Nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG) neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception.+-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the Emax without altering the EC50 for either compound.Compared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP)-evoked calcitonin gene-related peptide (CGRP) release, reaching approximately 300% at the highest concentration tested (100 ng/ml). Also, NGF and GDNF each augmented anandamide (AEA)- and arachidonyl-2-chloroethylamide (ACEA)-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM KTaken together, our results illustrate that NGF, GDNF and BDNF differentially alter TG sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release in culture. In particular, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis than GDNF. These findings are discussed in relation to possible therapeutic roles for growth factors or their modulators in pathological pain states, especially as these relate to the trigeminal system. During development, NGF, GDNF and BDNF, along with neurotrophin-3 (NT3), support the survival of subpopulations of sensory neurons through their cognate trk receptors -3. In th4 (IB4, ).NGF is thought to play a primary role in the development and maintenance of several pro-algesic states. NGF produces sensitization of nociceptive responses in vivo and increases responsiveness to chemical stimuli associated with nociceptive neurotransmission in vitro -12. NGF GDNF also plays a role in modulating nociceptive processing, but with contrasting in vitro and in vivo effects. In cultured DRG neurons, GDNF increases CAP sensitivity and TRPV1 expression ,26 and iInflammation and nerve injury both increase BDNF content in DRG neurons and in the spinal cord, and this increase in BDNF is associated with the maintenance of a hyperalgesic state -36. FurtIn addition to CAP, a number of other TRPV1 agonists have been described. Among these are compounds that are also cannabinoid receptor agonists, including the endogenous cannabinoid anandamide  on 8-well culture slides  did not significantly alter TRPV1 mRNA levels after 5 days of treatment Fig. . To eval+-evoked CGRP release and total CGRP content in TG neuronal cultures. NGF treatment significantly and concentration-dependently increased 50 mM K+-evoked CGRP release at every concentration, peaking (10.5-fold increase vs. control) at 100 ng/ml  at 100 ng/ml. On the other hand, BDNF did not alter K -evoked CGRP release.We next examined the effect of growth factor treatment on K/ml Fig. . Similar4. Representative photomicrographs for each of these conditions are shown in Figure 4 are shown in Table 4-binding neurons not likewise immunoreactive for NF-H. We then utilized the proportion of CGRP-expressing neurons under NGF, GDNF or BDNF treatment conditions to normalize K+- evoked CGRP release to the number of CGRP-immunoreactive neurons under each condition, respectively . Following this transformation, NGF treatment still augmented K+-evoked CGRP release at every concentration tested, with the magnitude of the effect reaching 18 times that of control  supplementation significantly enhanced CAP-evoked CGRP release over the same concentration of BDNF Fig .To determine to what extent the enhancement of CAP-evoked CGRP release in response to growth factor treatment might derive from increased neuronal survival and/or up-regulation in the proportion of neurons expressing TRPV1, release data were normalized to the number of TRPV1-immunoreactive neurons Fig. .50 (41 nM for GDNF or NGF and 47 nM for no growth factor). GDNF (100 ng/ml) or NGF (100 ng/ml) treatment caused a significant, five-fold increase in the Emax of the CAP concentration-response curve . In contrast, BDNF supplementation did not augment AEA- or ACEA-evoked CGRP release, while a small, yet significant, increase in CAP-evoked CGRP release was observed with increasing BDNF concentration ) ; however, when compared with non-growth factor-treated TG neurons, the Emax was significantly augmented . Removal of astroglial support promotes apoptosis in cerebral neuronal cultures, an effect which is reversed by addition of GDNF or BDNF, but not NGF, to the culture medium . The add4-binding sites were assessed with attention to how these proportions changed in the presence of NGF, GDNF and BDNF. Notably, the proportion of sensory neurons in vitro that expressed CGRP (~65%) was much larger than the known proportion of CGRP-containing neurons in native TG .The proportions of TG neurons in culture that expressed CGRP- or TRPV1-immunoreactivity or IBG (~35%, ). This l ~35–60% ,59 make 4-binding neurons following peripheral inflammation [We observed that a greater proportion of TRPV1-immunoreactive neurons were present with GDNF supplementation (nearly 30% increase), again indicating either that GDNF preferentially supports the survival of TRPV1-expressing sensory neurons or that neurons that do not normally express TRPV1 begin expressing TRPV1 when GDNF is included in the culture medium. The latter proposition is supported by the finding that peripheral treatment with anti-GDNF antibodies suppresses the novel expression of TRPV1 in IBammation . The perammation . This fi+-evoked CGRP release after normalization. This difference in the ability of GDNF and NGF to enhance individual neuronal neuropeptide content and CAP responsiveness could partially explain why NGF induces hyperalgesia [We have also illustrated that chronic application of NGF or GDNF, but not BDNF (except at high concentrations, and to a much lesser degree), increases CAP-evoked CGRP release from TG neurons in vitro. NGF and GDNF each increased TRPV1 protein, and both upregulated the CGRP content of TG neurons. Both the NGF- and GDNF-induced upregulation of TRPV1 appears to be translationally regulated, as neither of these growth factors altered TRPV1 mRNA levels. NGF, in the setting of inflammation, is known to increase TRPV1 protein, but not mRNA, through activation of the p38/MAP kinase pathway ,26, and ralgesia ,12,19,61ralgesia ,28,29. OWhile it has been shown, using a variety of dependent measures, that both NGF and GDNF increase CAP responsiveness ,17, thisSimilarly to CAP-evoked CGRP release, the ability of AEA and ACEA to evoke release was concentration-dependently augmented by NGF or GDNF supplementation but was unaltered by BDNF. Moreover, the respective potencies of NGF and GDNF to enhance the CGRP release in response to CAP, AEA and ACEA were equivalent. This suggests that the pharmacology of CAP, AEA and ACEA at TRPV1, at least with respect to neuropeptide secretion, is similarly regulated in the presence of either of these growth factors. Insofar as NGF and GDNF have been implicated in the development of nociceptor sensitization in a number of pathological states, it should be considered that endocanninoids are apt to have enhanced TRPV1-mediated peripheral neuromodulatory effects under these conditions.Taken together, our results illustrate that NGF, GDNF and BDNF differentially alter sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release of TG neurons in culture. GDNF and, to a lesser extent, BDNF promote survival of TG neurons and GDNF enhances the proportion of neurons that exhibit TRPV1-immunoreactivity. GDNF or NGF enhanced CAP-, AEA- and ACEA-evoked CGRP release from TG neurons in vitro and increased TRPV1 protein, likely through translational regulation, and overall CGRP content. On the other hand, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis, providing a possible explanation for why NGF promotes thermal hypersensitivity in vivo while GDNF apparently does not. Although the present studies were conducted on cultured neurons under artificially controlled conditions, these findings contribute to a growing body of work concerning neurotrophin modulation of the properties of nociceptors. Thus the results described here have implications for advancing our understanding of how this system might be advantageously manipulated in a therapeutic setting especially as it concerns the TG system.N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide, in water soluble emulsion), and ACEA (N-(2-chloroethyl)-5Z,11Z,14Z)-eicosatetraenamide were from Tocris (Ellisville, MO). Recombinant rat GDNF and recombinant human (100% homologous to rat) BDNF were from Sigma (St Louis, MO). Rat NGF was from Harlan (Indianapolis, IN). All other chemicals were from Sigma, unless otherwise stated.Capsaicin (CAP, 8-methyl-N-vanillyl-trans-6-nonenamide) was from Fluka-Aldrich (St Louis, MO). AEA . TGs were enzymaticaly digested for 30 min with 5 mg/ml collagenase followed by 25 min with 0.1% trypsin type IX supplemented for the last 10 min with 10 units of DNase I (Roche, Indianapolis, IN). TG cell suspensions were then centrifuged at 2000 RPM for 2 min, vortexed briefly and centrifuged again. They were then resuspended in basal culture medium containing high glucose Dulbeco's Modified Eagle's Medium (DMEM, Gibco), 1X pen-strep (Gibco), 1X glutamine (Gibco) and 3 μg/mL 5-FDU and 7 μg/mL uridine as mitotic inhibitors. TG cell suspensions were gently triturated with a Pasteur pipette followed by successive triturations through 19- and 23-gauge needles. TG cell suspensions were then transferred to a separate container and adjusted to the total volume needed for plating at a density of ~5000 neurons / well. The appropriate growth factors were added to this suspension prior to plating.Adult, male Sprague-Dawley rats weighing 250–300 g were used in this study. All procedures utilizing animals were approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio and were conducted in accordance with policies for the ethical treatment of animals established by the National Institutes of Health. Animals were euthanized by decapitation and their TGs were rapidly dissected (~30 s) and placed in ice-cold Ca4-conjugated to Alexa-Fluor-488 (1:1000, Molecular Probes) was then added overnight at 4°C. CGRP or TRPV1 antisera were followed by goat anti-rabbit Alexa-Fluor-594 (1:300) for 1 hr at room temperature. All images were acquired using a Nikon E600 microscope (Melville, NY) equipped with a Photometrics SenSys digital CCD camera (Roper Scientific, Tucson, AZ) connected to a computer equipped with Metamorph V4.1 image analysis software . Twenty 20X images were taken of each well to capture the complete cellular area for neuron counts. For these experiments, all neurons displaying fluorescent signal above background were counted as positive for the specific marker. This was defined by scaling the image, using Metamorph's built-in scaling feature, to an average pixel value for negative neurons in the case of TRPV1, CGRP and IB4  and establishing all neurons as positive that were above that threshold. For double labeling experiments, the thresholded images were then overlaid and analyzed for the presence of both signals to assess colocalization.TG cultures were first washed in PBS and fixed for 1 hr in 3.7% formaldehyde in PBS. Next, TG cultures were washed 3 times in PBS and permeabilized in PBS containing 10% normal goat serum (NGS, Gibco) and 0.2% Triton X-100 (Sigma) for 1 hr. Finally, TG cultures were blocked 3 × 10 min in PBS containing 10% NGS and then exposed to NF-H mouse-monoclonal antibody  overnight at 4°C. Primary antisera were then washed off and goat anti-mouse Alexa-Fluor-488 (1:300, Molecular Probes, Eugene, OR), or goat anti-mouse Alexa-Fluor-594 (1:300) was applied for 1 hr at room temperature. For double-labeling either CGRP rabbit polyclonal (1:750, Peninsula Labs) or TRPV1 guinea pig polyclonal (1:3000, Neuromics) or IB4-binding neurons in the same wells to generate the proportion of neurons expressing these markers. The total for the 2 matching wells were summed to yield the final proportion. At least 500 NF-H-immunoreactive neurons were counted under every growth factor condition to calculate proportions of neurons expressing the three markers examined. In no cases were CGRP-, TRPV1- or IB4-binding neurons not likewise positive for NF-H. Colocalization is presented only as an overall percentage.TG cell suspensions (~5000 neurons/well) were added to 8-well poly-D-lysine Lab-Tek II chamber slides . Neuron density for plating was determined by counting neurons with a hemacytometer with Neubauer rulings and the cultures for each slide were generated independently. Neurons were easily distinguished from other cell types for plating density measurements due to their large size and opaqueness. Medium was changed after 24 hr and 72 hr, and on day 5, ICC was performed (as described above). In total, 12 chamber slides were utilized. The experimental design is shown in figure TG cultures were prepared on 48-well poly-D-lysine pre-coated plates (Becton Dickinson, Franklin Lakes, NJ) at an initial density of ~5000 neurons/well. For each 48 well plate 12 wells received no growth factor, 12 received 100 ng/ml NGF, 12 received 100 ng/ml GDNF and the remaining 12 received 100 ng/ml BDNF. A total of 3 plates were utilized and each culture plate was generated independently. Following 5 days of culture, RNA was extracted from TG cultures using a ToTALLY RNA (Ambion, Austin, TX) total RNA extraction kit and each of the 12 wells per condition were pooled together to yield sufficient RNA. RNA samples were subsequently treated with DNA-free DNAse (Ambion) for removal of trace amounts of DNA. RNA concentrations were determined by UV absorbance, and RNA samples were then diluted to equal concentrations in TE buffer. For realtime PCR assessment of TRPV1 mRNA levels, samples were loaded in triplicate in 96-well reaction plates with each sample containing 150 ng RNA, 2X RT-PCR Taqman Master Mix, 40X Multiscribe and RNase Inhibitor solution, forward primer (tcc agt caa gcc cca cat c), reverse primer (tcc gag tca ccc ttc cca) and Taqman probe (6FAM tca cta cca gga gtc gta ccc ggc ttt TAMRA)  in a final reaction volume of 50 μL. Controls were run concomitantly using the same reaction recipe with a rodent GAPDH control kit (Applied Biosystems), primers and probe at 50 nM. Reactions were run on an ABI Prism 7700 (Applied Biosystems) with an initial RT step of 48°C for 30 min followed by a 95°C 10 min denaturation step and then a repeating denaturation, extension cycle of 95°C for 15 s and 60°C for 1 min for 55 cycles in order to reach a full plateau for all samples. All data were normalized to GAPDH mRNA levels to account for any variation in RNA concentrations between samples.TG cultures were prepared on 48-well poly-D-lysine pre-coated plates at an initial density of ~5000 neurons/well. For each 48 well plate 12 wells received no growth factor, 12 received 100 ng/ml NGF, 12 received 100 ng/ml GDNF and the remaining 12 received 100 ng/ml BDNF. A total of 3 plates were utilized and each culture was generated independently. After five days, total protein was extracted by first lysing cells with lysis buffer (1 mM Na pyrophosphate, 50 mM HEPES, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 5 mM EDTA and 1 mM Na orthovanadate) supplemented with 1% protease inhibitor cocktail and then homogenizing the combined lysate from the 12 wells per condition by pumping it through a 25 gauge needle 20 times. Extracted proteins were cleared of nuclei and cellular debris by spinning the homogenate at 1000 × g for 5 min at 4°C. Protein levels were then measured by the Bradford method. Proteins were run at a concentration of 20 μg/lane on a 12.5% SDS-PAGE gel and transferred to Immobilon – P membranes (Millipore). Membranes were blocked in 5% dry milk for 1 hr and then exposed to rabbit anti-TRPV1 antibody (Neuromics, Minneapolis MN), at a concentration of 1:1000, overnight at 4°C. Membranes were then incubated with donkey anti-rabbit horseradish peroxidase-linked secondary antibody (Amersham, 1:5000) for 1 hr followed by ECL Western blotting detection for 1 min (Amersham). Blots were next exposed to film and subsequently scanned on a flatbed scanner. To control for protein loading, membranes were then stripped and reblotted for β-actin. Images were assessed for changes in TRPV1 protein using NIH image and normalized to β-actin protein levels.+ buffer (containing 2.5 mM CaCl2, 50 mM KCl, 1.2 mM MgCl2, 90 mM NaCl, 25 mM NaHCO3, 1 mM NaH2PO4, 10 mM dextrose, 15 mM Hepes, 16 uM thiophan and 0.1% BSA at pH = 7.4), after which the CGRP-containing supernatant was removed and transferred to glass culture tubes (Fisher). Content was assessed by hypotonic lysis with deionized H2O supplemented with 1% protease inhibitor cocktail (Sigma) for 30 min. CGRP release or content for each well was subsequently measured by radioimmunoassay.All experiments were performed in 48-well poly-D-lysine pre-coated plates. Data shown are representative of at least 3 independently conducted CGRP release experiments with consistent results and neurons were plated at the same density as indicated for survival, ICC, realtime PCR and Western blotting experiments (~5000 neurons/well). Culture medium was changed at 24 and 72 hr, and all CGRP assays were performed on day 5. TG cultures were washed free of culture medium by 2 successive washes with release buffer  supplemented with 10.9 mM HEPES, 4.2 mM sodium bicarbonate, 10 mM dextrose and 0.1% bovine serum albumin (BSA), pH 7.4). Growth factors were not included in the release buffer. Following washing, TG cultures were exposed for 10 min to the indicated concentrations of CAP, AEA or ACEA or to 50 mM KI]-CGRP28–37 (approximately 20000–25000 cpm) and 50 μL of goat anti-rabbit antibody conjugated to ferric beads were added. Following another 24 h, bound peptide was separated from free peptide via immunomagnetic separation (PerSeptive Biosystems, Framingham, MA, USA). All incubations were carried out at 4°C. The minimum detection limit for this assay is approximately 1–2 fmol per tube, with 50% displacement occurring at 20–40 fmol per tube. To account for the possibility of any nonspecific effects on the RIA, all drugs used in the release experiments were included in separate standard curves for the purposes of data analysis. We did not observe any alterations in the standard curve for any of the compounds utilized in these studiesFollowing culture release assays, individual aliquots of the superfusate (0.5 ml) were incubated with a C-terminally directed anti-CGRP antiserum (kindly donated by Dr Michael Iadarola, NIDCR, NIH, Bethesda, MD, USA). After 24 h, 100 μL of [125All data are presented as mean ± SEM unless otherwise stated. When normalizations for CGRP release or content to neuron numbers for either CGRP or TRPV1-immunoreactive neurons were made the equation shown in figure 4: isolectin B4, ICC: immunocytochemistry, NGF: nerve growth factor, SP: substance P, TG: trigeminal ganglion, TRPV1, transient receptor potential receptor vanilloid type 1ACEA: arachidonyl-2-chloroethylamide, AEA: anandamide, BDNF: brain-dervied neurotrphic factor, CAP: capsaicin, CGRP: calcitonin gene-related peptide, DRG: dorsal root ganglion, GDNF: glial cell-line derived neurotrphic factor, IBTJP performed and conceived (or participated in their conception) experiments in each section, analyzed all data and authored the manuscript. MDL performed immunocytochemistry and neuron counts. DCS conducted the intitial CAP-evoked CGRP release study. GOD assisted in conceiving the experiments and performing the pilot studies. NAJ conducted the Western blots. AP assisted in conducting the CGRP release experiments. AD assisted in real-time PCR experiments. AAT assisted in generating cultures and in conducting pilot studies for CAP-evoked CGRP release. KMH assisted in conceiving the experiments and gave critical readings of the manuscript. CMF supervised all studies and conceived the original experimental designs as well as critically editing the manuscript.


Impaired physical performance is a common but not obligate result of a low carbohydrate diet. Lessons from traditional Inuit culture indicate that time for adaptation, optimized sodium and potassium nutriture, and constraint of protein to 15–25 % of daily energy expenditure allow unimpaired endurance performance despite nutritional ketosis. In the opinion of most physicians and nutrition scientists, carbohydrate must constitute a major component of one's daily energy intake if optimum physical performance is to be maintained . This coDuring the time that this consensus view of the necessity of carbohydrate for vigorous exercise was forming, the last pure hunting cultures among the peoples of North America finally lost out in competition with expanding European cultural influences. Between 1850 and 1930, the routine consumption of carbohydrates spread north from the U.S. Plains States through central Canada, where the indigenous peoples had heretofore made at most seasonal use of this nutrient class. However the last of these groups to practice their traditional diet, the Inuit people of the Canadian and Alaskan Arctic regions, were luckily observed by modern scientists before their traditional dietary practices were substantially altered. The reports of these early scientists imply that the Inuit people were physically unhampered despite consuming a diet that was essentially free of identifiable carbohydrate.Given this juxtaposition of clinical research results favoring carbohydrate against observed functional well-being in traditional cultures consuming none, it is an interesting challenge to understand how these opposing perspectives can be explained. This paper will review the observations of early explorer scientists among the Inuit, track the controversy that they stimulated among nutritionists in the last century, and utilize some of the forgotten lessons from the Inuit culture to explain how well-being and physical performance can be maintained in the absence of significant dietary carbohydrate.Until the development of agriculture over last few millennia, our human ancestors' consumption of dietary carbohydrate was opportunistic. As some groups adapted to hunting and fishing for their sustenance, they were able to move into temperate and then arctic regions, where limited access to wild grain, nuts, and fruit dictated sustained dependence upon fat and protein as primary sources of dietary energy.With the development of agriculture came the ability to grow and store grain, allowing societies to remain in a stable physical location, build permanent dwellings, and potentially stimulating the development of written language (those early stone tablets would have been difficult to transport from camp to camp on a dog sled). Starting from locations in the Middle East and Asia, cultures based upon agricultural wheat and rice spread over 5 millennia to dominate Europe, Africa, and the Americas.With its ability to support a non-nomadic life style, greater population density, and permanent communities; there were clear advantages of agriculture-based societies over those based upon hunting and fishing, particularly as agricultural communities built the infrastructure to support trade and transport. Given its success in this competition of cultures (and by implication, the competition of their diets), it is an easy assumption that a grain-based diet is functionally superior to one based upon the meat and fish (fat and protein) of the hunting societies that they superseded.th Century, numerous comparative studies were undertaken to assess differences between diets. Although there were some advocates of low carbohydrate diets (eg, the Banting diet of the 19th Century, promoted for weight loss and diabetes control), the prevailing premise for these studies was that carbohydrate was a necessary nutrient for optimum human health and function. Among studies confirming this view, a classic was the 1939 study by two Danish scientists, Christensen and Hansen [As the science of nutrition developed in the early 20During the Second World War, another oft-cited study was performed, this time examining the practicality of pemmican (a mixture of dried meat and fat) as a light-weight emergency ration for soldiers. This experiment by Kark et al  involvedWith the resurgence of biomedical science in the 1960's came development of the percutaneous needle biopsy, facilitating assessment of intra-muscular fuel stores and metabolism. This led to the concept of muscle glycogen as the limiting fuel for high intensity exercise  and to tAlthough high-carbohydrate diets might be more effective in short-term tests of high-intensity exercise, there are multiple clues in the published literature that the debilitating effects of ketogenic diets are overstated. Not only is there the demographic evidence that whole populations of people lived for millennia as hunters, but there are many reports of Europeans crossing over to live within the cultures of these hunting societies without apparent impediment.New York Herald and the American Geographical Society, departed from the west coast of Hudson's Bay in April of 1879 with 4 Caucasians, 3 families of Inuits, and 3 heavily laden dog sleds. Totaling 18 people, they started out with a month's supply of food  and a prodigious supply of ammunition for their hunting rifles. After covering over 3000 miles on foot over ice, snow and tundra, all 18 members of the original party plus their 44 dogs returned to Hudson's Bay in March of 1880. Once their initial provisions were depleted, the expedition's only source of additional food was hunting and fishing, as there were no other sources of supply along their route.One of the earliest documented demonstrations of physical stamina during a ketogenic diet was the Schwatka 1878–80 expedition in search of the lost Royal Navy Franklin expedition. The Schwatka expedition, sponsored by the New York Herald in the Fall of 1880, but his written diary was lost for 85 years until its discovery and publication by the Marine Historical Association of Mystic CT in 1965 [The leader of this expedition, Lt. Frederick Schwatka, was a graduate of both West Point and Bellevue Hospital Medical College. His summary of the expedition was published as a news article in the In one notation, Schwatka provides an interesting insight into his weaning from their initial supply of carbohydrate-containing food.When first thrown wholly upon a diet of reindeer meat, it seems inadequate to properly nourish the system, and there is an apparent weakness and inability to perform severe exertive fatiguing journeys. But this soon passes away in the course of two or three weeks.""This observation, written a century before the current author first came to grips with the issue of "keto-adaptation", offers an early clue to resolve the dichotomy between impaired performance with low carbohydrate diets in the laboratory and their lack of debilitating effects when taken among people practiced in their use. That Schwatka was not impaired by his prolonged experience eating meat and fat is evidenced by his diary entry for the period 12–14 March 1880, during which he and an Inuit companion walked the last 65 miles in less than 48 hours to make a scheduled rendezvous with a whaling ship and complete his journey home.Twenty-six years later, a Harvard-trained anthropologist named Vilhjalmur Stefansson entered the Arctic with the purpose of studying the Inuit language and culture. Having been born in 1879 in Manitoba and grown up in North Dakota, it is unlikely that Stefansson was aware of the Schwatka expedition or its reported technique of extended dogsled travel while living by hunting. However when separated from his expedition and thus his source of supply over the winter of 1906–7, Stefansson was taken in by a group of Inuit on the Canadian Arctic coast. With the arrival of spring in June of 1907, he both spoke their language and had acquired their skill of living and traveling by dogsled on a hunter's diet.For the next decade, Stefansson traveled extensively over the arctic mainland and among the islands to the north. During this period, he was away from the outposts of European settlement for periods of up to 18 months at a time, and in the remote regions of the Canadian Arctic he lived with groups of Inuit for whom he was the first European they had met.Stefansson wrote extensively about these experiences in both the scientific literature and in books for the lay public . One of Subjected to great criticism and even scorn, Stefansson agreed to recreate the Inuit diet under scientific observation. Therefore, for the calendar year of 1929 he and a colleague from his arctic explorations ate a diet consisting of meat and fat for 12 months. This experiment, supervised by Dr. Eugene DuBois, was conducted at Bellevue Hospital in New York. For the first 3 months of this study, the two explorers were under constant observation to guarantee dietary compliance, after which they were allowed more freedom of movement but with frequent tests to document that they remained in ketosis. This study was reported in multiple peer-reviewed publications, the primary reports being published in the Journal of Biological Chemistry in 1930 , As noteIt is interesting to note from the careful observations published from the Bellevue study that Stafansson ate relatively modestly of protein, deriving between 80–85% of his dietary energy from fat and only 15–20% from protein . This waIt is also interesting to conjecture that the vigorous defense of his arctic observations by Stefansson may have led indirectly to the development of the carbohydrate loading hypothesis. Stefansson was a polarizing influence in the field of nutrition, and his advocacy of pemmican as an emergency ration for troops during the Second World War led directly to the Kark study quoted above, which in turn was a predecessor to many comparative dietary trials performed in Europe and the U.S. in later decades.There was a resurgence of interest in very low calorie ketogenic diets for weight loss in the 1970's, followed closely by the complications (including sudden death) associated with the Liquid Protein diet popularized in 1976. However, the fatigue and apparent cardiac dysfunction caused by this collagen-based fad diet stood in stark contrast to the published experience of arctic explorers such as Schwatka and Stefansson. In addition, physicians who monitored patients following very low calorie diets observed wide variations between the exercise-tolerance of these individuals.Given that the elegant research on the metabolism of total fasting by Dr. George Cahill and colleagues had demonstrated that full adaptation of nitrogen, fat, and carbohydrate metabolism required a number of weeks , it seemTo test this hypothesis, the current author (under the mentorship of Drs. Ethan Sims and Edward Horton at the University of Vermont) undertook a study of subjects given a very low calorie ketogenic diet for 6 weeks in a metabolic research ward . The pro2max) after a 2-week weight maintenance baseline diet, and again after 6 weeks of the ketogenic weight loss diet. Endurance time to exhaustion was quantitated at 75% of the baseline VO2max. This endurance test was repeated again after one week of weight loss and finally after 6 weeks of weight loss. Other than these tests, the subjects did no training exercise during their participation in this study. To compensate for the fact that the average subject had lost over10 kg, the final endurance treadmill test was performed with the subject carrying a backpack equivalent in weight to the amount lost.Treadmill performance testing of these subjects included determinations of peak aerobic power (VOThe energy expenditure data (expressed as oxygen consumption) and exercise times across this 8-week inpatient study are shown in Table This question of improved efficiency notwithstanding, it is clear that our subjects experienced a delayed adaptation to the ketogenic diet, having reduced endurance performance after one week followed by a recovery to or above baseline in the period between one and six weeks. Given the reduced energy cost of the exercise despite the backpack, the extent of this adaptation cannot be determined from this study. To explain this improved exercise efficiency, we can speculate that humans are more efficient carrying weight in a modern backpack than under their skin as excess body fat. It is also possible that these untrained subjects became more comfortable with prolonged treadmill walking by their third test, and therefore improving their overall efficiency.Given the uncertainties of this study caused by the subject's weight loss and potential for improved technique with multiple tests, the current author undertook a second study under the mentorship of Dr. Bruce Bistrian at MIT in Cambridge MA ,15. The This second study utilized competitive bicycle racers as subjects, confined to a metabolic ward for 5 weeks. In the first week, subjects ate a weight maintenance  diet providing 67% of non-protein energy as carbohydrate, during which time baseline performance studies were performed. This was followed by 4 weeks of a eucaloric ketogenic diet (EKD) providing 83% of energy as fat, 15% as protein, and less than 3% as carbohydrate. The meat, fish, and poultry that provided this diets protein, also provided 1.5 g/d of potassium and was prepared to contain 2 g/d of sodium. These inherent minerals were supplemented daily with an additional 1 g of potassium as bicarbonate, 3 grams of sodium as bouillon, 600 mg of calcium, 300 mg of magnesium, and a standard multivitamin.40K counting) revealed a 2% reduction in the first 2 weeks (commensurate with the muscle glycogen depletion documented by biopsy), after which it remained stable in the 4th week of the EKD. These results are consistent with the observed reduction in body glycogen stores but otherwise excellent preservation of lean body mass during the EKD.The bicyclist subjects of this study noted a modest decline in their energy level while on training rides during the first week of the Inuit diet, after which subjective performance was reasonably restored except for their sprint capability, which remained constrained during the period of carbohydrate restriction. On average, subjects lost 0.7 kg in the first week of the EKD, after which their weight remained stable. Total body potassium , which revealed marked reductions in the use of these carbohydrate-derived substrates after adaptation to the EKD.The results of physical performance testing are presented in Table 2max) endurance performance were fully restored. In both studies, one with untrained subjects and the other with highly trained athletes who maintained their training throughout the study, there was no loss of VO2max despite the virtual absence of dietary carbohydrate for 4–6 weeks. This whole-body measure of oxidative metabolism could not be maintained unless there was excellent preservation of the full complement of functional tissues including skeletal muscle  mass, circulating red cell mass, and cardiopulmonary functions.Examining the results of these two ketogenic diet performance studies together indicates that both groups experienced a lag in performance across the first week or two of carbohydrate restriction, after which both peak aerobic power and sub-maximal  and/or improved acclimation to the endurance test procedure. Such acclimation would not be expected in the second study, as the highly trained bicycle racers were well conditioned to the stationary ergometer at the start of the study. It is also worth noting that the bicycle racers remained weight stable  across the 4 weeks of the EKD, which was equi-caloric with the baseline diet. Although 4 weeks is a relatively short period to assess small differences in energy efficiency between diets, this observation implies that there was no great reduction in the efficiency of energy metabolism after keto-adaptation.As a final note in this section, neither the Vermont study nor the MIT study has been refuted in the 2 decades since their publication. Understandably given the expense of human metabolic ward studies and the orthogonal conclusions of these two studies, neither study has been corroborated by a similar human study. However two subsequent animal studies examining physical performance after keto-adaptation have yielded results consistent with those presented above ,17.There are three factors that can help us explain the paradox presented by studies showing superior performance with high carbohydrate diets versus the present author's two studies noted above.The most obvious of these is the time allotted (or not) for keto-adaptation. In this context, the prescient observation of Schwatka (that adaptation to "a diet of reindeer meat" takes 2–3 weeks) says it all. None of the comparative low-carbohydrate versus high-carbohydrate studies done in support of the carbohydrate loading hypothesis sustained the low carbohydrate diet for more than 2 weeks , and mosThere are to date no studies that carefully examine the optimum length of this keto-adapataion period, but it is clearly longer than one week and likely well advanced within 3–4 weeks. The process does not appear to happen any faster in highly trained athletes than in overweight or untrained individuals. This adaptation process also appears to require consistent adherence to carbohydrate restriction, as people who intermittently consume carbohydrates while attempting a ketogenic diet report subjectively reduced exercise tolerance.The second factor differentiating the author's studies from many others is optimized mineral nutriture, which has benefits for both cardiovascular reserve in the short term and preservation of lean body mass and function over longer time periods. The Inuit people lived much of the year on coastal ice , and much of their food consisted of soup made with meat in a broth from this brackish source of water. When they went inland to hunt, they traditionally added caribou blood  to their soup. With these empirically derived techniques, the Inuit culture had adapted the available resources to optimize their intakes of both sodium and potassium.When meat is baked, roasted, or broiled; or when it is boiled but the broth discarded, potassium initially present in the meat is lost, making it more difficult to maintain potassium balance in the absence of fruits and vegetables. Because our research subjects were accustomed to eating meat, fish, and poultry prepared as something other than soup, we chose to give them most of their sodium separately as bouillon and a modest additional supplement of potassium as potassium bicarbonate. With these supplements maintaining daily intakes for sodium at 3–5 g/d and total potassium at 2–3 g/d, our adult subjects were able to effectively maintain their circulatory reserve  and effective nitrogen balance with functional tissue preservation.An example of what happens when these mineral considerations are not heeded can be found in a study prominently published in 1980 . This wagaining potassium. As noted in a rebuttal letter published soon after this report [A clue to what was happening in this "Turkey Study" could be found in the potassium balance data provided in this report. Normally, nitrogen and potassium gains or losses are closely correlated, as they both are contained in lean tissue. Interestingly, the authors noted that the protein only diet subjects were losing nitrogen but s report , this ans report , this "TThe third dietary factor potentially affecting physical performance is adjusting protein intake to bring it within the optimum therapeutic window for human metabolism. The studies noted herein -15,20 de2max over a 3 month period on a ketogenic diet, whereas subjects given 1.5 g/kg-d maintained VO2max.The effects of reducing daily protein intake to below 1.2 g/kg reference weight during a ketogenic diet include progressive loss of functional lean tissue and thus loss of physical performance, as demonstrated by Davis et al . In thisAt the other end of the spectrum, higher protein intakes have the potential for negative side-effects if intake of this nutrient exceeds 25% of daily energy expenditure. One concern with higher levels of protein intake is the suppression of ketogenesis relative to an equi-caloric amount of fat . In addition, Stefansson describes a malady known by the Inuit as rabbit malaise . This prBoth observational and prospectively designed studies support the conclusion that submaximal endurance performance can be sustained despite the virtual exclusion of carbohydrate from the human diet. Clearly this result does not automatically follow the casual implementation of dietary carbohydrate restriction, however, as careful attention to time for keto-adaptation, mineral nutriture, and constraint of the daily protein dose is required. Contradictory results in the scientific literature can be explained by the lack of attention to these lessons learned (and for the most part now forgotten) by the cultures that traditionally lived by hunting. Therapeutic use of ketogenic diets should not require constraint of most forms of physical labor or recreational activity, with the one caveat that anaerobic (ie, weight lifting or sprint) performance is limited by the low muscle glycogen levels induced by a ketogenic diet, and this would strongly discourage its use under most conditions of competitive athletics.2max – maximum aerobic capacityVORQ – respiratory quotientEKD – eucaloric ketogenic dietNone declared.


During translation, the ribosome reads an inosine as a guanosine; thus, an A-to-I edit in RNA can even cause an amino acid change in the resulting protein. A new study in this issue by Stefan Maas and colleagues shows that A-to-I editing is remarkably widespread among human genes, and commonly targets a ubiquitous repetitive sequence, the Alu repeat.The so-called “central dogma” of biology—DNA makes RNA makes protein—is a simple statement that subsumes a wealth of complexity. In particular, the past decade has shown that after RNA is made, it is run through a gauntlet of processes that strip it of introns and splice its exons, add a cap and tail, and even chemically modify one or more bases along the way. This last possibility includes deamination—removal of an NHA-to-I editing has been recognized for several years, but the known targets have been few, far fewer than the number predicted by measuring the inosine content in messenger RNA. To identify more targets, Alekos Athanasiadis, Alexander Rich, and Maas compared genomic sequences to cDNA sequences. Adenosines are unchanged in genomic DNA, while in complementary DNA (cDNA), which is derived from reverse transcribing mRNA, any adenosines that were converted to inosines during RNA editing show up as guanosines. Thus, A-to-G discrepancies revealed candidate editing sites. To reduce the number of false positives, the researchers confined their search to regions with multiple A-to-G discrepancies. In an initial screen of 3,000 cDNAs, they found 26 A-to-I edited genes. In all but one case, the editing occurred in an Alu sequence.There are approximately 1.4 million Alu sequences in the human genome, each about 300 base-pairs in length, which together comprise about 10% of the entire genome. Not all of them occur in genes, but those that do are typically found in transcribed but untranslated regions (introns), either upstream (3′) or downstream (5′) of the translated region. In many genes, they are found in pairs, ordered head to head or tail to tail, separated by a short intervening sequence. Once transcribed, the Alu sequences can pair up, forming a stretch of double-stranded RNA that makes an ideal target for the A-to-I RNA editing machinery, called ADAR (adenosine deaminase acting on RNA).A typical gene contains between one and two dozen Alu sequences. Based on this and the frequency of editing found when analyzing more than 100,000 mRNAs in the human transcriptome, Athanasiadis, Rich, and Maas estimate that the probability that any particular mRNA undergoes A-to-I editing is between 85% and 95%.While the bulk of edited Alu sites are in introns, a small fraction of them are in exons. Here they can lead to alternative forms of the same protein, expressed in different cell types or at different times; this appears to be especially common in the nervous system. Alu editing can also convert introns to exons, and vice versa, through creation or destruction of splice sites. It is possible A-to-I editing may be used to reduce the creation of deleterious new exons, although more work will be needed to explore this possibility, as well as what role, if any, A-to-I editing plays in promoting new exon creation.


Low physical activity is known to be a potential risk factor for cardiovascular disease. With high prevalence of cardiovascular diseases in the Portuguese urban population, little is known about how sedentary this population is and what factors are associated to sedentary lifestyles. This study's objective was to examine sedentary lifestyles and their determinants through a cross-sectional study.2134 adults (18 years and older) were interviewed using a standard questionnaire, comprising of social, behavioural and clinical information. Time spent in a variety of activities per day, including: work, household chores, sports, sedentary leisure time and sleep, were self-reported. Energy expenditure was estimated based on the related metabolic equivalent (MET) and time spent in each activity (min/day). Those with less than 10% of energy expenditure at a moderate intensity of 4 METs or higher were categorised as sedentary. The proportion of sedentary people and 95% Confidence Intervals (CI) were calculated, and the magnitude of associations, between sedentary lifestyles and the population characteristics, were computed as age-adjusted odds ratios using logistic regression.Sedentarism in both genders during leisure time is high at 84%, however in full day energy expenditure, which includes physical activity at work, sleeping hours and household chores, 79% of males and 86% of females are found to be sedentary. In leisure-time only, increased age is associated with higher odds of being sedentary in both genders, as well as in women with increased BMI. In comparison, in full-day energy expenditure, sedentarism is more likely to occur in those with higher levels of education and in white-collar workers.A high prevalence of sedentarism is found in the study participants when measuring leisure-time and full-day energy expenditure. The Portuguese population may therefore benefit from additional promotion of physical activity. Physical activity has been defined by World Health Organization  as comprPhysical activity tends to be associated with lower cardiovascular morbidity and mortality and an overall improved quality of life. Some studies -9 have dPortugal has the highest stroke mortality rates in Western Europe and cardiovascular diseases cause approximately 40% of deaths ,13. The Data was obtained as part of an ongoing cross-sectional health survey of adults living in the city of Porto, Portugal. Random digit dialling was used to select a single person over 17 years old from each household, without allowing for substitution of refusals. A participation rate of 70% was achieved . Using aAs reported in an earlier publication , educatiParticipants completed a physical activity questionnaire designed to estimate usual individual daily energy expenditure, focused on the activity in the past year. Time spent in a variety of activities per day, including: work, transport to or from work, household chores, sports, sedentary leisure time and sleep, was self-reported and activity intensity categorised as very light, light, moderate and heavy with a corresponding average of 1.5, 2.5, 5.0 and 7.0 METs respectively, where one MET is equal to the energy expended at the basal metabolic rate or at rest . Due to In the exploration of the population studied it was found that a significant difference between men and women was noted in most baseline characteristics other than in the age distribution and the hours of sleep . A higher proportion of household work was undertaken by women (95.5% versus 55.5% of men), a higher proportion of men were married (84.3% versus 61.3% of women), 68.3% of women and 55.7% of men reported no chronic disease. 31% of all participants worked between 20 and 40 hours a week, with a higher percentage of men working greater than 40 hours (31.1% versus 15.2% of women). It is also worthy to note that a high percentage of both genders reported not undertaking regular leisure-time sports and exercise (69.3% of women and 58.9% of men). All subsequent analyses were performed separately for males and females.Overall sedentary lifestyle percentages Table  and 2 arFew differences were found in the level of sedentarism in adults when considering differences in population characteristics. Younger participants tend to have lower percentages of sedentarism compared to older participants. In the leisure-time only estimation unmarried men  and female white-collar workers  tend to be more active. Women and men tend to be more active in leisure-time with increasing years of education, changing from 94% sedentarism in those with less than five years of education to 84% for those with five to eleven years of education and 72.5% for men and 70.6% for women with greater than 11 years of schooling. However, when the full-day energy expenditure is used as the estimate, these differences are no longer found and the trends with education and occupation are reversed. The lowest levels of sedentarism are found in males  with less education, and men who have a high energy intake  as well as, both male  and female  blue-collar workers.When further examining the results of the age-adjusted associations between the population characteristics and sedentarism it is found that few factors were associated with an increased proportion of sedentarism. Marital status, physician visits in the last year and tobacco consumption, once adjusted for age, were not associated with differences in energy expenditure and sedentarism. In the leisure-time only estimation, increased age was associated with higher odds of being sedentary in both males and females. Sedentarism increased with increased BMI in women , as well as in women with a high energy intake . Sedentarism also increased in males who were ex-drinkers when compared to non or occasional drinkers (OR 2.6 95%CI: 1.1–6.2). Following what was noted earlier, higher levels of education were associated with higher levels of activity in leisure-time in both males  and females (OR 0.4 95%CI: 0.2–0.6 and OR 0.2 95%CI: 0.1–0.3, respectively) and blue-collar workers were more likely to be sedentary .In comparison, in the full-day energy expenditure estimation, increased odds in age were not as strong for men and evidence of an association was not apparent with increased BMI or calorie intake for women. However, the reverse association was identified where those with higher amounts of education tended to be more sedentary in both males  and females (OR 1.2 95%CI: 0.8–1.8 and OR 2.2 95%CI: 1.4–3.6, respectively) and blue-collar workers were found to be significantly less sedentary (OR 0.2 95%CI:0.1–0.3). Relationships with energy intake also were identified in men with those consuming less than 2300 Kcal/day on average having a higher odds of being sedentary (OR 1.6 95%CI: 1.0–2.5) and those consuming greater than 2900 Kcal/day having a lower odds of being sedentary (OR 0.6 95%CI:0.4–0.9).Our results highlight the primarily sedentary nature of this adult urban population, with 70% of women and 60% of men not undertaking any regular physical activity or sports during leisure time. Similar studies, which have only evaluated leisure time physical activity, have identified comparable levels of sedentarism, as well as associations between sedentarism and certain population factors. In a European Union study  conducteThe questionnaire, which was used to collect data on physical activity, was developed according to the European Prospective Investigation into Cancer and Nutrition study questionnaire, which showed acceptable repeatability and validity . Formal The above mentioned studies only recorded and based results on leisure-time energy expenditure, excluding the potential input of physical activity that is undertaken at work or on household tasks. As presented in the results of this study, the inclusion of work-time energy expenditure shows that those less educated and those with manual occupations are less sedentary, which reverses the association seen in the leisure-time only estimation. The differences between the associations of the two separate measurements, leisure-time energy expenditure versus full-day energy expenditure, therefore demonstrate the potential for work-related and household-related physical activity to significantly affect the proportion of sedentarism, and the associations between sedentarism and the factors studied. Efforts, therefore, need to be made to include all components of daily physical activity and energy expenditure and to study the effects of this energy expenditure as a whole, on cardiovascular disease and other health benefits of moderate and high-intensity energy expenditure, which has also been highlighted by Salmon et al . HoweverThe European Society of Cardiology has outlined, in a recent position paper, the need for physical activity to be prescribed in primary and secondary prevention and to implement successful strategies to reduce cardiovascular risks . It has The urban Portuguese population has a very high prevalence of reported sedentarism potentially contributing to the high levels of cardiovascular disease in the country. Caution, however needs to be taken in the classification of individuals as sedentary when considering leisure-time versus full-day energy expenditure, as work and household-related activities can account for a large portion of the energy spent. Including these measures may also affect the overall associations found between sedentarism and the population characteristics.BMI Body Mass IndexMET Metabolic EquivalentRMR Resting Metabolic RateWHO World Health OrganisationThe author(s) declare that they have no competing interests.DG designed the study, performed the statistical analysis and drafted the manuscript. ACS participated in the design of the study and in the interpretation of the results. HB conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Escherichia coli, which reproduces without a juvenile phase and with an apparently symmetric division. We show that the cell that inherits the old pole exhibits a diminished growth rate, decreased offspring production, and an increased incidence of death. We conclude that the two supposedly identical cells produced during cell division are functionally asymmetric; the old pole cell should be considered an aging parent repeatedly producing rejuvenated offspring. These results suggest that no life strategy is immune to the effects of aging, and therefore immortality may be either too costly or mechanistically impossible in natural organisms.In macroscopic organisms, aging is often obvious; in single-celled organisms, where there is the greatest potential to identify the molecular mechanisms involved, identifying and quantifying aging is harder. The primary results in this area have come from organisms that share the traits of a visibly asymmetric division and an identifiable juvenile phase. As reproductive aging must require a differential distribution of aged and young components between parent and offspring, it has been postulated that organisms without these traits do not age, thus exhibiting functional immortality. Through automated time-lapse microscopy, we followed repeated cycles of reproduction by individual cells of the model organism  Detailed time lapse photography reveals that organisms that divide symmetrically, such as the bacterium E. coli, can indeed age and consequently that no organism is immune to mortality That populations survive indefinitely while individuals grow old and die requires that young offspring be produced at the expense of old parents, and this has classically been explained in terms of an immortal germ line passing through a transient and disposable soma, or body . HoweverSaccharomyces cerevisiae and the bacterium Caulobacter crescentus) share the traits of a visibly asymmetric division and an identifiable juvenile phase [Schizosaccharomyces pombe has reinforced these apparent characteristics of aging organisms. The same study that found indications of aging in this organism also found that, contrary to expectations, its cell division process was visibly asymmetric, resulting in enlarged mother cells after only a few divisions, possibly indicating that S. pombe development is similar to that of budding yeast [One difficulty with this distinction is that an asymmetry sufficient for aging may not necessarily result in a visible difference during cell division. Therefore, what may be a more useful common feature of the organisms that have currently been found to age is the presence of a juvenile phase  in their life cycle. Juvenile cells are either smaller or undifferentiated and must go through a period of growth or differentiation before becoming capable of reproducing. This is similar to the idea that the critical requirement for aging in unicellular organisms is the presence of a parent cell that provides for a smaller offspring cell . Consistle phase ,5,6. Theng yeast .Escherichia coli, that is, in cells that do undergo a morphologically symmetrical division and do not exhibit a juvenile phase, in order to determine if such organisms age. Previous studies of senescence in E. coli have mostly focused on the loss of viability over time during nutrient depletion  [E. coli grows in the form of a rod, which reproduces by dividing in the middle. One new end (or pole) per progeny cell is produced during this division event  . Howeveron event . TherefoCells were grown from one cell into a monolayer microcolony that contained up to 500 cells, and time-lapse images see . At eacht-test) includes cells of all division ages and shows that the average growth rate of the old pole progeny cell is 2.2% (+/−0.1%) slower than that of the new pole cell. This analysis, performed on 7,953 cell pairs, conclusively demonstrates (p < 0.00001, t = 14.40, df = 7952) that the cell that inherits the old pole grows slower than the new pole cell produced in the same division. Two factors from this same dataset demonstrate the lack of a juvenile phase in E. coli. First, comparison of the progeny cells shows that the new pole cell is slightly larger on average  than the old pole cell (the contrary would be expected in the presence of a juvenile phase). Second, the young pole cell is marginally more likely to divide sooner than the old pole cell , which is also not consistent with a phase where the young cell must grow or differentiate before reproduction. These differences are consistent for all generations during steady-state growth (data not shown). Therefore, while a juvenile phase is absent, there is a consistent functional asymmetry between the two progeny cells that is disadvantageous to the old pole.The pattern of fast and slow growth rates in this average lineage gives striking evidence for reproductive asymmetry between the progeny cells, as the cells that show a cumulatively slowed rate of growth (shorter lines) are those cells that have more often inherited an old pole during their ancestry. To verify that this pattern in the average lineage is actually due to a difference in growth rate between new and old pole cells, we performed a pairwise comparison of every set of sister cells that was produced at the eighth generation in each of the films. As sister cells share temporal and spatial surroundings, this comparison controls for potential environmental variation within the microcolony. The comparison . Equivalently, each new pole cell can be assigned a number of divisions that it sequentially divided as the new pole cell. Comparing these values with the growth rates of the cells, we find that the older the old pole of a cell is, the slower the growth rate of that cell, while cells with more consecutive new pole divisions exhibit increasing growth rates A. Furthep < 0.00001, t = 9.29, df = 1565). Therefore, it appears that the slower growth rate of the old pole cells also results in a longer-term decreased ability to produce offspring biomass. Another long-term effect of aging is the probability of survival of the organism over time. During the growth of the microcolonies, sixteen cells were observed to cease growing; these cells never resumed growth during the course of the experiment. We have defined these cells as potentially dead cells and have analyzed their locations in the lineages. While these apparent deaths may ultimately be due to stochastic events, they show a statistically significant bias  is almost 400 times more frequent.To determine the longer-term effect of inheriting the old pole, we performed a second pairwise analysis, comparing the total length of offspring produced by sister cells from the fifth generation until the end of tracking (this generation was chosen as each cell has the opportunity to progress through about three divisions). As the bacteria are rod shaped, the total length of cells produced is proportional to the biomass of the offspring. The results show that old pole cells produce less offspring biomass compared to their new pole sisters , reduced offspring biomass production, and an increased chance of death. Thus, nescence , where aS. cerevisiae, the daughter cell of a young mother cell has a greater reproductive life span compared to the daughter cell of an old mother cell [(Drosophila melanogaster), several generations of daughters are required to recover from the effects of chromosome damage [That the two cells are not equal, despite appearances, indicates a functional asymmetry that may be explained by a number of mechanisms, such as those that result in the polar localization of cell components ,17. In ther cell . A likelher cell , and posher cell . Additioe damage . In our C. crescentus  is of about the same magnitude as the decline in growth rate measured here [S. cerevisiae, the rate of offspring production also declines, resulting in cell cycle times that are as much as five times longer for mother cells that have divided 30 times than for young cells. This is an accelerating decline, however, and there is no detectable difference in cell cycle time for the first ten divisions [On the other hand, the observed rate of decline in old pole cells  would result in a complete cessation of growth after about 100 divisions. While the behavior of cells with more than seven old pole divisions cannot be determined from these data, it is interesting to note that the observed rate of decline in offspring production in red here . In S. civisions .E. coli, which is essential for the process of aging. The cost of the aging process in lost growth to the population under these conditions is about 2%. In competition, these cells would be rapidly displaced by competitors that did not age, but only if the cost of avoiding senescence were not equal or greater. It has been proposed that one such cost is the rigorous level of maintenance and repair that would be required to prevent the decline and eventual extinction of a perfectly symmetrical organism [Our results show that a juvenile phase is not required for the process of aging any more than the presence of a germ line or a visibly asymmetric division is. In contrast, we demonstrate the presence of a physiological asymmetry in organism . The phyE. coli provides an excellent genetic platform for studying the fundamental mechanisms of cellular aging and may provide insight into the costs and evolutionary roots of repair, maintenance, and longevity. Simple model organisms such as yeast and nematodes have already proven their value in identifying evolutionarily conserved pathways that regulate life span in higher organisms [E. coli will also be conserved in other forms of life.The use of the model organism rganisms ,25. As hE. coli, MG1655 [The sequenced wild-type strain of , MG1655 , was mod, MG1655 ). Cells Up to seven fields containing one to four cells each were manually identified at the start of the experiment, and stored in the MetaMorph microscope control software . Fluorescent images were recorded at each field with time points taken generally every 4 min for the first 160 min, then every 2 min for the remaining time. A subset of six colonies was recorded every 40 s for improved time resolution. The excitation light did not affect the cellular growth rate (data not shown). Images were taken with a CoolSNAP HQ (Princeton Instruments, Trenton, New Jersey, United States) at 100X magnification; the resulting images have a spatial dimension of 0.064 μ per pixel. Excitation light (480 to 520 nm) was limited to 5% of the output of a 100-W Hg vapor lamp, with an exposure of 2 s. Emission wavelengths were 505 to 565 nm (filters from Chroma, Rockingham, Vermont, United States). See The custom analysis software (BHV) was developed by integration of open-source software under a central shell scripted in Python . For pixt-test. Since the sibling cells occupy similar points in space and time, this eliminates influences coming from overall changes in the colony growth rate or potential variations in environment across the colony. Second, we created control datasets (data not shown) from the colonies in which all the properties of the cells and lineages are preserved, with the exception of pole identity, which is randomly assigned at each division. In this case, we tested the null hypothesis that the pole age of a cell is not a factor in cell physiology by comparing the observed result with the expected normal binomial distribution derived from the null hypothesis. In both cases, we determined from these tests the probability that the old pole effect is due to random fluctuations, expressed as a p-value. We determined if the apparently dead cells were biased toward divisions as old pole cells by examining their complete recorded division histories, which describe how many times each cell divided as an old pole or new pole cell. We compared the average number of old pole divisions of the population of 16 dead cells (mean = 3.44) with the distribution of averages (mean = 2.75, SD = 0.29) from 1,000,000 randomly generated sets of 16 cells with the same number of total divisions . This yielded the one-tailed probability (the hypothesis was that these cells would be enriched in old pole divisions, based on our observations of growth rates) of such an old pole bias arising by chance. All error ranges in the text and figures represent the standard error of the mean. The values for We verified that our results are not the effect of bias unrelated to pole age in two ways. First, we compared pairs of daughters of the same cell using a pairwise Dataset S1The lineage is represented as a series of letters, where “O” indicates a division as an old pole cell, and “N” a division as a new pole cell. The first cell in the tree is labeled “–.” The number of cells indicates how many individual cell growth rates comprise the average rate. At early points in the lineage, there are many more than 94 cells (the number of films), as each initial cell gives rise to two lineages , and some films start with more than one cell. The average growth rates approximately correspond to microns per minute.(52 KB XLS).Click here for additional data file.Video S1This film shows 305 min (114 frames) of the growth of a microcolony condensed to 7 s. For the first 40 frames (approximately 3 s), images were taken every 4 min; for the remaining frames, images were taken every 2 min. The complete lineage history of the entire microcolony from the single initial cell in frame 1 to all 505 cells in frame 114 has been tracked and recorded, allowing pole ages to be assigned to every cell.(286 KB WMV).Click here for additional data file.


Many medical specialities have reviewed the statistical content of their journals. To our knowledge this has not been done in general practice. Given the main role of a general practitioner as a diagnostician we thought it would be of interest to see whether the statistical methods reported reflect the diagnostic process.British Medical Journal , British Journal of General Practice and Family Practice over a one-year period (1 January to 31 December 2000).Hand search of three UK journals of general practice namely the A wide variety of statistical techniques were used. The most common methods included t-tests and Chi-squared tests. There were few articles reporting likelihood ratios and other useful diagnostic methods. There was evidence that the journals with the more thorough statistical review process reported a more complex and wider variety of statistical techniques.BMJ had a wider range and greater diversity of statistical methods than the other two journals. However, in all three journals there was a dearth of papers reflecting the diagnostic process. Across all three journals there were relatively few papers describing randomised controlled trials thus recognising the difficulty of implementing this design in general practice.The  For example, the use of the naïve Bayes' discriminant function (and from it the derivation of likelihood ratios) is appropriate. Proponents of Bayes' argue for its simplicity and ease of interpretation,6. In coMany medical journals, both generalist,11 and sBritish Medical Journal (BMJ) , British Journal of General Practice (BJGP) and Family Practice. These three journals were chosen because they reflected the main primary care journals in the UK. The journals were hand searched for original research articles over a one-year period (1 January to 31 December 2000). Articles were classified for both their statistical content and methods of design according to criteria laid down elsewhere[Three statisticians (MJC, ASR and GKA)  including one Professor, one Senior Lecturer and one Lecturer each reviewed one leading UK journal in general practice. The fourth author (NS) is a Primary Care Physician. The journals chosen were the elsewhere,20. TablThe main study was preceded by a pilot phase in which a random sample of 10 articles was classified both by statistical content and study design by the three statisticians. Where there were differences of opinion, consensus was reached by discussion. We met once to discuss our classification system, and to iron out differences of opinion. One problem lay in how we actually classified study design. For example, one of use used the phrase 'cross-sectional survey' while another used the phrase 'questionnaire survey' when both meant the same in terms of study design. Another problem was that we missed some of the statistical techniques (where there were many) and this required much more careful reading of the articles when we carried out the main survey. We did not carry out a formal reliability study of the pilot phase but instead relied on our experiences both as statisticians, and as journal reviewers. Similarly we chose not to carry out a formal reliability analysis in the main study.BMJ  (n = 79), BJGP (n = 145) and Family Practice (n = 81).The total number of articles reviewed over a one year period was as follows: BMJ, BJGP and Family Practice respectively (Table Family Practice (21.0% compared to an average of 11.8%). Qualitative studies included those encompassing terms such as 'focus groups' and 'semi-structured interviews' for example. Figure Helicobacter pylori for the detection of peptic ulcer). Examples of more unusual study designs include those based on video recordings, literature reviews and quasi-experimental designs.The most common design was that of a cross-sectional survey being found in 24.1%, 39.3% and 35.1% of articles in the BMJ shows a greater range and breadth of articles than Family Practice. More sophisticated techniques are reported more often in the BMJ than either of the other two journals. In the BMJ, the two most common statistical methods used were logistic regression (n = 14, 17.7%) and the Chi-squared test (n = 13, 16.5%). The two least common were the Mantel-Haenszel statistic (n = 1, 1.3%) and Cronbach's alpha (n = 1, 1.3%). Relatively new innovations such as random effects models were seen in both the BMJ and the BJGP. The least sophisticated statistical methods appeared in Family Practice. Methods based on likelihood ratios were seldom found in either the BMJ or BJGP and not at all in Family Practice. Nonparametric tests were often unspecified but where they were included Mann-Whitney U test, Spearman's correlation coefficient and the Wilcoxon matched-pairs signed ranks test. Multiple comparisons included Bonferonni techniques and Scheffe's contrasts. Survival analysis included Kaplan-Meier curves and Cox regression.The range of statistical methods reported can be seen in Table One-third of all articles reported no statistics or simple summaries (for example, mean, median, percentage, standard deviation, interquartile range). No journal article with a qualitative design had any statistical content.BJGP. This was due to a wide range of statistical methods being reported only once. Examples include time series, multilevel modelling and factor analysis. In others, we could not decipher which statistical techniques had been used.A large number of articles reported other statistical methods, in particular the Table BJGP and Family Practice. Papers in the BMJ and the BJGP used more sophisticated statistical methods than Family Practice . It is perhaps too easy for us to lay blame at the Editors door for this lack of sophistication. Statisticians are relatively rare, and review, for the most part, is unpaid.Two-thirds of all journal articles relied on some type of statistical analysis beyond descriptive statistics Table . The Chice Table . While byears ago. Despitelsewhere,,15,17,20lsewhere,. The BJGAlthough these three journals publish a large proportion of the research in general practice within the UK, they by no means represent 100% of it. To look at this further we examined the year 2000 and undertook a MEDLINE search using the key indexing phrase 'General Practice'. We found over 800 articles in a diversity of journals. Articles were published in the fields of rheumatology, medical ethics, obstetrics, public health, clinical pharmacology, clinical neurology and telemedicine to name but a few.We chose to look at the year 2000. Would our results be different had we selected a different year? The published literature suggests otherwise. In a 20 year old study, Emerson and Colditz found t-Now let us turn to study design. The gold standard research design is considered to be the randomised controlled trial (RCT). It has been acknowledged that carrying out RCTs in general practice are difficult,24. In oWhat are the issues here? Are they really that different from secondary care? A recent publication posed the question 'What do residents really need to know about statistics?'. The autFor all three journals there was a dearth of articles reflecting the diagnostic process. Why is this? It has already said that diagnosis is the Achilles Heel of GPs. If it iThe author(s) declare that they have no competing interests.Three authors (ASR, GKA and MJC) carried out the literature review while all four authors contributed to the writing.The pre-publication history for this paper can be accessed here:


The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins.We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells. Genome analyses of a range of organisms have lead to the identification of an increasing number of putative RNA-binding proteins (RBPs) whose function is still unknown. RBPs have been found to act as integral part of ribonucleoparticles (RNPs) controlling gene expression at different levels . RNPs arTrypanosoma brucei [Drosophila [Recently, research has mainly been focused on identifying protein-protein interactions using two-hybrid interactions ,5, immuna brucei , Drosophosophila  and mammosophila . It has osophila .Besides the purification method, the way to lyse cells is also crucial. In yeast, different lysis methods are in use. Glass bead milling has been applied to identify RNAs from immunoprecipitated RNPs . Both FrBreaking yeast with a glass bead mill is a common method to produce cell lysates. The principle is based on the physical rupture of the yeast's cell wall and cell due to the friction produced by glass beads rapidly moving through the cell suspension. One of the advantages of this method is the high lysis efficiency.We lysed two different yeast strains using a "bead-beater" bead mill in the presence of RNase inhibitors (100 U/ml SuperaseIn and 20 mM Ribonucleoside Vanadyl Complex, RVC) as described in Methods. We used a strain where Nrp1p, a putative RNA-binding protein that contains one RRM (RNA Recognition Motif) , has beePDA1) or by direct staining of ribosomal RNAs after blotting with methylene blue  and extracted the RNA. As shown in Figure PDA1 and ASH1 mRNAs is already detected in the crude lysate. Whereas no further degradation of PDA1 mRNA is detected during subsequent centrifugation, ASH1 mRNA is heavily degraded, indicating that there might not be a protective effect of RNA-binding proteins like She2p. In addition, when the extract was incubated with the IgG-coupled beads for 2 hours degradation becomes evident also for PDA1 mRNA , since the following purification steps might become an additional source of RNA degradation. This idea is also supported by our results Figure  where, eOur data suggest that mechanical breakage of frozen yeast cells at ultra-low temperatures is the lysis method of choice for purification of intact mRNP complexes. Since in recent approaches ,16 diffeStrains used in this work are isogenic with W303. The relevant genotypes are listed in Table TAP-tagged strains were generated by transformation with PCR products using the lithium acetate method . The pri600 nm/ml of 2–3. Cultures were harvested at 4°C and washed with ice-cold sterile water.Yeast cultures were grown at 30°C in YPD media  up to an600 nm (5 l cultures with an OD600 nm/ml of 2) were harvested, washed with cold sterile water and immediately lysed or the wet pellet snap-frozen in liquid nitrogen. For lysis, harvested cells were resuspended in 30 ml lysis buffer (10 mM K-Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc (Biomol, Germany), 0.7 μg/ml pepstatinA, 100 U/ml SuperaseIn (Ambion, UK), 20 mM Ribonucleoside Vanadyl Complex (RVC, NEB, UK)) and immediately broken. Frozen cells were stored at -80°C until used. Then, they were thawed in 30 ml lysis buffer and immediately lysed. Cells were lysed in a glass bead mill (Bead-beater, Biospec) in 3 cycles (3 min breaking and 5 min cooling). During lysis, cells were kept cold by a water-ice bath.Cell pellets from 10000 OD600 nm (2.5 l cultures with an OD600 nm/ml of 2) were harvested, washed with cold sterile water and immediately lysed or the wet pellet snap-frozen in liquid nitrogen. For lysis, harvested cells were resuspended in 15 ml lysis buffer (10 mM K-Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA, 100 U/ml SuperaseIn, 20 mM RVC) and immediately broken. Frozen cells were stored at -80°C until used. Then, they were thawed in 25 ml lysis buffer and immediately broken. Lysis was performed as described [Cell pellets from 5000 ODescribed  with a c600 nm/ml of 2–3, were frozen by pressing the cell pellet through a 50 ml syringe directly into liquid nitrogen [2, 0.30% Igepal CA-630, 120 mM NaCl, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA, 200 U/ml SuperaseIn, 40 mM RVC) per gram.Cell pellets corresponding to 3–15 l cultures, with an ODnitrogen  and stor2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA) with the addition of adjusting buffer . The resulting sample was again spun for 20 min at 20000 × g and the supernatant (S20) finally spun at 200000 × g for 20 min. The supernatant was collected (S200) and the resulting pellet (P200) was resuspended in 10 ml IgG binding buffer .After cell lysis, cell extracts were subjected to serial centrifugations at 4°C. First, the crude lysate (CL) was spun 3 min at 1200 × g to pellet cell debris. The total extract (S1) was further spun for 20 min at 7500 × g and the supernatant collected (S7). Salt and buffer concentration were adjusted to IgG binding conditions  was spun 3 min at 1200 × g to pellet cell debris. Total extract (S1) was further spun for 20 min at 7500 × g and the supernatant (S7) was collected. The resulting pellet (P7) was resuspended in 10 ml 0.5 × lysis buffer. Supernatant (S7) was again spun for 20 min at 20000 × g and the supernatant (S20) finally spun at 200000 × g for 20 min. The supernatant was collected (S200) and the resulting pellet (P200) was resuspended in 10 ml 0.5 × lysis buffer.For TAP purifications of Sub2p, total extract (S1) was directly centrifuged at 100000 × g for 1 h (S100).The lipid phase was discarded from all fractions.100–300 μl aliquots from the different fractions were extracted immediately 3–4 times with phenol:chloroform:isoamylalcohol (pH 4), precipitated with sodium acetate and ethanol and stored at -80°C until analyzed.2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA. Samples were taken before (IgG input) and after IgG immunopurification (flow through, IgG FT) and immediately extracted, precipitated and stored at -80°C as described above.Glycerol  was then added to those samples used for further TAP purification steps. These samples were snap-frozen in liquid nitrogen and stored at -80°C for a maximum of 2–3 days. Aliquots were taken before freezing and immediately after thawing to control for degradation during freeze-thawing. These aliquots were extracted, precipitated and stored as described above. TAP purification was performed up to the IgG immunopurification step (first step of the TAP protocol) as described , using a260 nm/A280 nm measured and equivalent amounts of RNA were loaded onto 1.2% agarose-formaldehyde gels. Samples loaded on gels for direct visualization were supplemented with 0.2 μg/μl of ethidium bromide in loading buffer. For Northern blot analysis gels were blotted onto nylon membranes, the membrane stained with methylene blue for determining transfer efficiency and probed against ASH1 and/or PDA1 mRNA .Total RNA was isolated by direct phenol:chloroform extraction from the same strains used in the study as described , precipiThe abbreviations used are: RNP, ribonucleoprotein; RBP, RNA-binding protein; TAP, tandem affinity purification; RVC, ribonucleoside vanadyl complex; RRM, RNA recognition motif; UTR, untranslated region; ORF, open reading frame; DEPC, diethylpyrocarbonate; TCA, trichloroacetic acid; TEV, tobacco etch virus protease.MLdH carried out the studies and drafted the manuscript. RPJ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


To determine the prevalence of severe acute maternal morbidity (SAMM) worldwide (near miss).Systematic review of all available data. The methodology followed a pre-defined protocol, an extensive search strategy of 10 electronic databases as well as other sources. Articles were evaluated according to specified inclusion criteria. Data were extracted using data extraction instrument which collects additional information on the quality of reporting including definitions and identification of cases. Data were entered into a specially constructed database and tabulated using SAS statistical management and analysis software.A total of 30 studies are included in the systematic review. Designs are mainly cross-sectional and 24 were conducted in hospital settings, mostly teaching hospitals. Fourteen studies report on a defined SAMM condition while the remainder use a response to an event such as admission to intensive care unit as a proxy for SAMM. Criteria for identification of cases vary widely across studies. Prevalences vary between 0.80% – 8.23% in studies that use disease-specific criteria while the range is 0.38% – 1.09% in the group that use organ-system based criteria and included unselected group of women. Rates are within the range of 0.01% and 2.99% in studies using management-based criteria. It is not possible to pool data together to provide summary estimates or comparisons between different settings due to variations in case-identification criteria. Nevertheless, there seems to be an inverse trend in prevalence with development status of a country.There is a clear need to set uniform criteria to classify patients as SAMM. This standardisation could be made for similar settings separately. An organ-system dysfunction/failure approach is the most epidemiologically sound as it is least open to bias, and thus could permit developing summary estimates. Severe acute maternal morbidity (SAMM), also known as "near miss", is defined as "A very ill pregnant or recently delivered woman who would have died had it not been that luck and good care was on her side" ,2. This This study was undertaken to systematically review all available studies on SAMM with a view to establishing the global prevalence and examining the usefulness as a maternal health indicator.This study is a part of a bigger systematic review undertaken by the UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP), Department of Reproductive Health and Research at the World Health Organization (WHO) to obtain prevalence/incidence data on maternal mortality and a range of morbidities including SAMM. The methodology of the systematic review followed an a priori protocol and involved an extensive search of all relevant published/unpublished data from 1997 to 2002. The methodology of the systematic review and the search strategy have been described elsewhere . In brieA data extraction instrument was used to extract data from included studies. This instrument includes 48 items distributed in five modules three of which were relevant to this analysis. Modules were designed to collect information on (i) the general study level characteristics such as design, population, setting, (ii) prevalence/incidence of maternal morbid conditions, and (iii) quality assessment of morbidity reports. Reporting of definitions and of the procedures used for identification of cases for morbidities were part of quality assessment. We did not assign quality scores to articles, but preferred to present available information on variables regarded as quality components (including reporting of definitions, case-identification criteria, characteristics of setting and participants).Nearly 65 000 reports were screened initially by titles and/or abstracts of which more than 4500 were retrieved for full-text evaluation. More than 2500 of these were included in the review. Data extracted were entered into a specifically constructed database and tabulated using SAS software.A small number of the articles in the review report on SAMM, near miss or a similar definition such as severe morbidity, critically ill obstetric patient. Although we included other articles reporting on individual severe morbidities within their own category of conditions (e.g. severe hypertension within hypertensive disorders of pregnancy), this particular article is concerned with those papers which define a separate entity of SAMM or similar, or those which report on the most recognised end-points for SAMM (i.e. emergency hysterectomy and obstetric admissions to intensive care units).We later conducted an updated search of MEDLINE and Popline using the keywords 'near miss morbidity' and 'severe maternal morbidity' to find out about any recent publications that could be of importance, but were not included in the database due to the end date of the original search (2002) for the bigger systematic review. The date of this complementary search was June 2004.We describe below the included studies with an emphasis on the different definitions used and criteria for identification of the cases.A total of 30 reports of SAMM are included in the systematic review . Study designs are mainly cross-sectional and most of them are conducted in tertiary care hospitals see . Most ofThere are essentially two types of definition of severe acute maternal morbidity; one describing what the authors meant by a near miss; and the other describing a response to an event such as hysterectomy or admission to intensive care unit (ICU). Fourteen studies define a specific SAMM or near-miss 'condition' ,2,7-18 wIn the majority of articles there is an intuitive agreement on what a near miss means – a woman who almost died but survived. Identification of cases, however, is complex and varies widely across studies. We listed the articles in Individual prevalence rates for SAMM vary between 0.80% – 8.23% in the first category of studies that use disease-specific criteria. Case-finding criteria differ significantly within this category as well. In addition, some of the studies with similar criteria use mainly clinician's evaluation for identification , while oThe range is 0.38% – 1.09% in the group of reports that use organ-system based criteria and include unselected women. Rates are lower (0.01% – 2.99%) and variation is lesser in the category of studies using management-based criteria.An expected finding is the difference between resource-poor and more advantageous settings in the prevalence of SAMM. In resource-poor settings, 4–8% of pregnant women who deliver in the hospitals will experience SAMM when case-identification criteria are based on specific diseases. This rate is around 1% when the organ failure is considered. In more developed country settings, the rates are around 1% with disease-specific and 0.4% with organ-system based criteria, respectively. The results also suggest that the use of organ-system based criteria is more specific in identifying the real SAMM cases.Due to the wide variation in identification of the cases, it is not possible to pool data and make a summary estimate for SAMM. Because of the variation in case identifications in the three categories of identification criteria as well as variation within each category  it is difficult to make comparisons as well. Nevertheless, it is evident that the prevalence of SAMM is higher in studies conducted in less developed country settings. Rates seem to be higher also in studies that use disease-specific criteria as compared to those using organ-system based criteria for similar settings. This finding suggests less specificity of disease-specific criteria in identifying real SAMM cases.Although less specific, the use of disease-specific criteria has some advantages; it is easy to interpret, cases can be identified retrospectively, and the quality of care for that particular disease can be assessed ,35. HoweUse of organ-system based criteria allows for identifying all severe morbidities and then investigating the primary cause, thus does not discard any particular condition. It is the most reproducible across similar areas and criteria can be defined according to resources available. High technology requiring laboratory and haemodynamic investigations can be avoided. However, it is the most labour-intensive for identifying cases, hence criteria for inclusion as near miss must be strict. Bias can be introduced if data collection is incomplete and prevalence can be underestimated.Two approaches are used as potential methods of assessing the care SAMM cases receive. "Mortality Index-MI" is defined as the ratio of maternal deaths among the SAMM cases to the sum of maternal deaths and SAMM cases ,37. It rIt is clearly illustrated in the studies that more SAMM cases are likely to die in resource-poor settings than in more developed country settings. For example, the studies conducted in Niger, Benin and Malaysia give the morbidity to mortality ratio as 11–12 ,15,17 whConsidering all complexities in definition and case-identification of SAMM, it is necessary that studies clearly describe their identification criteria for the cases. There is a clear need to set criteria to identify SAMM cases. Use of organ-system based criteria seems to be a more useful approach in identifying cases as variation in defining criteria can be avoided, particularly for similar settings. It would then be easier to establish summary estimates for SAMM prevalence which could serve as a measure of maternal health and quality of care indicator.None declared.AMG and LS coordinated the conduct of the systematic review of maternal morbidity and mortality. RCP and LS outlined the manuscript. LS reviewed the studies and wrote the initial draft. AMG and RCP substantially improved the manuscript.Table describing important variables of all studies included in the systematic reviewClick here for file