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Evaluation of the In Vitro Efficacy of Artemisia annua, Rumex abyssinicus, and Catha edulis Forsk Extracts in Cancer and Trypanosoma brucei Cells

The current drugs against sleeping sickness are derived from cancer chemotherapeutic approaches. Herein, we aimed at evaluating the in vitro effect of alcoholic extracts of Artemisia annua (AMR), Rumex abyssinicus (RMA), and Catha edulis Forsk (CEF) on proliferation/viability of 1321N1 astrocytoma, MCF-7 breast cancer, THP-1 leukemia, and LNCaP, Du-145, and PC-3 prostate cancer cells and on Trypanosoma brucei cells. Proliferation of tumor cells was evaluated by WST-1 assay and viability/behaviour of T. brucei by cell counting and light microscopy. CEF was the most efficient growth inhibitor in comparison to AMR and RMA. Nevertheless, in LNCaP and THP-1 cells, all extracts significantly inhibited tumor growth at 3 μg/mL. All extracts inhibited proliferation of T. brucei cells in a concentration-dependent manner. Microscopic analysis revealed that 95% of the T. brucei cells died when exposed to 33 μg/mL CEF for 3 hrs. Similar results were obtained using 33 μg/mL AMR for 6 hrs. In case of RMA, however, higher concentrations were necessary to obtain similar effects on T. brucei. This demonstrates the antitumor efficacy of these extracts as well as their ability to dampen viability and proliferation of T. brucei, suggesting a common mechanism of action on highly proliferative cells, most probably by targeting cell metabolism.

Introduction
Many anticancer reagents, including nucleotide analogues and other DNA synthesis inhibitors (e.g., methotrexate), aim at exerting a specific activity against rapidly proliferating cell types. Because of the capacity of trypanosomes for rapid growth within mammals, they have been linked to some types of cancer cells. D,L--Difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, was originally developed as a potential anticancer reagent before it has been proved useful in trypanosomiasis therapy. Furthermore, suramin, melarsoprol, and pentamidine which are drugs licensed for the treatment of human African trypanosomiasis (HAT) are known to arise from cancer research studies [1][2][3]. If left untreated, sleeping sickness patients die within months when infected with T. brucei rhodesiense (acute form of the disease in East and Southern Africa) or within years when infected with T. brucei gambiense (chronic form of the disease in West and Central Africa) [4]. It is estimated that at least 300 000 ± 500 000 people are presently infected [5]. Above this burden of the disease, all the available drugs have a number of shortcomings during treatment because of resistance development against them, allergic reactions, undesirable effects in the urinary tract, and reactive encephalopathy (with 3-10% fatality), and some regimens are strict and difficult to apply [6]. This raises an urgent need for novel, safe, rapidly-acting, and inexpensive agents for the treatment of HAT [7].
Our project selects three plants called Catha edulis Forsk (CEF), Artemisia annua (AMR), and Rumex abyssinicus (RMA), which are locally endemic to Ethiopia and widely used in the community for various purposes. The pharmacological effect of the selected plants on different cell lines and the known similarity of chemotherapeutics against trypanosomes and cancer initiated us to continue investigation of drugs to be commonly effective against these two important diseases.
CEF leaves were commonly used as a stimulant; that was formerly believed to be due to its norpseudoephedrine content. However, it might be cathinone, a newly discovered potent alkaloid with a pharmacological profile closely resembling that of amphetamine, which is responsible to this effect [8]. It can be said that CEF is an amphetamine-like plant material, the effects of which can be accounted to the "khatamines" including phenethylamines cathinone and, to a lesser extent, norpseudoephedrine. In addition to these two "khatamines, " the leaves also contain the cathedulins [8],an interesting group of substances awaiting pharmacological elucidation.
AMR contains artemisinin, an endoperoxide consisting of sesquiterpene lactone [9]. Unlike other peroxide compounds, artemisinin can be stored at room temperature for many years without noticeable decomposition. Semisynthetic derivatives include artemether, arteether, and artesunate [10]. Interestingly, artemisinin is essentially nontoxic to normal cells [11]. Zheng [12] reviewed the cytotoxic activity of artemisinin, and recently it has been shown to be effective in killing cancer cells by inducing apoptosis and inhibition of nuclear factor -B, an important activator in cancer development and progression [13,14]. With regard to its antiprotozoal effect, artemisinin has shown a promising antileishmanial activity [15], and currently an artemisinin-based combination therapy (ACT) is recommended for the treatment of Plasmodium falciparum malaria [16,17].
RMA is widely used in folklore medicine for treatment of headache, hemorrhoid, ascaris, scabies, leprosy, fungal skin infection, wounds, eczema, and sore-throat and to control mild forms of diabetes. The decoction of the root or leaf powder of the plant is used as vermifuge [18,19]. Studies have also shown its effect against leukaemia cells by initiating cell death through apoptosis [20]. Unlike the other species of this plant, studies done on the effect of RMA against cancer cells, however, are preliminary.
The objective of this study was, therefore, to evaluate the in vitro effect of the three aforementioned plant extracts against cancer cells and Trypanosoma brucei cells and to determine the prospect of their common application against these two respective diseases.

Plant Extracts.
Three different medicinal plants (Catha edulis Forsk, Artemisia annua, and Rumex abyssinicus) were deposited in and identified by the National Herbarium, Faculty of Science, Addis Ababa University in Ethiopia. The plants were selected mainly on the basis of their extensive usage in the community and/or on their local medicinal knowledge. The air-dried and powdered plant materials were extracted with 95% methanol at 60 ∘ C for 8 hrs using Soxhlet apparatus. The obtained methanol extract was vacuumfiltered and evaporated by using a rotary evaporator and freeze-dried (D. Piatkowski, Munich, Germany). The dried extracts were stored at −20 ∘ C until used. Stock solutions were prepared in DMSO just prior to the start of experiments so that its final concentrations did not exceed 1% during the experiment.

Tumor Cells Vitality
Test. Proliferation/vitality of tumor cells was assessed using the WST-1 assay according to manufacturer's instructions. This colorimetric assay was used to determine cell proliferation/cytotoxicity in 96 well plates (5000 cells/well). Increasing concentrations of plant extracts (3 to 333 g/mL) were added after preincubation of cells for 24 hrs (start value). A volume of 12 L stock solution of WST-1 was added to each well plate, and was incubated for 4 hrs at 37 ∘ C. As a control, DMSO was added instead of the plant extracts. The absorbance was directly recorded on an ELISA reader/96-well multiscanner at a wavelength of 450 nm.

Impact of Exposure of Plant Extracts on
Survival of Trypanosoma brucei Cells. Time dependency test was done in 24 well plates where 1 mL of cell suspension (2 × 10 5 cells) plus 100 L of plant extracts giving a total volume of 1.1 mL/well were incubated for 24 hrs at 37 ∘ C in humidified environment containing 5% CO 2 . To count the cells first the plate was slowly shaken in circular motion and well mixed using a 100 L pipette to help the cells float well in the media just before pipetting for counting on a Neubauer hemacytometer. Ten microliters of the cell suspension has been taken, and the number of moving cells was then recorded after 1 hr, 3 hrs, 6 hrs, and 24 hrs of incubation. Tests have been performed in triplicates, and the average values were obtained for two repeated experiments ( = 6). The controls were seeded in a 1.1 mL of fresh medium containing 1% DMSO and handled in exactly the same way as the exposed cells.

Microscopic Observation of Trypanosoma brucei Cells'
Viability. This test was done using 24 well plates containing 1 mL of cell suspension (2 × 10 5 cells) plus 100 L of plant extract. Cells were exposed to three different concentrations of the respective plant extracts, and the activity of cells was evaluated by monitoring (i) movement of live cells, (ii) morphology of the moving and nonmoving cells, and (iii) the number of live moving cells recorded at the 2nd, 3rd, 6th, and 24th hr of incubation at 37 ∘ C in humidified environment containing 5% CO 2 and compared with the controls.

Statistical Analysis.
Count and percentage of dead/viable cells has been estimated to compare effect of plant extracts on exposed cells and the corresponding controls. The optical density reading was used to determine the relative rate of cell proliferation to determine the IC 50 of plant extracts using a linear interpolation. The statistical significance of difference of two means in cancer cells experiment was calculated using unpaired -test, and values of < 0.05 were considered to be statistically significant. The mean and standard deviation values are also employed for comparison. The data analysis, all figures, and tables presented were done using GraphPad Prism (version 4) statistical software.

In Vitro Evaluation of Anticancer
Effect. CEF showed concentration-dependent inhibitory effect in all cancer cells tested, and a statistically significant inhibition of proliferation ( < 0.05) was observed at 3 g/mL concentration in LNCaP, astrocytoma 1321N1, MCF-7, and THP-1 cells. When compared with the other cancer cell types tested, the lowest recorded IC 50 was found in case of LNCaP prostate cancer cells (2.4 g/mL). In addition to LNCaP prostate cancer cells, CEF best inhibits THP-1 leukemia and MCF-7 cells when applied at concentrations ≥33 g/mL. In all cases, the maximum concentration applied resulted in nearly 100% inhibition. In PC-3 cells, however, maximum constant inhibition has been reached at 66 g/mL ( Figure 1).
AMR exerts a concentration-dependent effect on all cancer cells undertested except in PC-3 cells in which case it looks to have a provital effect at lower concentrations ≤33 g/mL ( Figure 2). In LNCaP, 1321N1, and THP-1 cells it revealed a statistically significant inhibition ( < 0.05) at a concentration starting from 3 g/mL. In the remaining three cancer cell types, however, a significant inhibition has reached concentrations greater than or equal to 100 g/mL. The lowest IC 50 (3 g/mL) has been recorded in LNCaP cells when compared with the remaining cancer cells employed in our experimental study ( Figure 2).
In case of RMA, the concentration-dependent inhibitory effect has also been well documented against growth of all cancer cell lines. A statistically significant growth inhibition ( < 0.05) was observed at 3 g/mL in LNCaP, 1321N1, and THP-1 cells. Unlike the other two plant extracts, however, the degree of growth inhibition by Rumex abyssinicus extract was low as it has not resulted in a complete inhibition of multiplication even at the maximum concentration (333 g/mL) in all cancer cells tested. The lowest IC 50 (29 g/mL) was recorded in THP-1 cells. In these cells the maximum dilution had resulted in only 70% inhibition (Figure 3).

In Vitro Evaluation of Antitrypanosomal Effect.
The proliferation of T. brucei cells has been assayed using Alamar blue, an indicator for metabolic cell function, evaluated as a colorimetric dye. However, when the Alamar blue was mixed with the growth media, the deep red or yellow coloration of the extracts at higher concentrations interferes with the absorbance measurement. For this reason, we have employed the time dependency test by counting the number of actively moving cells after exposing the cells to the plant extracts for 24 hrs at 37 ∘ C in 24 well plates. Cell counting has been done at the start of seeding, and at 1 hr, 3 hrs, 6 hrs and 24 hrs incubation periods. Microscopic observation on the relative speed of cellular motility and their morphological changes has also been recorded.
Results demonstrated that CEF inhibited proliferation of T. brucei cells at the last two higher dilutions (166 and 333 g/mL), and its initial effect was observed within the first hour of incubation, and its maximum effect was seen at the 6th hr (Figure 4(a) and Table 1). At concentrations of 3 and 8 g/mL, no inhibitory effect was observed. At 33 g/mL, however, cytostatic effect was observed where the number of cells remains almost constant within the following 24 hrs of incubation (Figure 4(a)).
AMR similarly inhibits growth of cells at concentrations of 33 g/mL, 166 g/mL, and 333 g/mL. The inhibitory effect was detected following the first hour of incubation and continues until it kills all the cells within the next 24 hrs of incubation (Figure 4(b) and Table 1). At 3 g/mL and 8 g/mL concentrations, however, no inhibition had been observed whereby it resulted in a cytostatic effect at 8 g/mL, as the number of cells kept constant over a time elapse of 24 hrs (Figure 4(b)).
RMA also results in 100% inhibition at ≥166 g/mL after 24 hrs of incubation. At 33 g/mL, the cell number had decreased to a value of about 30%, but no inhibitory effect at 3 or 8 g/mL was observed (Figure 4(c)). Table 1 lists the phenotypic changes recorded upon monitoring cells exposed to the various concentrations of CEF, AMR, and RMA extracts.  Figure 1: Inhibition of cancer cell growth by Catha edulis Forsk (CEF) extract. A colorimetric assay was used to determine proliferation of tumour cells (5000 cells/well in a 96 well plate) exposed to different concentrations of CEF extract (3-333 g/mL) using a 33 mg/mL stock solution. WST-1 was added to each well plate and incubated for 4 hrs at 37 ∘ C. As a control, DMSO was added instead of the plant extract (zero value). The absorbance was measured at a wavelength of 450/620 nm by an ELISA reader. The reduction in cell number was calculated from the absorbance given in percent. Statistical significance was calculated using unpaired -test, at 95% CI ( * ).  Figure 2: Inhibition of cancer cell growth by Artemisia annua (AMR) extract. A colorimetric assay was used to determine proliferation of tumour cells (5000 cells/well in a 96 well plate) exposed to different concentrations of AMR extract (3-333 g/mL). After incubation periods of 24 hrs, 12 L stock solution of WST-1 was added to each well plate and incubated for 4 hrs at 37 ∘ C. As a control, DMSO was added instead of the plant extract (zero value). The absorbance was measured at a wavelength of 450/620 nm by an ELISA reader. The reduction in cell number was calculated from the absorbance given in percent. Statistical significance was calculated using unpaired -test, at 95% CI ( * ).  Figure 3: Inhibition of cancer cell growth by Rumex abyssinicus (RMA) extract. A colorimetric assay was used to determine proliferation of tumour cells (5000 cells/well in a 96 well plate) exposed to different concentrations of RMA extract (3-333 g/mL). After incubation periods of 24 hrs, 12 L stock solution of WST-1 was added to each well plate and incubated for 4 hrs at 37 ∘ C. As a control, DMSO was added instead of the plant extract (zero value). The absorbance was measured at a wavelength of 450/620 nm by an ELISA reader. The reduction in cell number was calculated from the absorbance given in percent. Statistical significance was calculated using unpaired -test, at 95% CI ( * ).

Discussion
Although cancer is not a single disease, and Trypanosoma are eukaryotic parasites presenting a high level of genotypic variability, the growth of the parasites in their host and that of cancer cells share at least one common feature, that is, their mutual capacity for rapid cell division [21]. Such cells require high polyamine concentration, and the inhibition of polyamine metabolism highly affects their survival. For example, the drug eflornithine (DFMO) is known to affect the polyamine synthesis in both Trypanosoma and cancer cells.
Another area of interest linking these two cell types is the glycolytic pathway. Tumor cells gain their energy mainly from aerobic glycolysis, and trypanosomes solely depend on glycolysis which is critical to their survival. For example, silencing the hexokinase 1 (TbHK1) gene in T. brucei induced parasite death, and a similar effect has been observed using the hexokinase inhibitor lonidamine, both in cancer and trypanosome cells [22].
For the treatment of sleeping sickness only four drugs, of which three were developed 50 years earlier, are available. Eflornithine (DFMO), a selective inhibitor of ornithine ISRN Biochemistry DL-decarboxylase, is the only new drug for chemotherapy of sleeping sickness. It was first used in 1990 and is only effective against T. b. gambiense [23]. With the exception of the prodrug pafuramidine maleate (DB289), an orally bioavailable diamidine about to enter Phase II clinical trials [24], no other compounds are in clinical development, indicating that no new antisleeping sickness drug will become available in the next few years [25]. In the light of declining investment in drug development for parasitic diseases, new strategies for the chemotherapy of HAT are urgently required [26]. Among these strategies it is promising to employ drugs developed as potential antitumour agents in the fight against HAT. The current antitrypanosoma drugs including suramin, pentamidine, melarsoprol, and eflornithine are good examples supporting this strategy [23,[25][26][27]. Similarly, (−)-epigallocatechin-3-gallate, a constituent of green tea, has antioxidant, anti-inflammatory, antimicrobial, anticancer, and antitrypanocidal activities [28]. Investigation of other plants with antitrypanosoma and anticancer activities is the easiest way to evade the long and costly process of development of new antisleeping sickness drugs. Here we, therefore, present the pharmacological activity of plants Artemisia annua, Rumex abyssinicus, and Catha edulis Forsk against T. brucei and different cancer cells.
By comparing the efficacy of these three plant extracts in inhibition of proliferation of cancer cell lines, we have observed that CEF was more effective than AMR and RMA, showing a statistically significant inhibition in four of the cancer cell types at the lowest concentration used (3 g/mL). On the other hand, when the complete inhibitory effect of the maximum dilution was considered, RMA was not as effective as AMR and CEF. Among all cell lines tested, LNCaP prostate cancer and THP-1 leukemia cells were found to be most susceptible to the inhibitory effects of all the three plant extracts, where their IC 50 s lay below 33 g/mL. In addition, it was observed that extracts of CEF, AMR, and RMA displayed antiproliferative effects on T. brucei cells. Best inhibitory effects were observed at concentrations ranging between 33 g/mL and 333 g/mL. Similarly, T. brucei cells proved to be most susceptible to CEF and AMR ( Figure 4 and Table 1). As it was observed in a microscope, the effect of the latter two extracts was detected after 2 hours of incubation at concentrations ≥33 g/mL (Table 1). Overall, all the three plant extracts exerted their inhibitory effect, even though with variable efficacies, both in cancer and trypanosome cells. This result is in accordance with previous findings using various cancer cell lines and trypanosome species [8,[29][30][31].
Our study on T. brucei cells has demonstrated that 98% of cells died after 6 hrs at 33 g/mL of the CEF extract (Table 1). A study done by Dimba et al. [32] described a novel mechanism of CEF-induced cell death by apoptosis involving induction of caspases in HL-60 leukemia cell lines. They had also shown that its effect is rapid resulting in cell death within 2 hrs. A similar study done by Rosenkranz and Wink [33] has also shown that alkaloids induce apoptosis in blood stream form T. brucei. Alkaloids are known to affect protein biosynthesis, interact with DNA, and disturb membrane fluidity and/or microtubule formation. This might be the reason for the rapid cell death observed in T. brucei Table 1: Efficacy of alcoholic extracts of Rumex abyssinicus (RMA), Artemisia annua (AMR), and Catha edulis Forsk (CEF) on Trypanosoma cells. T. brucei cells were seeded in 96 well plates (2 × 10 4 cells/well), and extracts of the different plants were added in increasing concentrations. The cells were inspected at the 2nd, 3rd, 6th, and 24th hrs of incubation by microscopic observation according to the following operational definitions: live cells: cells moving either fast or slowly; dead cells: no moving cells are observed in the medium; actively moving: cells move actively swimming from place to place in the medium within the microscopic field; weakly moving: a live cell that moves slowly in a fixed position; clamping: cells sticking together in groups of a minimum of 5 cells per clamp. cells in our study. However, the type of cell death process in our experiments must be clearly demonstrated in future studies. The current experimental study similarly had shown a good inhibitory effect of CEF on THP-1 leukemia cells, starting from 33 g/mL, which is in agreement with the study done by Dimba et al. [32]. In the other cancer cell lines used in the current study, CEF extract efficiently inhibited cell proliferation even at the lower concentration, supporting its activity as an anticancer and antitrypanosomal drug. This result, therefore, is in agreement with in vitro antimicrobial property studies against periodontal pathogens done by Al-Hebshi et al. [34].
As reviewed by Sinclair et al. [35] on the pharmacological effect of artemisinin on cancer cells, it is indicated that its mechanism of action is done by affecting the transferrin receptor in iron metabolism that is pertinent for survival in almost all cancer cells. Transferrin receptors are highly expressed in cell surfaces of breast cancer, brain tumour glioblastoma and meningo-glioma as well as in myelogenous leukaemia (CML) cells compared to their corresponding normal cells. Leukaemia cells were found to be most sensitive to artesunate. Accordingly, our result shown in Figure 2 was partly in support of these findings whereby a significant cell growth inhibition was observed both in 1321N1 and THP-1 cells at the lowest applied concentration. The MCF-7 cells were also affected significantly, but at a higher concentration than in case of the previous two cell lines. This warrants further investigation to identify the reason for their lower susceptibility albeit the high expression of transferrin receptors on their surfaces.
Artemisinin contains an endoperoxide bridge (R-O-O-R ) that interacts with Fe (II) to form toxic free radicals and is required for its antimalarial as well as anticancer activity. It is believed to exert its cytotoxic effect via Fe (II)mediated cleavage of the endoperoxide bridge [11,36]. An intact endoperoxide is crucial since artemisinin derivatives lacking an endoperoxide bridge are devoid of antimalarial activity [17]. In the case of bloodstream forms of T. brucei, iron is delivered by host transferrin to a heterodimeric, glycosylphosphatidylinositol-anchored transferrin receptor [37]. The trypanosomal transferrin receptor is homologous to the N-terminal domain of the variant surface glycoprotein (VSG) and bears no structural similarity with the human transferrin receptor [37]. This structural difference to the human transferrin receptor makes this pathway a good target to develop chemotherapy against human trypanosomiasis. A study done by Mishina et al. [38] also confirmed the inhibitory effect of artemisinin in the in vitro growth of T. cruzi and T. b. rhodesiense in low micromolar range. It targets the calcium dependent-ATPase activity in T. cruzi cell membrane. With respect to this fact, the strong efficacy of AMR to inhibit T. brucei cells' proliferation revealed during the current study is quite appreciated, where nearly 50% of cells died within 3 hrs of incubation (Table 1) and 100% after 24 hrs of incubation at 33 g/mL (Figure 4(b)).
An in vitro experiment done by Maksimović et al. [39] on the fruit of the yellow dock Rumex crispus indicated that it has a specific antioxidant effect. It has also been identified that ethyl acetate extracts from the roots of Rumex dentatus contained four spectroscopically separable compounds and two of which were novel. From these four identified compounds chrysophanol exerted a better inhibition of growth in MCF-7 cell lines [40]. In the current study, Rumex abyssinicus selectively showed better inhibition in THP-1 and LNCaP cells that corresponds with the study done by Wagiera et al. [20] using Rumex dentatus. However, our findings did not agree with regard to the effect of this plant extract on MCF-7 cells when compared with the study done by Zhang et al. [40]. Otherwise, our study revealed that methanol extract of Rumex abyssinicus was not a good candidate of therapy mainly against MCF-7 and PC-3 cells, where by a statistically significant inhibition ( < 0.05) has been seen only at the maximum concentration applied (333 g/mL).

Conclusions
Our present study demonstrates the efficacy of CEF, AMR, and RMA in inhibiting growth of different cancer cell lines as well as T. brucei cells. The CEF extract was relatively the best effective extract in comparison to AMR and RMA against proliferation of all cell lines. In all cases, LNCaP and THP-1 cells were found to be the most susceptible cell lines among others. It has been recalled that almost all of the drugs against human African trypanosomiasis are derived from drugs for cancer. Our current results will also strengthen this consent and can be applied towards developing drugs to be commonly used in sleeping sickness and cancer. This area of integrated "cancer and neglected tropical diseases (NTDs)" research should further be promoted so that NTDs management suffering from various problems like drug resistance, insecticide resistance of transmitting vectors, and financial shortage of production of new drugs can probably be solved.


extfieldsofstudy: Biology Medicine

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Growth Inhibition of Hepatocellular Carcinoma Huh7 Cells by Lactobacillus casei Extract

Purpose Lactobacillus casei (L. casei) is known to exert anti-proliferation effects on many types of cancer cells. However, the effect of L. casei on liver cancer has not been reported. Accordingly, the aim of this study was to determine the anti-cancer effect of L. casei extract on Huh7 cells. Materials and Methods L. casei ATCC393 extract was prepared and purified. After the treatment of L. casei extract on Huh7 cells, cell viability, cell cycle arrest and cell death were analyzed by flow cytometry. The expression levels of tumor necrosis factor-α receptor 1 (TNFR1) and death receptor 3 (DR3) mRNA related with extrinsic apoptosis were assessed by reverse transcription polymerase chain reaction. Additionally, P21 and P27 cell cycle proteins as well as Caspase-3, -8, -9, phospho-Bad and Bcl-2 apoptosis proteins were analyzed by western blot analysis. To determine the effect of L. casei extract on cancer stem-like cells, we analyzed changes in side population fraction through flow cytometry. Results The cell viability of Huh7 cells treated with L. casei extract was decreased by 77%, potentially owing to increases in the rates of Huh7 cells arrested in the G2/M phase (3% increase) and that underwent apoptosis (6% increase). The expression levels of TNFR1 and DR3 mRNA, as well as P21 and P27 cell cycle proteins, were increased. Meanwhile, the expressions of caspase-8, -9, phospho-Bad and Bcl-2 proteins decreased. However, in the case of side population cells, no remarkable changes were observed. Conclusion L. casei extract exerts a potent anti-tumor effect on the viability of liver cancer cells, although not on cancer stem-like cells.

INTRODUCTION
Lactobacillus casei (L. casei) is a gram-positive non-pathogenic, rod-shaped anaerobic lactic acid bacterium, which is widely distributed in nature and commonly present in the human mouth, intestines and reproductive tracts. L. casei is now used widely in nutritional supplements and beneficially affects the host by improving its intestinal microbial balance in the human body. Previously, L. casei was shown to induce improvement in murine models of inflammatory disorders such as tract, 20 g of glucose, 1 g of sorbitan monooleate complex (Tween 80), 5 g of ammonium acetate, 5 g of sodium acetate, 2 g of K 2 HPO 4 , 0.2 g of MgSO 4 -7H 2 O, and 0.2 g of Mn-SO 4 -4H 2 O. The pH of the growth medium was adjusted to 6.5 after mixing the components.
Preparation and purification of Lactobacillus casei extract L. casei was grown in 1 liter MRS medium for 24 hrs up to the mid-exponential phase, an A.600 value of about 0.5. After three washings with 1X phosphate buffered saline (PBS), bacterial cell walls were disrupted with a Sonic dismembrator (Fisher scientific, Rockingham County, NH, USA) for 1 minute on ice. The extracted protein concentration was assayed by the Quick Start Bovine Serum Albumin (BSA) Standard kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's guidelines.

Assessment of cell growth rate
Huh7 cells were seeded into cell culture plates at a density of 5×10 5 cells. The effect of L. casei extract on Huh7 cells viability was compared and determined by inverted microscopy (Olympus Model IX51, Tokyo, Japan), comprising a DP50 camera system (Olympus, Tokyo, Japan), using a 0.4% trypan blue dye exclusion method 72 hours after treatment with the 0.1 mg concentration. Cell survival was evaluated on the basis of the percentage of the total number of Huh7 cells (viable plus nonviable). Mean survival rate was determined by counting randomly selected non-overlapping fields four times. Each culture dish represents an n=1 determination with each experiment replicated independently 4 to 6 times using different cultures.

Cell cycle analysis
Huh7 cells were treated with 0.1 mg L. casei extract for 72 hours, harvested, fixed with 70% ethanol for 15 minutes, thawed and centrifuged at 1200 rpm for five minutes, washed arthritis, type I diabetes and systemic lupus, as well as murine chronic colitis and ileitis. 1,2 Oral administration of L. casei exhibits anti-tumor activity by augmentation of the host's immune system because of the strain's immunomodulatory activity. 3 Furthermore, a cell wall-derived polysaccharidepeptidoglycan complex in one L. casei strain plays a role in its anti-inflammatory actions in chronic intestinal inflammatory disorders. 4 Moreover, L. casei has the ability to moderate inflammatory responses in damaged liver tissue. 5 In a previous study, children treated with L. casei after liver transplantation experienced beneficial effects limited to the period of bacteria intake. 6 Several Lactobacillus strains have been shown to exert potent anti-tumor effects in rodents. 7 L. casei was reported to potentiate systemic immune responses in colon cancer mouse models, 8 and also to reduce tumor induction upon oral administration. 9 In another study, L. casei induced an immune response and tumoricidal growth inhibition upon intraperitoneal (i.p.) inoculation. 10 Orally administered L. casei is also known to be useful in preventing the recurrence of superficial bladder cancer. 11,12 Previous studies demonstrated that L. casei may retain its anti-cancer effects in human cancers. However, the anti-tumor activity of L. casei against liver cancer has not yet been reported.
Hepatocellular carcinoma is one of the most common cancers in the world and its prognosis is very poor. Nevertheless, administration of food supplements may be an easy and effective way of decreasing the incidence of liver cancer. L. casei is widely used in supplemental health foods, and has been shown to alleviate liver injury in rat models and maintain the normal structure of hepatocytes. 13 In this study, we investigated the anti-tumor activity of L. casei against human liver cancer cells. In doing so, we validated the efficacy of L. casei in Huh7 human hepatocellular carcinoma cells directly treated with L. casei extract. Through this study, L. casei extract was found to inhibit the growth of Huh7 cells. . Equal amounts of proteins were loaded onto a sodium dodecyl sulfate poly-acrylamide 4-12% gradient gel (Invitrogen, Carlsbad, CA, USA), electrophoretically separated for 2 hours at 180V with a X-cell kit (Invitrogen, Carlsbad, CA, USA), transferred onto a polyvinylidene difluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA), incubated with 5% w/v nonfat dry milk in tris-buffered saline tween 20 (TBST) buffer for 1 hour at room temperature, and probed with primary antibodies overnight at 4°C. After incubation with horseradish peroxidase conjugated goat anti-mouse and anti-rabbit secondary antibodies (1 : 2000) (Cell Signaling Technology, Danvers, MA, USA), peroxidase activity was detected by a LumiGLO chemiluminescent substrate system (KPL, Gaithersburg, MD, USA), according to the manufacturer's instructions. 17 Human actin levels were used as a control.

Analysis of the effect of L. casei extract on side population cells
Side population cells were analyzed as previously described. 18 To determine the effect of L. casei extract on side population cells, Huh7 cells were cultured and harvested as described previously. To analyze side populations, cells were incubated with Hoechst 33342 5 μg/mL (Sigma, St. Louis, MO, USA) or Hoechst 33342 5 μg/mL and verapamil 50 μmol/L (Sigma, St. Louis, MO, USA) for 90 minutes at 37°C. Cells were vortexed every 15 minutes. Propidium iodide 1 μg/mL (Sigma, St. Louis, MO, USA) was added to identify dead cells at the end of the incubation period. Side population with 1 mL of ice-cold 1X PBS, centrifuged again, and then treated with 100 μg/mL of RNAase A (Sigma, St. Louis, MO, USA) for 90 minutes at 37°C in a humidified 5% CO 2 atmosphere. Cells were stained with 25 μg/mL propidium iodide (Sigma, St. Louis, MO, USA) to analyze cell cycles. Cells were analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA, USA), which indicated the distribution of the cells into their respective cell-cycle phases based on their DNA content. G0/G1 cells were 2n, S-phase cells were >2n, but <4n, and G2/M cells were 4n. All experiments were performed in triplicate.

Apoptosis measurement by Annexin V staining
Huh7 cells were cultured as described above. Annexin V staining was done using an Annexin V-FITC Apoptosis kit (BD Pharmingen, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. Annexin V-positive cells were measured at a fluorescence intensity of 1×10 4 cells using the FACSCalibur system (Becton & Dickinson, San Jose, CA, USA) within 1 hour.

L. casei extract induces apoptosis in Huh7 cells
To determine whether the effect of L. casei extract on growth inhibition was due to cell death, as well as cell cycle arrest, the extent of cell death was determined by staining with Annexin-V-FITC and propidium iodide together, 72 hours after the treatment of Huh7 cells with L. casei extract. After treating cells with L. casei extract, 2% of cells were found in early apoptosis (lower, right) and 4% of cells were in late apoptosis (upper right and left). The cell death rate was cells were detected by FACS Aria (Becton & Dickinson, San Jose, CA, USA), using a 515-nm side population filter (Hoechst blue).

Statistical analysis
All experiments were performed independently in triplicate. All data were calculated using the R-statistics program. Data were presented as mean±standard error, and Student's t-test was used to evaluate differences between the treated and control experiments. p-values <0.05 were considered statistically significant.

L. casei extract inhibits the growth of Huh7 cells
To investigate the effect of L. casei extract on liver cancer cell lines, Huh7 cells were treated with an optimal concentration of L. casei extract of 0.1 mg, and the growth rate was evaluated using a 0.4% trypan blue dye exclusion method. Huh7 cells treated with L. casei extract were not significantly different compared with the controls after 24 hours. As shown in Fig. 1, the growth rate of L. casei extract-treated Huh7 cells was considerably lower (77%), compared to the controls. Thus, L. casei extract was specifically effective against liver cancer cells.

L. casei extract arrests liver cancer cells in the G2/M phase
To study the effect of L. casei extract on the different phases of the cell cycle, the DNA contents of Huh7 cells treated with L. casei extract were analyzed by flow cytometry after staining with propidium iodide and compared to that of the  CTL Lacto found to be increased by 6% in the treated group, compared to the control group (Fig. 3).

L. casei extract increases the mRNA expression levels of TNFR1 and DR3
Tumor necrosis factor-α receptor 1 [TNFR1; also known as death receptor 1 (DR1)] and DR3 (also known as APO-3, TRAMP) play roles of inducing apoptosis. These TNF receptors induce apoptosis by extracellular signals. Compared to the control group, L. casei extract-treated Huh7 cells exhibited significant increases in mRNA expression levels of TNFR1 and DR3 (Fig. 4). Thus, L. casei extract increased apoptosis in Huh7 cells through multiple extracellular related death receptors.
L. casei extract increases P21 and P27 cell cycle protein L. casei extract increased the expression levels of P21 and P27 proteins in Huh7 cells (Fig. 5A). P21 is a cyclin-dependent kinase inhibitor and inhibits the activity of cyclin-dependent kinase (CDK)-1, -2 complex. The function of P21 is responsible for inhibiting cell growth and inducing cells to apoptosis. Expression of P21 is dependent on extrinsic stimulus and thus prevents cell proliferation. P27 also acts as a tumor suppressor protein that inhibits CDK complexes and plays a role as a cell cycle regulator. L. casei extract was found to decrease the viability of Huh7 cells by inducing P21 and P27-dependent cell cycle arrest.

L. casei extract induces apoptosis
Caspase-3, caspase-8 and caspase-9 play important roles in mitochondria-related intrinsic and death receptor-related ex-

A B
cellular extracts of Lactobacillus strains was found to inhibit cancer cell proliferation. 19 Taken together, we concluded that the cell wall components of L. casei exhibit anti-tumor activities. Both chemotherapy and radiotherapy provide beneficial effects and improve quality of life and survival for two thirds of cancer patients. However, these therapies are temporal in many cases because of remission or subsequent relapses of cancer. 21 Recently, evidence has suggested that tumors are composed of a heterogeneous cell population with a small subset of CSC that sustain tumor formation and growth. The remission of drug resistance has recently been explained, by the discovery that CSC have resistance against anti-cancer drugs. 22 CSC are found to be enriched in a side population of cells that can recognized by fluorescence-activated cell sorting (FACS). 18 To date, side population cells have been detected in various solid tumor cells. 23 In the present study, after the treatment of L. casei extract, we used dual-wavelength flow cytometry to detect Huh7 side population cells on the basis of the ability of these cells to efflux the fluorescence DNA-binding dye Hoechst 33342. We found in this study that the fraction of side population cells in the treated group was higher than that in the control group (Fig. 6). This means that L. casei extract did not affect cell viability in side population cells. Side population cells are known to have high drug efflux capacity with strong expression of the ATP-binding cassette transporter protein ABCG2/Bcrp1. 24 Accordingly, the bacterial extracts might not have affected the Huh7 side population cells because of the active efflux pump. Previous studies reported that both P21 and P27 are cell cycle arrest related proteins. 25 P21 is known to inhibit cell growth and be related to apoptosis. P27 acts as a cell cycle regulator. The elevation of these proteins indicates the induction of cell cycle arrest. In Huh7 cells treated with L. casei extract, the expression levels of P21 and P27 proteins were increased, indicating that the treatment of L. casei extract is related to P21 and P27 dependent cell cycle arrest.
We also found that the expression of DR3 mRNA is increased by L. casei treatment in Huh7 cells. This evidence demonstrated that L. casei induced apoptosis is mediated through DR3 dependent pathways. TNFR1, 26 DR3 15 is a protein expressed in various cell lines and mediates the effect of TNF-a. TNFR1 mRNA was also increased in Huh7 cells treated with L. casei extract, indicating that TNFR1 is also related to extrinsic apoptosis pathways. Thus, L. casei extract increases the apoptosis of Huh7 cells through multiple extra-trinsic pathways of apoptosis. Caspase-3 is the final executioner of apoptosis, while caspase-8 and -9 are mediators of the intrinsic mitochondrial pathway of apoptosis. As shown in Fig. 5B, caspase-3 levels were unchanged, while both caspase-8 and -9 levels decreased after treatment of Huh7 cells with L. casei extract. Additionally, phospho-Bad, an anti-apoptotic protein associated with the cleavage of procaspase-9, and Bcl-2, an apoptosis regulator protein, were also decreased. Thus, Huh7 cells treated with L. casei extract were induced apoptosis by caspase-8, -9, phospho-Bad, Bcl-2 decreasing manner.

L. casei extract increase side population fraction
Side population (SP) cells have been identified as cancer stem-like cells (CSC) in leukemia and solid cancer cells. SP cells demonstrate specific characteristics of self-renewal, differentiating, tumor initiating and drug resistance. To determine the effect of L. casei extract on the viability of side population cells, we analyzed changes in side population fractions through Hoechst 33342 staining by flow cytometry before and after treatment of L. casei cellular extract in Huh7 cells. Accordingly, L. casei extract was found to increase side population fractions by 0.5% in the treated group (2.3%), compared to the control (1.8%). Thus, L. casei extract failed to inhibit CSC in Huh7 cells.

DISCUSSION
Lactic acid bacteria, such as Lactobacillus strains, have been reported to possess certain anticancer properties. 19 However, the anti-tumor activity of L. casei again human liver cancer has not been reported. So, in the present study, we evaluated the inhibitory effects of L. casei on Huh7 human hepatoma cells to demonstrate the causes of such effects by the trypan blue dye exclusion method and flow cytometry after staining with Annexin-V-FITC and propidium iodide together. We found that L. casei extract decreased the cell viability of Huh7 cells by 77%, compared to the controls, due to both cell cycle arrest in the G2/M phase and apoptosis.
In the present, we used the whole cellular extract of L. casei to evaluate anti-tumor activity, without differentiating cytoplasm components and cell wall components. Peptidoglycans and membrane components derived from the cell wall of L. casei were reported to have anti-cancer activities. 20,21 Additionally, the soluble polysaccharide fraction of cellular mechanisms.
Caspase-3, -8 and -9 play crucial roles in the apoptotic pathway. In the present study, caspase-3 was not significantly decreased after treatment of L. casei extract in Huh7 cells, while caspase-8 and -9, as well as phospho-Bad and Bcl-2 were. Thus, Huh7 cells treated with L. casei extract were induced by apoptosis pathway by caspase-8, -9, phospho-Bad, Bcl-2 decreasing mechanisms.
Finally, we demonstrated that L. casei extract is a promising candidate for treating hepatocellular carcinoma, and some of the small molecules of L. casei may be of use in anti-cancer drug components.


s2fieldsofstudy: Biology Medicine

text:
Pleiotropic effects of simvastatin in physically trained ovariectomized rats

This study tested the hypothesis that simvastatin treatment can improve cardiovascular and autonomic functions and membrane lipoperoxidation, with an increased effect when applied to physically trained ovariectomized rats. Ovariectomized rats were divided into sedentary, sedentary+simvastatin and trained+simvastatin groups (n = 8 each). Exercise training was performed on a treadmill for 8 weeks and simvastatin (5 mg/kg) was administered in the last 2 weeks. Blood pressure (BP) was recorded in conscious animals. Baroreflex sensitivity was evaluated by the tachycardic and bradycardic responses to BP changes. Cardiac vagal and sympathetic effects were determined using methylatropine and propranolol. Oxidative stress was evaluated based on heart and liver lipoperoxidation using the chemiluminescence method. The simvastatin-treated groups presented reduced body weight and mean BP (trained+simvastatin = 99 ± 2 and sedentary+simvastatin = 107 ± 2 mmHg) compared to the sedentary group (122 ± 1 mmHg). Furthermore, the trained group showed lower BP and heart rate compared to the other groups. Tachycardic and bradycardic responses were enhanced in both simvastatin-treated groups. The vagal effect was increased in the trained+simvastatin group and the sympathetic effect was decreased in the sedentary+simvastatin group. Hepatic lipoperoxidation was reduced in sedentary+simvastatin (≈21%) and trained+simvastatin groups (≈57%) compared to the sedentary group. Correlation analysis involving all animals demonstrated that cardiac lipoperoxidation was negatively related to the vagal effect (r = -0.7) and positively correlated to the sympathetic effect (r = 0.7). In conclusion, improvement in cardiovascular and autonomic functions associated with a reduction of lipoperoxidation with simvastatin treatment was increased in trained ovariectomized rats.

Introduction
Cardiovascular diseases remain the leading cause of death among women in the most developed areas of the world (1). Studies have demonstrated that menopause can induce metabolic, endothelial and autonomic dysfunctions and increase oxidative stress in women, thus increasing cardiovascular risk (2)(3)(4)(5). Similarly, rats subjected to ovarian hormone deprivation presented increased blood pressure (BP) and sympathetic tonus and reduced baroreflex sensitivity (BRS) compared to control female rats (6).
On the other hand, simvastatin treatment reduces both mortality and coronary events in humans, and also prevents and controls atherosclerosis (7,8). The beneficial pleiotropic effects of simvastatin treatment may be due to the endothelial protection, oxidative stress reduction, sympathetic decrease, and control of inflammatory reactions it seems to offer (9)(10)(11)(12).
In addition, exercise training has been recognized as a non-pharmacological treatment for cardiovascular disease. A recent systematic review of randomized, controlled trials reported benefits of regular exercise after menopause (13). Furthermore, previous studies performed in our laboratory have demonstrated that exercise training induced a reduction in body weight, BP and cardiac oxidative stress and an increase in BRS in an experimental model of menopause (6,14,15).
However, there are few data in the literature concerning the effects of either pharmacological or non-pharmacological Correspondence: K. De Angelis, Laborato´rio de Fisiologia Translacional, Universidade Nove de Julho, Rua Vergueiro 235/249, 2 o subsolo, Mestrado, 01504-001 Sa˜o Paulo, SP, Brasil. E-mail: prof.kangelis@uninove.br approaches on hemodynamic, autonomic or oxidative stress profile changes induced by ovarian hormone deprivation. Also, there are no data on the effects of the association of simvastatin treatment with exercise training, two common approaches used in postmenopausal women in clinical practice.
Therefore, the purpose of the present study was to test the hypothesis that short-term simvastatin treatment (2 weeks) can induce cardiovascular and autonomic improvement and reduce oxidative stress when evaluated by membrane lipoperoxidation. Moreover, we hypothesized that simvastatin treatment can induce additional benefits in physically trained ovariectomized rats.

Animals and groups
Experiments were performed on 24 female Wistar rats (215-230 g) from the animal house of the Universidade Sa˜o Judas Tadeu, Sa˜o Paulo, Brazil. The rats were given standard laboratory chow and water ad libitum and were housed in individual cages in a temperature-controlled room (226C) with a 12-h dark/light cycle. All rats were treated similarly in terms of daily manipulation. The experimental protocol was approved by the institutional Animal Care and Use Committee of Universidade Sa˜o Judas Tadeu and the investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Ovariectomized rats were randomly assigned to the following groups: sedentary (SO, n = 8), sedentary treated with simvastatin (SSO, n = 8), and exercise trained treated with simvastatin (STO, n = 8).

Ovariectomy
At 10-12 weeks of age, animals were anesthetized (80 mg/kg ketamine and 12 mg/kg xylazine), and a small abdominal incision was made. The ovaries were then located, and a silk thread was tightly tied around the oviduct, including the ovarian blood vessels. The oviduct was sectioned and the ovaries removed. The skin and muscle wall were then sutured with silk thread. After surgery, the animals received an injection of antibiotics (40,000 U/kg penicillin G procaine, im) (6,14,16).

Exercise training
Exercise training was performed on a motor treadmill at low-moderate intensity (<40 to 65% maximal running speed) for 1 h a day, 5 days a week, for 8 weeks, with a gradual increase in speed from 0.3 to 1.2 km/h. All animals were adapted to the procedure (10 min/day, 0.3 km/h) for 1 week before beginning the exercise training protocol. This adaptation period began 24 h after ovariectomy (6,14). All groups were subjected to a maximal treadmill test, as described in detail in a previous publication (17,18). The tests were performed three times: 1) at the beginning of the experiment, 2) in the fourth week, and 3) in the eighth week of the training protocol. The purpose was to determine maximal physical capacity and exercise training intensity.

Simvastatin treatment
Simvastatin treatment (5 mg kg -1 ?day -1 ) was performed daily by gavage in the last 2 weeks of sedentarism or training. The SO group was submitted to a daily gavage of water. The choice of a simvastatin dose of 5 mg kg -1 ?day -1 was based on a pilot study in which we compared the dose of 20 mg kg -1 ?day -1 to a dose of 5 mg kg -1 ?day -1 in male rats. The results of these experiments showed that the dose of 20 mg kg -1 ?day -1 induced further reduction in triglycerides and total cholesterol in treated male rats, which could induce changes in cardiac and autonomic functions, independently of the pleiotropic effects of simvastatin. Considering these results and the fact that the dose of 5 mg/kg did not induce changes in the metabolic parameters evaluated, for the present experiments we chose the simvastatin dose of 5 mg/kg.

Metabolic evaluations
Body weight was measured at the beginning and at the end of the protocol. Blood glucose and triglycerides were evaluated after 4 h fasting at the beginning and at the end of the protocol using the Roche GCT Accutrend (Roche, Brazil).

Cardiovascular assessments
After the last training session, 2 catheters filled with 0.06 mL saline were implanted into the carotid artery and jugular vein (PE-10) of anesthetized rats (80 mg/kg ketamine and 12 g/kg xylazine) for direct measurement of BP and for drug administration, respectively. The rats were conscious and allowed to move freely during the hemodynamic experiments. The arterial cannula was connected to a strain-gauge transducer (Blood Pressure XDCR, Kent Scientific, USA), and blood pressure signals were recorded over a 30-min period by a microcomputer equipped with an analog-to-digital converter board (CODAS, 2-kHz sampling frequency; Dataq Instruments, Inc., USA). The recorded data were analyzed on a beatto-beat basis to quantify changes in mean BP (MBP) and heart rate (HR). Increasing doses of phenylephrine (0.25 to 32.0 mg/kg, iv) and sodium nitroprusside (0.05 to 1.6 mg/kg, iv) were given as sequential bolus injections (0.1 mL) to produce pressure responses ranging from 5 to 40 mmHg. A 3-to 5-min interval between doses was necessary for blood pressure to return to baseline. Peak increases or decreases in MBP after phenylephrine or sodium nitroprusside injection and the corresponding peak reflex changes in HR were recorded for each dose of the drug. BRS was evaluated by a mean index relating changes in HR to the changes in MBP to allow for separate analysis of gain for reflex bradycardia and reflex tachycardia. The mean index was expressed as bpm/ mmHg, as described elsewhere (14,15).
Vagal and sympathetic effects were studied by injecting methylatropine (3 mg/kg, iv) and propranolol (4 mg/kg, iv) at a dose of 0.1 mL/100 g body weight.
Resting HR was recorded while the rats were in their cages in an unrestrained state, and methylatropine was injected immediately after the recording. Because the HR response to these drugs reaches its peak within 5 min, this time interval was allowed to elapse before the HR measurement was made. On the following day, the sequence of injection was inverted, with propranolol given first. The sympathetic effect was determined as the difference between the basal HR and the lowest HR after propranolol. The vagal effect was obtained by the difference between the maximum HR after methylatropine injection and the basal HR (19).

Evaluation of membrane lipoperoxidation
The animals were killed by decapitation and the heart (ventricles) and liver were immediately removed, rinsed in saline, and trimmed to remove fat tissue and visible connective tissue. These tissues were cut into small pieces, placed in ice-cold buffer, and homogenized in an ultra-Turrax blender with 1 g tissue per 5 mL 150 mM KCl and 20 nM sodium phosphate buffer, pH 7.4. The homogenates were centrifuged at 600 g for 10 min at -26C. Tissue membrane lipoperoxidation was evaluated by chemiluminescence. The chemiluminescence assay was carried out with an LKB Rack Beta liquid scintillation spectrometer 1215 (LKB Producer AB, USA) in the out-of-coincidence mode at room temperature (256 to 276C). The supernatants were diluted in 140 mM KCl and 20 mM sodium phosphate buffer, pH 7.4, and added to glass tubes, which were placed in scintillation vials; 3 mM tert-butylhydroperoxide was added, and chemiluminescence was determined up to the maximal level of emission (14,20,21).

Statistical analysis
Data are reported as means ± SE. Comparisons between the 3 groups were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Pearson's correlation was used to determine association among variables. The level of significance was established at P , 0.05.

Results
There were no differences in body weight between groups at the beginning of the protocol (SO = 207 ± 2.5 g). At the end of the training period, SSO and STO animals (SSO = 306 ± 5 g; STO = 308 ± 8 g) had a smaller increase in body weight compared to SO animals (323 ± 4 g). No difference in blood metabolic parameters was observed between groups at the beginning of the protocol. After 4 h of fasting, blood glucose (SO = 89 ± 2 mg/dL) and triglycerides (SO = 97 ± 5 mg/dL) did not differ between groups at the end of the protocol.
Maximal physical performance was evaluated by the response to the maximal treadmill test. At the beginning of the experiment, the physical performance was similar for all groups (SO = 2 ± 0.08 km/h). However, the animals submitted to the exercise training protocol (STO = 2.4 ± 0.09 km/h) showed an increase in maximum running speed compared to the SO (2 ± 0.09 km/h) and SSO (1.9 ± 0.07 km/h) groups after 8 weeks of exercise training.
Oxidative stress evaluated by membrane lipoperoxidation showed a significant reduction in liver homogenates from animals submitted to simvastatin treatment (<21%) and from animals submitted to exercise training associated with simvastatin treatment (<57%) compared to the sedentary group (SO). No difference in cardiac lipoperoxidation was observed between the two groups (Table 1).

Discussion
Important insights were obtained in the present study. Firstly, although the simvastatin-treated ovariectomized rats, either trained or not, showed unchanged blood triglycerides and glucose levels (after 4 h of fasting), they indeed presented decreased body weight, SBP, DBP, and MBP compared to sedentary ovariectomized rats. Secondly, simvastatin treatment of sedentary or trained ovariectomized rats induced improved bradycardic and tachycardic baroreflex responses compared to sedentary untreated rats, a fact possibly related to a decrease in cardiac sympathetic effect (sedentary group treated with simvastatin) or with an increase in vagal effect on the heart (trained group). Thirdly, simvastatin treatment of ovariectomized animals (SSO and STO groups) induced a reduction in membrane lipoperoxidation of the liver, the target organ for the lipid-regulating effect of this drug. Fourthly and importantly, the BP and liver lipoperoxidation reductions in the simvastatin-treated groups were more pronounced in trained rats and were accompanied by resting bradycardia and improved physical capacity. Finally, the levels of cardiac lipoperoxidation were correlated with the autonomic control of HR, suggesting that improved autonomic cardiac control could be associated with reduced oxidative stress in the heart.
The incidence of cardiovascular disease in women increases sharply after menopause and probably involves changes in BP and its regulation associated with estrogen loss (2)(3). In the present study, we showed elevated BP values in ovariectomized rats when compared to BP values previously found not only in intact female rats but also in male rats (6,16,(21)(22)(23). In fact, ovariectomized rats showed an increase in BP values, reaching values similar to the ones observed in our experiments (6). Moreover, some previous studies have not shown a decrease in BP values after simvastatin treatment in male rabbits, rats or humans (9,(24)(25)(26). In the present study, we showed a decrease in BP after simvastatin treatment, corroborating previous data that demonstrated BP reduction after longterm simvastatin treatment (27,28). Reduction in BP values after training was reported in normotensive postmenopausal women (13,29). Moreover, previous studies have shown that low-to moderate-intensity exercise training is an efficient nonpharmacological treatment for hypertension (30)(31)(32). This is the first study to demonstrate that simvastatin treatment in trained rats increases the BP reduction observed in rats only exposed to this pharmacological approach.
The mechanisms involved in BP reduction may be related to changes in cardiac output or peripheral resistance. It is well established that increased oxidative stress plays an important role in endothelial and cardiac dysfunctions, which are involved in the vasomotor and cardiac tonus. It is important to emphasize that an additional BP reduction was observed in STO animals.  This change may be associated with the resting bradycardia observed in this group, which could induce a reduction in cardiac output. In fact, some investigators (33) have reported that exercise training causes a reduction in cardiac output in hypertensive subjects, whereas others (30) have observed a decrease in total peripheral resistance in humans. The resting bradycardia verified in the trained ovariectomized rats treated with simvastatin can be attributed to the increased cardiac vagal tonus obtained in this group as previously documented in males and ovariectomized females (6,15,19). BRS dysfunction has been documented in cardiovascular diseases as an important predictor of mortality and survival (34). Thus, the identification of the mechanism underlying a depressed BRS, as well as the study of therapies to improve this cardiovascular reflex, have important clinical implications. Davy et al. (35) have reported that physically active postmenopausal women present higher BRS when compared to age-matched less active women, providing insights into a possible cardioprotective mechanism in physically active postmenopausal women. Pliquett et al. (9) have also shown baroreflex improvement in rabbits with heart failure after statin treatment without changes in total plasma or high-density cholesterol. In the present study, we demonstrated that simvastatin treatment, associated or not with exercise training in an experimental model of ovarian hormone deprivation, induced an improvement in BRS for both the bradycardic and tachycardic responses evoked by BP rises and falls, respectively.
The improvement in this important cardiovascular reflex could be due to changes in the efferent pathways, since the vagal effect was increased in the trained group and the sympathetic effect was decreased in the sedentary simvastatin group. Moreover, the BP reduction may have contributed to improving the sensitivity of this reflex. However, additional changes in afferent pathways (36) or in the central component of this reflex should not be excluded.
Studies from our laboratory have shown an increase in cardiac vagal activity in trained mice and rats (6,15,19). Corroborating these data, in the present study we showed an increased vagal effect in the group subjected to both treatments (simvastatin+ +exercise). Other studies, however, have reported that exercise training decreased or had no effect on sympathetic activity in spontaneously hypertensive rats and in healthy and ovariectomized female rats (6,15,23,31). Moreover, Gentlesk et al. (37) have demonstrated a reduction in cardiac death after simvastatin treatment, which was attributed to changes in sympathetic modulation. In the present study, we observed a decrease in sympathetic effect in simvastatin-treated rats.
Importantly, these beneficial changes in cardiac autonomic control observed in the simvastatin groups, trained or not, were related to reduced cardiac oxidative stress as evaluated by membrane lipoperoxidation. Exercise training (14,21,32) and simvastatin (38) have been reported as therapies, which improve the antioxidant mechanism and reduce oxidative stress. Corroborating these data, we observed reduced hepatic lipoperoxidation in the simvastatin-treated groups (trained or not) compared to the untreated group, but no significant changes were obtained in cardiac lipoperoxidation. Two aspects deserve a special note: first, the trained group presented about 50% reduction in hepatic lipoperoxidation compared to the sedentary group treated with simvastatin alone. This suggests an additional effect obtained by associating the therapies. Second, despite the fact that cardiac lipoperoxidation was similar in all groups studied, the correlations observed between cardiac lipoperoxidation and sympathetic and parasympathetic effects suggest that the animals presenting a decreased sympathetic effect and/or increased vagal effect also presented lower cardiac membrane lipoperoxidation. In the present study, correlation analysis (r 2 ) showed that cardiac lipoperoxidation variability explained 50-60% of cardiac sympathetic or vagal effect variability. This reinforces the role of oxidative stress in cardiovascular autonomic dysfunction as previously reported by our group (14,32).
Importantly, the trained females treated with simvastatin showed a marked increase in estimated physical capacity as evaluated by their response to the maximal exercise test, indicating that the training protocol used was effective. Significant correlation between oxygen consumption and maximum running velocity was reported in untrained rats (17,18). Furthermore, the findings for resting bradycardia and reduced body weight are also good indications of the efficacy of the exercise training protocol in promoting overall fitness. In a recent review, Asikainen et al. (13) have shown that 9 studies on postmenopausal women have reported improvement in exercise capacity and body weight reduction after regular physical activity.
The improvement in cardiac autonomic control after short-term simvastatin treatment in an experimental model of ovarian hormone deprivation in the present study was independent of the classical plasma lipid effects of this drug, thus reinforcing the hypothesis that statin would reduce cardiovascular risk in females. These autonomic pleiotropic effects of statins may account for the patients' outcome and should be further characterized. In this respect, compelling evidence suggests that statins have beneficial effects on survival, even for normal lipid levels (8,39). However, additional studies are needed to confirm the pleiotropic effects of long-term statin treatment as well as the additional effects of associated physical training on the autonomic dysfunction and on the outcome of women in the climacterium.


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The Iron Distribution and Magnetic Properties of Schistosome Eggshells: Implications for Improved Diagnostics

Background Schistosoma mansoni and Schistosoma japonicum are the most frequent causative agents of human intestinal schistosomiasis. Approximately 200 million people in the world are infected with schistosomes. Diagnosis of schistosomiasis is often difficult. High percentages of low level infections are missed in routine fecal smear analysis and current diagnostic methodologies are inadequate to monitor the progress of parasite control, especially in areas with low transmission. Improved diagnostic methods are urgently needed to evaluate the success of elimination programs. Recently, a magnetic fractionation method for isolation of parasite eggs from feces was described, which uses magnetic microspheres to form parasite egg – magnetic microsphere conjugates. This approach enables screening of larger sample volumes and thus increased diagnostic sensitivity. The mechanism of formation of the conjugates remains unexplained and may either be related to specific surface characteristics of eggs and microspheres or to their magnetic properties. Methods/Principal Findings Here, we investigated iron localization in parasite eggs, specifically in the eggshells. We determined the magnetic properties of the eggs, studied the motion of eggs and egg-microsphere conjugates in magnetic fields and determined species specific affinity of parasite eggs to magnetic microspheres. Our study shows that iron is predominantly localized in pores in the eggshell. Parasite eggs showed distinct paramagnetic behaviour but they did not move in a magnetic field. Magnetic microspheres spontaneously bound to parasite eggs without the presence of a magnetic field. S. japonicum eggs had a significantly higher affinity to bind microspheres than S. mansoni eggs. Conclusions/Significance Our results suggest that the interaction of magnetic microspheres and parasite eggs is unlikely to be magnetic in origin. Instead, the filamentous surface of the eggshells may be important in facilitating the binding. Modification of microsphere surface properties may therefore be a way to optimize magnetic fractionation of parasite eggs.

Introduction
Schistosomiasis is a helminth infection representing a major health burden for humans in tropical and developing nations. Some 200 million people are infected, and 600 million are currently estimated to be at risk of infection [1].
Recently, schistosomiasis control efforts have been increasingly focused on mass drug administration in endemic areas to alleviate morbidity in affected individuals [2]. Although it has been acknowledged that the goal to regularly administer chemotherapy to at least 75% of school-age children at risk of morbidity was not achieved by 2010, many countries are controlling schistosomiasis with increasing success using a combination of therapeutics and improved sanitation [3,4]. Sustained and effective drug therapy has the effect of pushing the disease into a state of low endemicity, where individuals carry low-level infections that are very difficult to detect. As a result, new efforts are required for parasite surveillance in regions of low endemicity [5].
It has become increasingly recognized that the evaluation and monitoring of control and elimination efforts for schistosomiasis is hindered by the lack of appropriate diagnostic techniques [5,6]. With a diagnostic limit of approximately 100 eggs per gram feces, the current WHO recommended test, the Kato-Katz method of fecal examination, is limited by poor sensitivity [7,8,9]. It is estimated that more than half of all infections with schistosomiasis are missed in cross sectional studies relying on the observation of only one fecal smear, necessitating the need to perform multiple smears [10]. Examining multiple fecal smears at different time points is logistically difficult, and time and labour intensive. There is, therefore, an urgent need to develop new diagnostic methodologies for intestinal schistosomiasis that are highly sensitive and applicable under field conditions [5]. There is also the need for a new gold standard method to which more sensitive, newly developed, simple and field applicable molecular and rapid diagnostic tests can be compared.
Recently, a novel method for Schistosoma egg detection based on magnetic fractionation of parasite eggs from fecal matter was developed [11]. For this method, termed Helmintex, magnetic microspheres are added to larger volumes of fecal samples (30 g). Parasite eggs and magnetic microspheres can then be co-purified from other fecal components through the application of a magnetic field and field gradient. The purified egg concentrates are more readily detectable by light microscopy. Teixeira and colleagues reported that magnetic microspheres coated with a variety of adsorbed molecules could be used to purify eggs of Schistosoma mansoni from fecal matter in a magnetic field [11]. The nature of the adsorbed molecules had no influence on the efficiency of the purification and even the use of uncoated microspheres resulted in the purification of Schistosoma mansoni eggs from feces in a magnetic field. The mechanism of interaction between parasite eggs and microspheres is unclear, yet it is important to characterize it further in order to optimize specificity and efficiency of the Helmintex method [12].
There are two possible explanations for the seemingly specific interaction of magnetic microspheres and Schistosoma eggs. Firstly, it could be that biochemical, chemical or physical surface properties of the eggs mediate the interaction. Secondly, the eggs could themselves be magnetic, leading to a magnetically mediated adhesion of the microspheres to the eggs. Since the interaction seems independent of the surface characteristics of the magnetic microspheres, the latter of these two possibilities seemed the more probable at the beginning of this investigation.
It has been shown that the eggshells of S. japonicum contain iron in concentrations detectable by energy dispersive X-ray spectroscopy in the transmission electron microscope [13]. The authors of that study suggested that iron assists in the formation of the biopolymer that makes up the eggshells. Eggshells are formed by polymerization of tyrosine-rich eggshell precursor proteins that are synthesised in the vitelline glands of the parasite. The tyrosine residues are oxidised by tyrosinases to o-quinones. Lysine and histidine residues on the same or adjacent eggshell precursors subject the o-quinones to nucleophilic attack, leading to a robust cross-linked polymer [14,15,16].
In other invertebrates, such as the bivalves Mytilus, DOPA-rich bonds in quinone-tanned protein polymers are stabilized by divalent metal-ions, including iron [17]. The vitelline glands of schistosomes are enriched with iron and the iron storage protein ferritin [18]. Thus, there is strong evidence for a role of iron in stabilizing the protein polymer that is the schistosome eggshell. Full verification of this hypothesis requires the refinement of methods for hydrolysis of the highly resistant shells [19].
Here we present results of experiments elucidating the elemental composition of the eggshell, with special focus on the organization of iron in the matrix and the resulting magnetic properties of the eggs. We performed a range of experiments to characterise the nature of magnetic microsphere interaction with parasite eggs and in order to investigate whether this interaction was a result of nonspecific binding of the microspheres to the surface of the eggs, or whether it was the result of magnetically aided adhesion of eggs and microspheres.

Materials and Methods
All work using animals was approved by the Animal Ethics Committee of the Queensland Institute of Medical Research (Project P1289). This study was conducted according to guidelines of the National Health and Medical Research Council of Australia, as published in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th edition, 2004 (www. nhmrc.gov.au). S. mansoni was maintained in Biomphalaria glabrata snails and S. japonicum was sourced from infected Oncomelania hupensis hupensis snails collected in Anhui Province, China. Both, S. mansoni and S. japonicum worm stages were maintained in outbred Swiss mice.
Eggs of both species were purified from livers of mice by digestion of liver parenchymal tissues with collagenase B in phosphate buffered saline (PBS) in the presence of ethylenediaminetetraacetic acid (EDTA) as an iron chelator. The eggs were incubated in enzyme for 8 h at 37uC with agitation. For further purification eggs were centrifuged in Percoll gradients as described by Dalton et al. in 1997 [20].

Scanning electron microscopy (SEM)
Samples of S. mansoni and S. japonicum eggs were fixed in 2% (v/v) glutaraldehyde, 1% (w/v) paraformaldehyde in PBS for 60 min at 4uC and washed twice with PBS (pH = 7.4) in 1.5 mL Eppendorf tubes. The samples were then serially dehydrated in ascending

Author Summary
In the present study, we investigated the mechanism underlying a novel diagnostic method for Schistosomaone of the most widespread and frequently occurring parasites infecting humans in tropical countries. In recent years, the world has seen significant reduction in the burden of Schistosoma infections in many countries due to improved control and sanitation. However, it is becoming increasingly difficult to evaluate and monitor the progress of control towards elimination. At the moment it is extremely difficult to determine whether the parasite has been eliminated from a region. This is due to the absence of a sensitive and inexpensive method to detect the parasite. A series of recent studies describes a method with vastly improved diagnostic sensitivity based on the magnetic fractionation of parasite eggs from fecal samples. However, the mechanisms of action of this new diagnostic are not currently known. To further optimize and improve this method, we studied the magnetic properties of parasite eggshells and their binding characteristics to magnetic microspheres.
concentrations of ethanol (33%, 50%, 66%, and 100% (dry)) followed by two further washes in dry ethanol using a PELCO Biowave microwave processor (TedPella Inc., Redding, CA, USA). Dehydrated samples were transferred onto circular polylysinecoated glass coverslips and critically point dried (Emitech 850 Critical Point Drier, Quorum Technologies, Ashford, UK). The coverslips were then attached to aluminum sample holders and coated with a 5 nm thick platinum coating for morphological analyses. SEM was performed using the in-lens detector of a Zeiss 1555 VP field emission scanning electron microscope operating at 15 keV (Carl Zeiss, Ü berkochen, Germany).

Cryopreparation and High Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM)
Eggs of both parasite species were fixed in 3% (v/v) glutaraldehyde in 0.1 M phosphate buffer. Eggs were trans- ferred to a solution of 20% (w/v) bovine serum albumin in PBS on a copper membrane and rapidly frozen in a Leica EM PACT2 High Pressure Freezer (Leica, Vienna, Austria). Subsequently, the membranes and samples were transferred in cryo-tubes under liquid nitrogen to a Leica EM AFS freeze substitution apparatus for fixation and dehydration in 2% (w/ v) osmium tetroxide and 0.5% uranyl acetate (w/v) in 100% anhydrous acetone. The tissues were cryo-substituted for 3 days, according to the following protocol: The temperature of the substitution chamber was increased from 2160uC to 285uC over 2 h, and maintained at 285uC for 48 h, after which the samples were brought to room temperature. After further washes in anhydrous acetone, the samples were infiltrated and embedded in Epon resin.
For HAADF-STEM, resin sections were cut from blocks at a thickness of 150 nm using an EM UC6 ultramicrotome (Leica, Vienna, Austria) and mounted onto 200 mm mesh carbon filmed copper grids for analysis at 300 kV using a JEOL JEM 3000F FEGTEM transmission electron microscope (JEOL, Tokyo, Japan). A ,1 nm probe size was used to image the mass variation within the sections, with areas of high mass appearing bright. Energy Dispersive X-ray Spectroscopy (EDS) data was combined with STEM imaging using an Oxford Instruments INCA detector (Oxford Instruments NanoAnalysis, High Wycombe, UK) to map the composition of features of interest.

Superconducting quantum interference device (SQuID) magnetic susceptometry
SquiD magnetic susceptometry is a technique to determine the magnetic properties of any given solid material. Samples are exposed to a desired sequence of magnetic fields at constant temperature or a sequence of temperatures at a constant magnetic field. The magnetization of the sample material resulting from this exposure is recorded for each point in a sequence. Using standard sequences, basic magnetic properties (e.g., whether a material is ferromagnetic, paramagnetic or diamagnetic (non-magnetic)) can be determined.
Lyophilized S. mansoni and S. japonicum eggs were placed in gel capsules for magnetic characterization in a 7 Tesla (T) magnetic property measurement system SQuID magnetic susceptometer (Quantum Design, San Diego, CA, USA). Magnetic hysteresis loops were acquired between 27 T and 7 T at 5 K. Zero-fieldcooled and field-cooled (ZFC-FC) magnetization versus temperature curves were obtained from 5 to 300 K, in a measurement field of 0.01 T.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES)
The concentration of iron, copper and silicon was determined for both types of eggs using ICP-AES. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) is an analytical technique used to determine the elemental composition of a material. It uses inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element which are then detected by a detector.
The same samples as in the SQuID measurements were used. For ICP-AES analysis, three replicate samples were digested in 70% HNO 3 at 95uC. The analysis was performed at the Marine and Freshwater Research Laboratory at Murdoch University, Murdoch, WA, Australia.

Exposure of eggs to magnetic fields with and without magnetic microspheres
In order to assess the ability to manipulate parasite eggs using a magnetic field, approximately 100 glutaraldehyde-fixed eggs of S. japonicum were floated on a 100% Percoll/water interface in a 5 mL cell culture dish. No spontaneous hatching of eggs was observed in these fixed eggs. A cylindrical neodymium-iron-boron magnet was brought close to the eggs so that they were exposed to a magnetic field of approximately 0.1 T and a magnetic field gradient of approximately 35 T/m while being observed under an optical microscope.
To assess the capability of the different egg species to bind magnetic microspheres, eggs of the two species were incubated with microspheres at egg/microsphere ratios of 1:100 and 1:500. Unbound microspheres were washed out using custom made filters after an incubation time of 10 min with agitation. Images of the conjugated microsphere/egg suspension were taken at a 100-fold magnification and the distributions of the number of observed microspheres per egg were recorded and compared with the Poisson distribution.

Analysis of magnetic susceptometry data
The SQuID magnetometry data was fitted to two functions, the Brillouin function and Curie's law. These functions are used to determine the atomic iron specific moment in the samples [21]. The Brillouin function is specifically used to describe the response of an ideal paramagnet to an applied magnetic field. The spin state and thus the oxidation state of the iron atoms in a material can be deduced from the Brillouin fit using Equation 1.
The function describes the dependency of the magnetization (M) on the applied magnetic field (B) in an ideal paramagnet mixed with diamagnetic (non-magnetic) atoms and gives the total angular momentum quantum number J of the microscopic paramagnetic moments of the material. N is the number of paramagnetic atoms in the sample; g is the electron g-factor (22.0023); m B is the Bohr Magneton (9.274610 224 J T 21 ); J is the total angular momentum quantum number for each paramagnetic atom; k B is the Boltzmann constant (1.380610 223 m 2 kg s 22 K 21 ); T is the temperature and B is the magnetic flux density. The factor A is the diamagnetic susceptibility of the sample holder and the diamagnetic components of the eggs.
Curie's law is used to describe the temperature dependency of magnetic susceptibility (x). Data for this analysis are often plotted as 1/x versus T. A linear relationship with line of best fit running through the coordinate origin indicates ideal paramagnetic behavior. In the present study, the magnetization versus temperature data were fitted with Curie's law shown in Equation 2.
x~M B~N where x is the magnetic susceptibility of the eggs and all other symbols correspond to those used in Equation 1.

Scanning electron microscopy
SEM images of S. mansoni and S. japonicum eggs are shown in Figure 1. The eggs exhibit the typical features of S. mansoni (large spine) and S. japonicum (oval shape, small spine) [22]. The fibrous matrix surrounding the egg, also typical for S. japonicum, can clearly be seen in Figures 1C and 1D [22]. Figures 1B and 1D show examples of eggs of both species where the eggshell has broken open and the miracidium is still inside the egg. It can be seen that the eggshell curls outwards after initial rupture. Figure 2 shows details of the surfaces of the eggs of both species at a higher magnification. The surface of S. mansoni is covered with evenly spaced structures previously termed microspines with a length of about 200-300 nm and a diameter of 60 nm [22,23]. The surface of S. japonicum is also covered with microspines, however they are considerably shorter. In addition a structure, previously termed the fibrous matrix covers the S. japonicum surface [22].

Transmission electron microscopy
The elemental composition and structure of the eggshells of both species was studied by HAADF-STEM and STEM-EDS ( Figure 3). The TEM images show that the eggshells are about 700 nm thick. There are regions where the shell is interspersed with small holes of variable size (50-200 nm). These holes have been shown to be empty in other studies [23]. In the present study we show for the first time that these holes are partially filled with a material containing iron, phosphorous and oxygen and we hypothesise that this material is an iron-phosphate that is retained more readily by cryo-fixation and subsequent freeze-substitution processing compared to conventional TEM sample preparation methods (Figure 3).

Magnetic measurements
The results from the magnetometry measurements of the eggs are presented in Figure 4. Both, S. mansoni and S. japonicum eggs exhibited paramagnetic behaviour with no hysteresis at 5K ( Figure 4A). The magnetic susceptibility versus temperature measurements were in nearly perfect agreement with Curie's Law ( Figure 4B). The magnetic moment per iron atom (measured in Bohr magnetons -m B ) obtained from fitting the Brillouin function with an additional diamagnetic contribution (Equation 1) to the magnetization versus magnetic field data ( Figure 4A) at 5K was 4.8 and 4.16m B for S. mansoni and S. japonicum respectively. It   Figure 4A shows the curve fits of the Brillouin function with diamagnetic contribution (Equation 1) to the magnetization versus magnetic field data (for whole eggs at a temperature of 5 K). It can be seen that ideal paramagnetic behavior is approximated with good precision. (R 2 .0.99) for both, Schistosoma mansoni and Schistosoma japonicum eggs. Figure 4B shows the curve was assumed that iron was the only paramagnetic material in the eggs. Similar values were obtained from fits of Curies's law to the magnetization versus temperature data (4.76 and 3.76m B for the S. mansoni and S. japonicum eggs respectively). These values agree but are slightly lower than what would be expected if all the iron in the sample were present as high spin Fe 2+ ions (typically 5.46m B ) or high spin Fe 3+ (typically 5.96m B ). We can therefore conclude that there is mix of high spin and low spin iron configurations present in the eggs. Further, more sophisticated measurements using, for example, Mössbauer spectroscopy, that can be used to detect the chemical environment around each iron atom, would be necessary to resolve the exact distribution of these configurations.
Elemental analysis using inductively coupled plasma atomic emission spectroscopy (ICP-AES) ICP-AES data are summarized in Table 1. S. mansoni and S. japonicum eggs contained approximately 0.74 mg/g and 1.26 mg/g of iron respectively (dry weight). By comparison, 1 g of blood contains 3.39 mg iron and 1 g of normal human feces approximately 0.3 mg iron (dry weight) [24]. The concentrations of copper and silicon were also determined.

Exposure of eggs to magnetic fields with and without microspheres
No movement of parasite eggs suspended at the Percoll/water interface was observed when the eggs were exposed to a strong magnetic field and a high magnetic field gradient, and imaged with light microscopy. However, when magnetic microspheres were incubated with parasite eggs they readily bound to a fraction of the eggs even without the presence of a magnetic field. The microsphere-egg conjugates were very susceptible to magnetic fields and field gradients as shown in Figure 5 and the video file provided as supporting information (Video S1). Figure 6 illustrates the binding characteristics of the microspheres to the parasite eggs. For both tested parasite egg to microsphere ratios (1:100 and 1:500) the fraction of S. japonicum eggs that bound microspheres was statistically significantly higher than the fraction of S. mansoni eggs that bound microspheres (54% versus 41%, p = 0.02 for the 1:100 ratio and 76% versus 30%, p,0.001, for the 1:500 ratio, unpaired t-test). In addition the number of microspheres which bound to individual S. japonicum eggs was significantly higher than that for S. mansoni ( Figure 6). The distribution of microspheres per egg was not well characterized by a single Poisson distribution, especially for the S. japonicum eggs but reasonable fits were obtained on the assumption that a fraction of the eggs had no binding capacity (for more details on the analyses using Poisson statistics please refer to the supporting Figures S1 and S2 as well as Text S1).

Discussion
The present study investigated the magnetic properties as well as iron localization and content of S. mansoni and S. japonicum eggs and especially the eggshells. This investigation was conducted to elucidate the processes underlying the success of a previously developed magnetic fractionation approach for the detection of parasite eggs in fecal samples, namely the Helmintex method [11]. In recent years, this method has been used in a series of diagnostic studies and has consistently shown improved sensitivity when compared with the conventional Kato-Katz method of fecal evaluation and the saline gradient method [11,25,26,27].
The most important question for optimizing the existing Helmintex method was whether the magnetic properties of the Schistosoma eggs were the cause for the adhesion of the magnetic microspheres to the surface of the eggs or whether this binding was of another biochemical or physical nature.
We show that the eggshells of S. mansoni and S. japonicum eggs contain iron in concentrations measurable by STEM-EDS and ICP-AES, and that the eggs are distinctly paramagnetic, meaning they magnetize in an applied magnetic field and demagnetize when the magnetic field is taken away. These are the principal characteristics required for magnetic fractionation. Interestingly, most of the iron seems to be accumulated as iron-phosphate in pores in the eggshell.
However, the magnetization of the eggs was comparatively weak and the iron content was low. Furthermore we did not observe any movement of the eggs in magnetic fields and field gradients of a similar order of magnitude to those used in the Helmintex protocol. Therefore, the interaction between the eggs and the magnetic microspheres, which is the basis of the success of the Helmintex method, is unlikely to be magnetic in origin.
The original Helmintex studies have shown that magnetic microspheres coated with a wide variety of different ligands could be used to purify parasite eggs [11]. Here, we show for the first time that microspheres physically bind to the eggs. The high surface area of the filamentous outer structure of the eggs may be part of the explanation as this large surface area may provide strong overall adhesion from relatively weak interactions. This hypothesis is further supported by the observation that S. japonicum eggs with their additional fibrous matrix bound significantly more microspheres than S. mansoni eggs, which do not have this matrix. However, the distributions of microspheres per egg observed in the binding studies suggest that a fraction of the eggs have very little, if any, binding capacity. Furthermore, it should be noted that the fixation using glutaraldehyde may lead to modified surface characteristics of the eggs. Further studies are required to investigate the impact of fixation on particle binding.
The present study provides the first magnetic characterization of S. mansoni and S. japonicum eggs. We report the discovery of an iron-containing material, presumably iron phosphate located in pores within the eggshell. We provide evidence that Schistosoma eggs are not magnetic enough to move in an applied magnetic field of a similar order of magnitude as the one used in the Helmintex method. We show that magnetic microspheres spontaneously bind to eggs of S. mansoni and, to a greater degree, to S. japonicum. Based on these results we conclude that the conjugation of magnetic microspheres and parasite eggs is mediated not by magnetism but by the surface properties of eggs and microspheres. Systematic fits of Curie's law to the magnetization versus inverse temperature data. Again, ideal paramagnetic behavior can be observed. For S. mansoni the total magnetic moments were approximated to 4.846m B using the Brillouin function and 4.766m B using Curie's law. For S. japonicum the total magnetic moments were approximated to 4.126m B using the Brillouin function and 3.716m B using Curie's law. doi: 10   quantification of the binding of microspheres that have different surface functionalizations to parasite eggs is likely to represent an opportunity to optimize the Helmintex magnetic fractionation method. Previous field studies have indicated that such an optimized Helmintex method may be developed into a new gold standard to validate future rapid diagnostic and molecular methods for Schistosoma detection [25,26,27]. Figure S1 Comparison of the calculated Poisson distribution (red) and measured distribution of microspheres per egg (black) at a microsphere to egg ratio of 100 microspheres per egg. Panel A shows the distribution of the number of microspheres bound to all S. mansoni eggs (including those eggs that had no microspheres bound). Panel B shows the distribution for S. mansoni when the eggs that had no microspheres bound to them were excluded. Panel C shows the distribution of the number of microspheres bound to all S. japonicum eggs (including those eggs that had no microspheres bound). Panel D shows the distribution for S. japonicum when the eggs that had no microspheres bound to them were excluded. (TIF) Figure S2 Comparison of the calculated Poisson distribution (red) and measured distribution of microspheres per egg (black) at a microsphere to egg ratio of 500 microspheres per egg. Panel A shows the distribution of the number of microspheres bound to all S. mansoni eggs (including those eggs that had no microspheres bound). Panel B shows the distribution for S. mansoni when the eggs that had no microspheres bound to them were excluded. Panel C shows the distribution of the number of microspheres bound to all S. japonicum eggs (including those eggs that had no microspheres bound). Panel D shows the distribution for S. japonicum when the eggs that had no microspheres bound to them were excluded. (TIF)

Supporting Information
Video S1 Movement of microsphere-egg conjugates when exposed to a magnetic field of approximately 0.1 T and a field gradient of approximately 35 T/m.

(WMV)
Text S1 Additional information on the statistical analysis of binding of magnetic microspheres to S. mansoni and S. japonicum eggs providing further explanations for figures S1 and S2. (DOC)