Complex magnetic field exposure system for in vitro experiments at intermediate frequencies

In occupational environments, an increasing number of electromagnetic sources emitting complex magnetic field waveforms in the range of intermediate frequencies is present, requiring an accurate exposure risk assessment with both in vitro and in vivo experiments. In this article, an in vitro exposure system able to generate complex magnetic flux density B‐fields, reproducing signals from actual intermediate frequency sources such as magnetic resonance imaging (MRI) scanners, for instance, is developed and validated. The system consists of a magnetic field generation system and an exposure apparatus realized with a couple of square coils. A wide homogeneity (99.9%) volume of 210 × 210 × 110 mm³ was obtained within the coils, with the possibility of simultaneous exposure of a large number of standard Petri dishes. The system is able to process any numerical input sequence through a filtering technique aimed at compensating the coils' impedance effect. The B‐field, measured in proximity to a 1.5 T MRI bore during a typical examination, was excellently reproduced (cross‐correlation index of 0.99). Thus, it confirms the ability of the proposed setup to accurately simulate complex waveforms in the intermediate frequency band. Suitable field levels were also attained. Moreover, a dosimetry index based on the weighted‐peak method was evaluated considering the induced E‐field on a Petri dish exposed to the reproduced complex B‐field. The weighted‐peak index was equal to 0.028 for the induced E‐field, indicating an exposure level compliant with the basic restrictions of the International Commission on Non‐Ionizing Radiation Protection. Bioelectromagnetics 34:211–219, 2013. © 2012 Wiley Periodicals, Inc.     

Current density in a model of a human body with a conductive implant exposed to ELF electric and magnetic fields

A numerical model of a human body with an intramedullary nail in the femur was built to evaluate the effects of the implant on the current density distribution in extremely low frequency electric and magnetic fields. The intramedullary nail was chosen because it is one of the longest high conductive implants used in the human body. As such it is expected to alter the electric and magnetic fields significantly. The exposure was a simultaneous combination of inferior to superior electric field and posterior to anterior magnetic field both alternating at 50 Hz with the values corresponding to the ICNIRP reference levels: 5000 V m^?1 for electric field and 100 µT for magnetic flux density. The calculated current density distribution inside the model was compared to the ICNIRP basic restrictions for general public (2 mA m^?2). The results show that the implant significantly increases the current density up to 9.5 mA m^?2 in the region where it is in contact with soft tissue in the model with the implant in comparison to 0.9 mA m^?2 in the model without the implant. As demonstrated the ICNIRP basic restrictions are exceeded in a limited volume of the tissue in spite of the compliance with the ICNIRP reference levels for general public, meaning that the existing safety limits do not necessarily protect implanted persons to the same extent as they protect people without implants. Bioelectromagnetics 30:591–599, 2009. © 2009 Wiley‐Liss, Inc.     

Development and evaluation of intermediate frequency magnetic field exposure system for studies of in vitro biological effects

We have developed an intermediate frequency (IF) magnetic field exposure system for in vitro studies. Since there are no previous studies on exposure to heating-frequency magnetic fields generated from an induction heating (IH) cook top, there is a strong need for such an exposure system and for biological studies of IF magnetic fields. This system mainly consists of a magnetic-field-generating coil housed inside an incubator, inside which cultured cells can be exposed to magnetic field. Two systems were prepared to allow the experiment to be conducted in a double-blind manner. The level of the generated magnetic field was set to 532 microT rms in the exposure space, 23 kHz, 80 times the value in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines, with a spatial field uniformity better than 3.8%. The waveforms were nearly sinusoidal. It was also confirmed that the parasitic electric field was 157 V/m rms and the induced electric field was 1.9 V/m rms. The temperature was maintained at 36.5 +/- 0.5 degrees C for 2 h. Furthermore, leaked magnetic flux density was 0.7 microT rms or lower at extremely low frequency (ELF) and IF in the stopped system when the other system was being operated, and the environmental magnetic flux density was 0.1 microT rms or lower at the center of the coils. As a result, it was confirmed that this system could be successfully used to evaluate the biological effects of exposure to IF magnetic fields.     

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1–42 in 1,1,1,3,3,3-hexafluoro-2-propanol

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aβ) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aβ peptides may be complicated. To understand the mechanisms of Aβ(1–42) oligomer formation in more detail, we have established a method to produce ¹⁵N-labeled Aβ(1–42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aβ aggregates must be removed. Here, we succeeded in fractionating a “precipitation-resistant” fraction of Aβ(1–42) from an “aggregation-prone” fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aβ(1–42). However, both Aβ(1–42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the “aggregation seed” was not completely monomerized during HFIP treatment. In addition, Aβ(1–42) dissolved in HFIP was found to display a monomer–dimer equilibrium, as shown by two-dimensional ¹H–¹⁵N NMR. We demonstrated that the initial concentration of Aβ during the HFIP pretreatment altered the kinetic profiles of Aβ fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aβ(1–42) peptide.     

Induced movements of giant vesicles by millimeter wave radiation

Our previous study of interaction between low intensity radiation at 53.37 GHz and cell-size system – such as giant vesicles – indicated that a vectorial movement of vesicles was induced. This effect among others, i.e. elongation, induced diffusion of fluorescent dye di-8-ANEPPS, and increased attractions between vesicles was attributed to the action of the field on charged and dipolar residues located at the membrane–water interface. In an attempt to improve the understanding on how millimeter wave radiation (MMW) can induce this movement we report here a real time evaluation of changes induced on the movement of giant vesicles. Direct optical observations of vesicles subjected to irradiation enabled the monitoring in real time of the response of vesicles. Changes of the direction of vesicle movement are demonstrated, which occur only during irradiation with a “switch on” of the effect. This MMW-induced effect was observed at a larger extent on giant vesicles prepared with negatively charged phospholipids. The monitoring of induced-by-irradiation temperature variation and numerical dosimetry indicate that the observed effects in vesicle movement cannot be attributed to local heating.     

The establishment of frequency dependent limits for electric and magnetic fields and evaluation of indirect effects

Summary The biophysical model described in this paper has been used as basis for the preparation of the German standards which determine and define limits of exposure to electric or magnetic fields below several MHz, including 50/60 Hz. The electric field strength within the tissue is considered decisive for the biological effect in the low frequency range. Threshold values of field strengths and current densities including biological effects are compared. It is possible to establish safe, dangerous and hazardous current density curves as a function of frequency. To define exposure limits, the field strength or current density causing injury should be reduced by a factor exceeding 100 in order to avoid well established biological effects. The electric and magnetic field strengths in the human environment are correlated with the corresponding electric current density induced in the human body. This enables safe, dangerous and hazardous levels of current density in the human body to be correlated with the external electric or magnetic field strength. Additionally to the direct field effects indirect effects must be considered. In the second part of this paper data on touch voltages and currents are summarized and evaluated with regard to their health risk. Furthermore, as an example for indirect effects the interference of electric and magnetic fields with pacemakers is considered.Review article by invitation of the editor     

Linkage rules for plant-pollinator networks: trait complementarity or exploitation barriers?

Recent attempts to examine the biological processes responsible for the general characteristics of mutualistic networks focus on two types of explanations: nonmatching biological attributes of species that prevent the occurrence of certain interactions (“forbidden links”), arising from trait complementarity in mutualist networks (as compared to barriers to exploitation in antagonistic ones), and random interactions among individuals that are proportional to their abundances in the observed community (“neutrality hypothesis”). We explored the consequences that simple linkage rules based on the first two hypotheses (complementarity of traits versus barriers to exploitation) had on the topology of plant–pollination networks. Independent of the linkage rules used, the inclusion of a small set of traits (two to four) sufficed to account for the complex topological patterns observed in real-world networks. Optimal performance was achieved by a “mixed model” that combined rules that link plants and pollinators whose trait ranges overlap (“complementarity models”) and rules that link pollinators to flowers whose traits are below a pollinator-specific barrier value (“barrier models”). Deterrence of floral parasites (barrier model) is therefore at least as important as increasing pollination efficiency (complementarity model) in the evolutionary shaping of plant–pollinator networks.     

Characterization of monoclonal antibodies by a fast and easy liquid chromatography–mass spectrometry time-of-flight analysis on culture supernatant

Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain mass spectrometry profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates three fragments of 25 kDa that can be analyzed by liquid chromatography–mass spectrometry time-of-flight (LC–MS TOF) in one run: LC, Fd, and Fc/2. This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process was successfully compared with high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE–LIF), currently considered as the “gold standard” method.     

Comparison of three local frame definitions for the kinematic analysis of the fingers and the wrist

Because the hand is a complex poly-articular limb, numerous methods have been proposed to investigate its kinematics therefore complicating the comparison between studies and the methodological choices. With the objective of overcoming such issues, the present study compared the effect of three local frame definitions on local axis orientations and joint angles of the fingers and the wrist. Three local frames were implemented for each segment. The “Reference” frames were aligned with global axes during a static neutral posture. The “Landmark” frames were computed using palpated bony landmarks. The “Functional” frames included a flexion–extension axis estimated during functional movements. These definitions were compared with regard to the deviations between obtained local segment axes and the evolution of joint (Cardan) angles during two test motions. Each definition resulted in specific local frame orientations with deviations of 15° in average for a given local axis. Interestingly, these deviations produced only slight differences (below 7°) regarding flexion–extension Cardan angles indicating that there is no preferred method when only interested in finger flexion–extension movements. In this case, the Reference method was the easiest to implement, but did not provide physiological results for the thumb. Using the Functional frames reduced the kinematic cross-talk on the secondary and tertiary Cardan angles by up to 20° indicating that the Functional definition is useful when investigating complex three-dimensional movements. Globally, the Landmark definition provides valuable results and, contrary to the other definitions, is applicable for finger deformities or compromised joint rotations.     

Magnetic field from spot welding equipment—is the basic restriction exceeded?

A point measurement of the magnetic field (MF) near a typical spot welding machine showed that the magnetic flux density was above the ICNIRP reference level. To investigate if the basic restrictions were exceeded, the induced body currents in a full 3D human model were calculated. It was found that at an operator position of 34 cm away from the machine the maximum induced current density was below the ICNIRP basic restriction. But if the operator was closer to the machine, the basic restriction was exceeded. An important finding of the present article is that the basic restriction can be exceeded although the MF spatially averaged over the whole body is well below the reference level.     

Seed in soil,with an epigenetic view

It is becoming increasingly evident that discrete genetic alterations in neoplastic cells alone cannot explain multistep carcinogenesis whereby tumor cells are able to express diverse phenotypes during the complex phases of tumor development and progression. The epigenetic model posits that the host microenvironment exerts an initial, inhibitory constraint on tumor growth that is followed by acceleration of tumor progression through complex cell–matrix interactions. This review emphasizes the epigenetic aspects of breast cancer development in light of such interactions between epithelial cells (“seed”) and the tumor microenvironment (“soil”). Our recent research findings suggest that epigenetic perturbations induced by the tumor microenvironment may play a causal role in promoting breast cancer development. It is believed that abrogation of these initiators could offer a promising therapeutic strategy.     

A preliminary chemotaxonomic study on the condensed tannins of green banana flesh in the Musa genus

Sixty percent acetone-soluble tannins were obtained from the flesh of 29 accessions of green bananas and plantains belonging to the AA, AB, AAA, AAB, and ABB genotypes. Their tannin contents were between 0.3 and 2.1% of fresh matter, except for the Pacific plantains (“Maoli” and “Pōpō’ulu” sub-groups) which were tannin-free. (−)-Epigallocatechin (EGC) was their major constituent (88–98%) followed by minor proportions of (−)-epicatechin (EC) (2–12%). Hierarchical average linkage clustering analysis using (EGC/EC) as the sole variable agglomerated all plantains (AAB), while the “Pisang Lilin” cultivar (AA, “Pisang Lilin” sub-group) was separated from the other AA diploids. Finally (EGC/EC) seemed to be independent of agropedoclimatic conditions of banana growth.     

Modeling bacterial immune systems: Strategies for expression of toxic – but useful – molecules

Protection of bacterial cells against virus infection requires expression of molecules that are able to destroy the incoming foreign DNA. However, these molecules can also be toxic for the host cell. In both restriction–modification (R–M), and the recently discovered CRISPR/Cas systems, the toxicity is (in part) avoided through rapid transition of the expression of the toxic molecules from “OFF” to “ON” state. In restriction–modification systems the rapid transition is achieved through a large binding cooperativity, and low translation rate of the control protein. On the other hand, CRISPR array expression in CRISPR/Cas systems involves a mechanism where a small decrease of unprocessed RNAs leads to a rapid increase of processed small RNAs. Surprisingly, this rapid amplification crucially depends on fast non-specific degradation of the unprocessed molecules by an unidentified nuclease, rather than on large cooperativity in protein binding. Furthermore, the major control elements that are responsible for fast transition of R–M and CRISPR/Cas systems from “OFF” to “ON” state, are also directly involved in increased stability of the steady states of these systems. We here discuss mechanisms that allow rapid transition of toxic molecules from the unproductive to the productive state in R–M and CRISPR/Cas systems. The main purpose of this discussion is to put relevant theoretical and experimental work in a perspective that points to general similarities in otherwise mechanistically very different bacterial immune systems.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

Codon sextets with leading role of serine create “ideal” symmetry classification scheme of the genetic code

The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator.     

Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system

Extremely low levels of “leaky” expression of genes in bacterial protein expression systems can severely curtail cell viability when expressed proteins are toxic. A general method for sensitive detection of such expression is lacking. Here, we present a method based on microscopic visualization of a fluorescent “reporter” protein (RFP–HU-A) constructed by fusing red fluorescent protein (RFP) to the N-terminus of a nucleoid-associated, histone-like DNA-binding protein, HU-A. Localization of RFP–HU-A within nucleoids facilitates detection, quantitation, and characterization of leaky expression at the single-cell level.     

An activity-based probe for high-throughput measurements of triacylglycerol lipases

Modulating the activity of lipases involved in the metabolism of plasma lipoproteins is an attractive approach for developing lipid raising/lowering therapies to treat cardiovascular disease. Identifying small molecule inhibitors for these membrane-active enzymes, however, is complicated by difficulties associated with measuring lipase activity and inhibition at the water–membrane interface; substrate and compound dynamics at the particle interface have the potential to confound data interpretation. Here, we describe a novel ELISA-based lipase activity assay that employs as “bait” a biotinylated active-site probe that irreversibly binds to the catalytic active-site serine of members of the triacylglycerol lipase family (hepatic lipase, lipoprotein lipase, and endothelial lipase) in solution with high affinity. Detection of “captured” (probe–enzyme) complexes on streptavidin-coated plates using labeled secondary antibodies to specific primary antibodies offers several advantages over conventional assays, including the ability to eliminate enzyme–particle and compound–particle effects; specifically measure lipase activity in complex mixtures in vitro; preferentially identify active-site-directed inhibitors; and distinguish between reversible and irreversible inhibitors through a simple assay modification. Using EL as an exemplar, we demonstrate the versatility of this assay both for high-throughput screening and for compound mechanism-of-action studies.     

Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention.     

“Turn off–on” phosphorescent biosensors for detection of DNA based on quantum dots/acridine orange

A “turn off–on” switch mode was established by using the interaction between acridine orange (AO) and DNA as an input signal and using the room temperature phosphorescence (RTP) reversible change of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs) as an output signal in biological fluids. AO was absorbed into the surface of Mn-doped ZnS QDs via electrostatic attraction and, thus, formed a ground-state complex through photoinduced electron transfer (PIET). This complex quenched the phosphorescence of Mn-doped ZnS QDs and then rendered the system into the “turn-off” mode. Along with the addition of DNA and embedded binding with DNA, AO was competitively induced to fall off from the surface of Mn-doped ZnS QDs and embed into the double helix structure of DNA. As a result, the RTP of Mn-doped ZnS QDs was recovered and the system consequently was rendered into “turn-on” mode. In this case, a new biosensor for DNA detection was built and has a detection limit of 0.033 mg L⁻¹ and a detection range from 0.033 to 20 mg L⁻¹. What is more, this kind of biosensor does not require complex pretreatments and is free from the interference from autofluorescence and scattering light. Thus, this biosensor can be used to detect DNA in biological fluids.     

Magnetic Navigation

Recent evidence suggests that some amphibians, reptiles and birds may be capable of homing using information about geographic position (“map” information) derived from subtle geographic gradients in the earth's magnetic field. The “magnetic map” hypothesis faces numerous theoretical difficulties, however, due to the extremely high level of sensitivity that would be necessary to detect natural magnetic gradients, and to the presence of spatial irregularities and temporal variation in the geomagnetic field that might make map coordinates derived from magnetic gradients unreliable. To date, the majority of studies carried out to test the magnetic map hypothesis have involved field observations of the effects on homing orientation of naturally occurring spatial or temporal variation in the geomagnetic field. While providing an important first step, these studies are subject to the criticism that the observed changes in homing orientation could result from effects on a magnetic compass, or some other unidentified component of the navigational system, rather than from effects on a magnetic map. The recent development of experimental systems in which navigational ability can be studied under controlled or semi-controlled laboratory conditions has opened up the possibility of using new experimental approaches to more rigorously test the magnetic map hypothesis. After briefly reviewing the available evidence of the geomagnetic field's involvement in the map component of homing, a simple graphical model is presented which describes how the home direction derived from a bicoordinate map varies as a function of the value of one of the map coordinates when the value of the second map coordinate is held constant. In studies of homing orientation in which the value of a specific magnetic field parameter (e.g., total intensity, inclination, etc.) can be varied independently of other putative map parameters, the graphical model can be used to generate qualitative predictions about the changes in the direction of homing orientation that should be observed if the magnetic field parameter being manipulated serves as one coordinate of a bicoordinate map. The relationship between the direction of homing orientation and the value of a putative magnetic map parameter can also be used to generate quantitative predictions about characteristics of the local gradient of that magnetic field parameter in the vicinity of the home site (i.e., the alignment and “home value” of the local gradient) which can then be compared with actual measured values. Together, the qualitative and quantitative predictions of the graphical model permit rigorous tests of whether one or both coordinates of a bicoordinate navigational map are derived from the geomagnetic field.