Digital biology: Specificity of the digitized molecular signal

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CHAPTER 17
THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL,
A PUZZLING PHENOMENON: THE CRITICAL
CONTRIBUTION OF JACQUES BENVENISTE
YOLÈNE THOMAS∗, LARBI KAHHAK AND JAMAL AISSA
Laboratoire de Biologie Numérique, 32 rue des Carnets, Clamart 92140, France
Abstract: Making a brief history of what is named the ‘Memory of Water’ is obviously not an easy
task. Trying to be as fair and accurate as possible is hampered by two main difficulties:
1) one of the main actors, Jacques Benveniste, recently passed away and 2) cutting edge
science creates many controversies, especially with those whose lifetimes have been spent
pursuing an unorthodox track. High dilution experiments and memory water theory may
be related, and may provide an explanation for the observed phenomena. As Michel
Schiff said: ‘the case of the memory of water may or not contribute to the knowledge
about water structure. Perhaps the tentative interpretation Jacques suggested will finally
have to be modified or even abandoned. Time and further research will tell, provided that
one gives the phenomena a chance (Schiff, 1995, p 45)’
Keywords: human neutrophil; guinea pig heart; coagulation; water; audio-frequency oscillator;
computer-recorded signals
Abbreviations: EMF: electromagnetic field; PMA: phorbol-myristate-acetate; ROM: reactive oxygen
metabolites; ACh: acetylcholine; H: histamine; DTI: Direct Thrombin Inhibitor; d-X:
digital EMF signal from the molecule
1. INTRODUCTION: THE EARLY HISTORY OF HIGH
DILUTIONS EXPERIMENTS / HISTORICAL CONTEXT
Jacques Benveniste gained an international reputation as a specialist on the
mechanisms of allergies and inflammation with the ‘Platelet Activating Factor’
(paf-acether) discovery in 1972 (Benveniste et al., 1972, 1974). Benveniste’s
∗present address: Institut Andre Lwoff IFR89, 7, rue Guy Moquet-BP8, 94 801 Villejuif Cedex, France.
email: yolene@noos.fr
325
G. Pollack et al. (eds.), Water and the Cell, 325–340.
© 2006 Springer.

326 CHAPTER 17
research into allergy has taken him deep into the mechanisms which create such
responses. Understanding that the smallest amount of a substance affects the
organism - ‘A person can enter a room two days after a cat has left it and still suffer
an allergic response’ – led Benveniste in the mid-eighties, to research how homeo-
pathic dilutions appear to have a real and material effect upon immune system
cells called basophils. After 5 years of research he and his collaborators empir-
ically observed that highly dilute (i.e., in the absence of any physical molecule)
biological agents triggered relevant biological systems. It is worth recalling that at
that time, two papers were submitted and published in peer review journals, the
European Journal of Pharmacology and the British Journal of Clinical Pharma-
cology (Davenas et al., 1987; Poitevin et al., 1988). Here, the work was treated
as conventional research like many other manuscripts from peer-reviewed journals
which can be found in the scientific literature on the effect of high dilutions
(Schiff, 1995, p 150; Elia et al., 2004).
In 1988, Benveniste’s laboratory (I.N.S.E.R.M U 200) and three external labora-
tories announced that their research showed that highly diluted antibodies could
cause the degranulation of basophils and that water has a memory. Briefly, the
experimental dilution (anti-IgE) and the control one (anti-IgG) has been prepared in
exactly the same manner, with the same number of dilution and agitation sequences.
They co-authored an article, which was submitted to Nature (Davenas et al.,
1988). Nature’s referees could not find any fault in Benveniste’s research. It was
G. Preparata. and E. Del Guidice (quantum physicists working at Milan University)
at a conference organized a few months before the Nature ‘affair’ erupted, who
brought the theoretical basis for what is known as ‘the memory of water’. They
have hypothesized that interactions between the electric dipoles of water and the
radiation fields of a charged molecule generate a permanent polarization of water
which becomes coherent and has the ability to transmit specific information to
cell receptors, somewhat like a laser (Del Giudice et al., 1988). Two weeks after
publication, the three-man fraud squad (John Maddox, James Randi and Walter
Stewart) sent by Nature spent 5 days in the laboratory. The investigation concluded
that Benveniste had failed to replicate his original study (Maddox et al., 1988).
This marked the beginning of the ‘Water Memory’ saga, which placed him in a
realm of ‘scientific heresy’. As Michel Schiff remarked: ‘INSERM scientists had
performed 200 experiments (including some fifty blind experiments) before being
challenged by the fraud squad. The failure to reproduce (Maddox et al., 1988)
only concerned two negative experiments (Schiff, 1995, p 88, 151). Benveniste
replied (Benveniste, 1988) and reacted with anger: ‘ – not to the fact that an
inquiry had been carried out, for I had been willing that this be done – but to
the way in which it had been conducted and to the implication that my team’s
honesty and scientific competence were questioned. The only way definitely to
establish conflicting results is to reproduce them. It may be that we are all wrong
in good faith. This is not crime but science – ’ In rebuttal, we simply refer the
reader to the article confirming the initial findings in Nature, which appeared in the

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 327
Comptes Rendus de l’Académie des Sciences de Paris in 1991 (Benveniste et al.,
1991), reporting the results of subsequent blind experiments entirely designed and
run by Alfred Spira, and his research I.N.S.E.R.M Unit of independent statistical
experts.
To date, since the Nature publication in 1988, several laboratories have attempted
to repeat Benveniste’s original basophils experiments. Importantly, a blind multi-
center trial of four independent research laboratories in France, UK, Italy and
Holland, confirmed that high dilutions of histamine modulate basophil activity
(Belon et al., 1999, 2004; Brown et al., 2001) Histamine solutions and controls were
prepared independently in three different laboratories. This trial was coordinated by
an independent laboratory led by M. Roberfroid at Belgium’s Catholic University
of Louvain, who coded all the solutions and collected the data, but was not involved
in the experiments. In addition, an independent statistician analyzed the resulting
data. Not much room, therefore, for fraud or wishful thinking. Three of the four
labs involved in the trial reported a statistically significant inhibition of the basophil
degranulation reaction by high dilutions of histamine compared with the controls.
The fourth lab gave a result that was almost significant, so the total result over
all four labs was positive for histamine high dilution solutions. ‘We are,’ the
authors say in their paper, ‘unable to explain our findings and are reporting them to
encourage others to investigate this phenomenon.’ Benveniste may well have been
right all along.
In the meantime, between the repetitions, Benveniste and his team, of which we
were members, found the time to do their part: research aimed at understanding
the physical nature of the biological signal. In particular, we asked ourselves
questions concerning the nature of the biological activity in high dilutions. We
suspected some sort of ordering involving electromagnetism. Indeed, in collab-
oration with an external team of physicists (Lab. Magnetisme C.N.R.S.-Meudon
Bellevue, France), we showed in twenty four blind experiments that the activity of
highly dilute agonists was abolished either by heating (70"C, 30 min) or exposure
to a magnetic field (50 Hz!15 ×10 −3T!15 min) which had no comparable effect
on the genuine molecules (Hadji et al., 1991). We could thus speculate that trans-
mission of this ordering principle was electromagnetic (EM) in nature. Furthermore,
it is not insignificant that a growing number of observations suggest the suscepti-
bility of biological systems or water to electric and low-frequency electromagnetic
fields (Tsonga, 1989; Frey, 1993; Blanchard et al., 1994; Novikov et al., 1997;
Vallée et al., 2005). Together, these considerations informed exploratory research
which led us to speculate that biological signalling might involve low frequency
waves potentially transmissible to cells or water by purely electromagnetic
means.
For the sake of simplicity, we shall present here only three salient biological
models. The detailed descriptions of the different models have also been reported
in publications, technical reports and patents, most of which are available on the
digibio website (www.digibio.com).

328 CHAPTER 17
2. MATERIALS AND METHODS
2.1 Reagents
Ultra-pure water (W), phenol red-free Hank’s balanced salt solution (HBSS) were
obtained from Biochrom; cytochrome c (horse heart, type III), 4-phorbol-12-b-
myristate-13-acetate (PMA), acetylcholine (ACh), histamine (H), bovine thrombin
and bovine fibrinogen were obtained from Sigma Chemicals. PMA was dissolved in
DMSO at 10 mM and stored at −20"C. Vehicle (DMSO from the same batch) was
also aliquoted and stored at −20"C. Immediately before use, the stock solutions was
diluted to appropriate working concentrations in W. Vehicle consisted of DMSO at
the same concentration as that present in the respective PMA solutions.
ACh and H was dissolved in water at 1 "M and stored at −20"C. Bovine thrombin
(1 U/ml) and bovine fibrinogen (24 mg/ml) were dissolved in W and NaCl 0.9%
respectively, then aliquoted and stored at −20"C. All plastic materials were sterile
and purchased from Becton-Dickinson.
2.2 Preparation of Human Neutrophils
Human blood from consenting healthy donors was anticoagulated with citric acid-
dextrose. Blood was sedimented for 30-45 min in 0.3% final gelatin. The supernatant
was layered on Ficoll-Hypaque and centrifuged. The cell pellet was resuspended
in 1 ml of washing buffer (HBSS supplemented with 0.25% (v/v) BSA, 1ng/ml
LPS and 20mM HEPES). Erythrocytes were lysed by adding 3 vol. distilled water
to the cell suspension, followed 40s. later by 1 vol. of NaCl 3.5% (w/v). Cells
were then washed twice, resuspended in washing buffer and counted. All prepara-
tions contained at least 98% neutrophils as determined by microscopic observation
after staining with May Grünwald-Giemsa (Leyravaud S et al., 1989). Before trans-
mission or addition of molecular agonists, neutrophils were suspended at 1×106/ml
in washing buffer and Ca2+(1.3 mM), Mg2+(1mM) and cytochrome c (80 uM)
were added to the cell suspension which was then aliquoted (1 ml) into Eppendorf
tubes (Thomas et al., 2000). Reactive oxygen metabolites (ROM) production
was measured as the reduction of cytochrome c using a spectrophotometer at
550 nm.
2.3 Heart Preparation (Figure 1)
Isolated hearts were perfused according to the classical Langendorff method
(Benveniste et al., 1983; Kim et al., 1983). Acetylcholine (ACh), histamine (H) or
water (W) was injected via a catheter just above the aorta. Variation in coronary flow
(CF) was measured every min for 30 min. During the same time, other mechanical
parameters (min. and max. tension, heart rate) were recorded using a dedicated
software (Emka Technonologies, Paris, France). Percent (%) increase in CF was
calculated as follows: [1 -(CF maximal value / CF time 0 value#$ ×100.

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 329
Figure 1. Langendorff heart perfusion system. Isolated hearts (male Hartley guinea-pigs, 300 g) were
perfused using Krebs-Henseleit buffer (pH 7.4) gassed with O2/CO2, 95/5%, at a pressure of 40 cm
H2O at 37"C. Samples are injected (2 ml) via a catheter just above the aorta
2.4 In Vitro Coagulation
During blood coagulation there is a complex series of molecular interactions. Two
of the molecules are thrombin and fibrinogen. These two can interact alone in
water without any of the other players normally found in the formation of a clot
(Greenberg et al., 1985). Thrombin is a serine proteinase that converts fibrinogen to
fibrin. At room temperature and within a short time, a clear clot will form. Addition
of a Direct Thrombin Inhibitor (DTI), such as melagatran (Gustaffson et al., 2003)
can delayed or even blocked entirely the thrombin–fibrinogen reaction. Coagulation

330 CHAPTER 17
Figure 2. Schematic drawing of the computer-recorded signals: capture, storage and replay. Shielded cylindrical chamber: composed of three
superposed layers: copper, soft iron, permalloy, made from sheets 1 mm thick. The chamber has an internal diameter of 65 mm, and a height of
100 mm. A shielded lid closes the chamber. Transducers: coil of copper wire, impedance 300 Ohms, internal diameter 6 mm, external diameter
16 mm, length 6 mm, usually used for telephone receivers. Multimedia computer (Windows OS) equipped with a sound card (5KHz to 44 KHz
in linear steps), (Sound Blaster AWE 64, CREATIVE LABS). HiFi amplifier 2x100 watts with an ‘in’ socket, an “out” socket to the speakers, a
power switch and a potentiometer. Pass band from 10 Hz to 20 kHz, gain 1 to 10, input sensitivity +/−V. Solenoid coil: conventionally wound
copper wire coil with the following characteristics: internal diameter 50 mm, length 80 mm, R =3%6 ohms, 3 layers of 112 tums of copper vire,
field on the axis to the centre 44 10−4T/A, and on the edge 25 10−4T/A. All links consist of shielded cable. All the apparatus is earthed

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 331
is assessed by spectrophotometry at OD620. Percent (%) inhibition coagulation was
calculated as follows: &1– 'OD620 DTI/OD620 W#$ ×100.
2.5 Transmission Apparatus: Audio-Frequency Oscillator
The device used for transmission comprised a standard audio amplifier (Kemo kit
105, West Germany) with magnetic coils connected respectively to the input and
output (impedance 8 ohms). Tubes whose contents were to be transmitted were
placed on the input coil and cells or water on the output coil. When the amplifier
was not connected to the output coil, its output, as viewed with an oscilloscope,
appeared to be noise with some 50 Hz contaminations from the French power grid.
However, when the amplifier was connected to the output coil, it behaved as an
audio-frequency oscillator and signal analysis revealed the emission of a stable
square wave with a frequency of about 3 kHz and voltage of approximately 7 V.
In the presence of a weak, mV range signal not only the amplitude but also
the frequency of the wave were modulated (WO patent-94-17406). During the
transmission procedure, the various parameters such as power, voltage, capacitance
and impedance remained constant, the nature of the source tube being the only
variable.
2.6 Computer-Recorded Signals: Capture, Storage and Replay
The characteristics of the designed apparatus are described in Figure 2 and in the US
patent-03-6541978. Briefly, the process is to first capture the electromagnetic signal
from a biologically active solution and store this digitized signal on a computer’s
hard drive: Thus, tubes containing ACh, H, DTI at 1"M or W were used as source.
After recording (6 sec, 16 bits in mono mode, 44 kHz) the signal is then “played
back” for 10 mins from the computer sound card through a solenoid coil containing
a tube of water (tension of 4 Volts). The digital signals were standard Microsoft
sound files (*.wav). The order of the conditions and their repetitions was always
randomized and blinded. For ease in the discussion, the terminology d-X refers to
the digital EMF signal from the molecules.
3. RESULTS
3.1 Mimicking the Effects of Molecules Using a Transmission
Apparatus: Audio-Frequency Oscillator
Between 1991 and 1996, using a standard audio amplifier that, when connected to
another coil, behaves as an audio-frequency oscillator, we performed a number of
experiments showing that we could transfer specific molecular signals to water or
directly to cells. For instance, we investigated whether molecular signals associated
with PMA could be transmitted by physical means to human neutrophils to modulate
reactive oxygen metabolite (ROM) production. Briefly, neutrophils were placed in

332 CHAPTER 17
9 10
01 2 3 4 5 6 7 8
20
40
60
80
100
First set of experiments
% transmission
11 12 13 14 15 16 17 18 19 20
–30
–10
10
30
50
70
90
110
Second set of experiments
% transmission
Figure 3. Effect of transmitted phorbol-myristate-acetate on neutrophil ROM production. For each trans-
mission sequence to neutrophils, the input coil coupled to the amplifier was operated at room temperature,
while the output coil was placed in a 37"C humidified incubator. The tube containing PMA, (1 uM) or
vehicle was placed on the input coil, and tubes (duplicate) containing neutrophils on the output coil. The
oscillator was then turned on for the 15 min transmission period. In each experiment, 4 simultaneous

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 333
a 37"C humidified incubator on one coil attached to the oscillator, while PMA or
vehicle was placed on another coil at room temperature. For most experiments four
oscillators were used simultaneously. The oscillator was then turned on for 15 min
after which cells were usually further incubated for up to 45 min at 37"C before
OD550 measurement. Additional check consisted of unexposed cells. The positive
control consisted of neutrophils directly stimulated by molecular PMA (1 pM to
10 uM). The procedure and the results of twenty consecutive blind experiments are
shown in Figure 3. One of the two series of experiments was performed in a different
laboratory, with randomization and coding of source tubes being performed by the
head of the laboratory (Dr. F. Russo Marie, INSERM U332). Exposing cells to
transmitted PMA (T-PMA) resulted in an OD increase of 37 ±4% 'mean ±S%E%M,
40 transmissions) compared to unexposed cells. By contrast, exposing cells to
transmitted vehicle (T-vehicle) resulted in a 4%1±1%8% change. In the absence
of cells, transmission of PMA or vehicle alone was without effect on cytochrome
c reduction. The effect of transmitted PMA was roughly equivalent to that of 0.1 nM
molecular PMA. Additional experiments indicate that ROM were not induced when
4(-phorbol 12,13-didecanoate (PDD), an inactive PMA analogue, was transmitted
in the same manner as PMA. The observation that T-PMA but not T-PDD stimulated
ROM production suggested the involvement of Protein kinase C (PKC), the main
target of PMA. Indeed, the impact of transmitted PMA was substantially reduced in
cells pretreated with two PKC inhibitors, GF109203X or H-7 (Thomas et al., 2000).
We next attempted to block the transmission effect: one parameter of the basic
design was modified in half of the transmissions. Either: 1) the oscillator was
turned off or 2) the PMA solution or the cells were shielded with Mu-metal
(an alloy designed to inhibit magnetic fields down to low frequencies). Data
of 12 independent experiments indicate that PMA transmission effect (42 ±8%)
was essentially suppressed when the amplifier was turned off (−1%8±1%4%) and
when either the PMA solution or the neutrophils were shielded with Mu-metal
(−4%3±2%7%,).
The statistical significance of the experiments was analyzed using the Student’s
t-test. Percent transmission (as defined in the legend of Figure 3) was computed for
each set of cells (cells exposed to T-PMA, T-vehicle, T-PDD or T-PMA oscillator
off). Differences between cells exposed to T-PMA and other experimental groups
(cells exposed to T-vehicle, T-PDD or T-PMA oscillator off) were calculated at 60
min (total incubation time). T-PMA cells were associated with a 33%6±3%4% OD
!
Figure 3. transmissions were performed, using 4 source tubes (2 PMA and 2 vehicles). These 4 source
tubes were prepared, randomized and blinded by coding at the beginning of each experiment. After
transmission, the oscillators were switched off and all cells were left in the incubator for the additional
45 min post-transmission incubation period, before OD measurement. Additional check consisted of
unexposed cells. Viability of all samples was assessed by trypan blue exclusion both before and after
incubation. For each individual experiment, percent (%) transmission was calculated as: 100×(OD550
exposed cells - OD550 unexposed cells) / OD550 unexposed cells. Each error bar corresponds to the
standard error estimated from 4 OD values of exposed cell-tubes. (black bar) T-PMA cells; (hatched
bar) T-vehicle cells

334 CHAPTER 17
increase, in contrast to 2%3±1%3% (n = 58 transmissions, p <10−3) for T-vehicle,
T-PDD and T-PMA oscillator off (Thomas et al., 2000).
Although, the precise physical mechanism(s) involved remain(s) unknown,
together, these results suggest that PMA molecules emit signals that can be trans-
ferred to neutrophils by artificial physical means in a manner that seems specific
to the source molecules. Along this line are other studies showing transmission
of thyroxine signal via electronic circuit using water as target for the transmitted
signal (Endler et al., 1995). Part of this work was published (Thomas et al., 2000).
Appended to this article were two affidavits, one from a French laboratory testifying
that they supervised and blinded the experiments we did in this laboratory; the
other from an US laboratory (W. Hsueh, Department of Pathology, Northwestern
University, Chicago) testifying that they did some preliminary experiments similar
to ours, without any physical participation on our part, and detect the same effect
as we described.
3.2 Mimicking the Effects of Molecules Using a Computer-Recorded
Signal
Because of the material properties of the oscillator and the limitations of the
equipment used, it is most likely that the PMA signal is carried by frequencies
in the low kilohertz range. Theses considerations led to the establishment in 1995
of a new procedure for the recording and retransmission of the molecular signals
(Figure 2). Briefly, the process is to first capture the EM signal from a biologically
active solution and store this digitized signal on a computer’s hard drive. The EM
signal is then “played back” through a sound card to a solenoid containing a tube
of water.
One of the biological systems, which can be used to detect digital files endowed
with biological activity, is the measurement of coronary flow (CF) in isolated
perfused guinea-pig hearts (Fig. 1). In particular, we investigated the effect of
digital EMF signals of acetylcholine (d-ACh) and histamine (d-H). Digital EMF
signal of water (d-W) and ACh or H, similarly were applied as negative and
positive controls respectively. The procedure and the results of consecutive blind
experiments performed between November 21, 1997 and April 14, 1998 are shown
in Table 1.
d-ACh, ACh, d-H and H increase CF compared to d-W. d-W induced effects
that were indistinguishable from spontaneous flow variations. The two comparisons
d-ACh vs d-W and d-H vs d-W are both significant (p <0%05, Student’s t test for
unpaired variates, Sigma plot 40, Jandel Scientific Corte, Madena, CA). Interest-
ingly, atropine, an ACh inhibitor, inhibited both the effects of the ACh and d-ACh
but not those of H and d-H. Mepyramine, an H1 receptor blocker, inhibited both H
and d-H but not ACh and d-ACh.
In 1996, a team from Northwestern University at Chicago recorded a group of
biological signals, either from bioactive solutions (ACh, Ovalbumin (OVA), % % %)
or control (water), on a computer with a sound card, (using a recording instrument

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 335
Table 1. Effects of digital acetylcholine and histamine on the coronary flow in isolated guinea-pig hearts
(Consecutive blind experiments performed: November 21, 1997-April 14, 1998)
Exp. d-W d-ACh ACh 1uM d-H H 1uM
A. Buffer 4%6±2%1 19%5±7%4 26%6±8%3 14%3±2%5 21%1±8%4
[28] [21] [16] [14] [5]
B. Buffer +atropine 4%2±1%3 7%3±2%8 8%8±3%3 14 ±2%1 23%6±4%3
[12] [10] [3] [3] [4]
C. Buffer +mepyramine 5%9±2%0 19%1±3%9 29%5±4%2 5%8±1%8 8%2±2%9
[9] [3] [5] [5] [6]
Acetylcholine (ACh), histamine (H) and water (W) were recorded as in Fig. 2. Files were digitally
amplified and the signal of digital EMF ACh (d-ACh), H (d-H) or W (d-W) was replayed as described
in Materials and Methods. Atropine is used to inhibit the action of ACh, and mepyramine, to inhibit
the action of H.
A. Water, appropriately exposed to d-ACh or d-H, was then infused to isolated hearts. d-W, ACh or H
at 1 uM were infused as negative and positive controls respectively.
B. Water, appropriately exposed to d-ACh or d-H, was then infused in the presence of atropine
(2 mg/ml), to isolated hearts. d-W, ACh or H at 1 uM were infused as negative and positive controls
respectively.
C. Water, appropriately exposed to d-ACh or d-H, was then infused in the presence of mepyramine
(5 mg/ml), to isolated hearts. d-W, ACh or H at 1 uM were infused as negative and positive controls
respectively.
Results are expressed as percent (%) increase in CF as defined in Materials and Methods. Data
are presented as mean ±SD, nb of experiments.
provided by us), and transmitted them to us, blinded, via Internet. Several months
of “fine tuning” the methodology by both teams (including determining the optimal
time interval and amplification of recording settings, the optimal settings for playing
back the signal, the way of handling the samples, sending the file via e-mail one
file a time, rather than sending all files together, using the same stock solutions,
etc.) had to be done in order to eliminate the variables which might interfere
with the recording and transmission of electromagnetic molecular signals. Although
the possibility exists that we were not completely successful in removing these
interfering variables, we could detect the transmitted biological activities with high
accuracy (% increase in CF). For instance: d-OVA: 24%0±1%4!n=30 compared to
d-water 4%4±0%3!n=58 (p = 4.5 e−17, Student’s t test for paired variates). OVA
0%1"M)28%9±3%7!n=19 is not statistically different compared to d-OVA.
In 1999, the Team developed an other biological system: inhibition of fibrinogen
coagulation by a Direct Thrombin Inhibitor (DTI). The hypothesis tested was
whether the reaction rate for coagulation between thrombin and fibrinogen could
be modulated by d-DTI. d-W and DTI (1uM) were used as negative and positive
controls respectively. As illustrated in a representative experiment (Figure 4),
addition of DTI and d-DTI result in a slower reaction rate as compared to W or
d-W. The results of twenty-two consecutive blind experiments performed between
April 16 and June 26, 2004 are shown in Table 2. In the majority of the experiments
d-DTI prolongs the clotting compared to d-W although to a lesser extent than 1 uM

336 CHAPTER 17
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 5 10 15 20 25 30 35 40 45 50 55 60
Time (min)
OD (620 nm)
d-DTI
d-W
DTI 1uM
W
Figure 4. Effects of a Direct thrombin inhibitor on thrombin induced fibrinogen coagulation. Direct
thrombin inhibitor (DTI) and water (W) were recorded. Files were digitally amplified and the signal of
digital EMF DTI (d-DTI) or W (d-W) was replayed for 10 min, as described in Materials and Methods.
Water, appropriately exposed to d-DTI is added to fibrinogen along with thrombin (Thr). W, d-W and
DTI (1 uM) were used as negative and positive controls respectively. After different time periods,
coagulation is assessed by spectrophotometry and expressed as OD620. One representative experiment
is shown
Table 2. Effects of direct thrombin inhibitor on thrombin induced fibrinogen coagulation (Consecutive
blind experiments performed: April 16–June 26, 2004)
Mean ±SD [n]
d-W 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0
0, 0, 1 0%09 ±0%29 [22]
d-DTI 38, 25, 39, 31, 36, 61, 44, 40, 35, 35, 65, 30, 26,
26, 31, 24, 28, 26, 81, 16, 35, 20 36%00 ±15%36 [22]
DTI 1uM 65, 70, 68, 72, 75, 75, 69, 71 70%62 ±3%42 [8]
Water, appropriately exposed to the digital EMF signal of DTI (d-DTI) is added to fibrinogen along
with thrombin. Water (W), digital EMF water (d-W) and DTI (1 uM) were used as negative and positive
controls respectively. Coagulation is assessed by spectrophotometry at OD620. Results are presented at
30 min and expressed as percent (%) inhibition coagulation as defined in Materials and Methods. Data
are mean ±SD, nb of experiments.
DTI. The comparison d-DTI vs d-W is highly significant (p =3%7 e−10, Student’s t
test for unpaired variates).
These results suggest that at least some biologically active molecules emit signals
in the form of electromagnetic radiation of less than 44 kHz that can be recorded
and digitized. The digitized signal can be replayed to water, target cells or organs
in a manner that seems specific to the source molecules.
However, our attempts to replicate these data in four other laboratories yielded
mixed results. We then realized the difficulty in ‘exporting’ a method, which is very

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 337
far from conventional biology. This may reflect key variables like, for instance,
the purity of water, its conductance, the purity of the chemicals, electromagnetic
environmental conditions. Also, individual variations of the operator’s performance
could explain some erratic results. In order to eliminate these uncontrolled param-
eters, the same reagents are always used and two shielded robots were built in order
to eliminate the distorting effects of human intervention. An external laboratory
where a team of scientists is currently attempting to replicate the experiments is
using one of those.
4. DISCUSSION: THE CURRENT STATE OF KNOWLEDGE
Among the various theoretical problems associated with such a signal, three appear
particularly pertinent. The first relates to background noise. Given the level of
electromagnetic noise present in the environment, it is necessary to postulate
ways in which the signal-to-noise ratio or the detection of specific signals, or
both, are enhanced. In fact, an appropriate level of noise enhances a specific
periodic signal rather than overwhelming it, a phenomenon known as stochastic
resonance (Wiesenfeld et al., 1995; Astumian et al., 1995; Pickard, 1995). The
relevance of this concept to the phenomena reported here remains to be deter-
mined. Second, the limitations of the equipment used here, suggest that the signal
is carried by frequencies in the low kilohertz range, many orders of magnitude
below those generally associated with molecular spectra (US patent-03-6541978).
The ‘beat frequency’ phenomenon may explain this discrepancy, since a detector,
for instance a receptor, will ‘see’ the sum of the components of a given complex
wave (Banwell, 1983). Third, how to explain the ability of water to carry and
memorize biological signals? Will Quantum ElectroDynamics (QED) provide these
answers (Del Giudice et al., 1988; Preparata et al., 1995)?. QED-based long-range
electromagnetic communication between molecules may represent the founding
theory able to unravel the nature of the molecular signal and the role of perimolecular
water in its transmission. The best is to let Preparata explain it himself (excerpts
from the proceedings of the meeting (14/12/1999) at the Institute of Pharmacology,
University of Rome ‘La Sapienza’, The role of QED in medicine : ‘The space-time
order in biochemistry cannot be the product of the chemical interactions whose
range is too short (a few Angstroms) to allow the molecules to detect each other
from afar and, moreover, when they are inside a crowd of other molecules, not
involved in the specific biochemical sequence. QED solves this problem completely,
since, within a coherent medium, molecules may interact through their common
coupling to the electromagnetic field and the intensity of the force depends inversely
upon the difference of their oscillation frequencies, so that molecules whose oscil-
lation frequencies are significantly different ignore each other, whereas resonant
molecules attract themselves strongly. We get thus a selective recognition code
based on the electromagnetic resonance, which could provide the dynamic basis to
the biochemical codes. Electromagnetic fields have a long range and then are able
to produce a recognition at a distance, also in a crowd of non-resonating molecules’.

338 CHAPTER 17
Alternative hypotheses have been proposed for explaining water memory. For
instance, one hypothesis predicts changes in the water structure by forming more
or less permanent clusters (Fesenko et al., 1995). Louis Rey using a technique
that measures thermoluminescence points to the unusual properties of water under
certain treatments suggesting that water does have a memory of molecules that
have been diluted away (Rey, 2003). Clearly, more theoretical and experimental
work is needed to unveil the physical basis of the transfer (and storage?) of specific
biological information either between interacting molecules or via an electronic
device.
5. CONCLUDING REMARKS
This story, exemplifies the fact that most if not all researchers, nowadays and in
the past, were misguided to apply existing reasoning and methods to a completely
new domain of research.
The debate on the memory of water started in 1988 and in 2005, i.e., 17 years
late, the majority of the scientific community rejects it, even though an increasing
number of scientists report they have confirmed the basic results made by Jacques
Beneveniste ’group.
As Isaac Behar, who has worked closely with Jacques Benveniste, pointed out:
‘a parallel can be drawn between the polemics on memory of water, presuming
that the action of molecules are mediated by an electromagnetic phenomenon, and
the polemics on the transmission of nerve influx. This debate started in 1921 with
the first experiments performed by Otto Loewi. The polemic was still active in
1949 i.e., 28 years after the first test assuming that transfer of nerve influx through
synapses are mediated by specific molecules, the neurotransmitters (Bacq, 1974).’
Since the very beginning we have placed a great deal of emphasis on carrying
out our work under the highest standards of methodology and great effort has been
made to isolate it from environmental artifacts. More difficulties most probably
lie ahead. Now that Jacques Benveniste is no longer with us, the future of the
‘digital biology’ is in the hands of those who have been convinced of the reality
of the basic phenomena. Most likely they will succeed if they combine full
biological and physical competences to understand the nature of the biological
signals (Ninham, 2005).
ACKNOWLEDGEMENTS
The authors express their sincere appreciation to the members of the laboratory
staff, past, present and future, whose valuable contributions have been essential to
the success of this scientific adventure. A special mention is given to Françoise
Lamarre who for 30 years has served as the executive secretary. Now she is
continuing her part through the ‘Association Jacques Benveniste pour la Recherche’
(http://jacques.benveniste.org). We are deeply grateful to supporters and financial
investors who have enabled the “Laboratoire de Biologie Numerique” to carry on

THE PHYSICAL NATURE OF THE BIOLOGICAL SIGNAL 339
the work thus far. We are also indebted to Dr. Wei Hsueh (Northwestern University,
Department of Pathology, Chicago, USA) for her valuable scientific contributions
and collaborations.
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