Making Landscapes Negotiable: Q-methodology as a Boundary-Spanning and Empowering Diagnostic.

Landscapes are conceptually fuzzy and rich, and subject to plural framings. They are places of inquiry and intervention for scientists and practitioners, but also concepts bound to peoples' dynamic identities, knowledge systems, inspiration, and well-being. These varying interpretations change the way landscapes function and evolve. Developed in the 1930s, Q-methodology is increasingly recognized for being useful in documenting and interrogating environmental discourses. Yet its application in the context of how integrated landscape approaches better navigate land-use dilemmas is still in its infancy. Based on our experience and emerging literature, such as the papers in this special collection, this article discusses the value of Q-methodology in addressing landscape sustainability issues. Q-methodology helps unravel and communicate common and contradicting landscape imaginaries and narratives in translational and boundary-spanning ways, thus bridging actors' different understandings of problems and solutions and revealing common or differentiated entry points for negotiating trade-offs between competing land uses. The methodology can be empowering for marginalized people by uncovering their views and aspirational values to decision-makers and policymakers. We argue that this potential can be further strengthened by using Q to identify counter-hegemonic discourses and alliances that combat injustices regarding whose knowledge and visions count. In this way, applying Q-methodology in integrated landscape approaches can become a key tool for transitioning toward just, inclusive, and sustainable landscapes.

Localization of the greater occipital nerve through palpation of bony landmarks: A cadaveric study.

OBJECTIVE: to provide anatomic confirmation that standard methods which practitioners skilled in palpation use, can reliably identify the most likely site of emergence of the greater occipital nerve in most patients. The location and frequency of subcutaneous emergence of the greater occipital nerve and occipital artery with respect to the external occipital protuberance-mastoid line are reported. METHODS: The external occipital protuberance and the mastoid processes were identified by palpation bilaterally on 57 body donors and the medial trisection point of a line connecting these bony landmarks was identified. A 4 cm circular dissection guide divided into 4 quadrants was centered on the trisection point and used to guide the removal of a circle of skin. The in-situ location of the nerve and artery were exposed by deep dissection within the circle. The frequency of the emergence and occurrence of the nerve and artery by quadrant were analyzed. RESULTS: In 114 total dissections the greater occipital nerve was found to emerge within the circle 96 times (84%) and the occipital artery 100 times (88%). The nerve (90%) and artery (81%) emerged from the two inferior quadrants most of the time with no difference noted between male and female donors. The greater occipital nerve and occipital artery were found to emerge together most commonly in inferior lateral quadrant. Branches of the nerve and artery traveled together most frequently through the two lateral quadrants. CONCLUSION: This study confirmed that the medial trisection point of the external occipital protuberance-mastoid line can be located via palpation and reliably used to pinpoint the subcutaneous emergence of the greater occipital nerve and occipital artery in most individuals. When relying on palpation alone to identify the trisection point in the clinic, infusion of nerve block inferior and lateral to this point is most likely to bathe the greater occipital nerve in anesthetic.

Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Improving healthcare professionals' ultrasound-guided peripheral vascular access ability utilizing self-assembled ultrasound phantoms: A prospective, observational quality improvement project.

BACKGROUND: Ultrasound guidance can reduce the number of attempts to gain peripheral IV access while improving the success rate and satisfaction in patients with difficult IV access. Education and simulation are effective tools for improving the skills and knowledge related to ultrasound-guided peripheral IV access. Ultrasound phantom models allow for skill development without the risk of patient harm. METHODS: Twenty-nine registered nurses and nurse practitioners were recruited for education and simulation regarding ultrasound-guided peripheral IV (USGPIV) placement. Participants completed a survey evaluating the efficacy of the phantom models in addition to pre- and post-intervention confidence, perceived competence, knowledge surveys, and a Directly Observed Procedural Skills Evaluation (DOPSE). The intervention included an educational PowerPoint and open practice session using the phantom models. RESULTS: Statistically significant improvements were found in participants' confidence (p < 0.001; 95% CI: 5.287, 9.499; d = 1.31), perceived competence (p < 0.001; 95% CI: 1.231, 2.742; d = 1.20), knowledge (p < 0.001; 95% CI: 1.079, 2.163; d = 1.47), and skills (p < 0.001; 95% CI: 2.499; 5.501; d = 1.29). Participants improved in maintaining needle visualization (p < 0.001; 95% CI: 0.272, 0.9; d = 0.79) and decreasing their cannulation attempts (0.045; 95% CI: 0.013, 1.022; d = 0.48). Participants with no and novice experience saw statistically significant improvement across all categories (p < 0.02) compared to those with intermediate, advanced, or expert experience with ultrasound. 96.5% of participants could perform ultrasound-guided peripheral IV cannulation independently or with indirect supervision following the intervention. CONCLUSIONS: At $36.52 per model, the self-assembled ultrasound phantom models provided a cost-effective and sustainable solution to teaching ultrasound-guided peripheral IV cannulations. Education and simulation for ultrasound-guided peripheral vascular access may benefit individuals with no or novice ultrasound experience.

Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study.

BACKGROUND AND AIMS: We have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions. METHODS: We identified, isolated, cultivated, and sequenced a unique novel strain (20220303-A2) of B. vulgatus found only in control subjects. Using a human gut organoid system developed from pre-celiac patients, we monitored epithelial phenotype and innate immune cytokines at baseline, after exposure to gliadin, or gliadin plus B. vulgatus cell free supernatant (CFS). RESULTS: Following gliadin exposure, we observed increases in epithelial cell death, epithelial monolayer permeability, and secretion of pro-inflammatory cytokines. These effects were mitigated upon exposure to B. vulgatus 20220303-A2 CFS, which had matched phenotype gene product mutations. These protective effects were mediated by epigenetic reprogramming of the organoids treated with B. vulgatus CFS. CONCLUSIONS: We identified a unique strain of B. vulgatus that may exert a beneficial role by protecting CeD epithelium against a gluten-induced break of epithelial tolerance through miRNA reprogramming. IMPACT: Gut dysbiosis precedes the onset of celiac disease in genetically at-risk infants. This dysbiosis is characterized by the loss of protective bacterial strains in those children who will go on to develop celiac disease. The paper reports the mechanism by which one of these protective strains, B. vulgatus, ameliorates the gluten-induced break of gut epithelial homeostasis by epigenetically re-programming the target intestinal epithelium involving pathways controlling permeability, immune response, and cell turnover.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Integrating local and scientific knowledge: The need for decolonising knowledge for conservation and natural resource management.

Integrating Indigenous and local knowledge in conservation and natural resource management (NRM) initiatives is necessary to achieve sustainability, equity, and responsiveness to local realities and needs. Knowledge integration is the starting point for converging different knowledge systems and enabling knowledge co-production. This process is also a key prerequisite towards decolonising the research process. However, power imbalances may perpetuate dominant forms of knowledge over others, obstruct knowledge integration, and eventually cause the loss of knowledge of the marginal and less powerful knowledge holders. Despite increasing interest in knowledge integration for conservation, NRM, and landscape governance, documentation of integration processes remains fragmented and somewhat scarce. This semi-systematic literature review contributes to filling this gap by synthesising methods, procedures, opportunities, and challenges regarding integrating and decolonising knowledge for conservation and NRM in Southern Africa. The findings demonstrate that despite an increasing number of studies seeking to integrate Indigenous and local knowledge and scientific knowledge relevant to conservation and NRM, methods, procedures, and opportunities are poorly and vaguely documented, and challenges and colonial legacies are often overlooked. Documentation, valuing Indigenous and local knowledge, addressing power relations, and collaboration across knowledge systems are missing steps towards efficient knowledge integration. The paper concludes that there is a need for further research and relevant policies. These should address methods and implications for equitable knowledge integration processes and move beyond knowledge sharing and mutual learning towards decolonising knowledge for conservation and NRM.

Functional characterization of CYP1 enzymes: Complex formation, membrane localization and function.

CYP1A1, CYP1A2, and CYP1B1 have a high degree of sequence similarity, similar substrate selectivities and induction characteristics. However, experiments suggest that there are significant differences in their quaternary structures and function. The goal of this study was to characterize the CYP1 proteins regarding their ability to form protein-protein complexes, lipid microdomain localization, and ultimately function. This was accomplished by examining (1) substrate metabolism of the CYP1s as a function of NADPH-cytochrome P450 reductase (POR) concentration, and (2) quaternary structure, using bioluminescence resonance energy transfer (BRET). Both CYP1As were able to form BRET-detectable homomeric complexes, which was not observed with CYP1B1. When activities were measured as a function of [POR], CYP1A1 and CYP1B1 showed a hyperbolic response, consistent with mass-action binding; however, CYP1A2 produced a sigmoidal response, suggesting that the homomeric complex affected its function. Differences were observed in their ability to form heteromeric complexes. Whereas CYP1B1 and CYP1A1 formed a complex, neither the CYP1A1/CYP1A2 nor the CYP1B1/CYP1A2 pair formed BRET-detectable complexes. These proteins also differed in their lipid microdomain localization, with CYP1A2 and CYP1B1 residing in ordered membranes, and CYP1A1 in the disordered lipid regions. Taken together, despite their sequence similarities, there are substantial differences in quaternary structures and microdomain localization that can influence enzymatic activities. As these proteins exist in the endoplasmic reticulum with other ER-resident proteins, the P450s need to be considered as part of multi-enzyme systems rather than simply monomeric proteins interacting with their redox partners.

The Influence of Lipid Microdomain Heterogeneity on Protein-Protein Interactions: Proteomic Analysis of Co-Immunoprecipitated Binding Partners of P450 1A2 and P450 3A in Rat Liver Microsomes.

Liver cytochrome P450s (CYPs) of the endoplasmic reticulum (ER) are involved in the metabolism of exogenous and endogenous chemicals. The ER is not uniform, but possesses ordered lipid microdomains containing higher levels of saturated fatty acids, sphingomyelin, and cholesterol and disordered regions containing higher levels of polyunsaturated fatty acid chains. The various forms of drug-metabolizing P450s partition to either the ordered or disordered lipid microdomains with different degrees of specificity. P450s readily form complexes with ER-resident proteins, including other forms of P450. This study aims to ascertain whether lipid microdomain localization influences protein-P450 interactions in rat liver microsomes. Thus, liver microsomes were co-immunoprecipitated with CYP1A2-specific and CYP3A-specific antibodies, and the co-immunoprecipitating proteins were identified by liquid chromatography mass spectrometry proteomic analysis. These two P450s preferentially partition to ordered and disordered microdomains, respectively. More than 100 proteins were co-immunoprecipitated with each P450. Segregation of proteins into different microdomains did not preclude their interaction. However, CYP3A interacted broadly with proteins from ordered microdomains, whereas CYP1A2 reacted with a limited subset of these proteins. This is consistent with the concept of lipid raft heterogeneity and may indicate that CYP1A2 is targeted to a specific type of lipid raft. Although many of the interacting proteins for both P450s were other-drug metabolizing enzymes, other interactions were also evident. The consistent CYP3A binding partners were predominantly involved in phase I/II drug metabolism; however, CYP1A2 interacted not only with xenobiotic metabolizing enzymes, but also with enzymes involved in diverse cellular responses such as ER stress and protein folding. SIGNIFICANCE STATEMENT: This work describes the protein interactomes in rat liver microsomes of two important cytochromes P450s (CYP1A2 and CYP3A) in drug metabolism and describes the relationship of the interacting proteins to lipid microdomain distribution.

OeBAS and CYP716C67 catalyze the biosynthesis of health-beneficial triterpenoids in olive (Olea europaea) fruits.

The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri-food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown. Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits. Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold ß-amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2α oxidation of the oleanane- and ursane-type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane- and ursane-type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes. Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

Co-producing theory of change to operationalize integrated landscape approaches.

Integrated landscape approaches that engage diverse stakeholder groups in landscape governance are increasingly promoted to address linked social-ecological challenges in tropical landscapes. Recent research suggests that a transdisciplinary approach to landscape management can help identify common research needs, enhance knowledge co-production, guide evidence-based policy development, and harmonize cross-sectorial integration. Meanwhile, guiding principles for landscape approaches suggest that identifying common concerns and negotiating a process of change are fundamental to implementation and evaluation efforts. As such, the use of decision support tools such as theory of change models that build ordered sequences of actions towards a desired, and agreed, future state are increasingly advocated. However, the application of the theory of change concept to integrated landscape approaches is limited thus far, particularly within the scientific literature. Here, we address this gap by applying the principles of landscape approaches and knowledge co-production to co-produce a theory of change to address current unsustainable landscape management and associated conflicts in the Kalomo Hills Local Forest Reserve No. P.13 (KFR13) of Zambia. The participatory process engaged a diverse range of stakeholders including village head people, local and international researchers, district councillors, and civil society representatives amongst others. Several pathways, actions, and interventions were developed around the themes of deforestation, biodiversity and wildlife conservation, socio-economic development, access rights, and law enforcement. To make the theory of change actionable, participants identified a need for enhanced cross-sector and multi-level communication, capacity development, and improved governance, while a lack of commitment towards coordinated knowledge exchange and access to information along with poor policy formulation and weak enforcement of rules were among potential impediments to action. Use of theory of change can both inform evidence-based policy design (by revealing place-based challenges and proposing solutions) and support policy mechanisms that promote integration between state and non-state actors (by clarifying actor rights, roles and responsibilities). Co-developing a theory of change for integrated landscape management is inherently context specific, but the process and outcomes of this study should hold relevance across a range of contexts faced with sustainability challenges related to reconciling both conservation and development objectives.

Proteomic and Bioinformatics Analysis of Membrane Lipid Domains after Brij 98 Solubilization of Uninduced and Phenobarbital-Induced Rat Liver Microsomes: Defining the Membrane Localization of the P450 Enzyme System.

The proteomes of ordered and disordered lipid microdomains in rat liver microsomes from control and phenobarbital (PB)-treated rats were determined after solubilization with Brij 98 and analyzed by tandem mass tag (TMT)-liquid chromatography-mass spectrometry (LC-MS). This allowed characterization of the liver microsomal proteome and the effects of phenobarbital-mediated induction, focusing on quantification of the relative levels of the drug-metabolizing enzymes._The microsomal proteome from control rats was represented by 333 (23%) proteins from ordered lipid microdomains, 517 (36%) proteins from disordered lipid domains, and 587 (41%) proteins that uniformly distributed between lipid microdomains. Most enzymes related to drug metabolism were mainly localized in disordered lipid microdomains. However, cytochrome P450 (CYP) 1A2, multiple forms of CYP2D, and several forms of UDP glucuronosyltransferases (UGT) 1A1 and 1A6) localized to ordered lipid microdomains. Other drug-metabolizing enzymes, including several forms of cytochromes P450, were uniformly distributed between the ordered and disordered regions. The redox partners, NADPH-cytochrome P450 reductase and cytochrome b5, localized to disordered microdomains. PB induction resulted in only modest changes in protein localization. Less than five proteins were variably associated with the ordered and disordered membrane microdomains in PB and control microsomes. PB induction was associated with fewer proteins localizing in the disordered membranes and more being uniformly distributed or localized to ordered domains. Ingenuity Pathway Analysis (IPA) was used to ascertain the effect of PB on cellular pathways, resulting in attenuation of pathways related to energy storage/utilization and overall cellular signaling and an increase in those related to degradative pathways. SIGNIFICANCE STATEMENT: This work identifies the lipid microdomain localization of the proteome from control and phenobarbital-induced rat liver microsomes. Thus, it provides an initial framework to understand how lipid/protein segregation influences protein-protein interactions in a tissue extract commonly used for studies in drug metabolism and uses bioinformatics to elucidate the effects of phenobarbital induction on cellular pathways.

Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.

Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

Subtelomeric assembly of a multi-gene pathway for antimicrobial defense compounds in cereals.

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat-the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a 'self-poisoning' scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.

Victim selection patterns of community-residing child molesters identified by a nationwide youth-serving organization.

The current study examines data extracted from revocation files of a national youth-serving organization (YSO) involving 7819 revoked individuals and 12,254 alleged child victims to better understand victim selection patterns of community-residing child molesters. These data demonstrate consistent patterns of victim selection based upon the age, gender, and YSO affiliation of each victim. We created two variables to explore whether the revoked individual was "likely pedophilic (LP)" or a "mixed offender (MO)" based upon their behaviors and patterns of victim selection. Compared with the non-LPs and non-MOs, LPs and MOs had more known victims, were more often involved in other YSOs, and were more likely to have had contact with law enforcement. We utilized a public health perspective to interpret these findings, suggesting that child sexual abuse in YSOs reflects a societal problem that is not a unique phenomenon specific to programs offering services to youth.

Complete chloroplast genome of Prunus fasciculata (Rosaceae), a species native to western North America.

Prunus fasciculata is a wild species of Prunus native to western North America. Here, we reported the complete chloroplast (cp) genome of P. fasciculata (GenBank accession number: MW160273). The cp genome was 157,986 bp long, with a large single-copy (LSC) region of 86,068 bp and a small single-copy (SSC) region of 19,166 bp separated by a pair of inverted repeats (IRs) of 26,376 bp. It encodes 129 genes, including 84 protein-coding genes, 37 tRNA genes, and eight ribosomal RNA genes. We also reconstructed the phylogeny of Prunus sensu lato using maximum-likelihood (ML) method, including our data and previously reported cp genomes of related taxa. The phylogenetic analysis confirmed the sister group relationship between P. fasciculata and the remaining subg. Prunus.