Effects of static magnetic field on magnetosome formation and expression of mamA,mms13, mms6 and magA in Magnetospirillum magneticum AMB‐1

Magnetotactic bacteria produce nanometer‐size intracellular magnetic crystals. The superior crystalline and magnetic properties of magnetosomes have been attracting much interest in medical applications. To investigate effects of intense static magnetic field on magnetosome formation in Magnetospirillum magneticum AMB‐1, cultures inoculated with either magnetic or non‐magnetic pre‐cultures were incubated under 0.2 T static magnetic field or geomagnetic field. The results showed that static magnetic field could impair the cellular growth and raise C_mag values of the cultures, which means that the percentage of magnetosome‐containing bacteria was increased. Static magnetic field exposure also caused an increased number of magnetic particles per cell, which could contribute to the increased cellular magnetism. The iron depletion in medium was slightly increased after static magnetic field exposure. The linearity of magnetosome chain was also affected by static magnetic field. Moreover, the applied intense magnetic field up‐regulated mamA, mms13, magA expression when cultures were inoculated with magnetic cells, and mms13 expression in cultures inoculated with non‐magnetic cells. The results implied that the interaction of the magnetic field created by magnetosomes in AMB‐1 was affected by the imposed magnetic field. The applied static magnetic field could affect the formation of magnetic crystals and the arrangement of the neighboring magnetosome. Bioelectromagnetics 30:313–321, 2009. © 2009 Wiley‐Liss, Inc.     

Static magnetic field exposure fails to affect the viability of different bacteria strains

The viability of the microbes Saccharomyces cerevisiae, Bacillus circulans, Escherichia coli, Micrococcus luteus, Pseudomonas fluorescens, Salmonella enteritidis, Serratia marcescens, and Staphylococcus aureus was tested under static magnetic field exposure up to 24 h in either a homogeneous (159.2 ± 13.4 mT) or three types of inhomogeneous static magnetic fields: (i) peak‐to‐peak magnetic flux density 476.7 ± 0.1 mT with a lateral magnetic flux density gradient of 47.7 T/m, (ii) 12.0 ± 0.1 mT with 1.2 T/m, or (iii) 2.8 ± 0.1 mT with 0.3 T/m. Even the longest period of exposure failed to produce any effect in the growth of bacteriae that could be correlated with static magnetic field exposure. Bioelectromagnetics 31:220–225, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of static magnetic fields at the cellular level

There have been few studies on the effects of static magnetic fields at the cellular level, compared to those of extremely low frequency magnetic fields. Past studies have shown that a static magnetic field alone does not have a lethal effect on the basic properties of cell growth and survival under normal culture conditions, regardless of the magnetic density. Most but not all studies have also suggested that a static magnetic field has no effect on changes in cell growth rate. It has also been shown that cell cycle distribution is not influenced by extremely strong static magnetic fields (up to a maximum of 10 T). A further area of interest is whether static magnetic fields cause DNA damage, which can be evaluated by determination of the frequency of micronucleus formation. The presence or absence of such micronuclei can confirm whether a particular treatment damages cellular DNA. This method has been used to confirm that a static magnetic field alone has no such effect. However, the frequency of micronucleus formation increases significantly when certain treatments (e.g., X-irradiation) are given prior to exposure to a 10 T static magnetic field. It has also been reported that treatment with trace amounts of ferrous ions in the cell culture medium and exposure to a static magnetic field increases DNA damage, which is detected using the comet assay. In addition, many studies have found a strong magnetic field that can induce orientation phenomena in cell culture.     

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real‐time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real‐time. Two heat parameters were considered in combination with sham‐ and 100 mT‐exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult. J. Cell. Biochem. 108: 956–962, 2009. © 2009 Wiley‐Liss, Inc.     

Hypothesis: The risk of childhood leukemia is related to combinations of power-frequency and static magnetic fields

We present a hypothesis that the risk of childhood leukemia is related to exposure to specific combinations of static and extremely-low-frequency (ELF) magnetic fields. Laboratory data from calcium efflux and diatom mobility experiments were used with the gyromagnetic equation to predict combinations of 60 Hz and static magnetic fields hypothesized to enhance leukemia risk. The laboratory data predicted 19 bands of the static field magnitude with a bandwidth of 9.1 μT that, together with 60 Hz magnetic fields, are expected to have biological activity. We then assessed the association between this exposure metric and childhood leukemia using data from a case-control study in Los Angeles County. ELF and static magnetic fields were measured in the bedrooms of 124 cases determined from a tumor registry and 99 controls drawn from friends and random digit dialing. Among these subjects, 26 cases and 20 controls were exposed to static magnetic fields lying in the predicted bands of biological activity centered at 38.0 μT and 50.6 μT. Although no association was found for childhood leukemia in relation to measured ELF or static magnetic fields alone, an increasing trend of leukemia risk with measured ELF fields was found for subjects within these static field bands (P for trend = 0.041). The odds ratio (OR) was 3.3 [95% confidence interval (CI) = 0.4–30.5] for subjects exposed to static fields within the derived bands and to ELF magnetic field above 0.30 μT (compared to subjects exposed to static fields outside the bands and ELF magnetic fields below 0.07 μT). When the 60 Hz magnetic fields were assessed according to the Wertheimer-Leeper code for wiring configurations, leukemia risks were again greater with the hypothesized exposure conditions (OR = 9.2 for very high current configurations within the static field bands: 95% CI = 1.3–64.6). Although the risk estimates are based on limited magnetic field measurements for a small number of subjects, these findings suggest that the risk of childhood leukemia may be related to the combined effects of the static and ELF magnetic fields. Further tests of the hypothesis are proposed. © 1995 Wiley-Liss, Inc.     

Effects of pulsed magnetic field on the formation of magnetosomes in the Magnetospirillum sp. strain AMB‐1

Magnetotactic bacteria are a diverse group of microorganisms which possess one or more chains of magnetosomes and are endowed with the ability to use geomagnetic fields for direction sensing, thus providing a simple and excellent model for the study of magnetite‐based magnetoreception. In this study, a 50 Hz, 2 mT pulsed magnetic field (PMF) was applied to study the effects on the formation of magnetosomes in Magnetospirillum sp. strain AMB‐1. The results showed that the cellular magnetism (R_mag) of AMB‐1 culture significantly increased while the growth of cells remained unaffected after exposure. The number of magnetic particles per cell was enhanced by about 15% and slightly increased ratios of magnetic particles of superparamagnetic property (size <20 nm) and mature magnetosomes (size >50 nm) were observed after exposure to PMF. In addition, the intracellular iron accumulation slightly increased after PMF exposure. Therefore, it was concluded that 50 Hz, 2 mT PMF enhances the formation of magnetosomes in Magnetospirillum sp. strain AMB‐1. Our results suggested that lower strength of PMF has no significant effects on the bacterial cell morphologies but could affect crystallization process of magnetosomes to some extent. Bioelectromagnetics 31:246–251, 2010. © 2009 Wiley‐Liss, Inc.     

Magnetic field direction differentially impacts the growth of different cell types

Magnetic resonance imaging (MRI) machines have horizontal or upright static magnetic field (SMF) of 0.1–3 T (Tesla) at sites of patients and operators, but the biological effects of these SMFs still remain elusive. We examined 12 different cell lines, including 5 human solid tumor cell lines, 2 human leukemia cell lines and 4 human non-cancer cell lines, as well as the Chinese hamster ovary cell line. Permanent magnets were used to provide 0.2–1 T SMFs with different magnetic field directions. We found that an upward magnetic field of 0.2–1 T could effectively reduce the cell numbers of all human solid tumor cell lines we tested, but a downward magnetic field mostly had no statistically significant effect. However, the leukemia cells in suspension, which do not have shape-induced anisotropy, were inhibited by both upward and downward magnetic fields. In contrast, the cell numbers of most non-cancer cells were not affected by magnetic fields of all directions. Moreover, the upward magnetic field inhibited GIST-T1 tumor growth in nude mice by 19.3% (p < 0.05) while the downward magnetic field did not produce significant effect. In conclusion, although still lack of mechanistical insights, our results show that different magnetic field directions produce divergent effects on cancer cell numbers as well as tumor growth in mice. This not only verified the safety of SMF exposure related to current MRI machines but also revealed the possible antitumor potential of magnetic field with an upward direction.     

Combined effect of X‐ray radiation and static magnetic fields on reactive oxygen species in rat lymphocytes in vitro

The aim of this study was to investigate the effect of static magnetic fields (SMF) on reactive oxygen species induced by X‐ray radiation. The experiments were performed on lymphocytes from male albino Wistar rats. After exposure to 3 Gy X‐ray radiation (with a dose rate of 560 mGy/min) the measurement of intracellular reactive oxygen species in lymphocytes, using a fluorescent probe, was done before exposure to the SMF, and after 15 min, 1 and 2 h of exposure to the SMF or a corresponding incubation time. For SMF exposure, 0 mT (50 µT magnetic field induction opposite to the geomagnetic field) and 5 mT fields were chosen. The trend of SMF effects for 0 mT was always opposite that of 5 mT. The first one decreased the rate of fluorescence change, while the latter one increased it. Bioelectromagnetics 34:333–336, 2013. © 2012 Wiley Periodicals, Inc.     

Exposure to strong static magnetic field slows the growth of human cancer cells in vitro

Proposals to enhance the amount of radiation dose delivered to small tumors with radioimmunotherapy by constraining emitted electrons with very strong homogeneous static magnetic fields has renewed interest in the cellular effects of prolonged exposures to such fields. Past investigations have not studied the effects on tumor cell growth of lengthy exposures to very high magnetic fields. Three malignant human cell lines, HTB 63 (melanoma), HTB 77 IP3 (ovarian carcinoma), and CCL 86 (lymphoma; Raji cells), were exposed to a 7 Tesla uniform static magnetic field for 64 hours. Following exposure, the number of viable cells in each group was determined. In addition, multicycle flow cytometry was performed on all cell lines, and pulsed-field electrophoresis was performed solely on Raji cells to investigate changes in cell cycle patterns and the possibility of DNA fragmentation induced by the magnetic field. A 64 h exposure to the magnetic field produced a reduction in viable cell number in each of the three cell lines. Reductions of 19.04 ± 7.32%, 22.06 ± 6.19%, and 40.68 ± 8.31% were measured for the melanoma, ovarian carcinoma, and lymphoma cell lines, respectively, vs. control groups not exposed to the magnetic field. Multicycle flow cytometry revealed that the cell cycle was largely unaltered. Pulsed-field electrophoresis analysis revealed no increase in DNA breaks related to magnetic field exposure. In conclusion, prolonged exposure to a very strong magnetic field appeared to inhibit the growth of three human tumor cell lines in vitro. The mechanism underlying this effect has not, as yet, been identified, although alteration of cell growth cycle and gross fragmentation of DNA have been excluded as possible contributory factors. Future investigations of this phenomenon may have a significant impact on the future understanding and treatment of cancer. © 1996 Wiley-Liss, Inc.     

0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane

Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved.     

Design,construction, and validation of a large capacity rodent magnetic field exposure laboratory

A magnetic field exposure laboratory has been constructed to support National Toxicology Program studies for the evaluation of the toxicity and carcinogenicity of pure, linearly polarized, 60 Hz magnetic fields in rodents. This dual corridor, controlled access facility can support the simultaneous exposure of 1200 rats and 1200 mice. The facility contains fully redundant electrical and environmental control systems and was constructed using non‐metallic materials to maintain low levels of background (ambient), stray, and cross‐talk magnetic fields. The exposure module design provides for large uniform exposure volumes with good control of stray and cross‐talk fields, while allowing the use of roll‐around cage racks for simplified animal husbandry. Stray fields and cross‐talk have been further reduced by the inclusion of “steering coils” in each exposure module. Ambient 60 Hz fields (less cross‐talk) in all exposure rooms are <0.1 μT (1 mG), and static magnetic fields have been mapped extensively. Magnetic field strength, waveform, temperature, relative humidity, light intensity, noise level, vibration, and air flow in all animal holding areas are tightly regulated, and are monitored continuously during all studies. Field uniformity in the animal exposure volumes is better than ±10%; a systematic program of cage, rack, and room rotation controls for possible positional effects within the exposure system. Magnetic fields are turned on and off over multiple cycles to prevent the induction of transients associated with abrupt field level changes. Total harmonic distortion is <3% at all field strengths. The facility has been used to study magnetic field bioeffects in rodent model systems in experiments ranging in duration from 8 weeks to 2 years. Bioelectromagnetics 20:13–23, 1999. © 1999 Wiley‐Liss, Inc.     

Magnetosome Formation and Expression of mamA, mms13, mms6 and magA in Magnetospirillum magneticum AMB-1 Exposed to Pulsed Magnetic Field

To investigate the effects of pulsed magnetic field on magnetosome formation in Magnetospirillum magneticum AMB-1, cultures inoculated with either mangetic or non-magnetic pre-cultures were incubated under 1 mT pulsed magnetic field. Magnetism of cells was measured by using spectrophotometer coupled with applied magnetic fields and the values were described as C _mag. Magnetosome in cells was counted by transmission electron microscopy observation. The results showed that pulsed magnetic field did not affect cellular growth, but enhanced magnetosome formation. The applied pulsed magnetic field might exceed the chain of magnetosomes and change the homogeneity of the magnetosome particles. The results implied that magnetite precipitation induced by the adjacent magnetosome was affected by pulsed magnetic field. Moreover, the applied pulsed magnetic field up-regulated the magA and mamA expression in cells, which might account for the increasing number and the exceeding chain of magnetosomes in cells.     

Magnetic field effects on pineal indoleamine metabolism and possible biological consequences.

In recent years, there has been a great deal of publicity concerning the possible health effects of electric and/or magnetic field exposure. One of the most frequently reported observations after the exposure of animals to either electric or magnetic fields relates to alterations in the metabolism of serotonin (5HT) to melatonin within the pineal gland. This review summarizes these results particularly in animals exposed to intermittently inverted, non-time varying magnetic fields, i.e., pulsed static magnetic fields. When exposure occurs at night, the conversation of 5HT to melatonin is typically depressed, not unlike that after light exposure at night. The mechanisms by which pulsed magnetic fields alter the ability of the pineal to convert 5HT to the chief pineal hormone melatonin remains unknown but may involve effects on any or all of the following: the retinas, the suprachiasmatic nuclei, the peripheral sympathetic nervous system, and the pinealocytes. Results to date suggest that induced electrical currents (eddy currents) produced by the pulsed magnetic fields are particularly detrimental to pineal indoleamine metabolism and may be an important causative factor in the metabolic changes measured. The physiological consequences of perturbations in the melatonin rhythm induced by magnetic field exposure remain unknown.     

ENHANCEMENT OF INVERTASE PRODUCTION BY Aspergillus niger OZ-3 USING LOW-INTENSITY STATIC MAGNETIC FIELDS

The aim of this study is to investigate the effect of low-intensity static magnetic fields (SMFs) on invertase activity and growth on different newly identified molds. The most positive effect of SMFs on invertase activity and growth was observed for Aspergillus niger OZ-3. The submerged production of invertase was performed with the spores obtained at the different exposure times (120, 144, 168, and 196 hr) and magnetic field intensities (0.45, 3, 5, 7, and 9 mT). The normal magnetic field of the laboratory was assayed as 0.45 mT (control). Optimization of magnetic field intensity and exposure time significantly increased biomass production and invertase activity compared to 0.45 mT. The maximum invertase activity (51.14 U/mL) and biomass concentration (4.36 g/L) were achieved with the spores obtained at the 144 hr exposure time and 5 mT magnetic field intensity. The effect of low-intensity static magnetic fields (SMFs) on invertase activities of molds was investigated for the first time in the present study. As an additional contribution, a new hyper-invertase-producing mold strain was isolated.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Influence of weak static and 50 Hz magnetic fields on the redox activity of cytochrome-C oxidase

The effects of static and 50 Hz magnetic fields on cytochrome-C oxidase activity were investigated in vitro by strictly controlled, simultaneous polarographic measurements of the enzyme's high- and low-affinity redox reaction. Cytochrome-C oxidase was isolated from beef heart. Control experiments were carried out in the ambient geomagnetic and 50 Hz magnetic fields at respective flux densities of 45 and 1.8 μT. The experimentally applied fields, static and time-varying, were generated by Helmholtz coils at flux densities between 50 μT and 100 mT. Exposures were timed to act either on the combined enzyme-substrate interchange or directly on the enzyme's electron and proton translo-cations. Significant changes as high as 90% of the overall cytochrome-C oxidase activity resulted during exposure (1) to a static magnetic field at 300 μT or 10 mT in the high-affinity range, and (2) to a 50 Hz magnetic field at 10 or 50 mT in the low-affinity range. No changes were observed at other flux densities. After exposure to a change-inducing, static or time-varying field, normal activity returned. © 1993 Wiley-Liss. Inc.     

Experimental studies on extremely low frequency pulsed magnetic field inhibiting sarcoma and enhancing cellular immune functions

The previous observation with an electron microscope showed that extremely low frequency (ELF) pulsed magnetic field (PMF) (with the maximum intensity of 0. 6-2. 0 T, gradient of 10-100 T. M-1, pulse width of 20-200 ms and frequency of 0. 16-1. 34 Hz) inhibited the growth of S-180 sarcoma in mice and enhanced the ability of immune cell's dissolving sarcoma cells. In this study, the DNA contents of nuclei were assayed by using Faulgen Staining method. With an electron microscope and cell stereoscopy technology it was observed that magnetic field affected the sarcoma cell's metabolism, lowered its malignancy, and restrained its rapid and heteromorphic growth. The magnetic field enhanced the cellular immune ability and the reaction of lymphocytes and plasma. Since ELF pulsed magnetic fields can inhibit the growth of sarcomas and enhance the cellular immune ability, it is possible to use it as a new method to treat cancer.     

Chick embryo development can be irreversibly altered by early exposure to weak extremely-low-frequency magnetic fields

Several reports have shown that weak, extremely-low-frequency (ELF), pulsed magnetic fields (PMFs) can adversely affect the early embryonic development of the chick. In this study, freshly fertilized chicken eggs were exposed during the first 48 h of postlaying incubation to PMFs with 100 Hz repetition rate, 1.0 μT peak-to-peak amplitude, and 500 μs pulse duration. Two different pulse waveforms were used, having rise and fall times of 85 μs (PMF-A) or 2.1 μs (PMF-B). It has been reported that, with 2 day exposure, these fields significantly increase the proportion of developmental abnormalities. In the present study, following exposure, the eggs were allowed to incubate for an additional 9 days in the absence of the PMFs. The embryos were taken out of the eggs and studied blind. Each of the two PMF-exposed groups showed an excess in the percentage of developmental anomalies compared with the respective sham-exposed samples. This excess of anomalies was not significant for the PMF-A-treated embryos (P = 0.173), whereas it was significant for the PMF-B-exposed group (P = 0.007), which showed a particularly high rate of early embryonic death. These results reveal that PMFs can induce irreversible developmental alterations and confirm that the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields. The data also validate previous work based on the study of PMFs' effects at day 2 of embryonic development under field exposure. © 1994 Wiley-Liss, Inc.     

Teratological evaluation of mouse fetuses exposed to a 20 kHz EMF

As a continuation of our previous study, we performed a teratological evaluation of the importance of gestational age with regards to the exposure of 20 kHz intermediate frequency magnetic field (IF) on pregnant ICR mice. The pregnant mice were exposed to a 20 kHz IF magnetic field for 8 h/day in a carousel irradiator at 30 µT which is the limit standard for occupational population in Korea. The animals were sacrificed on the 18th day of gestation and the fetuses were examined for mortality, growth retardation, changes in head size and other morphological abnormalities. We concluded that exposure to 30 µT with 20 kHz IF did not cause any observable adverse effects on mouse fetuses. Bioelectromagnetics 30:330–333. © 2009 Wiley‐Liss, Inc.     

Origins of electromagnetic hypersensitivity to 60 Hz magnetic fields: A provocation study

With increasing electrical device usage, social concerns about the possible effects of 60 Hz electromagnetic fields on human health have increased. The number of people with self‐attributed electromagnetic hypersensitivity (EHS) who complain of various subjective symptoms such as headache and insomnia has also increased. However, it is unclear whether EHS results from physiological or other origins. In this double‐blinded study, we simultaneously investigated physiological changes (heart rate, respiration rate, and heart rate variability), subjective symptoms, and perception of the magnetic field to assess origins of the subjective symptoms. Two volunteer groups of 15 self‐reported EHS and 16 non‐EHS individuals were tested with exposure to sham and real (60 Hz, 12.5 µT) magnetic fields for 30 min. Magnetic field exposure did not have any effects on physiological parameters or eight subjective symptoms in either group. There was also no evidence that the EHS group perceived the magnetic field better than the non‐EHS group. In conclusion, the subjective symptoms did not result from the 60 Hz, 12.5 µT magnetic field exposures but from other non‐physiological factors. Bioelectromagnetics 33:326–333, 2012. © 2011 Wiley Periodicals, Inc.     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.