The Arabidopsis thaliana SERK1 kinase domain spontaneously refolds to an active state in vitro

Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state

On the Origin of SERKs: Bioinformatics Analysis of the Somatic Embryogenesis Receptor Kinases

Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases involved in several, seemingly unrelated, plant-signaling pathways. In Arabidopsis thaliana, functional and genetic analysis of four SERK proteins has indicated that they are only partly redundant; their functions overlap but each performs a specific subset of signaling roles. The molecular basis for the functional specificity within this highly homologous protein family is currently not known. Sequence analysis of SERK proteins from different plant species indicates that the SERKs are a highly conserved protein family present in monocots, dicots, and non-vascular plants. Residues in the extracellular domain that are important for interaction with other receptor kinases are highly conserved, even among SERK members without a function in the corresponding pathways. SERK2, for instance, does not function in the brassinosteroid pathway, does not interact with BRI1, but is conserved in its BRI1-interacting domain. Further sequence analysis indicates that SERK3/BAK1 and SERK4/BKK1 have diverged from the original SERK protein in both their extracellular and cytoplasmic domains. Functional analysis of chimeric SERK proteins shows that different domains provide the SERK proteins with different functional specificity. For instance, the SERK1 or SERK2 extracellular domains are essential for SERK function in male sporogenesis, while the SERK3 extracellular and cytoplasmic domains are essential for SERK3 activity in brassinosteroid and flagellin signaling. The emerging picture is that SERKs are ancient genes, whose products have been recruited as co-receptors in the newly evolved signaling pathways. The SERK ligand-binding and protein–protein interaction domains are highly conserved, allowing all SERKs to form complexes, albeit with different affinity. However, specific functional residues must have been altered, in both the extracellular and intracellular domains, to allow for the observed differences in functionality

Decarbonising meat : Exploring greenhouse gas emissions in the meat sector

Consumption of meat is an important source of global greenhouse gas (GHG) emission and deep decarbonisation of the whole meat production chain is required to be able to meet global climate change (CC) mitigation goals. Emissions happen in different stages of meat production ranging from agricultural input production, feed production, livestock production to slaughtering, meat processing, and retail. An overview of direct emissions from processes in the meat sector themselves and indirect emissions from energy consumptions would provide a clearer picture for potential CC impact reduction. This paper explores the total GHG emissions and data availability within the meat sector of the pig, chicken, and cattle meat product system. Through statistical data provided by FAOSTAT and supplementary data from literature, the CC impacts of energy use and process GHG emissions in the pig, chicken and cattle meat life cycle are estimated. Cattle dominates, but pig and chicken meat have a sizable amount of GHG emissions with a relatively high contribution from agricultural inputs and post-farm processes. However, uncertainty and unavailability of data are large for the energy consumption, direct GHG emissions, and product flows of post-farm and agricultural input processes. In order to gain a more complete understanding of the total CC impacts of the meat sector, further research is necessary to reduce the uncertainty in the considered life cycle stages and to quantify the processes and meat products that have been excluded from this study

How much can combinations of measures reduce methane and nitrous oxide emissions from European livestock husbandry and feed cultivation?

In the EU28, the meat and dairy supply chains emitted 360 Mt CO2-eq or 80% of all agricultural CH4 and N2O emissions in 2016, which must be reduced to reach net-zero greenhouse gas emissions by 2050. Our research explores how far these emissions can be reduced by combining field tested mitigation measures for beef cattle, dairy cattle, swine, sheep, and synthetic fertilizers. Many mitigation measures targeting enteric fermentation, manure management, and fertilizer application have been experimentally tested; however, the impact of combining measures is relatively unexplored. To address this knowledge gap, we use graph theory to create combinations of measures for which we calculate the overall mitigation potential. From previous review studies, we identified 44 measures and formulated rules on impossible and mandatory combinations of measures. Based on the resulting sets of feasible cliques in the graphs and a simplified technological baseline, we estimate that the combinations with the highest reductions reduce CH4 and N2O emissions from beef cattle by 57%, dairy cattle by 47%, swine by 70%, sheep by 48%, and synthetic fertilizers by 44%. Together, they can reduce CH4 and N2O emissions in the EU28 from meat and dairy production by 54%, and for agriculture overall by 42%. This indicates that implementing more measures in the meat and dairy sectors can create room for further reduction than in the existing modelled pathways for the EU28. However, technical measures are incapable of fully eliminating agricultural CH4 and N2O, so there remains a need for CO2 removal technologies

Decarbonising meat : Exploring greenhouse gas emissions in the meat sector

Consumption of meat is an important source of global greenhouse gas (GHG) emission and deep decarbonisation of the whole meat production chain is required to be able to meet global climate change (CC) mitigation goals. Emissions happen in different stages of meat production ranging from agricultural input production, feed production, livestock production to slaughtering, meat processing, and retail. An overview of direct emissions from processes in the meat sector themselves and indirect emissions from energy consumptions would provide a clearer picture for potential CC impact reduction. This paper explores the total GHG emissions and data availability within the meat sector of the pig, chicken, and cattle meat product system. Through statistical data provided by FAOSTAT and supplementary data from literature, the CC impacts of energy use and process GHG emissions in the pig, chicken and cattle meat life cycle are estimated. Cattle dominates, but pig and chicken meat have a sizable amount of GHG emissions with a relatively high contribution from agricultural inputs and post-farm processes. However, uncertainty and unavailability of data are large for the energy consumption, direct GHG emissions, and product flows of post-farm and agricultural input processes. In order to gain a more complete understanding of the total CC impacts of the meat sector, further research is necessary to reduce the uncertainty in the considered life cycle stages and to quantify the processes and meat products that have been excluded from this study

How much can combinations of measures reduce methane and nitrous oxide emissions from European livestock husbandry and feed cultivation?

In the EU28, the meat and dairy supply chains emitted 360 Mt CO2-eq or 80% of all agricultural CH4 and N2O emissions in 2016, which must be reduced to reach net-zero greenhouse gas emissions by 2050. Our research explores how far these emissions can be reduced by combining field tested mitigation measures for beef cattle, dairy cattle, swine, sheep, and synthetic fertilizers. Many mitigation measures targeting enteric fermentation, manure management, and fertilizer application have been experimentally tested; however, the impact of combining measures is relatively unexplored. To address this knowledge gap, we use graph theory to create combinations of measures for which we calculate the overall mitigation potential. From previous review studies, we identified 44 measures and formulated rules on impossible and mandatory combinations of measures. Based on the resulting sets of feasible cliques in the graphs and a simplified technological baseline, we estimate that the combinations with the highest reductions reduce CH4 and N2O emissions from beef cattle by 57%, dairy cattle by 47%, swine by 70%, sheep by 48%, and synthetic fertilizers by 44%. Together, they can reduce CH4 and N2O emissions in the EU28 from meat and dairy production by 54%, and for agriculture overall by 42%. This indicates that implementing more measures in the meat and dairy sectors can create room for further reduction than in the existing modelled pathways for the EU28. However, technical measures are incapable of fully eliminating agricultural CH4 and N2O, so there remains a need for CO2 removal technologies

Needs of children with a chronic bladder in preparation for transfer to adult care

Item does not contain fulltextOBJECTIVE: Transfers to adult care can be problematic, resulting in postponement due to the protective nature of pediatric care and patient dependency. It is unknown whether these findings apply specifically to urology patients. Our department is taking part in a national general transition project. In this light, our aim was to investigate the specific needs of adolescent urologic patients, regarding their independence and transition. PATIENTS AND METHODS: 80 patients, born in 1975-1998, with a chronic bladder condition received a questionnaire. They were divided into pre- and post-transfer groups. Parents (n = 7) of post-transfer patients formed a third group. Questionnaires were based on those used in the national transition study, supplemented with urological questions. Pre-transfer patients were asked about their level of independence, what subjects were discussed during consultations, and their expectations and wishes regarding transfer. Post-transfer patients and parents were asked for their opinions on the transfer process. RESULTS: 73% (n = 58) responded (55 pre-transfer and 3 post-transfer patients plus parents). It appeared that the confidence built-up with the pediatric urologist impeded the transfer. An adequate level of disease-related knowledge was reported. Relationships, sexuality and fertility were hardly talked about (respectively n = 17, 16 and 18). Parents played an important role, which patients appreciated, confirming their dependency. Despite the 49% (n = 27) who stated they can arrange their urological care themselves, 44% (n = 24) felt ill-prepared for transfer. CONCLUSION: Although overall self-perceived knowledge is sufficient, the trust in and personal relationship with the pediatric urologist formed the greatest obstruction to successful transition. These findings have been used to improve support during transition by creating a transition protocol

The <em>Arabidopsis thaliana</em> SERK1 Kinase Domain Spontaneously Refolds to an Active State <em>In Vitro</em>

<div><p>Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein <em>in vitro</em>. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.</p> </div

Denaturant-dependent folding of SERK1-KD.

<p>(<b>A</b>) Urea induced unfolding of SERK1-KD followed by fluorescence spectroscopy. Fluorescence intensity of 0.24 µM of SERK1-KD is measured at 340 nm, upon excitation at 280 nm. The native baseline ranges from 0 to about 0.5 M urea. Above 2 M urea, the unfolded baseline commences. The transition region of urea-induced protein unfolding ranges from about 0.5 to 2.0 M. (<b>B</b>) Fluorescence emission of native, unfolded and of refolded SERK1-KD. Black: native SERK1-KD in 0.09 M urea. Dark grey: unfolded SERK1-KD in 6 M urea. Light grey: refolded SERK1-KD, obtained by unfolding the protein for a period of 6 hours in 4.5 M urea, and subsequent dilution of denaturant to 0.09 M urea. Refolding took place during a period of 30 min at room temperature. Refolded SER1-KD has a fluorescence maximum at 340 nm, indicative of folded protein. Approximately 80% of unfolded SERK1-KD properly refolds, according to the difference in fluorescence intensity between native and refolded protein. (<b>C</b>) Far-UV CD spectra of native, unfolded and of refolded SERK1-KD. Black: native SERK1-KD in 0.45 M urea. Dark grey: unfolded SERK1-KD in 4.5 M urea. Light grey: refolded SERK1-KD, obtained by unfolding the protein for a period of 6 hours in 4.5 M urea, and subsequent dilution of denaturant to 0.45 M urea. Refolding took place during a period of 30 min at room temperature.</p