An alternative angiosperm DGAT1 topology and potential motifs in the N-terminus.

The highly variable cytoplasmic N-terminus of the plant diacylglycerol acyltransferase 1 (DGAT1) has been shown to have roles in oligomerization as well as allostery; however, the biological significance of the variation within this region is not understood. Comparing the coding sequences over the variable N-termini revealed the Poaceae DGAT1s contain relatively high GC compositional gradients as well as numerous direct and inverted repeats in this region. Using a variety of reciprocal chimeric DGAT1s from angiosperms we show that related N-termini had similar effects (positive or negative) on the accumulation of the recombinant protein in Saccharomyces cerevisiae. When expressed in Camelina sativa seeds the recombinant proteins of specific chimeras elevated total lipid content of the seeds as well as increased seed size. In addition, we combine N- and C-terminal as well as internal tags with high pH membrane reformation, protease protection and differential permeabilization. This led us to conclude the C-terminus is in the ER lumen; this contradicts earlier reports of the cytoplasmic location of plant DGAT1 C-termini.

Clinicians' experience of the diagnosis and management of patellofemoral pain: A qualitative exploration.

BACKGROUND: Patellofemoral pain (PFP) is common and has a poor long-term prognosis. There is a lack of clarity about the clinical reasoning of recognised inter-disciplinary experts in the published literature. OBJECTIVES: To help identify best practice by exploring the clinical reasoning of a range of inter-disciplinary experts that regularly diagnose and treat PFP. DESIGN: Qualitative study with semi-structured interviews. METHOD: Recruitment resulted in a convenience sample for semi-structured interview, which were recorded and transcribed verbatim. Data were analysed until theoretical saturation, as determined by multiple investigators. FINDINGS: Interviews with 19 clinical experts (15 men, 4 women; mean experience 18.6 years ± 8.6) from four broad professions yielded four themes. Firstly, the assessment and diagnosis process should include a thorough history and examination to rule in PFP. Secondly, information provision should aim to increase patients' understanding, aid in controlling symptoms, and facilitate behaviour change. Thirdly, active rehabilitation, which was a salient theme and included advocacy of combined hip and knee exercise that is adapted to the individual. Finally, treatment adjuncts, which can be used selectively to modify symptoms, may include running retraining, taping, or foot orthoses. CONCLUSIONS: PFP should be diagnosed clinically, and tailored treatment programmes should be prescribed for people with PFP. Exercise was considered the most effective treatment and underlying psychological factors should be addressed to improve prognosis.

Development of Inhibitors of SAICAR Synthetase (PurC) from Mycobacterium abscessus Using a Fragment-Based Approach.

Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.

Patient experience of the diagnosis and management of patellofemoral pain: A qualitative exploration.

BACKGROUND: Patellofemoral pain (PFP) is common and long-term treatment outcomes are unsatisfactory. Qualitative exploration of diagnosis and management from the perspective of people with PFP is lacking. OBJECTIVES: To inform care and improve intervention delivery by exploring the experience of people with PFP regarding diagnosis and management. DESIGN: Qualitative study with semi-structured interviews. METHOD: Online recruiting yielded a convenience sample of participants with PFP for semi-structured interview. Interviews were recorded, transcribed verbatim, and analysed using thematic analysis until theoretical saturation by multiple investigators to determine themes and sub-themes. RESULTS: 12 participants were interviewed, with three themes identified; the value of diagnosis, the need for tailored (individualised) care, and the role of education. Participants viewed receiving a diagnosis as essential to guide management, yet one was rarely provided, causing uncertainty about pain mechanisms; "it's nice to be told what it is that's wrong". Interventions needed to be tailored to the individual as not all participants responded in the same way to treatment(s) or had the same needs; "everyone copes and reacts differently". Finally, participants viewed education as essential to empower them to understand and manage the condition; "if I'd have been given more information, I think I'd know how to deal with it more". CONCLUSIONS: The overarching narrative from three themes was a desire for clearly communicated personalised care that meets individual needs. People with PFP desire a diagnosis to explain their pain, tailored interventions, and appropriate education to optimise their experience and outcomes.

Non-attendance and utilization of a speech and language therapy service: a retrospective pilot study of school-aged referrals.

BACKGROUND: Non-attendance and inappropriate referrals affect the effective and efficient running of healthcare services. Non-engagement with speech and language therapy (SLT) services may lead to negative long-term consequences for children in need of SLT intervention. Currently there is a dearth of research on non-attendance and non-engagement with SLT services. AIMS: To identify factors associated with (1) non-attendance and (2) parents' non-engagement with SLT services. METHODS & PROCEDURES: Demographic data were collected from 140 case files of children (aged 5;0-17;11 years) discharged from a public community SLT service (November 2011-October 2013) with no intervention provided. Logistic regression analyses explored relationships between demographic data and (1) non-attendance and (2) non-engagement with the SLT service. OUTCOMES & RESULTS: There was an increased probability of non-attendance during winter (i.e. September-February inclusive; OR = 3.14; p = 0.028) relative to summer, and with each month a child waited for SLT assessment (OR = 1.19; p = 0.066). There was decreased probability of non-attendance with children referred for speech (OR = 0.08; p = 0.011) or language difficulties (OR = 0.15; p = 0.050) relative to dysfluency. The probability of non-engagement with the SLT service increased in each of the following conditions: with each month a child waited for assessment (OR = 1.27; p = 0.004); in urban (OR = 2.40; p = 0.066) relative to rural locations; during winter (OR = 2.65; p = 0.021) relative to summer; and with referrals made by occupational therapists, physiotherapists, psychologists and social workers (OR = 18.65; p = 0.016) relative to doctor referrals. CONCLUSIONS & IMPLICATIONS: Non-attendance is influenced by wait times, season and the reason for referral. Location (urban versus rural), referral source, wait times and season are factors related to non-engagement with SLT services. Targeted policies to improve efficiency and effectiveness of SLT services could be designed around these study findings.

In vivo packaging of triacylglycerols enhances Arabidopsis leaf biomass and energy density.

Our dependency on reduced carbon for energy has led to a rapid increase in the search for sustainable alternatives and a call to focus on energy densification and increasing biomass yields. In this study, we generated a uniquely stabilized plant structural protein (cysteine [Cys]-oleosin) that encapsulates triacylglycerol (TAG). When coexpressed with diacylglycerol O-acyltransferase (DGAT1) in Arabidopsis (Arabidopsis thaliana), we observed a 24% increase in the carbon dioxide (CO2) assimilation rate per unit of leaf area and a 50% increase in leaf biomass as well as approximately 2-, 3-, and 5-fold increases in the fatty acid content of the mature leaves, senescing leaves, and roots, respectively. We propose that the coexpression led to the formation of enduring lipid droplets that prevented the futile cycle of TAG biosynthesis/lipolysis and instead created a sustained demand for de novo lipid biosynthesis, which in turn elevated CO2 recycling in the chloroplast. Fatty acid profile analysis indicated that the formation of TAG involved acyl cycling in Arabidopsis leaves and roots. We also demonstrate that the combination of Cys-oleosin and DGAT1 resulted in the highest accumulation of fatty acids in the model single-cell eukaryote, Saccharomyces cerevisiae. Our results support the notion that the prevention of lipolysis is vital to enabling TAG accumulation in vegetative tissues and confirm the earlier speculation that elevating fatty acid biosynthesis in the leaf would lead to an increase in CO2 assimilation. The Cys-oleosins have applications in biofuels, animal feed, and human nutrition as well as in providing a tool for investigating fatty acid biosynthesis and catabolism.

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes.

Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca(2+) signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca(2+)-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17-yellow fluorescent protein (YFP) and CPK34-YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34-YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca(2+)-activation mechanism of CPK34 (CPK34-YFP-E465A,E500A), providing in vivo evidence linking Ca(2+) activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca(2+) signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.