50 °C and 80 W constant voltage. The gels were stained with silver nitrate solution and developed in sodium carbonate, as described by The binary data obtained from RAPD and AFLP were used to estimate allele frequencies based on a correction proposed by The ten primers chosen produced 78 bands in the six populations, with most (69.23%) of the bands being polymorphic. The minimum and maximum number of bands per primer was, respectively, 3 and 12 . Figure The genetic characteristics of the six populations based on the allele frequencies (assuming Hardy-Weinberg equilibrium for the 205 loci studied) are shown in ST value was 0.303 for RAPD, while for AFLP it was of 0.263 (p < 0.001); both markers indicated a highly significant structuring of the genetic variability in these populations  while significant similarity was observed among the populations from Mogi-Guaçú and São Carlos (0.0139) .J. decurrens populations studied here. Both findings support a model of partially isolated islands that evolved independently in genetic space.The spatial pattern determined by the Mantel test based on 5000 permutations was 0.37 (p = 0.1006) and -0.16 (p = 0.2082) for AFLP and RAPD, respectively. This finding indicated that there was no significant matricial correlation between geographic and genetic distances. These results clearly indicate that geographic distance alone does not explain the spatial pattern of genetic diversity among the populations. They also show that there is no spatial structuring of the genetic variability for AFLP and RAPD markers in the J. decurrens in the State of São Paulo. The high variability reported here indicates that these markers are useful for analyzing the genetic constitution of the six populations that were studied; this conclusion was supported by the number of polymorphic bands observed  reflects the probability that two random genes from two populations are identical by descent   since local adaptations tend to favor the establishment of a species in its natural habitat. With the exception of the Mogi Guaçú population, which is located on the Mogi Guaçú Biological Reserve, all of the other populations are located on private lands that have experienced different degrees of human intervention. Since it is likely that in the near future these populations will cease to exist because of increased land use, we suggest that samples of the material analyzed here be preserved in a germplasm bank.The results of this study may be useful in establishing conservation strategies of endemic species such as


In First Nations communities of northwestern Ontario, where rates of type 2 diabetes mellitus are some of the highest in the world, ascertaining wild food dietary practices is extremely challenging owing to seasonal availability, environmental factors, life circumstances and language/cultural barriers. The purpose of this study was to determine whether analysis of isotopic and fatty acid (FA) profiles could provide more comprehensive information to discriminate between three categories of wild food consumption  in two isolated First Nations communities of northwestern Ontario. In addition, this analysis also highlights whether wild food consumption as practiced in these two communities can increase circulating levels of polyunsaturated FAs (PUFAs), which provide a number of important metabolic benefits that could impact the prevention/treatment of T2DM.13C enrichment , 15N enrichment (in hair) and FA profiles in plasma phospholipids (phospholipid fatty acid (PL-FA)) were quantified in men and in women consuming various amounts of wild food. 13C/12C ratios were lower and 15N/14N ratios were higher in participants consuming wild food at least once a week. In addition, FA results indicated that the relative contributions of 20:4 Ω-6 and 22:6 Ω-3 to total PL-FAs were higher and 18:2 Ω-6 lower in wild food consumers.Together, these findings confirm that isotopic and lipid markers discriminate between the different wild food categories in these two First Nations communities. Knowing the close relationship between dietary intake and the potential role of PUFA in the prevention/treatment of obesity and obesity-related diseases, it is critical to accurately measure the composition of diet for individuals in their specific environments. In this context, locally harvested foods remain a key source of nourishment for these communities, making its incorporation into diet an important strategy for reducing high rates of obesity and type 2 diabetes mellitus. Locally harvested plants and animals are significant sources of dietary protein, essential minerals, vitamins and polyunsaturated fatty acids (PUFAs),9 particularly long-chain Ω-3 fatty acids (FAs). These Ω-3 FAs have protective benefits against cardiovascular disease, obesity and diabetes11 which continue to be much more prevalent in aboriginal populations.13 The environment around remote communities of northwestern Ontario provides access to a wide range of fish, mammals, birds and wild berries that can be consumed throughout the year.14 However, these wild foods are far from being accessible in sufficient amount to all community members. Ascertaining the extent to which these foods are consumed is challenging owing to their seasonal availability, environmental factors and life circumstances that impact access to land-based foods. This unique context makes accurate and objective assessments of overall food consumption difficult, which is exacerbated by language/cultural barriers and reporting bias.In research and clinical perspectives, adequate assessments of dietary practices are essential for the prevention/treatment of type 2 diabetes mellitus. This holds particularly true in Canada's First Nations communities of northwestern Ontario where rates of type 2 diabetes mellitus are some of the highest in the world.19 For example, some studies have used stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) to estimate the relative consumption of sugars and sweeteners made from corn and sugar cane20 and the consumption of specific types of proteins between groups of individuals.21 Similarly, intervention studies in humans and in other animals showed that dietary FAs are reflected in the FA profile of membrane phospholipids (PLs).25 Traditionally, the hunter/gatherer diet north of the 49th parallel was rich in proteins ), rich in lipids (∼43% E), and low in carbohydrates .26 The CHO consumed were mainly from C3 photosynthesis plants low in carbon-13 as sugars from corn and sugar cane were not available.28 Proteins and lipids were obtained from hunting and fishing and constituted at least 70% of their subsistence.26 Proteins from wild meats, especially fish, are naturally enriched in 15N (∼14 δ15N‰) compared with market meats (∼4 δ15N‰).21 Moreover, high wild meat consumption provides a greater diversity of FA intake.9 For example, PUFA intake of hunter–gatherer societies was estimated at 27 g·day−1 with 42% of linoleic acid, 41% of alpha-linolenic acid, 11% of 20–22C Ω-6 FAs and 6% of 20–22C Ω-3 FAs for a Ω-3 PUFA balance of 47%.29 Whether the combined measurements of isotopic enrichment (13C and 15N) and FA profiles would allow researchers to discriminate between dietary practices in First Nations communities of Northwestern Ontario remains to be determined.Methods to assess dietary intake using isotopic and lipid markers may assist in mitigating challenges with communication and reporting. It is well established that isotopic enrichment and FA profiles of consumed foods are reflected in the organism and can be quantified in blood, expired gases and tissue biopsies.13C enrichment , 15N enrichment (in hair) and FA profiles in plasma PLs were quantified in men and in women consuming various amounts of wild food. Based on the combined results obtained by a number of researchers,30 we anticipated that wild food consumers would show lower 13C enrichment in all compartments measured, higher 15N enrichment and higher PUFA levels in plasma PL.The purpose of this study was to quantify differences in the quality of CHO, lipid and protein consumed in two Oji-Cree communities of northwestern Ontario. More specifically, This study was conducted according to the guidelines in the Declaration of Helsinki and all procedures involving human subjects/patients were approved by the University of Ottawa Research Ethics Board and Health Canada Ethics Board. Written informed consent was obtained from all volunteers. The study was conducted in Wapekeka (Angling Lake) and Kasabonika Lake First Nations in northwestern Ontario, Canada. Inclusion criteria required that individuals be Aboriginal, over 18 years, non-pregnant and free of type 1 diabetes. The number of individuals participating in each community (39 in Wapekeka and 32 in Kasabonika) represents ∼9% of the eligible population in Kasabonika and 24% of the eligible population in Wapekeka at the time of the study. In order to potentiate differences in dietary behaviour between individuals, data collection was performed over a period of 2 months when wild food consumption was known to be high in these communities (late September to early November 2007). Participants were recruited using a mixed-method ethnological approach combining semi-structured interviews, food frequency questionnaires, 3-day dietary records and 24-h dietary recalls. As presented in 2, participants exhaled in a 10 ml vacutainer tube containing no additives. Tubes were sealed with a rubberized cap and secured in place with paraffin tape for transport to the University of Ottawa . Blood samples were taken from the antecubital vein (left arm) in 4 ml vacutainers containing an anticoagulant (ethylenediaminetetraacetic acid). Upon collection, blood samples were immediately placed on ice and centrifuged at 3500 g. Plasma and red blood cells were separated and frozen at −20 °C. At the end of the study, samples were transported frozen to a −80 °C freezer at University of Ottawa  until analysis. Hair samples were collected from the nape of the neck as near as possible to the scalp using stainless steel scissors, and were sealed separately in labelled bags for safe transportation to the University of Ottawa. Hair was then cleaned by soaking in a 2:1 chloroform:methanol solution to remove any residues and then rinsed several times with distilled water. Samples were thoroughly dried before performing any analysis.Volunteers were asked to report at the nursing station (Kasabonika) and clinic (Wapekeka) located in each community at ∼0900 following 24 h without strenuous excercise and a 12 h fast. First, height and weight were measured and used to calculate body mass index (BMI) by dividing body weight  by square height . For the collection of expired CO13C/12C ratio in expired CO2. 13C enrichment in expired CO2 was measured at the GEOTOP UQAM/McGill Stable Isotope Laboratory  in a Prism mass spectrometer . In plasma glucose. 1 ml of plasma was deproteinized, and the glucose was then separated by double-bed ion exchange chromatography by running the supernatants through superimposed columns , as previously described by Peronnet et al.31 Following evaporation, glucose was combusted (60 min at 400 °C) in the presence of copper oxide. CO2 was recovered and measured at the GEOTOP UQAM/McGill Stable Isotope Laboratory  in a Prism mass spectrometer . Isotope ratios in hair. 13C and 15N enrichment in hair was measured at the G.G. Hatch Stable Isotope Laboratory, University of Ottawa. Cleaned hair samples (∼0.6 milligram) were cut, weighed and placed into tin capsules (8 × 5 millimetres (mm), Isomass Scientific . Isotopic enrichment was determined by the analysis of CO2 and N2 produced by flash combustion at 1800 °C on a CE 1110 Elemental Analyzer followed by gas chromatographic separation and on-line analysis by continuous-flow with a DeltaPlus Advantage isotope ratio mass spectrometer coupled with a ConFlo interface. Data were normalized using internal standards previously calibrated with International standards IAEA-CH-6, IAEA-NBS22, IAEA N1, IAEA-N2, USGS-40 and USGS-41.2 and in CO2 obtained from plasma glucose was expressed as δ values (‰) compared with the V-PDB standard for 13C and atmospheric N2 for 14N, respectively:33Isotopic composition in hair samples, in expired CO13C and 15N and R is the corresponding ratio of 13C/12C and 15N/14N in the sample and standard, respectively.where X is 34 Extracted lipids were then suspended in chloroform in which PLs were separated by filtration on Superclean solid-phase extraction tubes  as previously described35 and then eluted with methanol. Two hundred micro liters of margaric acid  was added as an internal standard and PLs were then trans-esterified with acetyl chloride in methanol for 2 h at 90 °C. After evaporation, the newly formed FAs methyl ester were dissolved in 60 μl isooctane and 2 μl were injected in a Hewlett-Packard gas chromatograph (HP 9890 with HP 7683B autosampler) equipped with a flame-ionization detector and a 60 m fused silica column . The carrier gas was nitrogen and detector gases were hydrogen and air. Injection port temperature was set at 220 °C and the detector at 240 °C. The temperature was 185 °C for 35 min after injection, raised to 210 °C at a rate of 5 °C min−1, and kept at 210 °C for an additional 10 min. Exact retention times of each FA were determined with pure standards (Sigma-Aldrich). Plasma PL concentrations were measured by colorimetry using the PL-C microtiter procedures kit . Because samples were not store in transported in N2 filled vacutainers, it is possible that some hydrolysis of PL occurred resulting in a release of FAs. These FAs would not have been included in the PL fraction following the filtration through the solid-phase extraction tubes (described above). However, it was assumed that the hydrolysed PL would release FAs in the same proportion as what remained in the non-hydrolysed PL fraction. FA profiles were presented as per cent contribution of individual FA to total plasma PL-FA. Only FAs accounting for >5% of total PL-FA were presented in this analysis. Degree of unsaturation of PL-FA was calculated using the following formula:36Circulating lipids were extracted twice in chloroform–methanol (2:1 v/v) from 200 μl of plasma collected in the fasting state according to the Folch method.13C/12C ratios, 15N/14N ratios and relative contributions of individual FAs to total PL-FA. Bonferonni's multiple comparisons post hoc test was used, where applicable. A two-tailed P-value of <0.05 was considered significant. All analyses were performed using SPSS for Windows  or GraphPad Prism version 5.00 for Windows . All parameters were normally distributed (according to the Shapiro–Wilk test) and the homogeneity of variance between our groups was equal, based on the Levene's test. Despite limited sample size, the effect size for the differences observed between our groups was large . Because 25 of the 71 participants suffered from type 2 diabetes mellitus, we conducted a chi-square test and Pearson correlations between HOMA-IR to determine whether this chronic disease could influence the results of this study. We found that proportion of type 2 diabetic individuals did not differ between food groups . In addition, isotopic and FA markers did not correlate with HOMA-IR scores (P=0.47–0.98). Based on these two results, we confirm that our findings remain the same regardless of whether type 2 diabetic individuals are included in the analyses.Data were expressed as mean+s.e. A one-way ANOVA with between-subjects design was used to analyse differences in P=0.13) and body mass index (P=0.10) were found between groups, WF1 were 1.8 years and 12.1 years younger than WF2 (P=0.02) and WF3 (P=0.02), respectively.Differences in wild food categories, age, sex ratios, weight and body mass index between individuals and within each food behaviour group are presented in 13C/12C ratios for expired CO2, plasma glucose and hair for WF1, WF2 and WF3 are presented in 13C/12C ratio was found in expired CO2 with δ13C values averaging −23.4±0.2‰ for WF1, −23.3±0.2‰ for WF2 and −23.9±0.2‰ for WF3 (P=0.16). In contrast, the average plasma glucose 13C/12C ratio was lower in WF1 (δ13C of −24.1±0.5‰) than in WF3 (δ13C of −26.3±0.8‰ P=0.05). Values in WF2 for this compartment (δ13C of −24.3±0.8‰) were not different from WF1 (P=0.16) and WF3 (P=1.00). Similar results were found for 13C/12C ratios in hair where WF1 displayed a lower average (δ13C of −19.2±0.1‰) than in WF3 (δ13C of −19.5±0.1‰ P=0.019). No differences were observed between WF2 (δ13C of −19.2±0.1‰) and WF1 (P=0.13) and between WF2 and WF3 (P=1.00).Differences in 15N/14N ratios found in hair in WF1, WF2 and WF3 are presented in 13C/12C ratios in plasma glucose and hair, a significant difference was observed for the 15N/14N ratio in WF1 (δ15N of 9.6±0.1‰) compared with WF3 (δ15N of 10.1±0.1‰) (P=0.006). Values in WF2 (δ15N of 9.9±0.1‰) were not different from WF1 (P=0.90) or WF3 (P=0.116).Differences in P=0.76), 18:0 (P=0.18), 18:1 Ω-9 (P=0.22) and 20:3 Ω-9 (P=0.34) to total PL-FA between WF1 , WF2  and WF3  (P<0.001). Conversely, per cent contribution of 20:4 Ω-6 to total PL-FA increased overall from WF1 (9.6±0.3%) to WF3 (11.2±0.5%) (P=0.03). No difference in this FA was found between WF2 (10.4±0.5%) and WF1 (P=0.60) or WF3 (P=0.57). Finally, per cent contribution of 22:6 Ω-3 to total PL-FA was lower in WF1 (4.1±0.2%) when compared with WF3 (6.6±0.6%) (P<0.0001). However, no difference for 22:6 Ω-3 was observed between WF2 (5.6±0.4%) and WF1 (P=0.27) although this FA was lower in WF2 than in WF3 (P=0.04). As shown in P=0.001). No difference in degree of unsaturation was found between WF2 (1.62±0.03%) and WF1 (P=0.38) or WF3 (P=0.12).Differences in the FAs composition and degree of PL-FA unsaturation for WF1, WF2 and WF3 are shown in ctively) . The rel13C/12C and 18:2 Ω-6) and fish consumption markers (15N/14N and 22:6 Ω-3). For store-bought food markers, 13C/12C ratios and the relative contribution of 18:2 Ω-6 to total PL-FA decreased overall from WF1 to WF3 . For fish consumption markers, 15N/14N ratios and the relative contribution of 22:6 Ω-3 to total PL-FA increased overall from WF1 to WF3 . In addition, 13C/12C ratios and the relative contribution of 18:2 Ω-6 to total PL-FA as well as 15N/14N ratios and the relative contribution of 22:6 Ω-3 to total PL-FA were significantly correlated .13C/12C ratios were lower and 15N/14N higher in participants consuming wild food at least once a week .38 In contrast, traditional hunter–gatherer diet consists of plants relying on C3-photosynthesis with a lower natural abundance of 13C (δ13C of −25‰). Based on these important differences in 13C enrichment, many have advocated the value of carbon isotope ratios (13C/12C) as a means to differentiate dietary preferences/behaviours.20 In our study, results indicated that differences in the quality of CHO consumed between participants were sufficient to obtain significant differences in average 13C/12C ratios in blood or hair between low-  and high-wild food consumers . However, 13C enrichment was not sensitive enough to discriminate between the quality of CHO consumed in WF1 and WF3 from the intermediate wild food consumers . In addition, contrary to what was anticipated, breath CO2 did not allow us to differentiate the three levels of wild food consumption. This might not be that surprising as 13CO2 abundance in breath CO2 is not only influenced by diet but also by the 13C abundance of the substrate being oxidized.39 As fuel selection was not quantified in this study, it is difficult to speculate on whether the lack of differences in 13C/12C between groups is due to differences in substrate utilization.39 Nevertheless, findings from this study show that 13C/12C ratios in blood glucose and in hair are the most reflective of dietary practices in these communities.The traditional First Nations diet continues to be undermined by the access to low quality market foods in northern Canadian First Nations communities.13C/12C ratios, nitrogen isotope ratios (15N/14N) have proven to be a powerful dietary marker for wild food and fish consumption in this study. This outcome corroborates several other studies that have shown hair δ15N to track animal protein consumption among vegans, vegetarians and omnivores.18 Compared with vegans and vegetarians, omnivores are feeding at the highest trophic level and thus have a greater enrichment of δ15N in their hair due to higher animal protein intake. As expected, in our study, individuals consuming greater amounts of wild fish and hunted meats display higher hair δ15N values. More specifically, community members that consumed wild meat more than once a week (WF3) had higher δ15N values than participants who consumed wild meats less than once a month (WF1). However, as for 13C/12C ratios in blood and hair, variations in diet were not sufficient to find differences between WF2 and the two other groups. It is important to note that higher δ15N values observed in the WF3 are likely attributed to the high consumption of wild fish. In these two communities, wild fish is a staple food available in large amounts most of the year.In addition to 15N isotope, wild foods are particularly rich in 20–22C Ω-3 FAs.41 Previous work has shown that PL-FA were highly reflective of FA intake for 3–6 weeks prior to the blood draw.43 This is mainly due to the fact that proportion of PUFA in plasma PL is tightly regulated and approximate 40% of total FA.44 Here, results showed that 22:6 Ω-3 (6.6±0.6% PL-FA) and 20:4 Ω-6 (11.2±0.5% PL-FA) were, respectively, 1.6-times and 1.2-times higher, whereas 18:2 Ω-6 was 1.2-times lower (18.8±0.9% PL-FA) in WF3 when compared with WF1 . In the intermediate WF2, measurements of PL-FA were sensitive enough to discriminate the relative contribution of 18:2 Ω-6 from that of the two extreme groups. For 22:6 Ω-3, values for WF2 were different from those found for WF3 (In addition to higher levels of  for WF3 . In addi48store-bought food is highly processed and is particularly rich in linoleic acid (18:2 Ω-6) derived from soybean oil and rich in refined sugar derived from corn and sugar cane containing high level of 13C label.49 In contrast, wild foods have higher 20–22C Ω-3 FAs, much lower linoleic acid and lower α-linolenic acid29 and contain virtually no refined sugars (δ13C∼−25‰). In addition, proteins from wild meats, especially fish, are naturally enriched in 15N (δ15N ∼14‰) compared with market meats (δ15N ∼4‰).21 Although historical diet was entirely land based, most individuals living in remote First Nations communities today have to rely on store-bought foods to sustain their dietary needs and complement wild food consumption. Interestingly, the parallel increase in 22:6 Ω-3 and δ15N of values strongly suggest that fish is a key source of meat for wild food consumers in these communities; a finding confirmed by the information obtained during the interviews. Clearly, our study showed that isotopes and FA measurements can be used to establish significant differences in the relative contribution of store-bought vs wild food consumption in First Nations communities. However, it seems that this is only possible when extremes in wild food consumption are considered; that is, wild food more than once a week or wild food less than once a month. Results show that only 22:6 Ω-3 and δ13C can be differentiated between intermediate WF2 and high wild food consumers in WF3. This finding exposes the importance of carefully recruiting participants in research attempting to quantify the potentially beneficial effects of wild food consumption on rates of chronic diseases. Typically, studies interested in estimating dietary pattern within a population would use epidemiological methodologies involving excessively large cohorts (ex: >10 000 samples) and food frequency questionnaires.51 However, this approach is difficult in remote, isolated First Nations communities where available cohorts are small and where it is difficult to assess precisely the dependence on locally harvested/hunted foods.46 In this context, many researchers interested in assessing dietary intake rely on self-reporting by participants which has obvious limitations. Dietary assessment tools such as individual interviews, food frequency questionnaires and dietary recalls are essential to understanding dietary behaviours; however, they can be problematic owing to, among other things, under-reporting,30 individual privacy, bias, cost, and time required of both participants and researchers.55 Although dietary marker-based methods provide less qualitative information, they are relatively free of any bias56 and can help to validate dietary surveys by identifying inconsistencies between tissue concentrations and dietary intake.When compared with westernized foods obtained in stores and restaurants, adherence to land based foods is often recognized to provide several health benefits for indigenous populations.21 The use of these methods can be directly applied to assessing dietary behaviours within and between First Nations communities. Knowing the close relationship between dietary intake and the development/prevention of obesity and obesity-related diseases, it is critical to accurately measure the composition of diet for individuals in their specific environments. This information will be key for the development of region specific chronic disease prevention strategies.Stable isotopes and FA profiles as dietary markers have been used widely in both human and animal dietary studies, however none have successfully combined these methods to assess well-defined dietary behaviours in northern Canadian First Nations communities. With large differences in dietary behaviours observed within First Nations communities, analysis of stable isotopes and FA profiles of PLs can provide quantitative information about an individual's diet and can contribute greatly to Aboriginal health and nutrition studies by providing a ‘non-invasive, simple, yet powerful tool for monitoring dietary pattern'.


Arabidopsis thaliana β-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 °C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co2+ and inhibited by Mn2+. The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min−1 mg−1 mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.Each plant genome contains a repertoire of β-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of  Arabidopsis thaliana, mannan-type polysaccharides are detected in low levels in most tissues, but appear to be more abundant in flowers, siliques and inflorescence stems. Secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibers contain higher amounts of mannans . These enzymes are responsible for catalyzing hydrolysis of the β-1,4-linked backbone within different mannans. In the CAZy (carbohydrate-active enzymes) database 8 barrel fold structure and a retaining mechanism with conserved catalytic residues . All plant mannanases belong to family GH5, specifically subfamily 7 (GH5_7) , cucumber (Cucumis sativis), poplar (Populus trichocarpa), rice (Oryza sativa) and Arabidopsis . In the genome of the model plant Arabidopsis thaliana, the endo-β-mannanases family consists of eight members, but one is likely a pseudo gene  are expressed in germinating seeds and affect the germination time (t50)  has been detected in three different tissues , but not in geminating seeds  is presented in the current study.Only a few plant mannanases have been enzymatically characterized. LeMAN4a involved in tomato fruit development has been reported to possess both hydrolytic and transglycosylation activities   and ELM (http://elm.eu.org/) , the open reading frame of AtMan5-1  without signal peptide was amplified by PCR using Phusion polymerase (Finnzymes) and the forward primer 5′-CACCATGGTAAAGACAGGCT-3′ and the reverse primer 5′-TTCTGCACTATGTGTGACCA-3′. The forward primer contained a CACC overhang needed for the TOPO® cloning procedure. The PCR products were cloned into the pENTR/SD/D-TOPO entry vector (Invitrogen). The cloning reaction mixture was transformed into chemically competent E. coli TOP10 cells. The extracted constructs were recombined with the pET-DEST42 destination vector  (Invitrogen) by using the LR Clonase mix II (Invitrogen). To express the target gene in P. pastoris (AtMan5-1p), the catalytic module without the signal peptide were PCR amplified using the forward primer 5′-ATTATCGCGGCCGCCGTA AAGACAGGCTTTG-3′ and the reverse primer 5′-GGCGGCTCTAGATGTTCTGC ACTATGTGTGAC-3′. The constructs were combined with the pPICZα-C vector  by ligation. To confirm the presence of correct gene, DNA sequencing was performed by a commercial service (Eurofins MWG Operon).Plasmid DNA containing the AtMan5-1 full-length cDNA was obtained from RIKEN  transformants were grown in Terrific Broth with 100 μg/mL ampicillin and 30 μg/mL chloramphenicol at 37 °C. The culture was induced by adding 0.5 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG) at OD600 of 0.5–0.8 and grown at 16 °C overnight. Biomasses were harvested by centrifugation . The cell pellets were resuspended in phosphate buffer  with NaCl (0.5 M) and lysed by performing French press. Crude extracts were separated from cell debris by centrifugation  and then used for protein purification.To obtain expressed AtMan5-1e, the P. pastoris SMD1168H transformants were grown in BMGY medium at 30 °C to an OD600 of 2–6. The cells were harvested by centrifugation  and resuspended in BMMY medium to OD600 of 0.5. Cultures were grown at 25 °C up to 72 h and feed with methanol to a final concentration of 1 % every 24 h. Culture supernatants were harvested by centrifugation  and then used for protein purification.To obtain secreted AtMan5-1p, the 6-tag by immobilized metal affinity chromatography (IMAC) using an ÄKTA purifier system  with a BioRad Profinity IMAC Ni-Charged Resin . Then, the protein was purified further by size-exclusion chromatography (SEC) using a HiPrep 26/60 Sephacryl S-200 column  in 20 mM tris–HCl (pH 8.1), which was followed by ion exchange chromatography using HiTrap Q XL column . The eluted protein was washed and concentrated by 10 kDa cutoff Amicon Ultra centrifugal filters (Millipore) using sodium citrate buffer . AtMan5-1p was purified via its C-terminal His6-tag by IMAC as described above, and followed by buffer exchanging to sodium citrate .AtMan5-1e was purified via its C-terminal HisTo evaluate the purity and size of enzyme preparations, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by using 10 % precast polyacrylamide gels  and 1× Tris–Glycine buffer. Deglycosylation of AtMan5-1p was performed using the Glycoprofile™ II, Enzymatic In-solution N-deglycosylation kit (Sigma) following the manufacturer’s protocol. For identification of the generated recombinant enzymes, proteins bands of interest were excised, trypsin digested, and peptide samples were analyzed by mass spectrometry (MS)  in 50 mM sodium citrate buffer  containing 3 mg/mL carob galactomannan at 35 °C for 30 min. The control reaction was performed under the same conditions as above but without addition of any metal ion. To determine apparent kinetic parameters, enzymatic activity was measured in 50 mM sodium citrate buffer  containing carob galactomannan (1.5–6 mg/mL) at 35 °C  reducing sugar assay Miller . Total vC Miller .3,64-α-d-galactosyl-mannopentaose (G2M5), mannohexaose (M6). 0.26 μM AtMan5-1p enzyme was added into the polysaccharide reactions, whereas the oligosaccharide reactions contained 0.13 μM enzyme. The mixtures were incubated at room temperature for 7 days. Oligosaccharide analysis was carried out by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an ICS-3000 system . 10 μL of enzyme incubations were injected in a PA200 column with an isocratic flow of 0.5 mL/min of 45 mM NaOH at 30 °C during 15 min. Peak assignation was performed by comparison of the retention time with a series of manno-oligosaccharides  from Megazyme (Ireland).To confirm the hydrolytic capability of AtMan5-1, samples were prepared using different substrates including 2 mg/mL suspensions of carob galactomannan, konjac glucomannan, guar gum, spruce galactoglucomannan, arabinoxylan, hydroxyethylcellulose, xyloglucan and 1 mM of mannotriose (M3), mannotetraose (M4), mannopentaose (M5), 6To detect possible transglycosylation reactions, 0.22 μM of purified AtMan5-1p was mixed with 5 mM M5/M6 or 5 mM M5/M6 and 1 M sodium chloride, and these reactions were incubated at 35 °C for 24 h. The final products were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) for molar mass analysis. The reaction products and the matrix solution  were pre-mixed in a ratio 1:1 and spotted directly on a standard steel MALDI plate prior to analysis. The mass spectra were collected using a LaserToF LT3 Plus instrument  in the reflectron mode with positive ionization.Coffea arabica; GenBank accession CAC08208, CAC08442), sitka spruce  and tomato  were added to the dataset. The Arabidopsis mannanases were redesignated so that the GH family number is included in their names  and spikemoss (Selaginella moellendorffii) proteins  and (E319), as well as other well conserved residues within the GH5 family Fig.  Jenkins. Most am6 tag was successfully expressed in both E. coli Rosetta (DE3) cells (denoted AtMan5-1e) and P. pastoris SMD1168H cells (denoted AtMan5-1p). The purification yield was higher for AtMan5-1p (12 mg/L) than for AtMan5-1e (0.7 mg/L). The recombinant AtMan5-1e was purified by IMAC followed by gel filtration and ion exchange. The recombinant AtMan5-1p was purified in a single step using IMAC. According to SDS-PAGE, AtMan5-1e migrated as a single distinct band of approximately 48 kDa  was slightly higher for the E. coli produced AtMan5-1 .The kinetic parameters of AtMan5-1 were determined by using carob galactomannan as substrate under optimal conditions. The Michaelis constant Pichia-produced recombinant protein was included in the study since the yield of soluble AtMan5-1e was relatively low. The major products from carob galactomannan depolymerization were M2, M3 and a galactomanno-oligosaccharide that can be assigned as a GM3. From konjac glucomannan hydrolysis the main identified oligosaccharide was M2, although M3 was also moderately abundant. Two abundant hydrolysis products could not be identified using HPAEC-PAD, although these fragments most probably consist of short glucomanno-oligosaccharides , whereas M2 and M3 were identified as the main products of M5 although not in high amounts. The digestion of M6 released M2, M3 and M4 among which M3 was the most abundant product. Comparison of the relative amounts of digested M5 and M6 revealed that the catalytic rate of AtMan5-1 towards M6 was 2.6 times higher than that of M5. The absence of products with a higher degree of polymerization (d.p.) than the examined oligosaccharides indicated that no transglycosylation reaction occurred. However, when in the substrate concentration of M5 was increased in order to favor transglycosylation the larger manno-oligosaccharides M6, M8 and M9 were observed indicating that recombinant AtMan5-1 is able to catalyze transglycosylation reactions in vitro at higher substrate concentrations  or P. pastoris (AtMan5-1p). Effects of pH and temperature on enzyme activity were similar for both recombinant enzyme variants. The obtained pH optima close to 5.0 is in agreement with the reported pH optima for the characterized plant mannanases LeMAN4a and GmMAN1  of AtMan5-1e was slightly higher than for AtMan5-1p. Again, the apparent differences are probably a consequence of AtMan5-1p glycosylation. The observed catalytic efficiency for AtMan5-1 using carob galactomannan as substrate was approximately ten times higher compared to what was reported for GmMAN1  was approximately 3–5 orders of magnitude lower compared to the catalytic efficiency numbers obtained for most fungal mannanases  and xyloglucan were tested as substrates, but all mannan polysaccharides except guar gum were efficiently degraded with M2, and M3 as major hydrolysis products. The carob galactomannan hydrolysis product pattern obtained was similar to the hydrolytic cleavage profile of the same substrate incubated with a purified barley mannanase  (data not shown). None of the other Arabidopsis mannanase genes are as distinctly up-regulated by these factors as AtMan5-1. Probably, AtMan5-1 modifies cell wall mannans in response to elevated levels of ABA caused by drought and high salinity, but whether the mode of action in vivo includes hydrolysis and/or transglycosylation remains to be investigated.The examination of AtMan5-1p hydrolysis products confirmed the results from the substrate specificity analysis and also demonstrated that the mannanase is a specific The acquired detailed knowledge of the catalytic properties of the Arabidopsis mannanase, AtMan5-1 belonging to GH5 subfamily 7 presented in this study, is a first step towards understanding the enzymatic degradation and regulation of mannan-type polysaccharides in Arabidopsis. The results indicate that AtMan5-1 is adapted to hydrolyze linear stretches of mannans and glucomannans, can catalyze transglycosylation reactions at high substrate concentrations, and the importance of glycosylation for thermal stability was revealed by comparing recombinant production of AtMan5-1 in two different expression systems. Since only a minor portion of plant mannan-active enzymes have been biochemically characterized, the present findings are not only valuable for the Arabidopsis community, but crucial for further exploration of the plant mannanase portfolio.


To assess interfraction translational and rotational setup errors, in patients treated with image-guded hypofractionated stereotactic radiotherapy, immobilized by a thermoplastic mask and a bite-block and positioned using stereotactic coordinates.x, θy, θz) were calculated for the matching results of bone matching algorithm.37 patients with 47 brain metastases were treated with hypofractionated stererotactic radiotherapy. All patients were immobilized with a combination of a thermoplastic mask and a bite-block fixed to a stereotactic frame support. Daily cone-beam CT scans were acquired for every patient before the treatment session and were matched online with planning CT images, for 3D image registration. The mean value and standard deviation of all translational  and rotational errors  were respectively 0.0 degrees+/- 1.3 degrees (θx) (range -6.0 degrees and 3.1 degrees); -0.1 degrees +/- 1.1 degrees (θy) (range -3.0 degrees and 2.4 degrees); -0.6 degrees +/- 1.4 degrees (θz) (range -5.0 degrees and 3.3 degrees). Median and 90th percentile of rotational errors were respectively within 0.1 degrees and 1.4 degrees (θx), 0.0 degrees and 1.2 degrees (θy), 0.0 degrees and 0.9 degrees (θz). Mean +/- SD of 3D vector was 3.1 +/- 2.1 mm (range 0.3 and 14.9 mm); median and 90th percentile of 3D vector was within 2.7 mm and 5.1 mm.A total of 194 CBCT scans were analyzed. Mean +/- standard deviation of translational errors  were respectively 0.5 +/- 1.6 mm (range -5.7 and 5.9 mm) in X; 0.4 +/- 2.7 mm (range -8.2 and 12.1 mm) in Y; 0.4 +/- 1.9 mm (range -7.0 and 14 mm) in Z; median and 90th percentile were respectively within 0.5 mm and 2.4 mm in X, 0.3 mm and 3.2 mm in Y, 0.3 mm and 2.2 mm in Z. Mean +/- standard deviation of rotational errors (θHypofractionated stereotactic radiotherapy have the significant limitation of uncertainty in interfraction repeatability of the patient setup; image-guided radiotherapy using cone-beam computed tomography improves the accuracy of the treatment delivery reducing set-up uncertainty, giving the possibility of 3-dimensional anatomic informations in the treatment position. Brain is a common site for metastases. Stereotactic radiosurgery (SRS) and hypofractionated stereotactic radiotherapy (HSRT), due to their ability to deliver very high doses to a small volume with high tumour control rates , may be The aim of this study was to assess inter-fraction translational and rotational set-up errors, in patients treated with IGRT-HSRT immobilized by a thermoplastic mask and a bite-block and positioned using stereotactic coordinates.Between April 2008 to September 2010, 37 patients with 47 brain metastases were treated with IGRT-HSRT at the Radiation Oncology Therapy Unit of the University of Rome, Tor Vergata. Inclusion criteria were: maximum 3 brain metastases, ≤ 3.5 cm of diameter and good performance status (Karnofsky performance status ≥ 70). Table ®) fixed to a stereotactic frame support ; the frame for stereotactic coordinate generation  was applied over the mask  and a bite-block  with a 1.25 mm slice thickness was acquired in axial mode for planning. All patients had an MRI study that was used for image registration in the planning; CT images were transferred to Ergo stereotactic treatment planning system  in order to contour the gross tumour volume (GTV), that consisted of the radiographically evident contrast-enhancing gross disease, and to determine the stereotactic localization of the isocenter. CT images with a marker point (isocenter) and GTV contours were transferred from Ergo to Pinnacle ; CT and MRI images were automatically registered on Syntegra software . The CT-contoured GTV was corrected on CT-MRI registration; planning target volume (PTV) and organs at risk (OARs) were delineated. The PTV was defined as the GTV plus a 3 mm isotropic margin. Treatment plans were produced on Pinnacle3 version 8.0 m ; multiple (5-9) no-coplanar beams (6 MV photons) were used to perform the treatment plan. The median isodose at the periphery of the PTV was 95%, the mean isodose 95.6% (range 91%-100%); for every lesion the total dose was prescribed to the isodose at the periphery of the PTV. Different fractionation schedules were used, based on tumour size and site: 3 fractions of 6 Gy (6 lesions), 4 fractions of 6 Gy (4 lesions), 5 fractions of 6 Gy (6 lesions), 2 fractions of 15 Gy (1 lesion), 4 fractions of 8 Gy (29 lesions), 5 fractions of 8 Gy (1 lesion). For every organ at risk we started from the Biologic Effective Dose (BED) definition to calculate the equivalent max dose for every fractionation scheme:A contrast-enhanced CT scan  equipped with Beam ModulatorTM (leaf width of 0.4 cm at the isocenter) and a kilovolt (kV) imaging system capable of acquiring 3D X-ray volume images based on kV cone-beam computed tomography (CBCT). In particular CBCT has the tube and flat panel imager both mounted on retractable arms that extend from the accelerator's drum structure. The kilovoltage system is mounted in an orthogonal direction to the MV system sharing a common axis of rotation. We used the "head and neck protocol" with the following parameters: 100 kV, 36.1 mAs, nominal scan dose 0.9 mGy, FOV (Field Of View) at the isocenter 260.4 mm. All the images were stored and processed on a control work station (XVI).Patients were treated with Linac Elekta SynergyFor every patient, planning CT images, with OARs, PTV and marker isocenter were transferred from Pinnacle to XVI.3.Daily CBCT scans were acquired for every patient before the treatment session. Projections are acquired during a single rotation of the gantry (about 113 s) and processed with Elekta XVI software  that with a back-projection algorithm reconstructs the 3D volumetric images. Reconstruction of about 625 projections is performed on a Intel Xeon (TM) 3.06 GHz processor and it takes about 30 s to reconstruct a 410 × 264 × 410 voxel matrix with voxel dimension 1 × 1 × 1 mmPlanning CT images were matched online with the daily CBCT images using bone-matching algorithm for 3D image registration (chamfer matching).An alignment clip-box for volumes matching was defined by the physicians. The registration was checked by a physician using a "cut" display modality. After 3D registration the XVI software calculates translational and rotational set-up errors. On-line corrections were performed before each treatment. The translational errors were corrected using the mechanical movements of the couch; the rotational errors were corrected using the knobs of the stereotactic head frame support.If the translational and rotational errors in the first set-up exceeded respectively 6 mm and/or 3 degrees, the patient was repositioned.x, θy, θz) were calculated for the matching results of bone matching algorithm. It was calculated the length of the translational correction 3D vector using the formula3D vector = (X2+Y2+Z2)1/2.The mean value and standard deviation of all translational  and rotational errors , cranio-caudal (Y) and antero-posterior (Z)] considering the totality of the data obtained for all patients and all fractions. The same parameters were evaluated for rotational set-up errors (θx), (θy), (θz).For the 194 CBCT, mean, median, standard deviation (SD), 90The results of the corrections obtained by XVI software are listed in Table th percentile of translational errors were respectively within 0.5 mm and 2.4 mm (X axis), 0.3 mm and 3.2 mm (Y axis), 0.3 mm and 2.2 mm (Z axis).Median and 90x, θy, θz) were respectively 0.0° ± 1.3° (θx) (range -6.0° and 3.1° mm); -0.1° ± 1.1° (θy) (range -3.0° and 2.4°); -0.6° ± 1.4° (θz) (range -5.0° and 3.3°).Mean ± SD of rotational errors , 0.0° and 1.2° (θy), 0.0° and 0.9° (θz). Mean ± SD of 3D vector was 3.1 ± 2.1 mm (range 0.3 and 14.9 mm); median and 90th percentile of 3D vector was within 2.7 mm and 5.1 mm.Median and 90Stereotactic radiotherapy is a treatment modality for brain metastases to deliver very high doses to a small volume and to obtain high tumour control rates. Hypofractionated stereotactic radiotherapy has the advantage to deliver large doses - probably more effective at killing radioresistant tumours- with the radiobiological features of fractionation ,7; in faThe combination of two relocatable immobilization devices, such as thermoplastic mask and bite-block, is more effective for head treatment-position reproducibility. Recently, Masi et al. evaluated set-up errors measured by a CBCT for patients immobilized by a thermoplastic mask and a bite-block; when patients were completely repositioned, the set-up corrections were significantly larger than those measured when patients were left immobilized, and set-up errors measured with mask alone were larger than those obtained with a mask and a bite-block, but not with statistically significant difference. In the analysis of 131 CBCT for 57 patients receiving SRT, they obtained an overall mean and standard deviation for the 3D vector of 3.0 ± 1.4 mm and a maximum standard deviation of 2.4 mm that was observed along the cranio-caudal direction .r ≥ 0.88) and thus the great accuracy of daily repositioning with image-guidance based on the bony anatomy of the skull that can be used as a surrogate for the actual target position [In the study of Boda-Heggemann et al.  the accuposition .In the present study we evaluated set-up accuracy using automatic image registration between on-board kV CBCT and planning CT scan, based on a clipbox involving the base of the skull; a system based on mask and bite-block immobilization was used. A total of 194 CBCT have been examined. We obtained an overall mean and standard deviation for the 3D vector of 3.1 ± 2.1 mm, that is comparable with data obtained by other authors, with a range of the 3D vector between 0.3 mm and 14.9 mm; we registered the widest range of set-up errors in the cranio-caudal direction (between -7 mm and 14 mm). The analysis of daily repositioning errors shows that errors related to mask-making technique and patient compliance have to be considered for a stereotactic treatment; in our series in 17 cases we had a translational error exceeding 6 mm and/or a rotational error > 3 degrees and we repeated all the set-up procedure assuming that this difference was attributable to the wrong position of the head into the mask, most of all due to the movements of the joint between the occipital bone and the first cervical vertebra, or to radiation therapist random errors.The accuracy of automatic 3D-3D matching for evaluation of patient set-up is better than manual 2D-2D matching; for Elekta XVI automatic registration, phantom studies have shown errors less than 0.5 mm ,13. The In our analysis stereotactic fractionated radiotherapy have the significant limitation of uncertainty in inter-fraction repeatability of the patient set-up; IGRT using cone-beam computed tomography improves the accuracy of the treatment delivery reducing set-up uncertainty, giving the possibility of 3-dimensional anatomic informations in the treatment position.The authors declare that they have no competing interests.GI analyzed data and drafted the manuscript; RM analyzed data and drafted the manuscript; DF collected and analyzed data; EP collected and analyzed data; MB collected data; RB revised literature; LDM revised literature; EG collected data; FS collected data; RS revised the manuscript. All authors read and approved the final manuscript.


Shiga-like toxin 2 (Stx2) is one of the most important virulence factors in enterohaemorrhagic Escherichia coli (E. coli) strains such as O157H7. Subtypes of Stx2 are diverse with respect to their sequence, toxicity, and distribution. The most diverse Stx2 subtype, Stx2f, is difficult to detect immunologically, but is becoming more frequently associated with human illness.A purification regimen was developed for the purification of Stx2f involving cation exchange, hydrophobic interaction, anion exchange, and gel filtration. The molecular weight of Stx2f B-subunit was approximately 5 kDa, which appeared significantly smaller than that of Stx2a (6 kDa) on a SDS-PAGE gel, although the size of the A subunit was similar to Stx2a (30 kDa). Stx2f was shown to be active in both cell-free and cell-based assays. The 50% cytotoxic dose in Vero cells was 3.4 or 1.7 pg (depending on the assay conditions), about 3–5 times higher than the archetypical Stx2a, while the activity of Stx2f and Stx2a in a cell-free rabbit reticulocyte system was similar. Stx2f bound to both globotriose-lipopolysaccharide (Gb3-LPS) and globotetraose-LPS , but its ability to bind Gb4-LPS was much stronger than Stx2a. Stx2f was also much more stable at low pH and high temperature compared to Stx2a, suggesting the toxin itself may survive harsher food preparation practices.E. coli strain isolated from a feral pigeon. Information obtained in this study will be valuable for characterizing Stx2f and explaining the differences of Stx2a and Stx2f in host specificity and cytotoxicity.Here, we detail the purification, biochemical properties, and toxicity of Stx2f, from an  Escherichia coli (E. coli) subtypes are responsible for many recent food illness outbreaks. A subset of these pathogens, Shiga toxin-producing E. coli (STEC), can generate symptoms from bloody diarrhea to the potentially life-threatening hemolytic uremic syndrome (HUS) E. coli O157:H7, but many non-O157 serotypes can result in severe disease and food contamination outbreaks, including the deadly 2011 outbreak of O104:H4 in Germany A group of major and potentially deadly bacterial contaminants, enterohemorrhagic 5 category of bacterial toxins, along with cholera and pertussis toxins N-glycosidase that cleaves a single adenosine residue from the 28S rRNA and inactivates 28S ribosomal subunits E. coli are Stx1, which are almost identical to the toxin from the Shigella genus, and Stx2. Stx1 and Stx2 can be found independently or together in O157:H7 and other serotypes of STEC E. coli but also to non-pathogenic E. coli in infected intestines E. coli is lysed by the phage Stxs consist of a catalytic A subunit with a pentameric B subunit protein complex for targeting and binding the host receptor. They therefore fall into the ABE. coli-associated gastroenteritis, there are vast differences in toxicity among Stx variants and subtypes. The nomenclature recently proposed by Scheutz et al in 2012 was used in this study Although any Stx can exacerbate E. coli, these would be preserved. Any other bacterial factors that associate with Stx may also persist during a purification regimen. Purification of the prototype Stx2a and closely-related Stx2c, Stx2d, and Stx2g subtypes have been previously demonstrated While production and purification of recombinant Stx and its subunits have been well-documented, there are additional benefits to obtaining naturally-released toxin directly from wild type bacterial cultures. If there are any modifications that occur to the toxin while it exits the E. coli used are included in E. coli strain was used as a non-toxin-producing control for the growth curve.All strains of 4SO4 to 70% saturation. The mixture was then centrifuged , and the pellet was resuspended in phosphate buffered saline . The Stx2a-containing pellet was then buffer exchanged using a 4 mL Zeba desalting column (Thermo Scientific) to 50 mM sodium acetate (NaOAc) pH 4.3 in preparation of cation exchange chromatography.Stx2a was precipitated from 250 mL of cell supernatant by adding solid NHStx2f culture supernatant pH was set to 4 using glacial acetic acid (approximately 10 mL of 14.3 M acetic acid), then the mixture was centrifuged  and sterile filtered  for clarity. This pH 4 cell supernatant was added directly to a 5 mL SP-HP cation exchange column on the Äkta FPLC (GE Life Sciences) that was previously equilibrated with 50 mM sodium acetate (NaOAc), pH 5. After a wash with 50 mM NaOAc, pH 5, toxin was eluted with a 20 mL NaCl gradient (0 to 0.6 M) in 50 mM NaOAc, pH 5.3, at 3 mL/min, and collected in 1 mL fractions. Stx2a protein was purified in the same manner, but without pH-fixing and using 50 mM NaOAc, pH 4.3 instead of pH 5.4) +1 M NH4SO4, pH 7 using a 4 mL Zeba desalting column. Approximately 3 mL of partially (cation exchange) purified toxin was thus injected onto a 5 mL Phenyl HP column, equilibrated with 50 mM NaPO4+1 M NH4SO4, pH 7, on the Äkta FPLC. Toxin was then eluted with a 25 mL NH4SO4 gradient (1 to 0 M) in 50 mM NaPO4, pH 7, at 3 mL/min, and collected in 1 mL fractions.Based on ELISA results, fractions from CEC containing Stx2f were pooled, and buffer was exchanged to 50 mM sodium phosphate , pH 9, using a 4 mL Zeba desalting column. Approximately 3 mL were loaded on a 1 mL Q HP anion exchange column, equilibrated with 20 mM 13DAP. Toxin was eluted with a 10 mLNaCl gradient (0 to 1 M) in 20 mM 13DAP, pH 9, at 1 mL/min, and collected in 0.5 mL fractions.Based on ELISA results, fractions from AEC containing Stx2f were pooled, and approximately 0.7 mL was injected onto a Sephacryl 100 16/60 gel filtration column pre-equilibrated with PBS on the Äkta FPLC. Column was eluted at 0.5 mL/min. Fractions containing pure toxin (by Coomassie staining) were collected, concentrated by an Amicon Ultra filter (10 kD pore size) (Millipore), and sterilized by a 0.2 µm PVDF filter.All samples for SDS-PAGE analysis were incubated for 5 min at 75°C prior to loading onto the gel, with the exception of the cell pellets (95°C for 10 min.). Gels (4%–12% NuPAGE Novex Bis-Tris mini gels from Invitrogen) were run following the manufacturer’s specifications and stained with SimplyBlue Safe Stain (Invitrogen) for visualization of proteins. For Western blots, after PAGE, the proteins were electroblotted to a PVDF membrane . The membrane was blocked with 2% ECL Prime blocking agent  in PBS-Tween-20 (0.05%) (PBST). Stx’s on the membrane were detected with either mouse anti-Stx2f antibodies (prepared as indicated below) and anti-mouse IgG-HRP (Promega), or rabbit anti-Stx2a-HRP conjugate  and then developed with Lumigen TMA-6 (Lumigen) substrate. Western blots were visualized with a 2 minute exposure using a FluorChem HD2 (Alpha Innotech).rd injection, sera were collected using a tail vein bleed. To develop a Stx2a polyclonal antibody, a rabbit was immunized with catalytically inactive Stx2a toxoid (E167Q mutation) and serum was collected by cardiac puncture with exsanguination (procedures performed by Pacific Immunology).To develop the Stx2f polyclonal antibody, a His-tagged Stx2f A subunit construct was developed using the pTrcHis2 TOPO cloning kit (Invitrogen). His-tagged Stx2f A subunit expressed in TOP10 cells (Invitrogen) was induced with 1 mM IPTG and purified using a Ni-NTA affinity column (Qiagen). 5 µg of Stx2f in 2 mL PBS was used to reconstitute a vial of MPL-TDM (Sigma) adjuvant and injected intraperitoneally into a Balb/c mouse 3 times at 2 week intervals. One week after the 3All procedures with animals were carried out according to institutional guidelines for husbandry approved by the Animal Care and Use Committee of the U.S. Department of Agriculture, Western Regional Research Center (USDA ACUC Protocol #09-J-10).http://web.expasy.org/compute_pi/). When calculating the pI of the toxin complex, the amino acid sequence of the A subunit followed by five repeats of the B subunit sequence were used as the input.Estimated molecular weights and isoelectric points (pI) were calculated using the ExPASy Compute pI/MW tool (2SO4. Absorbance was measured at 450 nm on a Victor II plate reader (Perkin Elmer). Between each step plates were washed 3 times with 200 µl PBST.ELISAs for monitoring Stx2a and 2f purifications were direct-well binding assays. Fractions collected were diluted 1/25 in a final volume of 100 µL in PBS and bound directly to a flat-well Nunc maxisorp plate overnight at 4°C. After blocking the plates with 200 µl 5% milk in PBS-Tween 20 (0.05%) (PBST), they were then incubated with 100 µl primary antibody: Sifin 2A  for Stx2a; mouse polyclonal serum for Stx2f. Both antibodies were diluted 1/10,000 in blocking buffer for 1 hour at RT. Goat anti-mouse IgG-HRP (Promega) was used as a secondary antibody, and the plates were developed with TMB substrate (Pierce), followed by addition of 100 µl 0.3 N HE. coli cells expressing globotriose-lipopolysaccharide  or globotetraose-LPS  or control E. coli cells  600 in carbonate buffer  and 100 µl was bound to the wells of a black 96-well Nunc Maxisorp plate by incubating at 50°C until all liquid had evaporated. Wells were then blocked with 200 µl 5% milk/PBST for 1 hour at RT. Stx2f or Stx2a toxin was then diluted in blocking buffer to the indicated concentrations and incubated on the plate for 1 hour at RT. Anti-Stx2f mouse or anti-Stx2a rabbit polyclonal serum was then added at a 1/5,000 dilution in blocking buffer and incubated for 1 hour at RT. Goat anti-mouse IgG HRP or goat anti-rabbit IgG HRP (Promega) was then added  and incubated for 1 hour at RT. Signal was detected with 100 µl SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and read on a Victor II plate reader (Perkin Elmer). Between each step plates were washed 3 times with 200 µl PBST.The Gb3/Gb4-LPS binding assay was conducted similar to a traditional sandwich ELISA. Formaldehyde-fixed Cell-free translation assays were conducted with a previously described rabbit reticulocyte lysate protocol The absorbance of pure Stx2f at 280 nm at 200, 100, and 50 µg/mL was analyzed by Nanodrop (Thermo Scientific) in triplicate. These values were plotted and, using a linear standard curve, extrapolated to estimate an extinction coefficient for Stx2f at 1 mg/mL.2). The cells were trypsinized, diluted to 105 cells/mL, and then distributed onto 96-well cell-culture-treated plates. 24 hours later, the cells received toxin and were diluted in DMEM +10% FBS  in one of two formulations. In the first, cells were incubated at 4°C for 1 hour, 100 µl/well toxin-containing media was removed and replaced by fresh media, and cells were returned to the incubator to grow for 24 hours. In the second, toxin was simply added to the cells and incubated for 24 hours. The cells were then lysed using 100 µl/well 1/5 dilution of CellTitre-Glo reagent (Promega), and luminescence was measured using a Victor II plate reader. A 50% cytotoxic dose (CD50) represents the amount of toxin necessary to kill 50% of the attached monolayer of cells in the wells. CD50’s were approximated by plotting three points within the linear portion of the graph, and solving for 50% (0.5). This was typically 2.72(0.5−a)/b for semi-log, where a and b are determined by the plot. All photographs were taken using a Leica DM IL microscope at 200x magnification.Vero (African green monkey kidney) cells To pH treat Stx2f and Stx2a, 500 ng/mL toxin was diluted in NaOAc buffer (250 mM) at various pH values and allowed to incubate at RT for 1 hour. To heat treat Stx2f and Stx2a, 500 ng/mL toxin was incubated in NaOAc buffer at indicated temperatures for 1 hour. After treatment, the mixture was diluted 100-fold with DMEM/FBS culture media, and incubated 100 µl/well with Vero cells for 1 hour at 4°C, and then the media was replaced with 100 µl fresh DMEM/FBS for 24 hours. Cells were lysed and luminescence measured using a 1/5 dilution of 100 µl/well CellTitreGlo reagent and a Victor II plate reader, as above. Treating Vero cells with a 2.5 mM, pH 1.5 NaOAc in DMEM/FBS control had a negligible toxicity (data not shown).E. coli strains  on the surface was measured by “sandwich” ELISA.  Gb3-LPS , it exhi Gb3-LPS .in vitro assay N-glycosidase activity. An in vitro translation assay conducted on rabbit reticulocyte lysate (Promega) confirmed that intact (A+B pentamer) purified Stx2f is capable of inactivating ribosomes, resulting in the halting of protein synthesis (50) for inhibition of translation was 3.5 µg/mL, a little higher than that of Stx2a (2.5 µg/mL). Although purified Stx2f effectively kills Vero cells (50) of Stx2f was shown to be 3- or 5-fold higher than that of Stx2a, indicating that Stx2f is considerable less toxic compared to Stx2a  have been associated with human illness 5 toxoid, both the A and B subunits should be detectable. If a Stx2f toxoid was available for comparison, it would also be interesting to note if the B subunit of the toxoid is smaller than that of Stx2a. This phenomenon  amino acid sequence identity to Stx2a 5 complex itself might be more stable, and requires harsher conditions to cause it to fall apart. Fascinatingly, a similar phenomenon is evident for a pH 2 treatment , Stx2f-encoding STEC strains are not yet considered a major health concern due to the lack of severity and rarity of infections. However, since most Stx immunoassays are poor at detecting Stx2f, it is becoming clearer that Stx2f infections are more common than we realize Figure S150 values in Vero cell toxicity curves of purification steps used to calculate the CD. The X-axis is total nanograms of protein added per well.(TIF)Click here for additional data file.Figure S2Vero cells are more sensitive to pH 2-treated Stx2f than to pH 2-treated Stx2a. Photographs are of Vero cells used in (TIF)Click here for additional data file.