Virtual Reality (VR) provides a unique medium suited to the achievement of several requirements for effective rehabilitation intervention. Specifically, therapy can be provided within a functional, purposeful and motivating context. Many VR applications present opportunities for individuals to participate in experiences, which are engaging and rewarding. In addition to the value of the rehabilitation experience for the user, both therapists and users benefit from the ability to readily grade and document the therapeutic intervention using various systems. In VR, advanced technologies are used to produce simulated, interactive and multi-dimensional environments. Visual interfaces including desktop monitors and head-mounted displays (HMDs), haptic interfaces, and real-time motion tracking devices are used to create environments allowing users to interact with images and virtual objects in real-time through multiple sensory modalities. Opportunities for object manipulation and body movement through virtual space provide frameworks that, in varying degrees, are perceived as comparable to similar opportunities in the real world. This paper reviews current work on motor rehabilitation using virtual environments and virtual reality and where possible, compares outcomes with those achieved in real-world applications. One of the major goals of rehabilitation is to make quantitative and qualitative improvements in daily activities in order to improve the quality of independent living. Three determinants of motor recovery are early intervention, task-oriented training, and repetition intensity  while a The successful integration of virtual reality into multiple aspects of medicine, psychology, and rehabilitation has demonstrated the potential for the technology to present opportunities to engage in behaviors in challenging but safe, ecologically valid environments while maintaining experimental control over stimulus delivery and measurement [for review see ,7]. MoreSeveral features distinguish virtual environments from other forms of visual imaging such as video and television. A key feature of all VR applications is interaction. Virtual environments (VE) are created that allow the user to interact with not only the VE but also with virtual objects within the environment. In some systems, the interaction may be achieved via a pointer operated by a mouse or joystick button. In other systems, a representation of the user's hand (or other body part) may be generated within the environment where movement of the virtual hand is "slaved" to the user's hand allowing a more natural interaction with objects. Finally, while many applications of VR allow the user to control the viewpoint on the screen, third-person views or images of the users themselves that appear as players in the environment also provide the opportunity for interaction with the VE.A broad range of visual interfaces are used to create varying degrees of immersion in a VE ranging from conventional desktop monitors to head mounted displays. Increasingly complex, fully immersive VR systems, such as the Cave Automatic Virtual Environment (CAVE) developed at the University of Illinois at Chicago, provide the illusion of immersion by projecting stereo images on the walls and floor of a room-sized cube. Several persons wearing lightweight stereo glasses can enter and walk freely inside the CAVE. A head tracking system continuously adjusts the stereo projection to the current position of the leading viewer. In order to integrate the movement of the user with that of the VE and virtual objects, user position and motion must be tracked so that virtual images can be updated in real-time. Motion tracking approaches include color subtraction technology, video frame subtraction as well as magnetic and infrared tracking devices. Technical advances in the development of these interfaces have minimized the once lengthy lag times responsible for some of the earlier reports of cybersickness.To date, rehabilitation applications have primarily used visual and auditory sensory input while the addition of haptics is less developed. Haptic interface devices including gloves, pens, joysticks and exoskeletons provide users with a sense of touch and allow the user to feel a variety of textures as well as changes in texture. There is increasing evidence that haptic information is an effective addition towards the accomplishment of certain treatment objectives such as increasing joint range of motion and force . Haptic Another cardinal feature of virtual reality is the provision of a sense of actual presence in, and control over, the simulated environment . The senOne important consideration with the application of virtual reality and movement in virtual environments is the behavior or movement characteristics of subjects in virtual environments . Recent In contrast, we have demonstrated significant differences between functional lateral reach performances when performed in the real environment versus in a virtual environment delivered on a flatscreen . The VR Improving the functional abilities of patients is commonly achieved by using tasks of increasing difficulty in combination with physical and/or verbal guidance of the patient's movements or actions. Thus, integrating the means to modulate the level of difficulty within a VR task is of crucial importance. A virtual reality system (VIVID GX) was used to provide independent leisure opportunities to adults with cerebral palsy and severe intellectual disabilities who were non-speaking and who used wheelchairs for mobility . The parCentral to the issue of virtual environments as a training medium is the issue of transfer of training; does task improvement or learning transfer reliably from a VE to a real environment? Virtual environments and VR interventions should not only be used to augment current ability or to provide exposure to "other" therapeutic possibilities, but importantly to demonstrate distinct carryover to real-life functional tasks. One major challenge is identifying effective and motivating intervention tools that enable transfer of the skills and abilities achieved during rehabilitation to function in the "real" world. For example, recent studies stress that simple repetitive movements of an affected limb are not productive for the reorganization process but that it is action related to skill acquisition which contribute to the desired effect .Rose and colleagues studied the transfer of training of a simple sensorimotor virtual task to performance on the "real world" equivalent . The reaSeveral systems have been used in studies of balance including a combined HMD display system combined with a fixed bicycle, a flatscreen VR system providing primarily 2D visual information and more recently an immersive dynamic virtual environment combined with a posture platform.Kim et al  reportedMultiple applications of flatscreen VR for balance training have been reported that have used video capture technology from VividGroup's GX or IREX systems [see for example, ,10,24-26We have used a similar approach with a significantly larger study population of community-living individuals with traumatic brain injury see ,10,26 fo fo26 forMore recently, Keshner and colleagues  have uniPatients with Parkinson's disease akinesia have little difficulty stepping over objects in their path even when they are totally unable to initiate a step on open ground . A virtuA locomotor interface, GaitMaster2 (GM2), intended to provide the user with the sense of forward movement while his/her actual position in space is constant, has been tested with two individuals with hemiplegia following a stroke . The useA VR-enhanced orthopedic appliance for use with individuals with spinal cord injuries has also been developed and links a gait-inducing exoskeleton to a HMD providing binocular visual displays . BrieflyA novel VR application for locomotor rehabilitation couples a three dimensional visual scene with a self-paced treadmill . BrieflySeveral upper and lower extremity VR applications have been developed using different technologies. Preliminary data suggest potential benefits of various systems. For example, a report based on two case studies using the Vivid GX video capture technology demonstrates improvements in upper extremity function . The firHolden and colleagues  developePiron et al.  used a vThe Rutgers ankle and hand systems, both incorporating the haptic sense, were developed as assessment and intervention tools although there are limited clinical data available at this time regarding efficacy see ,36]. The. The36]]Chuang et al  comparedPositive outcomes of virtual reality as a distractive technique have also been reported for physiotherapy treatment sessions. Hoffman and colleagues  report dAlthough the majority of VR environments that have been developed for assessment to date focus on daily living skills such as meal preparation , spatialAssessments can be developed using VR technologies that will provide objective, repeatable and quantitative results. Standardized instructions, non-varying environmental cues, tasks and feedback can be achieved. In the extreme condition, interactions are limited to those between the patient and a virtual assessor. Since the devices are programmable, varying the complexity of assessment tasks is relatively trivial allowing for batteries of simple and more complex tasks to be developed. For example, an upper extremity assessment scale may include tasks requiring self-selected motion as well as responses to force perturbations permitting assessment of feedback limb control.We have tried in the past to have him involved in things but he seemed uninterested. With these exercises  he was trying to explain what he was doing, he was interested in what he was doing, he was looking forward to going.The degree of functional movement outcome achieved by therapy is often sub-optimal since intensive therapy is limited by resource allocation and access. For many individuals, such as traumatic brain injury survivors, access to therapy is terminated once a level of function is achieved even if residual deficits remain. For other individuals, even when therapy is available such as during in-patient neurological rehabilitation, low levels of interaction between the patient and environment have been reported ,46. For An exponentially increasing number of distinct VR applications are being developed for intervention and assessment of a broad range of motor rehabilitation needs including upper and lower extremity function, balance and locomotion. Although the initial VR rehabilitation applications that were developed, in particular applications using video capture technologies and most HMDs, were subjected to relatively prohibitive entry level costs associated with the technology, recent developments in technology have made the number of low-cost multisensory VR applications increasingly available. Significant decreases in the costs associated with HMDs and motion trackers, desktop computers and certain haptic devices, are facilitating the development of low cost off-the-shelf applications.The applications reviewed in this paper have demonstrated improvements of specific motor function with certain populations. It is clear that many of the applications that have been developed, for example gait trainers, will serve a specific rehabilitation niche. These devices have the potential to significantly extend our current understanding of movement and therapy and may substantially impact delivery of rehabilitation interventions. Critical for continued successful integration of virtual reality in motor rehabilitation is the need for the ongoing development and use of the technology to be based on clear understanding of the complexity of voluntary movement . Sensori


The retrieval of mitochondrial DNA (mtDNA) sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution. Analysis of mitochondrial DNA from four Neandertal fossils and five "modern human" contemporaries excludes any large genetic contribution of Neandertals to the gene pool of modern humans Despite intense research efforts, no consensus has been reached about the genetic relationship between early modern humans and archaic human forms such as the Neandertals. While supporters of “multiregional evolution” argue for genetic exchange or even continuity between archaic and modern humans , proponeTo explore the genetic relationship between early modern humans and Neandertals in spite of these difficulties, we made use of the fact that the four Neandertal mtDNA sequences determined to date can easily be distinguished from those of modern humans . This alThe preservation of endogenous DNA in fossils is correlated with the amount, composition, and chemical preservation of amino acids . We findIf low amounts of DNA are preserved in a specimen, some extracts will fail to contain DNA molecules by chance . TherefoWhen amplified with the hominoid primers, all Neandertal and all early modern human remains yielded modern human DNA sequences see . In addiWhen amplified with Neandertal-specific primers, Neandertal-like mtDNA sequences were amplified from two independent extractions from all Neandertal fossils see . For oneThus, all Neandertal remains analyzed yielded mtDNA sequences that are not found in the human mtDNA gene pool today but are similar to those found in four previously published Neandertals  see Fig. This isp < 0.007) that one or more of the five early modern humans analyzed here are among these few ancestors of current humans, the five Upper Pleistocene individuals can be added to the ancestors of the current mtDNA gene pool to allow us to ask what extent of Neandertal mtDNA contribution to early modern humans can be statistically excluded using the coalescent. Under the model of a constant human effective population size  under conditions that separate the different amino acids as well as their stereoisomers. Eight amino acids are analyzed and their respective concentration measured: D- and L-alanine, glycine, D- and L-aspartic acid, serine, glutamic acid, valine, D- and L-leucine, and isoleucine.About 10 mg of bone were removed from each specimen and analyzed as in Escherichia coli using the TOPO TA cloning kit (Invitrogen, Leek, The Netherlands), and ten or 30 clones of each amplification were sequenced on a ABI 3700 .DNA extractions were performed in a laboratory dedicated to ancient DNA work. In this laboratory, positive air pressure is maintained with filtered air at all times, and all areas and equipment are treated with UV light when the laboratory is not used. A maximum of six bone or teeth samples were processed together with two blank extractions. Neandertal samples were always processed together with early modern human samples or cave bear samples. For each extraction, the samples were ground and between 30 mg and 120 mg of bone powder was extracted as in Tm with a  population of Neandertal individuals K, where c represents the Neandertal genetic contribution to the merged population. If none of n mtDNA sequences sampled in the merged population is Neandertal-like, we can exclude (at the 5% level) contributions that give a probability smaller than 0.05 of observing only modern human sequences, i.e., (1 − c)K < 0.05. The number of ancestors of n samples at the time t is represented by a probability distribution, An(t). Thus, the probability of observing only one kind of sequences in n samples becomes:Given that previous analyses of mtDNA sequences have rejected a model of complete panmixia between Neandertals and early modern humans , we focuuals see  from whiK vary from 1 to n. For a population of constant size over time, Pr(An(t) = K) has been derived in n samples at time t as the expected value of An(t), E(An(t)), according to this model and calculate the probability of observing only human sequences for different values of c.where The entire hypervariable region I sequence was determined from this specimen using amplifications and clones given in Figure S1. Its sequence is identical to the sequence previously determined from individual Vi-75 from Vindija . We coulFigure S1(30 KB PDF).Click here for additional data file.Figure S2n individuals (x axis) is estimated at 20,000, 25,000, and 30,000 years ago. For example, 150 humans living today have approximately seven ancestors 20,000 years ago.The number of ancestors of (56 KB PDF).Click here for additional data file.Figure S3c (x axis) under a model of constant population size .Click here for additional data file.Table S1The bones were analyzed by high performance liquid chromatography for their amino acid content see . The ext(54 KB PDF).Click here for additional data file.


PLoS Medicine. “This first field efficacy trial was an important milestone in the progression of new recombinant vectored vaccines to deployable products,” says Adrian Hill (University of Oxford, United Kingdom), the lead investigator of the study. “The safety profile was excellent and the efficacy data provide a first indication of the levels of cellular immunogenicity that will be required for preventing infection,” he says.A malaria vaccine called ME-TRAP, which targets the pre-erythrocytic stage of the disease, was not effective at reducing natural infection rates in semi-immune African adults, according to the report of a randomized controlled trial published this month in Hill and his co-workers used a heterologous prime–boost vaccination technique. They gave the volunteers two vaccines—a DNA priming vaccine followed by a modified vaccinia virus Ankara (MVA) that acted as a booster. The DNA and MVA vaccines both had the same insert coding for thrombospondin-related adhesion protein  and a string of T cell epitopes .Plasmodium falciparum infection.Hill's team had previously shown that ME-TRAP vaccines given in prime–boost sequence could induce large T cell responses in healthy volunteers from the UK and could delay parasitemia in a sporozoite challenge test (Nat Med 9: 729–735). The next step was to do a randomized controlled trial in Gambia to determine whether this vaccination strategy could provide protection against natural P. falciparum infections.The researchers recruited volunteers from 13 Gambian villages that were close to the alluvial flood plain and so were at high risk of developing malaria. They randomly assigned the 372 volunteers to receive either two doses of the DNA ME-TRAP vaccine followed by a single dose of MVA ME-TRAP, or three doses of rabies vaccine. This three-dose schedule is similar to the one used by the World Health Organization/United Nations Children's Fund Expanded Program on Immunization. Two weeks before the third dose was given, all the volunteers received antimalarial drugs to clear blood-stage The time to first infection, the primary end point of the study, was similar in the two groups, with an estimated vaccine efficacy of only 10%. However, the effector T cell response to the TRAP antigen T9/96, measured one week after the third vaccination, was 80 times higher in the DNA/MVA vaccine group than in the rabies vaccine group.P. falciparum, and partly because this is a fertile area for trying out new techniques, such as DNA vaccines, where there is still a lot to learn.”“It is absolutely crucial that results like these are published, since the failures, as well as the successes, need to be documented if we are to move towards rational strategies for optimizing malaria vaccines,” says Tom Smith from the Swiss Tropical Institute, who was not involved in the study. “At the same time, it makes sense to move on quickly without shedding too many tears, in a field that is moving much faster than it was before the recent injections of money from the Gates Foundation, but where it is still impossible to second-guess the results of field trials. This is partly because we do not have any good proxy measures of effective immunity in Hill is planning to do further trials that address the important question of whether this type of vaccine can prevent the symptoms of malaria. “The next step,” says Hill, “is to assess newer vaccine regimes that employ two viral vectors rather than DNA and to study prevention of malaria rather than infection.”


We describe a patient who underwent transurethral resection of the prostate for urinary obstructive symptoms and had histological findings of adenocarcinoma of the prostate with prostatic localization of chronic lymphocitic leukemia (CLL).The contemporary presence of CLL, adenocarcinoma of the prostate and residual prostatic gland after transurethral resection has never been reported before and the authors illustrate how they managed this unusual patient.3 and the abdomen ultrasound found a modest splenomegaly and no peripheral lymphadenophaty. The patient underwent transurethral resection of the prostate and had a pathological finding of adenocarcinoma in the prostate with a Gleason Score 4 (2+2) of less than 5% of the material , associated with a diffused infiltration of chronic lymphocitic leukemia elements.A 79-years-old white man, presented with acute urinary retention, had a peripheral blood count with an elevated lymphocytosis (21.250/mL) with a differential of 65.3% lymphocytes and the prostate-specific antigen (PSA) value was 3.38 ng/mL with a percent free PSA of 8.28%. The transrectal ultrasound (TRUS) indicated an isoechonic and homogenic enlarged prostate of 42 cmThe incidental finding of a prostatic localization of a low-grade non-Hodgkin's lymphoma does not modify eventually further treatments for neither prostate cancer nor lymphoma. The presence of a low-grade and low-stage lymphoma, confirmed by a hematological evaluation, and the simultaneous evidence of an adenocarcinoma after transurethral resection of the prostate for acute urinary retention do not require any immediate treatment due to its long-term survival rate and the follow-up remains based on periodical PSA evaluation and complete blood count. The incidence of non-Hodgkin's lymphoma among prostate cancer patients is extremely low 0.2%), and, even more the leukemic infiltration of the prostate ,2. It ha%, and, e3 and the abdomen ultrasound found a modest splenomegaly and no peripheral lymphadenophaty. The peripheral blood count showed an elevated lymphocytosis (21.250/mL) with a differential of 65.3% lymphocytes, while other parameters as platelet count, number of erythrocytes and lactate dehydrogenase hormone level were all normal. The prostate-specific antigen (PSA) value was 3.38 ng/mL with a percent free PSA of 8.28%. Due to his age and symptoms, the patient underwent transurethral resection of the prostate. The pathological examination of the tissue revealed an adenocarcinoma in the prostate with a Gleason Score 4 (2+2) of less than 5% of the material , associated with a diffused infiltration of chronic lymphocitic leukemia elements  was 21. The transrectal ultrasound (TRUS) indicated an isoechonic and homogenic enlarged prostate of 42 cmet al.,) to 0.8%  [The prostatic involvement by non-Hodgkin's lymphomas is an unusual finding. The simultaneous presence in the prostate of CLL and adenocarcinoma has been reported in a percentage variable from 0%  . The incThe author(s) declare that they have no competing interests.RB and PB managed the patient, edited the manuscript and coordinated the submission. LR carried out the literature search, SC performed the surgical procedure and MZ performed the pathological examination and realised the figures. WA revised the manuscript for scientific content. All authors contributed to the preparation of the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LNvs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LNv-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LNv-specific neuropeptide, because the molecular oscillations are gradually lost when 01Pdf mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the 01Pdf mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LNvs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LNvs alone are insufficient to support the circadian program. Based on the damping results with 01Pdf mutant flies, we propose that LNvs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include  Circadian rhythms are characterized by robust molecular oscillations, which are shown here to require a brain region-specific neuropeptide, PDF, for maintenance and coordination Circadian rhythms of diverse organisms are based on similar intracellular molecular feedback loops . Based oDrosophila, circadian clocks have been identified in a diverse range of cell types throughout the head and the body (Drosophila “core pacemaker” is believed to maintain robust oscillations for a long time in constant darkness (DD) with little or no damping, such that circadian behaviors can persist under such conditions (In the body . Howeverthe body . In manythe body . This isthe body . In contnditions . Indeed,Drosophila brain are well characterized (vs), as the best “core pacemaker” candidate for the following reasons: (1) in the developmental mutant disco, the presence of LNvs correlates with the maintenance of behavior rhythmicity , and the 01Pdf-null mutant loses behavioral rhythmicity under DD conditions  LNvnditions ; (3) genty in DD ; and (4)ys in DD , in cont rhythms . Consisttide PDF . Our purvs can support free-running circadian locomotor activity rhythms independently of other functional clock cells, we restricted pacemaker activity to these few PDF-expressing cells. CYCLE (CYC) is a bHLH–PAS protein  and timeless (tim) mRNAs are essentially undetectable  were examined by two independent criteria. First, molecular oscillations were assayed by in situ hybridization with a tim probe (tim RNA levels undergo robust cycling in wild-type flies, with a trough at ZT3 and a peak at ZT15 (tim mRNA cycled in the LNvs (tim mRNA-negative (vs under these light–dark (LD) conditions. The observed oscillations are also not passively driven by light, since they persisted in DD, at least in the s-LNvs  still cycle robustly at this time (vs fail to maintain oscillations at the beginning of DD (vs “adapt” to constant conditions by becoming rhythmic once again after about 2 days in DD (data not shown). These results clearly distinguish the brain from the eyes and other peripheral tissues, which rapidly lose coherent molecular oscillations under free-running conditions  advance in the peak of tim RNA and also confirmed the reduced cycling amplitude . Taken together, the results indicate an excellent quantitative correspondence in phase and amplitude between the tim RNA rhythms and the behavioral rhythms in all clock neurons of the 01Pdf strain.Despite the gradual fading of locomotor activity rhythms in DD, a significant fraction of  d of DD . By trac d of DD . Quantitmplitude D. In ordcryptochrome (cry) mRNA oscillations by in situ hybridization. cry is expressed in a similar clock neuron pattern to tim, but it has a peak expression at ZT2 and a trough at ZT14  clock and eclosion rhythms suggests that the LNvs also control the PG clock via PDF signaling  epitope by PCR cloning. CYC–HA was subsequently cloned into pUAST to generate pUAS–CYC–HA. The transformation plasmid was used to generate transgenetic flies. A third chromosome insertion line (UAS–CYC–HA15) was used subsequently. All wild-type flies and specimens were taken from a Canton-S stock.Full-length pdf–GAL4  and imaged on a Leica laser scanning confocal microscope. Optical sections were taken at 1–2 μm intervals and used to construct a maximum projection image for each brain.To visualize the axon projections from circadian neurons, a tim and cry was done as described previously . The analysis was done by using a signal processing toolbox  conditions before release into DD. Locomotor activities of individual flies were monitored using Trikinetics  toolbox . Autocor toolbox . In someDrosophila PER protein (a gift from P. Nawathean). In addition, a new N-terminus biotinylated PDF peptide was chemically synthesized de novo (Sigma-Aldrich). Identical results were obtained with the two PDF peptides, and no specific signal was obtained with the two control peptides.Biotinylation of the PDF peptide was with EZ-Link Sulfo–NHS–LC–Biotin reagent (Pierce Biotechnology, Rockford, Illinois, United States), following the manufacturer's instruction. Excess biotinylation reagent was removed by prolonged incubation in Tris–HCl buffer (1 M [pH 7.5]) followed by protein purification through a Polyacrylamide 1800 desalting column (Pierce Biotechnology). A control neuropeptide, allatostatin I (Sigma-Aldrich, St. Louis, Missouri, United States), was biotinylated using the same method. A second control was a synthetic, biotinylated peptide derived from the Drosophila, brains were dissected in PBS and fixed in 3.7% paraformaldehyde in PEM for 30 min. After they were rinsed in PBS plus 0.3% Triton and blocked using 1% FBS or BSA, biotinylated peptide was incubated with the brains at a final concentration of 0.2 μg/ml. The brains were washed thoroughly with TNT (0.1 M Tris–HCl [pH 7.5], 0.15 M NaCl, 0.05% Tween 20). The bound peptide was subsequently detected through the biotin label using streptavidin–HRP  and fluorescent tyramides (NEN LifeScience). A detailed protocol is provided as To detect the binding of the neuropeptide in the CNS of Figure S1y w; pdf–GAL4;UAS–CYC,01cyc/01cyc was released into DD after entrainment and assayed by tim whole-mount in situ hybridization on the fourth day of DD. A Pdf probe was used to label the LNv group. Brains were taken at two opposite timepoints, CT3 (top panels) and CT15 (bottom panels). From left to right are Pdf (green, FITC labeled), tim (red, Cy3 labeled), and an image overlay. The lower arrows point to the s-LNvs and the upper arrows to l-LNvs. Whereas the l-LNvs show barely visible tim RNA oscillations under these conditions, the s-LNvs are obviously cycling. This difference suggests that the l-LNvs might damp more rapidly or be more light-dependent than the s-LNvs in this unusual genotype. (7.1 MB PDF).The “rescued” mutant Click here for additional data file.Protocol S1(23 KB DOC).Click here for additional data file.


Emberiza bruniceps. Each experiment employed three groups (N = 6–7 each) of photosensitive birds that were held under 8 hours light: 16 hours darkness (8L:16D) since early March. In the experiment 1, beginning in mid June 2001, birds were exposed to natural day lengths (NDL) at 27 degree North (day length = ca.13.8 h, sunrise to sunset) for 23 days. In the experiment 2, beginning in early April 2002, birds were exposed to 14L:10D for 22 days. Beginning on day 4 of NDL or day 1 of 14L:10D, they received 10 (experiment 1) or 13 (experiment 2) daily injections of both melatonin and prolactin (group 1) or prolactin alone (group 2) at a dose of 20 microgram per bird per day in 200 microliter of vehicle. Controls (group 3) received similar volume of vehicle. Thereafter, birds were left uninjected for the next 10 (experiment 1) or 9 days (experiment 2). All injections except those of melatonin were made at the zeitgeber time 10 ; melatonin was injected at ZT 9.5 and thus 0.5 h before prolactin. Observations were recorded on changes in body mass, testicular growth and feather regeneration.Little is known about how hormones interact in the photoperiodic induction of seasonal responses in birds. In this study, two experiments determined if the treatment with melatonin altered inhibitory effects of prolactin on photoperiodic induction of seasonal responses in the Palearctic-Indian migratory male redheaded bunting Under NDL (experiment 1), testis growth in birds that received melatonin 0.5 h prior to prolactin (group 1) was significantly greater (P < 0.05, Student Newman-Keuls test) than in those birds that received prolactin alone (group 2) or vehicle (group 3). Although mean body mass of three groups were not significantly different at the end of the experiment, the regeneration of papillae was dramatically delayed in prolactin only treated group 2 birds. Similarly, under 14L:10D (experiment 2) testes of birds receiving melatonin plus prolactin (group 1) and vehicle (group 3) were significantly larger (P < 0.05, Student Newman-Keuls test) than those receiving prolactin alone (group 2). Also, birds of groups 1 and 3, but not of group 2, had significant  gain in body mass. However, unlike in the experiment 1, the feather regeneration in birds of the three groups was not dramatically different; a relatively slower rate of papillae emergence was however noticed in group 2 birds. Considered together, these results show that a prior treatment with melatonin blocks prolactin-induced suppression of photoperiodic induction in the redheaded bunting, and suggest an indirect role of melatonin in the regulation of seasonal responses of birds. In many birds, day length regulates seasonal changes in fattening and body mass gain, gonadal growth and development, molt, and plasma levels of several hormones, including luteinizing hormone (LH), prolactin and melatonin -4. TherePrevious studies on how hormones interact in photoperiodic induction of seasonal responses in birds have yielded conflicting results. A number of early findings show prolactin acting both as pro- and anti-gonadal in birds exposed to stimulatory day lengths -11. HoweEmberiza bruniceps), in which melatonin is not directly involved in the photoperiodic time measurement based on circadian rhythm of photosensitivity [-1 suppresses ovarian response in buntings subjected to long days [The production and secretion of melatonin encodes a photoperiodic calendar to birds as they exhibit changes in both the duration and amplitude of melatonin secretion corresponding to the duration of night length/ day length ,14,15. Asitivity ,23. Prevong days . In thisWe used adult male redheaded bunting caught in late February from the overwintering flock at 25°N. Buntings are migratory finch that breed in summer in west Asia and east Europe (~ 40°N) and overwinter in India. Birds were held outdoors and acclimatized to captive conditions for two weeks, and then brought indoors and maintained on short days (8 hours light: 16 hours darkness, 8L:16D) until subjected to experiments. Under short days, buntings do not fatten, and remain reproductively immature and responsive to photostimulation ; birds pThis experiment began in mid June 2001. Photosensitive birds maintained indoors on 8L:16D since early March were brought outdoors in the aviary and exposed to natural day lengths (NDL) at 27°N (day length = ~ 13.8 h, sunrise to sunset). After three days of acclimatization, they were divided in three groups (N = 6 each). Beginning on day 4, they received subcutaneous injections once daily for 10 days as follows: group 1- first melatonin and 0.5 h later prolactin; group 2- prolactin alone; group 3- vehicle (control). After 10 consecutive injections (days 4–13), birds were left uninjected for the next 10 days (day 14–23). The experiment was terminated on day 24.To confirm results of the experiment 1, we performed the experiment 2 under artificial conditions providing light-dark (LD) cycles corresponding to that was available outdoors (NDL) to birds of the experiment 1. This experiment began in the second week of April 2002. Three groups (N = 6–7) of photosensitive birds were subjected to 14L:10D. Beginning on day 1, they received 13 injections (days 1–13) as in the experiment 1: group 1- first melatonin and 0.5 h later prolactin; group 2- prolactin alone; group 3- vehicle (control). Thereafter, birds were left uninjected for the next 9 days (days 14–22). The experiment was terminated on day 23.Melatonin was administered at zeitgeber time 9.5  in view of our previous study  and seve-1 day-1 in 200 μl injection volume. Prolactin was obtained from Sigma Chemical Co. USA  and melatonin from Genzyme Fine Chemicals Ltd., Haverhill, Suffolk, UK). Melatonin injections were prepared as described by Kumar [In both experiments, melatonin and prolactin were administered each at a dose of 20 μg birdby Kumar . Brieflyab2, where a and b denote half of the long and short axes, respectively. Feather papillae regeneration was recorded as follows. On day 1 of the experiment, feathers in a specific area on the left chest were plucked. A permanent ink marker marked an area on bare epidermis measuring 1 cm2. Beginning 24 h after the first injection, the number of papillae emerged from the epidermis were counted daily throughout the experiment 1 or till 4 days after the last injection in the experiment 2, and scored subjectively as outlined by Boswell [We measured the effects on changes in body mass, testis size and regeneration of feather papillae. Body mass and testis size were measured at the beginning (day 0, the day before injections began), in the middle (body mass only) and at the end of the experiments. Birds were weighed on a top pan balance providing accuracy nearest to 0.1 g. In view of the findings that the fattening accounts for the most of the gain in body weight in photostimulated passerine birds ,28, in t Boswell . Brieflyad libitum. In an artificial LD cycle, light was provided by white compact fluorescent lamps at ~ 500 lux. Data are presented as mean ± SE. They were analyzed using one-way analysis of variance (1-way ANOVA) with or without repeated measures, followed by post-hoc tests if ANOVA indicated a significance of difference. 1-way repeated measure ANOVA was used to compare data generated from the same group as a function of time, and 1-way ANOVA was used to compare data of different groups at one observation. Two-way (2-way) ANOVA was used to analyze data when two factors were considered together, for example the effect of the treatment and duration of the treatment. Significance was taken at P < 0.05. In the experiment 1, one bird of group 1 and two birds of group 2 died, and their data are excluded from the statistical analyses.Food and water were available F(2,12) = 4.656, P = 0.0319; 1-way ANOVA). Testes were significantly larger (P < 0.05, Newman-Keuls test) in birds that received melatonin prior to prolactin (group 1) compared to those that received prolactin alone (group 2) or vehicle (group 3)  = 3.3.19, P = 0.0701; 2-way ANOVA) although in group 1 birds the emergence of the first papilla was delayed by at least a day  = 15.26, P < 0.0001; 2-way ANOVA) and group 3 (F(17,144) = 16.49, P < 0.0001; 2-way ANOVA)  Fig. . The ratday Fig. . Whereascf. Fig. .F(2,10) = 12.16, P = 0.0021; group 3: F(2,12) = 6.978, P = 0.0098; 1-way RM ANOVA; Fig. F(2,12) = 0.2839, P = 0.7379; 1-way RM ANOVA; Fig. F(2,17) = 3.857, P = 0.0416; 1-way ANOVA). Although testes were stimulated in all groups  = 4.343, P = 0.0299; 1-way ANOVA). Testes grew to full size in birds of groups 1 and 3, and hence were significantly larger (P < 0.05, Newman-Keuls test) than those of group 2 in which they grew to less than half-maximal size  = 3.9550, P < 0.0485; 2-way ANOVA) and group 3 (F(14,165) = 24.76, P < 0.0001; 2-way ANOVA)  than those received prolactin alone (group 2) or vehicle (group 3). A role of melatonin in enhancing responsiveness of the photoperiodic response system is shown in an experiment on the blackheaded bunting , an allied species that shares breeding and wintering grounds with the redheaded bunting. In blackheaded buntings exposed to 11.75L:11:25D of red light (650 nm), testes grew significantly larger in individuals that carried implants filled with melatonin compared with those that carried empty implants [A second possibility is that exogenous melatonin changes phase-relationship between daily rhythms of endogenous endocrine rhythms, and this somehow enhances sensitivity of the circadian response system to stimulatory effects of long day lengths. Testicular response in birds of the experiment 1 supports this Fig. . Birds timplants .P < 0.05, Student t-test) smaller testes than those of the experiment 2. This occurred perhaps because of one or both of the following reasons. First, there was a difference in the lighting environment between two experiments, both in the shape of LD cycle (saw-tooth shape in NDL versus square-wave shape in 14L:10D) and intensity of light period  versus a continuous ~ 500 lux intensity for 14 h within the photoperiodic chambers in 14L:10D). It is reported that the duration of photoperiod and light intensity do affect photoperiodic induction of body mass and testis recrudescence in the blackheaded bunting [However, one observation of the present study is not entirely consistent. Controls of the experiment 1 had significantly ( bunting . Second, bunting , similarCoturnix coturnix), for example, melatonin given in drinking water influences the reproductive cycle [Estrilda amandava) [Psittacula cyanocephala) and the Indian weaver bird (Ploceus philippinus) [Ploceus philippinus) [The present study indicates that melatonin could be involved indirectly in regulation of photoperiod-induced seasonal responses in the redheaded bunting by modulating effects of other hormones such as the prolactin. This appears consistent with another finding on this species  suggestive cycle . Daily mmandava) , and cauippinus) . Inhibitippinus)  althoughippinus) . DiffereThe present results show that a prior treatment with melatonin blocks prolactin-induced suppression of photoperiodic induction in the migratory redheaded bunting. How melatonin acts to negate the effects of prolactin is unclear. Whatever is the actual mechanism of action, the current result provide evidence that melatonin modulates photoperiodic induction of seasonal responses in birds by interacting with prolactin. Whether such effect will vary during different seasons of the year remains to be investigated.AKT and SR carried out the experiments and prepared the first draft of the manuscript. VK supervised the experiments and the final version of the manuscript. The study was conceived by VK but then the experiments were discussed jointly. All the authors approved the final manuscript.


Protein-protein interactions play a critical role in protein function. Completion of many genomes is being followed rapidly by major efforts to identify interacting protein pairs experimentally in order to decipher the networks of interacting, coordinated-in-action proteins. Identification of protein-protein interaction sites and detection of specific amino acids that contribute to the specificity and the strength of protein interactions is an important problem with broad applications ranging from rational drug design to the analysis of metabolic and signal transduction networks.In order to increase the power of predictive methods for protein-protein interaction sites, we have developed a consensus methodology for combining four different methods. These approaches include: data mining using Support Vector Machines, threading through protein structures, prediction of conserved residues on the protein surface by analysis of phylogenetic trees, and the Conservatism of Conservatism method of Mirny and Shakhnovich. Results obtained on a dataset of hydrolase-inhibitor complexes demonstrate that the combination of all four methods yield improved predictions over the individual methods.We developed a consensus method for predicting protein-protein interface residues by combining sequence and structure-based methods. The success of our consensus approach suggests that similar methodologies can be developed to improve prediction accuracies for other bioinformatic problems. Protein-protein interactions play a critical role in protein function. Completion of many genomes is being followed rapidly by major efforts to identify experimentally interacting protein pairs in order to decipher the networks of interacting, coordinated-in-action proteins. Identification of protein-protein interaction sites and detection of specific residues that contribute to the specificity and strength of protein interactions is an important problem -3 with bComputational efforts to identify protein interaction surfaces -6 have bThere is an acute need for multi-faceted approaches that utilize available databases of protein sequences, structures, protein complexes, phylogenies, as well as other sources of information for the data-driven discovery of sequence and structural correlates of protein-protein interactions ,5. By exe.g. antibody-antigen complexes is currently under study and will be published later. It should be noted, however, that prediction of binding sites for other types of protein complexes, especially those involved in cell signaling, is likely to be more difficult than for the hydrolase-inhibitor complexes.Here we are using a dataset of 7 hydrolase complexes from the PDB, together with their sequence homologs. The application of our consensus method to other types of complexes, S. griseus in a complex with turkey ovomucoid inhibitor . The inThe primary amino acid sequence for proteinase B chain and the interface residue prediction results for the four individual methods and the consensus method are shown in Figure p); and the average over the total number of residues, i.e., the average on a "per residue" basis (<...>r).The prediction results for all methods are shown in Table Amino acid sequences are conserved for many different reasons related to the structure and function of proteins: for stability ,20, enzyTo identify protein residues that are conserved – perhaps due to their functional role in forming specific protein-protein interactions – we use ClustalX  multiplesensitivity+ and specificity+ measures are especially useful because they more clearly reflect the ability of a method to detect "positive" interface residues. (See the Methods section for definition and further discussion of performance measures). Note that even though Figure As shown in Figure To detect structurally conserved residues that are possible binding sites we have used the Conservatism of Conservatism method (CoC) developed by Mirny and Shakhnovich We use sWe have generated a support vector machine (SVM) classifier to determine whether or not a surface residue is located in the interaction site using information about the sequence neighbors of a target residue. An 11-residue window consisting of the residue and its 10 sequence neighbors (5 on each side) is chosen empirically. Each amino acid in the 11 residue window is represented using 20 values obtained from the HSSP profile of the sequence. Each target residue is therefore associated with a 220 (11 × 20) element vector. The SVM learning algorithm is given a set of labeled examples of the form (X, Y) where X is the 220 element vector representing a target residue and Y is its corresponding class label, either interface or non-interface residue. The SVM algorithm generates a classifier which takes as input a 220 element vector that encodes a target residue to be classified and outputs a class label. Our previous study , we trais by the hydrophobicity values of its amino acids hi obtained in this factorization and protein structure by the contact matrix Γ. The problem of finding the best alignment of a query sequence s with a structure having contact matrix Γ is to find the transformation from s to s' that optimizes the energy function. The optimum s' is the dominant eigenvector 0 vof the contact matrix Γ. There is a strong correlation between a protein sequence and the dominant eigenvector of its native structure's contact matrix. Here the transformation we seek is obtained by maximizing the correlation between s' and 0v. This is an alignment problem, and a dynamic programming method from sequence alignment has been adapted to solve this problem [Structural threading was performed for the set of 7 protein complexes using a recently developed threading algorithm , which w problem .For each sequence, threading is performed against structures in our template database and alignment results used only when the score exceeds a length-dependent threshold. From the alignments, residues involved in contacts at the interface are identified using a scale based on the number of times a particular residue is indicated and the strength of the threading score. The predicted binding sites for 3sgb_E by the threading method are marked in Figure Based on the results from the predictions with the four independent methods, we have developed a simple consensus method to obtain a better prediction. In the consensus method results presented here, an amino acid is considered to be an interface residue if any of the following conditions are met:i) at least three independent methods classify it as an "interface residue"ii) any two methods (except the Phylogeny-Threading pair) predict itFor this set of proteins, the parameters for combining results in the consensus method have been empirically determined without a systematic comparison of the strengths and weaknesses of each method. We employ this simple approach because it provides demonstrable improvement in prediction performance over the individual methods. The consensus interface residue predictions are indicated by an "E" in Figure Each of the four prediction methods presented in this paper sheds a different light on the conservation and prediction of protein interaction sites, but none of the methods taken separately is as powerful as the combination of all four methods. The simple consensus approach presented here could perhaps be improved by generating an ensemble predictor with more detailed probabilities. Our current work is directed at this approach. It is clear that the present subject is an active field of research -38.The dataset of 7 hydrolase-inhibitor complexes used in this work has been derived from a larger dataset of 70 protein heterocomplexes extracted from PDB by Chakrabarti and Janin  and used2 [Surface and interface residues for the proteins were identified based on information in the PDB coordinate files as previously described ,3. Brief2 . In the Based on the ASA definitions, 41% of the residues in the set of 7 proteins were surface residues, corresponding to a total of 631 surface residues. Among these surface residues, 166 were defined as interface residues and 465 as non-interface residues (i.e. surface residues that are not in the interaction sites). Thus, on average, interface residues represent 26% of surface residues, or 11% of total residues for proteins in our dataset.k during lineage l from amino acids i to j (i,j = 1,...20) can be expressed as λkl (i,j) = ck × alk × Q(i,j|k), where ck accounts for the rate variation among sites, alk for site-specific lineage (or subtree) effect caused by functional divergence [Q(i,j|k) is the (site-specific) model for amino acid substitutions. The likelihood function for a given tree can be determined according to a Markov chain model [Many computational tools have been developed for identifying amino acids that are important for protein function/structure, but there is no consensus regarding the best measure for evolutionary conservation . Evolutivergence , and thein model . We havein model ) that cain model  to restrThe phylogeny-based conservation of residues relies on sequence homology. It is well known, however, that many non-homologous proteins share similar folds . It is tFor each protein, we first calculate the sequence entropy at each position within a family of related sequences from the HSSP database  is the frequency of the class i of residues (for each of the six classes) at position l in sequence in the multiple sequence alignment. Then we use the FSSP database to obtain the structural alignment. The structural superimposition of different families was used to calculate the conservatism of conservatism (CoC)where sm(l) is the intrafamily conservatism within the family m at position l, and M is the number of families. The CoC is the measure of the evolutionary conservation of the specific sites within the protein fold. Because the CoC method does not distinguish between residues at the protein surface evolutionarily conserved for functional reasons and residues inside the protein core that are conserved because of their importance to the folding process, we use solvent accessibility data for the unbound molecules to eliminate those conserved residues located inside the protein core.where representative data training set to extract complex a priori unknown relationships, e.g., sequence correlates of protein-protein interactions. Examination of the resulting classifiers can help generate specific hypotheses that can be pursued using molecular and biophysical methods. For example, a classifier that is able to identify protein-protein interface residues on the basis of sequence or structural features can provide insights about sequence characteristics that contribute to important differences in function. The data mining approach for binding site identification consists of the following steps:Recent advances in machine learning  or data • Identify the surface residues in each protein.• Label each residue in each protein as either an interface residue or a non-interface residue based on appropriate criteria for defining residues in interaction sites.• Use a machine learning algorithm to train and evaluate a classifier to categorize a target amino acid as either an interface or a non-interface residue. Different types of information about the target residue  can be supplied as input to the classifier. A variety of machine learning algorithms ,54 can b• Evaluate the classifier  on independent test data (not used to train the classifier).• Apply the classifier to identify putative interface residues in a protein, given its sequence (and possibly its structure), but not the sequence or structure of its interaction partner.Here we have used a support vector machine (SVM) learning algorithm because SVMs are well-suited for the data-driven construction of high-dimensional patterns and are especially useful when the input is a real-valued pattern . In addiα atoms on the two interacting chains are less than 15 Å apart, reasonably well connected fragments suitable for threading studies are obtained. In the second stage, after identifying a set of candidate template structures, threading is performed to examine the probability that a given model resembles the real interface. The threading algorithm is described in Cao et al. [In phylogenetic and data mining approaches, the properties of the protein-protein interface are deduced by concentrating on the sequence information contained in the protein pair under investigation. However, it is well accepted that the physical origin of the specificity of protein-protein interactions comes predominantly from their structures. Thus, in any thorough investigation of protein-protein interactions, it is essential to include information from structural studies. Here we have adapted methods employed in protein structure recognition -63 to tho et al. . The thrDifferent approaches for identifying binding sites from amino acid sequence information yield different (sometimes contradictory, sometimes complementary) results. In such cases, approaches for combining results from multiple predictors have a potential importance. The key idea is that results obtained by using different approaches, which we will call classifiers henceforth, may be correlated  due to a variety of reasons including the use of a common dataset for constructing or tuning classifiers, use of intermediate variables for encoding input to the classifiers, and similarities between methods . Regardless of the source of statistical dependency, the goal is to develop methods for weighting the output of each classifier appropriately for the purpose of producing more accurate predictions. Our method takes as input the binary  output of each classifier (e.g., SVM, CoC) and produces as output a probability that the residue under consideration is an interface residue, using the outputs produced by each of the classifiers. Algorithms for learning Bayesian (or Markov networks) can be then used to learn the network of dependences and the relevant conditional probabilities.TP denote the number of true positives – residues predicted to be interface residues that are actually interface residues; TN the number of true negatives – residues predicted not to be interface residues that are in fact not interface residues; FP the number false positives – residues predicted to be interface residues that are not interface residues;FN the number of false negatives – residues predicted not to be interface residues that actually are interface residues. Let N = TP+TN+FP+FN. Sensitivity  and Specificity (precision) are defined for the positive (+) class as well as the negative (-) class. Sensitivity+ = TP/(TP+FN), Sensitivity-= TN/(TN+FP), Specificity+ = TP/(TP+FP), Specificity- =TN/(TN+FN). Overall sensitivity and overall specificity correspond to expected values of the corresponding measures averaged over both classes. The performance of the classifier is summarized by the correlation coefficient, which is given byLet The correlation coefficient ranges from -1 to 1 and is a measure of how predictions correlate with the actual data . It is iCY, VH and DD performed data mining calculations. XG performed phylogenetic calculations. KMH, CZW, YI, DD, and HC worked on threading. TZS, AK, and RLJ worked on the implementation of CoC and the development of consensus methodology. Every author contributed to the final draft of the paper.Comparison of individual methods for interface residue prediction for bovine α-chymotrypsin (1acbe).Click here for fileComparison of individual methods for interface residue prediction for porcine pancreatic trypsin (1avwa).Click here for fileComparison of individual methods for interface residue prediction for porcine pancreatic elastase (1flee).Click here for fileComparison of individual methods for interface residue prediction for kallikrein(1hiaa).Click here for fileComparison of individual methods for interface residue prediction for subtilisin BPN' (2sice).Click here for fileComparison of individual methods for interface residue prediction for carboxypeptidase A (4cpa).Click here for file


Genetic control of drug metabolism was well established by the 1960s, and pharmakokinetic-based individualized therapy was in use by 1973.Adjusting drug therapy to the individual, a common approach in clinical practice, has evolved from 1) dose adjustments based on clinical effects to 2) dose adjustments made in response to drug levels and, more recently, to 3) dose adjustments based on deoxyribonucleic acid (DNA) sequencing of drug-metabolizing enzyme genes, suggesting a slow drug metabolism phenotype. This development dates back to the middle of the 20 Despite tremendous advances in the science and technology of drug development, as well the emergence of guidance and consensus building among scientists, many clinicians, pharmacists, and consumers remain uninformed regarding the scientific basis of establishing bioequivalence, the generic-drug approval process, and the issues related to individualizing therapy in general ,8. The crelative risk reduction [RRR]) of therapy and thereby decrease the treatment's net benefit. Not prescribing an agent will effectively nullify the potential benefit to individuals, and when repeated frequently enough, failure to prescribe the agent will significantly decrease the benefit to the population as a whole. At other times, doctors tend to prescribe a drug more generally than clinical trials dictate. The treatment of a population with lower outcome prevalence (OP) decreases net benefit and may lead to harm. Overdosing may increase treatment-related harm, and underdosing may erode efficacy; both will result in diminished treatment benefits. Finally, noncompliance on the part of the patient may lead to a decrease in efficacy and a requisite decrease in net treatment benefit. If a patient reduces the dose without totally eliminating the drug, the risk of non-dose-related side effects of treatment may remain.Recent information  indicateThe relationship between the terms mentioned above, which govern treatment success, can be expressed mathematically as follows -12:Net Benefit = RRR * OP - HarmThe graphical representation in Figure equivalence/similarity, pharmaceutical equivalence/therapeutic equivalence, and bioequivalence/bioavailability. Problems are further compounded by the increasing use of generic drugs and the interpretation of such terms as prescribability and switchability.The difficulties in drug prescription that are mentioned above are not related to the physicians' training or experience but result instead from difficulties in relying with confidence on label claims of efficacy, safety, and interchangeable use of new drugs "within class." The common denominator among these problems seems to be an insufficiently well crystallized knowledge base regarding the proper use and interpretation of general terms such as Bioavailability (BA) indicates a measurement of the rate and amount of therapeutically active drug that reaches the general circulation and its presumed site of action [f action .Bioequivalence is the absence of a significant difference in the rate at which, and the extent to which, the active ingredients in pharmaceutical equivalents become available at the site of drug action in the body when administered under similar experimental conditions in an appropriately designed study. A product may also be considered bioequivalent to an innovator product if (a) the difference in rate of drug absorption between the two products is intentional and (b) no significant difference is found in the extent of absorption of the two products when they are evaluated under similar experimental conditions -17.Bioequivalence requirement refers to a requirement, imposed by the Food and Drug Administration (FDA), of in vitro and/or in vivo testing of specified drug products that must be satisfied as a condition of marketing [arketing .Pharmaceutical alternatives are drug products that contain the identical therapeutic moiety, or its precursor, but not necessarily in the same amount or dosage form as the same salt or ester. Each such drug product individually meets either the identical or its own respective compendial or other applicable standards of identity, strength, quality, and purity, including potency and, where applicable, content uniformity, disintegration times, and/or dissolution rates [on rates .To be considered pharmaceutically equivalent, two drug products must (a) contain identical amounts of the same active ingredients in the same dosage form, (b) be formulated to meet the same compendial or other applicable standards of quality and purity, and (c) generally be labeled for the same indications. However, pharmaceutical equivalents may differ in the excipients (e.g., flavors, preservatives) that they contain, as well as in their shape, scoring, packaging, and in certain circumstances, their labeling .Average bioequivalence involves assessment of pharmacokinetic parameters such as area under the curve (AUC) and peak concentration (Cmax), as well as calculation of a 90% confidence interval for the ratio of the averages of these parameters for the two products that are compared, usually a test product (T) against a reference product (R). The calculated confidence interval should fall within a conventionally established BE limit of 80% to 125% for the ratio of the product averages [averages . The cliSince the implementation of the FDA Bioavailability and Bioequivalence Requirements in 1977, the assessment of bioequivalence has been a subject of continuous debate ,20-22. Tpopulation and individual approaches reflect differences in the objectives of BE testing at various stages of drug development. These differences are embodied in the concepts of prescribability and switchability (interchangeability) [The average BE reflects comparison of population averages and therefore fails to assess the subject-by-formulation interaction variance . In contrast, the newer ability) -22.These concepts underscore the difference between the population and individual bioequivalence approaches. Population bioequivalence assesses total variability of the measure in the population, and it becomes important when physicians are initially prescribing a medication and they need to rely on the average performance of the drug product . In contPrescribability refers to the clinical setting in which a practitioner prescribes a drug product to a patient for the first time. In this setting, the prescriber relies on the understanding that the average performance of the drug product has been well characterized and relates in some definable way to the safety and efficacy information from clinical trials. Switchability refers to the setting in which a practitioner transfers a patient from one drug product to another. This situation arises with generic substitution, as well as with postapproval changes by an innovator or a generic firm in the formulation and/or manufacture of a drug product. Under these circumstances, both the prescriber of the drug and the patient should be assured that the newly administered drug product will yield safety and efficacy comparable to that of the product for which it is being substituted. However, such a switch may, in fact, occur without the patient's or clinician's knowledge, and this concern is addressed in equivalence studies designed to minimize the risk to the patient in both situations.Although average bioequivalence is the recommended parameter for most bioequivalence studies, the current FDA guide titled Statistical Approaches to Establishing Bioequivalence  recommenThe outcomes , can be measured as clinical or surrogate endpoints or as biomarkers. The relationship between exposure and outcomes, expressed as dose- or concentration-response curves that have efficacy and toxicity levels at their extremes, define the optimal dose and the therapeutic window . TheoretThe approach described above is a simplified framework of successive steps to be taken in phases I and II of the development of any drug, with the aim of generating data for drug labeling (i.e., data on dose-response [effectiveness and toxicity] relations of the new agent and how these depend on patient characteristics). The dose regimen of such a new agent, explored and established through phases I and II, is to be demonstrated in phase III as "safe and effective" for the claimed indication. Proper study design and research methodology, as well as appropriate statistical analysis, should be applied to ensure that the drug's effectiveness can be substantiated. Further, the outcome derived from two such randomized clinical trials should document that the estimate of the true treatment effect favors the new drug over the reference drug and that the toxicity of the new agent does not exceed acceptable limits. In other words, a first risk/benefit assessment would favor the new drug.external validity, meaning the extent to which the conclusions of a study would hold true for other persons, in other places and at other times. Examples from most therapeutic areas indicate that approved drugs may not work as expected when applied in broad community populations, in real-world settings, and among diverse practitioners operating under real-world constraints [Having come that far, a question justifiably arises: what logic leads to the conclusion that the new drug is likely to be effective in future patients? That is, are the treatment benefits generalizable to an actual clinical population? The question is extended to straints . In suchempiric confirmation at a conventional α level). However, despite all their methodological rigorousness, these trials may not entirely eliminate factors that influence bioavailability in earlier development stages and internal validity in later-phase studies .Conventionally, the dose-response trials mentioned above, exploring the behavior of biomarkers both cross-sectionally between individuals treated with different doses of an agent, and longitudinally within individuals as doses (or concentrations) change with time, supply the necessary information on the drug's pharmacologic action  -32.• Product-related factors, such as: physical and chemical properties of the drug , as well as variables related to manufacturing, formulation, or both (e.g., coatings, compression force, particle size, presence or absence of excipients) -33.To control for as many variables as possible, most bioequivalence trials are conducted with healthy volunteers as subjects, ,34,35 ancausal confirmation is much more complex than the empiric confirmation, even if the drug's previously established pharmacologic action is believed to ensure that the drug has the same intrinsic property that alters the clinical outcome – in the RCT patients – in a similar way and to a similar extent as in the previous patients.The A number of other causes, however, may interfere in the conduct phase of an RCT. These causes include• Confounding – a distortion of an association between an exposure and disease brought about by extraneous factors.• Interaction – the interdependent operation of two or more factors that produce an unanticipated effect.• Transience – an idiosyncratic property of a drug that displays its expected pharmacologic property when tested in one batch, but not in others.Many of the specific design and analysis features applied to RCTs  are meant to ensure that the possibility of confounding is minimized or eliminated. The same does not apply in the case of transience or interaction, for which independent evidence is needed to eliminate those possibilities.Drugs that tend to exhibit high degrees of variability in their pharmacokinetic profiles are known to complicate the assessment of bioequivalence ,36,37. TSmall changes in systemic concentration of such drugs can lead to marked changes in pharmacodynamic responses -22. A brA typical drug in this category is warfarin, which is widely used for its anticoagulant properties. For the most part, metabolism of S-warfarin occurs by means of the gene CYP2C9 . InhibitFactors that increase PT ratio or international normalized ratio (INR) response include:Endogenous factors: 11Exogenous factors: 117 specific drugs and 49 different classes of drugsFactors that decrease PT ratio or INR response include:Endogenous factors: 5Exogenous factors: 42 specific drugs and 24 different classes of drugsPhysician surveys have indicated that most clinicians favor more rigid bioequivalence guidelines for these types of drugs . Others Drug products suspected of having bioequivalence problems are listed in the Orange Book  . Such drIn light of serious concerns about risks incurred from using medical products, a variety of public and private agencies involved in health care are dedicating more attention to examining the current system of managing these risks. The main goal is to focus on the costs and value of better data concerning the incidence and causes of injuries from medical products and the roles of all stakeholders in risk management.The FDA has issued a concept paper  presenti• Important risk assessment concepts.• Generation and acquisition of safety data during product development.• Analysis and presentation of safety data in an application for approval.Risk assessment, defined as the process of identifying, estimating, and evaluating the nature and severity of risks associated with a product, should be continuous throughout the life cycle of any product, whereas the process of risk management intervention is intended to enhance the safe use of a product by reducing risk.systemic risk confrontation, the FDA, Center for Drug Evaluation and Research (CDER), and Center for Biologics Evaluation and Research (CBER) appear ready to develop and finalize, by fall 2004, guidance documents regarding risk assessment, clinical pharmacovigilance, and risk management. Table With regard to the current trend toward Determining the optimal initial dosage regimen (prescribability) and maintaining safety and efficacy outcomes when the regimen is changed in some way (switchability) demand careful decision making in the application of equivalence approaches. These approaches must be applied differently during the three phases of the drug development process, and the knowledge-base that is derived from this process must be transferred to and utilized by physicians and pharmacists to assist them in prescribing and dispensing medicines to patients. Consistent and appropriate management of equivalence approaches supports good assessment, management, and communication about risks associated with a therapeutic product, as expressed in product labeling, as well as in specifications and standards that control the quality of a therapeutic product in the marketplace.


Glucocorticoids are stress hormones that modulate a large number of physiological actions involved in metabolic, inflammatory, cardiovascular and behavioral processes. The molecular mechanisms and the physiological effects of glucocorticoids have been extensively studied. However, the involvement of glucocorticoid action in the etiology of the Metabolic Syndrome has not been well appreciated. Recently, accumulating clinical evidence and animal genetics studies have attracted growing interest in the role of glucocorticoid action in obesity and insulin resistance. This review will discuss the metabolic effects in the context of glucocorticoid metabolism and establish the association of glucocorticoid action with the features of the Metabolic Syndrome, especially obesity and insulin resistance. Special discussions will be focused on corticosteroid-binding globulin and 11β-hydroxysteroid dehydrogenase type 1, two proteins that mediate glucocorticoid action and have been implicated in the Metabolic Syndrome. Due to the complexities of the glucocorticoid biology and the Metabolic Syndrome and limited space, this review is only intended to provide a general link between the two areas with broad rather than in-depth discussions of clinical, pharmacological and genetic findings. Insulin resistance and hyperinsulinemia are often associated with a group of risk factors such as obesity, dyslipidemia, hypertension and impaired glucose tolerance. This cluster of metabolic abnormalities, first defined as Syndrome X by Reaven in 1988 ,3, is noGlucocorticoid (GC) excess has been linked to clinical observations associated with the Metabolic Syndrome. In Cushing's syndrome , increasin vitro but it is mainly a reductase in vivo [Cortisol, the principal active GC in humans, is secreted by the adrenal gland and is converted to cortisone, the inert GC, primarily in kidney -19. Two  in vivo . Since G in vivo -19. In aThe circulating cortisol level undergoes circadian variations peaking in the early morning at approximately 800 nM and reaching a nadir of about 200 nM at midnight . The plaBoth cortisol and cortisone are metabolized in liver first by the A-ring reductases followed by several steps of further structural transformation catalyzed by other enzymes . The finGC action is mediated by GR, a nuclear receptor that regulates physiological events through activation or repression of target genes involved in inflammation, gluconeogenesis and adipocyte differentiation ,36. UponAccumulating clinical evidence has demonstrated the association of abnormal GC metabolism and the Metabolic Syndrome. The plasma cortisol levels were increased in an elderly cohort with one or more features of the Metabolic Syndrome . Furtherob/ob or db/db mice slowed body weight gain [The clinical correlation studies raised the possibility that GC action could play a role in the origin of the features of the Metabolic Syndrome. This notion was further established and supported by animal studies to address the metabolic effects of GCs. Adrenalectomy in young ght gain . Upon GCght gain . Likewisght gain . The adrght gain . As the ght gain . Similarght gain . These eght gain , suggestThe metabolic effects of GCs are mediated by several mechanisms that are physiologically relevant to hepatic and peripheral insulin resistance, dyslipidemia, obesity and hyperglycemia. Events driven by these mechanisms take place across the tissues contributing to the abnormalities in the Metabolic Syndrome Fig. . In liveThese data, both physiologically and mechanistically, suggest that the metabolic effects of GCs are exerted in multiple tissues and increased GC action contributes to the etiology of the Metabolic Syndrome. Through molecular and genetic studies, more information has become available to dissect the role of tissue-specific GC action in the features of the Metabolic Syndrome. Genetic studies with the main players in GC action have been most revealing. Since GR has been well reviewed in other publications, this review will only discuss CBG and 11β-HSD1.CBG is not only in the blood but also found in tissues ,116. SinDespite the compensatory response by the HPA axis to balance the plasma free cortisol or corticosterone concentrations under conditions of CBG reduction or deficiency, tissue-specific GC excess can still occur. This is especially important with respect to GC-stimulated differentiation of pre-adipocytes and insulin resistance of mature adipocytes, with the former effect increasing fat content and the latter reducing the tissue sensitivity to insulin. For instance, preadipocytes from an individual with CBG deficiency had increased proliferation and enhanced differentiation compared to normal cells , which mBoth 11β-HSD1 and 11β-HSD2 are located at the endoplasmic reticulum (ER) but with distinct topologies. 11β-HSD1 has one short N-terminal transmembrane region with the catalytic domain protruding into the ER lumen; in contrast, the N-terminus of 11β-HSD2 is lumenal with the catalytic domain facing the cytoplasm -125. Theob/ob mice have reduced hepatic 11β-HSD1 activity but higher corticosterone level in liver due to their elevated plasma corticosterone [db/db mice [ob/ob mice, the 11β-HSD1 activity is decreased in liver but increased in omental fat in obese Zucker rats [Dysregulation of tissue-specific 11β-HSD1 expression and activity has been observed in obese diabetic animal models and humans. Compared with their lean littermates, osterone . As a re/db mice . As in oker rats ,130. Altker rats ,132, theker rats ,133,134.ker rats . In-situker rats . The assker rats . In a grker rats . This asker rats . The actker rats . These aker rats . It is iker rats . The conker rats . Interesker rats . These fGenetic studies using animal models support the findings in the clinical studies. In mice deficient in 11β-HSD1 generated through targeted gene disruption, there was no conversion of the inert 11-dehydrocorticosterone to corticosterone and attenuation of the hepatic activities of PEPCK and G6Pase, two key gluconeogenic enzymes . These mGiven its important role in the Metabolic Syndrome, antagonizing GC action has been taken as an approach to treat some features of the Metabolic Syndrome. Targeting GR is a direct approach to antagonize the GC action. The global effect on GC action by this approach could lead to the activation of the HPA axis as well as blocking the anti-inflammatory function of GCs. Inhibition of 11β-HSD1 activity offers more tissue specificity due to the limited expression pattern of this enzyme. Inhibitors for both 11β-HSD1 and GR include naturally occurring and pharmaceutically developed compounds.The expected effects of 11β-HSD1 inhibition include reduced hepatic PEPCK and G6Pase expression to reduce hepatic glucose output; reduced adiposity and improved peripheral insulin sensitivity. Since 11β-HSD1 mediated GC action inhibits glucose-dependent insulin secretion  and the GR antagonists were developed on the rationale that activated GR stimulates PEPCK and G6Pase, the two key enzymes in hepatic gluconeogenesis that increases the hepatic glucose output ,98,174. GCs are stress hormones with a wide spectrum of physiological effects and have been implicated in the pathophysiology of the Metabolic Syndrome. This notion has been supported by animal studies and clinical findings. The GC action appears to mediate certain aspects of the Metabolic Syndrome. In that regard, targeting key players in the GC action is expected to be a viable approach to treat some or all the features of the Metabolic Syndrome. However, cautions should be taken because the GC metabolism is regulated by the HPA axis and inhibition of GC pathways could lead to the activation of HPA axis and elevated adrenal cortisol secretion. To avoid the compensatory feedback response, efforts to separate the effect of GC modulators from HPA activity is needed. Although challenging, this could be achieved by tissue-specific modulation of GC action by targeting drugs to tissues of interest while sparing others, especially the CNS where HPA activation occurs. The availability of small molecule compounds will facilitate this type of studies in animal models to further dissect the regulatory function of the HPA axis and help assess whether tissue selective modulation of GC action without triggering the HPA axis is achievable.The author is an employed researcher in a biopharmaceutical company.


Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in  Drosophila spermatocytesTime-lapse confocal microscopy reveals a potential role for noncentrosomal microtubules nucleated near the nuclear envelope in spindle assembly in  Xenopus egg extracts  marker, we have been able to demonstrate such migration in living spermatocytes  are found at the periphery of these cells. The MTOCs of control cells at this stage can be seen near the nuclear membrane  marker γ-Tub23C  of the cells is anastral and establishes no contact with the membrane-bound centrosomes , 22% behaved like the cell shown in These observations strongly suggested that microtubules can nucleate in a centrosome-independent manner and assemble a spindle-like array in e before . Like ine asters , 11 min,e asters , 15 min,asp, and colcemid-treated spermatocytes is summarised in asp and in colcemid-treated wild-type spermatocytes, seems to be largely unaffected, suggesting that the feeble spindle checkpoint of these cells . As mentioned before, congression occurs  strain so that both chromosomes and microtubules could be visualised in the same cell ; Video 5n occurs ; Video 6n occurs  and end n occurs . In contn occurs ; Video 8n = 19) of asp cells. These cells, which contain unconnected centrosomal asters and anastral spindles, provide a valuable experimental system to assess the contribution of asters and spindle to specifying the place of cleavage. To this end, we plotted the angles between the line defined by the two asters, the major spindle axis, and the plane of cleavage in wild-type and asp spermatocytes . Although the bulk of microtubules has been described by time-lapse confocal microscopy to form over the chromatin  in 1 M sucrose. Upon dissection, their testes were prepared for in vivo imaging as described above. Once under the microscope, microtubules were allowed to repolymerise by a 1-s pulse of 350 nm light that inactivates the drug. For simplicity, this entire procedure of exposure to colcemid followed by light inactivation of the drug is referred to through this manuscript as ‘colcemid treatment.’asp (CG6875), Axs (CG9703), cnn (CG4832), γ-Tub23C (CG3157), His2AvD (CG5499). The GeneBank accession numbers discussed in this paper areGFP (U57609.1), Ran (NM_006325), RCC1 (D00679), TPX2 (BC020207), and YFP (U57609.1).The FlyBase accession numbers discussed in this paper are α-tubulin 84B (CG1913),


Malaria infections are often genetically diverse, potentially leading to competition between co-infecting strains. Such competition is of key importance in the spread of drug resistance.Plasmodium chabaudi. Mice were infected simultaneously with a drug-resistant and a drug-sensitive clone and were then either drug-treated or left untreated. Transmission was assessed by feeding mice to Anopheles stephensi mosquitoes.The effects of drug treatment on within-host competition were studied using the rodent malaria In the absence of drugs, the sensitive clone competitively suppressed the resistant clone; this resulted in lower asexual parasite densities and also reduced transmission to the mosquito vector. Drug treatment, however, allowed the resistant clone to fill the ecological space emptied by the removal of the sensitive clone, allowing it to transmit as well as it would have done in the absence of competition.These results show that under drug pressure, resistant strains can have two advantages: (1) they survive better than sensitive strains and (2) they can exploit the opportunities presented by the removal of their competitors. When mixed infections are common, such effects could increase the spread of drug resistance. Malaria infections often consist of more than one parasite genotype -3. Humande novo mutations arise, and when hosts acquire resistant and sensitive strains from one mosquito bite simultaneously or from different mosquito bites. In the absence of drug treatment, the transmission success of the resistant strain will depend on its intrinsic fitness and competitive ability. However, if drug treatment does occur, the resistant strain has two potential fitness advantages. First, it will better survive the drug than the sensitive strain. Second, treatment can remove drug-sensitive competitors, thus freeing up ecological space for the resistant strains to occupy; this would increase the relative transmission of the drug-resistant strain. This second effect, well recognized in theory, has the potential to greatly enhance the rate of spread of drug resistance in a population [Resistant and sensitive strains will co-occur in the same host both when pulation . HoweverPlasmodium chabaudi. This parasite is commonly used as a model for human malaria [Competition between drug-sensitive and -resistant malaria clones was studied using the rodent malaria  malaria , and has malaria . In the  malaria . Here, cPlasmodium chabaudi chabaudi clones were used: an AS clone resistant to the antifolate drug pyrimethamine [6 R parasites, and two groups with 106 R + 106 S parasites, as described elsewhere [Two genetically distinct ethamine , and AJ,lsewhere . One groAsexual parasite densities and gametocyte densities – the latter being the transmission stages to the mosquito – were monitored using microscopic examination of thin blood smears and determination of red blood cell densities using flow cytometry (Beckman Coulter), as described elsewhere . Real-tiAnopheles stephensi mosquitoes to feed on 3 mice from each group on each of days 7, 14, and 21 PI, as described elsewhere [e.g. [In the second experiment, two groups of nine mice were infected as above with either R parasites or R+S parasites. The subsequent transmission success of clone R was assayed by allowing batches of 30 starved re [e.g. ]. Eight All procedures were regulated and carried out under the British Home Office Animals (Scientific Procedures) Act 1986.Two untreated mice infected with R+S parasites died on days 10 and 11 PI respectively, and were excluded from the analysis.1,14 = 14.4, p = 0.002). Thus, R parasites were competitively suppressed in mixed infections in untreated mice, but this suppression was negated when mice were treated with pyrimethamine, which effectively removed S parasites.In untreated mice, there were far fewer R parasites during the acute phase in mixed infections with clone S than in R-only infections Figures . However1,14 = 13.8, p = 0.002).During the chronic phase, clone R was more numerous in untreated mice in mixed infections than in single-clone infections (due to the parasite peak around day 21; Figures The large peak of R parasites in the chronic phase in the untreated mixed infections around day 21 Figure  coincideThe second experiment assessed transmission to mosquitoes on days 7, 14 and 21 PI. It was found that the resistant clone R infected far fewer mosquitoes from mixed infections than from single infections (figure de novo [These results show that drug treatment of malaria infections can severely affect ecological interactions between co-infecting strains. The drug-resistant clone was competitively suppressed by the drug-sensitive clone in untreated mice, in terms of both within-host growth and transmission to the mosquito vector. However, drug treatment removed that competitive suppression, and allowed the resistant clone to fill the ecological space emptied, giving it a substantial and additional fitness benefit in addition to the simple survival advantage conferred by resistance. Thus, under drug pressure, resistant strains can have two advantages: they survive better than sensitive strains and they can exploit the opportunities presented by the removal of their competitors, thereby increasing their relative transmission. Competition was studied between two unrelated clones, and thus did not reflect the situation in which a resistant clone arose de novo , but it Competitive release following drug treatment will greatly enhance the spread of drug resistance . Also, wOf course, this is an argument for judicious use of drugs, not their non-use. Clearance of drug-sensitive strains from mixed infections might enhance the spread of drug resistance, but this has to be offset against the short-term public health benefits, such as reducing overall malaria prevalence. In these experiments, the drug-sensitive clone was also the more virulent clone , and wheIn this experiment, mice were drug-treated before symptoms occurred, resulting in competitive release. This situation perhaps best mimics the case of prophylactic drug use, or what might occur to new co-infections in high transmission areas where drug use is common. A battery of more complex experiments will be necessary to determine if competitive release occurs when treatment follows symptoms, and when drugs are used to treat semi-immune individuals. The facilitation observed in chronic infections Figures  suggestsP. chabaudi is now firmly established [Within-host competition in ablished ,15,16. Eablished . In oldeablished . Given tablished ,18 oftenablished , and theablished , it is hablished .JCdR and RC designed and performed the first experiment, while JCdR and ASB performed the second experiment. JCdR analysed the results and drafted the manuscript. ASB developed the real-time PCR assays for analysis of parasite populations inside mosquitoes. AFR assisted in designing both experiments and writing the manuscript. All authors read and approved of the final version of the manuscript.None declared.