Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex wit

7‑hydroxymitragynine is an active metabolite of mitragynine and a key mediator of its analgesic effects

Mitragynina speciosa, more commonly known as kratom, is a plant native to Southeast Asia, the leaves of which have been used traditionally as a stimulant, analgesic, and treatment for opioid addiction. Recently, growing use of the plant in the United States and concerns that kratom represents an uncontrolled drug with potential abuse liability, have highlighted the need for more careful study of its pharmacological activity. The major active alkaloid found in kratom, mitragynine, has been reported to have opioid agonist and analgesic activity in vitro and in animal models, consistent with the purported effects of kratom leaf in humans. However, preliminary research has provided some evidence that mitragynine and related compounds may act as atypical opioid agonists, inducing therapeutic effects such as analgesia, while limiting the negative side effects typical of classical opioids. Here we report evidence that an active metabolite plays an important role in mediating the analgesic effects of mitragynine. We find that mitragynine is converted in vitro in both mouse and human liver preparations to the much more potent mu-opioid receptor agonist 7-hydroxymitragynine, and that this conversion is mediated by cytochrome P450 3A isoforms. Further, we show that 7-hydroxymitragynine is formed from mitragynine in mice and that brain concentrations of this metabolite are sufficient to explain most or all of the opioid-receptor-mediated analgesic activity of mitragynine. At the same time, mitragynine is found in the brains of mice at very high concentrations relative to its opioid receptor binding affinity, suggesting that it does not directly activate opioid receptors. The results presented here provide a metabolism-dependent mechanism for the analgesic effects of mitragynine and clarify the importance of route of administration for determining the activity of this compound. Further, they raise important questions about the interpretation of existing data on mitragynine and highlight critical areas for further research in animals and humans.</p

Agricultural intensification in Nepal, with particular reference to systems of rice intensification

This thesis deals with agricultural intensification in Nepal. The initial focus of the study was the System of Rice Intensification (SRI), as introduced in Nepal from 2001. The multiple factors affecting SRI adoption, modification and dissemination together with the option to apply SRI in different combinations of its components result in a variety of SRI applications. For the same reason the effect of SRI on overall agricultural and livelihood development of Nepalese farmers has to be evaluated within the variety of farming systems in which it is applied. Despite government policies to promote rice cultivation, national rice production is declining. Farmer livelihood strategies, as reflected in rice farming systems, and field management strategies were influenced by several agro-ecological and socio-economic factors. Livelihood and field management strategies of rice farmers are interconnected. In the study presented here four livelihood strategies and three kinds of field management strategies are distinguished. Two livelihood strategies can be characterized as more intensive and more productive; the other two are less intensive and less productive. Livelihood strategies are more family resource-based strategies, while farmers’ field management strategies are more context-dependent. Field management strategies were characterized by forms of nutrient management. Intensive management strategies had most similarities with SRI. But rice intensification is not achievable as a general strategy. Government policies (fertiliser subsidies) encourage increased fertiliser use. Study results didn't show any significant effect of volume of fertilisers on rice yield but the combined use of organic manure and mineral fertilisers resulted in the highest average rice yields. Irrigation management is another important factor for rice production. Field management is influenced by the reliability of water which was better in farmers' managed irrigation system. Choice of rice varieties influenced the overall rice farming system and cropping intensity and preference of varieties for rice cultivation by scientists and by farmers were different in eastern Nepal. Most popular varieties were those not recommended by science and policy and were disseminated farmer to farmer. The introduction of SRI in Morang district resulted in several changes in rice farming, but only part of the farmers have adopted such technologies, and adoption has been only in part of their fields. Other farmers have incorporated some SRI practices in their conventional practices. After the introduction of SRI, farmers further tested, re-packaged or hybridized SRI methods to make SRI ideas suitable for their agro-ecological and socio-economic environments. In order to reform Nepalese rice farming, we need to recognize that different farmers, with different livelihood strategies, and with access to different kinds of fields, need different forms for agricultural intensification. High-intensive farmers prefer to use modified SRI methods where there is good irrigation and drainage facilities. There are many possibilities for improvement of the existing nutrient management practices of rice farmers in Nepal. Nutrient management will be useful to increase rice production because the majority of farmers currently use fertilisers non-judiciously. The SRI-recommended practices (younger seedlings, early weeding, use of organic manure, and alternate wetting and drying (AWD) irrigation) will be useful to improve the nutrient use efficiency of rice farmers. Cost-reduction strategies and less labour-intensive cultivation practices will be appropriate options to improve existing rice farming system of Nepal. Participatory cultivar selection and dissemination will be better strategies to introduce new, promising rice cultivars among rice farmers

Control of Protein Function through Optochemical Translocation

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applicationslight-triggered transcription and light-triggered protease cleavageboth based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology

Control of Protein Function through Optochemical Translocation

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applicationslight-triggered transcription and light-triggered protease cleavageboth based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology

Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA_CUA^Pyl pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA_CUA^Pyl in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA_CUA^Pyl pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNA_CUA^Pyl pairs to be site-specifically incorporated into proteins in yeast

Control of Protein Function through Optochemical Translocation

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applicationslight-triggered transcription and light-triggered protease cleavageboth based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology