A new peptidic vector for molecular imaging of apoptosis, identified by phage display technology

Phosphatidylserine (PS) exposure on the cell surface is an early marker of apoptosis. To select PS binding peptides as vectors of contrast agents to image apoptosis, a phage library has been exposed to perfused mouse livers. Phages not retained on control livers during the first perfusions were used for selections on apoptotic livers in a second series of perfusions. Four selected phages were further evaluated for binding to PS-coated enzyme-linked immunosorbent assay (ELISA) plates. They presented an apparent affinity constant (Ka app) for PS ranging from 6.08x10(10) M to 1.62x10(11)M. These phages did not bind to phosphatidylcholine, and competition with annexin V confirmed their specific interaction with PS. The phage with the highest affinity-bound PS in ELISA with a Ka app=(1.6+/-0.2)x10(11)M. It carried the TLVSSL peptide that was synthesized. Specific competition with annexin V and with the synthetic peptide was performed and confirms the specificity of the interaction.     

Ecosystem Services and Biodiversity in a Rapidly Transforming Landscape in Northern Borneo

Because industrial agriculture keeps expanding in Southeast Asia at the expense of natural forests and traditional swidden systems, comparing biodiversity and ecosystem services in the traditional forest–swidden agriculture system vs. monocultures is needed to guide decision making on land-use planning. Focusing on tree diversity, soil erosion control, and climate change mitigation through carbon storage, we surveyed vegetation and monitored soil loss in various land-use areas in a northern Bornean agricultural landscape shaped by swidden agriculture, rubber tapping, and logging, where various levels and types of disturbance have created a fine mosaic of vegetation from food crop fields to natural forest. Tree species diversity and ecosystem service production were highest in natural forests. Logged-over forests produced services similar to those of natural forests. Land uses related to the swidden agriculture system largely outperformed oil palm or rubber monocultures in terms of tree species diversity and service production. Natural and logged-over forests should be maintained or managed as integral parts of the swidden system, and landscape multifunctionality should be sustained. Because natural forests host a unique diversity of trees and produce high levels of ecosystem services, targeting carbon stock protection, e.g. through financial mechanisms such as Reducing Emissions from Deforestation and Forest Degradation (REDD+), will synergistically provide benefits for biodiversity and a wide range of other services. However, the way such mechanisms could benefit communities must be carefully evaluated to counter the high opportunity cost of conversion to monocultures that might generate greater income, but would be detrimental to the production of multiple ecosystem services.     

热休克预处理提高猪成肌细胞自体正常肌肉移植后的生存率

研究表明成肌细胞移植有可能用于治疗Duchenne型肌营养不良(Duchenne muscular dystrophy, DMD)、心衰和严重肌肉损伤. 移植后成肌细胞快速大量的死亡是影响该方法疗效的一个主要难题. 细胞在受到各种刺激后会过度表达热休克蛋白(heat shock protein, HSP). 体外和体内实验均已证实HSP能帮助细胞对抗多种有害刺激. 本实验针对HSP的过度表达能否提高猪成肌细胞自体正常肌肉移植后的存活率进行研究. 猪成肌细胞经过42℃, 1 h的热休克后会过度表达HSP. 通过Western blot 和流式细胞仪来定量检测猪成肌细胞中HSP的表达; 同时分析细胞中的CD56和结合蛋白含量, 从而了解休克后细胞的肌源性有无变化. 利用放射性H³定量分析移植后细胞的生存率. 借助慢病毒载体(Lentivector Lac Z)监测移植后成肌细胞与宿主细胞的融合情况. 结果表明热休克预处理后成肌细胞的HSP70显著增高(P<0.01), 并且细胞的肌源性无变化. 成肌细胞体内移植后(n=7), 24 h时热休克组和非热休克组的细胞存活率无显著性差异((67.69 ± 8.35)% vs (58.79 ± 8.35)%, P>0.05). 48 h时热休克组细胞存活率比非热休克组的高2倍((53.32 ± 8.22)% vs (28.27 ± 6.32)%, P<0.01). 120 h时热休克组细胞存活率比非热休克组的高3倍((26.33 ± 5.54)% vs (8.79 ± 2.51)%, P<0.01). 组织学检查显示; 热休克细胞和非热休克细胞均能与宿主细胞融合. 这些结果提示热休克预处理能增强成肌细胞HSP70的表达, 并可提高成肌细胞自体移植后的生存率. 体外热休克预处理是一个简单有效的能改善猪成肌细胞自体移植生存率的方法. 这也许是一个解决成肌细胞移植后存活率低的方法.     

Eco-floristic sectors and deforestation threats in Sumatra: identifying new conservation area network priorities for ecosystem-based land use planning

Biogeographical studies are a necessary step in establishing conservation area networks. Determining the ecological factors influencing vegetation is also a basic principle for hierarchical ecological classifications and a necessary prerequisite for ecosystem-based land use planning. Eco-floristic sectors (EFS) have already been identified for the Indonesian island of Sumatra, combining both approaches, dividing it into 38 EFSs representing unique ecosystems in terms of tree flora and environment (Laumonier 1997). The impact of deforestation on individual EFSs has been highly varied and in some cases extreme. We assigned one of five ‘extinction risk categories’ to each EFS based on the percentage of forest lost between 1985 and 2007. Eighty-five percent of all forest loss (10.2 million ha) occurred in the eastern peneplain, western lowland regions and swamps. In 2007, only 29% of forests were protected by conservation areas, only nine of the 38 EFS had more than 50% of their remaining forest cover protected. 38% of remaining forest was “critically endangered”, “endangered” or “vulnerable” EFSs (5 million ha) but only 1 million ha (20%) were protected. Sumatra’s existing network of conservation areas does not adequately represent the island’s ecosystems. Priorities for a new conservation area network can be formulated for integration into Sumatra’s new land use plans at provincial and district level. Decision makers can now use EFSs to locate new conservation areas so they represent and maintain the whole range of the island’s diversity.     

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.     

Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treatment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and massive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42°C for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and nontreated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analysis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treatment.     

From phage display to magnetophage, a new tool for magnetic resonance molecular imaging

Phage display, an extremely promising technology in the context of molecular imaging, allows for the selection of peptides interacting with virtually any target from a heterogeneous mixture of bacteriophages. In this work, we propose the concept of magnetophages, obtained by covalent coupling of ultrasmall particles of iron oxide (USPIO) to the proteins of the phage wall. To validate magnetophages as a magnetic resonance imaging contrast agent (MRI), we have used as a prototype the clone E3 because of its specific affinity for phosphatidylserine, a marker of apoptosis. Enzyme-linked immunosorbent assay showed that E3 magnetophages incubated with phosphatidylserine retained the properties of the nonmagnetically labeled phages. The usefulness of magnetophages as an MRI contrast agent was estimated by incubation with phosphatidylcholine and phosphatidylserine or with apoptotic and control cells. Under these conditions, E3 magnetophages allow the discrimination of phosphatidylserine from phosphatidylcholine and of apoptotic cells from control ones. Injected in vivo, magnetophages are rapidly cleared from the blood stream and internalized by the phagocytic cells of the liver. To abrogate this problem, USPIO were pegylated to obtain stealthy E3-PEG-magnetophages, invisible to phagocytic cells, which were successfully targeted to apoptotic liver. If this feature demonstrated for E3 magnetophages can be extrapolated to other phage display selected entities, magnetophages become an original system which allows validation of the candidate binding peptides before their synthesis is considered. The concept of the magnetophage could be extended to other imaging modalities by replacing USPIO with an adequate reporter (i.e., radiolabeled phages).     

Identifying spatial patterns in the tropical rain forest structure using hemispherical photographs

This work emphasises the usefulness of hemispherical photography for identifying spatial patterns in the tropical rain forest structure. Structural variability was investigated at a local (intra-site) scale, in relation to the forest mosaic, and at a regional (inter-sites) scale, for its implication in forest typology. Four primary forest sites, from 0.6 to 1 ha, were investigated in Central Sumatra, Indonesia.In a first instance, a qualitative analysis of the forest 3-D structure was found to be very helpful in the interpretation of quantitative results related to forest dynamics. The quantitative analysis was undertaken through the assessment of three structural characteristics: (1) the canopy openness (CO), or visible sky as seen from all directions of the hemisphere, (2) the spherical variance (SV), which quantifies the spatial dispersion of gaps and, (3) the plant area index (L_p), defined as half the surface area of canopy elements per unit ground area. At the local scale, maps of the CO values gave an indication about disturbance location and extent, providing an interesting document for studies on forest dynamics. At the regional scale, between-sites comparisons of CO, SV and L_p values added new information on forest structural differences when compared to dendrometric measurements. These results emphasised the fact that rain forest may exhibit high structural variability, even within a same bioclimatic region and a narrow altitude range. Hemispherical photographs could be a quick means of further investigating this spatial variability and it's relation to physical environment, thus providing information that is crucial for the refining of forest typology in the area.