METHOD FOR GENERATING CYTOTOXIC ELECTROMAGNETC SIGNALS
FIELD OF THE INVENTION
The present invention relates to the field of electromagnetic field interaction with biological systems.
BACKGROUND OF THE INVENTION
Each of the references cited herein are expressly incorporated herein by reference, for their teaching of the state of the art, including both techniques and specifics of technology, aspects of the present technology not expressly recited, and support for the written description for the claims presented herein, enablement for persons of ordinary skill in the art to practice the invention, and otherwise to provide disclosure.
The relationship of electromagnetic signals and biological systems is well established, for certain applications. For example, biological cells maintain different concentrations of intracellular ions than in the extracellular environment. This results in an electrical potential, the Nernst potential, at the cell membrane. Cells are able to modulate conductivity through membrane pores or transport proteins, resulting in electrical currents and corresponding magnetic fields. Organs of multicellular organisms typically communicate through electrical signals, and nervous tissue in particular exploits ion conductivity to convey information. The range of these potentials is typically below 100 mV, though in the case of some organisms, such as electric eels, 600V potentials are possible. The frequency of these signals is typically below 3 Hz, though the frequency spectrum may include components several orders of magnitude greater.
It is well known that various molecules, biological and otherwise, have bonds which form and break, with corresponding electrochemical activity. The range of potentials involved in covalent bonds is up to about 20 eV, though when including ionic bonds, the bonding energy extends through zero to negative (repulsive) levels. The frequency (and corresponding wavelength) of an electromagnetic wave corresponds to its energy, and therefore the range of emissions available from chemical environments in the environment ranges from ultraviolet and beyond to ultra-low frequencies, less than 100 Hz.
Disorganized energy emission or absorption represents noise, and such noise in equilibrium has no net emission or absorption of energy. However, above absolute zero temperature, various forms of energy are available, and can be emitted, with a corresponding reduction in
temperature. Even at static temperature, and no net input of energy, a system may emit electromagnetic energy, for example a glowstick (neglecting possible exothermic reactions).
Thus, a simplistic analysis of the laws of thermodynamics to conclude that a system to which appears to be at equilibrium, and which appears to have no net receipt of energy or decrease in temperature, could not appear to emit electromagnetic radiation, is not always accurate and correct. Rather, especially at very low energy levels, one must move beyond analysis of appearances, to a rigorous energy balance including all forms of energy, in order to understand the full system state and transition.
With this in mind, a further appreciation of a large number of reports and analysis of electromagnetic signals emitted from apparently stable systems is available. That is, if one presumes that a measured electromagnetic signal obeys the laws of thermodynamics, then the molecular and chemical interactions within the system can be analyzed as the source of the electromagnetic signal.
There have been some reports of effects of external electromagnetic signals, even those of low frequency, and therefore of "non-ionizing" level, on complex biological systems. For example, there are hypotheses that 60 Hz signals from high tension power lines cause various diseases.
Other reports have suggested that electromagnetic signals, even well below the microwave energy level, can have biological effect. Indeed, there is some recognition by the US Federal Communications Commission that various radio devices must be limited in their radio frequency emissions, to avoid adverse health effects. However, the model employed is generally based on a thermal model, wherein the heating effect of the radio frequency energy on a user is calculated.
However, while the art suggests a range of bio-electromagnetic signal interactions, even at low frequencies, and ultra-low frequencies (e.g., in a range of 1 Hz-1 MHz), the effects are not well understood, and often denied, leading to difficulties in formulating and testing hypotheses and therefore producing useful results.
US patents 6,150,812, and 7,477,053, US patent application 20040222789, and EP 2239586 Al , each of which is expressly incorporated herein by reference, relate to measurement of low frequency electromagnetic signals. The motion of the electrons within a single isolated atom or molecule generates electromagnetic fields which can be detected external to the boundaries of the atom or molecule. The magnitude and frequency of such external fields depends mainly upon the following factors: (i) the angular momentum of the electron as it spins on its axis (=electron spin angular momentum), (ii) the angular momentum of the electron as it moves in
quasicircular orbital paths around the nucleus (=electron orbital momentum), (iii) the quantized energy states of the electron orbital paths and angular spin velocities, (iv) interactions between intraatomic and intramolecular electron motions as governed by Lenz's law, (v) rate of individual transitions between quantized energy states and the frequency of transitional events, (vi) interactions between electron orbital and spin angular moments and nuclear magnetic moments, and (vii) intensity, frequency and direction of externally imposed magnetic fields. Other than bonding interactions, nuclei also have spin, linear and angular momentum, as well as interactions with nearby nuclei and electrons. The electromagnetic fields generated by electron motion within atoms or molecules are accompanied by the simultaneous emission of photons whose energies are characteristic of the frequencies of the associated intraatomically- or intramolecularly-generated external electromagnetic fields. The range of atomic and molecular electromagnetic frequencies extends from microwave and even lower-frequency energies, up to ultraviolet and even higher-frequency energies. Of interest are the lower energy interactions, which are typically in the "conduction" valence band of linked atoms, spins and motions of atoms and molecules, but typically not representing changes in electron states between lower valence states, corresponding to higher energies. It is noted that through resonance and multiple photon capture, relatively lower energy states may be converted into higher ones. In particular, large numbers of interacting atoms, such as water molecules which solvate a biological macromolecule, and in the aggregate possess a significant amount of energy, with no one molecule having high energy. However, under some conditions, there can be a coordinated transfer of energy. Indeed, biological macromolecules in the form of enzymes (catalysts) typically serve to concentrate available energy in the medium at an active site to supply an activation energy to achieve a transition state to permit a biochemical reaction to proceed.
Intraatomic changes in electron position, energy state, acceleration, deceleration, in addition to various interatomic interactions are observable through associated electromagnetic fields.
Detection of low frequency electromagnetic waves and phenomena is facilitated through detection of the associated B field, for example with a conductive loop or coil (solenoid, SQUID, Hal effect sensor), rather than an H field sensor (antenna, microstrip, etc.).
It is generally believed that no net magnetic field at low (e.g., 0 to < 10,000 Hz) frequency can be recorded, except over very short intervals, from macroscopic aggregates of atoms or molecules at rest in their ground state. This is because the magnetic moments of the individual atoms or molecules in such aggregates will on average find orientations whose resultant external magnetic field intensities are for all practical purposes zero. However, if the molecules or their
aggregates are not at rest, are not in their ground state, or are subject to an external coercive agency, then external low frequency signals can be recorded. It is noted that the presumptions of "rest" and ground state are violated above OK, at least to some extent. Further, the presence of chemical energy (high energy bonds and alternate possible bonds of lower energy) and organizations of molecules in other than a highest entropy state, also violate the presumptions required for an absence of low frequency magnetic emissions. Finally, environmental electromagnetic signals, which can be extremely hard to completely filter, and therefore, obtaining a condition that represents an absence of external coercive force is nearly impossible. Therefore, while under "ideal" conditions, one might not expect to see low frequency electromagnetic signals emitted from seemingly homogeneous solutions in which no chemical reaction is apparently occurring, ideal conditions may be difficult to achieve. Electromagnetic signal emissions, therefore may be due to "rare" conditions, such as highly dilute compositions, free radical excited molecules, or the like.
On the other hand, theory does not hold that it is difficult to influence a sample with externally applied electromagnetic fields, and indeed electromagnetic fields are well known to interact with ionic solutions, dipole molecules or nuclei with magnetic moments, etc.
Various studies have been conducted seeking to determine the biological effects of low frequency electromagnetic signals. A review of a portion of the literature reveals that few researchers have carefully considered and controlled for information that may be contained within the low frequency electromagnetic signals, and rather presume that the effect is based on or available from sinusoidal waves or intermittent sinusoidal waves. Even those that consider the source or information contained within the low frequency electromagnetic signals have to date not fully considered resonances, information communication, and implications for informational biopolymers, such as DNA.
C F Blackman, "Treating cancer with amplitude-modulated electromagnetic fields: a potential paradigm shift, again?", British Journal of Cancer (2012) 106, 241-242.
doi:10.1038/bjc.2011.576 www.bjcancer.com discusses various biological effects of electromagnetic fields (EMFs). Barbault et al (2009 infra) describes how they obtained the specific frequencies for different tumor diagnoses, which are then used in the amplitude- modulated (AM)-EMF treatment of those patients to stabilize the disease beyond normal expectations. Costa et al (201 1) reported clinical benefits from using the specific AM-EMF signals to treat advanced hepatocellular carcinoma, stabilizing the disease and even producing partial responses up to 58 months in a subset of the patients. Zimmerman et al have examined
the growth rate of human tumor cell lines from liver and breast cancers along with normal cells from those tissues exposed to AM-EMF. Reduced growth rate was observed for tumor cells exposed to tissue-specific AM-EMF, but no change in growth rate in normal cells derived from the same tissue type, or in tumor or normal cells from the other tissue type. The growth rate inhibitory response was field-strength (SAR) and exposure-time dependent. In ancillary tests, they observed reduction in gene expression and increases in mitotic spindle dysfunction only for the AM-EMF exposure that reduced the cell growth rate. Bawin et al, 1975, with independent replication by Blackman et al, 1979, demonstrated that biological effects could be caused by certain AM frequencies on a carrier wave but not other frequencies. See also Adey, 1992;
Blackman, 1992. This growing collection of reports demonstrating AM-EMF-induced biological effects led to recognition by national and international authorities that this modality needed to be considered in hazard evaluation, in addition to field-induced heating as a cause for health concern. The National Council on Radiation Protection and Measurements (1986) recommended a reduction in the allowable exposure intensity limits for AM radiation above a certain level, and the World Health Organization (1993) explicitly acknowledged AM as a future issue to be examined in setting exposure guidelines. Barbault et al (2009) identifies relevant treatment frequencies can be seen to have direct clinical and medical relevance in determining the characteristics of a new modality that may prove useful in cancer treatment.
Zimmerman et al demonstrates the fundamental requirement for a biological 'information content' code (i.e., the AM spectral profile) that can affect tumor cells from the tissue of origin, while apparently being ignored by normal cells from various tissues and tumor cells from different tissues of origin. By exposing HCC cells to 27.12DMHz RF EMF sinusoidally amplitude-modulated at specific frequencies, which were previously identified in patients with a diagnosis of hepatocellular carcinoma (HCC) (Barbault et al, 2009) and result in therapeutic responses in patients with HCC (Costa et al, 201 1), a robust and sustained anti-proliferative effect was demonstrated. This effect was seen within SARs ranging from 0.03 to l .ODWDkg-1. HCC-specific modulation frequencies began to hinder cell proliferation after 7 days of exposure and the anti-proliferative effect increased over a 7-week period. The anti-proliferative effect HCC-specific modulation frequencies were observed only in HCC cells, but not in breast cancer cells or normal hepatocytes. Two sets of similar modulation frequencies (breast cancer-specific and randomly chosen) within the same range (from 100□ Hz to 21 DkHz) did not affect the proliferation of HCC cells. Similarly, the proliferation of breast cancer cells was affected only by breast cancer-specific modulation frequencies, but neither by HCC-specific nor by randomly
chosen modulation frequencies.
Modulation of the signal appears to be a factor in the response of biological systems to electromagnetic fields (Blackmail, 2009). The amount of electromagnetic energy delivered is far too low to break chemical bonds or cause thermal effects. Several theories have been put forth to explain biological responses to electromagnetic fields. Some reports have shown that low levels of electromagnetic fields can alter gene expression and subsequent protein synthesis by interaction of the electromagnetic field with specific DNA sequences within the promoter region of genes (Blank and Goodman, 2008; Blank and Goodman, 2009). Such changes have been demonstrated in the family of 'heat shock' proteins that function in the cell stress response (Blank and Goodman, 2009). Zimmerman et al. interrogated gene expression changes in cells exhibiting decreased cell proliferation, using high-throughput sequencing technologies to sequence the cells' cDNA. Tumor cell GI was associated with downregulation of PLP2 and XCL2 as well as with disruption of the mitotic spindle. Exposure of HCC cells to the same RF EMF modulated at slightly different modulation frequencies did not result in changes in gene expression, which demonstrates that inhibition of cell proliferation is associated with changes in gene expression levels. Very low levels of 27.12DMHz radio frequency electromagnetic fields were shown to inhibit tumor cell growth when modulated at specific frequencies.
Martin Blank, Reba Goodman, "A mechanism for stimulation of biosynthesis by
electromagnetic fields: Charge transfer in DNA and base pair separation", Journal of Cellular Physiology, Volume 214, Issue 1, pages 20-26, January 2008, DOI: 10.1002/jcp.21 198 (2007), considers possible mechanisms for the biological effect of low frequency electromagnetic fields. Electrons have been shown to move in DNA, and a specific DNA sequence is associated with the response to EM fields. In addition, there is evidence from biochemical reactions that EM fields can accelerate electron transfer. Interaction with electrons could displace electrons in H- bonds that hold DNA together, leading to chain separation and (in cellular systems) initiating transcription. The effect of charging due to electron displacement on the energetics of DNA aggregation shows that electron transfer would favor separation of base pairs, and that DNA geometry is optimized for disaggregation under such conditions. Electrons in the H-bonds of both DNA and the surrounding water molecules fluctuate at frequencies that are much higher than the frequencies of the EM fields studied. The characteristics of the fluctuations suggest that the applied EM fields are effectively DC pulses and that interactions extend to microwave frequencies.
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Each of the references mentioned above and below are expressly incorporated herein by reference in their entirety.
SUMMARY OF THE INVENTION
The present technology proceeds from an understanding that biological nucleic acids contain information, which is a part of their structure. The structure, in turn, corresponds to various types of waves and resonances, which are information-coding sequence dependent.
Further, the same waves and resonances correspond to the biological nucleic acids, and their respective information sequences. Therefore, by conveying the electromagnetic signals that correspond to a biological nucleic acid, its information content can be conveyed.
The present technology is supported by data which shows that signals from highly diluted biological nucleic acids from particular sources emit electromagnetic signals, and that these signals, whether immediately amplified and presented, or recorded and amplified and presented to a specimen container which holds nucleic acid precursors, but starts without nucleic acids, results in production of the corresponding nucleic acid. Further, the signals may selectively exert toxic effects on certain cell types, but not others, which may result from for in situ formation of the nucleic acids corresponding to the signals in the cells.
It is noted that the DNA which emits electromagnetic signals typically comes from natural living sources, and therefore may include epigenetic modifications, free radical effects and adducts, and other chemical modifications that cause it to be incompletely described by its base pair sequence.
The present technology further provides a simple procedure for transducing DNA from some bacterial pathogens into living cells in culture, with induction of cytopathic effect in these cells. The actual mechanism by which this cytopathic effect, which is selectively dependent on both the source DNA being transduced, and the target cells, is not known; however, it is believed that the signals themselves are not merely representative of a biological nucleic acid, but rather the organization of the water and perhaps other solutes in the solution around the nucleic acid.
Likewise, the strength of the signal implies that the source is not a single sequence of DNA, and the basis for synchronization of emissions by a plurality of emission sources is not known. The signal represents a resonance with respect to a stable arrangement of water molecules, and that when a water sample is subjected to the electromagnetic signals, the corresponding resonance is established, and in a medium where the nucleic acid precursors are present, the emitted electromagnetic signals from a first sample of biological nucleic acids can induce formation of the corresponding biological nucleic acid in another sample.
This procedure opens the way to pinpoint in pathogenic organisms some DNA sequences which play a specific role in chronic diseases, even when the pathogenic agent have not yet been
identified. That is, since the signals correspond to biological DNA in a bidirectional manner, the electromagnetic signals emitted by a sample may be analyzed to yield information about biological nucleic acids within the sample, and part of this analysis may include determining the biological effect of the electromagnetic signals on cellular systems.
It is noted that present data reveals that not all DNA emits electromagnetic signals, and that
DNA that emits electromagnetic signals appear to emit different signals. However, the reason for these distinctions is not yet known.
New therapeutics targeted towards these DNA sequences may be derived. For example, it may be that electromagnetic signal transduction of DNA is biologically relevant in nature, and thus that physical contact between a source DNA molecule and a targeted effector is not necessary in order to generate an observable effect. However, the paucity of prior data demonstrating this effect in the absence of specialized instruments tends to indicate that the effect is not significant in nature, and that careful capture of the signals, amplification, and repetition over a long direction, may be required in order for significant effects to be observed. One type of therapeutic regimen involves subjecting a patient or organ of a patient to electromagnetic signal emissions from a particular source corresponding to a biological nucleic acid, which may be both high intensity and prolonged duration. Another type of therapy involves administration of agents that can disrupt or interrupt the effect of the signals on a biological system. For example, various compounds may interfere with the transduction of the electromagnetic signal into a biologically active nucleic acid. A further type of therapy involves emitting a signal that interferes with an electromagnetic signal, and thus interrupts its effect. Further therapies are possible as well.
In some previous patents (US 8,736,250; US 8,405,379), patent applications (US
20130224788, US 20130217000, US 20130196939, US 20130143205, US 20120024701 , US 201 10076710, US 20110027774, US 20100323391, WO2012142568, WO201.3113000), and published papers (Montagnier, Luc, et al. "Electromagnetic signals are produced by aqueous nanostructures derived from bacterial DNA sequences." Interdisciplinary Sciences:
Computational Life Sciences 1.2 (2009): 81-90., Montagnier, Luc, et al. "DNA waves and water." Journal of Physics: Conference Series. Vol. 306. No. 1. IOP Publishing, 201 1., Montagnier, Luc, et al. "Electromagnetic detection of HIV DNA in the blood of AIDS patients treated by antiretroviral therapy." Interdisciplinary Sciences: Computational Life Sciences 1.4 (2009): 245-253.; Montagnier, Luc. "Electromagnetic signaling from DNA: a new biomarker of chronic infection." Chinese Bulletin of Life Sciences 3 (2010): 015.,) the inventors and others
have described the possibility of capturing and recording electromagnetic signals of low frequency (EMS) emitted by DNA of pathogenic viruses and bacteria. Each of the foregoing references is expressly incorporated herein by reference in its entirety.
These emissions are produced at certain dilutions of DNA in water upon excitation by lower wave frequencies of natural or artificial origin. That is, the signals are emitted based on energy provided to the sample either from a variety of environmental sources, or a laboratory electromagnetic signal source. Prior work has also shown that agitation of the sample may be a source of energy for emissions of EMS for a period thereafter.
The EMS are believed to convey information representing the specific sequence of the DNA, since, from their digital recording, the DNA sequence can be reproduced in distant laboratories by Polymerase Chain Reaction (PCR). We describe this phenomenon as photonic transduction of DNA.
In experiments conducted by the inventors, not all DNA sequences appear to produce EMS which have known significance. In certain cases, the PCR-derived DNA amplicon was itself able to emit EMS which could be recorded and transmitted at a distance. This is particularly the case of an amplicon derived from the 16S ribosomal DNA sequence of Borrelia burgdorferi, the agent of Lyme disease, whose PCR (947 base pairs) and nested PCR (499 base pairs) primer sequences are as follows: Inner
BORR16S inS 5'-CAATCYGGACTGAGACCTGC (SEQ ID NO: 1) and
BORR16S inAS 5 '-ACGCTGTAA ACGATGC AC AC (SEQ ID NO: 2).
One aspect of the present invention describes a set of new PCR primers for detecting a 400 bp DNA sequence uniquely present in the red blood cells of HIV infected patients, whatsoever their geographical location and their ethnic origin. This 400 bp DNA sequence has not been detected in the red blood cells of HIV negative individuals. The 400 bp sequence has some sequence homology with the "Gypsy" retrotransposon sequence of human genomic DNA (e.g., 70-80%). The sequences of the primers are the following:
pRICK 1 S5' - CCT GAG A AG AGA TTT A AG AAC AAA (SEQ ID NO:4)
pRICK I AS 5 ' - CCA TAT ACT GCT TCT ARY TGC T
The optimal conditions for detecting the 400 bp amplicon by PCR in red blood cells are: annealing temperature of 56 degrees Celsius, with 50 cycles of amplification (up to about 70 cycles) in a thermocycler. However, this sequence appears to be part of the human genome, as it is detected also by the same primers in a 99% homologous sequence located in the p region of human chromosome 1 (using BLAST against a human genome databank), a region distant from
that of the 237 bp sequence (located in the q region), discussed in U.S. Patent Application No. 13/752,003 (Montagnier), US Pat. Pub. 2013/0196939, also located in human chromosome 1 (See Example 2).
The specificity of the Borrelia burgdorferi primers was checked first on the Borrelia burgdorferi DNA and then in patients suffering of Lyme disease. In 8 Lyme patients of chronic Lyme disease of the East Coast of the USA, all showed emissions of EMS from DNA derived from their plasma according to the procedure defined in US 20120024701; see also L.
Montagnier, J. Aissa, S. Ferris, Jl. Montagnier, and C. Lavallee. "Electromagnetic Signals Are Produced by Aqueous Nanostructures Derived from Bacterial DNA Sequences" Interdiscip Sci Comput Life Sci. 1 :81-90 (2009); and L Montagnier, J Aissa, E Del Giudice, C Lavallee, A Tedeschi and G Vitiello . "DNA waves and water". J. Phys.: Conference Series Volume 306 Number 1. 012007 (2011), Luc Montagnier, Emilio Del Giudice, Jamal Ai'ssa, Claude Lavallee, Steven Motschwiller, Antonio Capolupo, Albino Polcari, Paola Romano, Alberto Tedeschi, Giuseppe Vitiello, "Transduction of DNA information through water and electromagnetic waves", Electromagn Biol Med, 2015; 34(2): 106-112, informahealthcare.com/ebm, ISSN:
1536-8378 (print), 1536-8386 (electronic), doi: 10.3109/15368378.2015.1036072 (2015),.each of which is expressly incorporated herein by reference in its entirety, and from the 499 bp band (amplicon) obtained by PCR from 16S ribosomal DNA of Borrelia burgdorferi. This suggests a persistence of the microbial agent in these patients in the chronic phase of the disease, and a possible pathogenic role of the nano-structures present in the blood circulation.
The recording of the electromagnetic signals (EMS) associated with this amplicon, named BB16, has been successfully used to transduce the corresponding DNA in water tubes, according to the procedure reported in Montagnier et al., "DNA waves and water", J. Phys.: Conference Series Volume 306 Number 1. 012007 (201 1)
It was also sent over to and reproduced in a distant laboratory (Gottingen, Germany). The
DNA sequence was reconstituted from water nanostructures, by using all the ingredients of PCR, using the protocol disclosed in Montagnier et al, "DNA waves and water", J. Phys.:
Conference Series Volume 306 Number 1. 012007 (201 1), and US 20120024701 , and US 61/476,1 10 ("Remote Transmission of Electromagnetic Signals Inducing Nanostructures Amplifiable into a Specific DNA Sequence", 4/15/201 1), which are expressly incorporated herein by reference, which show that the TAQ polymerase used in PCR was able to read and synthesize the sequence from the specific water nanostructures induced by EMS.
The characteristics of the EMS which have biological effect have not been elucidated, and
statistical tests and other forms of analysis have not revealed distinct significant differences from EMS not associated with biological effects. However, the full analysis is not completed, and of course the digital sequences which represent the EMS are of course not the same.
It is therefore an object to provide a system and method for the "teleportation" of some DNA sequences by electromagnetic waves into living cells. This is evidenced in selected cases by the ability to measure DNA associated with a source of the signal in the living cells, though cytotoxicity may result in death of those cells. The DNA is not measured in cells not subject to the treatment, is dependent on the type of DNA used as a source, and occurs selectively in certain cell types.
A system is provided for inducing cytotoxicity, comprising: a receiver configured to receive an electromagnetic signal from a container, using a receiver configured to capture
electromagnetic emissions from the container over a frequency range; an amplifier configured to amplify the received electromagnetic signal; and an emitter configured to emit the amplified electromagnetic signal in proximity to living cells.
A method of producing cytotoxicity is provided, comprising: amplifying DNA from a source, e.g., a pathogen, using polymerase chain reaction (PCR) technology; purifying the amplified DNA; serially diluting and mixing the purified DNA in water, to generate a dilute DNA sample in a container; receiving an electromagnetic signal from the container, using a receiver configured to capture electromagnetic emissions over a frequency range; optionally recording the received electromagnetic signal; amplifying the received electromagnetic signal; and emitting the amplified electromagnetic signal in proximity to living cells.
The serially diluting may comprise obtaining a portion of a prior sample, diluting the portion of the prior sample with medium containing no DNA, and mixing the diluted portion until uniform. The diluting conveniently comprise diluting 1 :9, to result in 10 fold dilutions. The medium may be at least one of water, and a water-ethanol mixture. The serial dilutions are conducted over a range of, e.g., 10"2 to 10"15. Typically, the 10"15 dilution will be negative, and may serve as a control instead of or in addition to pure medium. The signals may require solutions in excess of 10"2 to be observed. Therefore, dilutions of 10"1, 10"2, 10"3, 10"4, 10"5, 10"6, 1(T7, 1(T8, 1(T9, 10"10, l O"1 1, 10"12, 10"13, 10"14, 10"15, 10"16, 10"17, 10"18, etc. may be obtained.
The received signal may be obtained over a band of 1.5-20 kHz, 400-4000 Hz, 100 Hz-10 kHz, 20 Hz-20 kHz, or <10 Hz to > 22 kHz. The signal may be recorded for, e.g., 6 seconds, though a range of recording times of 1 , 2, 3 , 4, 5 , 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 60, 102, 2- 102, 4- 102, 8-102, 1.5- 103, 3-103, 6-103, 1.2- 104, 1.8- 104, 3- 104, 6- 104 sec, or more, is possible.
The pathogen may comprise, for example, a Borrelia or a Ricketsiales.
Serial dilutions of the purified DNA may be analyzed for significant electromagnetic emissions by comparison with control samples that do not have DNA, and an amplitude of emissions within a band of 1500-2000 Hz is compared with control sample. Significant electromagnetic emissions may be determined by having an amplitude of emissions in a band of 1500-2000 Hz of at least 10% over a control sample.
According to one prototype embodiment, 1 the pathogen comprises Borrellia burgdorferi and the living cells comprise HL60 cells (ATCC CCL-240™), or SUM- 159 cells (Flanagan L, Van Weelden K, Ammerman C, Ethier SP, Welsh J., "SUM-159PT cells: a novel estrogen independent human breast cancer model system"; Breast Cancer Res Treat. 1999 Dec;58(3):193- 204; Forozan F, Veldman R, Ammerman CA, Parsa NZ, Kallioniemi A, Kallioniemi OP, Ethier SP (1999) Molecular cytogenetic analysis of 1 1 new breast cancer cell lines. Br J Cancer 81 : 1328-1334) or U937 cells (histiocytic lymphoma, ATCC CRL 1593.2™) or MCF7 cells (breast cancer, ATCC HTB-22™), each of which exhibits a cytopathic effect when exposed to EMS derived from Borrellia burgdorferi, e.g., the BB16 EMS signal.
The amplified electromagnetic signal may be emitted by transducing the amplified signal with a copper coil having 3 layers of 420 spirals of copper wire over a bobbin length of 80 mm, an internal diameter of 50 mm, and a resistance of about 6 Ohms. The amplifying may comprise amplifying over a pass band from 10 Hz to 20 kHz, with a variable output power of up to 140 W RMS. The living cells may be exposed to the amplified electromagnetic signal having a field strength of about 5 microTesla. The exposure may be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or more days. In the prototype, cytotoxicity was observed in 3 days, and cell death seen at 5 and was complete by 8 days.
It is believed that the signal sequence (e.g., defined by the source of the EMS), magnetic field strength and duration are relevant. Due in part to the relatively low frequencies, the transducer may receive the magnetic field of a sample placed in the hollow core of a solenoid.
When live cells are continuously exposed to the EMS for several days, e.g., the BB16 EMS signal, a test was conducted to changes in the cells. It was found that, despite the absence of Borrelia in the sample, PCR was able to produce an amplicon from the cells that included a DNA sequence identical to the DNA which was the origin of the BB16 EMS signal, which was not found in sterile controls. At the same time, there was a strong growth inhibition of the cultured tumor cells, followed by observed death of the majority of the tumor cells.
The effect appeared specific for tumor cells of various types, e.g., HL60 cells, SUM- 159
cells, U937 cells, and MCF7 cells. Non-tumor cells, such as mesenchymatous stem cells, fibroblasts, activated lymphocytes from healthy blood donor were tested, and did not display the cytopathic effects, and no amplification of Borrelia DNA by PCR was observed.
Because of this differential sensitivity of neoplastic as compared to normal cells, this technology may be used as a therapy for various neoplastic diseases. According to one embodiment, an apparatus is provided for exposing small animals (whole body) to the EMS.
The apparatus comprises a solenoid having a square section that has an aperture providing a space suitable for placing two standard size plastic mice cages (290x220x140 mm), and about 80 cm long, configured to provide a magnetic field strength in a frequency range of about 20 Hz to at least 10,000 Hz of at least 5 microTesla and in some embodiments exceeding 180 milliTesla. A current of between about 2-10 Amperes (RMS) circulates in the coil, resulting in a magnetic field of 180 milliTesla. Under these conditions, no disturbing heat is released into the tunnel.
Similar to the in vitro experiment, the animals (in plastic cages devoid of metal pieces ) are exposed continuously to the BB16 EMS emitted from the coil for a period of 12 days. During this 12 day exposure, the cages are taken out of the tunnel only for short term animal care.
Exposure to the same magnetic signals of healthy mice non-inoculated with tumor cells does not alter their physical behavior nor their blood cell count.
An apparatus for treatment of small animals comprises, for example, a laptop computer (e.g., a Sony laptop running Windows 8.1) which digitally stores recorded signals derived from a PCR amplified and aqueous solution (e.g., distilled water) diluted sample of a 499 BP fragment of the 16S ribosomal DNA of the B31 strain of Borrelia burfdorferi (ATCC 35210™), or other DNAs from pathogenic bacteria. The output may be the internal digital to analog converter of the laptop, or an external device (USB connected), such as the Creative Soundbalster X-Fi HD, or X-Fi Surround 5.1 Pro. A 20X amplifier is employed, e.g., from Conrad.
Because these frequencies are similar to the human audio spectrum, advantageously, an audio amplifier may be used, which typically provide power outputs of 50-150 or higher Watts per channel, into 4 or 8 Ohms, over a range of 20Hz-20kHz, with less than 1% total harmonic distortion. It is believed that the relevant frequency range for the EMS extends from about 50 or 100 Hz to 2500 Hz, with peaks observed in the 1500 Hz range, and therefore electronic equipment that handles at least this range may be used.
Similarly, because the target EMS has these characteristics, audio equipment may be used to acquire and process the signals, such as so-called "sound cards" and other computer-audio interfaces. Typically, the inputs of such devices sample at about 44 kHz or 48 kHz, and
therefore are above the Nyquist frequency of the signals of interest. Likewise, the digitizers have 14-16 bits or higher resolution, which is believed to be more than adequate. As discussed above, Matlab may be used as a tool to analyze the signals, but this is by no means the only available software. Other available packages include Octave, Scilab/Xcos, NumPy/Python, SciPy/Python, Julia, and R, for example.
Similarly, a device for treatment of humans may be provided, which may have
characteristics similar to magnetic resonance imaging field magnets, though the field strength need not be as high as used in MRI. It is not believed that the field uniformity need by high, as is a requirement of traditional MRI. Likewise, while MRI employs perturbed static fields, the present technology employs a dynamic field. Sensing coils are not required according to the present technology. The coil may encompass the entire human body, or provide localized treatment, such as the cranium. It is believed that the therapy may be intermittent, and thus continuous exposure to the EMS over several days is not required, and rather the therapy may be provided for several hours per day over a duration of days or weeks.
A device for human brain tumor treatment is provided, in which the coil is configured to surround the head of the patient. The generator of magnetic field is composed of two Helmoltz coils which are placed symmetrically close to the temporal sides of the patient's head. The magnetic field in the middle of the head is in the order of 100 mTesla (milliTesla). The helmet supporting the two coils may be either fixed on a mobile stand (patient sitting down in a chair) or fixed to a wall (patient lying in a bed). In some cases, the coil and apparatus can be sufficiently portable to permit the patient to stand and walk. For example, a lithium ion battery pack may permit untethered operation for minutes to hours, while tethered operation may permit operation indefinitely. Exposure of the patient to the magnetic field is preferably continuous, to the extent feasible, and maintained until complete disappearance of the tumor (per MRI). Gaps in therapy, such as for bathing, diagnostic tests, etc, are acceptable. Other treatments (radiotherapy, chemotherapy) are preferably discontinued during the period of EMS exposure, as it is believed that actively dividing tumor cells are more sensitive to the magnetic signals. Similarly, a whole body device may be provided for treatment of tumors in other parts of patient's body.
The exposed living cells may be analyzed for DNA from the source by PCR using primers adapted to amplify the DNA from the pathogen, e.g., primers specific for a 16S gene of the pathogen. The effect is not limited to DNA corresponding to 16S genes. Tests may be performed comparing DNA from different sources, e.g., signals derived from different pathogens, or different target living cells. It is of course noted that the source DNA is not
limited to DNA from pathogens, and DNA from other organisms, or even synthetic DNA sequences, may be employed. DNA from pathogens, however, has been found to selectively produce a cytotoxic effect on certain target cells. A differential effect on different target cells types may be determined. In some cases, the source of the DNA may by a pathogen that harbors DNA from another organism, for example, a Rickesiales is found in humans infected with HIV that carries certain human genetic sequences.
The DNA may be of various lengths, such as less than 100 bp, 150 bp, 180 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, etc.
An analyzer may be provided to analyze an amplitude of electromagnetic emissions. The analyzer may be configured to determine whether the electromagnetic emissions exceed an amplitude threshold within a define bandwidth. The defined bandwidth may comprise 1 ,500 Hz to 2,000 Hz, or consist essentially of 1,500 Hz to 2,000 Hz.
These and other object will become apparent after a review of the disclosure herein, and these objects and the preferred embodiments are not intended to be limiting on the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic diagram of a system which transduces EMS from DNA and produce a cytotoxic effect in cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1
The conditions described above for remote induction of Borrelia burgdorferi 16S RNA using the BB 16 recorded EMS were artificial, and could not establish that the same phenomenon could exist in nature in living cells. The present technology covers precisely the missing link between laboratory conditions and natural conditions, showing that the same process could occur in living structures.
The detailed procedure used is as follows:
1) Capture and Recording of the EMS:
The 16S ribosomal DNA partial sequence of Borrelia burgdorferi was amplified in a thermocycler (Eppendorff) at 40 cycles with an annealing temperature of 61 °C.
This optimal annealing temperature was optimized on a pure DNA sample of Borrelia burgdorferi obtained from ATTC. Initial denaturation was at 95 °C for 5 minutes. Each
Thermocycle included 30 seconds at 95 °C, 30 seconds at 61 °C and 60 seconds at 70 °C. Final extension was at 70 °C for 10 minutes.
The amplified DNA (amplicon) was separated in an agarose gel electrophoresis apparatus, and the 499 bp band was extracted from the gel by using a Qiaquick gel extraction kit (Qiagen).
The DNA concentration was adjusted to 2 ng/ml and diluted in ten-fold dilutions in 1 ml of pure water in Eppendorf plastic polyethylene conic tubes, under a laminar flow hood.
Each serial dilution was strongly shaken for 15 seconds in a Vortex shaker, before being used for the subsequent dilution. Dilutions were from 10"2 to 10"15 and each tube was placed on top of a copper coil; the electric signal was recorded twice for 6 seconds each by a micro- computer, after 500X amplification and digitization by a sound card (SoundBlaster X-FI HD, Creativelabs) as previously described (US 201 10027774, US 20120024701, US 20130143205, expressly incorporated herein by reference).
The signal was recorded in 201 1 from Borrelia DNA using the specific primers described above, SEQ ID NO: 1 and SEQ ID NO: 2. The amplicon showed typical emission over the background in the range of 1500-2000 Hertz. The amplitude of the overall recording was measured with the custom written routines for Matlab computer software (Mathworks, Natick MA), which revealed a significant increase in signal above background.
The increased amplitude over the background was measured according to the formula:
% of signal power (dB/Hz) = Avg. Power from positive sample dilutions - Avg. Power of negative unfiltered dilutions x 100
Average power of negative unfiltered dilutions: A result lower or equal to 10% is considered as negative.
The electromagnetic signals (EMS) of the 16S ribosomal DNA of Borrelia burgdorferi prepared according to the above method shows more than 20% increase over background (the standard error of the background being +/- 2.5 %), and is therefore considered a positive response.
2) EMS-mediated transduction of 16S BB DNA in HL60 cells.
HL60 is a continuous cell line derived from a patient with myeloblasts leukemia (Gallagher R, Collins S, Trujillo J, et al. (1979). "Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia", Blood 54 (3): 713-33. PMID 288488) registered at ATCC (CCL-240™, promyeloblast cells from acute promyelocytic leukemia). HL60 Cells were grown in RPMI 16-40 medium supplemented with 10% fetal calf serum, without antibiotics, in 25 ml Falcon flasks held vertically in a 37 °C
incubator with 5% C02 / air circulation. The culture medium was changed every 4 days.
Cells were transferred to an incubator containing a copper coil with the following characteristics: bobbin length 80 mm; internal diameter 50 mm; R = 5.93 ohms; 3 layers of 420 spirals of copper wire,
The copper coil was connected to the output of an amplifier (see Fig. 1) having the following characteristics: pass band from 10 Hz to 20 kHz; gain: 1 to 20; input sensitivity 250 mV; output power 140 W RMS into 8 ohms. This amplifier was connected to a digital-analog converter (SoundBlaster sound card, Creative Labs Inc.), receiving a digital signal from a micro-computer playing the Borrelia EMS file BB16.
The cell flask (Falcon 25 mis) containing HL60 cells 1 00 000 in 8 mis of RPMI medium supplemented with 10% of fetal calf serum, was placed inside the copper coil receiving a maximal output of 4 volts from the amplifier, in order to prevent any heating of the flask. The magnetic field inside the coil under these conditions was 5 microTesla (50 gauss).
Control experiments were performed using a blank EMS file recorded from pure water, which was uncontaminated by DNA, and kept physically and magnetically isolated from EMS derived from DNA.
When the HL60 cell culture was exposed to the BB16 EMS file for 5-8 days, two effects could be observed: at day 3 following the beginning of the exposure, an inhibition of cell growth occurred, and at day 5 complete cell death was observed. (SUM- 159 cells, derived from human breast cancer, as also sensitive to the Borrelia BB 16 EMS.)
At day 8, the culture was interrupted and the DNA extracted from 200 microlitres of the cell suspension. An analysis by PCR (70 cycles) of the HL60 sample, using the specific 16S primers for Borrelia burgdorferi 16S RNA showed on gel electrophoresis the specific 499 bp band of 16S DNA amplicon. Centrifugation experiments (2000 rpm, 5 minutes) show that this DNA is associated with the cell pellet and is not present in the culture supernatant.
A control sample of the HL60 cells with the blank EMS file did not produce the band under the same circumstances, and cell growth inhibition and cell death were not observed, even during 8 days of culture inside the coil with the 5 microTesla signal continuously emitted.
A control sample of human macrophage cells subjected to with the BB16 EMS also did not produce the band under the same circumstances, and cell growth inhibition and cell death were not observed.
In particular, a culture of T-lymphocytes from a human healthy donor, which were activated by Phytomagglutinin (PHA) and Interleukin 2, was exposed similarly to the BB16 EMS. There
was no cytopathic effect nor any sign of 16S DNA presence by PCR even after 8 days of culture.
These result would indicate, for example, that normal human differentiated cells do not have the capacity to transform the message carried by BB 16S EMS or by their derived water nanostructures into 16S DNA.
3) EMS- mediated transduction of other DNAs in HL60 cells.
In order to see if this effect was specific to the Borrelia 16SDNA, or was a general property of other amplicon-produced EMS, similar HL60 cultures were exposed to stored recordings of some other amplicons.
Indeed, cytopathic effects and specific DNA reconstitutions were obtained with EMS from the 700 bp amplicon of the 16S ribosomal DNA from a bacterium (similar to a Ricketsiales) associated with HIV infection (See US20130196939 and WO 2013/1 13000, expressly incorporated herein by reference), the 400 bp amplicon from the same bacterium is always associated with HIV infection, see US 61/903,182 ("System And Method For The Detection and Treatment of Infection by a Microbial Agent Associated With HIV Infection", expressly incorporated herein by reference). This amplicon corresponds to a sequence of human genomic origin (chromosome 1) but is carried by the bacterial co-factor present in red blood cells.
However, the 194 bp LTR amplicon from HIV 1, which is also an emitter of EMS, and was shown by the inventors and also in other distant laboratories (see,
www.waterjournal.org/uploads/vol5/supplement/Montagnier.pdf, expressly incorporated herein by reference) to be transduced by its own EMS, did not induce cytopathic effect in HL60 cells, nor any DNA synthesis in these cells.
In addition, the 16S DNA amplicon of Sutterella (See, US 20120207726, WO/2013/139861, expressly incorporated herein by reference) which was shown by the inventors in earlier work to be present in the blood of autistic children, did not induce EMS nor any effects on HL60 cells.
Therefore, a method is now provided to transmit through recordable and digitizable electromagnetic signals, a DNA sequence in living cultivated cells, wherein the signal and/or the
DNA sequence have a specific biological effect.
EMS have been detected in prepared samples from clinical specimens from patients suffering from certain chronic diseases. This would indicate that these EMS may play a role or be indicative of a process related to the persistence of the infectious agents and may contribute to their pathogenic effects. There is some evidence that DNA extracted from some tissues in certain chronic diseases has free radical modifications, and as discussed above, a number of prior researchers have associated free radical effects with EMS interactions.
Moreover, the successful transduction in living cells of the DNA specified by the EMS would indicate that such cells do possess the enzymatic capacity (DNA polymerase) to read the water nanostructures which represent the DNA sequence which is used to create the EMS. This property is therefore not a unique characteristic of the TAQ polymerase used in PCR, and may play a role in natural living organisms under physiological and pathogenic conditions.
Various modifications and variations of the described methods, procedures, techniques, and compositions as the concept of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed is not intended to be limited to such specific embodiments. Various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art, are intended to be within the scope of the following claims.
Each document, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety.
What is claimed is: