A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

Allocating community-level payments for ecosystem services initial experiences from a REDD pilot in Tanzania

Payments for ecosystem services (PES) typically reward landowners for managing their land to provide ecosystem services that would not otherwise be provided. REDD—Reduced Emissions from Deforestation and Forest Degradation—is a form of PES aimed at decreasing carbon emissions from forest conversion and extraction in lower-income countries. A key challenge for REDD occurs when it is implemented at the community rather than the individual landowner level. Whilst achieving this community-level reduction relies on individuals changing their interaction with the forest, incentives are not aligned explicitly at the individual level. Rather, payments are made to the community as a single entity in exchange for verified reduced forest loss, as per a PES scheme. In this paper, we explore how community level REDD has been implemented in one multiple-village pilot in Tanzania. Our findings suggest that considerable attention has been paid to monitoring, reporting, verification, and equity. Though no explicit mechanism ensures individual compliance with the group PES, the development of village level institutions, “social fencing,” and a shared future through equal REDD payments factor into community decisions that influence the level of community compliance that the program will eventually achieve. However, few villages allocate funds for explicit enforcement efforts to protect the forest from illegal activities undertaken by outsiders.