Biomechatronics is the integration of biological components with artificial devices, in which the biological component confers a significant functional capability to the system, and the artificial component provides specific cellular and tissue interfaces that promote the maintenance and functional adaptation of the biological component. Based upon functional performance, muscle is potentially an excellent mechanical actuator, but the larger challenge of developing muscle-actuated, biomechatronic devices poses many scientific and engineering challenges. As a demonstratory proof of concept, we designed, built, and characterized a swimming robot actuated by two explanted frog semitendinosus muscles and controlled by an embedded microcontroller. Using open loop stimulation protocols, the robot performed basic swimming maneuvers such as starting, stopping, turning (turning radius ~400 mm) and straight-line swimming (max speed >1/3 body lengths/second). A broad spectrum antibiotic/antimycotic ringer solution surrounded the muscle actuators for long term maintenance, ex vivo. The robot swam for a total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum. The development of functional biomechatronic prototypes with integrated musculoskeletal tissues is the first critical step toward the long term objective of controllable, adaptive and robust biomechatronic robots and prostheses. Many technological barriers exist for the implementation of life-like mobility in robotic and prosthetic systems. Included among these barriers are (1) the availability of high-energy density storage media, (2) the availability of adequate muscle-like actuators, and (3) the availability of biologically inspired sensory technologies. As a possible resolution to these challenges, we consider in this investigation the use of living muscle tissue as a viable actuator for synthetic devices.Although important research has been conducted to advance a synthetic actuator technology with muscle-like properties, engineering science has not yet produced a motor system that can mimic the contractility, energetics, scalability and plasticity of living muscle tissue ,2. Musclanimal-derived muscle as an actuator for artificial devices in the millimeter to centimeter size scale. Perhaps researchers in the past did not consider muscle tissue a viable mechanical actuator because of tissue maintenance and control difficulties. The objectives of this study are to identify, and to begin to address, the many technical challenges related to maintaining and controlling explanted muscle tissues in the context of a robotic platform. To this end, we construct a hybrid swimming robot comprising a synthetic elastomeric tail actuated by a single pair of whole muscle explants from frog semitendinosus muscle. We anticipate that basic swimming maneuvers such as straight-line swimming and turning can be performed by alternately modulating electrical signals to each muscle actuator across two electrode pairs, one on each muscle near the neuromotor junction. We further anticipate that a multi-day robotic maintenance or lifespan can be achieved by surrounding the muscle actuators with a specific bath of amphibian ringer's solution comprising antibiotic and antimycotic agents. To test these ideas, we construct two robotic build-ups, each comprising a freshly dissected pair of explanted semitendinosus muscles. For each build-up, pilot data are collected to characterize the robot's swimming mechanics and lifespan.In this investigation, we examine the feasibility of using Rana pipiens) were pithed, and both semitendinosus muscles were dissected free and removed with tendons intact. Before removal of the tissues from the animal, the length of each muscle belly was measured at an equilibrium or rest length. The resting length measurement was conducted on the intact muscle specimen with the limb positioned at an anatomically neutral position . Briefly, adult frogs ; MEM Vitamin Mixture, 1:100 dilution (GIBCO # 1120052); Creatine, 1 mM; DL-Carnitine, 1 mM; Ferric Chloride, 0.9 μM; Human Serum Transferrin, 1.35 μM; Insulin, 1 mU/ml; L-Glutamine, 1:100 dilution; Sigma Chemical #A9909, 1:50 dilution (an antibiotic/antimycotic). A broad-spectrum, antibiotic/antimycotic was added out of necessity for long-term maintenance of the muscles, ex vivo. We observed, for periods greater than 24 hours, septic degradation of the muscle specimens in the absence of the antibiotic/antimycotic agents. After each muscle was placed within a Petri dish, a small volume of ringer solution was used to surround each muscle, the balance being used in the test tank for the swimming robot evaluations. The total amount of time between muscle removal from the animal to finalizing the muscle installation into the robotic swimmer was approximately 1 hour.Shortly before harvesting the muscles, two fresh liters of amphibian ringer solution were prepared according to a protocol specifically designed for frog organ culture ,15. The The test tank was constructed from 6 mm (1/4") thick cast acrylic sheet, welded together with methylene chloride, with silicone fish tank adhesive being applied to form a water-tight seal at each joint. The test tank was 30 cm square and 6 cm deep.The robotic platform Figure  was specThe single part silicone RTV (Dow Corning type 734 flowable silicone) tails were cast using a 5-part virgin Teflon mold machined to form a single solid tail assembly with all of the features shown in Figure The onboard electronics were based upon a previously published design for an implantable muscle stimulator , and thuStimulation was controlled remotely via a unidirectional infra red (IR) link from a hand-held command module. The on-board fixed stimulation parameters were: amplitude = 5.8 V  , frequenThe electrodes were fashioned from medical grade TFE coated 40 AWG stainless steel multi-strand electrode wire (Cooner Wire). The distal ends were stripped to allow the electrode wire to be wrapped around each muscle, as described previously . The finDuring robotic swimming operations, the fuel sources were a glucose-bearing ringer solution (~2 g/L glucose), and lithium batteries to power the embedded microcontroller and stimulator system. Due to the micro-power electronic design, the estimated battery life for the system was ~21 days (assuming a 10% stimulation duty cycle) ,17.The semitendinosus muscle was selected primarily due to its convenient size and tendon anatomy. It is easily dissected with both proximal and distal tendons attached. The proximal tendons of each semitendinosus muscle were sutured to the rigid Delrin head piece, and the distal tendons were sutured to the lateral mounting flange on each side of the tail base using 5-0 braided silk suture Figure . The musTwo robotic platforms were evaluated in terms of muscle actuator performance, swimming efficiency and locomotory maneuverability. Each robotic platform was designated "B1x", where "x" indicated the build-up, serialized as "a, b, c, ..." for each subsequent pair of explanted frog muscles. Two build-ups were constructed, B1a and B1b, each with a separate pair of freshly explanted frog semitendinosus muscles.Muscle Removal and Maintenance) were poured into the test tank, providing a fluid depth of approximately 2.1 cm, enough for the robot to swim without touching the bottom of the tank. The tank temperature was measured but not controlled, and was allowed to stabilize at room temperature, approximately 22°C for the duration of each experiment. The ringer solution was aerated with unfiltered room air using 4 standard porous stone fish tank aerators, one placed at each corner of the tank, and connected to an aquarium aeration pump via silicone tubing. Aeration was discontinued briefly before each test run to minimize turbulence in the test tank. For each robotic build-up, or for each pair of explanted semitendinosus muscles, the test tank ringer solution was not replaced or replenished for the entirety of the robotic experimental session.Prior to swimming evaluations, two liters of ringer solution . After installation was complete, the muscles were allowed to acclimate for a period of approximately 5 minutes before stimulation. The robot was manually placed to allow forward motion through the bath, and muscle stimulus parameters, specifically stimulus train duration and dwell time, were varied manually until the maximum swimming velocity was achieved. To increase swimming speed, dwell period was decreased and train duration was increased until further decreases in dwell time or further increases in train duration did not result in additional increases in forward swimming speed. During experimentation, swimming speed was determined by measuring the amount of time required for the robot to swim across a known, fixed distance. Once the maximum swimming speed was achieved, the ventral view of the swimming robot was filmed , and the film was then digitized to determine tail-beat frequency, tail amplitude, and the wave speed and wave length of the propulsive body wave. In addition to forward straight-line swimming, muscle stimulus parameters were varied to investigate turning maneuvers. At a maximum forward swimming speed, the robot's open loop, alternating stimulation pattern between the antagonistic actuators was switched to a continuous one-sided muscle activation for steering control, causing the robot to turn in the direction of the single stimulated muscle . Here again, the ventral view of the swimming robot was filmed, and the film was then digitized to determine the maximum turning radius.For each tail-beat period, at least 10 video frames were captured, separated in time by 33 ms, depending on the swimming speed of the robot. A customized software program was used to digitize 10 points on each side of the outline of the ventral silhouette of the robot, for a total of 20 points for each image. A series of cubic spline functions were used to draw the best-fit line along these points ,19, and To estimate the overall mechanical swimming efficiency of each robotic build-up, we calculated the robot's slip value, a dimensionless velocity . A high Muscle Removal and Maintenance). Still further, to estimate muscle-shortening velocity at the maximum swimming speed, the measured linear-distance change between muscle attachment points was divided by the time required for the tail to re-position from a neutral, straight position to the tail amplitude posture measured during straight-line swimming. This time period was measured from the digitized films and was equal to approximately one quarter of a tail-beat period.To estimate muscle actuator performance at the maximum swimming speed, muscle strain and shortening velocity were estimated using the tail-beat frequency and amplitude measurements taken from the digitized films. After the swimming experiments were finalized, the change in linear distance between the robot's muscle attachment points was measured when the robot's tail was re-positioned from a neutral, straight position to the tail amplitude posture measured during straight-line swimming. As an estimate of peak muscle shortening strain, this linear-distance change was then divided by the muscle's resting length, or the muscle belly length when the tail was held straight  and isotonic contraction experiments  were conFor the B1a and B1b robotic swimmers, the locomotory performance parameters at maximum swimming velocity are summarized in Table Each robotic build-up was capable of the following controlled maneuvers: forward accelerations, decelerations, steady state gliding, and turning to the right or left. The robot was capable of surface swimming only, so all maneuvers were restricted to 2-dimensions. Turning was accomplished after forward momentum had been established by continuously activating only one actuator. The minimum gliding turn radius was 400 mm as estimated from the digitized video images of the robot's midpoint trajectory.After swimming the full length of the test tank, the robot was manually repositioned to the opposite end of the tank where it began, once again, to swim across the tank width. Typically, a period of swimming activity (~3 min) was followed by a period of swimming inactivity (~30 min). Due to muscle fatigue, periods of inactivity were required to restore the robot's peak swimming velocity to at least 75% of its maximum value measured during the first session of robotic swimming (first 10 minutes of the robot's lifespan). Robot B1a swam for a sum total of 45 minutes over a 7.5 hour lifespan (10% duty cycle), after which its swimming velocity degraded below 75% of its maximum value even after a 30 minute period of swimming inactivity. In distinction, robot B1b swam for a much longer period – a sum total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum value following a 30 minute period of swimming inactivity.To compare the overall swimming efficiency of each robotic build-up, we calculated the propeller efficiency using the measure of slip (swimming velocity/ propulsive wave speed) Table . In a stmax, of 78 ± 3 mm s-1 (Mean ± S.E., N = 6 muscles) in isotonic contractions. At the maximum swimming speed, the muscle actuators within robots B1a and B1b experienced a shortening velocity of 25 mm s-1  produced a maximum shortening velocity, Vanimal-derived muscle as an actuator for artificial devices. We construct a simple robotic platform powered by explanted living amphibian muscle and controlled by an embedded microcontroller via an infra red data link. Using an open loop control and a simple interface design, we present preliminary data that suggests that living muscle might one day be employed as a practical, controllable actuator. Hybrid robot B1b remained active for up to 42 hours, and during that time, performed basic swimming maneuvers such as starting, stopping, turning and straight-line swimming at speeds exceeding 1/3 body lengths per second. The muscle-actuated swimming robot also offered a reasonable swimming efficiency, as indicated by a slip value of 0.32 , both for controllability and for tissue phenotype maintenance.The frog semitendinosus muscles employed in the robot were comprised predominantly of fast-twitch muscle fibers, and therefore provided higher mechanical power, at the expense of being considerably more fatigable, than would have been achievable using a slow-twitch muscle of comparable size. Ideally, a biomechatronic swimming robot would incorporate several muscle fiber types to permit both explosive as well as low-power locomotion and maneuvering. For the robotic platform of this investigation, it is important to note that the stimulation was non-physiologic in many ways. Each muscle was stimulated in bulk, with all fibers being subjected to approximately the same electric field. In living muscle in vivo, individual motor axons innervate one or more muscle fibers, establishing the fundamental neuromotor functional unit: a in vitro generally fall into one of the following categories: (1) core necrosis due to lack of oxygenation/capillary perfusion and large diffusion distances, (2) sepsis, (3) exogenous toxicity, (4) electro-chemical damage resulting from excessive electrical stimulation, (5) accumulated contraction-induced injury, (6) sarcomeres heterogeneity leading to loss of thick and thin filament overlap in regions of muscle fibers , and (7) direct mechanical damage to the muscle from external sources, such as the robot frame, attachment hardware, or electrodes.In this study, the performance of the muscle actuators eventually degraded to the point where they were no longer effective mechanical actuators. Several factors contributed to the observed tissue degradation. To begin with, explanted muscle generally has a very finite functional life expectancy ,15, usuaex vivo tissue maintenance are necessary. These include loss of muscle excitability and mass, phenotypic drift, and de-differentiation of the muscle from desired adult muscle phenotypes.For the tissue-actuated device of this investigation, several design considerations were made to minimize many of these failure modes. The bath was aerated to assist oxygen delivery to the tissues, although this strategy would only be helpful to the outer shell of muscle fibers no greater than ~200 μm from the surface. In addition, the level of muscle cell depolarization was kept to a minimum in order to limit electro-chemical damage . Still fEven though organogenic mechanisms are poorly understood, it is nonetheless possible to engineer functional muscle organs from individual cells in culture -26, but The results of this investigation, although preliminary, suggest that some degree of ex vivo robustness and longevity is possible for natural muscle actuators if adequate chemical and electromechanical interventions are supplied from a host robotic environment. Clearly, an important area of future research will be to establish processes by which optimal intervention strategies are defined for a given hybrid-machine task objective. Another important area of research will be tissue control. It has been established that natural muscle changes in size and strength depending on environmental work-load, and when supplied with appropriate signals, changes frequency characteristic or fiber type -11. HencIn this paper, we ask whether muscle tissue explants can be employed as mechanical actuators for robots in the millimeter to centimeter size scale. Using a very simple control and interface design, we present preliminary data that suggests that living muscle might one day be employed as a practical, controllable actuator. The robot of this investigation remained active for up to 42 hours, and during that time, performed basic swimming maneuvers such as starting, stopping, turning and straight-line swimming at speeds exceeding 1/3 body lengths per second. It is our hope that this work will lead to further studies of tissue actuated robots and prostheses that will result in an even wider range of biomechatronic machine capabilities.


Another study casts doubt on the value of early treatment and treatment interruptions. What are the implications of this study for our understanding of HIV pathogenesis, treatment, and vaccine development? The devastating effects of HIV infection worldwide are reason enough for AIDS researchers to grasp at thin rays of hope. But seldom has a single anecdotal case stimulated as much hope as the 1999 report of an acutely infected patient who appeared to control HIV replication after two short treatment interruptions [Consistent with this hypothesis was the prior finding that early antiretroviral therapy led to induction of HIV-specific proliferative responses similar to those that had been observed in patients with long-term, non-progressing HIV . This leA series of discoveries from clinical trials began to chip away at the enthusiasm for both early treatment of HIV infection and supervised treatment interruptions (STIs) as a way to boost the immune response.Several small trials of STIs in chronically infected patients were carried out , buoyed Larger trials clearly showed that STIs were of little if any benefit in chronic infection and that when therapy was stopped, viral loads invariably returned to pre-treatment levels . Other sPLoS Medicine that found that in 14 patients who were treated early and who had controlled viral loads for at least 90 days, the virologic control was only transient [Now comes a study in this month's ransient . While oThere was a disconnect between the low viral loads and an unexpectedly high rate of CD4+ T cell decline in several patients. While the small number of patients and the single-arm nature of the study preclude definitive comparisons, it is possible that the early treatment and STIs did not result in a delay in CD4+ T cell decline  beyond what would have occurred had the patients received no early treatment.This study raises important questions in our understanding of HIV pathogenesis, treatment, and vaccine development.First, why is it that early antiretroviral treatment, even if it does lead to better control of viral replication, does not protect against CD4+ T cell depletion? It is possible that by the time patients present with acute retroviral syndrome their CD4+ T cell reserves (in gut and lymphoid tissues) have been severely depleted, despite the fairly normal CD4+ profile of their peripheral blood. Thus, even low-level viral replication is then sufficient to deplete the remaining central and peripheral reserves .Second, how do these findings affect treatment guidelines during acute infection? None of the current treatment guidelines in either resource-rich or resource-poor settings recommend early antiretroviral therapy. In the light of these new data , there dbefore any HIV replication (a prophylactic vaccine) might be better able to control virus replication. Far be it for us to stop grasping at rays of hope.Finally, is this good or bad news for HIV vaccine development? Since most current vaccine strategies are based upon the hypothesis that induction of T cell immunity will lead to control of viral replication, it is difficult to be optimistic when a strong and broad immune response is unable to prevent disease progression. However, one must recall that phenotypic and functional assessments of HIV-specific T cell responses, even in antiretroviral-treated patients, show that these responses clearly differ from responses against viruses that are normally cleared or controlled by the immune system . Therefo


As human beings, aging and death are an inevitable part of our lives. As we pass through each decade, the concrete signs of aging—greying hair, aches and pains, the gradual failure of one organ system after another—and the realization that we are mortal increasingly occupies our thoughts.Saccharomyces cerevisiae (baker's yeast), for example, the function of individual cells gradually declines with time, and each yeast cell has a finite life span. In organisms like this, it has been proposed that reproduction by asymmetric division is a prerequisite for aging. In other words, for a unicellular organism to age, when it divides, it must give rise to a “parent” cell and a smaller offspring cell (as in yeast), which then has to go through a juvenile phase of growth or differentiation before it divides. At each cell division, the parent cell becomes older until it reaches its natural life span and dies.All other multicellular animals and plants also show clear signs of aging, as do some single-celled organisms. In the yeast Escherichia coli, a bacteria that reproduces without a juvenile phase and with an apparently symmetric division.But what about organisms that produce two apparently identical cells when they divide? Do such organisms age? The assumption has been for some years that cells that divide symmetrically do not age and are functionally immortal. Eric Stewart and colleagues have now tested this idea by analyzing repeated cycles of reproduction in E. coli is a rod-shaped organism that reproduces by dividing in the middle. Each resultant cell inherits an old end or pole and a new pole, which is made during the division. The new and the old pole contain slightly different components, so although they look the same, they are physiologically asymmetrical. At the next division, one cell inherits the old pole again (plus a brand new pole), while the other cell inherits, a not-quite-so-old pole and a new pole. Thus, Stewart and co-workers reasoned, an age in divisions can be assigned to each pole and hence to each cell.E. coli cell. In all, the researchers built up a lineage for 35,049 cells in terms of which pole—old or new—each cell had inherited at each division during its history. They found that the cells inheriting old poles had a reduced growth rate, decreased rate of offspring formation, and increased risk of dying compared with the cells inheriting new poles. Thus, although the cells produced when E. coli divide look identical, they are functionally asymmetric, and the “old pole” cell is effectively an aging parent repeatedly producing rejuvenated offspring.The researchers used automated time-lapse microscopy to follow all the cell divisions in 94 colonies, each grown from a single fluorescently labeled E. coli should now provide an excellent genetic platform for the study of the fundamental mechanisms of cellular aging and so could provide information that might ameliorate some of the unpleasantness of the human aging process.Stewart and his colleagues conclude that no life strategy is immune to the effects of aging and suggest that this may be because immortality is too costly or is mechanistically impossible. This may be bad news for people who had hoped that advances in science might eventually lead to human immortality. Nevertheless,


Fatigue is a crucial sensation that triggers rest, yet its underlying neuronal mechanisms remain unclear. Intense long-term fatigue is a symptom of chronic fatigue syndrome, which is used as a model to study the mechanisms underlying fatigue.Using magnetic resonance imaging, we conducted voxel-based morphometry of 16 patients and 49 age-matched healthy control subjects.We found that patients with chronic fatigue syndrome had reduced gray-matter volume in the bilateral prefrontal cortex. Within these areas, the volume reduction in the right prefrontal cortex paralleled the severity of the fatigue of the subjects.These results are consistent with previous reports of an abnormal distribution of acetyl-L-carnitine uptake, which is one of the biochemical markers of chronic fatigue syndrome, in the prefrontal cortex. Thus, the prefrontal cortex might be an important element of the neural system that regulates sensations of fatigue. Chronic fatigue is common and is reported in more than 20% of people seen in primary care . HoweverCFS is a clinically defined condition characterized by severe disabling fatigue and a combination of symptoms, the prominent features being self-reported impairments in concentration and short-term memory, sleep disturbances and musculoskeletal pain . The diaSixteen CFS patients  and 49 age-matched healthy control subjects  were enrolled in the study. They were recruited from the outpatient fatigue clinic in Osaka University Hospital  where more than 430 CFS patients, who met the diagnostic criteria of CFS [Voxel-based morphometry (VBM)  was perf2 = 0.46, P = 0.004; Figure We observed a significant reduction in gray-matter volume in the bilateral prefrontal areas of CSF patients Figure . The affThe present study provides the first report of focal gray-matter atrophy in the prefrontal cortex of CFS patients. Previous MRI studies of CFS revealed non-specific abnormalities: hyperintense small punctuated subcortical white-matter foci were observed predominantly in the frontal lobes  and theiPrefrontal pathology has been reported in MS with pathological fatigue . RoelckeIn the present study, right dorsolateral prefrontal-cortex atrophy was significantly correlated with the severity of fatigue, as measured by the performance-status scores. As the performance status rates the daily activities that trigger or aggravate fatigue, this correlated volume reduction might reflect a functional deficiency that makes patients susceptible to fatigue.A single site in the dorsolateral prefrontal cortex revealed the parallel between volume reduction and fatigue severity. This does not necessarily mean that it is fatigue-specific; instead, this area might be the part of the network that, when functioning sub-normally, results in pathological fatigue. Fatigue is also a symptom of diseases that affect the basal ganglia, and that interrupt the connection between the prefrontal cortex and thalamus . Hence, The dorsolateral prefrontal cortex has dense widespread subcortical and cortical connections . A serieThe dorsolateral prefrontal cortex also has widespread reciprocal corticocortical connections with posterior temporal, parietal and occipital association areas . FurtherThese anatomical and functional characteristics of the frontal-subcortical circuits suggest that the large decrease in acetyl-L-carnitine uptake in the dorsolateral prefrontal, anterior cingulate and temporal cortices  represenThe results of the present study suggest that the dorsolateral prefrontal cortex might be an important component of the neural substrates that regulate the sensation of fatigue.BA, Brodmann's area; CMRGlu, cerebral metabolic rate of glucose; CFS, chronic fatigue syndrome; MRI, magnetic resonance imaging; VBM, voxel-based morphometryThe author(s) declare that they have no competing interests.TO carried out the MRI scanning, data analysis and drafted the manuscript. MT conducted MRI scanning and participants' coordination. HK conducted the medical evaluation of the participants. YW and NS participated in the study design. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Plasmodium falciparum infection. The feasibility of studying the P. falciparum in vivo transcriptome to understand parasite transcriptional response while it resides in the human host is presented.Both host and pathogen factors contribute to disease outcome in P. falciparum 3D7 laboratory strain chromosome 2 sequence was used to detect in vivo P. falciparum transcripts. This study analyzed transcripts from total RNA derived from small blood samples of P. falciparum infected patients and compared the in vivo expression profile to the in vitro cultivated 3D7 strain transcriptome.A custom made oligonucleotide array with probes based on the in vivo transcription can be studied from a small blood sample, despite the abundance of human RNA. The in vivo transcriptome is similar to the 3D7 ring stage transcriptome, but there are significant differences in genes encoding a sexual stage antigen and surface proteins.The data demonstrated that P. falciparum can be carried out successfully and further studies in selected patient cohorts may provide insight into parasite in vivo biology and defense against host immunity.Whole genome transcription analysis of  Plasmodium falciparum infection remains a major health problem worldwide. Its complex life cycle has hampered standard methods for the study of pathogenesis. New approaches to elucidate parasite biology using whole genomic methods have provided insight into gene function, transcriptional regulation and stage specific biology [in vivo biology of this pathogen, through adaptation of a whole genome approach, would provide insight into the host-parasite relationship, parasite virulence factors and inform new strategies for intervention. Genomic scale transcriptional profiling of P. falciparum during a natural infection is presented. Small amounts of parasite RNA, isolated from a few milliliters of a blood sample are found to be sufficient for whole genome transcriptional analysis. This data show that several genes are differentially expressed in vivo, indicating differences between the transcriptional program of 3D7 laboratory strain parasites growing in culture and naturally occurring infections in the human host. biology -4. Charain vivo conditions [Pasteurella multocida in vivo as compared to in vitro conditions [P. falciparum gene expression patterns, particularly the subset of genes that are specifically expressed in the in vivo state may identify unique parasite biology when it resides in the host environment. Processes involving parasite metabolism, immune evasion and transmission may be altered in the highly specialized environment of the human host as compared to in vitro conditions. In addition, approximately 12% of P. falciparum's predicted genes have not been found to be expressed in any of the life cycle stages previously studied [in vivo may reveal the unique expression of such genes in vivo, providing additional targets for intervention.Whole genome expression has been used in studies of bacterial pathogenesis to identify genes that are specifically transcribed under nditions -7. For enditions . Similar studied . Whole gP. falciparum chloroquine resistance study in Senegal [P. falciparum malaria gave consent for the study and were enrolled at an outpatient health clinic. Patients underwent venipuncture using K3 EDTA coated Vacutainers (Beckton Dickinson) and from this sample, 1.6–2.5 ml of whole blood was collected and passed through a white cell depletion filter using a 20 ml syringe. The filtered sample was centrifuged for 5 minutes at 3,200 rpm in a clinical centrifuge and placed in Tri-Reagent BD (Molecular Research Center). The samples were vortexed and stored at minus 70°C. Samples were thawed in a room temperature bath one month later and isolation of RNA was completed per Molecular Research Centers protocol. Three samples obtained in Senegal that had the highest parasitemia and largest blood volume are presented. A 14 ml blood sample from a P. falciparum infected traveler from Nigeria was similarly processed in Boston.This study was conducted as part of an ongoing  Senegal . PatientLabeling and hybridization of total RNA was performed as described . Expressin vivo sample and 3D7 ring stage total RNA using Super Script 1st Strand synthesis system (Invitrogen). Duplicate reactions using real time PCR were performed with 1 μl cDNA with gene specific primers in 50 μl reaction volume using fluorescent dye SYBR Green (SYBR Green PCR Master Mix, Applied Biosystems). The reactions were carried out on an ABI PRISM model 7700-sequence detector and all PCR reactions amplified a single product as determined by dissociation curve analysis (Dissociation Curve Software, Applied Biosystems).To confirm array data, a subset of genes (PFB0120, PFB0100, PFB0270 PFB0355, and PFB0065) that vary from high to low level abundance by array were quantified using real time PCR from cDNA. PFB0120: forward primer 5'-CAG CCC TCT TAG CTC TCA ACT TC-3', reverse primer 5'-AGC AAC AGC AGA GGC TAT AGA ACT-3', PFB0100: forward primer 5'-CAC CAA ATG GCT ATG CTT ATG GA-3', reverse primer 5'-TTC CAG GAG CAC CAT TAA ATC CT-3', PFB0270: forward primer 5'-ACA CTT ACT GGT ATT TCG GAA TTT-3', reverse primer 5'-TAA TTG TCC ATA TTC TTC AAT ATA T-3', PFB0355: forward primer 5'-ATT GTA AGA AAT AGT TGG GGT-3', reverse primer 5'-TAT ATC ATG CTC CTT CTT ATC A-3', PFB0065: forward primer 5'-CGT TGG TAG TGC GTT CCT TAC AA-3', reverse primer 5'-GTT CCT GCT ATA TCA GGA GCA CCA-3'. Sequence analysis confirmed the identity of the amplification products. 3D7 strain parasites were cultivated under standard conditions and synchronized with 5% sorbitol to obtain ring stage parasites for extraction of total RNA . cDNA waPlasmodium genes between the 10th and 90th percentile with at least six probes. Analysis based on rank was a second method used; in each experiment the probe intensity was ranked and this resulted in equivalent quantile distribution for all probes between two experiments. This method is more conservative and will define relative rank changes between experiments and is independent of potential normalization artifacts.Variation between samples was assessed using Kruskal-Wallis method (non-parametric ANOVA) to test the null hypothesis. To normalize samples, the mean gene expression level was calculated for all Patient blood samples were collected after informed consent was obtained. The study was approved by the institutional review boards at Harvard School of Public Health, Brigham and Women's Hospital and Cheikh Anta Diop University.in vivo isolated samples a denaturing RNA gel was carried out . The most abundant transcript of human origin in the in vivo was haemoglobin RNA . Further analysis was performed on the Nigerian sample. Only one parasite line was detected in this sample through DNA genotyping of the K1, MAD20, RO33 alleles of msp1 and FC27 and IC1 alleles of msp2 using primers and methods previously reported [Plasmodium genes on the array [P. falciparum chromosome 2 expression levels utilizing 15 μg to 30 μg or 15 μg to 60 μg cRNA were 0.95 and 0.92, respectively. However, as cRNA concentration was increased to 120 μg, the background to noise ratio increased significantly, resulting in a decreased correlation coefficient (R = 0.72)  Figure  and 2c.in vivo sample are listed in Table in vivo sample compared to 3D7 ring stage previously reported using the Kruskal-Wallis method [in vivo.The most abundant transcripts detected from the Nigerian s method . Notablyin vivo overexpression of a SERA antigen (PFB0355), the relative expression of five genes that had varying transcript abundance by array was carried out using real time PCR of cDNA generated from total RNA isolated from the Nigerian in vivo sample and a 3D7 in vitro ring stage sample. There is good correlation between the array results and those obtained by real time PCR (Figure in vivo and in vitro samples, the data is normalized to cDNA of PFB0120 to account for differences in starting parasite cDNA, secondary to human cDNA. The in vivo sample contained 0.15 ng cDNA of PFB0355c and 3D7 ring stage cDNA had 0.09 ng by real time PCR. When PFB0355c is normalized to PFB0120c, it was found to be ten fold overexpressed in vivo as compared to in vitro, consistent with the array results.To confirm the accuracy of the results from the oligonucleotide array and to confirm the igen PFB055, the rin vivo whole genomic expression can be performed despite the potential technical challenges of scarce RNA contained in a small blood volume sample and presence of abundant human RNA. Tri-Reagent BD was used to stabilize RNA and the samples were stored at -80°C before transport to the US. The denaturing gel suggests that the RNA remains intact using this reagent. Notably Kyes et al. reported that minus 80°C rather than 4°C or minus 20°C is the optimal temperature to store field sample RNA for detection of long transcripts such as var [in vitro sample [in vivo samples as compared to the 3D7 in vitro sample. Although human red cells are used for culturing in the in vitro system, reticulocytes which contribute to the haemoglobin detected are not abundant in in vitro samples. This is most likely due to the observation that reticulocyte levels display a 75% loss at 48 hours, when placed at 37°C, which is the condition of in vitro culture [in vivo samples and may be secondary to white cell contamination. Cross hybridization of human RNA to parasite probes may occur. However, Zhou et al have shown that human genomic DNA does not highly cross hybridize to the parasite probes on this custom array [in vivo parasite transcripts when using these probes.This data confirms that h as var . Surpris culture . In addiom array . The higom array ,19. Due in vivo samples it was not possible to determine how much parasite RNA is required for whole genome analysis. The samples from Senegal were of low parasitemia and small volumes, whereas the 14 ml blood sample with 0.4% parasitemia was sufficient to detect a greater number of chromosome 2 transcripts. Four to five mls of packed blood in a patient with a greater than 2% parasitemia should provide sufficient material for whole genome analysis using these methods. The data demonstrates the reproducibility of the method by independent hybridizations and that maximal senstivity can be achieved with up to 60 μg of cRNA, using this array.The amount of blood volume necessary for comprehensive detection of transcription depends on level of parasitemia and method of microarray analysis. This analysis utilized the Affymetrix system which requires very little starting RNA as compared to other methods . Due to in vivo transcript in all samples encoded on chromosome 2. This gene is a member of a recently described gene family, etramps, expressed at early ring stage encoding a protein thought to be involved in erythrocyte remodeling; this was also the most highly expressed transcript in in vitro ring stage cultures [in vivo expressed genes from the Nigerian sample was compared to that of the in vitro 3D7 ring stage chromosome 2 transcriptome [in vivo ring stage transcriptome and the 3D7 ring stage transcriptome. Most of the genes that are expressed in vivo are also expressed in vitro particularly those involved in cellular function and genes encoding hypothetical proteins (Table in vivo samples. Previously this gene has been demonstrated to be expressed and transcribed only in the sexual transmission stage [in vivo samples may have more parasites that are undergoing or are committed to sexual development than is detectable by microscopy. Several genes encoding membrane proteins, including SERA and rifin genes, were also found to be differentially expressed. These genes are members of multigene family that encode surface proteins which are thought to be involved in immune evasion [in vivo could be due either to genetic or transcriptional modulation of the parasite's defense repertoire or geographic variation. Differences between the in vivo and in vitro expression of SERA antigens may be due to differences in the in vivo stage of development as this gene family is transcribed at later stages in the in vitro life cycle [in vitro samples due to higher parasite counts and a subset of genes were found to be overexpressed in vitro after normalization. This analysis focussed on genes overexpressed in vivo as these results would not be influenced by normalization algorithms. Overall, this data demonstrates that P. falciparum transcripts can be detected from in vivo samples, and that there are potentially important differences between trancription of in vivo samples and that of the 3D7 in vitro trancription profile.PFB0120w is the most abundant cultures ,22. The on stage . Since n evasion ,24. The fe cycle . There wP. falciparum can be studied in vivo from a small blood sample of an infected patient. The in vivo sample however contains human RNA, whose quantity may vary from sample to sample and therefore differenes in parasite transcript level between samples must be reported relative to a reference transcript. Despite the abundance of human RNA the genomes are sufficiently different with resultant probes specificity. Predictably, there was a high correlation of in vivo expression with the in vitro ring stage 3D7 transcriptome [in vivo and in vitro expression levels in genes typically found in transmissable forms and encoding variant surface proteins. Evaluation of trancription of genes specific for gametocyte development in specific patient populations may uncover the in vivo conditions that favor development of transmissable forms. Similarily, a whole genome analysis can comprehensively characterize expression of multigene families that encode variant surface proteins under in vivo conditions. Further exploration of the in vivo biology of P. falciparum using specific probes to all annotated genes will be undertaken to confirm and explore other important biological differences. This new approach will further the understanding of the host-pathogen interaction and may result in the development of new strategies to combat this disease.In summary, this study provides evidence that whole genome gene expression in criptome . Importa


Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells.Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS.Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions Multiple large-scale analytical techniques to assess gene expression in defined cell populations have been developed. These include microarray analysis, EST enumeration, SAGE and MPSS. Each of these techniques offers unique advantages and disadvantages. Technique selection largely depends on the expertise of the investigator, the cost, the availability of the techniques, the amount of RNA/DNA that is available, and the existence of the genome databases. The human genome dataset is the best annotated one available ,2- makinHuman ES cells have been isolated relatively recently and ES cell genes are underrepresented in current databases. More importantly, recent evidence has suggested that mouse ES and human ES cells differ significantly in their fundamental biology ,4 and onWe selected MPSS for this analysis as it offers some unique advantages over other methods including SAGE ,16. MPSSWe have utilized MPSS using a pooled sample of three human ES cell lines grown in feeder-free culture conditions over multiple passages ,18 to asA pooled sample of undifferentiated human ES cell lines H1, H7, and H9 grown in feeder-cell free conditions  was usedPooled mRNA of the three hESC lines was subjected to MPSS analysis at Lynx Therapeutics (Hayward, CA), generating 22,136 distinct and significant signature sequences from a total of 2,786,765 sequences  that were elevated in ES cells when compared to neural stem cells examined in a similar manner was used. This list is provided in supplementary materials . A list Recently we and others have begun examining hESC with EST scan  and micrComparison with an EST scan analysis of 37,081 EST sequenced from a similar pooled sample of hESC ,10 also Richards et al  have useOur results provide a global overview of the gene expression pattern of undifferentiated human ES cells and allow comparisons with other data sets. These results suggest the hESC are an actively dividing population of cells that exhibit high metabolic activity. Our analysis detected expression of approximately 10,600 unique transcripts, a figure that about a third of the total number of mapped genes. Unlike other cell types, however, a much larger fraction of unknown or novel genes was present. This high ratio likely represents the paucity of information available in existing libraries on this relatively newly characterized cell population rather than the possibility that ES cells use radically different pathways for self-renewal, survival, proliferation and differentiation.Our results confirm the reported differences between rodent and human ES cells. We confirm the absence of expression of ERAS, Ehox and the orthologs PEPP1 and 2. The apparent lack of LIF requirement of hESC is reflected by the absence or low tpm levels for genes of the LIF pathway and high tpm for suppressors of LIF mediated signalling (see supporting information). The high level of expression of genes in the FGF pathway likely reflects the requirement of hESC for bFGF. The high level of FGFR1 expression suggests that FGFR1 is an important signal transducer and that FGF's other than FGF4 are important in hESC self-renewal. The high tpm of the fibronectin receptor also suggest that fibronectin or vitronectin are likely useful substitutes for matrigel and that activation of ras mediated signalling is likely critical, as has been described in the rodent ES cell analysis .Comparing data from the MPSS analysis with microarray, SAGE and EST scan analyses suggest that MPSS is a powerful alternative to these techniques. MPSS identified virtually all of the genes highlighted as genes common between six different human ES cell lines surveyed by microarray. We noted that most genes detected by microarray were expressed at high tpm indicating that MPSS is more sensitive than microarray analysis. MPSS however appeared to be able to identify genes detected by microarray. Analysing an additional 400 markers detected by MPSS using focused microarray or RT-PCR confirmed their expression ,  we predict that even a 5% contamination can confound results or detailed comparisons across different laboratories.We note also that gene transcription from both the X and Y chromosome is observed indicating that at least subtle differences will exist between male and female lines even in the undifferentiated state. Sex-based gene expression, along with MHC gene expression and ratio of expression of imprinted genes could serve to distinguish between different ES cell populations. The present results further suggest that analysing embryoid bodies that differentiate stochastically or analysing tissue samples (with variable proportions of cells) by MPSS will prove more difficult and that results will be variable. We suggest that variability can be reduced by pooling samples, normalizing by careful testing for known markers of differentiation, by semi quantitative PCR, or by focused microarray analysis.While MPSS is cost-effective and sensitive, it is by no means perfect. MPSS is limited by the requirement that DpnII sites (GATC) be present in a gene and be present in a unique locus such that the signature obtained is unique. For example, SNRF expression could not be assessed directly, as no GATC site is present. The signatures for ZFP42 are ambiguous and map to multiple transcripts. Although MPSS can distinguish between alternate transcript termination sites, MPSS cannot distinguish between alternative splicing events and possible incomplete digestion during the sample preparation process. Signature lengths are relatively short and it is possible to have to select between multiple genome hits  that had been maintained in feeder free culture conditions and evaluated for the presence of ES cell markers and absence of markers of differentiation. The mRNA was converted to cDNA and digested with DpnII. The last DpnII site and the downstream 16 bases were cloned into Megaclone vectors and their sequences determined according to the MPSS protocol ,16,25. Avirtual signatures") from the human genome sequence, the human Unigene sequences, and human mitochondrion. Each virtual signature was ranked, as outlined in the table To generate a complete, annotated human signature database, we extracted all the possible signatures . By thiTotal RNA was isolated from cell pellets using RNAeasy Qiagen mini protocol and kit. cDNA was synthesized using 100 ng of total RNA in a 20-μl reaction. Superscript II (Gibco-BRL), a modified Maloney murine leukemia virus RT, and Oligo (dT)12–18 primers were used according to the manufacturer's instructions (Gibco-BRL). The list of primers used for RT-PCR and annealing conditions are described previously ).RB, IK and MR were primarily responsible for the data analysis and writing and editing the manuscript. ST generated the ES cell samples and verified their quality, TM performed the RT-PCR and JC performed the immunocytochemistry. RP generated the microarray data and TV and JL provided support and supervision for RB, IK, ST.The document describing details of MPSS analysis of the HuES cells performed at Lynx, and algorithm for initial MPSS signature annotation and classification.Click here for fileThe file contains MPSS data for 22,136 significant and reliable signatures, annotated with genome version hg15 (April 2003, Golden Path, UCSC) and the human Unigene build Hs.161.Click here for fileThe file contains MPSS data for 22,136 significant and reliable signatures, annotated with genome version hg16 (July 2003, Golden Path, UCSC) and the human Unigene build Hs.169.Click here for filetable containing data for 8679 unigene clusters, 11 mitochondrial genes, and including 1991 signatures that did not map to unigene but uniquely matched genomic sequences .Click here for fileTop 200 rows from the table HuES17_onetpmHs169hg16.xls.Click here for fileList of tpm of genes identified by Sperger et al [11] present in the MPSS dataset.Click here for fileList of tpm of genes identified by Richards et al [8] present in the MPSS dataset.Click here for fileList of tpm of genes identified by MPSS in the BG02 dataset.Click here for fileThis file lists the genes located on the X and Y chromosome for which a MPSS signature sequence was found in the HuES cells. The X and Y chromosome genes are listed in separate worksheets. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband Signature: MPSS signature sequence Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Seq_id: Repeat if hit genome more than 100 locations; UniGene cluster ID if hit one or more UniGene clusters; Chromosome number if hit one genome location; MultiGenome if hit genome multiple times; Description: description of annotation (see Appendix B for more details).Click here for fileList of all UniGene clusters (release 161) located on the X and Y chromosome. The X and Y chromosome genes are listed in separate worksheets. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband Locusid: Locus Link identifier UniGene cluster: UniGene cluster ID Description: description derived from UniGeneClick here for fileGraphical representation of hotspots and coldspots for all human chromosomes. Cytobands with significantly higher number of genes in HuES cells are marked with '+' and cytobands with lower number of genes in HuES cells are marked with '-'. The number of '+' or '-' signs is proportional to the difference, with one '+' or '-' representing the fold difference. For example '++' represents a 2-fold higher number of genes expressed in HuES cells compared to the expected number based on the number of genes known to be located in the same cytoband.Click here for fileList of all chromosal regions that appeared statistically distinct.Click here for fileList of genes expressed in HuES cells and located in the hotspots and coldspots. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband Signature: MPSS signature sequence Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Seq_id: Repeat if hit genome more than 100 locations; UniGene cluster ID if hit one or more UniGene clusters; Chromosome number if hit one genome location; MultiGenome if hit genome multiple times; Description: description of annotation.Click here for fileList of all UniGene clusters located in the hotspot and coldspot regions. Chromosome: X or Y chromosome Cytoband: Giemsa staining cytoband UniGene ID: UniGene cluster ID Locusid: Locus Link identifier Description: description derived from UniGene;Click here for fileThis file contains the mapping information of MPSS signatures to the EST scan genes for major signaling pathways. Each pathway is presented in a separate worksheet Columns: Locusid: Locus Link identifier Pathway: Pathway the gene has been assigned to. Signature: MPSS signature sequence Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Stdev: standard deviation of the mean abundance from multiple MPSS runs Hit genome: HitGenome – Numbers of genomic locations a signature maps to HitUniGene: Numbers of UniGene Clusters a signature maps to; Seq_id: UniGene cluster ID ('Hs.' Has been omitted). NULL if there is no MPPS signature but an EST from the EST scan. Class: signature class (see Appendix B for more details); Title: description, derived from UniGeneClick here for fileThis file contains additional lists of potential homeodomain proteins, genes of unknown function, zinc finger proteins in separate worksheets. Tpm: mean abundance for a signature derived from all MPSS runs for the sample, in transcripts per million Stdev: standard deviation of the mean abundance from multiple MPSS runs Hit genome: HitGenome – Numbers of genomic locations a signature maps to HitUniGene: Numbers of UniGene Clusters a signature maps to; Seq_id: UniGene cluster ID. Class: signature class (see Appendix B for more details); Title: description, derived from UniGene.Click here for fileList of tpm of potential 400 ES enriched genes in other cell lines examined.Click here for fileList of all tpm of potential 13 ES novel genes in all tissues and cell lines examined.Click here for file


Retrovirology provide possible molecular mechanisms for the pro-apoptotic effect of Tat, which involve the cell's microtubular network and the mitochondrial pathway of apoptosis.The Tat protein of HIV-1 is a powerful activator of viral gene expression. Besides this essential function at the HIV-1 promoter, the protein also exerts a remarkable number of other biological activities, among which the induction of cellular apoptosis. Two papers now published in  Although more than 20 years have passed since the identification of HIV as the cause of AIDS, several essential questions about its pathogenicity remain as yet unanswered. In particular, a central, still unresolved issue is the mechanism underlying the progressive development of immunodeficiency. It is now well established that HIV infection determines a rapid turnover of infected CD4 cells ,2; consiIn this context, the finding that the virus-encoded Tat protein is released by the infected cells and can be taken up by neighboring, uninfected cells via an endocytic mechanism ,7 has loA link between microtubule polymerization and the pro-apoptotic effect of Tat has first been suggested a few years ago in the observation that Tat directly interacts with the αβ-tubulin dimers and polymerized microtubules in the cytoplasm of the cell . The funRetrovirology extend the link between the microtubule network, the mitochondrial pathway of apoptosis, and Tat. De Mareuil and coworkers show that Tat enhances tubulin polymerization into microtubules, an effect similar to that exerted by the MTPAs, and physically associates with the polymerized microtubuli [Two papers now published in rotubuli . As oppoThe accompanying manuscript by Epie and coworkers describes the identification of a microtubule-associated protein, LIS1, which specifically binds Tat . In the What might be the relevance of these findings in the context of HIV-1 infection? They clearly provide a mechanism for CD4 T-cell apoptosis and for the extension of the apoptotic effect to bystander, uninfected cells in the lymph node. Moreover, the interaction of Tat with the microtubular network might explain the occurrence of neuropathogenesis accompanying the progression of HIV disease, since many human neurodegenerative conditions are elicited by a reorganization of the neuronal cytoskeleton . Thus, tAs commonly happens in biology, the findings reported in these manuscripts raise more questions than answers. First, the Tat domains involved in the described interactions are different, a surprising finding given the very small size of Tat. This observation might possibly suggest that Tat is part of a large multi-molecular complex associated with the tubular network, making multiple contacts with different proteins. This issue can be experimentally addressed biochemically, or even within the cell, by taking advantage of the biophysical techniques available to investigate protein-protein interactions in vivo . SecondlMTPAs: microtubule-targeted, tubulin-polymerizing agentsCTD: carboxy-terminal domainThe author(s) declare that they have no competing interests.


GGPP synthase gene in Coleus forskohlii Briq.Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.The open reading frame for full-length This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots. Coleus forskohlii Briq. (Lamiaceae) [C. forskohlii has been used as an important folk medicine in India. Futher, forskolin has been found to be a potent activator of adenylate cyclase [Forskolin, a labdane diterpene, is a major active compound isolated from tuberous roots of miaceae) . C. fors cyclase , leading cyclase ,4.5) is a common metabolic precursor of all isoprenoids. Recently, several groups have demonstrated that two distinct pathways synthesise IPP in plants. The mevalonate (MVA) pathway occurs in the cytoplasm, and an alternative mevalonate-independent  pathway occurs in plastids [Isoprenoids are essential for the normal growth and development processes in all living organisms. Isopentenyl diphosphate IPP; C is a complastids .Geranylgeranyl diphosphate (GGPP) synthase catalyses the consecutive condensation of an allylic diphosphate with three molecules of IPP to produce GGPP, an essential linear precursor for biosynthesis of diterpenes, carotenoid, retinoids and side chain of chlorophyll . GGPP syGGPP synthase genes have been cloned in a number of organisms including; Arabidopsis thaliana [Taxus canadensis [Helianthus annuus [Scoparia dulcis and Croton sublyratus [Sulfolobus acidocaldarius [Neurospora crassa [thaliana ,10, Taxunadensis , Heliants annuus , Scopariblyratus , Sulfoloaldarius , Neurospa crassa , and moua crassa . Amino aa crassa . Futherma crassa ,18.crt genes) of Erwinia uredovora was elucidated [crt gene cluster is composed of six genes; crtB (phytoene synthase), crtE (GGPP synthase), crtI (phytoene desaturase), crtX (zeaxanthin β-glucosidase), crtY (lycopene cyclase) and crtZ (β-carotene hydroxylase). Consequently, the production of carotenoids using E. coli harbouring the crt gene cluster can be used for the determination of GGPP synthase activity.Carotenoids arise from the coupling of two molecules of GGPP. The carotenoid biosynthetic gene cluster , Arabidopsis thaliana (73%), Sinapis alba (72%), Croton sublyratus (69%), Scoparia dulcis (67%) and Mentha piperita (64%)  for full-length C. forskohlii GGPP synthase was predicted to be localized in chloroplasts by the ChloroP 1.1 Prediction Server. In an effort to determine the localization of C. forskohlii GGPP synthase, the sequence coding for the 80 amino acid sequence at the N-terminus of C. forskohlii GGPP synthase was fused to the N-terminus of the GFP reporter gene and transformed into BY-2 tobacco cells. The pattern of putative localization signal of C. forskohlii GGPP synthase was identical to the positive chloroplast targeting signal [35SΩ-pt-sGFP(S65T)]  in order to produce a lacZ fusion protein. pGGPPS was then transformed into E. coli DH10B carrying the plasmid pACCAR25ΔcrtE in which the crtE encoding GGPP synthase had been deleted. The yellow color of carotenoid was observed in the transformant, indicating that pGGPPS carried the gene substituting the function of the crtE gene  [crtE and a pBluescript II KS- (pBS) vector (negative control). This result suggested that the coding region of a cDNA of C. forskohlii GGPP synthase encodes a functional GGPP synthase.Functional activity of expressed GGPP synthase was investigated by genetic complementation with the carotenogenic the gene . Herein,ene Fig. . Carotencontrol) , and witGGPP synthase gene was investigated by RT-PCR in different organs of C. forskohlii. Total RNA extracted from the roots, stems and leaves of an eight-month-old plant were analysed. The C. forskohlii GGPP synthase gene was strongly expressed in the leaves, whereas expression was decreased in stems and barely expressed in roots  enzyme in the non-mevalonate pathway were applied to the ned Fig. . Treatme13C-glucose feeding experiment using 13C-NMR analytical methodology suggested the biosynthetic pathway of forskolin via a non-mevalonate pathway [C. forskohlii [A recent  pathway . In addirskohlii .C. forskohlii GGPP synthase was cloned and its subcellular localization was determined. The N-terminal region contained a signal which was localized in chloroplasts. Functional expression of GGPP synthase was investigated by genetic complementation with the carotenogenic crt gene cluster. Carotenoids were produced when the crtE gene was replaced with C. forskohlii GGPP synthase. GGPP synthase is thought to be involved in biosynthesis of forskolin, which is primary synthesised in the leaves, subsequently distributed to stems and finally accumulated in stem bases and roots.C. forskohlii plantlets were cultured in hormone-free MS (Murashige and Skoog) medium at 25°C under a 16 hours light cycle. The light intensity was 3000 lux and the relative humidity was 60%. Shoot cuttings (10 mm in length) propagated by shoot tip culture were successively cultivated in vermiculite. BY-2 tobacco single cell suspension [-1 of 2,4-D (2,4-dichlorophonoxy acetic acid) under dark conditions at 25°C on an orbital incubator. Restriction enzymes, ligase, and PCR-polymerase were purchased from Takara Shuzo Co., Ltd. (Tokyo, Japan) and Toyobo Co., Ltd. (Tokyo, Japan). Fosmidomycin (FR-3154) was purchased from Molecular Probes (Oregon, USA). Chemical reagents were purchased from Sigma Chemical Company (St. Louis, USA) and Nacalai Tesque Inc. (Tokyo, Japan)spension  was cultE. coli TOP10F' and E. coli DH10B carrying the plasmid pACCAR25ΔcrtE were used in the present investigation. The pUC119 vector was used for cDNA cloning and sequencing. The pBluescript II KS- vector was used as a GGPP synthase expression plasmid. The 35SΩ-sGFP(S65T) plasmid was used as a green fluorescent protein (GFP) reporter plasmid. The pBI121 plant vector and Agrobacterium tumefaciens LBA4404 were used for transformation of GFP and GFP-fusion genes to plant cells.C. forskohlii culture using the acid guanidium-phenol-chloroform extraction procedure [Total RNA was prepared from roots of the rocedure . Single rocedure . A 470 bE. coli TOP10F', clones harboring inserts were sequenced using a Model 310 Genetic Analyzer (PE Biosystems) using a BigDye Terminator Cycle Sequencing Kit.All amplified cDNA fragments were purified and digested with restriction enzymes at sites introduced via the PCR primers, and cloned into the vector pUC119. After transformation to The amino acid sequence deduced from the nucleotide sequence was compared with sequence databases in the Genome Net WWW server using the FASTA program. Multiple amino acid sequence alignment was performed using the CLUSTALW Multiple Sequence Alignment in the GenomeNet CLUSTALW Server.C. forskohlii GGPP synthase was PCR-amplified using primers P and Q and the PCR product was digested and cloned into the SalI-NcoI site of the 35SΩ-sGFP(S65T) plasmid. 35SΩ-pt-sGFP(S65T) was used as the positive control for chloroplast targeting [GGPP synthase-GFP fusion and pt-GFP fusion with CaMV35SΩ promoter and NOS3' terminator [35SΩ-sGFP (S65T), 35SΩ-GGPP synthase-sGFP (S65T) and 35SΩ-pt-sGFP (S65T), respectively] were subcloned into the HindIII-EcoRI site of the pBI121 vector and then transformed into A. tumefaciens LBA4404. The transformants were cultured at 28°C for two days in YEB liquid medium containing 25 μg/ml of kanamycin and 25 μg/ml of rifampicin. The transformants were washed twice and re-suspended in YEB medium. Agrobacterium transformants (108 cells) were applied to four ml of five-day-old BY-2 suspension culture. The culture was incubated at 28°C for two days under dark conditions. GFP and GFP fusion protein were analysed by fluorescence microscopy using Nikon Eclipse TE2000-U model. Cells were observed at a 400 × magnification.A 240 bp fragment of the N-terminal region of argeting ,30. GFP,C. forskohlii GGPP synthase was amplified by PCR using specific primers M and O. A PCR product was digested; purified and cloned into the KpnI-SalI site of pBluescript II KS- vector, namely pGGPPS. This plasmid was transformed into E. coli XL1-Blue MRF' for over-expression. The transformants were cultured in LB liquid medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol. The culture was induced with 1 mM isopropyl-1-thio-β-D-galactoside (IPTG) and incubated for six hours at 37°C. The cells were harvested and washed with 50 mM Tris-HCl pH 8.0 by centrifugation. The pellet was re-suspended, lysozyme was added and the mixture was incubated for 30 minutes. The mixture was then sonicated for four cycles of 15 seconds at one minute intervals. The soluble fraction was obtained after centrifugation at 10,000 × g for 10 minutes. SDS-PAGE was conducted in order to detect the proteins [The coding region of a cDNA of proteins .crtE plasmid contains the gene cluster crtB, crtI, crtX, crtY and crtZ encoding carotenoid biosynthetic enzymes with the exception of crtE (encoding GGPP synthase). The plasmid pBAA containing mouse GGPP synthase (positive control plasmid) and E. coli DH10B carrying the plasmid pACCAR25ΔcrtE was provided by Dr. M. Kawamukai, Shimane University, Japan [- vector, pBS, was used as negative control. pGGPPS, pBAA and pBS were transformed into E. coli DH10B carrying the plasmid pACCAR25ΔcrtE. All transformants were plated on LB agar medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol and then incubated for two to three days at 25°C.The pACCAR25Δy, Japan . pBluescC. forskohlii was analysed in twelve separate parts; leaf (L1–L4), stem (S1–S5) and root (R1–R3). The numbering is based on the maturation of organs. Total RNA was extracted from each part of plant. One microgram of total RNA was used as the template for the synthesis of the first strand cDNA (using SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen). Primers M and O, the first strand cDNA and KOD-polymerase were used for the amplification of C. forskohlii GGPP synthase with the condition of denaturation, 98°C, 15 seconds; annealing, 60°C, 2 seconds and extension, 74°C, 5 seconds. The 18S rRNA fragment used as an internal control was amplified using primers R and S under the same conditions of C. forskohlii GGPP synthase amplification. The amplified PCR products were analysed by 1.0% agarose gel electrophoresis.An eight-month-old C. forskohlii plantlets were treated with various concentrations of fosmidomycin and then investigated for forskolin content using the HPLC method as previously described [escribed . Forskolcrt, carotenogenic gene; FARM, first aspartate-rich motif; GFP, green fluorescent protein; GGPP, geranylgeranyl diphosphate; MEP, 2C-methyl-D-erythritol 4-phosphate; MVA, mevalonate; SARM, second aspatate-rich motifSE carried out the molecular genetic studies, participated in the sequence alignment, forskolin analysis and drafted the manuscript. TF participated in the design of the study and coordination. MK participated in genetic complementation and coordination. YS conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


Endopolyploidy is developmentally regulated. Presence of endopolyploidy as a result of endoreduplication has been characterized in insects, mammals and plants. The family Orchidaceae is the largest among the flowering plants. Many of the members of the orchid family are commercially micropropagated. Very little has been done to characterize the ploidy variation in different tissues of the orchid plants during development.Spathoglottis plicata were examined by flow cytometry. Sepals, petals and ovary tissues were found to have only a 2C (C, DNA content of the unreplicated haploid chromosome complement) peak. Columns, floral pedicels of newly open flowers and growing flower stems were observed to have an endopolyploid 8C peak in addition to 2C and 4C peaks. In developing floral pedicels, four peaks were observed for 2C, 4C, 8C and 16C. In root tips, there were 2C, 4C and 8C peaks. But in the root tissues at the region with root hairs, only a 2C peak was observed. Nuclei extracted from young leaves shown three peaks for 2C, 4C and 8C. A similar pattern was found in the vegetative tissues of both greenhouse-grown plants and tissue-cultured plantlets. In mature leaves, a different pattern of ploidy level was found at different parts of the leaves. In the leaf tips and middle parts, there were 2C and 4C peaks. Only at the basal part of the leaves, there were three peaks for 2C, 4C and 8C.The DNA contents and ploidy level of nuclei extracted from various tissues of a tropical terrestrial orchid Spathoglottis plicata from field-grown plants and in vitro cultures was identified. The implication of the findings was discussed.Systemic variation of cellular endopolyploidy in different tissues during growth and development of  Arabidopsis [In the classical cell cycle, the nuclear DNA contents vary only within the range of 2C and 4C, where C is the haploid DNA content per nucleus. When mitotic DNA replication in somatic cells is not followed by cell division , variation of cellular ploidy levels (designated as somatic polyploidy or endopolyploidy) can result . Endopolbidopsis  and brasbidopsis ,9. Presebidopsis ,11.Vanilla [Vanda seedlings [Spathoglottis plicata, a common tropical terrestrial orchid species, from field-grown plants and in vitro cultures was investigated.In orchids, endoreduplication has been described in the raphid crystal idioblasts of Vanilla  and in peedlings ,14. The eedlings . Many ofeedlings . The expeedlings ,17. Howeeedlings . In thisFlow cytometry analysis of nuclear preparations from entire young leaves of 1–3 cm in length revealed that there were three peaks of fluorescence corresponding to 2C, 4C and 8C DNA content of somatic cells Fig. . About 5More detailed analysis was done on mature leaves of 48 cm in length. Tissues taken from different regions of the mature leaves showed that the pattern of ploidy levels was different at different regions. For tissues taken from the basal (petiolar end) of the leaves, there were three fluorescence peaks corresponding to 2C, 4C and 8C nuclear DNA content Fig. . HoweverIn the young root tips, 2C, 4C and 8C peaks were observed Fig.  and the Preparations from floral pedicels Fig. , columnsS. plicata were found to have 2C, 4C and 8C nuclei  and root tips from greenhouse-grown plants. Similarly, endopolyploid cells were found in protocorms, young leaves and root tips from S. plicata seedlings in tissue culture. The common feature for protocorms, young leaves of 1–3 cm and root tips are that they are young and active in cell division and growth. In other orchids such as Dendrobium, endopolyploidy was found in root tips and newly expanded young leaves [S. plicata, endopolyploidy was neither found in tissue-cultured plants nor it was detected in greenhouse-grown plants. These results imply that the presence of the endopolyploidy during S. plicata development is an intrinsic programme, and it is not much affected by the growth condition.The patterns of endopolyploidy may be affected by plant growth conditions in some plants. For example, leaves of eenhouse ,23. Howeg leaves . In the Dendrobium species and cultivars [S. plicata. Furthermore, when the tip, middle and basal parts of the mature leaf were examined, different patterns of ploidy levels were obtained. Endopolyploidy was found only in leaf base part of mature leaves. A mature leaf represents a continuous developmental system, with the young, less green meristem cells at the basal petiolar end and the older, photosynthetically active cells at the tip [S. plicata are fully developed. Endoreduplication in these tissues is unlikely since it would lead to further cell expansion. The pattern of DNA ploidy variation within the mature leaf is closely associated with the developmental status. The mechanism that resulted in endopolyploidy, however, remains unclear.Besides root tips and newly developing young leaves, endopolyploidy was observed in mature leaves in a few ultivars . Endopol the tip . Previou the tip ,27. The S. plicata, endopolyploidy was present in some floral tissues such as columns, growing flower stems and pedicels of both un-open and freshly open flowers. However, other floral tissues like sepals, petals and ovary tissues were found to have only 2C nuclei. In the growing un-open flower pedicels, the highest ploidy level even reached 16C. In cabbage, endopolyploidy was reported in cabbage flowers [Arabidopsis, endopolyploidy was found in hypocotyls, cotyledonary leaves, rosette leaves, stems of bolting plants and floral leaves, but was not found in inflorescences [Arabidopsis floral buds, and the small population of endopolyploid nuclei found in columns and pedicels of S. plicata in this study, the minute population of the endopolyploid nuclei could easily be neglected when the whole floral buds were used for sampling. In S. plicata, it was found that the average size of nuclei was larger in columns and pedicels that have a measurable amount of endopolyploid cells than in other flora tissues without endopolyploidy (unpublished results). Similarly, a correlation was found between cell size and ploidy levels during cabbage petal development [Dendrobium, the post-pollination physiological changes were found to be different between floral tissues such as columns, ovary tissues, sepals and petals [In  flowers , and det flowers . In cabb flowers . In the  flowers . This st flowers . In Arabescences . Given telopment . In Dendd petals . Edgar ad petals  suggesteArabidopsis hypocotyls [Drosophila melanogaster and some small genome plants like Arabidopsis, it is thought that endoreduplication is developmentally regulated [Further evidence was found in legumes where cell differentiation to a specialized function as pod wall tissues was accompanied by endoreduplication, and higher ploidy levels coincided with maximum pod growth . During pocotyls . In cabbegulated ,10,32.S. plicata raises the question of its possible implications. In tobacco, it was reported that the morphogenetic response of the tissues culture was related to the nuclear DNA content variation within stem explants of different ages [Oncidium Gower Ramsey, a hybrid orchid, only root tips, cut surfaces of stem segments and young leaves were able to form callus in tissue culture. Other explants such as old leaves and the roots without meristem tips could not form any callus [The systemic endopolyploidy revealed within different tissues of ent ages . In Onciy callus . Moleculy callus . In variy callus ,9,32. ThThe role of endoreduplication in plant development is still not well understood. The presence of endopolyploidy was proposed to be associated with several factors, such as taxonomic position of a species, life cycle, genome size, and organ type . Recent S. plicata as revealed in this study suggests that endopolyploidy and DNA content variation in explants might be a cause for somaclonal variation in tissue culture derived orchid plantlets. Thus, the pre-knowledge about the ploidy variation in different explant tissues is valuable for clonal propagation or for deliberate induction of variants in culture. Further systemic investigation of the relationship between somaclonal variation and type and endopolyploid level of source explants will provide indepth knowledge for micropropagation of orchids.This research may also have an impact on the orchid industry. Orchidaceae is the largest family of the flowering plants, and many of its members are commercially hybridized . Clonal Spathoglottis plicata from field-grown plants and in vitro cultures was developmentally regulated. Pattern of endopolyploidy is a character of the developmental and physiological properties of the tissue. This finding provides useful information for understanding of the plant development and for industrial propagation of orchids.Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata L. is a common tropical terrestrial orchid. The plants were grown in pots and placed in the greenhouse at 28 ± 4°C without artificial lighting. The following materials were taken for analysis: a) young leaves (1–3 cm in length), b) mature leaf (48 cm in length), c) root tips (2 cm including the tip), d) root segments from region with root hairs (2 cm away from the root tip), e) newly opened flowers, f) growing flower stems and g) developing floral pedicels of un-open flower (4–6 days before flowering).-1m-2 s-1). The following materials from in vitro cultures were examined: a) protocorms (6 weeks after germination), b) young leaves (1–2 cm in length) from seedlings, c) root tips (1 cm segments from tips), d) root segments with root hairs (at least 1 cm away from the root tips).Seedpods were surface-sterilized for 20 min in 20% (v/v) Clorox™ solution and subsequently rinsed 3 times with autoclaved water. Seeds were germinated aseptically in 9 cm diameter petri dishes containing 25 ml Knudson C orchid medium (Duchefa, Netherlands) with 2% (w/v) sucrose and 0.8% (w/v) agar. All cultures were incubated at 25 ± 2°C under a 16 h photoperiod (light intensity: 54 μm4, 5 mM KCl, 0.5 mM HEPES, 1 mg/ml dithiothreitol, 2.5 mg/ml Triton X-100, pH 8.0) and extracted for 45 minutes. After filtering through a 45 μm Falcon cell strainer, 100 μl of propidium iodide (1 mg/ml) and 2.5 μl of 500 μg/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN) were added to each sample followed by 30 min incubation at 37°C.Extraction of nuclei and staining of DNA were performed according to the method of Arumuganathan and Earle ,45 with ® Elite ESP with 15 mW 488 nm Cyonics Argon air-cooled laser flow cytometer was used to measure the relative fluorescence of nuclei. For each sample, at least 10,000 nuclei were analyzed. Data were analyzed with WinMDI27B software (Joseph Trotter™).A Coulter EPICSC, DNA content of the unreplicated haploid chromosome complementPI, propidium iodidePMT4, photo multiplier tubeMY carried out experiments in the project. CSL and MY prepared the manuscript.