Conlon and Raff propose that mammalian cells grow linearly during the division cycle. According to Conlon and Raff, cells growing linearly do not need a size checkpoint to maintain a constant distribution of cell sizes. If there is no cell-size-control system, then exponential growth is not allowed, as exponential growth, according to Conlon and Raff, would require a cell-size-control system.A reexamination of the model and experiments of Conlon and Raff indicates that exponential growth is fully compatible with cell size maintenance, and that mammalian cells have a system to regulate and maintain cell size that is related to the process of S-phase initiation. Mammalian cell size control and its relationship to growth rate–faster growing cells are larger than slower growing cells–is explained by the initiation of S phase occurring at a relatively constant cell size coupled with relatively invariant S- and G2-phase times as interdivision time varies.This view of the mammalian cell cycle, the continuum model, explains the mass growth pattern during the division cycle, size maintenance, size determination, and the kinetics of cell-size change following a shift-up from slow to rapid growth. Conlon and Raff have described experiments that they claim casts doubt on a basic assumption regarding the way mammalian cell size is maintained during proliferation . The keyIn an article accompanying the work by Conlon and Raff , a quoteThe experiments of Conlon and Raff  are pres"We show that proliferating rat Schwann cells do not require a cell-size checkpoint to maintain a constant cell size distribution, as, unlike yeasts, large and small Schwann cells grow at the same rate, which depends on the concentration of extracellular growth factors."A reanalysis of the experiments and reasoning of Conlon and Raff, presented here, leads to a very different view of cell size control and cell size maintenance. It is first shown that there is no problem with either linear or exponential mass increase for size maintenance. Size maintenance does not depend on which pattern of cell mass increase occurs within a cell cycle. The preferred–and experimentally and theoretically supported–pattern of mass increase during the division cycle is exponential growth or exponential mass increase. An exponential growth pattern poses no problem for size maintenance. Constancy of cell size is fully compatible with an exponential pattern of mass increase as well as the hypothetical linear pattern of mass increase. No major difference between the size control systems of yeast, mammalian, or bacterial cells need be postulated to account for the constancy of cell size during the growth of cell cultures. In contrast to the proposed absence of a cell size control system in mammalian cells, it is shown that mammalian cells do have a very simple size control system. The formal elements of this system are similar to that found in the control of the bacterial cell cycle.Can cells grow exponentially during the division cycle and maintain a constant cell size? Consider two possible cases of exponential growth for cells with variable cell sizes. For the first case Fig. , three cLinear cell growth during the division cycle Figs.  and 1d cThe patterns shown in Figs. To be precise, it is not proposed that cells always divide at "exactly" the same size. There is a statistical variation in mass increase and interdivision times that can lead to variations in cell size at division . The impFurther, it is not proposed that a large, newborn cell "controls" its mass increase to have a slower rate of mass increase  thus compensating for the initial larger cell size. Nor do small cells increase their rate of mass synthesis to compensate for their initial mass deficit. Mass increase variation is postulated to have some inherent statistical variation  but withThere can be variability in cell mass increase with the rate of mass increase being independent of cell size. A large newborn cell could have a faster than average rate of mass increase. In this case, the interdivision time would be even shorter to compensate for both the larger initial newborn cell size and the greater than average rate of mass increase.The size maintenance pattern is illustrated in Fig. It may appear that this simple analysis is merely begging the question by not indicating how the cell "knows" to divide at a particular size. This question will be answered below. But first, two issues should be dealt with. An initial discussion will clarify the relationship of cell mass increase to cell size. This will be followed by a discussion of problems with the proposal of linear growth during the division cycle.What is meant by the Conlon and Raff proposal that, in yeast culture, large cells grow faster than small cells? And conversely, that in mammalian cells, large and small cells grow at the same rate? There are four different meanings that can be given the notion of the rate of mass increase and its relationship to cell size. These different meanings lead to some verbal confusion that requires clarification.One meaning of the proposal that large cells make mass faster than small cells is that given two cells of disparate sizes, the absolute rate of increase in cell mass is greater in the larger cells. A cell of size 2.0 might add, in some time interval, 0.2 units of cell mass, while a cell of size 1.0, in that same time interval might add only 0.05 units of cell mass. This pattern is a clear and unambiguous difference in the rate of mass increase that is related to cell size.A second meaning of cell size affecting the rate of mass increase considers that a cell of size 2.0 adds 0.2 unit of mass and a cell of size 1.0 adds 0.1 unit of cell mass over the same time interval. Of course, this case could arguably be said to be a constant rate of mass increase, as the rate of mass increase is proportional to the amount of extant mass. This second proposal is equivalent to mass increasing exponentially. This is because as extant mass changes during the cell cycle the absolute rate of mass increase also changes to reflect the newly added cell mass. After the cell of size 1.0 grows to size 1.1, in the next time interval, rather than 0.1 units of mass being added, there are 0.11 units of mass added to the cell mass. Just as interest is compounded in a bank account, and the funds grow exponentially, so mass in this second example increases exponentially.A third meaning of the variation in mass increase with cell size is that the rate of mass increase is determined at birth and continues throughout the cell cycle, unaffected by continued cell size increase. A relatively small newborn cell could have a rate of addition of "X" units per time interval, and this rate would remain constant even as the cell increases its cell mass. The larger cell would add more than "X" units each time interval and not change this rate during the cell cycle. This pattern of increase would be called linear synthesis during the cell cycle.It is interesting to think about these different meaning when considering the theoretical graph drawn by Conlon and Raff  to illusBut no indication of the length of the division cycles is given in Fig But if the absolute rates of mass increase were the same, then the smaller cell would have a much longer interdivision time than the larger cell. If, over a unit time, 1.0 unit of cell mass were added to the larger newborn cell, and that cell divided at size 11.0, then the interdivision time would be that unit time. The smaller cell, however, would require 10 time units for its interdivision time, because that is the time required to reach size 11.0 as 1.0 unit of material is added to each small cell during each unit of time. The smaller cell grows for a longer time before division. After this first division the new daughter cells produced by each of the initial cells would be the same size. By allowing interdivision time variation, cell size uniformity is restored in one generation.A similar analysis can be made for exponential mass increase . If the cell of size 10.0 added 1.0 unit of mass in a unit of time, then the small cell would add 0.1 unit of mass in that same time interval. In this case, there would be even more time required for the small cell to reach the division size of 11.0. In any case, exponential growth coupled with interdivision time variation can allow size maintenance because both the large and the small newborn cells will divide at the same size, as described in Fig. Of course, the example given by Conlon and Raff Fig  as discuRobert Brooks  notes that in some of his experiments cell size is observed as very variable. He states that in experiments with Shields they found that size varied over a range of at least 6-fold. In response it can be pointed out that recent careful measurements of the size variation during the division cycle of cells grown under ideal conditions indicates that size variation is not broad -7. Helmsresults from the growth rate. This idea, the fourth way of looking at the relationship of cell size and mass increase, will be illustrated below in the analysis of bacterial patterns of DNA replication and cell size maintenance.The fourth part of our verbal analysis of mass increase as a function of cell size relates to bacterial cells. As will be seen below, one of the most important results in bacterial physiology is that as growth rate speeds up, cells get larger. As the growth rate of a cell is continuously varied by increasing the richness of the medium, there is a continuous variation in bacterial cell size with the faster growing cells being larger than slower growing cells . BacteriAs we shall see, in bacterial cells a constant period for DNA replication and a constant time between termination of replication and cell division explains the variation in bacterial cell size as a function of growth rate. This same explanation also applies to mammalian cells: the rate of growth determined by external conditions determines cell size. Rather than taking the results of Conlon and Raff and concluding that larger cells when placed in medium with more serum now grow faster, it is better, as with bacteria, to say that when cells are placed in a condition that provides faster growth (i.e., a shorter interdivision time), the cells grow larger. While this may appear, at first sight, to be a trivial and semantic difference, this distinction actually lies at the heart of the problem and is the key to the solution of size determination and size maintenance. Rather than thinking that cell size produces cells with a particular growth rate (e.g., large cells grow fast), it is preferable to think that a particular growth rate produces cells of a particular size (e.g, fast growing cells are made larger than slower growing cells).There are problems inherent and unavoidable in any proposal of linear cell growth during the division cycle. Linear growth means that during the division cycle, as a cell proceeds from size 1.0 to size 2.0, cell mass is added at a constant amount per unit time. If a cell grows linearly, over tenths of a cell-cycle time, a cell increases its size from size 1.0, to 1.1, to 1.2, and so forth.The main problem with linear growth  is that as the cell gets larger, the cytoplasm becomes inefficient. Inefficiency is defined here as producing less mass per extant mass compared to more efficient use of the extant mass. Efficient mass increase would exist when extant cell mass makes new mass as fast as possible. As a cell grows, more cytoplasm is present. With linear growth the extra cytoplasm does not increase the absolute rate of cell mass synthesis. In essence, the new cytoplasm does not work to make new mass. There is a decrease in the relative rate of mass increase (i.e., mass synthesis per extant mass) which means that the ribosomes, after some growth, are not working as efficiently as before there was growth.One mechanistic model explaining the postulated absence of a change in the absolute rate of mass increase to produce linear growth is to propose that the new mass does not enter into active participation in mass synthesis until a cell division; new mass will not be "activated" to enter into mass synthesis until the next division. From this viewpoint, there is a constant rate of mass increase based on the original mass. As a cell approaches division, the efficiency of mass making new mass tends toward half that of the efficiency of the initial, newborn cell mass. An alternative mechanistic proposal to explain linear growth is that during a cell cycle the amount of material able to be taken up by a cell is constant, and only upon cell division is there an "activation" of the new cell surface so that there is an increase in the ability of the cell to take in material.Even more important and troublesome is the result that if a cell grows linearly, at the instant of cell division there must be a sudden saltation or jump in the synthetic activity of the cytoplasm. Toward the end of the cell cycle, 1.9 units of cell mass make 0.1 unit of cell mass to achieve a cell mass of 2.0. Given linear growth, at the instant of division the 2.0 units of cell mass, now apportioned into two daughter cells, must now make, during the next time interval, 0.2 units of cell mass or twice as much as in the previous time interval. When the cell of size 2.0 divides, linear growth implies that the two new daughter cells now immediately activate the "quiescent" cytoplasmic material (or activate the previously inert cell surface uptake capabilities). Irrespective of mechanism, considering the two daughter cells together, linear growth during the division cycle inevitably implies that at division there is a sudden doubling in the rate of mass increase.There is no known biochemical mechanism for these proposals to produce linear cell growth, or the sudden jump in the rate of mass increase. As currently understood, the new cytoplasm joins right in to make new mass. And there is no mechanism known to allow new cell surface to remain inert until a cell division. While the absence of any identification of these mechanisms does not mean that these mechanisms do not exist, there is no need to propose the existence of these mechanisms if cells grow exponentially.The experimental evidence favors exponential mass increase during the cell cycle. In bacteria the evidence for exponential growth is extremely strong ,10. AnalWhat of the experiments presented by Conlon and Raff  that celConlon and Raff realized that it is extremely difficult to distinguish linear from exponential growth over one doubling time. Therefore they measured mass increase over a longer period of time . The problem with this experiment is that the inhibited cells do not allow an exponential increase in cell number as DNA synthesis is inhibited. Therefore the experiment is subject to the critique that aphidicolin inhibition produced the observed results. The results may not, and very likely do not, reflect the situation in normal, uninhibited, and unperturbed cells. For example, there could have been exponential growth during the first "virtual cell cycle". Then the limitations of DNA content would lead to the observed linearity of growth as measured over the extended period of analysis. But this linearity should not be taken as an indication that during the normal cell cycle the cell mass increases linearly.a priori there should be evidence of an approach to exponential mass increase. If the rate of mass increase during the first cycle is 1.0, during the second cycle it should be 2.0, during the third cycle 4.0, and so on. Thus, even on its own terms, with linear mass increase during the division cycle, the experiments of Conlon and Raff [Even if cells grow linearly during the division cycle, if the rate of mass increase is measured over a number of cell cycles with uninhibited cells, then a priori, approach an exponential pattern (i.e., rate of 1, to 2, to 4, to 8 as cells multiply), and therefore the only meaningful discussion of the linear vs. exponential growth pattern relates to growth within the cell cycle. Regarding application of liver growth following starvation and refeeding, this complex situation seems particularly inapplicable to discussions of cell growth in cell culture as there are so many complicating factors. A detailed analysis of the proper systems for cell-cycle analysis has been presented [Raff  disputes this interpretation of the aphidicolin experiments, proposing that "while the aphidicolin-arrest strategy is certainly artificial, it is not unrealistic...as many cells, including Schwann cells, grow a great deal after they have stopped dividing. Moreover...hepatocytes grow linearly, independent of their size, if a mouse is re-fed after it has been starved for a couple of days." As noted in Fig. 3/cell increasing to ~9,500 μm3/cell) but the protein increase was only 2.93-fold (~0.16 ng/cell increasing to ~0.47 ng/cell). Until these differences are resolved, it is difficult to accept these experiments as supporting linear cell growth–or any other pattern of cell growth–during the normal division cycle. The discrepancies pointed out here suggest that the quantitative measures of cell size by Coulter Counter may not be able to distinguish different growth patterns.The experiments of Conlon and Raff also show some internal inconsistencies that weaken the actual data. A comparison of cell volume increase and protein per cell increase in the same cells over a 96 hour period  shows that the volume increase was 4.75-fold  has argued against this analysis, noting that the cells starved for 24 hours appeared to show "no turnover" as the line for this graph  was flat. But in the text in the legend to their Fig.4(b) Conlon and Raff state, "The shallowness of the curve for the 24-hour-arrested cells is likely to be the result of the lower than expected value at 0 hours." This explanation comes from the initial counts in Fig.4(a) where it can be seen that there is some apparent error in the zero time value for the 24 hour starved cells in their Fig.4(b).1-X, where X is the cell age during the cell cycle; X varies between 0.0 and 1.0 (newborn cells are age 0.0 and dividing cells are age 1.0). At age 0.0 the relative number of newborn cells is 2.0 (21-0 = 21 = 2) while the relative cell number of dividing cells is 1.0 at age 1.0 (21-1 = 20 = 1). This distribution of cell ages means that any incorporation measurement on asynchronous cells must, and will, yield an exponential pattern of uptake. This is illustrated in Fig. But an even more egregious error in analysis precedes even these technical problems. The cells studied by Conlon and Raff were not synchronized. The cells were not aligned and were in all phases of the cell cycle. Theoretically, it is impossible to determine the pattern of mass synthesis during the cell cycle on cells that are not synchronized. (For complete details see ). This iRobert Brooks  argues that this critique is incorrect because "they  started How can one determine whether mass increases exponentially or linearly during a normal, unperturbed, division cycle? To illustrate one approach to determining the pattern of mass increase during the division cycle, consider the following experiment. Grow cells for many generations in a radioactive amino acid (e.g., C-14 labeled amino acid) so that cell protein is totally labeled. Then add a pulse of a counter-labeled amino acid (e.g., H-3 labeled). As shown in Table To summarize this critique of the aphidicolin-inhibition results, the experiments of Conlon and Raff do not measure the mass increase during the cell cycle. The experiments using inhibition of DNA replication merely measure the pattern of mass increase in a perturbed experimental situation on cells that are not synchronized. This experiment is not supportive of any particular pattern of mass increase during the normal division cycle. More important, as shown in Fig. Escherichia coli) as well as the relationship of cell size to control of DNA replication were worked out [This analysis presented here explicitly deals with animal or eukaryotic cells. However, it is relevant to bring to bear on this problem the experience and results obtained regarding cell-size determination in bacteria. In 1968 the rules for the replication of DNA in a simple bacterium (rked out -20. The 1. A round of DNA replication is invariant (40 minutes) over a wide range of growth rates -19,21.2. The time between termination of replication and cell division is invariant (20 minutes) over a wide range of growth rates -19,21,223. At the time of initiation of replication, the cell mass per origin is a constant ,20,23.These rules are illustrated in Figs. The important consequence of Figs. The ideas of the bacterial cycle can be directly applied to animal cells. Cells of different growth rates are shown in Fig. As will now be seen, this variation in size is related to, and determined by, the growth rate.faster growth leads to larger average cell sizes.  that the richer a medium is (e.g., more serum rather than less serum), the faster the cells will grow. The faster a cell grows, the larger it will be Fig. . The varConlon and Raff  supply eThe analysis presented above explains the variation of cell size as function of growth rate as observed by Conlon and Raff . Furthermore, the analysis can also explain the maintenance of cell size, even with exponential mass increase during the division cycle, as shown in Fig. Thus, we now have an answer to the question  as a starting point Fig. . By defiThis analysis explains how cell size is maintained through a combination of interdivision time variation and cell growth rate variation. Exponential growth is possible and allowed during the division cycle, in contrast to the proposal of Conlon and Raff . The ideIt may be best to summarize these two contrasting views of size maintenance by looking at cell growth in a simple manner, and asking how the rate of mass increase is related to the passage of the cell through the cell cycle. The model of Conlon and Raff looks at the events of passage through the cell cycle as occurring independently of mass increase. The problem then remains as to how mass increase fits into, or coordinates with, the pattern or timing of passage through the cell cycle. It is as though the cell moves through the cell cycle without considering the mass problem, and then the mass of the cell looks at the cell cycle and says "I must grow at some rate so that I do not get too big or too small." In the Conlon/Raff model, a control exists that coordinates mass increase with the rate of cell division.The model presented here–in contrast to the model of Conlon and Raff–situates mass as the driving force of the cell cycle. Mass increases at some rate that is determined by external conditions (medium, growth factors, pH, etc.). As the mass increases, the accumulation of mass starts or regulates passage through the cell cycle. A cell cannot grow to an abnormal larger size because at a certain cell size or cell mass the S phase is initiated and this event starts a sequence of events leading to mitosis and cytokinesis. A cell cannot get too small because if mass grows slowly (or even stops growing) then the later events of the cell cycle (S-, G2-, and M-phases) are delayed (or do not occur) until mass increases sufficiently to start S phase. A cell cannot get too large because at a certain size the cell initiates S phase leading to the relatively early cell division. A cell cannot get too small because if mass accumulation is inhibited then S phase initiations are also inhibited.Thus, there is no problem relating mass increase and the cell cycle. Cell mass growth and cell cycle passage cannot be dissociated because one (mass increase) is the determinant of the other (S-phase initiation). For this reason one needs neither checkpoints nor control elements outside of mass increase.The time for mass to double in a particular situation determines the doubling time of a culture. This is because initiations of S phase occur every mass doubling time, and cell divisions similarly occur every mass doubling time. Thus total mass increases at the same rate as total cell number.The model presented here explains size determination, size maintenance, and the relationship of mass increase and cell number increase in a growing, exponential, unperturbed, mammalian cell cultures.


In daily use for centuries by hundreds of millions of people, nicotine has only lately been investigated for its therapeutic potential in a long list of common ills There's a cheap, common, and mostly safe drug, in daily use for centuries by hundreds of millions of people, that only lately has been investigated for its therapeutic potential for a long list of common ills. The list includes Alzheimer disease, Parkinson disease, depression and anxiety, schizophrenia, attention deficit hyperactivity disorder (ADHD), and even pain and obesity. Why has interest in this potential cure-all been slow to develop? One reason: in its current forms the drug offers pharmaceutical companies no possibility of substantial profit. Another, perhaps more important: the drug is reviled as the world's most addictive. The drug, of course, is nicotine.Nicotiana tabacum, which was smoked or chewed in the Americas for thousands of years before European invaders also succumbed to its pleasures and shipped it back to the Old World. Nicotine has always been regarded as medicinal and enjoyable at its usual low doses. Native Americans chewed tobacco to treat intestinal symptoms, and in 1560, Jean Nicot de Villemain sent tobacco seeds to the French court, claiming tobacco had medicinal properties and describing it as a panacea for many ailments. Higher doses are toxic, even lethal—which is why nicotine is used around the world as an insecticide. Yet few of the horrendous health effects of smoking are traceable to nicotine itself—cigarettes contain nearly 4,000 other compounds that play a role. Until recently, nicotine research has been driven primarily by nicotine's unparalleled power to keep people smoking, rather than its potential therapeutic uses.Nicotine is an alkaloid in the tobacco plant Nicotine locks on to one group of receptors that are normally targeted by the neurotransmitter acetylcholine. Nicotinic acetylcholine receptors (nAChRs) are ion channels threaded through cell membranes. When activated, either by acetylcholine or by nicotine, they allow selected ions to flow across the cell membrane. In vertebrates nAChRs are all over the autonomic and central nervous sytems and the neuromuscular junction. A nAChR is composed of five polypeptide subunits , but theIt is now conventional wisdom that acetylcholine and nicotine act at these receptors to alter electrochemical properties at a variety of synapses, which can in turn affect the release of several other neurotransmitters. This wisdom exists thanks in part to work by Lorna Role and her colleagues at Columbia University in New York City. “In 1995, we turned people's attention to how nicotine works as a modulator, tuning synapses and increasing the gain on transmitter release,” Role recalls. Although all nAChRs are activated by nicotine, other drugs could be found or designed that affect only a subset of these receptor types. “If you can dissect out the important players with respect to which nicotine receptors are tuning [a] particular set of synapses, then that provides another way to potentially target the therapeutics.”People with depressive-spectrum disorders, schizophrenia, and adult ADHD tend to smoke heavily, which suggested to researchers that nicotine may soothe their symptoms. Common to all these disorders is a failure of attention, an inability to concentrate on particular stimuli and screen out the rest. Nicotine helps. Researchers at the National Institute on Drug Abuse have shown via functional magnetic resonance imaging that nicotine activates specific brain areas during tasks that demand attention . This maUsing functional magnetic resonance imaging, scientists at the National Institute on Drug Abuse provided the first evidence that nicotine-induced enhancement of parietal cortex activation is associated with improved attention. They compared brain activity during a task demanding sustained attention—rapid visual information processing (RVIP)—with that during an undemanding sensorimotor control task . Group rNicotine may be the link between two genes that appear to figure in schizophrenia. Sherry Leonard and Robert Freedman of the University of Colorado in Denver, Colorado, have shown that expression of the gene for the alpha 7 neuronal nicotinic receptor is reduced in schizophrenics, and have argued that alpha 7 abnormalities lead to attention problems. Researchers in Iceland and elsewhere have shown that a different gene, for the growth factor neuregulin, also appears to figure in the disease. Neuregulin, Role and her colleagues have shown, governs the expression of nAChRs in neurons and helps to stabilize the synapses where they are found. The researchers are currently studying interactions between neuregulin and alpha 7, which Role thinks will prove important.Smokers also have lower rates of neurodegenerative disorders, and nicotine improves cognitive and motor functioning in people with Alzheimer disease and Parkinson disease. The prevailing hypothesis is that nicotine increases release of neurotransmitters depleted in those diseases. Dani and his colleagues have recently shown that acetylcholinesterase inhibitors—which block the degradation of acetylcholine and hence prolong its action—used to treat Alzheimer disease also stimulate dopamine release. They suspect that malfunctioning of the dopamine system may be affecting noncognitive aspects of dementia such as depressed mood, and that this might be alleviated by nicotine.Paul Newhouse and his colleagues at the University of Vermont in Burlington, Vermont, are studying nicotine drugs as potential therapeutic agents for cognitive dysfunction. Newhouse, a long-time nicotine researcher, is heading the first study ever to examine the efficacy and safety of nicotine patches for treating mild cognitive impairment, thought to be a precursor of Alzheimer disease. The researchers hope to see a positive effect on attention and learning. Newhouse also heads two studies of nicotinic stimulation in ADHD, using the patch, nicotine blockers, and some novel drugs that activate nicotine receptors.Nicotine's salutary effects in patients with neurodegenerative and mental disorders have been studied a lot and are fairly well known. Two much newer topics of academic research are nicotine's potential for pain relief and for treating obesity.Nicotine itself has provided modest pain relief in animal studies. Although the analgesic effect of drugs that mimic acetylcholine were originally attributed to a different class of receptors, it is now clear that nAChRs play an important role in the control of pain. For instance, epibatidine, a drug that is extracted from the skin of an Ecuadorian frog and that acts at nAChRs, has been shown to be 200 times more potent than morphine at blocking pain in animals. Current animal research is aimed at discovering just where, how, and which classes of nAChRs work against pain, with the aim of developing more selective drugs.Meanwhile, nicotine is also being investigated as an analgesic in humans. For example, Pamela Flood, an anesthesiologist at Columbia, is investigating nicotine's future as a postoperative analgesic. She recently completed a pilot study of 20 women undergoing gynecological surgery. All the women had access to unlimited morphine and also got either a single 3-mg dose of nicotine nasal spray or a placebo. The placebo group had peak pain scores of eight out of a possible ten in the first hour after surgery. Women who got nicotine averaged a pain score of five. Despite the small sample size, Flood says, the results were highly significant. “As far as I know this is the first clinical study to use nicotine for analgesia, and it was much more successful than I ever would have imagined.”“The nice thing about nicotine and drugs like nicotine is that they have opposite side effects to anesthetics. Instead of being respiratory depressants, they are respiratory stimulants. Instead of being sedating, they increase alertness. So theoretically this class of drugs is actually the perfect thing to add to an opioid regimen. The fact that they're synergistic was a fortuitous thing that we had never looked at, and neither had anybody else.”Nicotine may be the most effective drug around for weight control. As ex-smokers know, to their rue, one of the worst things about quitting cigarettes is putting on pounds—as much as 10% of body weight. “Something about being addicted to nicotine and then going off it causes massive increase in weight,” Role points out.Young-Hwan Jo in Role's lab is looking at a particular brain circuit involved in motivational behavior, especially feeding behavior. It is lodged primarily in the lateral hypothalamus but has projections all over the cortex, especially the nucleus accumbens, which is the center of reinforcement. “This is where information that has come in to the thalamus and the hypothalamus is relayed to cortical areas with some sense of salience or remembrance. It presumably is involved in changing perception and motivation for eating. It's not, ‘I have to eat this,’ it's, ‘I want to eat this,’” says Role.Jo has been comparing the synaptic effects of nicotine, which reduces appetite, to those of cannabinoids, which stimulate it. “Control of these projection neurons seems to be oppositely regulated by these two,” Role notes. “It doesn't necessarily mean we've found the root of the munchies, but it at least points to pathways that these things have in common.” Jo is also examining how nicotine and cannabinoids modulate these pathways in genetically obese mice, and also their interactions with leptins. Role says tuning these pathways up or down might be a reasonable aim. “If that could be done in a selective fashion, maybe that could be introduced in appetite control. Certainly I see…antagonism of some of these pathways that nicotine activates or the complementary activation of the cannabinoid pathways as very important targets for therapeutics with respect to the anorexia that's associated with chemotherapy.”Ming Li and his colleagues at the University of Texas in San Antonio, Texas, are studying nicotine's effects on weight and on expression of genes that nicotine upregulates orexin and neuropeptide Y and, more recently, that it also regulates leptin signaling. All three molecules regulate feeding behavior controlled by the hypothalamus. In the weight study, nicotine-treated rats not only lost weight, they lost about 20% of their body fat compared to saline-treated controls. The researchers suggest that, among its other effects, nicotine alters fat storage.The University of Texas researchers have scoured the literature for genes related to nicotine, and they are developing microarrays to study the expression of these genes . While nDani predicts that weight control is likely to be one of the earliest nicotine-based therapies. “There's a very good chance that the first drug is unlikely to be…nicotine itself, but will take advantage of nicotinic receptors in the therapy,” he says. “I know there are drugs now being tested by drug companies just for that purpose.”Developing new drugs that selectively target specific subtypes of nicotine receptors is an expensive, albeit potentially lucrative, proposition. And therein lies a question. Will nicotine-based therapy consist mostly of costly new drugs from the pharmaceutical industry? Or can less expensive nicotine products like the patch, chewing gum, and nasal spray—which are generally intended for smoking cessation but widely available, usually without prescription—find their way into the world's medicine cabinets?“It's a little early to call whether nicotine will be used itself as a therapeutic agent or whether these more specific drugs that are being produced or maybe even used in combination with other drugs may be the most important way to go,” says Dani. But he doesn't see the medicinal use of plain nicotine as very likely. Dani points out that the body's own agent, acetylcholine, acts over milliseconds to activate nicotinic receptors, whereas nicotine itself stimulates these receptors for hours. That lengthy action means that, although nicotine activates the receptors, it then often turns particular receptor subtypes off again, a process called desensitization. “It's hard to predict inside of a body what you're getting. Am I getting an activation or am I turning the receptors off?”Yet much of the work to date showing nicotine's effectiveness on a huge range of disorders has involved products available at any drugstore and intended to help people quit smoking. Newhouse is using patches for mild cognitive impairment. Flood has demonstrated pain relief with nasal spray and will use patches in her next study. And Role feels that gum hasn't been adequately explored for its therapeutic potential. Nicotine gum, she notes, is a better imitator of smoking than the patch because it delivers brief hits rather than a steady supply. She's also uncertain whether natural nicotine has been studied enough. But Role also points out that nicotine has its serious problems—addictive potential, cardiovascular damage, and  cancer.Dani says, “People are probably going to have to find creative ways to understand which subtypes of nicotinic receptors they're turning on and which ones they're desensitizing. Maybe drug delivery methods will matter. Maybe subtype specificity will matter. It's less than a decade that we've known how important nicotinic receptors are. Now we have to move forward from there.”“We've made an enormous amount of progress on understanding the biology of these receptor systems and how to target them. What has been trickier has been to develop an appropriate pharmacology that allows one to selectively target agents for particular therapeutic purposes with an adequate safety index,” Newhouse says. “But some of the drugs that are coming on in human trials now are very promising. So I'm cautiously optimistic that we're on the road to developing some useful nicotinic therapies.”


Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling.Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells  Fetal development and morphogenesis of various tissues and organs such as hair follicles, teeth, salivary glands, mammary glands, kidneys, liver, pancreas, and lungs depend on epithelium-mesenchyme interactions ,2. Such Epimorphin is a mesenchymal cell surface-associated protein that modulates epithelial morphogenesis in embryonic mouse organs including lungs and skin . EpimorpIn cultured mammary epithelial cells, epimorphin augmented expression of matrix metalloproteinase 2 (MMP-2), an important proteinase in matrix degradation, and both epimorphin and MMP-2 were required for mammary gland morphogenesis . During The epimorphin gene is highly conserved among mouse, rat, and human ,28: the All histologic slides from open or thoracoscopic lung biopsies in the files of the Department of Pathology, Kumamoto University Hospital, from 2000 to 2003, were evaluated. From among these samples, 17 patients were selected, with 8 fulfilling the histologic and clinical criteria for NSIP and 9 showing UIP. To confirm the histologic diagnosis, two pulmonary pathologists reviewed the slides; they were informed only of the age and sex of each patient and the presence of bilateral pulmonary disease determined by chest posteroanterior radiography and high-resolution computed tomography. All patients had had no steroid therapy before the biopsy. Normal control lung tissues were obtained from the normal areas of lungs that had been surgically removed from eight patients because of cancer. Table 1 shows the characteristics of the nine patients with UIP, eight patients with NSIP, and eight normal control subjects. In addition to the histologic diagnosis, we confirmed that the clinical data, pulmonary function test results, bronchoalveolar lavage findings, chest radiographic findings, and follow-up information obtained from the patients were consistent with the diagnoses. Diagnostic criteria of the American Thoracic Society/European Respiratory Society consensus classification system were applied . We alsoNcoI and SpeI sites of the pGEM-T Vector (Promega, Madison, WI), and authenticity was confirmed by sequencing. Total cellular RNA was isolated from lung tissues from each case by using the acid guanidinium-isothiocyanate-phenol-chloroform method. The RNA samples (10 Î¼g/lane) were fractionated by electrophoresis on 1% agarose-formaldehyde gels under denaturing conditions and were transferred to Nytran by capillary action. The blots were then probed with the 339-bp NcoI/SpeI fragment of the human epimorphin cDNA labeled with [32P]dCTP (3000 Ci/mmol) by means of the Nick Translation System kit (GIBCO/BRL, Grand Island, NY). After hybridization, blots were washed and were processed by autoradiography. Washed blots were analyzed by using a Fujix BAS2000 Bio-Imaging analyzer system (BASTM, Fuji Photo Film Co., Ltd., Tokyo, Japan) and were visualized on Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY). Signal intensity was quantified in digital images via BASTM analysis (FUJIX BAStation, Fuji Photo Film Co., Ltd.). To control for differences in gel loading, for each sample the RNA hybridized with the epimorphin probe was normalized to the expression of Î²-actin mRNA in the same sample.Human epimorphin DNA fragments were isolated by reverse transcriptase-polymerase chain reaction. Strand cDNA was synthesized with random primers from human lung total RNA. Polymerase chain reaction assays were carried out at 95Â°C for 1 minute, 64Â°C for 1 minute, and 72Â°C for 1 minute for 35 cycles. The following primer pairs were used: human epimorphin forward 5'-GGA ACC GGA CTT CAG TGG ATC-3' and reverse 5'-CAGC CAA TGA TTA GAG CCA GGA-3'. Polymerase chain reaction products were subcloned into the 2 antibody  used at a dilution of 1:1000 in Tris-buffered saline with 0.05% Tween 20 for 2 hours at room temperature. The bound antibody was detected by using the enhanced chemiluminescence method  according to the manufacturer's protocol.Although the monoclonal antibody to mouse epimorphin (MC-1) has been reported to cross-react with human epimorphin ,32, we pA portion of each lung specimen was fixed immediately in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), after which samples were sequentially washed for 4 hours in 10% sucrose in 0.01 M phosphate-buffered saline (pH 7.4, 4Â°C), 4 hours in 20% sucrose in phosphate-buffered saline, and overnight in 30% sucrose in phosphate-buffered saline. They were then snap-frozen in OCT embedding medium and stored at -80Â°C.2O2. Counterstaining was with Mayer's hematoxylin. Lung tissue of normal adult mice was stained as a positive control. Nonspecific labeling of primary antibody was evaluated with normal rat serum. Each tissue sample was also stained for keratin , Î±-smooth muscle actin , and MMP-2 . Serial sections were also stained with hematoxylin-eosin (H&E) and Alcian blue-periodic acid-Schiff (AB-PAS).Frozen tissues were cut into 4-Î¼m sections, which were incubated for 30 minutes with a biotin blocking system (Dako Corporation, Carpinteria, CA) and for 30 minutes with 0.3% hydrogen peroxide in methanol to eliminate endogenous peroxidase activity. After treatment with normal goat serum, tissues were incubated overnight at 4Â°C with 10 Î¼g/ml rat anti-mouse monoclonal epimorphin antibody and were then incubated for 1.5 hours at 37Â°C with 1 Î¼g/ml biotinylated goat anti-rat IgG (ZYMED, San Francisco, CA) and streptavidin-biotin-horseradish peroxidase complex (Dakopatts, Glostrup, Denmark). Bound antibody was visualized after incubation for 10 minutes in a Coplin jar with 100 ml of Tris-HCl buffer (pH 7.6) containing 20 mg of diaminobenzidine and 17 ml of 30% HTo localize epimorphin as precisely as possible, epithelial and stromal cells were double labeled by means of immunofluorescent probes, and the distribution of epimorphin was compared with that of keratin and vimentin . Briefly, after sections were exposed to primary antibodies, they were exposed to secondary antibodies: for the first analysis: fluorescein isothiocyanate-conjugated goat anti-rat IgG  or Texas Red-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR); for the second analysis: fluorescein isothiocyanate-conjugated goat anti-rat IgG  or Texas Red-conjugated goat anti-mouse IgG (Molecular Probes). The nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (Vector Laboratories, Inc., Burlingame, CA). Specimens were examined under a confocal laser scanning microscope , based on an upright microscope (DMRB, Leica Lasertechnik) equipped with a krypton/argon laser . The excTo compare the precise localization of MMP-2 and epimorphin in NSIP, a confocal microscope was used for immunohistochemical study of lung tissues from patients with NSIP, by means of the same technical immunohistochemical staining and analysis method as that described previously for epimorphin and keratin detection . Mouse a2 and 95% air.Human lung-derived alveolar epithelial cells (A549), an epithelial cell line derived from lung adenocarcinoma , were cultured in RPMI (GIBCO/BRL) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, and 50 Î¼g/ml streptomycin. All cells were maintained at 37Â°C in a humidified incubator containing 5% COEscherichia coli strain BL21 , or Type IV collagen from human placenta collagen , after which samples were dried at room temperature. After A549 cells were washed two times with phosphate-buffered saline, the cells (5 Ã— 105) were suspended in RPMI medium with 2% fetal calf serum and were incubated at 37Â°C on the chamber slides coated with recombinant epimorphin, BL, or Type IV collagen. After incubation for 9 h, culture dishes were placed on ice, medium from each dish was collected, and cells were washed twice with phosphate-buffered saline. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 5 minutes at -20Â°C and then were washed in phosphate-buffered saline and permeabilized in acetone for 5 minutes at -20Â°C. Cells were again washed in phosphate-buffered saline and incubated with a blocking solution containing 1% bovine serum albumin/phosphate-buffered saline for 1 hour at room temperature. Immunohistochemistry was performed via the same technical method described above for epimorphin detection. The primary antibody was 5 Î¼g/ml mouse anti-human MMP-2 monoclonal antibody, and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Amersham Pharmacia Biotech UK Limited).Four-well chamber slides  were coated with human recombinant epimorphin fragment , negative control solution prepared from non-transfected The A549 culture supernatant collected after incubation for 9 hours on the chamber slides coated with recombinant epimorphin, BL, and Type IV collagen was used to determine the amount of MMP-2 activity by means of the MMP-2 Biotrack Activity Assay System (Amersham Pharmacia Biotech UK Limited), according to the manufacturer's instructions. All samples were assayed in triplicate, and assays were repeated three times.Densitometric analysis of the bands of Northern and Western blotting was performed with Macintosh G4 computer (Apple Japan, Inc., Tokyo, Japan) using high-resolution scanner and NIH image software . The data are expressed as a ratio of a standard normal control subject to each case's band density units (arbitrary units) and reported as mean Â± standard error of the mean (SEM). Statistical significance was established using the one-way analysis of variance test followed by Tukey-Kramer multiple intergroup comparison test. Probabilities less than 0.05 were considered significant.P < 0.05)  Figure .Western blotting showed that rat anti-mouse epimorphin monoclonal antibody (MC-1) recognized the 150-kd bands in the extracted samples from both mouse lung and normal human lung Figure , similarIn the lung tissue homogenates of patients with NSIP, UIP and of control subjects, Western blotting with antibody for epimorphin also recognized the 150-kd bands Figure  and the In lungs of all patients with NSIP, alveolar walls were thickened with edema, fibrosis, and inflammatory cell infiltration; the appearance was typically uniform Figure . There wIn lungs of all patients with UIP, the fibrotic zones showed temporal heterogeneity, with dense acellular collagen and scattered fibroblastic foci with intervening nearly normal alveoli. Most fibrotic zones had honeycombing with complete destruction of the architecture Figure . Though Double-labeled confocal fluorescent images confirmed the presence of epimorphin in early fibrotic lesions and keratin-positive epithelial cells overlying the lesions Figure  and 6B. Areas of early fibrosis in lung tissues from patients with NSIP showed epimorphin immunoreactivity Figure , and regNine hours after plating of BL or Type IV collagen-coated chamber slides with cells of the human lung-derived alveolar epithelial cell line A549, the cells had spread and formed a loose sheet Figure .This is the first study to examine in detail the distribution and magnitude of epimorphin expression in normal human lungs and lungs from patients with NSIP or UIP. We found clear expression of epimorphin mRNA and protein in lung samples from normal control subjects and patients with NSIP or UIP.Consistent with previous reports , epimorpWe found, by means of Northern blotting assays, distinct expression of epimorphin mRNA and protein in normal lungs and lung samples of patients with NSIP or UIP, with significantly higher epimorphin expression in lungs of patients with NSIP than in other lung samples. Also, serial sections and double-labeled immunohistochemistry analyses of NSIP samples demonstrated strong expression of epimorphin in mesenchymal cells situated within active intra-alveolar fibrotic lesions and the presence of epimorphin in the ECM in the vicinity of these cells. These findings are consistent with our previous report that epimorphin was synthesized by mesenchymal cells and localized to these cells and the ECM of early intra-alveolar fibrotic lesions in a murine bleomycin-induced pulmonary fibrosis model . They arIt is well known that regeneration of alveolar and bronchiolar epithelial cells is crucial for repair of pulmonary fibrosis, and the ECM in early fibrotic lesions provides fibronectin and other adhesion molecules as ligands for regenerating epithelial cells to ensure successful repair ,4,39-41.We observed strong epimorphin immunostaining in early fibrotic areas with a few Î±-smooth muscle actin-positive cells in NSIP, as well as weak epimorphin immunostaining in fibroblastic foci with many Î±-smooth muscle actin-positive cells and desquamative regenerated alveolar epithelial cells in UIP. It is believed that in UIP, a failure of re-epithelialization of fibroblastic foci maintains fibroblast/myofibroblast activity and ECM synthesis ,44. We sTo understand the role of epimorphin in the repair process in the lungs of patients with NSIP, we evaluated whether epimorphin enhanced expression of MMP-2 (gelatinase A). Recombinant epimorphin induced formation of some monolayer cell islands in addition to a loose cellular sheet and augmented expression of MMP-2 in cultured human alveolar epithelial cells, as demonstrated by immunohistochemistry and the MMP-2 Activity Assay System. We suggest that epimorphin-induced monolayer cell islands are a type of formation of differentiated alveolar epithelial cells, like the epimorphin-induced spheroid formation of rat hepatocytes, and suggest that the augmented expression of MMP-2 may be similar to the increased albumin production of rat hepatocytes.in vitro [-/- mice was markedly reduced compared with that of gelatinase A+/+ controls. Thus, MMP-2 was up-regulated and activated in regenerating alveolar epithelial cells, which may allow elongation and migration of these cells for successful repair of pulmonary fibrotic lesions [[We also demonstrated expression of MMP-2 in re-epithelialized cells overlying epimorphin-positive early fibrotic areas by means of double-labeled confocal fluorescent images. MMP-2, which is an important proteinase needed for matrix degradation, is secreted by type II alveolar epithelial cells in vitro . Fukuda in vitro  observedin vitro . Yaguchiin vitro  reportedin vitro  reportedlesions ,27, 47]., 47.in vWe therefore suggest that epimorphin expression in early fibrotic lesions in the lungs of patients with NSIP may contribute to the repair process in pulmonary fibrosis, in part by inducing MMP-2 expression. What may follow this MMP-2 expression may be elongation and migration of regenerating epithelial cells and degradation of ECM in alveolar spaces. Thus, epimorphin, as a component of proteolytic systems, may contribute to cell migration and tissue remodeling during repair of human lung fibrosis.Although epimorphin receptors have not yet been identified, binding ECM molecules has been suggested as involved in establishing epithelial polarity, thus helping form an organized basement membrane and cell-ECM junctional complexes . Our resIn conclusion, epimorphin expressed in lungs may have important roles as a morphogen not only in mice but also in humans, and epimorphin may contribute to the repair process in human pulmonary fibrosis via epithelium-mesenchyme interactions.Table 1. Characteristics of subjects, including pulmonary function test resultsClick here for file


Beyond variant discrimination, independent component analysis automatically mapped several fingerprints to their metabolic determinants. The approach is flexible and paves the way to large-scale fluxome profiling of any biological system and condition.We introduce a conceptually novel method for intracellular fluxome profiling from unsupervised statistical analysis of stable isotope labeling. Without  In contrast to such compositional information, molecular fluxes through intact metabolic networks link genes and proteins to higher-level functions that result from biochemical and regulatory interactions between the components . As such biology ,3. Intraeriments ,5, whicheriments ,7 and geeriments ,9. The periments .a priori knowledge on possible distributions of the tracer used within the network, and, more importantly, extensive labeling and physiological data to resolve all fluxes within a given model. The lack of such structural knowledge on metabolic pathways and the technical difficulty of acquiring sufficient data hamper studies of metabolism, in particular in higher cells with complex nutrient requirements and for exotic tracer molecules. Hence, fluxome analysis is largely restricted to few 13C-labeled carbon sources in microbes or plants cultivated in minimal medium [We set out to overcome a principal bottleneck in metabolism-wide flux (fluxome ) analysil medium ,11-16.a priori knowledge of the biochemical reaction network. To illustrate the applicability of this conceptually novel profiling method, we focused on the reactions of central metabolism in the model bacterium Bacillus subtilis, for which detailed flux data were available to validate the results [Here we discriminate mutants/conditions and assess their metabolic impact directly from 'raw' mass-isotope data by unsupervised multivariate statistics without  results ,11,14.B. subtilis. In particular, we chose 10 knockout mutants [13C] or [U-2H]glucose, [U-13C]sorbitol or [3-13C]pyruvate and the nitrogen sources ammonium or casein amino acids (CAA). As a proof of concept, we detected the isotopic labeling patterns in proteinogenic amino acids by gas chromatography MS (GC-MS), which provides direct access to several metabolic nodes in the network [Environmental and genetic modifications were used to perturb intracellular metabolic activities in  mutants  that wer mutants  with six network ,7,19  fragments in the profiles of the glcP and cggR mutants during growth on sorbitol with CAA; that is, high fractions of masses m0 and m3 and low fractions of m1 and m2 (where the subscripts denote the number of 13C atoms in each amino-acid fragment). While the S signature of the mdh mutant on sorbitol with CAA was also distinct, it was different from that in the above two mutants with low m1, m2, and m3 fractions  in glcP and cggR is evidence of a lower exchange with the glycine pool, when compared with the wild type [mdh mutant, the high fraction of uncleaved but unlabeled S (m0) reveals high incorporation of unlabeled serine from the CAA supplement, and thus low de novo biosynthesis from 13C-labeled sorbitol.For the visualization of metabolic effects, the corrected MS signals of the wild type were subtracted from those of the mutants Figures  and 4. Sild type ,20. In tsdhC mutant on glucose and sorbitol. Because the sdhC mutation disrupts the tricarboxylic acid (TCA) cycle, the wild-type flux through the cycle must be similar on these substrates, both with and without CAA  and glutamate (E) were also present in the CAA profiles of the other TCA cycle mutant mdh. Their absence on ammonium indicates activity of the malic enzyme-based pyruvate bypass [mdh mutant.As well as consistency with the data in the literature, the analysis also revealed new information on pathway activity and regulation that was not previously accessible. One example is the pronounced signatures of the A Figure . The sdhe bypass  in the mgapA operon, for example, appears to affect TCA cycle fluxes because the mutant profile matches those of the TCA cycle mutants sdhC and mdh . An illustrative example is the cggR mutant signatures for the pyruvate-derived amino acids valine (V), leucine (L) and, partially, alanine (A) , which is fully consistent with increased transcription of the glycolytic enolase in the cggR mutant on glucose [13C experimentsA significant extension beyond the canonical  glucose  that couFor large-scale profiling studies, automated mutant classification based on metabolic function without user supervision would be desirable. Initially, we used principal component analysis (PCA), which is often used for graphical representation of multidimensional variables from profiling experiments ,25, as wConsequently we used independent component analysis (ICA) for unsupervised, automatic recognition of conserved labeling patterns that are biochemically relevant. The underlying assumption is that these patterns result from the superposition of independent metabolic activities. Each activity causes a specific shift in the mass distributions of one or more intermediates. ICA seeks to separate the observed variables into non-gaussian components that are statistically as independent as possible . General13C]sorbitol with ammonium experiment, we explored the ICA results in more detail  or reduced availability of replicas. The bottleneck resides in the stochastic approach of most ICA algorithms, for which independent runs result in different ICs or ordering thereof. Instead, algorithmic and statistical reliability of the ICs can be evaluated by repeating the estimation several times either with randomly chosen initial guesses or by slightly varying the dataset (bootstrapping [Similarly, trapping ), respectrapping .de novo biosynthesis from the tracer relative to the uptake of a medium component. Similarly, uniformly deuterated substrates or 2H2O are valuable for simultaneously capturing a wide number of ICs that are affected by the release, binding and exchange of water or protons. Substrates that are labeled at specific positions, in contrast, enable deeper interrogation of particular sub-networks, for example, [1-13C]hexoses for the initial catabolic reactions [13C]aspartate to assess urea cycle activity.Two factors directly affect the results that can be obtained by comparative fluxome profiling: the detected analytes and the choice of isotopic tracer. As well as polymer-based analytes such as the proteinogenic amino acids monitored here, fluxome profiles can be detected in any set of intra- or extracellular metabolites, thereby widening the observable metabolic processes The choice of tracer depends, to some extent, on the metabolic subsystem of interest. Uniformly labeled substrates provide a more global perspective because they allow assessment of the scrambling of any carbon backbone and, in the case of experiments performed in rich media, also allow quantification of the fraction of eactions ,19 or [1pgi and yqjI mutant signatures were different from the wild type and from each other. On sorbitol, in contrast, the same mutants were very similar to the wild type, suggesting that both reactions are only marginally involved in catabolism of this sugar. Second, the Krebs cycle flux was similar on glucose and sorbitol (with and without CAA), as deduced from the similarly pronounced signatures of the sdhC mutant. Third, absence of the sdhC signatures in the Krebs cycle-derived amino acids aspartate and glutamate of the mdh mutant when grown with ammonium (but not CAA) indicates activity of the malic enzyme-based pyruvate bypass [ytsJ mutant were seen on all substrates. The gluconeogenic phosphoenolpyruvate synthetase Pps, in contrast, was inactive in the presence of the repressing glucose but active on pyruvate or sorbitol. Fifth, as discussed above the data reveal a Krebs cycle-promoting effect of the repressor CggR on sorbitol but not on glucose, most likely through the repression of glycolytic genes [The results also revealed new biological information on pathway activity, function or regulation. First, both glycolysis and the pentose phosphate pathway actively catabolized glucose in the presence of CAA, because the e bypass . Fourth,ic genes .in vivo flux. Interesting variants are then subjected to deeper interrogation of the specific metabolic phenomenon identified. Besides mere data mining, fluxome profiling also has the potential to identify complex functional traits in higher cells where current flux methods fail, and possibly even identify the underlying biochemical mechanism of discriminant mass isotope signatures.The comparative fluxome profiling presented here complements traditional flux analysis because it enables potentially rapid and automated identification of relevant mutants or conditions from large-scale datasets, for example from entire mutant libraries. The approach is quantitative in terms of the relative difference between variants, but qualitative with respect to the B. subtilis 168 (trpC2) [2H- or 13C-labeled isotopic tracers (3 g/l) and nitrogen sources were used: (i + ii) uniformly 13C-labeled [U-13C]glucose with either 0.5 g/l CAA (Sigma) or 1 g/l NH4Cl; (iii + iv) [U-13C]sorbitol with either 0.5 g/l CAA or 1 g/l NH4Cl; (v) [U-2H]glucose ([1,2,3,4,5,6,6-2H]glucose) with 1 g/l NH4Cl; and (vi) [3-13C]pyruvate with 1 g/l NH4Cl and twofold higher concentrations of phosphate to ensure pH buffering. [U-13C]glucose (Martek Biosciences), [U-13C]sorbitol , and [1,2,3,4,5,6,6-2H]glucose (Euriso-Top) were supplemented as 50:50 mixtures of labeled and unlabeled isotopomers. Pyruvate was supplied entirely as the [3-13C] isotopomer (Euriso-Top).Wild-type  (trpC2)  and knoc (trpC2)  were gro (trpC2)  at pH 7.4Cl, and with pyruvate were harvested after 10, 14 and 24 h, respectively. Labeling patterns in the analyzed proteinogenic amino acids are rather stable [Aerobic batch cultures were grown in silicone-covered, deep-well microtiter plates at 37°C and 300 rpm in a 5-cm orbital shaker . Frozen r stable ; hence dm0, m1, ..., mn, expresses the fraction of molecules that are labeled at 0,1, ...,n positions, depending on the total number n of carbon or hydrogen atoms present. Considering all corrected fragment vectors obtained per sample, a complete dataset typically consisted of about 260 and 330 single mass values from 13C and 2H experiments, respectively, depending on the quality of the MS measurement.Cell harvest, protein hydrolysis and GC-MS analysis of amino acids were done exactly as described before ,32. Aminprincomp function of the Statistics toolbox 4.0. No input vectors were eliminated from the dataset to filter outliers in PCA, because this operation affected only PCs with higher order but only marginally PC1 and PC2.To obtain a new representation of the multivariate MS data and to make their essential structure accessible, we applied PCA to the corrected fragment vectors. This approach projects the input variables in an orthogonal space that is spanned by the PCs. Among the infinite number of possibilities, each successive PC is selected to maximize the variance of the projected data and to be orthonormal to the previous ones . Consequm fundamental components S = (s1, ..., sm)T, each of which is represented by a vector of p MS-signals. We assumed that the experimental data X = (x1, ..., xn)T, with n vectors of p corrected MS signals for each mutant/condition, result from a linear combination of the m fundamental processes, given by xi = ai1s1 +...+ aimsm. In matrix notation, this leads to Xp×n = Ap×mSm×n, with A as the mixing or loading matrix. ICA seeks to estimate the unknown terms A and S from the observed values X but has different objectives from PCA. Briefly, ICA identifies statistically ICs by selecting those with maximum non-gaussianity [To reveal hidden information in the labeling patterns, the corrected MS vectors were subjected to ICA , which issianity . Hence, ssianity . In partssianity  to estim2H experiment; Additional Figure 2 shows mutant discrimination by PCA (less relevant than by ICA); Additional Figure 3 is a complete representation of the 660 ICs (10 ICs in 6 experiments for 11 strains). All the raw data is contained in six Excel tables in Additional data file The following additional data is available with the online version of this paper. Additional data file 2H experiment; Additional Figure 2 shows mutant discrimination by PCA (less relevant than by ICA); Additional Figure 3 is a complete representation of the 660 ICs (10 ICs in 6 experiments for 11 strains)Three additional figures (Additional Figure 1 shows the mass distribution in the Click here for additional data fileAll the raw data contained in six Excel tablesClick here for additional data file


Background to the debate: The World Health Organization (WHO) and its partners aim to treat 3 million people infected with HIV in poor and middle income countries with antiretroviral treatment by the end of 2005. The ambitious “3 by 5” initiative has had its supporters and its critics since its announcement in 2002. Jim Yong Kim argues that getting 3 million HIV positive people on antiretroviral treatment by the end of 2005 is the right strategy. Arthur Ammann believes this approach is too narrow There are no sure prescriptions against great plagues like HIV. We must “learn by doing,” quickly assessing the inevitable missteps and false starts and using this information to improve outcomes. Our best information about the HIV pandemic suggests four clear principles.First, treatment must be a core element. This does not mean treatment alone. Antiretroviral therapy (ART) is in no way more important than education, prevention of mother-to-child transmission, expanded access to testing and counseling, or other pieces of a comprehensive public health effort. But preventing mother-to-child transmission is a pyrrhic victory if, as the latest data suggest, AIDS will cause 15 million of those uninfected children to grow up as orphans . The draSecond, time is of the essence. Every data point in the arcs of HIV transmission, morbidity, and mortality represents an exponential increase in human suffering and social and economic disruption. Countries that were able to respond early and comprehensively to HIV now have mortality rates comparable to those of Europe and North America and have cut transmission dramatically. Early interventions can provide large cost savings to the public sector and prevent devastating losses of human capital . There mThird, clear consensus targets are indispensable. An effective response to HIV demands the resources and attention of every region, state, and community. Coordinating these many different stakeholders requires clarity of purpose. All the great public projects of the modern era, such as the first manned moon expeditions of the 1960s, began with the establishment of a ringing collective priority. Like the 3 by 5 initiative, these projects were in themselves only surrogate endpoints. But they provided focus for the energies of their many participants. To reach the ultimate goal of universal access to ART we must plan in stages.Fourth, the specific procedures developed to combat HIV must be codified and simplified. Because pandemics, by definition, afflict communities with a broad range of material resources and technical capacities, our methods must be suitable to the most disrupted and impoverished of them. This means effective training modules to increase the supply of health workers; it means inexpensive, rapid tests and diagnostics; and it means streamlined regimens and fixed-dose combinations that facilitate drug procurement and improve rates of adherence.These principles are at the heart of the 3 by 5 effort. When it was announced in 2002, the gap between the damned and the saved had finally begun to narrow after sharp declines in the price of ART and the emergence of large-scale financing through the Global Fund, the World Bank, and the President's Emergency Plan for AIDS Relief. AIDS is unusual in the history of epidemics because proven, effective ways of interrupting the course of the disease have existed since shortly after its emergence, yet those methods were foreclosed to most of the world's population. The announcement of 3 by 5 drew from a widespread sense that this inequality presented unacceptable economic, political, moral, and epidemiological consequences.Scaling up treatment is not only a possibility, but is already a reality. The number of patients on ART has nearly doubled in the last two years, mostly in countries where therapy had been limited to a privileged few . High-buThose who object to 3 by 5 must address this question: what would be the likely cost if it were never attempted? We can work exclusively to prevent the further spread of HIV, or aim to improve treatment access more slowly, but in the meanwhile high-burden countries will collapse at our feet. Or we can aim for 3 by 5 and move ourselves that much closer to the ultimate goal: preventing all unnecessary deaths from HIV.There is much that is exemplary about the basic principles outlined by WHO for treatment expansion. Improving access to life-saving ART is a moral imperative and everyone agrees that this will take lots of money ,7. So whThe debate lies in the strategy. Reviewing the epidemic over the past 20 years, you would have to conclude that the current international public health approach has failed and that an urgent change is required. There are three fundamental problems with the 3 by 5 approach.First, it is very narrow, and narrow strategies for tackling HIV often fail. For example, we have known for five years that a single dose of nevirapine can help prevent perinatal HIV transmission. The intervention is simpler and cheaper than the 3 by 5 initiative—yet today less than 5% of pregnant women infected with HIV receive any ART ,9. WHO sSecond, confusion about realistic costs, sources of funding, and the relation to other critical WHO initiatives abound. Initial WHO estimates of $5 billion annually for the cost of the entire 3 by 5 initiative have been revised upward to $6 billion annually ,11. It iThird, the 3 by 5 initiative is a “top down” unsustainable approach that, without a high level of government investment, fosters dependence on international aid. Countries that have taken ownership of their HIV programs have often been the most successful in tackling the epidemic ,14,15. BThe HIV epidemic is the worst pandemic in history. Why, then, is the international public health response so disparate from public health responses to other life-threatening infectious diseases? The 3 by 5 initiative, with no requirement for contact tracing, does not ensure the right of uninfected individuals to be protected or of infected contacts to gain access to treatment. It is time to acknowledge that the severity of the epidemic requires universal offering of HIV testing and counseling, contact tracing, and integration of sound public health prevention and treatment principles into all health-care delivery systems ,10.Thousands of existing and new teams of health-care workers should be trained in voluntary testing and counseling, and in treatment, using streamlined training courses. Mobile teams could be used to reach rural areas where as many as 50% of infected individuals may live. Cumbersome “top down” training and certification, as is currently planned, will only delay the very therapy that WHO seeks to make more available ,14,15.It is a paradox that HIV is one of the few diseases that is deemed to require exceptional international and national bodies overseeing access to medicines. The success of ART in developed countries was a result of making ART directly available to those who treat patients, not to governments. Widespread and timely access can only occur when health workers are able to provide ART without repressive procedures. We can distribute drugs more freely for other diseases—why not HIV?I agree with Arthur Ammann that the response of the international community to the HIV epidemic over the last 20 years has been grossly inadequate. I also agree that a more effective strategy must pay close attention to mother-to-child transmission, provide “opt out” testing and counseling integrated into key health-care settings, implement effective partner referral strategies, and train many thousands of health-care workers. These are stated WHO priorities—integral to the 3 by 5 initiative—as any attempt to investigate our position would reveal ,17.Dr. Ammann's other charges are also puzzling. Our latest cost projections (which he cites) in fact provide a two-year, not annual, range of $5.1 to $5.9 billion, depending on the price of drugs . The accFinally, I must point out a contradiction in Dr. Ammann's arguments. On the one hand, he urges high-burden countries to take ownership of their HIV programs and finance these themselves. On the other, he acknowledges that if the “true cost [of ART] is $6 billion annually, this is $2,000 per individual per year, which is surely not sustainable for resource-poor countries.” The crucial question is whether $6 billion annually is “sustainable” for the world community as a whole. In facing the worst health disaster in several centuries, we simply cannot wait for the poorest countries to self-finance HIV treatment—or else we will be guilty of standing idly by as millions die and societies collapse.There are time-tested prescriptions for halting epidemics, and HIV should be no exception. But “HIV exceptionalism” persists . CurrentPrevention of perinatal HIV transmission cannot be called a victory—not even a pyrrhic one—since less than 5% of pregnant women infected with HIV receive any ART. Further, in most perinatal HIV prevention programs sexual partners are generally not identified, treatment has only recently been offered, and HIV testing has not been universally implemented. Narrowly focused, top-down programs such as the 3 by 5 initiative face similar outcomes when they adopt yet another partial approach that is not fully integrated into countrywide general health care.The 3 by 5 initiative suggests that treatment is the major incentive for HIV testing. But getting tested has two incentives—treatment and prevention. Knowing one's HIV status is a means of saving lives, an incentive that should motivate every individual to be tested even when treatment is not available. We will destine ourselves to an ever-escalating epidemic if we delay universal testing until treatment is available.Both Jim Yong Kim and I want to treat 3 million individuals infected with HIV and more, and we agree that time is of the essence. But why take another decade to learn what we already know from epidemics such as tuberculosis? Lateral, comprehensive approaches that equip health-care workers with testing and the required drugs work best ,22. Proc


Mass spectrometry based proteomics result in huge amounts of data that has to be processed in real time in order to efficiently feed identification algorithms and to easily integrate in automated environments. We present wiff2dta, a tool created to convert MS/MS data obtained using Applied Biosystem's QStar and QTrap 2000 and 4000 series.,  and is also available from Applied Biosystems.Comparing the performance of wiff2dta with the standard tools, we find wiff2dta being the fastest solution for extracting spectrum data from ABIs raw file format. wiff2dta is at least 10% faster than the standard tools. It is also capable of batch processing and can be easily integrated in high throughput environments. The program is freely available via wiff2dta offers the possibility to run as stand-alone application or within a batch process as command-line tool integrated in automation and high-throughput environments. It is more efficient than the state-of-the-art tools provided. In tandem mass spectrometry proteins are identified by matching the measured fragment ion spectra derived from peptides with theoretical spectra calculated from known DNA or protein sequences, for example the NCBI sequence database . AlgoritApplied Biosystems mass spectrometers are controlled by a software called Analyst™. This software is used for data evaluation purposes, too. It offers a possibility to integrate extensions called "scripts". One of these scripts available from the manufacturer  is "ExpoAnother script named mascot.dll provides support for invocating Mascot™ as protein identification algorithm using Applied Biosystems Analyst™. Such a script does not exist for Sequest™. In most proteomics labs support for Mascot™ as well as for Sequest™ is needed, because these two algorithms are most commonly used in this research field. Although the additional information that can be stored in mzXML is needed in the case of quantitative proteomics experiments based on isotopic labelling of peptides  because ActiveX™ is provided as the one and only application programming interface (API) by Applied Biosystem's Analyst™ software. Therefore, this is needed for accessing the binary wiff files. Thus, this tool is operating system dependant and only runs on Windows™ (Microsoft Corp.) systems. We use the code provided by the Analyst™ software API in order to benefit from new releases and maintain coherence.The program has two modes of user interaction: one provides a graphical user interface (GUI) and requires user interaction (GUI-mode); the other uses command-line parameters and suppresses the GUI as no user interaction is required (batch-mode). In batch mode, automation of conversion processes can be achieved. The GUI is shown in figure The conversion itself can be controlled by entering appropriate values in the text fields displayed under the title "Parameters", shown in figure wiff2dta is able to be integrated in automation and high throughput environments. This can be achieved making use of the command line options. All parameters and modes can be controlled by command-line parameters. These are shown in figure The program can be started in multiple instances, resulting in parallel processing. Using this feature, it is possible to use several processors on one computer. Additional to this, wiff2dta is about 10% faster than the original tool provided by Applied Biosystems and about 20 times faster than mzStar of the Sashimi project. See table wiff2dta demonstrates improvements in reducing computation time by exploiting a range of optimizations in coding and using the COM interfaces to Analyst™. Useful features like the capability of being integrated in batch processes and mass data processing lead to immense time savings, too.wiff2dta has to be installed in the BIN directory of an installed Analyst™ version 1.3 or higher. The installation consists just of copying the file wiff2dta.exe into this directory. If desired, a link to the program file can be created that can be placed onto the desktop or into the start menu. and  for download.The program is freely available from Applied Biosystems (UK) upon request and freely available via API: application programming interfaceDNA: desoxyribonuclein acidDTA: file extension ms spectra data in Sequest™ formatER: enhanced resolutionESI: electron spray ionizationGUI: graphical user interfaceMS: mass spectrometry, mass spectrometerMGF: mascot generic format, file extension used for this formatNL: neutral lossPrec: precursor ionTOF: time-of-flightWIFF: file extension of Applied Biosystems raw data filesAB implemented the program and made a draft of the manuscript. RPG and AS contributed with ideas and proofread the manuscript. RPG supervised the final testing. All authors have read and approved the final manuscript.


In randomized, controlled trials, antidepressant medications have been shown to reduce the duration of major depressive episodes and to reduce the frequency of relapse during long-term treatment. The epidemiological impact of antidepressant use on episode duration and relapse frequency, however, has not been described.Data from two Canadian general health surveys were used in this analysis: the National Population Health Survey (NPHS) and the Canadian Community Health Survey (CCHS). The NPHS is a longitudinal study that collected data between 1994 and 2000. These longitudinal data allowed an approximation of episode incidence to be calculated. The cross-sectional CCHS allowed estimation of episode duration. The surveys used the same sampling frame and both incorporated a Short Form version of the Composite International Diagnostic Interview.Episodes occurring in antidepressant users lasted longer than those in non-users. The apparent incidence of major depressive episodes among those taking antidepressants was higher than that among respondents not taking antidepressants. Changes in duration and incidence over the data collection interval were not observed.The most probable explanation for these results is confounding by indication and/or severity: members of the general population who are taking antidepressants probably have more highly recurrent and more severe mood disorders. In part, this may have been due to the use of a brief predictive diagnostic interview, which may be prone to detection of sub-clinical cases. Whereas antidepressant use increased considerably over the data-collection period, differences in episode incidence and duration over time were not observed. This suggests that the impact of antidepressant medications on population health may have been less than expected. While primary prevention for this condition has remained an elusive goal, provision of treatment has been viewed as having the capacity to reduce its impact on population health. Randomized, controlled clinical trials confirm that treatment with antidepressant medications can favorably impact the course of major depressive disorder. Clinical practice guidelines recommend acute treatment to reduce episode duration, continuation treatment to prevent relapse and maintenance treatment for those at high risk of recurrence [Depressive disorders are among the most important contributors to disease burden at the population level currence .Direct generalization of clinical trial data to the general population results in an expectation that depressive episode frequency and episode duration should be reduced in those receiving antidepressant treatment. However, outcomes reported in clinical trials are not necessarily reflected in "real world" outcome data. Since treatments are not assigned randomly in clinical settings, those treated are likely to have more severe illness (both in terms of episode duration and recurrence risk) than those who do not seek or receive treatment. In such circumstances, the effect of treatment may become confounded with the effect of the underlying disorder itself, and/or its severity .Despite the plausible occurrence of confounding by indication and confounding by severity, population-based studies examining outcomes in relation to treatment status in the general population have not been reported. The objective of the current study was to estimate the incidence and duration of major depressive episodes in members of the general population who are receiving or not receiving antidepressant treatment. The NPHS sample consists of 17,262 subjects selected in 1994 and 1995 (hereafter denoted 1994/95) who have been followed prospectively with interviews every two years since. Data has been released for the first four cycles (1994/95 to 2000/01). The Canadian Community Health Survey (CCHS) is another general health survey conducted by Statistics Canada. The CCHS has a very large sample size (n = 130,880) and employed a cross-sectional study design. Data collection for the CCHS occurred in 2000.The National Population Health Survey (NPHS) is a longitudinal general health survey conducted by the Canadian government's statistical agency, Statistics Canada Both the NPHS and the CCHS utilized probability samples, based on the same sampling frame, from the Canadian general population. The sampling procedures incorporated both clustering and unequal selection probabilities. Valid inference therefore requires the use of sampling weights and statistical procedures accounting for non-independence within clusters. In order to deal with these methodological issues, a bootstrap procedure for variance estimation developed by Statistics Canada was employed in this analysis. This procedure accounts for the design effects.Both the NPHS and the CCHS recorded medication use with a series of self-report items. The item relevant to this analysis asked whether the respondent had taken antidepressant medications during the month preceding the interview. Each interview also included the Composite International Diagnostic Interview Short Form for Major Depression (CIDI-SFMD), which was developed and validated by Kessler et al. . This isIn calculating incidence, pairs of observations across NPHS longitudinal data collection cycles were used. There were three suitable intervals covered by the four data collection rounds: 1994/95 to 1996/97, 1996/97 to 1998/99 and 1998/99 to 2000/01.Attrition rates have generally been modest in the NPHS, with 76.6% of subjects having been successfully followed over the first four cycles [Estimates of the number of weeks depressed in the past year derived from the CCHS were computed from 126,715 subjects who provided valid responses both to the CIDI-SFMD (including the duration item) and the antidepressant use survey item. There were 9729 subjects with an episode of major depression in the year preceding the interview and 9508 (97.7%) of these provided valid duration data. Figure Figure Table Direct generalization of clinical trial data to the general population would lead to an expectation that subjects in the general population who report antidepressant treatment should have briefer episodes of major depression than those who do not receive such treatment. Similarly, one might hypothesize that subjects reporting antidepressant treatment should have a reduced incidence of episodes. In the current analysis of national survey data, neither expectation was found to hold true. Subjects reporting an episode of major depression and reporting receipt of antidepressant treatment reported more, rather than fewer, weeks depressed in the preceding year. Similarly, subjects who did not have an episode of major depression in the year preceding an interview but who nevertheless reported using antidepressant medications had a higher risk of having an incident episode than similar subjects who did not report taking antidepressants. The most obvious explanation for these findings is that of confounding by indication or severity.Some of these results may have been due to inadequacies involving the data sources. One of these is the brief, and therefore somewhat crude  nature oItems evaluating antidepressant use in each survey referred to use of the medications during the past month, whereas probable major depressive episodes occurring during the past year are detected by the CIDI-SFMD. Therefore, even though subjects who reported taking antidepressants were found to be more likely to have a subsequent episode of major depression, the data did not allow a determination of whether some of these subjects may have stopped or started antidepressants at some time during the follow-up interval.Another limitation of the data sources used in this project was the lack of comorbidity data. Many of the subjects taking antidepressants may have been doing so for indications such as the prophylaxis of migraine headaches or for treatment of anxiety disorders. Both migraine headaches ,10 and aWhile confounding by indication and severity offer, perhaps, the most appealing explanation for these results it is important to emphasize that the attractiveness of these explanations is based on a set of assumptions: that antidepressants are efficacious in reducing episode duration and the risk of relapse and recurrence. An altered set of initial assumptions leads to other possible interpretations. For example, some authors have hypothesized that antidepressant treatment may lead to a deterioration in the long-term course of mood disorders . This hyGenerally, the public health challenges associated with major depression have been conceptualized in "common sense" terms, and in a way that does not depend on underlying etiological features of the disorder. For example, the idea that preventing relapse (episode incidence) should translate into reduced prevalence and reduced disease burden seems on the surface to be a common-sense belief. However, it has been hypothesized that depression can assume an adaptive role . If an aMost of the literature concerned with major depression and public health has emphasized that the disorder is under-treated, although more recent studies have begun to move beyond the frequency of treatment to address the issue of treatment adequacy . There iCIDI-SFMD Composite International Diagnostic Interview Short Form for Major DepressionDSM-IV Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition.MDE Major Depressive EpisodeNPHS National Population Health SurveyCCHS Canadian Community Health Survey (iteration 1.1)The author(s) declare that they have no competing interests.