extfieldsofstudy: Biology Medicine

text:
An integrated multidisciplinary model describing initiation of cancer and the Warburg hypothesis

Background In this paper we propose a chemical physics mechanism for the initiation of the glycolytic switch commonly known as the Warburg hypothesis, whereby glycolytic activity terminating in lactate continues even in well-oxygenated cells. We show that this may result in cancer via mitotic failure, recasting the current conception of the Warburg effect as a metabolic dysregulation consequent to cancer, to a biophysical defect that may contribute to cancer initiation. Model Our model is based on analogs of thermodynamic concepts that tie non-equilibrium fluid dynamics ultimately to metabolic imbalance, disrupted microtubule dynamics, and finally, genomic instability, from which cancers can arise. Specifically, we discuss how an analog of non-equilibrium Rayleigh-Benard convection can result in glycolytic oscillations and cause a cell to become locked into a higher-entropy state characteristic of cancer. Conclusions A quantitative model is presented that attributes the well-known Warburg effect to a biophysical mechanism driven by a convective disturbance in the cell. Contrary to current understanding, this effect may precipitate cancer development, rather than follow from it, providing new insights into carcinogenesis, cancer treatment, and prevention.

The living cell is an extremely complex molecular network of tens of thousands of different types of molecules from ionic species and small molecules to large polymers and polymer networks. Naturally, the number of large, multi-nanometer size polymers is not as high as the smaller molecules, but a shift in their numbers can easily result in the emergence of a disease state. For example, variation in the number of genes expressed due to changes in the genome (aneuploidy), can indicate cancer. Many of these large polymers are enzymes, or chemical reaction catalysts. Typical molecular reactions in the cell are represented by the relation: where the symbols E, S, ES, P represent the enzyme, substrate, enzyme-substrate complex, and reaction product, respectively. The coefficients labeled by k's represent forward, reverse and enzyme-substrate decomposition rate constants as indicated by their subscripts. Obviously, if there is a huge abundance of S and limited amount of E, the reaction is rate limited by the concentration of E. But if the cell is malfunctioning and producing an excess of E when an abundance of S is present, then the cell will increase the P concentration by massively parallel reactions. This is governed by the well-known Michaelis-Menten reaction kinetics of saturable chemical reactions [8]. This effect is also described by the Le Chatelier principle of reaction dynamics [9]. It states that chemical reactions move forward or backward so as to reduce excesses in the quantity of reactants or products, respectively, introduced into in the reaction vicinity. Given the reaction dynamics afforded by Le Chatelier's principle, it is possible to imagine that an external concentration of, for example, glucose allowed entry into a cell would ripple through the molecular network of a cell and produce an excess of the appropriate glycolytic enzymes and other molecules associated with anaerobic processing. This would in turn produce an excess of lactic acid and a shift in the hydrogen ion concentration. Neither of these causes will necessarily induce a cell to transition to a higher entropy state (disorganization) of a cancer cell [10], but we argue that these reaction processes can disrupt the mitochondria and/or the cytoskeleton in part via the microtubule growth rate which is dependent on the pH value [11], a parameter known to change between normal and cancer cells. In particular, in cancer cells, intracellular pH is alkalized [12][13][14][15], and extracellular pH is acidified [12,14,15]. These biophysical changes in turn could contribute to mitotic failure and in rare cases aneuploidyproducing a stable cell exhibiting a glycolytic shift, i.e. the Warburg effect. Our hypothesis is schematically outlined in Figure 1.

The model
We argue that non-equilibrium thermodynamics is a driving force for the ultimate transition of a cell from the normal to the cancer state, and that the first step in the process is an increased operation of the cytoplasmic glucose processing machinery due to a non-equilibrium mechanism analogous to Rayleigh-Benard convection ( Figure 1) [16,17]. Our proposed scheme that describes the development of the cancer phenotype at a cellular level is essentially a biophysics model, which should clearly be considered in parallel with more biochemical schemes [18][19][20][21][22]. We begin by reviewing some relevant physics of nonlinear dynamics and thermodynamics.

Rayleigh-Benard Convection
To appreciate the relevant dynamics, we can consider a thin layer of oil, of thickness d, lying on top of a uniformly heated metal plate. On top of the oil is another plate of high heat conductivity. The apparatus has non-thermally conducting bounding walls to keep the oil confined. We thus have a thermal gradient from a hot zone to a cooler zone. Initially the heat transfer is strictly via conduction from the hot surface to the cooler surface. The upper plate is kept at temperature T o and the lower plate is kept at T o + ΔT (ΔT > 0). As long as the temperature difference remains small, frictional forces in the fluid, due to viscosity, will keep convective motion near zero. Nonetheless, the conduction results in a small upward vertical vector from the bottom layer, and due to gravity a small downward vertical vector from the top layer. As a result of this, there will be a phase transition leading to a thermal convection mode of transport and an ordered state. The characteristic time for the displacement of the small regions of the fluid is given by: where μis the dynamic viscosity of the fluid, α is the thermal expansion coefficient of the fluid, ρ is the mean fluid density, and g is the gravitational acceleration. The condition for sustained convection is given by: where D T is the thermal diffusion coefficient and the dimensionless constant R RB is known as the Rayleigh number. In this case we have used the subscript RB to remind us that these are the Rayleigh-Benard relations. The Rayleigh number is related to the Figure 1 Diagram of proposed transition from healthy to aneuploidy cell. An excess of external glucose causes a series of phase transitions starting with glycolytic oscillators which induce a pH imbalance precipitating in mitochondrial membrane breakage followed by microtubule depolymerization and mitotic failure.
temperature differential. Above this convection threshold, the fluid undergoes a phase transition to an ordered phasean example of a so-called symmetry breaking phenomenon characteristic of many critical phenomena in physics and other fields [23]. What is special about this example is that it is a non-equilibrium effect driven by the thermal gradient across the sample. At this point one can observe convection patterns known as Rayleigh-Benard convection rolls shown in Figure 2. These patterns are an observable manifestation of this non-equilibrium phase transition with an associated symmetry-breaking effect being the spatial periodicity of the rolls and a characteristic time scale representing an oscillation period. Davies, Demetrius and Tuszynski [10] provided a qualitative argument that cancer is a dynamical phase transition. Here, we argue, by analogy, that a similar phenomenon to Rayleigh-Benard (R-B) convection, a symmetry breaking non-equilibrium phase transition could occur for cells grown with an excess glucose gradient between the outside of the cell and the inside of the cell, or with a critical change in the internal glycolytic enzyme distribution. This could represent a pre-cancerous transformation in terms of biological consequences of this physical process. Analogous to the R-B characteristic time relation above, we may now propose that for a cell: where ΔC is the change in concentration, and using ν to represent the kinematic viscosity which has the units cm 2 sec -1 . Water has a viscosity of about 1 cP (cgs units) at ambient conditions. We will assume the viscosity inside a cell is about 1000 times higher (e.g. 10 P, Kalwarczyk et al. [25]). Instead of the force of gravity that has no appreciable effect on living cells at this scale, we will use energy density with units erg g -1 (with dimension cm 2 /sec 2 , instead of gravity's cm/sec 2 ), and represent it by the symbol, Φ. A similar parameter, energy-density-rate (erg g -1 sec -1 ) has been described by Chaisson [26] who discusses at length the application of energy-density-rate for multiple phenomena in the universe including evolution of life and evolution of stars. The parameter Φ is a universal parameter used to describe energy and the rate of energy flow; in other words, a specific metabolic rate, which represents the rate of energy transduction per unit mass. It is essentially a measure of the complexity of an open system and its rate of free energy utilization. In the case of small resting animals the value is on the order of 10,000 erg sec -1 g -1 . For a single cell it is on the order of a few hundred erg sec -1 g -1 . Obviously, it is a nonlinear function due to its relationship to metabolism. As has been well-known for many decades, metabolic rates scale with a fractional exponent in the range of 0.5 to 1.0, typically 0.75 [27]. For specific metabolic rates (metabolic rates per mass unit), these exponents become in the range from −0.5 to 0.0 [28]. Finally, we use α, which is the analog of the thermal expansion coefficient and therefore in this case the inverse of concentration, and we judiciously set its value to be 10 -4 . Keeping in mind it has the same units as inverse concentration but when multiplied with the concentration, as shown in Equation (4) the units cancel. We thus have the following units for Equation (4): Most of the remainder of the paper will be devoted to present arguments and analyses aimed at supporting the use of Equation (4) in the context of the Warburg effect.
As can be seen in Figure 3, an increase in the external concentration results in the time constant falling more rapidly and larger values of the energy-density also result in smaller time constants. As the concentration increases, the value for Φ decreases. As the cell transitions from oxidative phosphorylation to glycolytic processing, the complexity of the molecular network transitions from mitochondrial to substrate level processing. The first primitive cells were anaerobic and had a lower Φ than later cells with mitochondria. Therefore, the model suggests that mitochondria are not being heavily exploited at this stage in the glycolytic oscillation experiment described by Hess et al. [29].

Glycolytic oscillators
Several in vitro and in vivo experiments have already demonstrated that an increase in extracellular glucose [30,31] or lactic acid [32] concentration can lead to increased tumorigenesis. Further, hypoxia has been linked to causing the metabolic shift to glycolysis, and to cause malignant progression, with much experimental evidence [33][34][35][36][37][38].
Goldbeter [39] discusses at length glycolytic oscillations, and uses a series of differential equations to model the dynamics for some of the chemical species involved in the reactions. The observed oscillations come about as a result of the fact that there is a delay in manufacture of the intermediate nicotinamide adenine dinucleotide (NADH) and the fact that there is a finite number of molecular components available for the glycolytic processing. The cycle time has been observed in S. carsbegensis to be about 5 minutes using fluorescence of the glycolytic intermediate, NADH [40]. More interestingly, Hess et al. [29] measured the oscillation frequency for different doses of fructose or glucose as input. As shown in Figure 3, as the concentration of the glucose (or fructose) increases, the period of oscillation decreases. This can only come about from increased concentration in the number of enzymes to participate in the reactions. As the number of enzymes and the number of molecular components increase, so does the entropy because the increase in numbers enables more ways to dissipate free energy, in this case represented as chemical potential of a glucose gradient [41]. As the concentration increases the cycle time decreases indicating a more efficient processing of glucose per time unit.
More recently, Aromolaran et al. [42] describe, and show experimentally, glycolytically generated adenosine triphosphate (ATP) and Ca 2+ waves propagating through a cell from application of glycolytic inhibitors focally injected from a glass pipette with a 1.5 micron diameter tip. The authors discover that glycolytically generated ATP is likely a key modulator of Ca 2+ homeostasis. Of course, this has direct effect on the permeability of the mitochondrial wall, as shown by Yang et al. [43] who describe glycolytic oscillation depolarizing the mitochondrial membrane. The authors describe a model based on the logistic function showing there is a region where the oscillations are "too rapid for observation." Though they do not use the term 'chaos' , this is likely a chaotic state observed in the logistic functions and other "chaotic dynamical functions." In the case of Rayleigh-Benard convection rolls, as shown in Figure 2, the rolls can be modeled with a sine-circle map, for example, θ i+1 = f(θ i ) where the function is periodic in the angle [44,45]. There are some theoretical arguments for the glucose oscillators being embedded in the cell membrane. Demetrius et al. [27] argue that the enzymes' concentration would oscillate due to periodicities in the redox potential and the result could be modeled as harmonic oscillators. Further, Tyner et al. [46] measure electrical gradients in the cell, and we show that a relevant protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) associated with glucose processing in the cytoplasm accumulate at the membrane thus showing experimental support for our hypothesis that molecular oscillators accumulate at boundaries. Lastly, Pokorny [47] suggests Duffing oscillators [48] as a potential model of oscillatory states of a cell. In the following subsections we first provide some experimental validation for the differences in GAPDH localization. Then we follow that with some simplistic analytical modeling to show phase locking of oscillators potentially resulting chaos and disruptions of mitochondria.

Experimental validation for GAPDH distribution
These predictions of GAPDH distribution were tested by using antibodies against GAPDH in normal human mammary epithelial cells (HMECs) under normal culture conditions, purchased from Life technologies.
Briefly, HMEC cells were grown on coverslips and incubated at 37 C, 5% CO 2 until they reached 50% confluency. They were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton and blocked for 1 hour in 1%BSA/1X PBS. The cells were then incubated with 1º Antibody (GAPDH from Santa Cruz #sc-25778) at 1:100 for 3 hours at room temperature. After 3 washes with 1X PBS, cells were then incubated with 2º Antibody (Alexa Fluor 647 anti-rabbit from Invitrogen A21244) for 2 hours at room temperature. After 3 washes in 1X PBS, some cells were incubated for 5 minutes with a dilute solution (300 nm in PBS) of with 4',6-diamidino-2-phenylindole (DAPI) which is a fluorescent stain that binds strongly to A-T rich regions in DNA and used to demarcate the nucleus. After 3 washes with 1X PBS, coverslips were mounted on slides with Prolong Gold (Invitrogen P36934). Cells were then imaged using the Leica SP5 Confocal Microscope. As shown in Figure 4, intracytoplasmic GAPDH was present in highest concentrations in the cell periphery and virtually absent from the perinuclear regions of the cytoplasm.
In some experiments HMEC cells were seeded on glass slides or Cytoo Chips at a concentration of 30 K cells/ml and incubated overnight at 37 C. The next day, cells were glucose starved for 1 hour. Control cells were incubated in complete media. After 1 hour, cells were washed in 1X PBS and fixed for 10 minutes in 4% Paraformaldehyde, permeabilized in 0.1% Triton for 10 min, and blocked in 1%BSA/PBS for 1 hour. For the immunostaining procedure, cells were incubated in a 1:100 dilution of GAPDH at room temperature for 1 hour, followed by incubation with the secondary rabbit antibody, Alexa Fluor 594, at 1:500 for 30 minutes at room temperature. The cells were imaged with a Zeiss Confocal Microscope. Figure 4 shows intracellular localization of GAPDH in HMEC cells on glass slides with and without glucose while Figure 5 shows intracellular localization of GAPDH in HMEC cells on Cytoo Chips with and without glucose.
These clear experimental results support our conjecture that there is an increase in the glycolytic processing as a result of an external glucose gradient. We now describe some analytical models outlining the phase locking of the glucose oscillators and the implications.

Sine circle Map modeling
As suggested in the above experimental work the glucose oscillators likely accumulate at specific regions in the cell to make maximum use of the large glucose gradient. To model this phenomena, we will use an array of sine-circle maps in a grid, called a coupled map lattice, with a global coupling parameter representing the external glucose concentration. This global coupling will result in increases in oscillation as the coupling constant increases. The sine-circle map, represented as a difference equation, is given by As κ increases from 0.5 to 4.5 the output of the function transitions from a fixed point to 2-cycle, 4-cycle and finally chaos. This is a typical bifurcation diagram similar to that produced by the logistic function [49]. If we replace the 2 in the argument of the sine function with 3 or 4 it simply moves the bifurcation points on the θ-κ curve. This is shown in Figure 6.
There has been research showing a quasi-periodic route to chaos in biochemical systems [21,39,50,51]. The biochemical system, in these cases, glycolysis, starts at some given frequency and as external glucose is pulsed, starting at a frequency similar to the glycolysis value, the biochemical reactions begin to phase lock with the external pulses and first results in increased amplitude followed by period doubling as the external glucose pulse frequency increases. Finally, at a stochastic external glucose pulse frequency, the observed internal pulses are only about 3-4 X higher than the original glucose oscillations, but the actual sine wave appears as if there is a frequency and/or amplitude modulation. The oscillations observed in the cell extracts are not chaotic. Analytical models of these biochemical systems [52,53], based on differential equations, support a periodic-doubling route to chaos, though in the real biochemical systems chaos is rarely observed. This is likely due to the fact that a cell is not just "a bag of chemicals" but really a hybrid between a chemical network and molecular nanomachines. The equations of dynamics also apply to mechanical systems, yet real machines are not capable of being driven into chaotic state, because of friction and physical constraints in the machine. Similarly, nanomachines are not likely able to be driven into chaotic states because of secondary bonding effects, e.g. van der Waals forces, and steric hindrance and potential energy surfaces [54].
In the above, cell-free extract, experiments, the investigators monitored NADH to observe the oscillations in the range of 0.002 to 0.005 Hz. Yang, et al. [43] did experiments on tissue models of rabbit ventricular myocytes and observed adenosine diphosphate (ADP) oscillations. Of course, when combining these experimental observations it must be remembered that there are 2 NADH molecules and 4 molecules of ADP per glucose molecule in the glycolysis reaction. Yang et al. [43] observed oscillation increases from about 0.02 to 0.067 Hz; but like the cell-free experiments, they did not observe chaos. Since chaos has been observed in real-world chemical systems [55], the lack of observed chaos in these biochemical and tissue experiments can be explained as follows.
In the case of tissue-based experiments, the oscillators are operating at a fixed frequency all driven by chemical kinetics and Le Chatelier's principle. The observation of increase in frequency is likely the mean-field effect from an increase in the numbers of oscillators and this would also account for the observed effect on decrease of energydensity-rate as the concentration increases. This means the cell is adapting to the excess external glucose by producing more glycolysis oscillators. As will be shown shortly, these oscillators can phase lock with each other and produce oscillations at a frequency about 2 or 3 times higher.
In the case of the cell-free extract, it is not likely that more oscillatorcomponents are being produced on demand, so Le Chatelier's principle will not be modulating the overall molecular network. Instead, the existing molecular components for oscillator construction are fixed, and more in situ oscillators may form because of the excess glucose. Again these oscillators can phase lock and produce the observed frequencies.
We can describe the phase locking with a coupled map lattice. Since the glucose oscillators are modeled here as a sine-circle map, we build our coupled map lattice from these. The definitive reference on coupled map lattices is by Kaneko [56]. Coupled map lattices (hereinafter CML and not to be confused with chronic myelogenous leukemia), are lattice models with, usually, difference equation mapping relations in the cells comprising the lattice. The cell updates are given by x(i) ← f(x(i)). And to include "diffusion" or coupling between the cells one typically modifies the update equation as This is a one-dimensional CML, where the left and right neighbor of cell i are coupled to cell i. We use the sine circle map as the main function ƒ Rather than use a global coupling parameter, ε, as is usually done, we assume a self-regulatory threshold dynamics. The adaptive mechanism is triggered when a glycolytic oscillator exceeds a critical threshold x*, and excess is passed on to its neighbor. As observed in spin-glasses [57], we assume a symmetry breaking effect, so that only one neighbor actually receives the excess and which neighbor, (e.g. left or right) is preserved throughout the dynamical update. Our algorithm for a one-dimensional array is thus: 1. for each oscillator compute the current state Hence, the adaption is triggered when x(i) > x*. This causes unidirectional transport δ(i) = x(i) − x* ; x(i + 1) = x(i + 1) + δ(i). This algorithm has been shown to be capable of universal computation [58]. It does, however require careful tuning of the threshold and bifurcation parameters. For example, since the values of the sine function can reach 1.0 and if x* = 0.15 and κ = 1.0, then the map can blow up. The full phase diagram for x* and κ is given in Figure 7. As expected for any chaotic attractor, there are regions of fixed point, complex oscillations (e.g. 2-cycle, 4-cycle) and regions we label as undefined because one or more oscillators blew up.
Kaneko [56] discusses many options of coupling the oscillators. In all cases the phase diagram contains regions of fixed points, oscillations, and undefined or chaos. This is to be expected from the bifurcation of the function as shown in Figure 6. The hard boundary in our phase diagram at κ = 2 is to be expected as shown in Figure 7. The fact that the oscillations begin prior to 2.5 is due to the fact that the thresholding and signal transfer changes the dynamics. The regions in the phase diagram labeled n are for ncycle, or strange attractor [44].
In mapping this phase diagram to glycolytic oscillations we would not expect all threshold values to be valid. If we set the threshold to 0.5 then the bifurcation parameter represents the glucose dosage and we have 2 cycles over a small range until the 4cycles followed by continued increase in the glucose (bifurcation parameter) results in n-cycles. A 4-cycle in this phase space would look like a doubled 2-cycle, amplitude modulated glycolytic oscillator; and an n-cycle would look like an amplitude or frequency modulated 2-cycle or 4-cycle. These types of modulations have been observed by Hess et al. [29] and von Klitzing and Betz [59].

Mitochondrial transition
We now turn our attention to the mitochondria and explore the possible implications from glycolytic oscillations on mitochondrial stability. It is known that the glycolytic oscillations result in similar pH oscillations (e.g. Hess et al. [29]). But these oscillations are about π/2 out of phase. This has implications on the polarization of the mitochondrial membrane. The cell contains buffer mechanisms to minimize pH imbalance, but too much polarization of the mitochondrial membrane will cause the mitochondria to break down. The phase lag in pH can lead to a potential problem.
Galante et al. [20] showed that an increase of protons (decrease in pH) to an assay of heart mitochondria results in a decrease in the Michaelis-Menten rate constant K m , and an increase in the forward velocity, V f , of the reaction The ratio of V f /K m shows an actual phase transition at pH 7.5. The authors did not discuss the significance of this phase transition. Later work by Aromolaran et al. [42] showed waves of Ca 2+ ions traversing the cell as a result of a localized ATP perturbation. These waves are able to traverse the entire cell within 30 secondsfar faster than diffusion. Other work by Ramanujand and Herman [60] show a nonlinear scaling of glucose metabolism in normal and cancer cells, where the scaling exponent is different for both types of cells. This is analogous to our observed Φ variation as a function of glucose. Lastly, Aon et al. [61] describe experiments on percolation and criticality [62] in mitochondrial networks of a cell. The authors used a local perturbation induced by a two-photon laser excitation. They observed a cell-wide transition to take place within 4 seconds resulting in depolarization of the majority of the mitochondria in the cell. This depolarization of the mitochondria membrane also accompanies an ATP depletion. This can in turn effect the following reactions [63]: D-glucose + ATP → D-glucose-6-phosphate + ADP D-fructose-6-phosphate + ATP → D-fructose-1,6-bisphosphate + ADP 1,3-bisphosphoglycerate + ADP → 3-phosphoglycerate + ATP phosphoenolpyruvate + ADP → pyruvate + ATP with the following heats of formation, respectively: ATP is needed to maintain Ca 2+ . A lag in production of ATP, as the above reactions compete with other reactants and products in the overall molecular network, could induce changes in the cytoskeleton via pH effects on the growth dynamics of the microtubules.
The dynamics of the entire system of substrate metabolism of glucose is beyond the scope of this paper and has already been reported in the literature [64][65][66][67]. But even a small system of two coupled components, where one is phase lagged with respect to the other can give rise to very complex dynamics. For example, the system given by: can result in essentially positive feedback. We show this in Figure 8. Gehrmann et al. [68] model the complete glycolysis reaction system and shown that the enzyme, phosphofructokinase (PFK) is a key component for not only stabilizing ATP but key because it can also result in positive feedback. This in turn can result in a runaway oscillation system similar to that shown in Figure 6. The authors also show chaos, but that is likely an artifact of the modeling since real chemo-mechanical systems comprising nanoscale machines operating on molecules could not possibly result in chaos. More likely the system would break. In this case apoptosis would be the result, not chaos. It is interesting to note that our analysis suggests that ATP oscillations could effect 2phosphoenolpyruvate þ ADP→pyruvate þ ATP amongst other reactions. Using Le Chatelier's principle we can argue that forcing this reaction in the reverse direction by using 3-bromopyruvate will deplete the ATP and thus induce the cancer cell to enter into an apoptotic state. This type of behavior has in fact been reported by Ko et al. [69] and Mathupala et al. [70], who reported significant reduction in tumor volume in mice by treatment with 3-bromopyruvate.

Cytoskeleton disruption
The metabolic shift to glycolysis leads to acidosis, which subsequently results in an acidic extracellular pH [12]. Gillies has documented an acidic extracellular environment in numerous tumors [12]. Several studies link acidosis to genomic instability. Morita et al. show low pH leads to sister-chromatin exchanges and chromosomal aberrations [71,72]. Brusick, Cifone, and Cipollaro report that low pH environments (~pH 6.5) caused genomic toxicity [73][74][75]. Several studies link the tumor environment, with hypoxia and low pH as inducing genomic instability by DNA repair activity being reduced [76], and enrichment for mismatch-repair deficient cells [77]. Thus, convincing evidence exists that a hypoxic and acidic environment will lead to genomic instability due to impairment of DNA repair processes.
In addition, Suresh [78] has reviewed the biomechanics of normal and cancer cells and shows that transformed cells routinely have altered deformability. This effect is likely due to the differences in the actin microfilamentsthe major structural element of a cell [79,80].
The cytoskeleton also consists of microtubules that have a bending stiffness 2.6 X 10 -23 N m 2 which is about 1000 times stiffer than actin filaments [78]. Microtubules, unless stabilized by ligands or microtubule-associated protein, are in a constant dynamic instability process being polymerized and depolymerized with a halflife of about 2 minutes [81]. This dynamic instability phenomenon has been mathematically modeled in detail by Sept et al. [82], and Bolterauer et al. [83] among others. The ends of actin filaments and microtubules are caped with ATP and guanosine triphosphate (GTP), respectively. GTP is created by the citric acid cycle in the mitochondria. Obviously, a mitochondria failure may reduce the concentration of ATP and GTP, if other systems do not compensate for the mitochondrial failure. A reduction in GTP results in a higher rate of depolymerization of microtubules [84] and a decrease in ATP concentration reduces the rate of growth of actin filaments [85]. Too few microtubules and/or microtubules being too short to participate in proper spindle pole formation can lead to mitotic catastrophe and/or potentially lead to chromosome instability. Bakhoum et al. [86] and Thompson et al. [87] describe the mechanism for this instability. Persistent mis-oriented attachment of chromosomes to the spindle microtubules leads to severe chromosome segregation defects.

Summary
We have described an integrated system model for the progression of a healthy cell to a cancer state and some of the implications. The potentially aberrant state of the cell may start by an excess glucose or other nutrient external to the cell impacting the cell or by internal defects leading to metabolic enzyme redistribution processes. This excess nutrient is essentially a chemical potential difference between inside and outside of the cell creating stress. Through a process analogous to Rayleigh-Benard convection, stable molecular oscillators accumulate in the cytoplasm to exploit this chemical gradient. The continued activity of these oscillators results in mitochondrial destabilization, which may occur as a non-equilibrium phase transition. Once the mitochondria begin to perform aberrantly there will be a chemical imbalance in key components for microtubule assembly/disassembly. This imbalance is driven by Le Chatelier's principle. The disruption in microtubule lengths and/or microtubule count will lead to chromosomal instability via kinetochore-microtubule dynamics finally leading to mitotic failure. In unlucky cases this will result in chromosome mis-segregation and cancer if mitotic catastrophe does not occur.

Clinical implications
The above synthesis of ideas surrounding the subject of Warburg initiation, or the transition from aerobic glycolysis to anaerobic suggests not only an avenue for treatment but also an avenue for prevention of cancer.
We hypothesize that excess of glucose and sugar-like energy sources or metabolic enzyme abnormalities, through a non-equilibrium phase transition (a symmetry breaking phenomenon) analogous to the Rayleigh-Benard convection, may cause a cell to prefer to process this energy source using substrate glycolysis. Continued excess substrate glycolysis will cause further phase transitions to disrupt the mitochondria via depolarization and also disrupt microtubule dynamics. When a cell then passes through mitosis, the chance of mitotic failure is increased. When a cell enters mitotic failure, it may undergo an aneuploidy event [88,89]. All this suggests that a low glycemic diet would lower the incidence of cancer, and may suggest a mechanism why metformin, which lowers blood glucose levels, is associated with improved outcomes in diabetic cancer patients [90,91] and reduced risk of pancreatic cancer [92].
The above synthesis of ideas also supports targeting cells that have made the glycolytic switch. For instance, the work of Pedersen [93] and his colleagues (Ko et al. [69,94]; Mathupala et al. [70]) have used 3-bromopyruvate to inhibit glyceraldehyde 3phosphate dehydrogenase (GAPDH), which effectively inhibits glycolysis [95]. In addition, 3-bromopyruvate may force, via Le Chatelier's principle, some reverse reactions to essentially deprive the cancer cell of substrate-created ATP. This leaves the cell little choice except to enter apoptosis. We further hypothesize that a Br derivative of 3phosphoglycerate would similarly, though perhaps not as energetically, and perhaps not as toxically, facilitate via Le Chatelier's principle, a reverse reaction to deprive a cancer cell of ATP.
Further, since microtubule dynamics are dysregulated by glucose oscillations and its associated pH oscillations, we speculate that metronomic dosing of microtubule poisons (e.g. nocodazol, taxol, vinblastine) would be an effective treatment strategy for cancers.
In our list of "prescriptions" above many of these are already known or in use. These current practices are essentially "prediction" of our theory and were strictly based on biophysics with little detailed biochemical or cellular biochemistry being considered.


Exponential Megapriming Pcr (emp) Cloning—seamless Dna Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts. Copyright: ß 2012 Ulrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. AU acknowledges a predoctoral fellowship from the German Academic Exchange Service (DAAD). KRA was funded by the Lundbeck Foundation and a Sapere Aude DFF postdoc grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction
One of the most common tasks in molecular cloning is the insertion of a defined DNA fragment into a target plasmid. Traditionally this is achieved by restriction enzyme mediated sticky end [1][2][3][4] or blunt end [5][6] ligation, greatly facilitated by the advent of PCR [7][8][9]. Nonetheless the efficiency of blunt end ligation is low and sticky end cloning is limited by the availability of suitable restriction sites. To overcome these disadvantages restriction-free cloning techniques have been developed, albeit all with their own limitations. TA-cloning [10] needs special vector treatment and does not discriminate against reverse insertion of the insert. The high-throughput methods Gateway [11][12] and Creator cloning [13] use site-specific recombination, thus depend on specific sequence elements and, in addition, require specific vectors and expensive enzymes. In contrast, homologous recombination techniques rely on fusion of complementary sequences and thus do not need specific sequence elements. In vivo homologous recombination (reviewed in [14]) can be achieved by three mechanisms, all limited in one way or another. RecAdependent recombination [15] requires recA+ strains and long overhangs, RecA-independent recombination [16] is of low efficiency and Red/ET dependent recombination [17] depends on strains overexpressing RedE/RedT/Redc. Homologous recombination in vitro does not require special bacteria strains. Instead recombination is facilitated by in vitro generation of single strand overhangs. For this purpose ligation-independent cloning (LIC) [18] uses the 39-59 exonuclease activity of T4 DNA polymerase. Overhangs are typically generated from ,12 nt terminal sequences lacking one of the four nucleotides. The sequence restriction is necessary to avoid uncontrolled DNA digestion. In the case of sequence and ligation-independent cloning (SLIC) [19] overhangs are determined either by stopping the exonuclease reaction after a certain time, or by PCR. SLIC generated overhangs have no sequence restrictions other than being complementary to the target plasmid. SLIC requires linearizing the target plasmid by enzymatic cleavage or PCR, and the addition of RecA, for highest efficiency. Another recent in vitro recombination technique was presented by Gibson and coworkers [20]. Here double stranded DNA fragments of up to several hundred kilobases with overlapping sequences of 40 bp are assembled in a single reaction using 59 exonuclease, DNA polymerase and DNA ligase. This method can be used for the assembly of genes and entire genomes. A common weakness of all recombination-based cloning techniques is that cloning success cannot be monitored before obtaining colonies, since the intermediary steps are not quantifiable. A megaprimer-based method, restriction-free (RF) cloning [21][22][23], is also sequenceindependent and restriction-free, like SLIC, but in addition does not require enzymatic strand treatment and intermediary steps can be monitored and controlled. Traditionally, the megaprimer PCR method was used to introduce mutations, insertions and deletions into a linear DNA sequence [24][25] or to fuse DNA fragments [26]. In RF cloning the insert is amplified with primers containing overhangs matching a sequence of choice in the target plasmid. In a second PCR reaction the PCR product of the first reaction is used as a megaprimer for linear amplification of the target plasmid. The resulting product can be observed via agarose gel electrophoresis and, in case of success, be transformed. This method works reasonably well for inserts up to 5 kb in length, although in practice efficiency is reduced for inserts .2-3 kb. A major disadvantage of RF cloning is low product yield due to linear PCR amplification, which becomes prohibitive for larger inserts. In the 1 st EMP PCR a forward primer F1 and a reverse primer R1 with overhang exponentially amplify the insert of interest. In the 2 nd EMP PCR reaction the purified product is used as a megaprimer to exponentially amplify the target plasmid together with a forward primer F1 and reverse primer R2. (B) RF cloning also includes two PCR reactions. In the 1 st RF PCR two overhang containing primers F1 and R1 exponentially amplify the insert of interest. In the 2 nd RF PCR the purified product is used as a megaprimer to linearly amplify the target plasmid. Experimental procedures are described in detail in Materials and Methods and Tables S1 and S2. doi:10.1371/journal.pone.0053360.g001 To overcome the limitations of current cloning methods, we developed exponential megapriming PCR (EMP) cloning. EMP cloning shares the advantages of RF cloning, but lifts the size limits for the inserts. EMP requires two consecutive PCR steps, which are both designed to amplify the template exponentially rather than linearly. In the first step, the insert is amplified, and in the second step the insert is integrated into the plasmid (Fig. 1A). Both steps can also be combined into a single PCR reaction. Importantly, simultaneous insertion of several DNA fragments is possible with EMP cloning.

Exponential Megapriming PCR (EMP) Cloning
Overview of EMP cloning. The first step of EMP cloning is the exponential amplification of the insert of choice and the addition of a 39 overhang by PCR. The product of this 1 st EMP PCR is used in a second PCR reaction as a megaprimer to exponentially amplify the target plasmid together with a short reverse primer (Fig. 1A). The product of this 2 nd EMP PCR is then in vitro phosphorylated, ligated and transformed into competent E. coli cells. Single colonies are screened for proper plasmid generation by standard methods, i.e. analytical restriction digest, colony-PCR, or by sequencing.
Synthesis of the EMP megaprimer. For the synthesis of the megaprimer two primers are used in an exponential PCR reaction (1 st EMP PCR). The forward primer (F1) typically has 20-25 nt and matches the 59 region of the insert. The reverse primer (R1) is 40-50 nt in size and contains a 20-25 nt sequence with reverse complementarity to the region on the target plasmid immediately downstream of the insertion site, followed by the reverse complement of the 39 end of the insert. The 1 st EMP PCR reactions contains 16 HF Phusion buffer (NEB), 200 mM of each dNTP, 0.5 mM primer F1, 0.5 mM primer R1, 25 ng template DNA, and 0.02 U/mL Phusion DNA Polymerase (NEB) in a volume of 50 ml. In rare cases GC Phusion buffer and/or the addition of up to 3% (v/v) dimethylsulfoxide (DMSO) can increase the reaction efficiency. The PCR conditions are: initial denaturation step (30 s, 98uC), followed by 25 cycles of denaturation (10 s, 98uC), annealing (30 s, Tm (F1/R1) +3uC) and extension (15 s/ 1 kb, 72uC). A final extension cycle (5 min, 72uC) completes the PCR reaction. For the annealing temperature the lower of the calculated melting temperature of either F1 or the insert binding part of R1 is used. Tm values were calculated using OligoAnalyzer 3.1 (IDT). Product of the 1 st EMP PCR is analyzed by agarose gel electrophoresis and purified with a PCR purification kit (E.Z.N.A. Cylce Pure Kit, Omega bio-tek). See also Table S1.
Insertion of the EMP megaprimer. The megaprimer is used together with a 20-25 nt primer R2, reverse complementary to the region 59 of the insertion site on the target plasmid, to exponentially amplify the target plasmid. The amount of megaprimer is not critical and can be fairly low (25-400 ng), since the re-use of primer F1 again in the 2 nd EMP PCR ensures high product yield. In the first cycles the megaprimer and R2 generate a starting population of insert fused with target plasmid. After the megaprimer is depleted, primers F1 and R2 continue to exponentially amplify the linear product. The 2 nd EMP PCR contains 16 HF Phusion buffer, 200 mM of each dNTP, 0.5 mM primer F1, 0.5 mM primer R2, 25 ng-400 ng megaprimer, 25 ng template DNA, and 0.02 U/mL Phusion DNA Polymerase (NEB) in a volume of 50 ml. The amount of megaprimer should be screened. If F1 is added to the reaction 25-50 ng megaprimer are sufficient. In rare cases the addition of F1 leads to secondary PCR products and thus low product amounts. In those cases no F1 and megaprimer amounts between 100-400 ng should be tried, with the highest success rate at 200 ng in our tests. PCR optimization is performed as described for the 1 st EMP PCR. The 2 nd EMP PCR starts with an initial denaturation step (30 s, 98uC), followed by 25 cycles of denaturation (10 s, 98uC), annealing (30s, Tm (F1/R1/ R2) +3uC) and extension (30 s/1 kb, 72uC) with no final extension. For the calculation of the annealing temperature the lower Tm of either R2, F1, or the plasmid binding sequence of the megaprimer is used. The product of the 2 nd EMP PCR is analyzed by agarose gel electrophoresis and purified with a PCR purification kit. The product is eluted in 30 ml of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. See also Table S1.
In vitro ligation and transformation of EMP product. 16.5 ml product of the 2 nd EMP PCR is incubated in 16 T4 DNA ligase buffer (NEB) with 5 U T4 PNK for 30 min at 37uC to add a 59 phosphate. The product is circularized by ligation with 200 U of T4 DNA ligase for 1 h at room temperature. Remaining parental plasmid is digested by adding 10 U DpnI for 30 min at 37uC. 5 mL of the 20 mL reaction are used for the transformation into 50 mL of chemically competent E. coli DH5a cells.

One-step EMP Cloning
EMP cloning allows to couple megaprimer production and insertion into the target plasmid in a single PCR reaction. In a one-step EMP PCR reaction limiting amounts of the overhang containing primer R1 are used to reach primer depletion in the first cycles. The resulting small amount of megaprimer generates a starting population of product template for the exponential amplification through forward primer F1 and reverse primer R2. A typical reaction contains 0.02 mM primer R1, 0,5 mM primer W1 and 0.5 mM primer R2 in addition to 16 HF QuikChange and inverse PCR allow insertion of up to 50 bp, whereas RF and EMP cloning can accommodate inserts of up to 5 kb. QuikChange and RF cloning use linear amplification to obtain their product. This causes few PCR errors since potential mutations cannot be inherited in the next PCR cycle but also causes low product amounts. Inverse PCR and EMP cloning utilize exponential amplification to obtain high product amounts resulting in intensive, easily observable product bands, and large colony numbers. doi:10.1371/journal.pone.0053360.g002 Phusion buffer, 200 mM of each dNTP, 25 ng template DNA for the insert, 25 ng template DNA for the target plasmid, and 0.02 U/mL Phusion DNA Polymerase (NEB) in a 50 mL reaxtion. See also Table S2.   primer F1 anneals to the beginning insert ensuring that all inserts are getting amplified. Reverse primer R2 binds 59 of the insertion site. In vitro ligation and transformation are performed as in regular EMP cloning. See also Table S3.

Restriction-free (RF) Cloning
Overview RF cloning. The protocol is modified from the original protocol published in van den Ent and Löwe [22]. In the first step of RF cloning the insert of choice gets amplified and overhangs at 39 and 59 end are added. The product of this 1 st RF PCR is used in a 2 nd PCR as a megaprimer to linearly amplify the target plasmid (Fig. 1B). The product of this 2 nd RF PCR can either be directly transformed, or first in vitro ligated and then   Optimization is performed as for the 1st EMP PCR. For the calculation of the annealing temperature the lower Tm of either the insert binding part of F1 or the insert binding part of R1 is used. The product of the 1 st RF PCR is analyzed by agarose gel electrophoresis and purified with a PCR purification kit. See also  Table S4. Insertion of the RF megaprimer. The megaprimer is used to linearly amplify the target plasmid. The overhangs of the megaprimer bind 59 and 39 of the insertion site on the target plasmid. A new product strand does not contain a binding site for the reverse megaprimer and is therefore not a template for the next round of PCR, causing a linear rather than exponential amplification. The 2 nd EMP PCR contains 16 HF buffer (NEB), 200 mM of each dNTP, 100 ng-400 ng megaprimer, 25 ng template DNA, and 0.02 U/mL Phusion DNA Polymerase (NEB). The amount of megaprimer has to be screened. PCR optimization is performed as for the 1 st PCR reaction. The 2 nd RF PCR reaction starts with an initial denaturation step (30 s, 98uC), followed by 35 cycles of denaturation (10 s, 98uC), annealing (30s, Tm (F1/R1) +3uC) and extension (30 s/1 kb, 72uC) with no final extension. The product of the 2 nd RF PCR is analyzed by agarose gel electrophoresis and purified with a PCR purification kit. See also Table S4.
Ligation and transformation of the 2 nd RF PCR product. Van den Ent and Löwe [22] suggest to directly add DpnI to the finished 2 nd RF PCR reaction and incubate for 2 h to digest parental plasmid, followed by transformation in E. coli cells. Since the two complementary product strands can form a circular double stranded plasmid with two single strand nicks in vivo ligation can occur in E. coli cells, albeit with low efficiency.
We instead performed an in vitro ligation by incubating the purified 2 nd RF PCR product in T4 ligase buffer (NEB) with 5 U PNK for 30 min at 37uC, followed by 1 h incubation at room temperature with 200 U of T4 ligase and incubation for 30 min at 37uC with 10 U DpnI. The product is then transformed in E. coli DH5a cells.
The advantage of direct DpnI incubation and transformation of the 2 nd RF PCR reaction, as suggested in [22], is to save time and labor by not performing DNA purification, PNK and T4 ligase incubation. However, the in vivo ligation protocol has severe disadvantages. First, the efficiency of DpnI in PCR buffer is reduced compared to T4 ligase buffer or NEB buffer 4, resulting in higher background due to incompletely digested parental plasmid. Second, in vivo ligation of doubly nicked plasmids is inefficient.
To better compare EMP cloning to RF cloning by eliminating influences of differential product treatment after 2 nd PCR we decided to perform RF cloning with in vitro ligation.

General PCR Optimization
Difficulties during DNA amplification by PCR can arise from the nucleotide sequence of primers and template. Secondary structure motifs, such as intramolecular hairpins, in the primer sequence can inhibit annealing to the template. Formation of primer homo-dimers or hetero-dimers dilute the effective primer concentration and, if primers get extended, also the effective polymerase concentration and nucleotide concentration. These problems can be minimized by a careful primer design [27][28]. Tools for designing primers are available online. Other reasons for PCR failure are GC-rich sequences and base pair repeats in the DNA template [29][30]. GC-rich sequences can lead to the formation of stable, non-B form secondary structure motifs. The ability of the polymerase to amplify difficult templates can be improved by optimizing the PCR buffer by adding Mg 2+ , DMSO, glycerol or formamide [29][30] or using commercial buffers for GC-rich sequences. A general way to trouble-shoot PCR reactions is to vary the primer annealing temperature. Initially an annealing temperature close to the lower melting temperature of the used primers should be tested. If this leads to low product amounts a lower annealing temperature should be tried to allow primer binding. If it results in bands of unwanted products a higher annealing temperature should be tried to avoid unspecific priming [31][32]. PCR errors by the polymerase can be reduced to a minimum by using high fidelity polyermases such as Phusion Polymerase or Pfu Turbo II instead of Taq polymerase [33][34][35][36] and by reducing the number of PCR cycles. PCR error rates can be calculated with web tools like the fidelity calculator by Thermo Fisher (http://www.thermoscientificbio.com/webtools/fidelity/).

The Concept and Mechanism of Exponential Megapriming PCR (EMP) Cloning
The in vitro mutagenesis PCR method QuikChange [37][38] is the conceptual basis for RF cloning, just that it introduces single mutations or small insertions or deletions ,50 bp instead of entire genes. In the QuikChange PCR reaction a complementary primer pair binds to the target site leading to linear product amplification. Although the efficiency of these short manipulations is fairly high, PCR products can often not be observed due to low product amounts. In analogy to how RF cloning relates to QuikChange, EMP cloning relates to inverse PCR (iPCR) [39] (Fig. 2). iPCR can be used for the same manipulations QuikChange was developed for, but it uses non-overlapping primers for exponential target amplification, resulting in drastically higher product amounts and ultimately more positive clones. iPCR was originally problematic due to the relatively high error-rate of native, thermostable DNA polymerases, such as Taq. Since the introduction of genetically engineered polymerases with extraordinarily low error-rates, such as Phusion and PfuUltra II, there is no longer a disadvantage of PCR amplifying large pieces of DNA, such as vector backbones [33][34][35][36]. EMP cloning now uses the mechanism of iPCR to introduce complete genes.
In EMP cloning the insert is amplified with a forward primer (F1) without and a reverse primer with overhang (R1) (Fig. 1A). This leads to a product with 39 overhang complementary to a landing sequence downstream of the desired insertion site in the plasmid. In the second PCR reaction the overhang binds to the target plasmid at the landing site and a second short primer (R2) binds upstream of the insertion site, mimicking the two primers of an iPCR reaction, and resulting in exponential product amplification. After megaprimer depletion the re-used primer F1 and primer R2 continue to exponentially amplify the linear product. This allows relatively low amounts of megaprimer (25 ng) (Fig. 1A, Table S1 and Materials and Methods). In RF cloning in comparison, the first PCR reaction uses two overhang containing primers (F1 and R1) to amplify the insert and to create a megaprimer with two overhangs. In the second PCR this megaprimer binds 39 and 59 of the insertion site thus product amplification is linear. (Fig. 1B, Table S4 and Materials and Methods).
EMP cloning follows a simple protocol leading to bacterial colonies in one day (Fig. 3). The products of the 1 st EMP PCR and 2 nd EMP PCR can be analyzed on an agarose gel and purified with a PCR purification kit. The final product needs to be 59 phosphorylated with T4 Polynucleotide Kinase (PNK), ligated with T4 Ligase and incubated with the restriction enzyme DpnI to digest the parental plasmid. These three enzymatic steps take about 2 h. The product is then transformed into competent E. coli cells and colonies are obtained the next day.

Comparison of EMP Cloning and RF Cloning
To test the performance of EMP cloning we compared it directly to RF cloning. We designed 10 test cases with insert lengths from 0.3 to 5 kb, resulting in plasmids of 3.9 to 13.6 kb ( Table 1). First we compared the product intensities of the 2 nd PCR ( Fig. 4A and Fig. S1). Expectedly, EMP cloning consistently and reproducibly yielded more product than RF cloning, which can then be monitored more easily by gel electrophoresis. Higher product amounts let to more colonies (Fig. 4B). EMP yielded ,1500 colonies whereas RF about 5-fold less (,300 colonies). For inserts .2.5 kb the colony number dropped in both methods. Whereas EMP cloning still produced $ 30 colonies, for RF cloning the number of colonies dropped to an average of 8, and in three cases there were # three colonies. This drastically reduces cloning efficiency in the RF setup.
To compare the cloning efficiency, we isolated plasmids of 10 colonies per experiment, if available, and did a control restriction digest ( Fig. 4C and Fig. S2). Compared to RF cloning, the efficiency of EMP cloning was better in 9 cases, and equal in one case. On average the EMP cloning efficiency was more than twice as high as RF cloning. Again the advantage of EMP cloning versus RF cloning is especially pronounced for inserts . 2.5 kb. EMP cloning worked in all cases, RF cloning failed in three cases.

One-step Reaction and Insertion of Multiple Inserts
EMP cloning is suitable for coupling of megaprimer production and insertion in a single reaction and for adding several inserts into a plasmid simultaneously.
In order to further improve time efficiency of EMP cloning experiments we developed a one-step protocol for EMP. The experiment can be shortened by combining the 1 st and 2 nd EMP PCR in one reaction, followed by one purification. One-step EMP cloning uses limited amounts of overhang containing primer (R1). This generates a small population of megaprimers, sufficient to initiate the production of the desired product plasmid. Primer F1 and R2 then exponentially amplify the product plasmid (Fig. 5A, Table S2 and Materials and Methods). One-step EMP cloning was tested by inserting a 492 bp fragment into a vector using three different amounts of R1 primer. The PCR reaction worked in all cases (Fig. 5B) and correct clones were obtained (Fig. S3).
EMP is an ideal tool for efficient and precise plasmid assembly. To make EMP an even more suitable tool for demanding cloning procedures we developed a multi-insert EMP protocol, enabling simultaneous insertion of several consecutive DNA fragments into a vector (Fig. 6A). This is useful for the generation of plasmids for co-expression of proteins. Multi-insert EMP assembles independently produced megaprimers in a single reaction. Megaprimer 39 overhangs are designed such that they bind in nested manner. The terminal insert then binds to the plasmid backbone downstream of the insertion site. Again Primer F1 and R2 are used to exponentially amplify the product plasmid, where primer F1 binds to the 59 end of the first insert ensuring that all inserts are getting amplified. The reverse primer R2 binds upstream of the insertion site. (Materials and Methods and Table S3). In our test case, we used an empty plasmid and successfully added three ORFs (339 bp, 549 bp, 1155 bp), in one PCR reaction ( Fig. 6A and  6B).

Discussion
EMP is a fast, cost efficient method for seamless insertion of DNA fragments (up to 5 kb inserts tested) into any target plasmid (up to 10.2 kb tested). Using this technology, one can replace or add ORFs, tags or other DNA elements in a one-day procedure leaving no scars behind. Importantly, the method is suitable for most insert lengths of practical importance, when protein expression is concerned. In addition EMP cloning also allows the insertion of multiple sequences at once.
As shown in our comparison study EMP cloning is qualitatively and quantitatively superior to RF cloning by generating higher PCR product amounts, higher colony numbers and a higher ratio of positive clones over background. The improvement is most prominent for inserts . 2.5 kb, where EMP cloning is still very reliable while RF cloning is not.
An important feature of EMP cloning is the option to perform both PCRs in one reaction, which is mechanistically not possible with RF cloning. This speeds up the experimental procedure. The downside of one-step EMP is the missing control checkpoint after the 1 st EMP PCR, therefore a potential failure cannot be traced easily. Although one-step EMP is an attractive option, the standard two-step protocol is probably more reliable since less aberrant amplifications are possible.
The strongest advantage of EMP over contemporary recombination based cloning techniques like SLIC is the possibility to monitor the success of vector-insert fusion prior to transformation: a potential failure can be corrected at an earlier stage, saving time. Furthermore the intensity of the product band provides an estimate of the resulting clone efficiency and thus of the number of clones necessary for successful screening.
The high flexibility in choice of insert and vector, seamless insertion and the high efficiency of EMP cloning make this method an ideal tool for any application that requires the generation of plasmid libraries, for example expression libraries for structural biology. Last but not least, EMP cloning is automatable and should be easily applicable to high-throughput efforts. Figure S1 Agarose gels of 2 nd EMP and 2 nd RF PCRs of the 10 test cases. Products of 2 nd EMP and 2 nd RF PCRs are shown on agarose gels. Product lengths are indicated in parenthesis. The 2 nd PCRs of case 3 are shown twice since RF only worked on the second attempt. (TIF) Figure S2 Control digests of the 10 test cases. Agarose gels of control digests of the 10 test cases are shown. If 10 or more colonies were obtained in an experiment, 10 plasmids were isolated and digested with appropriate restriction enzymes. If less then 10 colonies were obtained, all colonies were analyzed. Circles around colony numbers indicate clones with the expected band patterns. Sizes of the expected products from restriction analysis are indicated to the left of the gels. (TIF) Figure S3 Colony PCR of one-step EMP reaction. Agarose gel of colony PCRs of 7 clones obtained by one-step EMP. 4 out of 7 clones show the right PCR product at 948 bp. (TIF)


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Psychology Medicine

text:
Baclofen into the lateral parabrachial nucleus induces hypertonic sodium chloride intake during cell dehydration

Background Activation of GABAB receptors with baclofen into the lateral parabrachial nucleus (LPBN) induces ingestion of water and 0.3 M NaCl in fluid replete rats. However, up to now, no study has investigated the effects of baclofen injected alone or combined with GABAB receptor antagonist into the LPBN on water and 0.3 M NaCl intake in rats with increased plasma osmolarity (rats treated with an intragastric load of 2 M NaCl). Male Wistar rats with stainless steel cannulas implanted bilaterally into the LPBN were used. Results In fluid replete rats, baclofen (0.5 nmol/0.2 μl), bilaterally injected into the LPBN, induced ingestion of 0.3 M NaCl (14.3 ± 4.1 vs. saline: 0.2 ± 0.2 ml/210 min) and water (7.1 ± 2.9 vs. saline: 0.6 ± 0.5 ml/210 min). In cell-dehydrated rats, bilateral injections of baclofen (0.5 and 1.0 nmol/0.2 μl) into the LPBN induced an increase of 0.3 M NaCl intake (15.6 ± 5.7 and 21.5 ± 3.5 ml/210 min, respectively, vs. saline: 1.7 ± 0.8 ml/210 min) and an early inhibition of water intake (3.5 ± 1.4 and 6.7 ± 2.1 ml/150 min, respectively, vs. saline: 9.2 ± 1.4 ml/150 min). The pretreatment of the LPBN with 2-hydroxysaclofen (GABAB antagonist, 5 nmol/0.2 μl) potentiated the effect of baclofen on 0.3 M NaCl intake in the first 90 min of test and did not modify the inhibition of water intake induced by baclofen in cell-dehydrated rats. Baclofen injected into the LPBN did not affect blood pressure and heart rate. Conclusions Thus, injection of baclofen into the LPBN in cell-dehydrated rats induced ingestion of 0.3 M NaCl and inhibition of water intake, suggesting that even in a hyperosmotic situation, the blockade of LPBN inhibitory mechanisms with baclofen is enough to drive rats to drink hypertonic NaCl, an effect independent of changes in blood pressure.

Background
The lateral parabrachial nucleus (LPBN), is a pontine structure located dorsal to the superior cerebellar peduncle (SCP). The LPBN is reciprocally connected to different areas, such as area postrema (AP) and medial portion of the nucleus of the solitary tract (mNTS) in the hindbrain and forebrain areas, such as the paraventricular nucleus of the hypothalamus, central nucleus of amygdala and median preoptic nucleus [1][2][3][4][5][6][7]. Cells in the LPBN are activated after ingestion of NaCl solutions by dehydrated rats or by rats that received intragastric load of hypertonic NaCl, suggesting that LPBN cells are activated by visceral or taste signals [8][9][10]. Therefore, the LPBN may convey signals that ascend from AP/mNTS to forebrain areas involved in the control of fluid and electrolyte balance.
Studies about the involvement of the LPBN in the control of fluid-electrolyte balance have investigated mainly the inhibitory mechanisms for water and NaCl intake and different neurotransmitters or receptors in the LPBN have been shown to be involved with these mechanisms. For example, bilateral injection of the serotonergic antagonist methysergide into the LPBN increased water and 0.3 M NaCl intake induced by different treatments [sodium depletion; subcutaneous treatment with the combination of furosemide (FURO) + captopril (CAP) and treatment with deoxycorticosterone (DOCA)], while injections of a serotonergic agonist (DOI) reduced these intakes [11][12][13]. Increase of FURO+CAP-induced sodium intake is also produced by the blockade of cholecystokinin (CCK) receptors or activation of GABA A and GABA B receptors into the LPBN [14][15][16]. Therefore, in the LPBN, neurotransmitters like serotonin (5-HT) and CCK activate an inhibitory mechanism, while activation of GABAergic receptors in the LPBN is suggested to deactivate inhibitory mechanisms present in this area increasing water and 0.3 M NaCl intake.
The LPBN is also related to the inhibition of sodium intake by increased plasma osmolarity. Previous studies have shown that the blockade of LPBN inhibitory mechanisms by injections of methysergide or moxonidine combined with an increase in plasma osmolarity (intragastric load of 2 M NaCl) induced an unexpected ingestion of hypertonic NaCl [17,18], a clear paradox considering that 2 M NaCl load usually suppresses sodium appetite [19]. In addition, a previous study showed that activation of GABA A receptors into the LPBN induced 0.3 M NaCl intake in cell-dehydrated rats, suggesting that the increase in plasma osmolarity subsequent to NaCl ingestion does not appear to prevent further salt intake when GABA A receptors in the LPBN are activated [20].
The γ -aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the mammalian central nervous system [21,22]. GABA receptors can be classified into two structurally and pharmacologically distinct subclasses: GABA A and GABA B [23,24]. Activation of GABA A receptors leads to opening of the channel and the conductance of Cl − ions and this effect may be antagonized by bicuculline [25]. GABA B receptors are metabotropic, G protein-coupled receptors that mediate presynaptic and postsynaptic inhibition by reductions in calcium conductance or increases in potassium conductance [24,26]. GABA B receptors are activated by baclofen and 2-hydroxysaclofen antagonizes these receptors [26,27].
A dense plexus of GABA-immunoreactive varicosities has been shown throughout the parabrachial nucleus (PBN) and Kolliker fuse (KF) complex [28]. Both GABA A and GABA B receptors are present in LPBN [29,30] and a previous study showed that baclofen injected into LPBN induced ingestion of 0.3 M NaCl and water in fluid replete rats [31]. In addition, injections of baclofen into the LPBN increased 0.3 M NaCl and water intake by rats injected subcutaneously with the diuretic furosemide combined with the angiotensin converting enzyme inhibitor captopril [16].
Considering the possibility that baclofen deactivates the inhibitory mechanisms and increases 0.3 M NaCl intake in cell-dehydrated rats like that produced by GABA A receptor activation in the LPBN, in the present study, we investigated the effects of bilateral injections of baclofen alone or combined with the GABA B receptor antagonist 2-hydroxysaclofen into the LPBN on 0.3 M NaCl and water intake in rats with increased plasma osmolarity and compared the responses with the effects of baclofen into the LPBN on sodium intake in fluid replete rats that received no pre-treatment. In addition, possible changes in cardiovascular responses to baclofen injected into the LPBN in cell-dehydrated rats were also investigated. Intragastric load of 2 M NaCl produces cell dehydration by increasing plasma osmolality and sodium concentration and also reduces plasma renin activity [32].

Animals
Male Wistar rats weighing 290-310 g were used. The animals were housed in individual stainless steel cages with free access to normal sodium diet (Guabi Rat Chow, Paulinia, SP, Brazil), water and 0.3 M NaCl solution. The position of the bottles containing water and 0.3 M NaCl was rotated daily to avoid place preference. Room temperature was maintained at 23 ± 2°C and humidity at 55 ± 10% in a 12:12 light-dark cycle with light onset at 07:30 AM. The Institutional Ethics Committee on Animal Care and Use of the School of Dentistry, Araçatuba-UNESP approved the experimental protocols used in the present study (Proc. CEEA No. 057/03) and followed the recommendations of the Brazilian College of Animal Experimentation (COBEA). The procedures were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, 1996, USA). All efforts were made to minimize animal discomfort and the number of animals used.

Cerebral cannulas
Rats were anesthetized with ketamine (80 mg/kg of body weight, Agener União, Embu-Guaçu, SP, Brazil) combined with xylazine (7 mg/kg of body weight, Agener União, Embu-Guaçu, SP, Brazil ) and placed in a Kopf stereotaxic instrument (Kopf, Tujunga, CA, USA). The skull was leveled between bregma and lambda. Bilateral stainless steel 23-gauge cannulas were implanted in direction to the LPBN using the following coordinates: 9.4 mm caudal to bregma, 2.2 mm lateral to the midline, and 3.9 mm below the dura mater. The tips of the cannulas were put into place at a point 2 mm above each LPBN. The cannulas were fixed to the cranium using dental acrylic resin and jeweler screws. A 30-gauge metal obturator filled the cannulas between tests. The experiments began 5 days later when the animals had fully recovered from the surgery.

Injections into the LPBN
Bilateral injections into the LPBN were made using 5-μl Hamilton syringe connected by polyethylene tubing (PE-10) to 30-gauge injection cannula. At the time of testing, the obturators were removed and the injection cannula (2 mm longer than the guide cannula) was carefully inserted into the guide cannula, and manual injection was initiated 15 s later. For bilateral injections, the first injection was initially performed in one side, the needle was withdrawn and repositioned on the contralateral side, and then the second injection was made. Therefore, injections were made~1 min apart. The injection volume into the LPBN was 0.2 μl in each site. The obturators were replaced after the injections, and the rats were put back into their cages.

Drugs
The drugs injected into the LPBN were (±)-baclofen and 2-hydroxysaclofen purchased from Sigma-Aldrich (Saint Louis, MO, USA) and were dissolved in 0.15 M NaCl. The doses of baclofen and 2-hydroxysaclofen were chosen based on a previous study [31]. Doses of baclofen produce a long-lasting action (at least for three hours) when injected into the LPBN. The dose of 2-hydroxysaclofen used was an effective dose that blocks GABA B receptors into LPBN as showed previously [31].

Water and 0.3 M NaCl intake
The rats were tested in their home cages. In addition to water and food pellets, rats had access to 0.3 M NaCl for at least 5 days before the experiments began. The effects of baclofen were tested under three different experimental conditions: 1) In the same group of rats (n=9), we evaluated if baclofen affect water and 0.3 M NaCl intake in rats that received no pre-treatment (fluid replete rats) and after intragastric load of 2 M NaCl (cell-dehydrated condition). First, fluid replete rats received injections of baclofen (0.5 nmol/0.2 μl) or saline into the LPBN and 15 min later rats were given water and 0.3 M NaCl in graduated (0.1 ml divisions) glass burettes. Cumulative water and 0.3 M NaCl intake was measured at each 30 min time interval in the next 210 min after the access to water and sodium solution. The same group of animals, received an intragastric load of 2 M NaCl, to induce cellular dehydration. Forty-five minutes later, half of the group received injections of baclofen (0.5 nmol/0.2 μl), and the other half received saline into the LPBN and 15 min later rats had free access to both water and 0.3 M NaCl (two-bottle test). Cumulative water and 0.3 M NaCl intake was recorded at each 30 min time interval in the next 210 min after the access to water and sodium solution. The same procedure was repeated in a second experimental session performed 3 days later in a counterbalanced design. All tests began between 13:00 p.m. and 16:00 p.m.
2) In another group of cell-dehydrated rats (n=11), the effects of two different doses of baclofen (0.5 and 1.0 nmol/0.2 μl) injected into the LPBN on water and 0.3 M NaCl intake were tested. Rats received an intragastric load of 2 M NaCl. Forty-five minutes later, half of the group received baclofen (0.5 or 1.0 nmol/0.2 μl), and the other half received saline injections into the LPBN. The same procedure was repeated in a second experimental session performed 3 days later in a counterbalanced design. Fifteen minutes after LPBN injections, the animals had free access to both water and 0.3 M NaCl. Cumulative water and 0.3 M NaCl intake was recorded at each 30, 60, 90, 120, 180, and 210 min after the access to water and sodium solution. All tests began between 13:00 p.m. and 16:00 p.m.
3) To test a possible participation of GABA B receptors in the effects of baclofen, another group of rats (n=6) received an intragastric load of 2 M NaCl 45 min before bilateral injections of baclofen (0.5 nmol/0.2 μl) or saline into the LPBN, and cumulative water and 0.3 M NaCl intake was measured at each 30 min in the next 210 min, starting 15 min after the injections of baclofen or saline into the LPBN. This group of rats also received bilateral injections of 2-hydroxysaclofen (5 nmol/0.2 μl) or saline into the LPBN, 15 min before baclofen or saline injections. This group of rats was submitted to four tests. In each test the group was divided in two subgroups that received different combinations of treatments into the LPBN. The sequence of the combinations of treatments into the LPBN in each rat in different tests was randomized, and at the end of the tests each rat received all the combinations of treatments into the LPBN. All tests began between 13:00 p.m. and 16:00 p.m.

Arterial pressure and heart rate recordings
Mean arterial pressure (MAP) and heart rate (HR) were recorded in unanesthetized rats. One day before recording, the rats were anesthetized with ketamine (80 mg/kg of body weight) + xylazine (7 mg/kg of body weight), a polyethylene tubing (PE 10 connected to a PE 50) was inserted into the abdominal aorta through the femoral artery for arterial pressure recording. The cannula was guided subcutaneously and exteriorized at the back of the rat. On the next day, the cannula was connected to a P23 Db pressure transducer (Statham Gould) coupled to a pre-amplifier (model ETH-200 Bridge Bio Amplifier, CB Sciences) connected to a Powerlab computer recording system (Powerlab 8SP, ADInstruments) to record MAP and HR. The animals received an intragastric 2 M NaCl load and forty-five minutes after this, one group of rats was tested for the effects of baclofen (1.0 nmol/0.2 μl) and another group for the effects of saline injected into the LPBN on MAP and HR. MAP and HR were recorded for the next 180 minutes after injections into LPBN and the maximum changes were analyzed. During MAP and HR recordings, the rats did not have access to water, 0.3 M NaCl and food.

Histology
On completion of the experiments, the animals received bilateral injections of 0.2 μl of 2% Evans blue dye into the LPBN. Then, they were deeply anesthetized with sodium thiopental (80 mg/kg of body weight) and perfused transcardially with saline followed by 10% formalin. The brains were removed, fixed in 10% formalin, frozen, cut in 60 μm serial coronal sections, stained with Giemsa, and analyzed by light microscopy to confirm the injection sites into the LPBN.

Statistical analysis
The results are reported as means ± S.E.M. Water and 0.3 M NaCl intake were analyzed by two-way analysis of variance (ANOVA) with repeated measures for both factors (treatments and times), followed by Newman-Keuls post hoc test. Significance was set at P < 0.05. The software used to analyze the data was SigmaStat for Windows, version 2.03 from SPSS Inc.

Histological analysis
Similar to previous reports [16,20,31], the LPBN injection sites were centered in the central lateral and dorsolateral portions of the LPBN. Figure 1 shows the typical LPBN injection sites. Injections reaching the ventral lateral and external lateral portions, as well as the Kolliker-Fuse nucleus, were observed in some rats and the results from these rats were included in the analysis. In some rats, injections also spread to the brachium (superior cerebellar peduncle), or slightly ventral to this structure, reaching the dorsal portions of the medial parabrachial nucleus (MPBN) uni-or bilaterally. There was no difference in the effects whether injections were restricted to the LPBN or also spread to brachium and dorsal portions of MPBN.

Effects of bilateral injections of baclofen into the LPBN on water and 0.3 M NaCl intake in fluid replete and celldehydrated rats
Bilateral injection of baclofen (0.5 nmol/0.2 μl, n=9) into the LPBN in fluid replete rats induced 0.3 M NaCl and water intake from 150 to 210 min of test (Figure 2A, C). In rats treated with an intragastric load of 2 M NaCl, baclofen injected into the LPBN increased 0.3 M NaCl intake from 150 to 210 min of test (Figure 2A, B) and reduced water intake from 30 to 150 min of test ( Figure 2C, D). ANOVA showed significant interaction between treatments and time for 0.3 M NaCl intake [F(18, 144) = 4.3; P < 0.001, Figure 2A, B] and water intake [F(18, 144) = 3.3; P < 0.001, Figure 2C, D) in fluid replete rats and cell-dehydrated rats that received baclofen into the LPBN.
A hypotonic mixture of water and 0.3 M NaCl was ingested throughout the experimental session when fluid replete rats received saline in the LPBN. On an average, the rats ingested 0.2 ± 0.  Figure 3A, B), and an inhibition of water intake from 30 to 120 min of test ( Figure 3C, D). There were no significant differences in 0.3 M NaCl intake [F(1,10) = 1.1; P > 0.05, Figure 3A, B] and water intake [F(1,10) = 2.8; P > 0.05, Figure 3C, D], in celldehydrated rats treated with different doses of baclofen (0.5 and 1.0 nmol/0.2 μl) into the LPBN.
Cell-dehydrated rats (n=11) that received saline in the LPBN ingested 1.7 ± 0.8 ml of 0.3 M NaCl and 9.2 ± 1.4 ml of water in 210 min. Considering the total intake, the result is a hypotonic solution (nearly 0.05 M). Celldehydrated rats treated with baclofen (0.5 nmol/0.2 μl) into the LPBN ingested 17.9 ± 4.2 ml of 0.3 M NaCl and 5.2 ± 1.9 ml of water in 210 min, which results in a hypertonic solution (nearly 0.23 M). Cell-dehydrated rats treated with baclofen (1.0 nmol/0.2 μl) into the LPBN also ingested a hypertonic solution (0.21 M).

Effects of the combination of 2-hydroxysaclofen and baclofen into the LPBN on water and 0.3 M NaCl intake in cell-dehydrated rats
ANOVA showed significant differences among treatments for 0.3 M NaCl intake by cell-dehydrated rats that received saline or 2-hydroxysaclofen combined with saline or baclofen into the LPBN [F(3, 15) = 9.5; P < 0.05, Figure 4A, B]. There was significant interaction between treatments and times for water intake [F(18, 90) = 2.2; P < 0.05, Figure 4C, D].
Bilateral injections of baclofen after pretreatment with saline into the LPBN induced 0.3 M NaCl intake from 150 to 210 min of the test with P values ranging from P < 0.005 at 150 min to P < 0.001 at 180 and 210 min ( Figure 4A). Baclofen induced an early inhibitory effect on water intake in the first 150 min of the test with P values ranging from P < 0.05 at 30 min to P < 0.001 at 60 to 150 min ( Figure 4C, D). Previous injections of 2-hydroxysaclofen (5 nmol/0.2 μl) into the LPBN increased 0.3 M NaCl intake induced by baclofen in the first 90 minutes of test (8.6 ± 3.6; 12.0 ± 4.3 vs, baclofen: 0.01 ± 0.01; 1.7 ± 0.7 ml; respectively for 60 and 90 min; P < 0.005, Figure 4A), and did not modify 0.3 M NaCl intake induced by baclofen injected in the same area from 120 to 210 min of test (P > 0.1).
Water intake after 2-hydroxysaclofen + baclofen into the LPBN did not differ from saline + baclofen into the LPBN (P > 0.5 for all times tested, Figure 4C, D). After saline + 2-hydroxysaclofen injection into the LPBN, water or 0.3 M NaCl intake did not differ from control test with saline injections into the LPBN (P > 0.1 at any of the times studied).

Specificity of injections into the LPBN to produce the effects on water and 0.3 M NaCl intake in cell-dehydrated rats
The specificity of the LPBN as the site where baclofen produced the effects on water and 0.3 M NaCl intake in cell-dehydrated rats was confirmed by results from rats in which the injections did not reach the LPBN (misplaced injections). Bilateral injections of baclofen (0.5 nmol), 2-hydroxysaclofen (5 nmol) or 2-hydroxysaclofen combined with baclofen outside the LPBN produced no effect on 0.3 M NaCl or on water intake (Table 1). ANOVA showed no significant difference among treatments for 0.3 M NaCl intake [F(3, 15) = 0.54; P > 0.05] or water intake [F(3, 15) = 1.69; P > 0.05] by celldehydrated rats that received injections in sites outside the LPBN (Table 1). Injections outside the LPBN reached different sites; the majority were dorsal or caudal to the LPBN, although a few were ventral or rostral to the LPBN.

Discussion
In the present study, we showed that bilateral injections of baclofen into the LPBN induced both 0.3 M NaCl and water intake in rats that received no pre-treatment. Bilateral injections of baclofen into the LPBN induced ingestion of 0.3 M NaCl (from 120 to 210 min of test) and inhibited the large increases in water intake in hyperosmotic cell-dehydrated rats. At the end of 210 min of test, fluid replete rats and cell-dehydrated rats ingested a similar amount of 0.3 M NaCl after administration of baclofen into the LPBN despite of the hyperosmolality, hypernatremia, reduction of plasma renin activity and normovolemia present in celldehydrated rats [32]. Results from rats with misplaced injections confirmed that baclofen produces effects on water and 0.3 M NaCl intake if injected specifically in the LPBN and not into the surrounding areas.
In a recent study [20], the authors also showed that the blockade of LPBN inhibitory mechanisms by injections of muscimol in this area also induced 0.3 M NaCl intake in fluid replete rats and in rats treated with intragastric 2 M NaCl load, suggesting that the increase in plasma osmolarity subsequent to NaCl ingestion does not appear to prevent further salt intake when GABA A receptors in the LPBN are activated. Therefore, the present results suggest that baclofen injected into the LPBN induces ingestion of 0.3 M NaCl irrespective of the initial physiological state of the rats. A possible conclusion from these results is that it is not necessary to activate the excitatory mechanisms by baclofen into the LPBN for NaCl intake, which differs from the mechanisms that have been proposed to explain increased sodium intake after the blockade of LPBN inhibitory mechanisms [11][12][13].
Peripheral administration of baclofen reduce water deprivation-induced water intake and reduce water intake in rats pretreated with a hypertonic solution of NaCl [33,34]. The results obtained in this study on the effects of baclofen injected into the LPBN on water intake in dehydrated rats are therefore similar to those previously reported [33]. Ebenezer et al. [34] also demonstrated that systemic administration of baclofen had no effects on water intake in fluid replete rats. Interestingly, in the present study was demonstrated that bilateral injection of baclofen into the LPBN induced water and 0.3 M NaCl intake (150 -210 min of test) in a twobottle test in fluid replete rats. Recently [31], we showed that injections of baclofen into the LPBN induced no water intake if only water was available. As indicated by the present results, ingestion of water usually increased after injections of baclofen into the LPBN when fluid replete rats simultaneously ingested 0.3 M NaCl. In spite of the simultaneous ingestion of water, this intake is not enough to compensate for the increased osmolarity produced by the ingestion of hypertonic NaCl. The fluid ingested (water and 0.3 M NaCl), after injections of baclofen into the LPBN, was hypertonic during most of the test. Therefore, it seems that the increase in plasma osmolarity due to the ingestion of hypertonic NaCl may be reinforcing the effect of baclofen on sodium intake instead of inducing water intake.
We suggest that the effect of baclofen injected into the LPBN on water and sodium intake was due to a direct effect of baclofen on the LPBN. Callera et al. [15] demonstrated that bicuculline injected into the LPBN 60 min after muscimol strongly reduced the effects of muscimol on 0.3 M NaCl and water intake, suggesting that muscimol and probably baclofen is acting at this moment releasing water and sodium intake. On the contrary, baclofen into the LPBN reduced water intake in cell-dehydrated rats (30-150 min of test). Therefore, the effects on 0.3 M NaCl and water intake were due to baclofen facilitating the actions on GABA receptors.
The natriorexigenic effect of baclofen might be a secondary effect of baclofen injected into the LPBN, and reductions in blood pressure or increases in renal excretion would be one possibility. However, baclofen injected into the LPBN produces non-significant effects on blood pressure and renal excretion in fluid replete rats and cell-dehydrated rats [31, present data], suggesting that the natriorexigenic response to baclofen injected into the LPBN is not secondary to cardiovascular responses or increased renal excretion. It seems that injections of baclofen, similar to the injections of muscimol into the LPBN [20], completely block the action of all the inhibitory mechanisms present in the LPBN, which combined with any residual facilitatory signal still present as the one produced by normal levels of ANG II may facilitate water and especially NaCl intake.
Recently [16], we showed that pretreatment of the LPBN with bilateral injections of the nonpeptide AT 1 receptor antagonist losartan reduced 0.3 M NaCl and water intake caused by baclofen injected into the same site in fluid replete rats, as well as the increase in water and 0.3 M NaCl produced by baclofen injected bilaterally into the LPBN in FURO+CAP-treated rats, suggesting that angiotensinergic mechanisms in the LPBN are essential for the dipsogenic and natriorexigenic responses induced by the blockade of LPBN neurons with baclofen in fluid replete rats or FURO+CAP-treated rats. It is possible that ANG II acting on AT 1 receptors in hyperosmotic cell-dehydrated rats is not sufficient to facilitate water intake produced by baclofen in the LPBN. The ingestion of 0.3 M NaCl after baclofen injections into the LPBN takes at least 2 h to start, which is a time enough for changes in the levels of ANG II that acting in the LPBN may intensify the effects of baclofen on LPBN neurons, a step necessary for the release of sodium intake. More studies are necessary to investigate the effects of previous injection of AT 1 receptor antagonist on water and sodium intake induced by baclofen injected into the LPBN in hyperosmotic cell-dehydrated rats. The present study also showed that blockade of the GABA B receptors with 2-hydroxysaclofen alone into the LPBN did not affect sodium intake in cell-dehydrated rats, suggesting that GABAergic mechanisms in the LPBN do not tonically inhibit or facilitate 0.3 M NaCl intake [15,20,31]. In addition, previous blockade of the GABA B receptors with 2-hydroxysaclofen injected into the LPBN produced no effect on late 0.3 M NaCl intake and inhibition of water intake induced by baclofen injected in the same area. Although still not completely clear, the blockade of GABA B receptor potentiated the natriorexigenic effect of baclofen in the first 90 min of the test. An involvement of GABA A and GABA B receptors in the natriorexigenic effect of baclofen has previously been reported [31]. The natriorexigenic effect of baclofen injected into the LPBN in fluid replete rats was reduced by the pre-treatment with the GABA B receptor antagonist 2-hydroxysaclofen or by the GABA A receptor antagonist bicuculline injected into the same area. Perhaps, the activation of GABA B receptors by baclofen injected into the LPBN together with a baseline activation of GABA A receptors by endogenous GABA produces a sufficient inhibition of LPBN mechanisms to release sodium intake. Or instead of endogenous GABA, a nonspecific activation of a limited number of GABA A receptors by baclofen together with the activation of GABA B receptors in the LPBN might inhibit the LPBN mechanisms releasing NaCl intake. In both cases, although only the activation of GABA B receptors produces no sodium intake, the activation of GABA B receptors in the LPBN may facilitate the effects of GABA A receptor activation on sodium intake. An involvement of GABA A receptors on baclofen effects was already reported. The inhibition of food intake by intraperitoneal administration of baclofen was abolished by previous treatment with bicuculline, suggesting a possible involvement of GABA A receptors in the effects of baclofen [35].
Thus, the present results show that the blockade of LPBN mechanisms with baclofen induces hypertonic NaCl intake and reduces water intake in cell-dehydrated rats and induces hypertonic NaCl intake and reduces urinary sodium excretion and diuresis in fluid replete rats [31], all responses consistent with an action of LPBN mechanisms against body fluid volume expansion as previously proposed for the serotonergic mechanism in the LPBN [36]. Thus, further studies in rats submitted to intracellular dehydration are needed to test the effects of GABA receptors of the LPBN on behavioral and renal functions that induce to volume expansion.

Conclusion
In conclusion, the results of our experiments show that fluid replete rats or hyperosmotic cell-dehydrated rats ingest similar amounts of 0.3 M NaCl after bilateral injections of baclofen into the LPBN. This observation means that, after the blockade of LPBN inhibitory mechanisms with baclofen, hypertonic NaCl intake occurs independent of whether plasma renin levels are normal or reduced and independent of changes in blood pressure.