Cl) was determined in red-sensitive, large single cones from the tiger salamander retinal slice.GABAergic inhibition and effects of intracellular chloride ions on calcium channel activity have been proposed to regulate neurotransmission from photoreceptors. To assess the impact of these and other chloride-dependent mechanisms on release from cones, the chloride equilibrium potential ) and then determined the reversal potential for the current component that was inhibited by the Cl- channel blocker, niflumic acid. With this method, ECl was found to average -46 mV. In a complementary approach, we used a Cl-sensitive dye, MEQ, to measure the Cl- flux produced by depolarization with elevated concentrations of K+. The membrane potentials produced by the various high K+ solutions were measured in separate current clamp experiments. Consistent with electrophysiological experiments, MEQ fluorescence measurements indicated that ECl was below -36 mV.Whole cell recordings were done using gramicidin perforated patch techniques to maintain endogenous ClCl is close to the dark resting potential. This will minimize the impact of chloride-dependent presynaptic mechanisms in cone terminals involving GABAa receptors, glutamate transporters and ICl(Ca).The results of this study indicate that E Cl) that is hyperpolarized with respect to the resting potential in many nerve cells, but depolarized in others [Cl in salamander rod photoreceptors is 25 mV more positive than the dark resting potential [Cl in cones have ranged from -65 mV to -36 mV [- conductances that help to shape their responses and synaptic output. As discussed below, the value of ECl in cones is an important parameter for determining the strength and polarity of these effects.Regulation of intracellular chloride levels results in a chloride equilibrium potential  must be negative to the resting potential in order for GABA disinhibition to depolarize a cone.It has been suggested GABAto cones . Under tto cones ] and agato cones ,13; see to cones ]) this h2+-activated Cl- currents (ICl(Ca)) [2+ through voltage-gated Ca2+ channels as well as by release of Ca2+ from intracellular stores [- flux through ICl(Ca) can be substantial: during a 1.4 sec depolarizing step, the charge movement accompanying activation of ICl(Ca) is estimated to be 8.5 times that produced by activation of ICa alone [Cl. If ECl is positive to the resting potential, activation of ICl(Ca) can boost depolarizing feedback responses from horizontal cells onto cones and produce prolonged, regenerative depolarizing responses lasting many seconds [Cl is negative to the resting potential, activation of ICl(Ca) can operate as a negative feedback mechanism to limit regenerative activation of Ca2+ channels [- can directly inhibit the open channel probability of single Ca2+ channels, presumably by modifying an anion binding site on the intracellular surface of the channel [Cl is positive to the resting potential, there is evidence for a negative feedback pathway between ICa and ICl(Ca) in which activation of ICa stimulates ICl(Ca) leading to a Cl- efflux that in turn inhibits Ca2+ channel activation [Cl in cones is negative to the membrane potential, then activation of ICl(Ca) would stimulate an influx of Cl- that would be expected to enhance Ca2+ channel open probability [Cones possess prominent CaICl(Ca)) -17 activr stores . Cl- fluCa alone . These l seconds ,18,19. Ochannels ,17. In a channel . In rodstivation ,20. If, bability .- channels [Cl. Furthermore, analogous to the negative feedback from ICl(Ca) onto ICa described above, the chloride current produced by activation of glutamate transporters in rods can cause a Cl- efflux that inhibits ICa [Cl(Ca) and ICa, the strength and polarity of this potential interaction in cones depends on ECl.Cone photoreceptors have presynaptic glutamate transporters that are coupled to Clchannels -23. The channels . Whetherbits ICa . As withCl in determining the impact of various feedback mechanisms in the photoreceptor terminal, we determined ECl in cone photoreceptors of the salamander retina using a combination of imaging with a chloride-sensitive dye and electrophysiological approaches.Given the importance of EIn control superfusate, dark resting potentials of cones from slices prepared under visible light averaged -46.0 ± 2.00 mV (n = 9) after correcting for the liquid junction potential. This is nearly identical to the dark resting potentials of salamander cones prepared under infrared illumination (-46.8 ± 2.03 mV, n = 18).Cl, ICl(Ca) was recorded using gramicidin perforated patch whole cell recordings and activated by applying a 500 ms step from -78 to -8 mV. This depolarizing step typically evoked a sustained inward tail current arising largely from activation of ICl(Ca) [Cl(Ca) in cones [[Cl(Ca) . If patch rupture had occurred, ECl would be expected to attain -89 mV with the low Cl- pipette solution and -20 mV with the original pipette solution.To measure E ICl(Ca) . Only cen cones [; niflumin cones ,26]. Sub. SubCl, Ca) Fig. . In the Cl(Ca), difference currents calculated from ramps applied before and during GABA application indicate that the GABA-evoked current reversed at -46.4 ± 2.7 mV (n = 9).Bath application of GABA evoked small reversible inward currents that averaged -3.4 ± 0.3 pA at the holding potential of -78 mV (not shown). The small size of these currents may be due to receptor desensitization . ConsistCl, we used a Cl-sensitive dye, MEQ, to examine the Cl- flux that accompanied cone depolarization evoked by bath application of various high K+ solutions (12, 22, 31, 41, 50 and 70 mM K+). In a separate set of experiments, we used gramicidin-perforated patch recording methods to measure the membrane potentials produced in cones by application of the different high K+ solutions. Slices used for MEQ experiments and for measurement of membrane potentials in different solutions were prepared using similar techniques under visible illumination; control experiments showed that the fluorescent illumination used during MEQ experiments did not produce any further changes in the cone resting membrane potential (n = 3). An example of a retinal slice loaded with MEQ is shown in Fig. +, which depolarized cones to -36 mV, produced a 1.2% decrease in MEQ fluorescence. Since MEQ fluorescence is quenched by Cl- ions this indicates that depolarization to -36 mV stimulated an influx of Cl- ions. Application of a solution with 70 mM K+, which depolarizes cones to -7 mV, produced a greater influx of Cl- as evidenced by the 10% decrease in MEQ fluorescence seen in a different cone . ECl was found to be -46 mV from block of ICl(Ca) by niflumic acid; small GABA-evoked currents reversed around the same potential. MEQ fluorescence changes produced by depolarization support these electrophysiological measurements by indicating that ECl is below -36 mV.The main finding of this study is that ECl within cells [Cl(Ca) is localized to the terminal region in rods (30) and these channels are probably also localized to the terminals of cones. Thus, the measurements in the present study are likely to provide estimates of ECl in the synaptic terminal and adjacent regions of the cone cell. Measurements of intracellular Cl- levels suggest that ECl in the inner segment is not significantly different from that measured in the soma [There can be local variations of Ein cells . Large sin cells . MEQ meathe soma .- equilibrium potential is close to the resting potential does not necessarily mean that Cl- is passively distributed. Electrophysiological experiments required that cells be voltage clamped at -70 mV for many minutes. Nonetheless, the value of ECl determined from these electrophysiological experiments in which cells were voltage clamped at -70 mV was similar to the value estimated from MEQ studies in which cells were not voltage clamped and thus at their resting membrane potential. Results from experiments on the prolonged depolarization in cones also suggest that ECl can be maintained indefinitely at a value above the membrane potential. The plateau phase of the prolonged depolarization, which largely reflects ICl(Ca) activation [Cl above the membrane potential may arise from activity of the Na/KCl cotransporter as shown in rods (20) as well as from other mechanisms (e.g., CLC-2) [The finding that the Cltivation ,19, coul in rods 0 as well, CLC-2) .Cl in cones has been estimated in a number of previous studies. The most positive value for ECl of -36 mV comes from calibration of MEQ fluorescence levels to determine the resting intracellular Cl- concentrations in cones isolated from the salamander retina (11). However, these measurements showed a large variability (range of S.E.M.: -26.5 to -46.6 mV). The most negative estimate of ECl comes from a study by Attwell et al [Cl is around -65 mV. However, more recent evidence questions whether the horizontal cell to cone feedback pathway thought to underlie this sign-reversing pathway from rods to cones is truly GABAergic [Cl similar to those found in the present study. 1) By examining the polarity of GABA-evoked currents after patch rupture with either 12 or 24 mM Cl- in the recording pipette, Kaneko and Tachibana [Cl to be around -47 mV in isolated turtle cones. 2) Based on the membrane potential attained by the plateau phase of the prolonged depolarization in turtle cones from the eyecup slice preparation, ECl was estimated to be at or slightly above the dark resting potential of -42 mV [after correction for a liquid junction potential of -2 mV; ref. [Cl was 2 mV more positive than the dark resting potential. 4) A slightly more negative value for ECl was found in ruptured patch recordings from goldfish cones by examining the voltage dependence of the ICl(Ca) tail current [Cl was ~-55 mV.Ell et al  showing ABAergic ,13,14. Oachibana  estimatemV; ref. ]. 3) In mV; ref.  found th current . By extr current  concludeCa in cones, like that of rods, can be inhibited by lowering extracellular Cl- [Ca produced by lowering extracellular Cl- appears to result from a reduction in intracellular Cl- which in turn causes a reduction in the open probability of single Ca2+ channels [Cl is positive to the resting potential, activation of Cl- channels leads to a Cl- efflux thereby producing an inhibition of Ca2+ channels [- channels when the cell is at its resting potential would produce minimal changes in intracellular Cl- in cones. Therefore, the feedback between ICa and ICl(Ca) postulated for rod photoreceptors [Iular Cl- . The inhchannels . In rodschannels ,11,20. Teceptors ,20 wouldCl is close to the dark resting potential is that the stimulation of Cl- channels associated with glutamate transporters by glutamate released from cone terminals [- efflux accompanying activation of glutamate transporters appears to contribute to a glutamate-mediated inhibition of ICa [Ca and ICl(Ca) considered in the previous paragraph, the finding that ECl is near the resting potential leads to the prediction that in darkness there would be no Cl- efflux accompanying glutamate transporter activation and therefore glutamate would not be expected to inhibit ICa.Another implication of the finding that Eerminals  would ten of ICa . As withCl(Ca) and ICa or the glutamate transporter chloride current and ICa would be expected to increase as a cone hyperpolarizes in response to light. By reducing glutamate release, these chloride-dependent negative feedback mechanisms might thus contribute to making post-synaptic responses more transient.Cones hyperpolarize to light, although with prolonged illumination the membrane potential recovers to near the dark resting potential. The impact of chloride-dependent negative feedback between ICl is near the resting potential of cones indicates that GABAergic disinhibition near the dark potential should produce little membrane potential change. This result is inconsistent with the postulated role for GABA in generating the feedback depolarization [The finding that Erization  and supprization ,13,14.Cl in salamander cones is close to the dark resting membrane potential. By minimizing the trans-membrane flux of chloride, this will minimize the presynaptic impact of GABAa receptors, ICl(Ca), and glutamate transporter chloride channels.Electrophysiological measurements, supported by experiments using chloride-sensitive dyes, indicate that ECl is positive to the resting potential of many neurons in the immature brain [Ambystoma tigrinum, 15–25 cm) used in these experiments are thought to be 2–7 years old out of a life span of ~12 years (34).Ere brain . Based oSalamanders were handled humanely in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Center. Chilled salamanders were rapidly decapitated, an eye was enucleated, and the front of the eye was removed. The resulting eyecup was cut into three or four pieces and a single piece was placed vitreal surface down onto a piece of filter paper (2 × 5 mm, Millipore type AAWP, 0.8 μm pores). After adhering to the filter paper, the retina was isolated under chilled amphibian superfusate and cut into 125 μm slices using a razor blade tissue chopper . The slices were rotated 90° to view the retinal layers when placed under a water immersion objective (60X, 1.0 NA) on an upright fixed stage microscope (EF 600, Nikon Inc., USA). Slices were prepared under visible light but recordings were performed in darkness. All experiments were done using red-sensitive large single cones selected by anatomical criteria .2, 0.5 MgCl2, 10 N-2-hydroxyethylpiperazine-N' 2-ethanesulfonic acid (HEPES), and 5 glucose (pH 7.8). The osmolarity was measured with a vapor pressure osmometer (Wescor, Logan, UT) and adjusted, if necessary, to 242 ± 5 mOsm. For high K+ solutions, various quantities of NaCl were replaced with equimolar KCl. Niflumic acid was diluted (1:10,000) from DMSO stock solutions. Unless otherwise specified, chemicals were obtained from Sigma/Aldrich/RBI (St. Louis, MO).Solutions were applied with a single-pass, gravity-feed perfusion system (1 ml/min). The normal amphibian superfusate contained (in mM): 111 NaCl, 2.5 KCl, 1.8 CaCl-, we obtained perforated patch whole cell recordings using the cation channel, gramicidin [3SO4 , 3.5 NaCH3SO4, 10 HEPES. The pH was adjusted to 7.2 with KOH. The liquid junction potential (LJP) of this solution was estimated to be -7 mV using the junction potential calculator of PClamp (Axon Instruments). Membrane potential values reported throughout this manuscript were corrected for the LJP. For experiments with niflumic acid or GABA, pipettes were typically filled with a solution containing (in mM): 54 CsCl, 61.5 CsCH3SO3, 3.5 NaCH3SO4, 10 HEPES (LJP = -8 mV). In some experiments, a low Cl- pipette solution was used containing: 115.5 mM CsCH3SO3, 3.5 NaCl, 10 HEPES (LJP = -10 mV). The pH of both solutions was adjusted to 7.2 with CsOH. The osmolarity of pipette solutions were also adjusted, if necessary, to 242 ± 5 mOsm. Recordings were made using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA) and PClamp 8 software (Axon Instruments). Cell input resistance calculated using a step from -70 to -90 mV averaged 695 ± 111 MΩ. Access resistance estimated from the peak of the capacitative transient averaged 30.7 ± 4.8 MΩ (n = 24).Patch pipettes were pulled on a PP-830 vertical puller (Narishige USA, New York) from borosilicate glass pipettes  and had tips of ~1 μm outer diameter with resistances of 10 to 15 MΩ. To maintain endogenous levels of intracellular Clamicidin . GramiciDigital fluorescent images were obtained with a cooled CCD camera (SensiCam, Cooke Corp., Auburn Hills, MI). Axon Imaging Workbench (AIW 2.2, Axon Instruments Inc., Union City, CA) was used to control the camera, filter wheel, and image acquisition. Pixel binning (2 × 2) of the images was used to decrease acquisition time to ≤1 s. Images were acquired at 5 to 10 s intervals during experimental trials.-]i we used the dye, 6-methoxy-N-ethylquinolinium iodide (MEQ, Molecular Probes, Eugene, OR) [- quenches MEQ. The slow exponential decay in MEQ fluorescence due to dye leakage and bleaching was determined from a 3 min. series of control measurements prior to drug application and subtracted before analysis [For measurements of [Clene, OR) . MEQ wasene, OR) . DiHMEQ analysis ,20.Variance is reported as ± S.E.M.WT conceived the study and drafted the manuscript. WT and EB participated in all aspects of the experiments but most recordings were performed by EB. Both authors have read and approved the manuscript.


Despite the growing use of complementary and alternative medicine (CAM) by consumers in the U.S., little is known about the practice of CAM providers. The objective of this study was to describe and compare the practice patterns of naturopathic physicians in Washington State and Connecticut.Telephone interviews were conducted with state-wide random samples of licensed naturopathic physicians and data were collected on consecutive patient visits in 1998 and 1999. The main outcome measures were: Sociodemographic, training and practice characteristics of naturopathic physicians; and demographics, reasons for visit, types of treatments, payment source and visit duration for patients.One hundred and seventy practitioners were interviewed and 99 recorded data on a total of 1817 patient visits. Naturopathic physicians in Washington and Connecticut had similar demographic and practice characteristics. Both the practitioners and their patients were primarily White and female. Almost 75% of all naturopathic visits were for chronic complaints, most frequently fatigue, headache, and back symptoms. Complete blood counts, serum chemistries, lipids panels and stool analyses were ordered for 4% to 10% of visits. All other diagnostic tests were ordered less frequently. The most commonly prescribed naturopathic therapeutics were: botanical medicines (51% of visits in Connecticut, 43% in Washington), vitamins (41% and 43%), minerals (35% and 39%), homeopathy (29% and 19%) and allergy treatments (11% and 13%). The mean visit length was about 40 minutes. Approximately half the visits were paid directly by the patient.This study provides information that will help other health care providers, patients and policy makers better understand the nature of naturopathic care. The number of Americans using complementary and alternative medicine (CAM) rose from 34% to 42% from 1990 to 1997, with annual spending for CAM therapies in excess of $21 billion -8.Although we now have some understanding of who seeks CAM care and why, relatively little is known about the actual practices of CAM practitioners including the type of patients they treat, the kinds of therapies they use, and the numbers of patients they see each day. In addition, it is unclear if the practice patterns of CAM practitioners vary by state. The objective of this paper is to describe and compare the practice of naturopathic physicians in two US states: Washington and Connecticut.Naturopathic medicine is a system of health care, based on the teachings of Benedict Lust , with a ... a distinct system of primary health care – an art, science, philosophy and practice of diagnosis, treatment and prevention of illness. Naturopathic medicine is distinguished by the principles upon which its practice is based. These principles are continually reexamined in the light of scientific advances. The techniques of naturopathic medicine include modern and traditional, scientific and empirical methods .Naturopathic physicians are trained as generalists with expertise in a variety of core treatment methods including nutrition, hydrotherapy, colonic irrigation, physiotherapy, naturopathic manipulation, botanical medicine, homeopathy, pharmacology and minor office surgical procedures. Some licensed naturopathic physicians are also trained in traditional Chinese medicine, acupuncture and Ayurvedic medicine as well as clinical specialties such as natural childbirth ,11-14. TAccurate estimates of the number of naturopathic physicians practicing in North America are difficult to obtain. A recent estimate (based on data from licensing bodies) indicated that in 2000 approximately 1300 licensed naturopathic physicians were practicing in the United States and another 500 were practicing in Canada. Naturopathic medicine is a licensed health care profession in twelve US states , Puerto Rico and four Canadian provinces (British Columbia, Manitoba, Ontario and Saskatchewan) ,15. In mLegal regulation of naturopathic medicine varies by state and province. Connecticut and Washington have relatively similar (and representative of the other ten states) requirements for licensure including: Graduation from an accredited four-year naturopathic medical school, successful completion of licensing examination , the submission of completed application forms and paying the required fee. In addition, Washington State requires that individuals be of good moral character with no history of unprofessional conduct .The legal scopes of naturopathic practice in Connecticut and Washington are similar and relatively broad compared to the other states that license naturopathic physicians. Naturopathic physicians in these two states can diagnose and treat disease, utilizing a wide range of modalities and specialties including: minor surgery such as suturing (limited in Washington); physical therapies including hydrotherapy, naturopathic manipulation, and physiotherapy; colonic irrigation; electrotherapy (for example TENs); diagnostic x-ray; venipuncture; obstetrics (in Washington a midwifery license is required); gynecology; botanical medicine; nutrition; and homeopathy. Naturopathic physicians in Washington may prescribe a limited range of drugs; however, this is not part of the scope of practice in Connecticut. The practice of acupuncture requires separate licensure in both states .The data derive from a parent study of licensed acupuncturists, chiropractors, massage therapists and naturopathic physicians . The stuLicensing Boards provided contact information for all licensed naturopathic physicians in Washington 1998) and in Connecticut (1999) with in-state addresses. In total, 142 licensed practitioners were identified in Washington and 63 licensed practitioners were identified in Connecticut. Providers without identifiable telephone numbers, and those not currently practicing were excluded. The proportion of excluded providers was 11% in Connecticut and 29% in Washington. See Table 998 and iTo maximize the accuracy of state-wide estimates, data collection efforts were concentrated on high-volume practitioners: those with at least 20 patient visits per week. About 60% to 70% of practitioners had a high-volume practice and accounted for roughly 85% to 90% of all visits to the profession. The remaining practitioners were categorized as low-volume providers. While all high-volume practitioners were asked to collect data on 20 consecutive patient visits, only the first 10 low-volume practitioners were asked to collect data. The rationale was to collect only enough data from low-volume practitioners to ascertain whether their practices differed markedly from those of high-volume practitioners. It was ultimately decided, however, to weight data for high- and low-volume practitioners in a manner that produced annual estimates of visits in each state.Naturopathic physicians who agreed to participate were asked to complete one-page encounter forms for 20 consecutive patient visits. No financial incentives were provided for participation in the study and the protocol was approved by the Group Health Human Subjects Review Committee and the Harvard Pilgrim Health Care "Committee on Clinical Investigations, New Procedures and New Forms of Therapy" prior to the commencement of data collection.Research assistants conducted telephone interviews with the randomly selected providers in Washington State in 1998 and in all eligible practitioners in Connecticut in 1999. . Information about individual demographic characteristics (e.g., age, gender, race/ethnicity); training characteristics (e.g., duration, location); practice characteristics ; and workload characteristics  was obtained for each provider who agreed to be interviewed. Among those eligible to be interviewed, the participation rate was 94% in Connecticut and 78% in Washington.Naturopathic physicians who were eligible to collect visit data (i.e., those who saw at least 20 visits per week plus a sample of those who saw at least 10 – 19 visits per week) were asked to complete encounter forms for 20 consecutive patient visits.  Those who agreed were mailed encounter forms that were modeled after those used by the National Ambulatory Medical Care Survey (NAMCS) . Each naDescriptive statistics (means, standard deviations, frequencies) were used to present the practitioner characteristics in Table Given the large sample sizes (631 and 1186), the weighted percentages presented in the tables have small standard errors, generally between 0.5 and 2.5 percentage points and rarely exceeding 3 percentage points. As a result, moderate to large differences between the states are also statistically significant and we do not include standard errors in the tables.The demographic characteristics of the naturopathic physicians in Washington State and Connecticut were remarkably similar (see Table The majority of practitioners (80%) in Washington State trained at Bastyr University in Seattle, while most practitioners in Connecticut trained at National College of Naturopathic Medicine in Portland, Oregon. Although naturopathic doctors in Connecticut were more likely to report advanced specialty training (such as homeopathy), those in Washington were more likely to be licensed in another health profession. Washington naturopathic physicians were most often also licensed in chiropractic (8%), nursing (7%), acupuncture (6%) and midwifery (5%), while Connecticut naturopathic physicians were most often also licensed in acupuncture (10%). Almost one-quarter (22.5%) of naturopathic doctors licensed in Washington had completed at least one year of post-graduate residency training compared with only 8.5% of those in Connecticut. (Residency training is not required for licensure in either state.)Both groups graduated from 4–5 year accredited full time programs of naturopathic medical education and the resulting naturopathic practice characteristics were reported to be very similar across the two states Tables  and 3. JAs was the case for the practitioners, patients of naturopathic physicians in the two states were remarkably similar Table . About 7More than 70% of visits included examinations or the ordering of diagnostic or screening tests Table , most ofTherapeutic and preventive modalities were used in most visits (96%) in both states. The most common naturopathic therapeutics used in both states were: botanical medicines, vitamins, minerals, homeopathy and allergy treatments Table . In addiThree-quarters of all visits in both states resulted in a recommendation that the patient return at a specified time Table . The mea Theories about what protomorphogens are, as well as their role in health and disease continue to be debated.)A wide range of generally similar commercially packaged products  were used by naturopathic physicians in the two states Table . ConnectGiven the broad scopes of practice of naturopathic medicine in Washington and Connecticut, the similarities in the practitioner characteristics, patient characteristics and practice characteristics in Washington State and Connecticut are remarkable. The finding that naturopathic medicine is primarily a white and female profession is consistent with other recent studies of naturopathic physicians in the United States and Canada ,20. SubsPatients who visit naturopathic physicians are primarily female, white, middle aged, non-smokers. In many ways they are demographically similar to the naturopathic physicians themselves. Patients present to naturopathic physicians with a broad range of complaints and are diagnosed by the practitioners with a wide variety of conditions. It should be noted that much of naturopathic practice is focused on chronic problems, particularly those exclusively or primarily presented by women (e.g., fatigue, headache, symptoms associated with menopause, depression). In fact 75% of all visits are made by women. The most common "reasons for visits" identified in this study were very similar to those noted in a Canadian study , suggestOther than physical examination, blood tests and stool analysis, naturopathic practitioners use few diagnostics tests. However, they commonly recommend commercially packaged natural products such as herbal medicines, vitamins and minerals. Naturopathic practice also involves significant amounts of patient counseling and education. Overall, naturopathic physicians spend more than twice as much time with patients as conventional physicians at each visit (40 minutes vs. 14 minutes) , permittData from this and previous studies ,20 indicOur results also have implications both for naturopathic physicians and the American health care system. They indicate that naturopathic physicians treat a broad range of health problems. The results from our study provide baseline data for tracking changes as the practice of naturopathic medicine evolves over time across the US in response to increasing research into the safety, efficacy and cost-effectiveness of naturopathic treatments, as well as to changes in the social, economic and regulatory environment.The major strengths of this study are its large sample size, relatively high participation rates, and the ability to compare naturopathic physicians from two geographically distant states. The main limitation is that despite the similarities of the states studied, it is not known if the results are representative of the other ten states where naturopathic medicine is licensed. These two states reflect typical licensing requirements prevalent throughout the US. Washington State has one of highest numbers of licensed naturopathic physicians in the US, while the number of naturopathic physicians in Connecticut is significantly smaller, and more representative of, most other licensed states.Another limitation of the survey was its inability to accurately capture the use of office-compounded tinctures and powders. Our results highlight the use of bottled products as a major part of naturopathic practice; however, the practitioners' use of their own combination preparations was difficult to record on the data collection forms. This aspect of practice is likely under-reported in our data.Naturopathic physicians and their practices in Washington and Connecticut, two geographically distinct states, are similar. This study provides baseline data for describing and tracking the practice patterns and scope of practice of naturopathic physicians over time and will help inform researchers and policy makers considering the regulation of this profession in other states. It also helps conventional physicians, other health care providers and patients better understand the nature of naturopathic care.The authors declare that they have no competing interests.HB conceptualized and drafted the manuscript. DC conceptualized the overall project, designed and directed collection of the original data and participated in the conceptualization of this manuscript, and analysis of this data. KS participated in the design of the overall project and this manuscript, and participated in the statistical analyses. JE helped to conceptualize the overall project, directed the data collection, quality control, and participated in the analyses for this paper. BM, JB, EC, and DE helped to conceptualize the overall project including the data collection. MS helped to conceptualize this paper. RD helped to conceptualize the overall project, design data collection procedures, and secure funding. All critically edited drafts of this manuscript and approved the final manuscript.The pre-publication history for this paper can be accessed here:


July correction PLoS Biology, volume 2, issue 8In Nicotine's Defensive Function in NatureAnke Steppuhn, Klaus Gase, Bernd Krock, Rayko Halitschke, Ian T. Baldwin10.1371/journal.pbio.0020217DOI: The Academic Editor was erroneously listed as Michael Levine. The Academic Editor for this paper is Joy Bergelson, Chicago University.This correction note may be found online at DOI: 10.1371/journal.pbio.0020382.


It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence  may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB;  An international team has systematically validated and annotated just over 21,000 human genes using full-length cDNA, thereby providing a valuable new resource for the human genetics community The draft sequences of the human, mouse, and rat genomes are already available . The nexPrevious efforts to catalogue the human transcriptome were based on expressed sequence tags (ESTs) used for the identification of new genes , chromosArabidopsis full-length cDNA project  project , Flybase project . In our http://www.h-invitational.jp), which stores all annotation results by the consortium. Free and unrestricted access to the H-Inv annotation work is available through the database. Finally, we summarize our most important findings thus far in the H-Inv project in Concluding Remarks.This manuscript provides the first report by the H-Inv consortium, showing some of the discoveries made so far and introducing our new database of the human transcriptome. It is hoped that this will be the first in a long line of publications announcing discoveries made by the H-Inv consortium. Here we describe results from our integrative annotation in four major areas: mapping the transcriptome onto the human genome, functional annotation, polymorphism in the transcriptome, and evolution of the human transcriptome. We then introduce our new database of the human transcriptome, the H-Invitational Database . For the remaining 978 unmapped cDNAs, we conducted cDNA-based clustering, which yielded 847 clusters. The clusters created had an average of 2.0 cDNAs per locus . The aveIn total, 21,037 clusters (20,190 mapped and 847 unmapped) were identified and entered into the H-InvDB. We assigned H-Inv cluster IDs (e.g., HIX0000001) to the clusters and H-Inv cDNA IDs (e.g., HIT000000001) to all curated cDNAs. A representative sequence was selected from each cluster and used for further analyses and annotation.In order to evaluate the H-Inv dataset, we compared all of the mapped H-Inv cDNAs with the Reference Sequence Collection (RefSeq) mRNA database  Figure . The RefFrom the comparison, we found that 5,155 (26%) of the H-Inv loci had no counterparts and were unique to the H-Inv. All of these 5,155 loci are candidates for new human genes, although non-protein-coding RNAs (ncRNAs) (25%), hypothetical proteins with ORFs less than 150 amino acids (55%), and singletons (91%) were enriched in this category. In fact, 1,340 of these H-Inv-unique loci were questionable and require validation by further experiments because they consist of only single exons, and the 3′ termini of these loci align with genomic poly-A sequences. This feature suggests internal poly-A priming although some occurrences might be bona fide genes. The most reliable set of newly identified human genes in our dataset is composed of 1,054 protein-coding and 179 non-protein-coding genes that have multiple exons. Therefore, at least 6.1% (1,233/20,190) of the H-Inv loci could be used to newly validate loci that the RefSeq datasets do not presently cover. These genes are possibly less expressed since the proportion of singletons (H-Inv loci consisting of a single H-Inv cDNA) was high (84%).On the other hand, 78% (11,974/15,439) of the curated RefSeq mRNAs were covered by the H-Inv cDNAs. These figures suggest that further extensive sequencing of FLcDNA clones will be required in order to cover the entire human gene set. Nonetheless, this effort provides a systematic approach using the H-Inv cDNAs, even though a portion of the cDNAs have already been utilized in the RefSeq datasets.It is noteworthy that H-Inv cDNAs overlapped 3,061 (17%) of RefSeq model mRNAs, supporting this proportion of the hypothetical RefSeq sequences. These newly confirmed 3,061 loci have a mean number of exons greater than RefSeq model mRNAs that were not confirmed, but smaller than RefSeq curated mRNAs. The overlap between H-Inv cDNAs and RefSeq model mRNAs was smaller than that between H-Inv cDNAs and RefSeq curated mRNAs. This suggests that the genes predicted from genome annotation may tend to be less expressed than RefSeq curated genes, or that some may be artifacts. All these results highlight the great importance of comprehensive collections of analyzed FLcDNAs for validating gene prediction from genome sequences. This may be especially true for higher organisms such as humans.The existence of 978 unmapped cDNAs (847 clusters) suggests that the human genome sequence  is not yet complete. The evidence supporting this statement is twofold. First, most of those unmapped cDNAs could be partially mapped to the human genome. Using BLAST, 906 of the unmapped cDNAs (corresponding to 786 clusters) showed at least one sequence match to the human genome with a bit score higher than 100. Second, most of the cDNAs could be mapped unambiguously to the mouse genome sequences. A total of 907 unmapped cDNAs  could be mapped to the mouse genome with coverage of 90% or higher. If we adopted less stringent requirements, more cDNAs could be mapped to the mouse genome. The rest might be less conserved genes, genes in unfinished sections of the mouse genome, or genes that were lost in the mouse genome. Based on these observations, we conclude that the human genome sequence is not yet complete, leaving some portions to be sequenced or reassembled.The proportion of the genome that is incomplete is estimated to be 3.7%–4.0%. The figure of 4.0% is based upon the proportion of H-Inv cDNA clusters that could not be mapped to the genome (847/21,037), while the 3.7% estimate is based on both H-Inv cDNAs and RefSeq sequences (only NMs). This statistic indicates that a minimum of one out of every 25–27 clusters appears to be unrepresented in the current human genome dataset, in its full form. Possible reasons for this include unsequenced regions on the human genome and regions where an error may have occurred during sequence assembly. If this is the case, this lends support to the use of cDNA mapping to facilitate the completion of whole genome sequences . For exaUsing the H-Inv cDNAs, the precise structures of many human genes could be identified based on the results of our cDNA mapping . The medAlu repeats in 5% of human cDNAs was reported previously  AS isoforms contained AS exons that overlapped protein-coding sequences. 1,660 out of 3,020 AS isoforms (55%) harbored AS exons that encoded functional motifs. Additionally, 1,475 loci encoded AS isoforms that had different subcellular localization signals, and 680 loci had AS isoforms that had different transmembrane domains. These results suggest marked functional differentiation between the varying isoforms. If this is the case, it would appear that AS contributes significantly to the functional diversity of the human proteome.As the coverage of the human transcriptome by H-Inv cDNAs is incomplete, it would be misleading to conjecture that our dataset comprehensively includes all AS transcripts from every human gene. However, the current collection is a robust characterization of the existing functional diversity of the human proteome, and it represents a valuable resource of full-length clones for the characterization of experimentally determined AS isoforms.In the cases where three-dimensional (3D) structures could be assigned to H-Inv cDNA protein products, we have examined the possible impact of AS rearrangements on the 3D structure. Our analysis was performed using the Genomes TO Protein structures and functions database (GTOP) . We founAn example of exon differences between AS isoforms is presented in We predicted the ORFs of 41,118 H-Inv cDNA sequences using a computational approach see , of whicAfter determination of the H-Inv proteins, we performed a standardized functional annotation as illustrated in To predict the functions of hypothetical proteins (Category IV and V proteins), we used 196 sequence patterns of functional importance derived from tertiary structures of protein modules, termed 3D keynotes . ApplicaThe mean and median lengths of predicted ORFs were calculated for the 19,574 H-Inv proteins. These were 1,095 bp and 806 bp, respectively . The valhttp://biobase.dk/embossdocs/cai.html) of H-Inv proteins were investigated (Of the 4,104 Category II proteins, 3,948 proteins (96.2%) were similar to the functionally identified proteins of mammals . This imstigated . The resEach H-Inv protein in the five categories was investigated in relation to the tissue library of origin . We founOver recent years, ncRNAs have been found to play key roles in a variety of biological processes in addition to their well-known function in protein synthesis . AnalysiTo identify ncRNAs, we manually annotated 1,377 representative non-protein-coding transcripts, which were classified into four categories see : putativWe defined a manual annotation strategy  that allArabidopsis thaliana, and Escherichia coli  numbers to 1,892 of the 19,574 H-Inv proteins based on matches to the InterPro entries and GO assignments and on the similarity to well-characterized Swiss-Prot proteins see . The num14C Histidine tracer study unexpectedly revealed that NEUT2 mice deficient in 10-formyltetrahydrofolate dehydrogenase (FTHFD) excrete urocanic acid in the urine and lack urocanase activity in their hepatic cytosol    were useild 117) . The denG  and T (termination) being 0.43 and 0.57, respectively. As seen in this example, the identification of SNPs within cDNAs provides important insights into the potential diversity of the human transcriptome. Thus, polymorphism data crossreferenced to a comprehensively annotated human transcriptome might prove to be a valuable tool in the hands of researchers investigating genetic diseases.SNPs located in ORFs were classified as either synonymous, nonsynonymous, or nonsense substitutions . We idenAmong the 19,442 representative protein-coding cDNAs, we identified a total of 2,934 di-, tri-, tetra-, and penta-nucleotide microsatellite repeat motifs . Interes7, (GAG)9, (GAG)6, and (GCC)6, in its 5′ UTR. These microsatellites are all located at least 98 bp upstream of the start codon, but they could still have pronounced regulatory effects on gene expression. Another example is the cDNA that encodes CAGH3 (AB058719). This cDNA has four microsatellites, (CAG)8, (CAG)6, (CAG)8, and (CAG)8, all of which are located within the ORF. These microsatellites all encode stretches of poly-glutamine, which are known to have transcription factor activity (There were 382 cDNAs that possessed two or more microsatellites in their nucleotide sequences. This is illustrated in RBMS1 (BC018951), a cDNA which encodes an RNA-binding motif. This cDNA has four microsatellites, (GGA)activity  and ofteactivity .We also searched for repeat motifs containing the same amino acid residue in the encoded protein sequences. We located a total of 3,869 separate positions where the same amino acid was repeated at least five times. The most frequent repetitive amino acids are glutamic acid, proline, serine, alanine, leucine, and glycine. The glutamine repeats of this nature were found in 160 different locations.Beyond the study of individual genes, the comparison of numerous complete genome sequences facilitates the elucidation of evolutionary processes of whole gene sets. Moreover, the FLcDNA datasets of humans and mice give us an opportunity to investigate the genome-wide evolution of these two mammals by using the sequences supported by physical clones. Here we compared our human cDNA sequences with all proteins available in the public databases. Focusing on our results, we discuss when and how the human proteome may have been established during evolution. Furthermore, the evolution of UTRs is examined through comparisons with cDNAs from both primates and rodents.E <10−10) and weak (E < 10−5) similarity levels  and as a result may be biased, animal species may conceivably share a similar protein-coding gene set.We conducted further BLASTP searches matching entries from the Swiss-Prot database against the H-Inv dataset itself. As a result, 12,813 (45.3%) of 28,263 vertebrate proteins had homologs in nonvertebrates at A unique feature of the Animalia proteome is, for example, the presence of apoptosis regulator homologs, which are found widely in the animal kingdom, whilst they are rare in the other phyla . Since aThe UTRs of mRNA are known to be involved in the regulation of gene expression at the posttranscriptional level through control of translation efficiency , mRNA stA replacement of the entire UTR may lead to drastic changes in gene expression, especially if a UTR having a posttranscriptional signal is replaced by another. We compared the evolutionary distances of UTRs between primate and rodent orthologous sequences. We based our analysis on the UTR sequence distances that contradicted the expected phylogenetic tree of relatedness. We could detect 149 UTR replacements distributed among different species. Some of the observed replacements may result from selection of different AS isoforms of a single locus in different species. This is particularly likely if an AS event involves an alternative first or last exon. It seems that UTR replacements are more frequent in rodents than in primates, but the difference is not statistically significant at the 5% significance level . We deteAll the results of the mapping of the FLcDNA sequences onto the human genome, the clustering of FLcDNA sequences, sequence alignments, detection of AS transcripts, sequence similarity searches, functional annotation, protein structure prediction, subcellular localization prediction, SNP mapping, and evolutionary analysis, as well as the basic features of FLcDNA sequences, are stored in the H-InvDB . The H-IWe constructed two kinds of specialized subdatabases within the H-InvDB. The first is the Human Anatomic Gene Expression Library (H-Angel), a database of expression patterns that we constructed to obtain a broad outline of the expression patterns of human genes. We collected gene expression data from normal and diseased adult human tissues. The results were generated using three methods on seven different platforms. These included iAFLP , DNA arrhttp://www.ncbi.nlm.nih.gov/LocusLink/) and OMIM (http://www.ncbi.nlm.nih.gov/Omim/, ver. Jan. 2003), with an additional 108 identified from GenAtlas (http://www.dsi.univ-paris5.fr/genatlas/, ver. Jan. 2003). Using the OMIM and GenAtlas databases in conjunction with the annotation results from the H-InvDB may accelerate the process of identifying candidate genes for human genetic diseases.The second subdatabase of the H-InvDB is DiseaseInfo Viewer. This is a database of known and orphan genetic diseases. We tried to relate H-Inv loci with disease information in two ways. Firstly, 613 H-Inv loci that correspond with known, characterized disease-related genes were identified by creating links to entries in both LocusLink  The 41,118 H-Inv cDNAs were found to cluster into 21,037 human gene candidates. Comparison with known and previously predicted human gene sets revealed that these 21,037 hypothesized gene clusters contain 5,155 new gene candidates.(2) The primary structure of 21,037 human gene candidates was precisely described. For the majority of them we observed that both first introns and last exons tended to be longer than the other introns and exons, respectively, implying the possible existence of intriguing mechanisms of transcriptional control in first introns.(3) We discovered the existence of 847 human gene candidates that could not be convincingly mapped to the human genome. This result suggested that up to 3.7%–4.0% of the human genome sequences (NCBI build 34 assembly) may be incomplete, containing either unsequenced regions or regions where sequence assembly has been performed in error.(4) Based on H-Inv cDNAs, we were able to define an experimentally validated AS dataset. The dataset was composed of 3,181 loci that encoded a total of 8,553 AS isoforms. In the 55% of ORFs containing AS isoforms, the pattern of alternative exon usage was found to encode different functional domains at the same loci.(5) A standardized method of human curation for the H-Inv cDNAs was created under the tacit consensus of international collaborations. Using this method, we classified 19,574 H-Inv proteins into five categories based on sequence similarity and structural information. We were able to assign functional definitions to 9,139 proteins, to locate function- or family-defining InterPro domains in 2,503 further proteins, and to identify 7,800 transcripts as good candidates for hypothetical proteins.(6) A total of 1,892 H-Inv proteins were assigned identities as one of 656 different EC-numbered enzymes. This enzyme library includes 32 newly identified human enzymes on known metabolic pathway maps and comprises the largest collection of computationally validated human enzymes.(7) Based on a variety of supporting evidence, 6.5% of H-Inv loci (1,377 loci) do not have a good protein-coding ORF, of which 296 loci are strong candidates for ncRNA genes.(8) We identified and mapped 72,027 SNPs and indels to unique positions on 16,861 loci. Of these, 13,215 nonsynonymous SNPs, 358 nonsense SNPs, and 452 indels were found in coding regions and may alter protein sequences, cause phenotypic effects, or be associated with disease. In addition, we identified 216 polymorphic microsatellite repeats on 213 loci, 25 of which were located in coding regions.(9) During human proteome analysis, it was suggested that the basic gene set of humans might have been established in the early stage of animal evolution. Our analysis of UTRs revealed that insertions or deletions near coding regions were rare when compared with substitutions, though in some cases drastic changes such as UTR replacements occurred.(10) A consequence of the annotation process and our related research was the development of the H-InvDB to contain our annotation work. H-InvDB is a comprehensive database of human FLcDNA annotations that stores all information produced in this project. As a subdivision of H-InvDB, we developed two other specialized subdatabases: H-Angel and DiseaseInfo Viewer. H-Angel is a database of gene expression patterns for 19,276 loci. DiseaseInfo Viewer is a database of known disease-related genes and loci co-localized with 694 orphan pathologies. These pathologies were mapped onto the genome but were not identified experimentally.In the H-Inv project, we collected as many FLcDNAs as possible and conducted extensive analyses concerning the quality of cDNAs, such as detection of frameshift errors, retained introns, and internal poly-A priming, under a unified criterion. Although these analyses are still in an elementary state, we store these results in H-InvDB to share this information with the biological community. We believe that this is an important contribution of our project, because it will provide a reliable way to control the quality of the cDNA clones. In the future, this information will be useful for improving the methods of clone library construction.cis-regulatory element units may help to define the boundary of other genes that are undetected by current gene prediction programs. Another area that remains to be explored is the identification of potential hidden RNA gene families that may play vital roles, such as the recently uncovered family of microRNA genes, which is involved in the regulation of expression of other genes . Thus, efforts should be continued by the H-Inv consortium and others to “fully” characterize the human transcriptome. For this purpose new technologies should be implemented that are more sensitive in detecting rarely expressed genes and AS transcripts. Nevertheless, there are unavoidable limitations for human cDNA collections, such the identification of embryo-specific genes, for which other approaches should be employed. One alternative is the use of ab initio predictions from genomic sequences, in conjunction with expression profiling studies, to identify rarely expressed genes that share structural similarity to known genes. Additionally, a better characterization of The proteome determination aspects of this project, including the identification of new enzymes and hypothetical proteins, should stimulate more focused biochemical studies. The functional classifications may allow definition of subproteomes that are related to different physiological processes. The H-Inv transcriptome based on the definition of a consensus proteome (the H-Inv proteins) links both the analysis of genomic DNA and direct proteome analysis with the study of expressed mRNA analysis from different tissues, cells, and disease states. It creates a standard for the comparison of disease-related alterations of the human proteome. Moreover, comparison with pathogen proteomes may yield many possible drug target proteins. Also, the annotation of ncRNAs raises the possibility of novel “smart” therapeutics that could either inhibit or mimic the mechanisms of these RNAs.The H-Inv project is the first ever comprehensive compilation of curated and annotated human FLcDNAs. The project may lead to a more complete understanding of the human transcriptome and, as a result, of the human proteome. The preceding examples of the importance of the H-Inv data in understanding human physiology and evolution represent just a small fraction of the research potential of the H-InvDB.In conclusion, the H-InvDB platform constructed to hold the results of the comprehensive annotations performed by our international team of collaborators represents a substantial contribution to resources that are needed for further exploration of both human biology and pathology.41,118 H-Inv cDNAs were sequenced by the Human Full-Length cDNA Sequencing Project  at the Hhttp://ftp.genome.washington.edu/RM/RepeatMasker.html) and Repbase 7.5. Then we used the cross_match program to mask the remaining vector sequences in each cDNA sequence. Any poly-A tails were also masked by using a custom-made Perl script. In the first step of the mapping procedure, we conducted BLASTN (ver.2.2.6) searches of all the sequences against the human genome sequence and extracted the corresponding genomic regions for each query sequence. Then we used est2genome (EMBOSS package ver.2.7.1) to align each sequence to the genomic region with a threshold of 95% identity and 90% coverage. Coverage of each cDNA sequence was calculated excluding those from the vector and poly-A tails that were masked in the previous step. If the sequences were mapped to multiple positions on the human genome, then we selected their best locus based on the identity, length coverage, and number of exons of those sequences. As a result, 77,315 sequences (including 40,140 cDNAs from the H-Inv project) were successfully mapped onto the human genome and were clustered into 38,587 clusters based on sharing at least 1 bp of an exon on the same chromosome strand. We used all the mapped sequences, including human RefSeq sequences, to compare the clusters that included H-Inv cDNAs with those that consisted of only human RefSeq sequences. 20,190 clusters out of 38,587 consisted of only H-Inv cDNAs or both H-Inv cDNAs and human RefSeq sequences. The rest of the clusters consisted of RefSeq sequences only. All of the mapped cDNAs and the overlap with the RefSeq sequences can be viewed using G-integra in the H-InvDB (http://www.jbirc.aist.go.jp/hinv/g-integra/html/). The mapping procedure for all the unmapped cDNAs against the mouse genome was also performed, using a threshold of 60% identity and 90% coverage.We have mapped human cDNA sequences to the human genome sequence corresponding to the NCBI build 34 assembly. The datasets we used were a set of 41,118 H-Inv cDNAs and a set of 37,488 human RefSeq sequences available on 15 July 2002 and on the 1 September 2003, respectively. All the revisions for H-Inv cDNA sequences until August 2003 were applied in the datasets. Before performing the mapping procedure, all the repetitive and low-complexity sequences in all the cDNA sequences were masked using RepeatMasker  between two sequences. Only when the E-value of the two sequences was calculated to be 0, did we assume that a significant level of similarity was detected between the two sequences.The sequences that were not mapped onto the human genome were clustered by a single linkage clustering method. The similarity search was performed among all the unmapped sequences. The program used was MegaBLAST version 2.2.6 . As withIn order to identify gene structure, we used only the representative H-Inv cDNAs. When detecting repetitive elements in cDNAs, RepeatMasker was conducted in a similar manner to the previous phase. We used curated cDNAs in which frameshift errors and remaining introns were removed.http://opal.biology.gatech.edu/GeneMark/)  . Prior tneMark/) . During Prior to the human curation, we performed two computational automated annotation processes to select the representative clone for each locus and to predict function of H-Inv proteins see . We thenhttp://flybase.bio.indiana.edu/), worm (http://www.wormbase.org/), budding yeast (http://www.pasteur.fr/externe), fission yeast (http://www.sanger.ac.uk/), plant , and a bacteria (ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Escherichia_coli_K12/). The H-Inv proteins and other nonredundant proteome datasets were assigned InterPro codes by InterProScan ver. 3.1  and two rodents  that corresponded to UTRs from Homo sapiens. In order to do this, we mapped the cDNAs to the human or mouse genome. The corresponding rodent cDNAs were determined by using a human–mouse genome alignment provided by Ensembl. cDNAs of the primates and rodents were retrieved from the DDBJ/EMBL/GenBank databases using the cut off date of 15 July 2002. Additionally, we used the FANTOM2 mouse sequences released on 5 December 2002, and 4,063 5′ ESTs of chimpanzees (We obtained the UTR sequences from three primates (mpanzees . CorrespDataset S1The dataset consists of 41,118 H-Inv cDNAs that were cloned from cDNA libraries derived from 182 varieties of cell and tissue.(33 KB XLS).Click here for additional data file.Dataset S2The allotted EC numbers are based on the corresponding DNA databank records, UniProt/Swiss-Prot and TrEMBL records that show sequence similarity to the proteins, and InterPro records that the proteins hit.(247 KB XLS).Click here for additional data file.Dataset S3(56 KB XLS).Click here for additional data file.Figure S1(A) Schematic diagram for the prediction of ORFs. This diagram illustrates the ORF prediction method used on all H-Inv cDNAs. The method was based upon the alignment of similarity searches using FASTY and BLASTX. Gene prediction was carried out using GeneMark. Prior to the prediction of ORFs, we judged if a sequence had any frameshift errors or remaining introns. During ORF prediction, we corrected those sequence irregularities computationally. Details of how sequence irregularities were predicted are described in (B) and (C).(B) Schematic diagram for prediction of unspliced introns. This schematic diagram illustrates the prediction method used for unspliced introns.(C) Schematic diagram for prediction of frameshift errors. Frameshift errors were inferred from cDNA–genome pairwise alignment gaps due to insertion or deletion, exception of multiple of 3 bp, or over 10 bp in either the query cDNA or genome.(D) The statistics for the predicted frameshifts and unspliced introns.(49 KB PDF).Click here for additional data file.Figure S2(A) Schematic diagram for determining a representative transcript for each locus. The procedure of computational autoannotation is illustrated. Prior to the human curation of the representative transcript of each H-Inv cluster, we performed computational autoannotation.(B) Schematic diagram for functional prediction of H-Inv proteins. This schematic diagram illustrates the H-Inv autofunctional annotation pipeline that can determine the most appropriate data source ID, avoiding the following keywords that suggest proteins without experimental verification in the description; (1) hypothetical, (2) similar to, (3) names of cDNA clones (Rik, KIAA, FLJ, DKFZ, HSPC, MGC, CHGC, and IMAGE) and (4) names of InterPro domain frequent hitters.(34 KB PDF).Click here for additional data file.Figure S3The size distribution of all H-Inv proteins among the five similarity categories.(24 KB PDF).Click here for additional data file.Figure S4A total of 4,104 H-Inv proteins were classified as Category II based on sequence similarity to functionally validated proteins. The table and figure show source species of proteins in public databases to which the Category II proteins were similar.(9 KB PDF).Click here for additional data file.Figure S5The images illustrate the metabolic pathways of KEGG database based on the EC number assignments to H-Inv proteins.(47 KB PDF).Click here for additional data file.Figure S6−5, white bars, and E < 10−10, black bars) were employed. The “animal” group does not include mammalian species. The “eukaryote” group represents eukaryotic species other than animals, fungi, and plants.Two thresholds (E < 10(9 KB PDF).Click here for additional data file.Figure S7H-Inv protein families were identified by clustering H-Inv proteins using the single-linkage clustering method. Then, the number of homologs for each H-Inv protein family was calculated. Mammalian species are excluded from the “animal” group. “eukaryote” represents eukaryotic species other than animals, fungi, and plants.Single-linkage clustering. All of the H-Inv proteins were compared with themselves by BLASTP and clustered with the thresholds of E-values of 10−30 and 10−50. The numbers of singleton families detected were 11,890 and 13,938 at the E-value of 10−30 and 10−50, respectively.(49 KB PDF).Click here for additional data file.Figure S8A FLcDNA (BC003551) is shown with its detailed annotations, e.g., gene structure, functional annotation, ORF predictions, protein structure prediction by GTOP, etc. The H-InvDB has links to other internal databases (red boxes) such as a genome map viewer (G-integra) and gene expression library (H-Angel). Green boxes show internal viewers for the results of clustering (Clustering Viewer showing results by H-Inv, STACK, TIGR, UniGene, etc.), the prediction of subcellular localization (TOPOViewer showing results of TMHMM, SOSUI, TargetP, and PsortII), and the disease-related information (DiseaseInfo Viewer linking to OMIM and GenAtlas). The H-InvDB also has links to many external public databases (black boxes), including DDBJ/EMBL/GenBank, RefSeq, UniProt/Swiss-Prot and TrEMBL, Genew, InterPro, 3D Keynote, Ensembl, GeneLynx, LocusLink, PubMed, LIFEdb, dbSNP, GO, and GTOP, and to homepages by original data producers of FLcDNA clones and sequences (blue boxes), including the Chinese National Human Genome Center (CHGC), the Deutsches Krebsforschungszentrum (DKFZ/MIPS), Helix Research Institute (HRI), the Institute of Medical Science at the University of Tokyo (IMSUT), the Kazusa DNA Research Institute (KDRI), the Mammalian Gene Collection (MGC/NIH), and the FLJ project.(2,650 KB PDF).Click here for additional data file.Figure S9(A) G-integra: A genome mapping viewer.(B) SOUP Locus annotation viewer.(C) SOUP cDNA annotation viewer.(D) SMO Viewer: The similarity, motif, and ORF information viewer.(2,022 KB PDF).Click here for additional data file.Table S1(A) Gene structure of the cDNAs.(B) The frequencies and varieties of repetitive sequences found in the cDNAs. A list of the 20,899 loci representing cDNAs that RepeatMasker showed contained repetitive elements.(C) The positions (5′ UTR, ORF, and 3′ UTR) of repetitive sequences in the protein-coding cDNAs. A total of 1,863 cDNAs contained repetitive sequences in their ORF, of which 549 had repetitive sequences within their most probable ORF. Repetitive sequences appeared in 2,240 and 5,401 cDNAs in their 5′ UTRs and 3′ UTRs, respectively.(20 KB PDF).Click here for additional data file.Table S2http://biobase.dk/embossdocs/cai.html).(A) CAI was measured for all H-Inv proteins. CAI is a measure of biased patterns for synonymous codon usage , worm , budding yeast , fission yeast , plant , and bacteria .(B) Codon usage in predicted ORFs of H-Inv proteins. Total tri-nucleotide frequencies (forward strand) for the sequences of each species are shown. Nonredundant proteome datasets for nonhuman species were obtained from the following sites: fly ((20 KB PDF).Click here for additional data file.Table S3The results of classification into five similarity categories for each of ten tissue classes.(A) Numbers of H-Inv proteins.(B) Histogram.(10 KB PDF).Click here for additional data file.Table S4Drosophila melanogaster; http://flybase.bio.indiana.edu/), worm , budding yeast , fission yeast , plant , and bacteria .The top 40 InterPro IDs identified in H-Inv proteins and proteins from other species are listed for all types (A) and for each type of family, domain, and repeat (B–D). Analyses were conducted by InterPro ver. 3.1. Nonredundant proteome datasets of other species were obtained from the following sites: fly ((36 KB PDF).Click here for additional data file.Table S5(A) Molecular function.(B) Cellular component.(C) Biological process.(74 KB PDF).Click here for additional data file.Table S6Homo sapiens.All these 32 H-Inv proteins were newly assigned enzyme numbers with the support of the KEGG pathway. These enzyme assignments were previously unrepresented in (33 KB PDF).Click here for additional data file.Table S7See also .(9 KB PDF).Click here for additional data file.Table S8(8 KB PDF).Click here for additional data file.Table S9One hundred and forty-seven UTR replacements distributed among different species were detected.(9 KB PDF).Click here for additional data file.Table S10(31 KB PDF).Click here for additional data file.Protocol S1(25 KB PDF).Click here for additional data file.


Strong regional heterogeneity and generally sub-optimal rates of measles vaccination in Italy have, to date, hampered attainment of WHO targets for measles elimination, and have generated the need for the new Italian National Measles Elimination Plan. Crucial to success of the plan is the identification of intervention priorities based upon a clear picture of the regional epidemiology of measles derived from the use of data to estimate basic parameters. Previous estimates of measles force of infection for Italy have appeared anomalously low. It has been argued elsewhere that this results from Italian selective under-reporting by age of cases and that the true measles force of infection in Italy is probably similar to that of other European countries. A deeper examination of the evidence for this conjecture is undertaken in the present paper.R0) and critical vaccination coverage (pc)) are calculated for each region and for each of 5 plausible contact patterns. An analysis of the spectra of incidence profiles is also carried out. Finally a transmission dynamics model is employed to explore the correspondence between projections using different estimates of force of infection and data on seroprevalence in Italy.Using monthly regional case notifications data from 1949 to the start of vaccination in 1976 and notifications by age from 1971–76, summary equilibrium parameters (force of infection (FOI), basic reproductive ratio (FOI estimates are lower than comparable European FOIs and there is substantial regional heterogeneity in basic reproductive ratios; certain patterns of contact matrices are demonstrated to be unfeasible. Most regions show evidence of 3-year epidemic cycles or longer, and compared with England & Wales there appears to be little synchronisation between regions. Modelling results suggest that the lower FOI estimated from corrected aggregate national data matches serological data more closely than that estimated from typical European data.Results suggest forces of infection in Italy, though everywhere remaining below the typical European level, are historically higher in the South where currently vaccination coverage is lowest. There appears to be little evidence to support the suggestion that a higher true force of infection is masked by age bias in reporting. The WHO target of eliminating indigenous measles in Europe by 2007 represents a challenge for public health systems. The requirements for success in this battle are summarised by Gay . Italy, In designing an optimal elimination strategy, it is crucial that planners have at their disposal a clear picture of the regional "geography" of the intensity of effort required for measles elimination as a preliminary step for ranking intervention priorities. This is especially in view of the claim that there could be spatial heterogeneity in transmission rates due to the large socio-economical differences existing in the country . HoweverR0, the mean number of secondary infections which would arise from the introduction of a primary infection in a wholly susceptible population). As primarily shown by Anderson & May [et al. [Here we deal with this pre-vaccination epidemiology of measles in Italy, taking inspiration from two distinct standpoints. The first is a very practical one, i.e. the need to summarise the degree of effort needed for measles elimination in the Italian regions. This is carried out using the fundamental parameters of the basic SEIR (i.e. Susceptible-Exposed-Infected-Recovered) transmission dynamics mathematical model of vaccine preventable diseases: forces on & May -11, prov [et al.  for the et al. [et al.[et al. [et al. [et al.[Edmunds et al.  have sho. [et al. - the so. [et al. - here r.[et al.  for scho [et al.  paper pr. [et al. that i) et al. [et al. [First we have systematically looked at the structure of the FOIs (and contact patterns, basic reproduction numbers, etc) for all the Italian Regions and Provinces. Since the national datum used by Edmunds et al.  to compu [et al. ), a deepSecond, we carried out a systematic time series analysis of measles periodicities in the Italian regions during the pre-vaccination era Fig  &2. Our Third, we add some results from our modelling work on measles in Italy.The results suggest that i) forces of infection estimated from case reports of measles in all the Italian regions are systematically and significantly lower compared with the EURO FOI with little, or only moderate, between regions heterogeneity; ii) regional periodicities mostly suggest a longer  inter-epidemic period compared to England & Wales, a fact which is consistent with lower transmission (and which, together with the lower FOIs, suggests lower vaccination coverage might suffice to achieve control and elimination); iii) predictions from a mathematical model using the EURO force of infection, even compared with those based on a FOI estimated from Italian case reports, poorly match the 1996/1997 Italian serological data .The geographic analysis of the epidemiology of vaccine preventable diseases is made difficult by complex correlations between local dynamics. A first, though clearly partial, step is characterization of local infection patterns by treating spatial sub-areas as autonomous epidemiological units. Two measures are used here to characterise the regional  "landscape" of measles in Italy in the pre-vaccination era: summary parameters from the SEIR model for vaccine preventable disease, and summary periodicities from time series analyses of measles incidence.R0; and iv) related critical vaccination coverages, pc, the proportion of a population needed to be successfully immunised at age zero with a 100% effective vaccine in order to eliminate the infection, where pc = 1-1/R0  = R0 (1- [1-1/R0]) = 1.0 contacts) [From the pre-vaccination age distribution of cases we computed, for each region, summary equilibrium parameters from the SEIR model: i) forces of infection, FOI; ii) mixing or contact ("who acquires infection from whom", WAIFW) matrices; iii) basic reproduction numbers, ontacts) .et al. [For comparison purposes we used the same age groups as in Edmunds et al. , i.e. 0–et al.  were comβij summarises the risk of acquiring infection for a susceptible individual in age group i due to contacts with infective individuals aged j. When estimating from age-structured data (e.g. serology or case reports), a mixing matrix with m age groups can have at most m distinct elements [βij entries are independent of age . Other types of mixing matrix are considered in the paper and are listed below. However not all forms of mixing are compatible with a given force of infection. As noted by Anderson & May [non feasible for the given FOI  and in such circumstances "the chosen matrix is inappropriate to the observed age dependence in the FOI". Hethcote [Mixing matrices Table  are usedelements . In the on & May , a mixinHethcote  further R0 to achieve its upper bound [Individuals of a given age are assumed to mix only with individuals of the same age, yielding a diagonal matrix. Given a specific FOI it is the form of mixing allowing er bound .et al. [β5) as that between adults [This matrix  has then adults .This matrix emphasises transmission between school age groups .βij = bibj j = 1,...m.Under PM  contactsh) weighted average of proportionate (PM) and fully assortative (DIAG) mixing: PREF = (1 - h) * DIAG + h* PM, 0<h<1 [PREF matrix has (m+1) distinct entries: the extra parameter h is usually estimated "ad hoc".The PREF matrix is a single parameter , and hence PREF is a weighted average between two limit cases. There is thus no clear relationship between, for instance, RDIAG & DEF on the one hand and PREF on the other. For this reason, we explored all forms that have been reported in the paper.The matrices RDIAG & DEF are defined "In contrast to the spatially well synchronised biennial England & Wales oscillation, visual inspection of Italian regional data did not suggest clear common patterns of oscillation or hence of the inter-epidemic period. Thus investigation of periodicities in regional measles incidence became necessary to provide satisfactory characterisation of the inter-epidemic period. Though incidence data might be seriously affected by under-reporting, recent work  suggestset al. [The cyclical behaviour in the monthly incidence time series of measles for each Italian region in the pre-vaccination period 1949–1976 was analysed in the frequency domain  with sp2π/T (T = series length); this is the basis of "harmonic" analysis. Such analysis may not identify exactly cyclical components where true frequency falls between two "harmonic" frequencies, e.g. it may suggest periodicity of either 3 or 4 observations when true periodicity lies between these values and hence is not identifiable exactly with this procedure. This problem may be overcome with "non-harmonic frequency domain" analysis where dependence of periodicities of cyclical components on series length is relaxed. Here the following procedure, as proposed in [λ, the quantityA drawback of periodogram analysis is the assumption that cyclical components of frequencies depend on the length of the observed series, i.e. that they are integer multiples of posed in , is usedposed in , by minixt denotes the detrended series .where K of the expected duration of the latent and infectious states is short compared to the life of the host (as is the case with measles), disease incidence will have a long-term oscillation around its endemic equilibrium with the period given, to an excellent approximation, by [A is the average age at infection and K can be taken to be 14 days in the case of measles.The results from the time series analysis were also compared with the prediction from the homogeneous mixing SEIR model that where the sum tion, by , where AAnalyses described above were carried out for all Italian regions .In addition to published official Italian data on births, and birth and death rates, monthly measles case reports were provided by the Istituto Nazionale di Statistica (ISTAT) for the period from the first available year, 1949, to 1976, together with regional age structured measles case reports from the first available year, 1971. Pre-vaccination FOIs at the regional level were estimated from data for the time window 1971–76, which encompassed about two full three-years long epidemic cycles .The spectral densities of the pre-vaccination (1949–76) Italian regional time series of measles incidence indicate, besides a well-pronounced annual cycle, the presence of a less pronounced longer term cycle of varying length, which is in contrast with the sharp biennial oscillation in England & Wales. Given the importance of the long term oscillation which is taken as representing the true "inter-epidemic period", the non-harmonic estimator was calculated .A); ii) the non-harmonic estimate TO of the period of the long-term oscillation; iii) the length of the inter-epidemic period from the homogeneous SEIR model via formula for T given in the Methods section.Table TO in table T predicted by the SEIR model range from a minimum of 2.8 (Puglia) to 3.3 years (Tuscany). The agreement between T and TO in some cases is not very good: North-Eastern regions show a higher average age at infection, A, compared to Southern regions and yet the shorter inter-epidemic periods in the former compared with the latter, as suggested by the time series analysis, would imply the reverse.Values of Compared to England & Wales there is also a surprising lack of synchronisation between regional cycles in the pre-vaccination period. A cross-spectral analysis suggests limited correlation between pre-vaccination long term cycles; coherencies greater than 0.7 were observed, as expected, only for a few neighbouring regions such as Emilia-Romagna and Marche, Marche and Umbria, Friuli and Trentino, Lombardia and Piemonte, Liguria and PiemontePre-vaccination age-distributed forces of infections in the Italian regions (we report a sample of results) can be well summarised by 3 clusters, North, Centre, South. These are shown in Fig. A , that coarsely summarises the force of infection, shows similar spatial patterns. For instance A is systematically smaller in the South (little above 5 years), around 7 years in the Centre, and takes intermediate values in the North  is, with the exception of Southern regions, higher than in the "young" (0–1 yr.). A verifiable consequence of this fact, is that "realistic assortative" mixing matrices (RDIAG) can be non-feasible (they can yield negative coefficients), and this indeed happens in all Central and several Northern regions. In other words, such mixing is not compatible, Southern Regions apart, with observed forces of infection. This may be a problem because "realistic assortative" is the mixing pattern that provides under a EURO-type FOI, the upper bound of "plausible" values of R0  and 8.5–12 in the Centre , with intermediate values in the North.This mixing yields an der EURO . Thanks R0 and critical coverages happen to be systematically higher. Critical coverages range from 80% in the North up to 88% in the South .It happens that this mixing pattern is never admissible for all Central Regions and for some Northern ones, as it can yield negative transmission rates when, as is the case in Central Italy, the FOI is rather low in the youngest age groups. Default mixing is, on the contrary, admissible in all Southern regions where R0. Predicted critical coverages ranged from 72% in the Centre, with intermediate values in the North, up to 83% in the South  are the type of mixing ,17 yieldh starting from h = 1 (proportionate mixing) and then [h until "minimally plausible" contact rates were achieved. Essentially, for all Italian regions , use of a moderate degree (h = 0.9) of assortativeness allowed the "minimal plausibility" threshold to be surpassed. This led, compared to the case of proportionate mixing, to increases of 1% to 3% in the corresponding critical coverages  may be needed to describe Italian pre-vaccination infection patterns.An issue is the degree of assortativeness, Hethcote ). AlthouThis also leads to the nub of the real gain provided by preferred matrices (just because they are "many") compared to "ad hoc" behavioural matrices. One could ask whether Hethcote's  preferenOur analysis of the pre-vaccination force of infection of measles in Italy formed a step toward developing a mathematical model for the transmission dynamics of measles in Italy. The model has the following features:et al. [i) it provides bounds for the uncertainty surrounding the estimate of the FOI Table , by takiet al. ;ii) it incorporates "realistic" demography in order to mirror the broad process of population ageing observed in Italy in the past 20 years (and which would be observed in the future if the present state should continue) as a consequence of the onset of sustained low fertility;iii) it considers two distinct options as regards interaction between the demography and the epidemiology: the first (D1) assumes no effects on transmission arise as a consequence of population ageing; the second (D2) assumes low fertility and population ageing could lead to a significant decline in transmission (e.g. a decline in the FOI), via proxy mechanisms such as a substantial decline in intra-family transmission which might occur as a consequence of contraction in average family size.By crossing the two FOI assumptions, EURO and UR , with the two assumptions D1, D2 we obtain four assumptions: EURO/D1, EURO/D2, UR/D1, UR/D2. The model simulations were carried out by adopting for the post-vaccination era what we considered the "best" approximation for the profile of routine vaccination coverages Fig. , obtaineFig. An attempt has been made to characterise the measles pre-vaccination epidemiological "landscape" in Italy, and also with reference to what we have referred to as the "EURO conjecture" (i.e. that age-selective under-reporting disguises underlying homology with FOIs elsewhere in Europe). There are points open to criticism in this analysis: first, the fact that by looking at "local" forces of infection, e.g. treating spatial sub-areas as autonomous epidemiological units, we disregard spatial correlations between the various local dynamics; this makes the present analysis, at best, a first step. Second, the present work relies only on case reports, the only available pre-vaccination data. Third, the increasing temporal distance between the present time and the pre-vaccination era (ending in 1976) seems to make more and more heroic the assumption that mixing patterns remain unchanged since then. Nevertheless several interesting facts emerge.Clearly, good data on immunisation coverage and reports of outbreaks of infection would provide some broad picture of patterns of susceptibility. However, as the level of vaccine-based immunity increases, the mean interval between outbreaks also increases so that case reports, even if unbiased, become much less useful as a source of timely information on patterns of susceptiblity. Also in Italy, historically, data on immunisation coverage has been very poor or in many instances completely absent; in many regions case reporting has also been very poor, forming an insensitive tool for detecting small pockets of infection, and large outbreaks, almost by definition, cannot be timely indicators of the presence of susceptibles. Hence the first motivation for the paper was to construct a map of the efforts needed for elimination of measles in Italy, which could then constitute a useful tool for prioritising regional intervention priorities in the context of the agreed WHO target for measles elimination and with the aim of avoiding such outbreaks. During the second half of the 1990's several attempts have been made in Italy to increase routine coverages, which the available data suggested had remained until then disappointingly low . Even though surveillance data suggests incidence has reached a historic minimum during 1999–2001, national coverage was still well below 70% in 2000, with much regional heterogeneity. It is unsurprising therefore that there have been substantial epidemics causing serious concern, especially in Campania (the region with the lowest coverages) during the spring of 2002, with an estimated 20,000 cases between January and May 2002 .From this standpoint the results here (i.e. basic reproductive ratios etc) suggest that the regions which seem to be the most demanding in terms of effort needed to eradicate the infection are systematically the Southern ones. This is rather problematic, as the Southern regions are those presently characterised by the lowest vaccination coverages ,3. The aThe "EURO conjecture" suggests that the measles FOI in Italy is, in fact, homologous to that observed in other European countries but that, in the case notifications data, this fact is camouflaged by a strong under reporting bias with age.Under-reporting of childhood diseases has been a major problem in Italy. It is known ,12 that et al [We therefore made an extensive investigation of forces of infection at the spatial level . Our feeling was that by going more deeply spatially it should have been possible to find some "footprint" of the presence of the presumed underlying EURO pattern (e.g. areas with a "high" FOI, or with significant heterogeneity in FOIs). As documented in the Results, we have not been successful in detecting such a "footprint". All "local" forces of infection are systematically substantially smaller compared to the EURO FOI, and surprisingly similar to each other in levels and shape: significant heterogeneity exists only in the youngest age groups. This raises the point, if the EURO conjecture were to be true, of what might be the social processes, if any, underlying the poor age reporting of measles cases, which are capable of so effectively camouflaging the true underlying force of infection. Indeed, in order to appear so stable over space and time, such processes should be profoundly rooted in social responses of families toward diseases of their children and/or medical practitioners' behaviour. In the absence of the relevant data any discussion of the nature of such processes must remain in the realm of speculation, but clearly there is room for bias at many points in the chain leading to reporting of a case -22Fig. : perhapset al  in recena priori (one possibility could be that there is an association between practitioners' reporting rates and the phase of the epidemic cycle, e.g. an epidemic year vs a non-epidemic year), it appears rather unlikely in most circumstances. Indeed, if the "true" percentage age distribution of cases is broadly stationary over time, as we would expect in the pre-vaccination era , then significant (and periodic) time changes in age-specific reporting rates would be needed to effectively mask true incidence patterns.We then investigated the measles incidence time series, encouraged by the fact that  reportinFinally, our modelling work on measles in Italy, shows that the EURO assumption matches rather poorly the serological profile observed for measles in Italy in 1996/7 in the European Sero-epidemiology Network (ESEN) survey ,25.et al. [R0 values estimated for the South suggest that, even with achievement uniformly of WHO targets across the regions, elimination of measles will take longer than in the North.Which then was the true force of infection acting in Italy during the pre-vaccination era: the higher EURO FOI, or the lower FOI emerging from Italian case notifications data, or indeed perhaps something in between? Rather than being essentially homologous, as implicit in the EURO conjecture, the possibility that patterns of infection by measles throughout Europe could be and have been largely different  is a stThe results suggest that critical vaccination coverages for elimination are likely to be higher in the south of Italy, precisely where the existing record of coverage is lowest. Substantial efforts are still required if there is to be a realistic hope of achieving WHO targets for measles elimination in Italy, particularly in the south. Notwithstanding this, the evidence does appear to suggest that the force of infection for measles in Italy is indeed somewhat lower than that applicable to other regions participating in the European Sero-Epidemiology Network. More particularly, the evidence suggests that it is probably unlikely that age biases in reporting (suggested elsewhere) could have led to an underestimate of the measles force of infection in Italy.The author(s) declare that they have no competing interests.PM conceived the work, provided the main theoretical analysis and drafted the manuscript, EMC conducted the time series analysis, JRW undertook the modelling work and SS and MCdA contributed epidemiological insight into the Italian public health context of the work. All authors read and approved the final manuscript.


Yet oxygen supplementation remains first line treatment in cardiovascular disease. We hypothesized that hypoxia improves and hyperoxia worsens myocardial performance.Low O2, 79% N2), 2) while inhaling a hypoxic gas mixture (≈11% O2, 89% N2), and 3) while inhaling 100% O2. Tissue Doppler recordings were acquired in the apical 4-chamber, 2-chamber, and long-axis views. Strain rate and tissue tracking displacement analyses were carried out in each segment of the 16-segment left ventricular model and in the basal, middle and apical portions of the right ventricle.Seven male volunteers (mean age 38 ± 3 years) were examined with echocardiography at respiratory equilibrium during: 1) normoxia (≈21% OHeart rate increased with hypoxia (68 ± 4 bpm at normoxia vs. 79 ± 5 bpm, P < 0.001) and decreased with hyperoxia (59 ± 5 bpm, P < 0.001 vs. normoxia). Hypoxia increased strain rate in four left ventricular segments and global systolic contraction amplitude was increased (normoxia: 9.76 ± 0.41 vs hypoxia: 10.87 ± 0.42, P < 0.001). Tissue tracking displacement was reduced in the right ventricular segments and tricuspid regurgitation increased with hypoxia (7.5 ± 1.9 mmHg vs. 33.5 ± 1.8 mmHg, P < 0.001). The TEI index and E/E' did not change with hypoxia. Hyperoxia reduced strain rate in 10 left ventricular segments, global systolic contraction amplitude was decreased (8.83 ± 0.38, P < 0.001 vs. normoxia) while right ventricular function was unchanged. The spectral and tissue Doppler TEI indexes were significantly increased but E/E' did not change with hyperoxia.Hypoxia improves and hyperoxia worsens systolic myocardial performance in healthy male volunteers. Tissue Doppler measures of diastolic function are unaffected by hypoxia/hyperoxia which support that the changes in myocardial performance are secondary to changes in vascular tone. It remains to be settled whether oxygen therapy to patients with heart disease is a consistent rational treatment. Low oxygen tension dilates coronary arteries and high oxygen tension is a coronary vasoconstrictor. Yet oxygen supplementation remains first line treatment in cardiovascular disease states such as myocardial infarction and pulmonary oedema.Endothelium-dependent vasodilation is reduced in patients with ischemic heart disease  and suchHyperoxia, a condition in which the total oxygen content of the body is increased above that normally existing at sea level, is associated with impairment of cardiac relaxation and increased left ventricular filling pressures in patients with and without congestive heart failure .Tissue Doppler imaging (TDI) objectively derives measurements of contraction and relaxation velocities directly from the myocardium and thus yields information not previously accessible by echocardiography . We usedSeven healthy men, (age 25–46 (mean 38) years) completed the study. All subjects were normotensive, non-smokers, on no medication, had a normal left ventricular function by 2-D echocardiography, and had no family history of ischemic heart disease. The local ethical committee approved the study.We used a system consisting of a ventilator, a gas analyser with pulse oximeter, and a computer. Computer programs control the experimental procedure and continuously collect data from the ventilator and gas analyser .Echocardiography was performed from the apical acoustic window. Tissue Doppler recordings were acquired as digital loops in the apical 4-chamber, 2-chamber, and long-axis views . To avoi2 97.9 ± 0.1 %), after 5 minutes of respiratory equilibrium during hypoxia (SpO2 77.6 ± 1.2 %), and after 5 minutes of respiratory equilibrium during hyperoxia (SpO2 99.0 ± 0.2 %). Heart rate was continuously recorded on computer by means of the pulse oximeter. Blood pressure was measured once during each of the three respiratory steady state situations by an automatic blood pressure measuring device based on the oscillometric method.TDI was performed when the subjects had rested for 15 minutes breathing room air (SpOiO2 (normoxia, hypoxia and hyperoxia) were made with a one-way repeated-measures analysis of variance. The Student-Newman-Keuls test was used post hoc to identify pairwise differences. Differences were considered statistically significant when P < 0.05.All data are presented as mean ± SEM. Comparisons of the responses to changes in F2 and FeO2 decreased with hypoxia and increased with 100% oxygen breathing (table 1). FeCO2 and tidal volume were unchanged in all three test situations, reflecting that the subjects were in respiratory steady state. There was a small, but statistically significant decrease in respiratory rate from normoxia to hypoxia, which might reflect that the subjects were more accustomed to the test situation at this stage.FiOThere was a significant increase in heart rate with hypoxia and a decrease with hyperoxia figure . NeitherA significant increase in strain rate was found in 4 segments figure . GSCA inHyperoxia worsened left ventricular function. Strain rate was reduced in 10 segments figure  with preThe main findings of the present study of healthy volunteers are: 1) hypoxia increased strain rate and tissue tracking displacement. 2) Hypoxia increased tricuspid regurgitation and reduced right ventricular tissue tracking displacement. 3) Hyperoxia reduced strain rate and tissue tracking displacement and increased the TEI index. The novelty of our findings, when compared to the literature, is the demonstration that longitudinal myocardial function, and thus the function of the subendocardium, is sensitive to moderate changes in inspired oxygen.Patients with ischemic heart disease and heart failure frequently encounter hypoxia but the consequences of hypoxia on left ventricular function remain a matter of controversy, in part because of differences in methodology and the measured parameters. In early studies using roentgenkymograms  and dye Hemoglobin saturation in healthy persons increase very little from breathing room air to breathing 100% oxygen. Nevertheless, profound cardiovascular effects were found. This is probably because of an anticipated increase in oxygen dissolved into plasma from 0.32% to 2.09% . HyperoxBecause of the finding of no change  or a disIn conclusion, hypoxia improves and hyperoxia worsens systolic myocardial performance in healthy male volunteers. TDI measures of diastolic function are unaffected by hypoxia/hyperoxia which support that the changes in myocardial performance are secondary to changes in vascular tone. It remains to be settled whether oxygen therapy to patients with heart disease is a rational treatment that may sometimes be harmful or whether supplemental oxygen consistently results in an overall gain in delivered oxygen.