Escherichia coli using three transcription factors, nearly two orders of magnitude more efficient in parts than prior digital implementations.We analyse the pros and cons of analog versus digital computation in living cells. Our analysis is based on fundamental laws of noise in gene and protein expression, which set limits on the energy, time, space, molecular count and part-count resources needed to compute at a given level of precision. We conclude that analog computation is significantly more efficient in its use of resources than deterministic digital computation even at relatively high levels of precision in the cell. Based on this analysis, we conclude that synthetic biology must use analog, collective analog, probabilistic and hybrid analog–digital computational approaches; otherwise, even relatively simple synthetic computations in cells such as addition will exceed energy and molecular-count budgets. We present schematics for efficiently representing analog DNA–protein computation in cells. Analog electronic flow in subthreshold transistors and analog molecular flux in chemical reactions obey Boltzmann exponential laws of thermodynamics and are described by astoundingly similar logarithmic electrochemical potentials. Therefore, cytomorphic circuits can help to map circuit designs between electronic and biochemical domains. We review recent work that uses positive-feedback linearization circuits to architect wide-dynamic-range logarithmic analog computation in  The average 10 μm human cell performs these amazing computations with 0.34 nm self-aligned nanoscale DNA–protein devices, with 20 kT per molecular operation (1 ATP molecule hydrolysed), approximately 0.8 pW of power consumption  and analog (graded) in their nature . The ‘1'The pioneers of digital computation, John von Neumann and Alan Turing, appreciated the great power of analog computation and were investigating it intensely to cope with the limitations of using only logic to compute. Near the end of their lives, they were working on understanding analog computation in brains  and in cSaccharomyces cerevisae (yeast) cells, namely that of adding two numbers in an analog versus a digital fashion. This simple example will help us to analyse general trade-offs w.r.t. energy consumption or w.r.t. the number of molecules needed to implement addition at a given level of computational precision. The analysis will show that, below a certain crossover computational precision, it is highly advantageous to compute in an analog fashion to reduce the energy, part count or number of molecules (and thus volume or space) needed for the computation. It is therefore not surprising that cells exploit analog computation to perform their moderate-precision computations. Other work has also shown that collective analog computation can use several moderate-precision interacting analog units to architect computations of arbitrary precision, e.g. 16-bit addition via four interacting 4-bit-precise analog computational units [In this article, we begin by describing analog circuit schematics for a single gene, which provide a foundation for the rest of the article and a quick introduction to the computational basics of cell biology. Then, we review the pros and cons of analog versus digital computation with a focus on computation in living cells. In particular, we extend a prior analysis ,14 to a al units , on an eal units . CollectIn analog computation, every signal and every device are not reliable, but important final or decisive outputs are. To attenuate noise, analog computation invariably relies on feedback loops, the wise use of energy, time or space resources to improve precision via averaging at critical state variables and reference inputs, learning and adaptation, nonlinearities, such as thresholding or digitization, or invariants and attractors in the analog dynamical system that cause it to equilibrate to restorative discrete outputs. Though noise limits information capacity, it can sometimes be beneficial for searching intractable combinatorial spaces and for improving the detection of signals by phenomena such as stochastic resonance.cytomorphic fashion, as Escherichia coli bacterial cells, which were inspired by log-domain electronic circuits, could efficiently compute logarithms, add, subtract, divide and do square-root-like computations with less than three genetic parts. By contrast, a prior in vitro 2-bit-precise digital square-root computation required 130 DNA-based parts [How do we transfer analog circuits in electronics to analog circuits in cells? Fortunately, there is a deep connection between electronics and chemistry, which greatly aids the design of analog circuit motifs and analog computation in synthetic biology. This deep connection arises because there are astounding similarities between the equations that describe noisy electronic flow in subthreshold transistors and the equations that describe noisy molecular flow in chemical reactions, both of which obey the laws of exponential thermodynamics ,21. Thered parts . Anothered parts  was ableed parts . The gened parts  may haveThe analog circuits approach described in this article may enable large-scale design and analysis in synthetic and systems biology, which is faithful to how messy analog biology works, quite different from clean, well-defined digital design. It may enable several applications in synthetic biology, wherein, just as in electronics today, all applications benefit from low-part-count, low energy usage and clever analog feedback loops to improve performance. For example, synthetic biological circuits will be increasingly important in finding cures for diabetes, cancer, autoimmune, infectious and other diseases  wherein Section 2 provides an analog circuit schematic of a gene that is useful for accurately representing steady-state, dynamic and stochastic phenomena. The success of analog circuit design arises in large part from having efficient pictorial representations that are more intuitive to humans than reams of differential equations. Intuition aids design. The analog circuit schematics nevertheless preserve most of the needed mathematical information in the equation as labelled parameters, such that analysis can aid design. Section 3 discusses the pros and cons of analog versus digital computation, first intuitively, then quantitatively. Section 4 discusses the deep similarities between electronics and chemistry, which can enable powerful cytomorphic circuit design in cells owing to the presence of a common logarithmic electrochemical potential. Section 5 provides examples of such circuit design using log-domain genetic circuits. Section 6 concludes with a summary of seven benefits of analog computation in synthetic biology.2.a shows an analog circuit schematic of a genetic circuit with two inputs: one activator, Vact, which enhances the rate of gene transcription (expression) and one repressor, Vrep, which suppresses the rate of gene transcription. The gene transcription initiates from a ‘promoter region’ on the DNA, Pact−rep, near which these molecules bind and interact with the DNA reading machinery of the cell. The diamond-shaped dependent current generator, Pact−rep, outputs an mRNA transcription current that is controlled by these ‘transcription factor’ inputs via an ‘analogic’ basis function with both linear analog and saturating digital regions of operation (the minimum and maximum expressions) [C  . The mRNa ‘translates’ VmRNA via ribosomal machinery in the cell to create a synthesis protein current, which accumulates on a capacitor to create Vprot, the molecular concentration. The resistor Rprot degrades the protein such that the synthesis translation current balances the protein degradation current and Vprot reaches a steady-state value. The equations in figure 1a define Av and βeff, which determine the gain from VmRNA to Vprot and a noise factor similar to the current gain of a bipolar transistor, respectively [βeff for determining noise in §3. The dynamics and stochastics (noise) of the circuit of figure 1a faithfully capture experimental dynamic data [βeff in figure 1a corresponds to a ‘burst factor’ that has been experimentally measured [figure 1a, create analogic basis functions that are mathematically exact representations of prior models [E. coli [The dependent current generator on the right end of figure 1mic data  as well mic data  providedmic data , is simpmeasured . Compactr models . They al[E. coli .Rprot is typically much greater than RmRNA, it is common to assume that the mRNA dynamics are relatively instantaneous compared with protein dynamics [figure 1b, which is a simplified representation of figure 1a without mRNA dynamics and without explicit mRNA state variables, is significantly more convenient and usually accurate. It allows one to focus on a protein-input–protein-output point of view. In this article, our noise analysis in §3 is based on the accurate figure 1a; all other genetic circuit schematics are drawn similar to that of figure 1b.As dynamics . Thus, t3.We begin with an intuitive comparison of the pros and cons of analog versus digital computation presented in (a)S. cerevisae. Hence, we shall use it as our representative cell. For any gene, the Poisson shot noise of molecular flux that produces mRNA from genetic DNA and of molecular flux that produces protein from the mRNA is averaged over the bandwidth of an RC-like protein degradation filter to generate a mean protein copy number Nprot [a, Nprot and Vprot, respectively.There are a lot of experimental measurements and numbers available for er Nprot ,31. It a Nprot [ . In figuNmRNA, are low (0.1 to 130 in S. cerevisae) [6 in S. cerevisae), and mRNA degradation time constants, τmRNA, are small (3–90 min in S. cerevisae) [τprot, are large (10–1000 min in S. cerevisae) [Av=Nprot/NmRNA or Vprot/VmRNA in figure 1a, which amplifies the mRNA noise content in the protein signal. However, owing to the averaging caused by an effective bandwidth reduction factor of τmRNA/τprot, there is also a reduction in mRNA noise content in the protein signal in figure 1a. The total noise of the protein signal is owing to the net mRNA noise content as well as that owing to the intrinsic shot noise of the Poisson protein molecular flux. That is,a show circuit parameters for βeff and Av. These circuit parameters reproduce equation , the molecular protein copy number Nprot and consequently the mRNA copy number need to increase in proportion with SN:Typically, mean mRNA copy numbers, revisae) –37, whilrevisae)  while prrevisae) . Thereforecision  is given(b)EmRNA≈30LmRNA [LmRNA is the length of the mRNA molecule in nucleotides, with a median value of LmRNA=1474 nucleotides for yeast [Eprot≈50Lprot [Lprot is the length of the protein molecule in amino acids, with a median value of Lprot=385 amino acids in yeast. Hence, the power consumption needed to charge or maintain a protein signal level of Nprot is given byProtein and mRNA molecules have to be constantly synthesized to counter their degradation, and this synthesis consumes power. The synthesis power is provided by the hydrolysis of several molecules of adenosine triphosphate (ATP), the universal energy-providing molecular currency of cells. ATP hydrolysis enables various processes in transcription and translation to move forward in a nearly irreversible fashion . The hyd≈30LmRNA , where Lor yeast ,36. Simi≈50Lprot , where L(c)Nprot in the boxed expression in equation (3.5) with its value in equation (3.4), we find another important relationshipresource–precision equation [τprot) and the precision (SN) of signals in a computation increase. Such resource–precision relationships are universal and can be found in neurobiological, electrical, mechanical and all systems [If we substitute for equation ,14 that  systems . For exaSN) and speed (1/τprot), there is an optimum value of βeff that reduces power consumption. Simple differentiation reveals that this optimum occurs whenβeff in equation (3.5) or in equation (3.6) to determine the power consumptionτprot, the optimum βeff can be attained either by varying the gain Av if the time constants are fixed, or by varying τmRNA if Av is fixed. If τmRNA is fixed, and 1/τprot is increased to improve the speed of the computation, then Av must be lowered to maintain βeff at its optimal value.Equation (3.6) suggests that, for a given precision Kd, the binding constant of the DNA promoter. A large Kd can be architected by using weak, mutated or multiple-binding-site promoters [To implement a molecular adder via analog computation is simple: we have the two inputs to be added; each regulates the expression of a common output protein from independent genetic promoters. During synthesis, the two molecular fluxes will automatically add to create the common output protein, which is the answer. To enable linear analog operation and avoid saturated digital operation, the input proteins must operate at a concentration that is well below romoters ,31. As tSN all molecular counts must be increased by a factor of 2 at the input and output, leading to a four times larger total molecular count for the same SN. Thus, the fundamental resource–precision equations for one signal, equations (3.4) and (3.5), cause the resource–precision equations for the molecular count, NA, and power consumption, PA, of the analog adder to beAnalogous to how mRNA noise flux propagates to protein outputs and increases noise, the input noise flux propagates to the adder output. Thus, the adder will have twice the squared noise as would be expected from its output molecular count alone. Therefore, to preserve the ribed in  can be u(e)N-bit digital molecular adder requires 1 half-adder logic circuit (binary addition without carry for the least significant bit), N−1 full-adder circuits (binary addition with carry for the N−1 other bits) and two N-bit inputs. Appendix A presents that the total number of logic gates, each of whose outputs requires energy-consuming molecular synthesis, as well as 2N inputs, adds up to 17 N−7 net protein (and associated mRNA) state variables for N≥2. If N=1, six protein state variables are needed.An NmRNA varies from 0.1 to 130 in yeast and protein copy numbers then vary proportionately [Kd variations among gates. The parameter Kd is an equilibrium chemical binding constant that effectively behaves as a threshold voltage in the digital operation.At high ‘1’ levels, protein synthesis occurs at a high rate, and at low ‘0’ levels, protein synthesis occurs at a lower rate. To minimize the power consumption, digital logic gates must have as small a protein copy number for high values as possible. At low values, the ‘leakage’ or ‘basal transcription’ in the genetic circuits will then limit practical implementations to 100-fold or 1000-fold lower protein copy numbers (ionately –37). HowKd of the logic promoter such that multiple logic gates can function together in fan-out or cascade configurations with good noise margins and in spite of Kd variations among gates. Note that Kd variations among gates can be static or dynamic owing to loading and crosstalk. As in all engineering systems, a robustness–efficiency trade-off exists: a large ‘1’ value will robustly guarantee saturation and signal restoration, good noise margins with low error rate, robust fan-out and robustness to Kd variations but will require higher power consumption to encode the ‘1’.A reasonable design choice is to use a ‘1’ value that is at least 10 times the Kd's for signalling proteins are usually in the 30 nM–10 μM range [Kd and at a ‘0’ value that is at least 200 times lower. As yeast is approximately 200 μm3 in volume [NdigHI=20 000, then NdigLO=20 000/200=100, which is about two times the lowest measured copy numbers in yeast [NdigLO does not matter too much as long as it is sufficiently low.Measured μM range , and somμM range . We shaln volume ,43, 200 n volume –37). Thuin yeast –37. The Kd; but, we could reduce the ‘1’ value to operate at 5Kd owing to higher digital slopes, thus halving molecular count and power consumption. Thus, we shall, for simplicity, ignore dimerization and polymerization effects that are, to first order, neutral w.r.t. resource consumption in our analysis. For simplicity, we shall also assume that any logic gate operates at saturated values that are the same as that of its inputs such that logic gates can be easily composed and cascaded similar to that in today's electronic systems. Otherwise, digital implementations will require molecular gain and attenuation between logic stages. So, our assumption is generous to digital implementations.Many signalling proteins require at least dimerization to bind, which doubles molecular count and power consumption for the same N+2)τprot. Therefore, for the same speed as a parallel analog adder, each logic stage of the digital adder must decrease its degradation time constant, τdigN, according toβeff given by equation τprot) and precision , we use equation , we can compare analog and digital computation w.r.t. their resource consumption of molecules or power consumption. The resource–precision equations for analog addition are described by equation , while tequation  to convea reveals that the costs of analog power consumption in ATP s−1 are significantly less than those of digital power consumption even at nearly 10 bits of precision. At this crossover precision, the power consumption is a staggering 10 million ATP s−1, so high that a yeast cell will be using almost its entire power budget to add two numbers at 10-bit precision! It is not surprising then that cells do not waste their energy doing highly accurate digital computation as computers do. The figure also implies that a synthetic biological circuit that just has 163 simple two-input deterministically robust logic gates is not practical. Thus, deterministic digital approaches to synthetic computation have barely scaled over a decade [NdigHI. A lower value of NdigHI will necessarily make digital computation less robust and more probabilistic as we discuss in §3g. Probabilistic ‘dithering’ converts digital black-and-white pixels in an image to greyscale pixels. Thus probabilistic digital operation will have similarities to noisy analog operation, which is in fact noisy precisely because many probabilistic digital Poisson events constitute its value [Figure 2a decade  and seemts value .Figure b shows that the costs of analog addition in total protein molecular number are also significantly less than those of digital addition even at nearly 8 bits of precision. At this crossover precision, a staggering 1 million protein molecules are involved in addition. This number is about 1 in every 1000 protein molecules in yeast [figure 2b, figure 2a shows that the corresponding analog power consumption is almost an order of magnitude less than digital power consumption.Figure 2in yeast , which min yeast , this 1/High molecular copy numbers and high power consumption are characteristic of high-precision computation. Both lead to toxicity in the cell: high protein copy numbers can cause inadvertent binding of high-copy-number molecules to molecules in essential pathways and interfere with their function via competitive binding. High power consumption generates reactive oxygen species that lead to oxidative stress, cellular damage and cell death. So, high power consumption is not just an undesirable feature in cells as it is in some electronic systems: it actually leads to death. In some electronic systems, especially digital ones, high power consumption can lead to catastrophic failure of portions of a chip via thermal runaway effects.(g)a,b but the exact crossover precision depends on the computational task. Tasks that can be performed with fewer analog parts push the crossover point to higher precision [Do the lessons learned from the example of addition generalize to other computations? Are these lessons sensitive to our parameter values and assumptions? All computations have analog–digital crossover curves similar to those in figure 2recision ,14. FeedNdigHI=2 K instead of 20 K such that digital power consumption is reduced by nearly 10 times. The analog–digital crossover point for power consumption will then be near 7 bits rather than 10 bits for addition, which is still very precise for a cell. In practice, there may be several problems associated with this lower value of NdigHI: with NdigHI=2 K, we require operation at Kd=1 nM for all gates in the digital adder. The low Kd requires significant DNA–protein engineering to improve chemical binding for all adder molecules, and also leads to a significant slowing of transcription-factor binding to DNA analogous to similar trade-offs seen in translation [NdigHI increases the bit error rate in each channel, making the overall digital computation more probabilistic. As the median protein copy number in yeast is nearly 2000 [NdigHI may also lead to significant crosstalk owing to competitive binding with background molecules. As βeff is not likely at its optimal value for all digital signal variables as we have assumed, each of them will be noisier, such that the lower NdigHI may significantly increase bit error rate. Finally, even with NdigHI at a 10 times lower value, nearly 10% of the cell's power budget would just be spent on 7-bit addition, compromising its other functions severely. At a 100 times lower value of NdigHI=200, digital operation will be highly probabilistic owing to insufficient noise margins and it would require significant engineering to reduce NdigLO to values near 20 to achieve on–off ratios of just 10: protein copy numbers below 50 are extremely rare in yeast [NdigHI=20 K to get yeast cells to perform digital addition with 163 gates in a deterministic and robust digital fashion appears to be reasonable. By contrast, as the analog approach only requires two genes, it is significantly easier to engineer and optimize.As an example of how parameters can shift the analog–digital crossover point, let us assume that nslation . Higher rly 2000 –37, the in yeast –37. So oN=Nbits in digital computation, e.g. in equation  [in vitro molecular computation has attempted to engineer neural-network-like computations [Hybrid analog–digital architectures termed as s (HSMs)  are usefs (HSMs) . These ms (HSMs) ,20. Indes (HSMs) , and in utations .4.a) and electronic current flow in the subthreshold regime of transistor operation (b): electron concentration at the source is analogous to reactant concentration; electron concentration at the drain is analogous to product concentration; forward and reverse current flows in the transistor are analogous to forward and reverse reaction rates in a chemical reaction; the forward and reverse currents in a transistor are exponential in voltage differences at its terminals analogous to reaction rates being exponential in the free-energy differences in a chemical reaction; increases in gate voltage lower energy barriers in a transistor increasing current flow analogous to the effects of enzymes or catalysts in chemical reactions that increase reaction rates; and the stochastics of the Poisson shot noise in subthreshold transistors are analogous to the stochastics of molecular shot noise in reactions. Some of these analogies have been listed in the There are striking similarities between chemical-reaction dynamics a and eleperation b: electrThe logarithmic dependence of the electrochemical potential in chemical concentration or of current enables one to map log-domain analog transistor circuit motifs in electronics to log-domain analog molecular circuit motifs in cells and vice versa.x/Kd) for x<Kd and saturating after x=10 Kd; or, if we look at the right-hand side, it may be viewed as being log-linear over the range 0.1Kd<x<10Kd where the sigmoid is linear. Hence, this basis function has a log-linear analog regime of operation over the central portion of the sigmoid and a saturating digital regime at the extremes of the sigmoid. Note that ln(Kd) is proportional to the free energy of the binding reaction. Highly compact eight-transistor log-domain analog differential-pair circuits generate almost exactly identical mathematical basis functions in either the linear current [x and u are vector state and input variables, respectively, the matrix coefficients are determined by chemical-reaction parameters, and the multiply operations refer to outer products derived from two-molecule chemical binding. The limitation to two-molecule binding is for practicality and leads to no loss of generality [If we examine the classic Michaelis–Menten enzyme–substrate binding basis function, we find that current   and is seen in many natural systems, including audition, vision and in cells .a shows a genetic circuit motif [figure 4b that simultaneously alleviates both these saturation problems to widen the dynamic range.If the concentration of a transcription factor is fixed, as the inducer increases in value, it will eventually be bound to all the available transcription-factor molecules and saturate the number of bound inducer–transcription-factor complexes that are possible. In addition, if the number of DNA-binding sites for a complex is limited, these sites will eventually all be bound by complexes and gene expression will saturate. These two sources of saturation limit the dynamic range of inducer operation. Figure 4it motif  and an aa, a positive-feedback loop increases the generation of transcription factor by catalysing its own production on a low-copy plasmid. As the amount of inducer increases, more transcription factors are created alleviating the saturation of complexes. The high-copy plasmid has several binding sites that also shunt away the complexes, thus alleviating the saturation of DNA-binding sites on the low-copy plasmid. The high-copy plasmid also serves to control the gain of the positive-feedback loop by altering the number of complexes available to the low-copy plasmid [Figure 5a shows that the log-linear dynamic range is almost four orders of magnitude.In figure 4 plasmid . Such coa actually exploits an idea from log-domain analog circuit design shown in figure 5b: expansive sinh-based linearization of compressive and saturating tanh-based differential-pair circuits causes them to widen their linear range of operation [sinh is achieved by positive feedback in figure 4a and the function of the tanh is analogous to that of the saturation of biochemical binding. Indeed, in the future, other linearization circuit motifs from log-domain circuit design, e.g. those in [figure 4a is achieved at an optimal value of positive feedback.The genetic circuit of figure 4peration . The funthose in , could athose in  models dthose in , the wida and its associated schematic in figure 6b show how to use logarithms to divide by using activating its production. The AHL input generates LacI on the low-copy plasmid, which then represses mCherry production. The IPTG inducer fine-tunes this repression gain. The RBS1 and RBS2 translation gains  also serve to fine-tune the gain of the positive logarithm w.r.t. the gain of the negative logarithm. The net result is that the logarithmic ratio of the two inducer molecules in figure 6c is obtained over almost four orders of magnitude. This ‘pRATIO’ circuit generalizes the concept of pH, a logarithmic ratio used to measure H+ concentration w.r.t. a reference, to any arbitrary ratio of two inputs w.r.t. one another. It may have applications for the wide-dynamic-range sensing of biomolecules by serving as a log differential amplifier.Figure 6et al. [figure 6a,b. Scaling is also implicit in figure 6a,b . The equivalent of addition, subtraction and scaling with logarithms corresponds to multiplication, division and power laws in the linear domain, much as in the operation of slide rules.In log-linear systems, the computational basis functions needed for universality besides the logarithm itself are addition, subtraction and scaling. Daniel et al.  shows hoa and its associated circuit schematic in figure 7b show how to compute a power law without any explicit use of logarithms: the high-copy plasmid produces a high level of LacI that strongly represses AraC production with AraC=gAraC(Kdf/LacI)(IPTG/Km)h; IPTG controls the strength of its de-repression according to the inherent power law h, and gAraC is the gain of the first-stage amplifier in figure 7b. As the low-copy production of AraC does not saturate the binding sites on the high-copy plasmid, the falling AraC reduces the level of LacI in a relatively linear fashion according to LacI=gLacI (AraC/Kd), with gLacI being the gain of the second-stage amplifier of figure 7b. The net consequence of the negative feedback is then that LacI must equilibrate at a consistent value, whereh/2, which in figure 7c is found to be about 0.7. The supplementary section in [in vitro circuit, which required 130 DNA parts to implement a square-root circuit with 2 bits of output precision [Figure 7ction in  providesrecision .Figure 6.Digital computation is a subset of analog computation that only operates at its saturated high or low extremes. Therefore, by allowing operation over the whole range of signal levels and by allowing exploitation of all the basis functions of biochemistry and biophysics, not just logic, horizons for computation are expanded. Digital computation will continue to be important for decision-making, signal restoration, communication and sequential operation.(1) At the moderate precision of computation seen in cells, analog computation is significantly more efficient in its use of energy, time and space (molecular count and part count) than digital computation. Therefore, analog and collective analog computation is likely to be more practical and scalable for synthetic biology than purely digital computation.(2) Probabilistic digital computation operates with analog probabilities, and behaves as noisy analog computation. As even digital computation in cells may be forced to be probabilistic to cope with constraints on part count, molecular count and energy, analog computation may be inevitable.(3) Problems such as loading and fan-out, which are limiting the scaling of synthetic biology, are part of the design process of analog computation, which uses concepts of input and output impedance and feedback design to cope with such problems. It can even exploit them for computation .(4) putation a,b.Analog design, which has been developed over several decades, provides accurate pictorial motifs with abstraction (b) unlike pure differential-equation design, but these abstractions are not oversimplified as in molecular logic design.(5) traction b unlike analog computation in the cell may have predated and pioneered analog computation in the brain.(6) The great similarities between neural and cellular computation suggest that deep cytomorphic connection between electronics and chemistry via the common electrochemical potential can enable log-domain analog electronic circuits to inspire new analog circuit motifs in cells and vice versa. Therefore, future work in synthetic biology or in systems biology will be likely to benefit from the cytomorphic mapping .(7) The  mapping a,b.We summarize by reviewing seven benefits of analog computation in cells, which are important for synthetic (and systems) biology.


To search for possible differences in its expression strategy with respect to HTLV-1, we investigated the pattern of HTLV-2 expression in infected cell lines and peripheral blood mononuclear cells (PBMCs) from infected patients using splice site-specific quantitative RT-PCR.Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are delta retroviruses with similar genetic organization. Although both viruses immortalize T-cells env transcripts was found to be subtype-specific. Time-course analysis revealed a two-phase expression kinetics in an infected cell line and in PBMCs of two of the three patients examined; this pattern was reminiscent of HTLV-1. In addition, the minus-strand APH2 transcript was mainly detected in the nucleus, a feature that was similar to its HTLV-1 orthologue HBZ. In contrast to HTLV-1, expression of the mRNA encoding the main regulatory proteins Tax and Rex and that of the mRNAs encoding the p28 and truncated Rex inhibitors is skewed towards p28/truncated Rex inhibitors in HTLV-2.A novel alternative splice acceptor site for exon 2 was identified; its usage in Our data suggest a general converging pattern of expression of HTLV-2 and HTLV-1 and highlight peculiar differences in the expression of regulatory proteins that might influence the pathobiology of these viruses. P. L. Green). Results showed that the env(1-2iso) mRNA was expressed at similar levels in both cell lines, while the canonic env(1–2) transcript was about 100-fold less abundant in BJAB-Gu cells , suggesting a subtype-specific usage of the 2 vs. 2iso 3’SS  mRNA which was readily detected in both cell lines and in the molecular clone pH6neo (data not shown). Selection of 1-2iso 3'SS as opposed to the 1–2 3'SS is not predicted to affect the coding potential of the env, tax/rex or 1-2-B and 1-2iso-B mRNAs, as both 3'SS are located in the non-coding portion of the transcripts. Interestingly, the 1-2iso splice site is not unique to only the BJAB-Gu cell line, as it is efficiently used in the patients, which are infected with subtype 2B (see next paragraph).Using two splice site-specific primer pairs, we quantified the levels of the alternative s Figure A. Interets . This is in contrast with HTLV-1, where tax/rex is much more abundant than the mRNAs coding for p30Tof and p21Rex, the orthologs of p28 and tRex, respectively  is skewed toward tax/rex in HTLV-1 and towards the inhibitors in HTLV-2; (ii) the p28/trex mRNA was expressed as a “late” transcript in HTLV-2 while mRNAs for HTLV-1 orthologs p30/Tof and p21Rex are expressed early in most patients examined, suggesting interesting differences in the fine-tuning of Rex function between the two viruses. Nevertheless, it is appropriate to be cautious in the interpretation of these data, since only three patients were examined. This limitation was due to the fact that the kinetics analysis required (i) large numbers of PBMC, (ii) a high proviral load, and (iii) no coinfection with HIV or HTLV-1, which severely limited the number of samples suitable for this analysis.Taken together with previous analyses of HTLV-1 expression, the present study suggests a converging expression strategy of the two viruses characterized by 2-phase expression kinetics of viral mRNAs and by marked nuclear localization of minus-strand transcripts. Our results also highlight the following differences between the 2 viruses: (i) the relative expression of the Future studies should be aimed at further investigating the functions of these regulatory proteins in HTLV-1 and HTLV-2 and at understanding how they may contribute to the different pathobiology of the two viruses.The authors declare that they have no competing interests.CB identified the novel splice acceptor site and carried out kinetics of expression in cell lines and patients. FR carried out nucleo-cytoplasmic fractionations and real-time RT-PCR assays; AC and PR set real-time RT-PCR and measured mRNA expression in cell lines; PR carried out cell lines and patients cultures; IC carried out cell culture and transfection; CC provided patient samples and supervised cell cultures; VC and UB designed the experiments and prepared the manuscript; and all authors contributed to the analysis and interpretation of the data. All authors read and approved the final manuscript. Table S1. List of primer sets and probes used in qPCR detection of HTLV-2 proviral load or HTLV-2 mRNAs expression.Click here for file Table S2. Clinical and immunological features of HTLV-2 infected patients.Click here for file


The growth signaling pathways downstream of β1 and β3 integrins are well characterized. However, new integrin pathways responsible for inhibiting apoptosis induced by hemodynamic overload are emerging. β1 and β3 integrins activate differential survival signaling, yet both integrins initiate survival signaling downstream of ubiquitination and the kinase pathway including phosphoinositol-3-kinase (PI3K)/Akt. Further characterization of these integrin-signaling mechanisms may lead to drug targets to prevent decompensation to heart failure.To compensate for hemodynamic overload of the heart, an event which stretches the myocardium, growth and survival signaling are activated in cardiac muscle cells (cardiomyocytes). Integrins serve as the signaling receptors of cardiomyocytes responsible for mechanotransduction toward intracellular signaling. The main integrin heterodimers on the cardiomyocyte surface are  Cardiac hypertrophy begins as a compensatory response to hemodynamic overload, such as from increased pressure with hypertension. Several cell types are present in the ventricular wall that provide diverse contributions for optimal organ performance. Cardiomyocytes are the muscle cells in the heart which make up myofibrils that contract to pump blood to the body. Like skeletal muscle cells, cardiomyocytes are striated, but only cardiomyocytes have intercalated discs, which connect adjacent cells and help them simultaneously contract. While all cell types in the heart differentially respond to hemodynamic changes, the cardiomyocytes ultimately regulate cardiac performance through pump function and must change intracellular signaling pathways most substantially. Hypertrophy is defined by the increase in protein synthesis, cardiomyocyte size, and cytoskeletal remodeling, which are important to increase the ventricle wall thickness to normalize against the mechanical stress  and optiHemodynamic overload of the heart instigates many stimuli received by cardiomyocytes for induction of growth, including neurohormonal changes, mobilization of growth factors, stretch of the myocardium, and extracellular matrix (ECM) remodeling. Many of these upstream events either utilize or synergize integrin receptors to initiate growth and survival signaling. Cardiomyocytes require tight regulatory processes to elicit signaling changes in response to hypertrophic stimuli. In addition, cytoskeletal dynamics must be able to change, but remain functional in order for each cardiomyocyte to enlarge, add sarcomeres, and extend their cell borders within the heart tissue . Targeteβ-adrenoreceptors and growth factor receptors which may elicit downstream signaling through synergistic integrins. GPCRs activate matrix metalloproteinases (MMPs), MMP-2, MMP-7, MMP-9, and a disintegrin and metalloproteinases (ADAMs), ADAM-12, and ADAM-17. MMPs cleave ECM proteins, resulting in activation of hypertrophic signaling and induction of fetal genes. One recent study showed angiotensin II-induced GPCRs induce MMP-7 which transcriptionally activates ADAM-12. This signaling axis is required for angiotensin-induced hypertrophic signaling [Other receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are known to be activated by autocrine and paracrine factors released during mechanical stress and can function in concert with integrins to activate downstream kinase pathways such as phosphotidylinositol-3-kinase (PI3K). For instance, humoral factors released during hemodynamic overload activate ignaling .α- and β-adrenoreceptors during pressure overload did not affect hypertrophic growth [However, mechanical stimulation alone also begets hypertrophic growth  through c growth . Therefoc growth .α and β subunits. Extracellular domains function for adhesion and ligand recognition, and upon activation, short cytoplasmic domains of β subunits, which lack kinase function, physically connect to the cytoskeleton and recruit proteins for signaling [α subunits and 8 β subunits, which in various combinations make up 24 different discovered integrin heterodimers [α5β1 and αvβ3. As physical links between the intracellular cytoskeleton and either the extracellular matrix or the adjacent cardiomyocyte, integrins are regulators of adhesion and mechanotransduction. Integrins are basally expressed in the intercalated disc [α5β1 only binds to fibronectin [β3 and β1 integrins have been identified at intercalated discs [β1 integrin translocates to the intercalated disc in damaged areas of the myocardium after infarct [β3 integrin is upregulated to the cell surface in RGD-stimulated adult cardiomyocytes [α5β1 has been shown to be important for adherence of cardiomyocytes [Integrins are a class of heterodimeric transmembrane proteins made up of the noncovalent interaction of ignaling . The introdimers . These hrodimers . The maited disc  and costted disc , 15. Intronectin . The impronectin , 18. Boted discs –20 and ced discs . To medi infarct  while βmyocytes . Additiomyocytes  and is nmyocytes –25. α5β1 and, mediate their signals by recognizing the specific amino acid sequence Arg-Gly-Asp (RGD) in ECM proteins, such as collagen, vitronectin, and fibronectin [α1, α5, and β3 subunits have RGD specificity [β1 subunit is more promiscuous, and its role in binding to RGD is dependent on the associated α subunit [α5 subunit, β1 integrin interacts primarily with the RGD motif of fibronectin. In intact myocardium, tortional stress induced in the tissue from increased blood pressure causes structural alterations of ECM proteins which results in exposure of their RGD motifs. These extracellular matrix proteins are also genetically upregulated and secreted with hypertrophic induction in the immediate early gene response of cardiomyocytes [Prior to mechanical stimulation, integrins remain freely mobile singular heterodimers on the cell surface, meaning they do not have a constitutive connection to extracellular matrix proteins or cytoskeletal proteins. Integrins that respond to mechanical stretch of the myocardium, namely, ronectin . Althougcificity . The β1 subunit . For examyocytes , 26, 27.myocytes . β1 and β3 integrins have roles in hypertrophic growth. During hypertrophic induction, several integrin subunits are upregulated and/or activated: α1, α5, β1, and β3 [β3 or β1 integrins inhibits pressure-induced hypertrophic signaling, resulting in reduced cardiac output with increased mortality and heart failure [β1 integrin plays a role in β-adrenergic receptor-induced hypertrophy [β1 integrin activates expression of the immediate early gene, atrial natriuretic factor (ANF). Conversely, blocking β1 integrin prevents ANF expression during α1-adrenergic stimulation [β1 knockout mouse subjected to isoproterenol exhibits less hypertrophic growth and more apoptosis (TUNEL-positive nuclei) compared to wild-type mice [β1 integrin is involved in another humoral hypertrophic pathway, as blocking antibodies for β1 integrin in a stretch model of adult rat cardiomyocytes reduced Ang II secretion [β1 integrin without the cytoplasmic domain in RGD-stimulated adult cardiomyocytes renders β1 integrin incapable of recruiting specific focal adhesion molecules, though focal adhesion complex (FAC) formation itself is not affected [β3 integrin, our recent work demonstrates that FAC formation and subsequent downstream signaling in pressure-overloaded myocardium occurs through this integrin activation [β3 integrin knockout mice results in a deficiency of compensatory hypertrophic growth, evidenced by stunted hypertrophic growth, decreased ventricular function and geometry, and increased mortality with compromised survival signaling [β3 integrin signaling but not with β1 integrin inhibition with specific blocking antibodies [Since integrins are positioned to sense stretch of the myocardium, both the  and β3 , 28, sug failure , 20. It ertrophy , 29–31. mulation . A heteraffected . In the tibodies . This obβ1 and β3 integrins initiate downstream survival signaling utilizing PI3K/Akt and ubiquitination activation [in vivo have been shown to initiate FAC formation [β3 integrin activates caspase inhibiting molecules for survival signaling in smooth muscle cells [Upon integrin activation from pressure-induced hypertrophy, multiple downstream signaling cascades mediate protein synthesis, survival, and gene expression: (i) there is evidence both tivation , 33, 34,ormation , 22 and ormation , (iii) βle cells , cardiomle cells , and hyple cells , 33, andle cells . The surβ integrin cytoplasmic tail upon stimulation [Once activated after recognition of extracellular matrix proteins, integrins cluster on the cell surface, associate with cytoskeletal proteins, and recruit signaling molecules onto the actin cytoskeleton to form a complex of scaffolding proteins and kinases called a FAC. Since the cytoplasmic tails of integrins do not possess kinase function on their own, recruitment of kinase molecules into the FAC is required for initiation of intracellular signaling and downstream gene expression (Reviewed in ). In thimulation . After amulation , Nck [42mulation . With reThe FAK/Src complex mediates hypertrophic growth and survival signaling in the adult rat heart activated by mechanical stimulation from load/pressure , 43. Eviβ1 integrin engages with several critical proteins  activation, which is mediated by the interference of β1 integrin with the matrix metalloproteinase-2 (MMP-2) [β phosphorylation and subsequent inhibition during pressure overload [ melusin . Genetic melusin . β-adren (MMP-2) ; in fact (MMP-2) . Similaroverload  while ovoverload .β1 integrin activation [β-adrenergic stimulation by isoproterenol. The ubiquitin is also taken in by the cardiomyocytes and is required for the PI3K-dependent antiapoptotic pathways activated [β-adrenergic receptor in adult cardiomyocytes induces apoptosis, measured by terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) staining, by activating GSK3β for subsequent JNK activation [β-adrenergic receptor inhibited apoptosis, JNK activation, and cytochrome c release; further PI3K activation was required [β. Though not fully investigated, these data indicate the possible link between β1 integrin and ubiquitin in antiapoptotic signaling.Both tivation  and ubiqtivation  can actictivated . As desctivation . Additiorequired , since Pβ3 integrin signaling, hypertrophic stimulation leads to FAC formation, ubiquitination of specific proteins, and NFκB activation [β3 integrin upregulates ubiquitination near intercalated discs of cardiomyocytes within the first week of pressure overload [κB (IκB) during pressure overload of the heart, NFκB is activated and transcribes the cellular inhibitor of apoptosis protein 1 (cIAP1) [κB is also regulated by FAK activation during mechanical stimulation of cardiomyocytes [κB functions as a survival transcription factor known to be required for hypertrophic growth [β3 integrin regulates cIAP1 expression. Consequently, the lack of cIAP1 expression correlates with increased caspase 3 activation [In tivation . Synonymoverload ; this isoverload , 19, 22. (cIAP1) . Accordimyocytes . NFκB fuc growth , and onec growth . E3 ligac growth . During tivation . A similtivation . Therefoc-Cbl is an E3 ligase for both RTKs and NTKs . Confocain vitro by embedding laminin-plated adult cardiomyocytes in a collagen matrix with the integrin-stimulating peptide RGD, referred to as a 3D model (β 3 and α5β1 [β3 integrin and focal adhesion proteins, such as Src, FAK, Nck, Shc, and p130Cas, as observed in the hypertrophic animal models [As mentioned, integrin activation and FAC formation can be recapitulated 3D model . This mo α5β1 . To deci α5β1 . Devoid  α5β1 , 32. The α5β1 , such as α5β1 . On the l models . Importal models , indicatl models . These cβ1 and β3 integrins are required for compensatory hypertrophic growth in response to pressure overload of the heart. Integrins interact with a complex network of proteins, which new studies continue to show are necessary for normal or hypertrophic cardiac function. The complex network of proteins transducing β1 integrin-mediated survival and growth signaling continues to grow. This includes a newly discovered interaction with ubiquitin, shown to activate PI3K/Akt signaling downstream of β1 integrin activation. Moreover, the importance of β3 integrins in ubiquitin-mediated removal of proapoptotic proteins for the survival of hypertrophying cardiomyocytes is a recent finding, and additional studies will likely decipher how this integrin subtype is tied into ubiquitination in the heart. Since integrins, PI3K/Akt, and ubiquitin signaling are each required for hypertrophic induction, continuing to study their interdependence will lead to better understanding of cardiac function during hemodynamic overload. Further study of integrin signaling may help decipher the various roles integrins play in cardiac performance and aid in the identification of potential drug targets against cardiac failure.Both


In the present study, we developed a robust splice site-specific real-time RT-PCR method to quantitate all HTLV-2 transcripts. Results of this analysis conducted on three different infected cell lines  showed that the most abundant mRNA was gag/pol followed by the accessory transcript 1-3, coding for the p28 and for p22/p20 proteins. The third most abundant mRNA was tax/rex.To investigate if different mRNAs produced by HTLV-2 are expressed at different levels upon viral reactivation, we studied the kinetics of viral expression in PBMCs from three subjects infected with HTLV-2B and cultured in vitro for 48 hours. The level of expression of the full length gag/pol transcript was the highest in all samples. The tax/rex mRNA was detected already at time zero and increased very rapidly following in vitro culture, reaching the highest copy number between zero and 2-4 hours. The minus-strand APH-2 mRNA, was expressed at high level. As observed in the infected cell lines, the 1-3 mRNA was expressed at high levels in all subjects. This finding is particularly intriguing, as it encodes two proteins that were shown to exert a powerful control on Tax and Rex function. This peculiar pattern of expression, which is in striking contrast with that of HTLV-1, might in part explain the differential pathogenicity of the two viruses.


Although most HTLV infections in Spain have been found in native intravenous drug users carrying HTLV-2, the large immigration flows from Latin America and Sub-Saharan Africa in recent years may have changed the prevalence and distribution of HTLV-1 and HTLV-2 infections, and hypothetically open the opportunity for introducing HTLV-3 or HTLV-4 in Spain. To assess the current seroprevalence of HTLV infection in Spain a national multicenter, cross-sectional, study was conducted in June 2009.A total of 6,460 consecutive outpatients attending 16 hospitals were examined. Overall, 12% were immigrants, and their main origin was Latin America (4.9%), Africa (3.6%) and other European countries (2.8%). Nine individuals were seroreactive for HTLV antibodies . Evidence of HTLV-1 infection was confirmed by Western blot in 4 subjects  while HTLV-2 infection was found in 5 . Infection with HTLV types 1, 2, 3 and 4 was discarded by Western blot and specific PCR assays in another two specimens initially reactive in the enzyme immunoassay. All but one HTLV-1 cases were Latin-Americans while all persons with HTLV-2 infection were native Spaniards.The overall prevalence of HTLV infections in Spain remains low, with no evidence of HTLV-3 or HTLV-4 infections so far. Four different types of human T-lymphotropic viruses (HTLV), named 1-4, have been described in humans. HTLV-1, the first human retrovirus was identified in 1980; it is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) . These iThe main routes of transmission of HTLV are from infected mothers to their newborns, especially through prolonged breast-feeding, sexual intercourse, blood transfusion and sharing of needles and syringes between intravenous drug users . HTLV-1 Several serological surveys conducted over the last decade have monitored the prevalence of HTLV infections in Spain . In the Recent estimates have pointed out that the number of immigrants legally registered as living in Spain has increased more than six-fold over the last 10 years . ImmigraA total of 6,460 consecutive adult outpatients attended during June 2009 at 16 distinct hospitals were screened for HTLV antibodies. The median age of the study population was 38 years and 40% were male. Although native Spaniards represented 88% of the total study population, 4.9% of subjects come from Latin America, 3.6% from Africa and 2.8% from other European countries.Nine specimens were repeatedly EIA reactive and were confirmed by Western blot as HTLV-1 positive (n = 4) and HTLV-2 (n = 5). Another two samples exhibited EIA reactivity close to the cut-off but could not be confirmed as HTLV positive by Western blot or PCR testing. No PBMCs could be obtained from the four HTLV-1 positive cases, which precluded further virological characterization of these samples.The prevalence of HTLV found in this study was 0.14%, being 0.06% for HTLV-1 and 0.08% for HTLV-2. Neither HTLV-3 nor HTLV-4 were found in this survey. Three of the HTLV-1 carriers had been born in Latin America  while the last one was a 31 years-old native Spanish woman who denied any significant risk behavior for HTLV exposure, including intravenous drug use, sexual promiscuity, transfusions or stages in Latin America. All HTLV-1 carriers but one were asymptomatic. The woman from the Dominican Republic was newly diagnosed with mild TSP/HAM  and similar to that found in previous serosurveys carried out in Spain % and sim. Only 9 The recognition of HTLV-1 infected persons in Spain has several clinical and public health implications. HTLV testing should be considered for a broader number of persons and conditions, including blood bank donors, organ transplantation, antenatal testing or sexually transmitted diseases clinics. Close relatives of infected individuals should be offered for HTLV testing. Given that most HTLV-1 carriers would remain asymptomatic life long, unaware silent transmission of the virus is the most worrisome. Wider HTLV testing may allow identification of carriers and help to reduce further transmission. Pregnant women with HTLV-1 infection should be advised against breast-feeding, which is the most effective way to prevent vertical HTLV-1 transmission. Moreover, physicians must monitor periodically asymptomatic persons known to be infected with HTLV-1, in order to facilitate early recognition of classical complications, such as TSP/HAM or ATL. Attention to mild symptoms or laboratory abnormalities, as in one of the current cases complaining minimal paraparesis, may permit early diagnosis and better treatment options.The rate of HTLV-2 infection seems to remain fairly stable in Spain and confined mainly to former intravenous drug users and their sexual partners. As most HTLV-2 carriers are intravenous drug users coinfected with HIV and intravenous drug use practices have dramatically declined in Spain , we shouIt is worth to note that this is the largest study carried out in Spain examining the prevalence of HTLV infections. We are confident that the 16 hospitals that participated in the study properly covered the whole country. However, testing was made on hospital outpatients and therefore bias exists in terms of extrapolation of prevalence rates to the entire population. Studies conducted in other populations, including pregnant women, blood donors, immigrants, sexually transmitted disease clinics, would help to define more accurately the extent of HTLV infections in Spain. Of note, despite testing a relatively large immigrant population from Western Africa, where HTLV-3 and HTLV-4 were described originally -7 no sinThis study supports that the prevalence of HTLV infection in Spain remains stable and low. However there is a slightly trend towards an increase in HTLV-1 mainly driven by the large immigration from endemic regions in Latin America over the last decades. The dramatic decline in intravenous drug use practices in recent years may result in a steadily decline in HTLV-2 in the near future. Altogether, HTLV-1 must be expected to take over HTLV-2 in coming years. Given that HTLV-1 is more pathogenic and that transmission often occurs from people unaware of their infection, HTLV testing should be considered for a broader number of persons.All adult outpatients attending the hospitals belonging to the HTLV Spanish Network, were invited consecutively during June 2009 to be tested for HTLV antibodies. In order to make sampling representative, the hospitals selected were distributed geographically across Spain and each of them recruited at least 350 samples. Every participating centre obtained approval from the corresponding ethical committee and informed consent was obtained from all recruited individuals.LTR and/or pol regions, using primers and conditions described elsewhere [Pools of five sera were screened for HTLV antibodies using a commercial enzyme immunoassay (EIA) . This strategy has been evaluated previously and considered as appropriated for HTLV antibody testing . Sera frlsewhere ,18,25. TResults are given as proportions and median values. Comparisons were made using the chi square test, with Fisher correction when appropriated. Differences were considered to be significant only when p values were lower than 0.05. All analyses were performed using SPSS version 11.0 .C. Rodríguez and J. del Romero ; C. Tuset, G. Marcaida and T. Tuset ; E. Caballero and I. Molina ; A. Aguilera, J.J. Rodríguez-Calviño, S. Cortizo and B. Regueiro ; R. Benito and M. Borrás ; R. Ortiz de Lejarazu and J.M. Eiros ; J.M. Miró, C. Manzardo, M.M. Gutiérrez and T. Pumarola ; J. García and I. Paz ; E. Calderón, F.J. Medrano and M. Leal ; F. Capote ; A. Vallejo, F. Dronda and S. Moreno ; D. Escudero ; E. Pujol ; M. Trigo, J. Diz, P. Álvarez and M. García-Campello ; M. Rodríguez-Iglesias ; A.M. Martín and A. Hernandez-Betancor ; J.M. Ramos, J. C. Rodríguez and F. Gutiérrez ; C. Gómez-Hernando ; A. Guelar ; G. Cilla and E. Pérez-Trallero ; J. López-Aldeguer ; J. Sola ; L. Fernández-Pereira ; J. Niubó ; S. Veloso ; J.L. Gómez Sirvent, L. Arroyo, M.M. Alonso, M.R. Alemán and D. García Rosado ; L. Force ; C. Cifuentes ; J. García ; S. Pérez ; C. Raya ; A. González-Praetorius ; A. Mena, J.L. Pérez and M. Peñaranda ; J.M. Montejo ; N. Margall, M. Gutiérrez, P. Domingo ; L. Roc and A. Martinez Sapiña ; I. Viciana ; T. Cabezas, A. B. Lozano and J.M. Fernandez ; I. García and G. Gaspar ; R. García and M. Gorgolas ; A. Treviño, P. Parra, C. de Mendoza and V. Soriano .The authors declare that they have no competing interests.HTLV Spanish Study Group designed and conceived the study. AT, AA, EC, RB, JME, AH, EC, MR, AT, JG, JMR, LR, GM, CR, MT, CG, ROL and VS collected and provided data. AT, PP and CM analyzed the data. AT, CM, VS wrote the paper. All authors read and approved the final manuscript.


We report a 21-year-old woman who was admitted because of unilateral facial paresis and then developed progressive ascending flaccid tetraparesis with generalized areflexia. Electrodiagnostic studies revealed acute motor axonal polyradiculoneuropathy (AMAN type of Guillain-Barré Syndrome). Further evaluations revealed severe leukocytosis, increased erythrocyte sedimentation rate (ESR), increased protein content and presence of a few lymphocytes in cerebrospinal fluid (CSF), and then presence of human T-cell lymphotropic virus type 1 (HTLV-I) in serum and CSF. Finally, biopsy of the enlarged lymph nodes resulted in the diagnosis of Adult T-cell Leukemia-Lymphoma. The HTLV-1 has been endemic to certain parts of Iran like Khorasan province in the northeast since 1985 with 2.3% prevalence rate of infection. Thus, some rare neurologic complications occasionally occur in this area as a result of being infected with HTLV-1. Adult T-cell Leukemia/Lymphoma (ATLL) is a non-Hodgkin T-cell Lymphoma that is linked to infection by the human T-cell lymphotropic virus type 1 (HTLV-1). It is characterized by skin and lung involvement, hepatosplenomegaly, lymphadenopathy and hypercalcemia. The clinical course has a terminal end in most of the acute cases. It was first described by Uchiyama and co-workers in 1977 in southwestern Japan . The . The 5].HTLV-I infection is endemic in tropical areas such as South America, Central Africa, Southern Japan, and Middle East like Khorasan province of Iran . Its preHealthy relatives of patients with ATLL have an especially high incidence of sero-positivity . It is r


S-glutathionylation (forming mixed disulfides with the protein–sulfhydryl groups), a potent mechanism for posttranslational regulation of a variety of regulatory and metabolic proteins when there is a change in the celluar redox status (lower GSH/GSSG ratio), has received increased attention over the last decade. GSH, as a single agent, is found to affect DNA damage and repair, redox regulation and multiple cell signaling pathways. Thus, seemingly, GSH does not only act as a radioprotector against DNA damage induced by X-rays through glutathionylation, it may also act as a modulator of the DNA-repair activity. Judging by the number of publications within the last six years, it is obvious that the field of protein glutathionylation impinges on many aspects of biology, from regulation of protein function to roles of cell cycle and apoptosis. Aberrant protein glutathionylation and its association with cancer and other diseases is an area of increasing interest.The tripeptide glutathione (GSH) is the most abundant intracellular nonprotein thiol, and it is involved in many cellular functions including redox-homeostatic buffering. Cellular radiosensitivity has been shown to be inversely correlated to the endogenous level of GSH. On the other hand, controversy is raised with respect to its role in the field of radioprotection since GSH failed to provide consistent protection in several cases. Reports have been published that DNA repair in cells has a dependence on GSH. Subsequently,  GRXglutaredoxinMPGmercaptopropionylglycineBSObuthionine sulfoximineNHEJnon-homologous end-joiningAP-1activator protein 1NF-κBnuclear factor kappa BSp-1Specificity Protein 1APE1apurinic endonuclease 1NPSHnon-protein bound sulphydrylsMEAβ-mercaptoethylamineAETS-2-aminoethylisothioureaTRXthioredoxinSSbsingle-strand breakdsbdouble-strand breakL-γ-glutamyl-L-cysteinyl-glycine; GSH) is the most abundant intracellular nonprotein thiol that reaches millimolar concentrations in most cell types. It is present predominantly in a reduced form (GSH), which is biologically an active form. It is involved in many cellular functions, including antioxidant defense via direct interaction with reactive oxygen/nitrogen species (ROS/RNS) or via activities of detoxication enzymes like GSH peroxidases and GSH-S-transferases [in vivo and also bring beneficial health effects through other mechanisms, including acting as inducers of mechanisms related to antioxidant defense [The tripeptide glutathione . The hydroxyl radical (•OH) is truly the mediator of much of the radiation-induced DNA damages that are initiated by abstraction of a deoxyribose hydrogen atom by the hydroxyl radical [A large amount of chemical changes in biomolecules, especially DNA, are made by free radicals that are generated by mutagenic agents, including ionizing radiation . However radical . Interes radical . It was  radical . TherefoTraditionally, GSH in the cell nucleus has been considered a protector against oxidative stress and radiation-induced damages in DNA and DNA-binding proteins . VariousS-2-aminoethylisothiourea (AET) and cysteine cause an increase in the free GSH content in the cell by triggering a release of the nucleo-protein bound GSH [−) human cells, which have only 6% GSH level of the proficient cells, it has been demonstrated that they are more sensitive to radiation than normal cells [In contrast to limited studies on the protective effect of GSH employing parameters of chromosome aberrations, data obtained on the role of GSH in the inherent cellular radiation protection mechanism are quite rich and informative. It has been demonstrated that GSH plays a significant role in the cellular detoxification process , regulatound GSH . In viewound GSH ,46. Theround GSH . With thal cells . Depletial cells ,50.Drosophila or chromosomal exchange aberrations in mammalian systems were not protected by GSH [It is interesting to note that in spite of the wider acceptance of the role of GSH as a radioprotector, there is some controversy with respect to its role in the field of radioprotection. As shown in d by GSH ,40. In ad by GSH . Moreoved by GSH .Available evidence suggests that GSH may not be an efficient protector of DNA due to its −1 net charge, which, on the basis of counter-ion condensation and co-ion depletion phenomena, may allow its dissociation from DNA . It was In a separate study , an atteControversial reports are also there with regard to the role of GSH in the induction of apoptosis which is dependent on cell types and pro-apoptotic stimuli ,66. SuchS-glutathionylation or reversible protein glutathionylation  or inac-Cys-118  and nucl-Cys-118 ).S-glutathionylation is a prevalent protein modification; however, its mechanisms of protein–SSG (PSSG) formation are not yet resolved. S-glutathionylation. Deglutathionylation is the process by which GSH moiety can be removed from protein–mixed disulfides, and it occurs when the environment becomes more reducing in an enzyme-dependent or -independent manner  and thioredoxins (TRX) [In fact, t manner . Enzymesns (TRX) . Glutarens (TRX) . They cans (TRX) . Since gns (TRX) .There is a series of DNA-repair pathways that are implicated in correcting different types of DNA damage, including single-strand breaks (ssbs) and double-strand breaks (dsbs), base damages, and DNA adducts. A distinct DNA damage is repaired by different pathways, and there is also an overlap and interaction among the various pathways . HoweverIt was suggested that ionizing radiation-induced DNA dsbs are critical lesions, which, if unrepaired or misrepaired, can cause chromosome aberrations, cell death, as well as mutations and cell transformation ,81. In ao-lymphocytes indicates the involvement of the non-homologous end joining (NHEJ) pathway in the misrejoining of DNA breaks. Usually, in quiescent mammalian cells, this process of misrejoining is prevalently taken up by the NHEJ process. It may be hypothesized that reduction in the frequency of exchange aberrations after exposure to Bleomycin + radiation combined treatment in BSO-treated samples indicates the involvement of the endogenous GSH in the illegitimate reunion of DNA dsbs [It was demonstrated by Preston  that if DNA dsbs .S-glutathionylation of procaspase-3 blocked its activation and regulated the activities of Ku indirectly [It has been demonstrated that GSH may be an important determinant of a cell’s ability to repair DNA damage and resist cell death . Ku is adirectly . It was directly . On the S-glutathionylation of Cys99; the human APE1 is S-glutathinoylated under conditions of oxidative stress both in the presence of glutathione in vitro and in vivo [Recently, it was reported that human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) reduced its endonuclease activity due to  in vivo .S-glutathionylation in a cell. Any modification of critical cysteine residues on enzymes, receptors, transport proteins, and transcription factors is recognized as an important mechanism of signal transduction and perturbation of the thiol–disulfide homeostasis is considered to be an important consequence of many diseases [S-glutathionylation must (a) be at a discrete site and functionally effective, (b) occur in a high GSH/GSSG ratio, i.e., normal physiological conditions, (c) occur in response to a physiological stimulus, (d) occur through an efficient mechanism for specific protein–SSG formation, and (e) be reversed through an efficient mechanism . The ratio of the GSH/GSSG redox couple provides means of diseases ,94. TherReports regarding protein glutathionylation are steadily increasing; however, they are yet to be documented in a physiologically relevant context. In many cases, protein glutathionylation was observed in an endogenous milieu, but its functional change and physiological impact were not studied. For example, in diabetic patients, glutathionylation of hemoglobin (HbSSG) has been reported. While an increase in HbSSG content is correlated with the increase in microangiopathy, whether or not this has any impact on redox homeostasis, or if it serves only as a biomarker of oxidative stress, is as yet unclear . GlutathS-glutathionylation, which is overcome by glutaredoxin-1 (GRX1), the latter under physiological conditions catalyzes deglutathionylation and enhances NF-κB activation [S-glutathionylation on Cys62, thereby inhibiting DNA binding [Jun and Fos families) regulates the expression of a large number of genes. Klatt et al. reported that binding of AP-1-c-Jun subunit to DNA depended on the cellular GSH/GSSG ratio [S-glutathiolation of c-Jun at Cys269 and sterically blocks DNA binding.NF-κB and AP-1 are the two pleiotropic transcription factors that play an important role in almost all aspects of human cancers. NF-κB belongs to the Rel family and is comprised of diverse subunits to form hetero- or homo-dimers frequently including one subunit of p65 (relA) and another subunit such as p50, c-rel or relB . The inativation . In anot binding . SimilarSG ratio . A decreThe tumor suppressor gene p53 is mutated in many human cancers and has been known to play an important role in maintaining the genomic integrity, cell cycle control, DNA repair, differentiation and apoptosis. There are 10 cysteine residues in p53 protein, all of which are present within the DNA-binding domain. It was reported that in human colon cancer cells glutathionylation was identified at the proximal DNA binding domain  of p53 protein . GlutathS-glutathionylation of caspases has gained much interest as a sensitive mechanism for caspase activation in apoptotic signaling. Glutathionylation of caspase-3 inhibits its function in HL60 cells [Apoptosis or the programmed cell death is a highly orchestrated and physiologically conserved mechanism of cell death. In the execution phase of apoptosis, a highly conserved family of proteins, the caspases (cysteine proteases), play an important role. The reversible 60 cells , whereas60 cells . Reports60 cells . It has 60 cells  and prot60 cells , while s60 cells . S-glutathionylation, a redox-dependent posttranslational modification of a protein, accounts for the reversible regulation of the structure and function of a variety of proteins that are integral to cell structure, signaling, and metabolism. Therefore, the deleterious consequences that GSH depletion shows under oxidative stress are not only attributable to the lack of an antioxidant, but also to the blockade of S-glutathionylation-dependent signaling mechanisms, just as the ATP depletion can affect phosphorylation-mediated signaling.The most exciting development over the last decade has been the emergence of glutathione as an important signaling molecule. There are evidences that highlighted the important role of a variety of redox signaling mechanisms in the control of a plethora of cellular processes. Indeed, the list of cellular processes that are dependent on or influenced by GSH in biological systems continues to grow. The most obvious conclusion to be drawn from this review is that numerous agents can modify the radiation damage in more than one way. It is likely that all radiation response modifiers affect multiple aspects of biology that are of possible relevance to the outcome. It is striking to know that GSH as a single agent is found to affect DNA damage and repair, redox regulation, and multiple cell signaling pathways. There is a need to design new classes of radioprotectors for clinical use. For this, the critical prerequisite is to understand the metabolic differences between the normal tissue and tumor cells. New radioprotectors should act on mechanistic aspects of radiation action, particularly forming DNA strand breaks rather than focusing on distal steps in a cellular response, like transport of signaling molecule or induction of pathways leading to cell death, including autophagy, apoptosis, and necrosis. Clearly, many questions remain unanswered. Could deglutathionylation of nuclear proteins play a role in the interaction of DNA double-strand breaks leading to form higher frequencies of chromosomal exchanges in the presence of a higher level of GSH? If it is so, then cancer cells with higher levels of GSH  can form


Closely related species often bear many similarities in outward appearance—think of the elongated snout of wolf and dog, or the opposing twigs on Norway and sugar maples. The parsimonious explanation for similar features is that they arise by similar developmental mechanisms, but an emerging concept in evolutionary development suggests this may not always be so. “Developmental systems drift” proposes that so long as the morphological feature itself doesn't change (thus preserving the organism's interaction with its environment), the underlying molecular pathway specifying it can be altered without evolutionary penalty.PLoS Biology, Xiaoyue Wang and Ralf Sommer elucidate those details to show that in two species of roundworm, similar sexual structures arise from marked differences in signaling pathways, differences involving both novel wiring and novel protein modifications.While it is an intriguing hypothesis, there have been few studies that offer evidence for its operation in nature, not surprisingly, given the degree of molecular detail needed to confirm or refute it. In this issue of Caenorhabditis elegans and Pristionchus pacificus are members of separate roundworm families whose last common ancestor lived at least 250 million years ago. Nonetheless, they share many features, including a vulva that arises from the same set of precursor cells. In C. elegans, the induction of the vulvar precursor cells is under the control of a signaling pathway employing epidermal growth factor and RAS, while in P. pacificus, induction relies on Wnt signaling. Furthermore, in P. pacificus, signaling emanates in part from the posterior body region, and involves a unique ligand-receptor interaction; neither of these features is found in C. elegans.P. pacificus, the authors explored the molecular effects of three genes: Ppa-lin-17, Ppa-lin-18, and Ppa-egl-20. Both lin-17 and lin-18 are membrane receptors, and bind egl-20. Binding of egl-20 to lin-18 induces vulvar formation. Not so for lin-17, however. Either suppressing lin-17, or overexpressing egl-20, leads to vulvar induction. This suggested to the authors that lin-17's role in vulvar induction is inhibitory, and its binding of egl-20 serves as a brake on lin-18/egl-20 signaling.To understand the details of vulvar induction in lin-17 and egl-20 were expressed in the posterior body region, consistent with the source of vulvar induction signaling in P. pacificus.Additional support for this model came when they found that both lin-17 mutation that drove ectopic multiple vulva formation. But this mutation was not a null allele, and its mechanism differed from that of the mull mutant. Instead, the mutation caused a 17-amino acid domain to be added to the intracellular end of the protein, and this extension served as an SH3 binding motif, a motif known as a common binding domain. Curiously, they found the same domain on lin-18.Further experiments shed light on other details of the signaling system. The authors found a lin-18 was most likely to bind an inhibitor to prevent Wnt signaling and vulva induction in the absence of egl-20. When the domain was expressed on lin-17, they speculate, it depleted the inhibitor from its normal position on lin-18, leading to excess Wnt signaling and ectopic vulva formation.The authors showed that the job of the domain on Finally, they showed that the normal signaling system requires the downstream participation of two proteins, Axin and a beta-catenin, both known members of other Wnt signaling pathways.C. elegans, the authors note, indicating the degree of developmental dissimilarity between the two species in accomplishing the same morphological result—the very definition of developmental systems drift. They also point out that the central role of the SH3 binding motif uncovered in this study suggests the potential role for evolution of small peptides in causing major developmental changes.None of the above is found in Wang X, Sommer RJ (2011) Antagonism of LIN-17/Frizzled and LIN-18/Ryk in Nematode Vulva Induction Reveals Evolutionary Alterations in Core Developmental Pathways. doi:10.1371/journal.pbio.1001110


HTLV-II seropositivity is confirmed by the presence of rgp46-II. However, numerous cases have been documented in which serum samples are reactive by EIA, but an incomplete banding pattern is displayed by subsequent confirmatory WB. Although the significance of these HTLV-I/II seroindeterminates is unclear, it may suggest a much higher incidence of exposure to HTLV-I/II than previously estimated.Human T-lymphotropic virus type I (HTLV-I) infects an estimated 15–20 million persons worldwide. A number of diseases have been associated with the virus including adult T-cell leukemia (ATL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-I uveitis, and HTLV-I-associated infective dermatitis. Once it was shown that there is an increased risk for developing HAM/TSP associated with blood transfusion, screening for HTLV-1 among blood banks was implemented in Japan, United States, France, and the Netherlands. This process includes detection by an enzyme immunoassay (EIA) followed by a confirmatory Western blot (WB) in which recombinant proteins specific for HTLV-I Env glycoproteins are incorporated into WB strips. HTLV-I seropositive results are defined by the presence of antibodies against either gp46 or gp62/68 (both Env protein bands) and either p19, p24, or p53 (one of the  Sincei.e., breast milk), and transfusion with infected blood [Among diseases associated with HTLV-I is a chronic, progressive, neurodegenerative disease termed HAM/TSP. Since the initial report of the association between HTLV-I and HAM/TSP, more associations were discovered between HTLV-I and diseases including HTLV-I uveitis, bronchopneumonitis, arthritis, polymyositis, and various other inflammatory conditions ,10,11. Aed blood . SpecifiDue to this increased risk of disease with blood transfusion, many screening methods have been employed to detect HTLV-I infected donors. Blood bank screening for the virus was implemented in Japan in 1986, the United States in 1988, France in 1991, and the Netherlands in 1993 ,11. Thes2.The WB used to confirm seropositivity in an EIA positive patient sample utilizes recombinant proteins specific for HTLV-I/II Env glycoproteins incorporated into the WB strips. These recombinant proteins increase the sensitivity of the blot, as well as differentiate between HTLV-I and HTLV-II. The specific sensitivity of an EIA assay is approximately 95% and the EIA specificity averages 98% while the sensitivity of the WB is 97.1% with a specificity of 97.5% ,14. A saGenerally, HTLV-I infected individuals such as asymptomatic carriers, HAM/TSP patients, and ATL patients exhibit the clear seropositivity illustrated in 3.As research progresses, reports of large numbers of HTLV-I/II seroindeterminates have come from Iran, Brazil, Japan, Argentina, Taiwan, the Caribbean, Central Africa, and the United States, indicating a higher prevalence than originally thought –21. Impo3.1.Plasmodium falciparum . P. falciparum is a lethal malaria parasite and is the most prevalent of malaria parasites infecting humans [P. falciparum and HTLV-I must contain regions of immunogenic epitope homology. It was hypothesized that this homology may be a result of mimicry of host tissue by the two organisms [P. falciparum [P. falciparum. Examination of data from a separate cohort showed cross-reactivity with HTLV-I could be blocked by erythrocyte lysates  infected with P. falciparum, however HTLV-I seroindeterminate samples from the United States failed to block antibody reactivities. This led to the assertion that the molecular mimicry may extend to epitopes other than the P. falciparum and plasmodial antigens [P. falciparum is extremely unlikely, such as the United States. Furthermore, HTLV-I/II seroindeterminate patterns are observed in normal, healthy blood donors, showing no sign of malaria or similar parasite infection [P. falciparum, this cannot explain the WB banding patterns found in areas where these organisms are extremely rare or nonexistent.It has been reported that anti-HTLV-I reactivity of sera can result from antibodies produced in response to g humans . Studiesrganisms . This sulciparum . It was antigens . Howevernfection . While aIt should also be noted that cross-reactivity between antibodies against severe acute respiratory syndrome (SARS) coronavirus and HTLV has been observed on occasion . The HTL3.2.gag, and pol regions of the virus. However, a later report demonstrated the tax region of prototype HTLV-I virus was amplified by nested PCR from one patient with an HTLV-I/II seroindeterminate WB that could not have been derived from the DNA sequence of an endogenous virus [Various other hypotheses have been presented regarding the cross-reactivity of anti-HTLV-I antibodies. In 1997, it was reported that HTLV I/II indeterminate serologies were observed as responses to two types of simian T-lymphotropic virus (STLV) . An HTLVus virus . HTLV-I/us virus . These nus virus .3.3.tax region from PBMCs of some HTLV-I/II seroindeterminates, but not other regions of the virus [in vitro long-term generated B-cell line tested positive by primary PCR. The virus infecting the seroindeterminate B cell line was then sequenced in an attempt to identify any mutations or other factors that may be associated with an HTLV-I/II WB seroindeterminate status. The results indicated that the virus was globally >97% homologous to prototypic HTLV-I on the nucleotide level. Fine analysis of the 5′ LTR indicated that the HTLV-I strain infecting the patient was of the Cosmopolitan subtype [Due to the typically negative PCR results and lack of antibody response to some of the HTLV-I antigens but reactivity to others, the most plausible suggestions seems to be that HTLV-I/II seroindeterminate blots may result from a low copy number of prototypic HTLV-I . This exhe virus ,28,29. T subtype . This wa subtype . Further subtype . Eight s subtype .The hypothesis that HTLV-I/II WB seroindeterminates  may be related to prototype HTLV-I is further supported by the recent observations that demonstrated HTLV-I PCR positive results in 12.5% of HTLV-I/II seroindeterminates from Iran  Table 1Table 1. 4.While the significance of these prevalent seroindeterminate HTLV-I/II blotting patterns remains uncertain, it is clear that further investigation is necessary. The sequencing of a virus with homology to prototype HTLV-I from a seroindeterminate cell line implicates exposure to the virus in at least some HTLV-I/II seroindeterminates. This finding may suggest that exposure to HTLV-I is greater than previously estimated. Of concern are reports that have demonstrated serum samples with negative HTLV-I/II results by EIA, which may also have seroindeterminate banding patterns by WB . Since tgag, env, or tax region of HTLV-I). Renilla luciferase-fusion proteins (Ruc-antigen) are obtained from cell lysates transfected with each plasmid. A preincubated serum/antigen mixture is added to a filter plate coated with immobilized protein A/G beads. This mixture is then washed to remove any fusion proteins that did not bind to the antibody. Luciferase activity is then measured in a microplate luminometer.One such technique is LIPS assay. The LIPS assay yields high throughput results while also generating quantitative values of anti-HTLV-I antibody responses . This crenv region of HTLV-I, inclusion of other synthetic peptides in serological assays, and optimization of an enzyme oligonucleotide assay (EOA) [The LIPS assay provides a promising high throughput method of anti-HTLV-I antibody detection. Further examination of immunogenic epitopes, such as HTLV-I Rex and HBZ, may enhance the specificity and sensitivity of the assay. Currently, a whole Gag protein is used for detection of antibody response; however, antibody responses for p19, p24, or other portion of the Gag protein may yield even more specific results. A panel of these quantitative antibody responses may be able to detect seroindeterminate samples in a fast, inexpensive, and efficient manner. LIPS may also detect seroindeterminates that tested negative by EIA, proving to be a more sensitive assay in terms of seroindeterminate detection. While the LIPS assay is under further development, other methods to improve detection and classification of seroindeterminates have also been proposed including: real-time PCR, molecular diagnostic analysis, detection of amino acid changes in the ay (EOA) –39.With further in depth research involving these newer methodologies, the significance of these HTLV-I/II seroindeterminate banding patterns will become clearer. As it becomes apparent that the prevalence of seroindeterminates is more widespread than originally thought, it becomes increasingly important to clarify the meaning of these results. If HTLV-I/II seroindeterminates (or subsets of these subjects) represent exposure to prototype HTLV-I or HTLV-II, the frequency of infection in the general population will be greater than previously believed. It remains to be seen whether the incidence of exposure is also associated with any clinical disease outcome.


Estrogens are potent nongenomic phospho-activators of extracellular-signal–regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously.2), estrone (E1), and estriol (E3).We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs  to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol  to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated  and attenuated  the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated.XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important . E2 is the predominant form driving sexual development and function of reproductive organs  and the menstrual cycle. E1 and E3 have important physiologic functions at discrete life stages such as pregnancy, and E1 is a major estrogen in the postmenopausal phase. These estrogens are produced mainly in the ovaries and in the placenta, although small amounts are also produced by the adrenal glands and brain , including extracellular-signal–regulated kinases (ERKs), play central roles in controlling such diverse cellular outcomes as cell proliferation, apoptosis, and maturation to perform distinct functional roles. Such kinases can also influence whether cells proliferate excessively and develop into tumors. Many different stimuli, including steroids, growth factors, cytokines, ligands for G-protein–coupled receptors, and carcinogens, can activate the ERK pathways; ERKs can therefore serve as signal integrators for all of these inputs , so that3/B6/F10) that we selected for its naturally high levels of membrane estrogen receptor-α (mERα) expression ] can disrupt physiologic estrogen signaling via ERKs when in combination. We studied all compounds at a wide range of low concentrations and at multiple time points because we expected variations in both their concentration dependence and response progression. We then examined which estrogen receptor (ER) subtypes participate in these ERK and proliferation responses. We used fixed cell-based immunoassays that allowed us to analyze many conditions simultaneously. By examining these mER-mediated cellular responses leading to the integrated activation of ERKs, we hoped to better understand the endocrine-disrupting impact of the alkylphenol class of environmental XEs.n-ethylphenol (EP), 4-n-propylphenol (PP), 4-n-octylphenol (OP), 4-n-nonylphenol (NP), and other chemicals were purchased from Sigma . GH3/B6/F10 cells were routinely propagated in DMEM containing 12.5% horse serum, 2.5% defined supplemented calf serum, and 1.5% fetal calf serum. Cells were used between passages 10 and 20.We purchased phenol red-free Dulbecco’s modified Eagle medium  from Mediatech , horse serum from Gibco BRL , and defined supplemented calf and fetal bovine sera from Hyclone . Paraformaldehyde and picric acid were purchased from Fisher Scientific . The antibody used to measure phosphorylated ERKs 1 and 2 was purchased from Cell Signaling Technology , and the ERα-selective agonist 4,4′,4″-[4-propyl-[1H]-pyrazole-1,3,5-triyl] trisphenol (PPT) and the ERβ-selective agonist 2,3-bis[4-hydroxyphenyl]-propionitrile (DPN) were purchased from Tocris . The G-protein–coupled ER (GPER) agonist G1 was a gift from Chemdiv Inc.  arranged by E. Prossnitz. Pertussis toxin, nystatin, BPA, 4-3/B6/F10 cells  containing 2% bovine serum albumin (BSA)/0.1% Triton X-100 for 1 hr at room temperature. Cells were then washed three times with PBS and treated with primary antibody against pERKs . After overnight incubation at 4°C, the cells were processed for signal development with the Vectastain ABC kit  following manufacturer recommendations. Biotin-conjugated secondary antibody was used at a 1:300 dilution. Plates were incubated in the dark for 20 min at 37°C for the generation of alkaline phosphatase product (para-nitrophenol) and then read at A405 in a model 1420 Wallace microplate reader . We used the crystal violet assay to estimate the number of cells in each well for signal normalization. Experiments were repeated at least two to three times using different passages of cells on different days.We developed this assay to assess levels of activated ERKs 1 and 2 in fixed GH10 cells . Briefly590 signal of the extract was read in a model 1420 Wallace microplate reader. This assay was also used for proliferation studies to approximate cell number.After ERK assays, the quantitative immunoassay reagents were washed from the multiwell plates and the cells were stained with 0.1% crystal violet for 30 min, destained with deionized water, and extracted (10% acetic acid) following the method of p < 0.05.Data from pERK studies were analyzed by one-way analysis of variance followed by multiple comparisons with the control group (Holm–Sidak method) using Sigma Stat . Significance was accepted at 1, E2, and E3 .Because women are exposed to environmental estrogens during life phases when different physiologic estrogens are prevalent, we studied the changes in pERK when each physiologic and environmental estrogen was present simultaneously. We previously detailed ERK (and other) responses of these cells to the physiologic estrogens E, and E3 ; those d, and E3 –3, showi, and E3 . We obse2, E1, or E3) in combination, the usual 2.5- to 5-min pERK peak caused by individual estrogens was often abolished or blunted. Instead, the combination of compounds usually caused a pronounced early dephosphorylation and then created a new time-delayed, augmented phosphorylation peak, just as the actions of individual estrogens waned. These new large activations  peaked instead in the intermediate 10- to 30-min time frame, demonstrating how combinations of estrogens with XEs that individually do not elicit a significant response at a given time point can cause a synergistic response. Then, as the ERK activation induced by individual estrogens again rose at 60 min, the level due to the combined estrogens usually declined, often far below the response to the individual estrogens.  Although the later (30–60 min) response period was usually inhibited, it depended somewhat on the efficacy of the XE at that time point. That is, stronger individual XE responses (resembling physiologic estrogen responses) tended to predict the ability of the XE to inhibit the actions of physiologic estrogens when in combination. Thus, a three-peaked oscillation caused by physiologic estrogens was transformed to a single major intermediate peak of activation in most cases.When pituitary cells were cotreated with XEs plus each of the physiologic estrogens (E−14 M) caused a two-phased oscillating response rather like the alkylphenol XEs . However, PP enhanced the estrogenicity of E3 at all but the lowest tested concentrations  or even enhanced (for E1 and E3).BPA showed the most striking nonmonotonicity in its dose–response pattern , as we h−14 M), including those highly selective for ERα. The ERβ agonist DPN had no effect at any selective concentration; it is known to be rather nonselective at higher levels  profile for inducing cell proliferation.Because ERK activation is often associated with the proliferative response, we also used these same receptor-selective compounds to evaluate their ability to evoke cell proliferation after 3 days of treatment . At sele1, E2, E3) and an XE class , and also allowed us to carefully document the combinatorial effects of XEs (both inhibiting and enhancing estrogenicity) on these physiologic estrogens that are functionally important to different life stages of women and men.Oscillating time-dependent and nonmonotonic concentration-dependent responses are typical for nongenomic estrogenic responses , and we 2) showed them to be weak , the paired physiologic estrogen response was inhibited; whenever an XE caused a weak or no response, its combination with physiologic estrogens usually caused a synergistic enhancement of pERK levels. This was particularly easy to observe for BPA, with its pronounced nonmonotonic dose curve, but we also observed it in the responses to the other alkylphenols. Cotreatments often resulted in out-of-phase oscillations compared with those caused by individual estrogens, with apparent shifts to later response times and less frequent oscillations. In another  cell type exhibiting a different functional response (dopamine efflux), we saw a similar pattern, especially with low concentrations of XEs in combination with E2 .We used very low doses of BPA in the present study compared with many others in the literature; this emphasizes the toxicity that can be caused by levels commonly present in our environment and food. BPA potentiated ERK activation at levels as low as picomolar concentrations, whereas femtomolar and nanomolar BPA generally disrupted all physiologic estrogen-induced ERK activities. Changes seen in later (60 min) ERK activation with several estrogen/XE combinations may indicate functional consequences for regulating cell proliferation  and othe2) are not known for GPER, these data suggest an inhibitory role for GPER at what would be an expected physiologic concentration of hormone acting on adult tissue (up to nanomolar concentrations). GPER’s sensitive activation by G1 does not match what is known about its affinity for G1 and its activation of other functional end points  but inactivating ERK at picomolar to nanomolar ranges. Although natural ligands  activations. MAPKs serve as signal integrators in most tissues , and exaExposed human and animal populations will probably have windows of vulnerability based on sex, life stage, and tissue differences in the expression and activities of the ERs involved in these actions. We have examined only limited cases, where ERα and perhaps GPER are involved in causing some kinase activations/deactivations. However, many other estrogen-induced signaling cascades  need to In