Tough as a rock-boring urchin: adult Echinometra sp. EE from the Red Sea show high resistance to ocean acidification over long-term exposures

Ocean acidification, a process caused by the continuous rise of atmospheric CO2 levels, is expected to have a profound impact on marine invertebrates. Findings of the numerous studies conducted in this field indicate high variability in species responses to future ocean conditions. This study aimed at understanding the effects of long-term exposure to elevated pCO2 conditions on the performance of adult Echinometra sp. EE from the Gulf of Aqaba (Red Sea). During an 11-month incubation under high pCO2 (1,433 µatm, pHNBS 7.7) and control (435 µatm, pHNBS 8.1) conditions, we examined the urchins' somatic and gonadal growth, gametogenesis and skeletal microstructure. Somatic and gonadal growths were exhibited with no significant differences between the treatments. In addition, all urchins in the experiment completed a full reproductive cycle, typical of natural populations, with no detectable impact of increased pCO2 on the timing, duration or progression of the cycle. Furthermore, scanning electron microscopy imaging of urchin tests and spines revealed no signs of the usual observed effects of acidosis, such as skeletal dissolution, widened stereom pores or non-smoothed structures. Our results, which yielded no significant impact of the high pCO2 treatment on any of the examined processes in the urchins studied, suggest high resistance of adult Echinometra sp. EE to near future ocean acidification conditions. With respect to other findings in this area, the outcome of this study provides an example of the complicated and diverse responses of echinoids to the predicted environmental changes.

Short- and long-term conditioning of a temperate marine diatom community to acidification and warming

Ocean acidification and greenhouse warming will interactively influence competitive success of key phytoplankton groups such as diatoms, but how long-term responses to global change will affect community structure is unknown. We incubated a mixed natural diatom community from coastal New Zealand waters in a short-term (two-week) incubation experiment using a factorial matrix of warming and/or elevated pCO2 and measured effects on community structure. We then isolated the dominant diatoms in clonal cultures and conditioned them for 1 year under the same temperature and pCO2 conditions from which they were isolated, in order to allow for extended selection or acclimation by these abiotic environmental change factors in the absence of interspecific interactions. These conditioned isolates were then recombined into 'artificial' communities modelled after the original natural assemblage and allowed to compete under conditions identical to those in the short-term natural community experiment. In general, the resulting structure of both the unconditioned natural community and conditioned 'artificial' community experiments was similar, despite differences such as the loss of two species in the latter. pCO2 and temperature had both individual and interactive effects on community structure, but temperature was more influential, as warming significantly reduced species richness. In this case, our short-term manipulative experiment with a mixed natural assemblage spanning weeks served as a reasonable proxy to predict the effects of global change forcing on diatom community structure after the component species were conditioned in isolation over an extended timescale. Future studies will be required to assess whether or not this is also the case for other types of algal communities from other marine regimes.

MARECHIARA-phytoplankton long-term time-series (1984-2006) at the fixed coastal station in the Gulf of Naples, Southern Tyrrhenian Sea

The "MARECHIARA-phytoplankton" dataset contains phytoplankton data collected in the ongoing time-series at Stn MC ( 40°48.5' N, 14°15' E) in the Gulf of Naples. This dataset spans over the period 1984-2006 and contains data of phytoplankton species composition and abundance. Phytoplankton sampling was regularly conducted from January 1984 till July 1991 and in 1995-2006. Sampling was interrupted from August 1991 till January 1995. The sampling frequency was fortnightly till 1991 and weekly since 1995. Phytoplankton samples were collected at 0.5 m depth using Niskin bottles and immediately fixed with formaldehyde (0.8-1.6% final concentration) for species identification and counts.

Infrastructure Development of Railway in Cambodia: A Long Term Strategy

Infrastructure development means for the making of living environment, transport and communications, disaster prevention and national land conservation, agriculture, forestry and fisheries, and energy production and supply. Transport infrastructure development in Cambodia involved with (1) road, (2) railway, (3) port, inland-water way and (4) aviation. All model of transport infrastructure have special different kinds of importance. Railway is different from other base important of railways are transport passengers and traffic freight especially transport for heavy goods in huge capacity and in long distance by safer and faster. Transport in Cambodia for traffic freight export import base from Thailand and other via Sisophon and Shihanoukvill port. Traffic is increasing rapidly during nowadays railway condition in adequate of demand required. This is why Railway is selected as the topic of this paper to prevent monopoly of road transport. This paper, does review about infrastructure development plan for Railway in Cambodia as a long term strategy by review and analysis forecast on the previous performance of Royal Railways of Cambodia (RRC) transport traffic involved with condition of infrastructure development of railway in Cambodia. And also review the plan of development RRC but just only detail a plan of rehabilitation that is immediately needed. Suggest some recommendation at the last part. As Cambodia is a member country of ASEAN and also Mekong sub-region. For make sure that transport networks work effectively with a progress of economic integration, we make clear what is important for infrastructure development of railway in Cambodia from the standpoint of the development plan of Mekong sub-region. This paper is organized by 4 sections. Section 1 review about Infrastructure Development of Railway in Cambodia (IDRC) Historical Background, Follow by Section 2 will review the Current Situation of IDRC and some analysis of transport performance from previous years, Then Section 3 review of the focusing on traffic transport of RRC in the future, Section 4 review Infrastructure Development of Railway in Cambodia Future plans in long term; at last conclusion and recommendation. In section 1 does review history background of RRC from the rail first begun. But why is needed to review? Because of history background is involved infrastructure development of RRC in present time. History background made big gaps constraint and obstacle for socioeconomic development and poverty reduction, also left Cambodia with tragedy and left developed behind. After that remain infrastructure development needs huge fund and long time for restoration, reconstruction, rehabilitation and development into new technology as most of world practice.

Understanding long-term hunting statistics: the case of Spain (1972-2007)

Hunting is assuming a growing role in the current European forestry and agroforestry landscape. However, consistent statistical sources that provide quantitative information for policy-making, planning and management of game resources are often lacking. In addition, in many instances statistical information can be used without sufficient evaluation or criticism. Recently, the European Commission has declared the importance of high quality hunting statistics and the need to set up a common scheme in Europe for their collection, interpretation and proper use. This work aims to contribute to this current debate on hunting statistics in Europe by exploring data from the last 35 years of Spanish hunting statistics. The analysis focuses on the three major pillars underpinning hunting activity: hunters, hunting grounds and game animals. First, the study aims to provide a better understanding of official hunting statistics for use by researchers, game managers and other potential users. Second, the study highlights the major strengths and weaknesses of the statistical information that was collected. The results of the analysis indicate that official hunting statistics can be incomplete, dispersed and not always homogeneous over a long period of time. This is an issue of which one should be aware when using official hunting data for scientific or technical work. To improve statistical deficiencies associated with hunting data in Spain, our main suggestion is the adoption of a common protocol on data collection to which different regions agree. This protocol should be in accordance with future European hunting statistics and based on robust and well-informed data collection methods. Also it should expand the range of biological, ecological and economic concepts currently included to take account of the profound transformations experienced by the hunting sector in recent years. As much as possible, any future changes in the selection of hunting statistics should allow for comparisons between new variables with the previous ones.

Midday measurements of leaf water potential and stomatal conductance are highly correlated with daily water use of Thompson Seedless grapevines

A study was conducted to determine the relationship between midday measurements of vine water status and daily water use of grapevines measured with a weighing lysimeter. Water applications to the vines were terminated on August 24th for 9 days and again on September 14th for 22 days. Daily water use of the vines in the lysimeter (ETLYS) was approximately 40 L vine−1 (5.3 mm) prior to turning the pump off, and it decreased to 22.3 L vine−1 by September 2nd. Pre-dawn leaf water potential (ΨPD) and midday Ψl on August 24th were −0.075 and −0.76 MPa, respectively, with midday Ψl decreasing to −1.28 MPa on September 2nd. Leaf g s decreased from ~500 to ~200 mmol m−2 s−1 during the two dry-down periods. Midday measurements of g s and Ψl were significantly correlated with one another (r = 0.96) and both with ETLYS/ETo (r = ~0.9). The decreases in Ψl, g s, and ETLYS/ETo in this study were also a linear function of the decrease in volumetric soil water content. The results indicate that even modest water stress can greatly reduce grapevine water use and that short-term measures of vine water status taken at midday are a reflection of daily grapevine water use

Implications of agricultural production, policy and land use changes on water resource assessment

Una gestión más eficiente y equitativa del agua a escala de cuenca no se puede centrar exclusivamente en el recurso hídrico en sí, sino también en otras políticas y disciplinas científicas. Existe un consenso creciente de que, además de la consideración de las cambiantes condiciones climáticas, es necesaria una integración de ámbitos de investigación tales como la agronomía, planificación del territorio y ciencias políticas y económicas a fin de satisfacer de manera sostenible las demandas de agua por parte de la sociedad y del medio natural. La Política Agrícola Común (PAC) es el principal motor de cambio en las tendencias de paisajes rurales y sistemas agrícolas, pero el deterioro del medio ambiente es ahora una de las principales preocupaciones. Uno de los cambios más relevantes se ha producido con la expansión e intensificación del olivar en España, principalmente con nuevas zonas de regadío o la conversión de olivares de secano a sistemas en regadío. Por otra parte, el cambio de las condiciones climáticas podría ejercer un papel importante en las tendencias negativas de las aportaciones a los ríos, pero no queda claro el papel que podrían estar jugando los cambios de uso de suelo y cobertura vegetal sobre las tendencias negativas de caudal observadas. Esta tesis tiene como objetivo mejorar el conocimiento de los efectos de la producción agrícola, política agraria y cambios de uso de suelo y cobertura vegetal sobre las condiciones de calidad del agua, respuesta hidrológica y apropiación del agua por parte de la sociedad. En primer lugar, el estudio determina las tendencias existentes de nitratos y sólidos en suspensión en las aguas superficiales de la cuenca del río Guadalquivir durante el periodo de 1998 a 2009. Desde una perspectiva de política agraria, la investigación trata de evaluar mediante un análisis de datos de panel las principales variables, incluyendo la reforma de la PAC de 2003, que están teniendo una influencia en ambos indicadores de calidad. En segundo lugar, la apropiación del agua y el nivel de contaminación por nitratos debido a la producción del aceite de oliva en España se determinan con una evaluación de la huella hídrica (HH), teniendo en cuenta una variabilidad espacial y temporal a largo de las provincias españolas y entre 1997 y 2008. Por último, la tesis analiza los efectos de los cambios de uso de suelo y cobertura vegetal sobre las tendencias negativas observadas en la zona alta del Turia, cabecera de la cuenca del río Júcar, durante el periodo 1973-2008 mediante una modelización ecohidrológica. En la cuenca del Guadalquivir cerca del 20% de las estaciones de monitoreo muestran tendencias significativas, lineales o cuadráticas, para cada indicador de calidad de agua. La mayoría de las tendencias significativas en nitratos están aumentando, y la mayoría de tendencias cuadráticas muestran un patrón en forma de U. Los modelos de regresión de datos de panel muestran que las variables más importantes que empeoran ambos indicadores de calidad del agua son la intensificación de biomasa y las exportaciones de ambos indicadores de calidad procedentes de aguas arriba. En regiones en las que el abandono agrícola y/o desintensificación han tenido lugar han mejorado las condiciones de calidad del agua. Para los nitratos, el desacoplamiento de las subvenciones a la agricultura y la reducción de la cuantía de las subvenciones a tierras de regadío subyacen en la reducción observada de la concentración de nitratos. Las medidas de modernización de regadíos y el establecimiento de zonas vulnerables a nitratos reducen la concentración en subcuencas que muestran una tendencia creciente de nitratos. Sin embargo, el efecto de las exportaciones de nitratos procedente de aguas arriba, la intensificación de la biomasa y los precios de los cultivos presentan un mayor peso, explicando la tendencia creciente observada de nitratos. Para los sólidos en suspensión, no queda de forma evidente si el proceso de desacoplamiento ha influido negativa o positivamente. Sin embargo, los mayores valores de las ayudas agrarias aún ligadas a la producción, en particular en zonas de regadío, conllevan un aumento de las tasas de erosión. Aunque la cuenca del Guadalquivir ha aumentado la producción agrícola y la eficiencia del uso del agua, el problema de las altas tasas de erosión aún no ha sido mitigado adecuadamente. El estudio de la huella hídrica (HH) revela que en 1 L de aceite de oliva español más del 99,5% de la HH está relacionado con la producción de la aceituna, mientras que menos del 0,5% se debe a otros componentes, es decir, a la botella, tapón y etiqueta. Durante el período estudiado, la HH verde en secano y en regadío representa alrededor del 72% y 12%, respectivamente, del total de la HH. Las HHs azul y gris representan 6% y 10%, respectivamente. La producción de aceitunas se concentra en regiones con una HH menor por unidad de producto. La producción de aceite de oliva ha aumentado su productividad del agua durante 1997-2008, incentivado por los crecientes precios del aceite, como también lo ha hecho la cantidad de exportaciones de agua virtual. De hecho, las mayores zonas productoras presentan una eficiencia alta del uso y de productividad del agua, así como un menor potencial de contaminación por nitratos. Pero en estas zonas se ve a la vez reflejado un aumento de presión sobre los recursos hídricos locales. El aumento de extracciones de agua subterránea relacionadas con las exportaciones de aceite de oliva podría añadir una mayor presión a la ya estresada cuenca del Guadalquivir, mostrando la necesidad de equilibrar las fuerzas del mercado con los recursos locales disponibles. Los cambios de uso de suelo y cobertura vegetal juegan un papel importante en el balance del agua de la cuenca alta del Turia, pero no son el principal motor que sustenta la reducción observada de caudal. El aumento de la temperatura es el principal factor que explica las mayores tasas de evapotranspiración y la reducción de caudales. Sin embargo, los cambios de uso de suelo y el cambio climático han tenido un efecto compensatorio en la respuesta hidrológica. Por un lado, el caudal se ha visto afectado negativamente por el aumento de la temperatura, mientras que los cambios de uso de suelo y cobertura vegetal han compensado positivamente con una reducción de las tasas de evapotranspiración, gracias a los procesos de disminución de la densidad de matorral y de degradación forestal. El estudio proporciona una visión que fortalece la interdisciplinariedad entre la planificación hidrológica y territorial, destacando la necesidad de incluir las implicaciones de los cambios de uso de suelo y cobertura vegetal en futuros planes hidrológicos. Estos hallazgos son valiosos para la gestión de la cuenca del río Turia, y el enfoque empleado es útil para la determinación del peso de los cambios de uso de suelo y cobertura vegetal en la respuesta hidrológica en otras regiones. ABSTRACT Achieving a more efficient and equitable water management at catchment scale does not only rely on the water resource itself, but also on other policies and scientific knowledge. There is a growing consensus that, in addition to consideration of changing climate conditions, integration with research areas such as agronomy, land use planning and economics and political science is required to meet sustainably the societal and environmental water demands. The Common Agricultural Policy (CAP) is a main driver for trends in rural landscapes and agricultural systems, but environmental deterioration is now a principal concern. One of the most relevant changes has occurred with the expansion and intensification of olive orchards in Spain, taking place mainly with new irrigated areas or with the conversion from rainfed to irrigated systems. Moreover, changing climate conditions might exert a major role on water yield trends, but it remains unclear the role that ongoing land use and land cover changes (LULCC) might have on observed river flow trends. This thesis aims to improve the understanding of the effects of agricultural production, policies and LULCC on water quality conditions, hydrological response and human water appropriation. Firstly, the study determines the existing trends for nitrates and suspended solids in the Guadalquivir river basin’s surface waters (south Spain) during the period from 1998 to 2009. From a policy perspective, the research tries to assess with panel data analysis the main drivers, including the 2003 CAP reform, which are having an influence on both water quality indicators. Secondly, water appropriation and nitrate pollution level originating from the production of olive oil in Spain is determined with a water footprint (WF) assessment, considering a spatial temporal variability across the Spanish provinces and from 1997 to 2008 years. Finally, the thesis analyzes the effects of the LULCC on the observed negative trends over the period 1973-2008 in the Upper Turia basin, headwaters of the Júcar river demarcation (east Spain), with ecohydrological modeling. In the Guadalquivir river basin about 20% of monitoring stations show significant trends, linear or quadratic, for each water quality indicator. Most significant trends of nitrates are augmenting than decreasing, and most significant quadratic terms of both indicators exhibit U-shaped patterns. The panel data models show that the most important drivers that are worsening nitrates and suspended solids in the basin are biomass intensification and exports of both water quality indicators from upland regions. In regions that agricultural abandonment and/or de-intensification have taken place the water quality conditions have improved. For nitrates, the decoupling of agricultural subsidies and the reduction of the amount of subsidies to irrigated land underlie the observed reduction of nitrates concentration. Measures of irrigation modernization and establishment of vulnerable zones to nitrates ameliorate the concentration of nitrates in subbasins showing an increasing trend. However, the effect of nitrates load from upland areas, intensification of biomass and crop prices present a greater weight leading to the final increasing trend in this subbasins group, where annual crops dominate. For suspended solids, there is no clear evidence that decoupling process have influenced negatively or positively. Nevertheless, greater values of subsidies still linked to production, particularly in irrigated regions, lead to increasing erosion rates. Although agricultural production has augmented in the basin and water efficiency in the agricultural sector has improved, the issue of high erosion rates has not yet been properly faced. The water footprint (WF) assessment reveals that for 1 L Spanish olive oil more than 99.5% of the WF is related to the olive fruit production, whereas less than 0.5% is due to other components i.e. bottle, cap and label. Over the studied period, the green WF in rainfed and irrigated systems represents about 72% and 12%, respectively, of the total WF. Blue and grey WFs represent 6% and 10%, respectively. The olive production is concentrated in regions with the smallest WF per unit of product. The olive oil production has increased its apparent water productivity from 1997 to 2008 incentivized by growing trade prices, but also did the amount of virtual water exports. In fact, the largest producing areas present high water use efficiency per product and apparent water productivity as well as less nitrates pollution potential, but this enhances the pressure on the available water resources. Increasing groundwater abstractions related to olive oil exports may add further pressure to the already stressed Guadalquivir basin. This shows the need to balance the market forces with the available local resources. Concerning the effects of LULCC on the Upper Turia basin’s streamflow, LULCC play a significant role on the water balance, but it is not the main driver underpinning the observed reduction on Turia's streamflow. Increasing mean temperature is the main factor supporting larger evapotranspiration rates and streamflow reduction. In fact, LULCC and climate change have had an offsetting effect on the streamflow generation during the study period. While streamflow has been negatively affected by increasing temperature, ongoing LULCC have positively compensated with reduced evapotranspiration rates, thanks to mainly shrubland clearing and forest degradation processes. The research provides insight for strengthening the interdisciplinarity between hydrological and spatial planning, highlighting the need to include the implications of LULCC in future hydrological plans. These findings are valuable for the management of the Turia river basin, as well as a useful approach for the determination of the weight of LULCC on the hydrological response in other regions.

Long Term Performance Study of a Direct Methanol Fuel Cell Fed with Alcohol Blends

The use of alcohol blends in direct alcohol fuel cells may be a more environmentally friendly and less toxic alternative to the use of methanol alone in direct methanol fuel cells. This paper assesses the behaviour of a direct methanol fuel cell fed with aqueous methanol, aqueous ethanol and aqueous methanol/ethanol blends in a long term experimental study followed by modelling of polarization curves. Fuel cell performance is seen to decrease as the ethanol content rises, and subsequent operation with aqueous methanol only partly reverts this loss of performance. It seems that the difference in the oxidation rate of these alcohols may not be the only factor affecting fuel cell performance.

Long-term dynamics of fast rotating tethers around planetary satellites

We derive a semi-analytic formulation that enables the study of the long-term dynamics of fast-rotating inert tethers around planetary satellites. These equations take into account the coupling between the translational and rotational motion, which has a non-negligible impact on the dynamics, as the orbital motion of the tether center of mass strongly depends on the tether plane of rotation and its spin rate, and vice-versa. We use these governing equations to explore the effects of this coupling on the dynamics, the lifetime of frozen orbits and the precession of the plane of rotation of the tether.

Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.

Running enhances neurogenesis, learning, and long-term potentiation in mice

Running increases neurogenesis in the dentate gyrus of the hippocampus, a brain structure that is important for memory function. Consequently, spatial learning and long-term potentiation (LTP) were tested in groups of mice housed either with a running wheel (runners) or under standard conditions (controls). Mice were injected with bromodeoxyuridine to label dividing cells and trained in the Morris water maze. LTP was studied in the dentate gyrus and area CA1 in hippocampal slices from these mice. Running improved water maze performance, increased bromodeoxyuridine-positive cell numbers, and selectively enhanced dentate gyrus LTP. Our results indicate that physical activity can regulate hippocampal neurogenesis, synaptic plasticity, and learning.

Specific therapy regimes could lead to long-term immunological control of HIV

We use mathematical models to study the relationship between HIV and the immune system during the natural course of infection and in the context of different antiviral treatment regimes. The models suggest that an efficient cytotoxic T lymphocyte (CTL) memory response is required to control the virus. We define CTL memory as long-term persistence of CTL precursors in the absence of antigen. Infection and depletion of CD4+ T helper cells interfere with CTL memory generation, resulting in persistent viral replication and disease progression. We find that antiviral drug therapy during primary infection can enable the development of CTL memory. In chronically infected patients, specific treatment schedules, either including deliberate drug holidays or antigenic boosts of the immune system, can lead to a re-establishment of CTL memory. Whether such treatment regimes would lead to long-term immunologic control deserves investigation under carefully controlled conditions.

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28K expression.

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the time course or size of a spike of intracellular calcium are cytosolic calcium binding or "buffering" proteins. To consider the role in memory formation of one of these proteins, calbindin D28K, which is abundant in many neurons, including the CA1 pyramidal cells of the hippocampus, transgenic mice deficient in calbindin D28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calcium-dependent intracellular signaling pathways underlying memory function.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.