Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution

Denial of long-term issues with agriculture on tropical peatlands will have devastating consequences

Lette

Tree species that ‘live slow, die older’ enhance tropical peat swamp restoration: Evidence from a systematic review

Funder: Arcus Foundation; Id: http://dx.doi.org/10.13039/100016681Funder: Darwin InitiativeFunder: European Association of Zoos and Aquaria; Id: http://dx.doi.org/10.13039/501100009167Funder: European Outdoor Conservation Association; Id: http://dx.doi.org/10.13039/501100013711Funder: Fundacion BioparcFunder: Ocean Parks Conservation Foundation Hong KongFunder: Orangutan Land TrustFunder: Save the OrangutanFunder: Taronga ZooFunder: The Orangutan ProjectFunder: Ministry of Environment & ForestryFunder: PT Rimba Makmur UtamaAbstract: Degraded tropical peatlands lack tree cover and are often subject to seasonal flooding and repeated burning. These harsh environments for tree seedlings to survive and grow are therefore challenging to revegetate. Knowledge on species performance from previous plantings represents an important evidence base to help guide future tropical peat swamp forest (TPSF) restoration efforts. We conducted a systematic review of the survival and growth of tree species planted in degraded peatlands across Southeast Asia to examine (1) species differences, (2) the impact of seedling and site treatments on survival and growth and (3) the potential use of plant functional traits to predict seedling survival and growth rates. Planted seedling monitoring data were compiled through a systematic review of journal articles, conference proceedings, reports, theses and unpublished datasets. In total, 94 study‐sites were included, spanning three decades from 1988 to 2019, and including 141 indigenous peatland tree and palm species. Accounting for variable planting numbers and monitoring durations, we analysed three measures of survival and growth: (1) final survival weighted by the number of seedlings planted, (2) half‐life, that is, duration until 50% mortality and (3) relative growth rates (RGR) corrected for initial planting height of seedlings. Average final survival was 62% and half‐life was 33 months across all species, sites and treatments. Species differed significantly in survival and half‐life. Seedling and site treatments had small effects with the strongest being higher survival of mycorrhizal fungi inoculated seedlings; lower survival, half‐life and RGR when shading seedlings; and lower RGR and higher survival when fertilising seedlings. Leaf nutrient and wood density traits predicted TPSF species survival, but not half‐life and RGR. RGR and half‐life were negatively correlated, meaning that slower growing species survived for longer. Synthesis and applications. To advance tropical peat swamp reforestation requires expanding the number and replication of species planted and testing treatments by adopting control vs. treatment experimental designs. Species selection should involve slower growing species (e.g. Lophopetalum rigidum, Alstonia spatulata, Madhuca motleyana) that survive for longer and explore screening species based on functional traits associated with nutrient acquisition, flooding tolerance and recovery from fire