Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline.

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.

Arabidopsis calmodulin-like proteins CML13 and CML14 interact with proteins that have IQ domains.

In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs) and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13 and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13 and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+ . Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13 and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.

Structural insights into TAZ2 domain-mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A.

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

A cross-sectional survey of the perspectives of older people in the Scottish Highlands on the management of their chronic pain.

BACKGROUND: Although there is evidence of suboptimal outcomes in older people with chronic pain, little emphasis has been placed on those in remote and rural settings. OBJECTIVE: To describe the perspectives of older people in the Scottish Highlands on their chronic pain management. DESIGN: Cross-sectional survey. SETTING: NHS Highland, the most remote and rural geographical health board in Scotland. SUBJECTS: Home-dwelling members of the public aged ≥70 years. METHODS: Anonymised questionnaires were mailed to a random sample of 1800 older people. Questionnaire items were demographics, nature of any chronic pain, management regimens and perceived effectiveness. Validated scales were the Pain Disability Questionnaire and the Tampa Scale for Kinesiophobia. RESULTS: Adjusted response rate was 39.3% (709/1755). One-quarter (25.0%, n = 177) were experiencing chronic pain, being more likely to live in deprived areas (P < 0.05). Median pain intensity was 6 (IQR 4-7, 10 high), causing distress (median 5, IQR 3-7). Respondents largely consulted GPs (66.1%, n = 117) with a minority (16.4%, n = 29) referred to a specialist pain clinic and few consulting other health professionals. Over three quarters (78.0%, n = 138) were receiving prescribed medicines, most commonly paracetamol, alone (35.6%, n = 63) or in combination with opioids (16.4%, n = 29). One-third (31.6%, n = 56) expressed a desire for more effective medicines; few reported using any non-pharmacological therapies. The median scores for the Pain Disability Questionnaire and Tampa Scale for Kinesiophobia were 74 (IQR 34-104.5, 150 high) and 40 (IQR 35-45, 68 high). CONCLUSIONS: Evidence of provision of appropriate integrated and person-centred chronic pain care is lacking.

Multiple Calmodulin-Binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12.

Ca(2+) signaling is critical to plant immunity; however, the channels involved are poorly characterized. Cyclic nucleotide-gated channels (CNGCs) are nonspecific, Ca(2+)-permeable cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca(2+) sensor calmodulin (CaM), and previous work has focused on a C-terminal CaM-binding domain (CaMBD) overlapping with the cyclic nucleotide binding domain of plant CNGCs. However, we show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic N and C termini, which is reminiscent of animal CNGCs and unlike any plant channel studied to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a conserved isoleucine-glutamine (IQ) motif in the C terminus of the channel, while Ca(2+)/CaM binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with a nonfunctional N-terminal CaMBD constitutively induced programmed cell death, providing in planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM binding to the IQ motif was required for channel function, indicating that CaM can both positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest exciting parallels between plant and animal channels.

N-Heterocyclic Carbene Self-Assembled Monolayers on Gold as Surface Plasmon Resonance Biosensors.

Surface plasmon resonance (SPR)-based biosensing is a powerful tool to study the recognition processes between biomolecules in real-time without need for labels. The use of thiol chemistry is a critical component in surface functionalization of various SPR biosensor surfaces on gold. However, its use is hampered by the high propensity for oxidation of the gold-thiol linkage even in ambient atmosphere, resulting in a short lifetime of SPR sensor chips unless strict precautions are taken. Herein, we describe an approach to overcome this limitation by employing highly robust self-assembled monolayers (SAMs) of alkylated N-heterocyclic carbenes (NHCs) on gold. An alkylated NHC sensor surface was developed and its biosensing capabilities were compared to a commercial thiol-based analogue-a hydrophobic association (HPA) chip-in terms of its ability to act as a reliable platform for biospecific interaction analysis under a wide range of conditions. The NHC-based SPR sensor outperforms related thiol-based sesnsors in several aspects, including lower nonspecific binding capacity, better chemical stability, higher reproducibility, shorter equilibration time, and longer life span. We also demonstrate that the NHC-based sensor can be used for rapid and efficient formation of a hybrid lipid bilayer for use in membrane interaction studies. Overall, this work identifies the great promise in designing NHC-based surfaces as a new technology platform for SPR-based biosensing.

Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300.

The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved ϕ-x-x-ϕ-ϕ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved ϕ-x-x-ϕ-ϕ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

Structure of the small Dictyostelium discoideum myosin light chain MlcB provides insights into MyoB IQ motif recognition.

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca(2+). However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca(2+) binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.

The calmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca(2+) sensor in Arabidopsis.

Many signalling pathways in plants are regulated by the second messenger calcium (Ca(2+)). In the standard model, Ca(2+)-sensor proteins, such as CaM (calmodulin), detect Ca(2+) signals and subsequently regulate downstream targets to advance the signal transduction cascade. In addition to CaM, plants possess many CMLs (CaM-like proteins) that are predicted to function as Ca(2+) sensors, but which remain largely uncharacterized. In the present study, we examined the biochemical properties, subcellular localization and tissue-specific distribution of Arabidopsis CML43. Our data indicate that CML43 displays characteristics typical of Ca(2+) sensors, including high-affinity Ca(2+) binding, conformational changes upon Ca(2+) binding that expose hydrophobic regions and stabilization of structure in the presence of Mg(2+) or Ca(2+). In vivo localization analysis demonstrates that CML43 resides in cytosolic and nuclear compartments. Transgenic plants expressing a CML43:GUS (ß-glucoronidase) promoter reporter gene revealed that CML43 promoter activity is restricted almost exclusively to root tips under normal growth conditions. GUS reporter activity in these transgenic plants was strongly increased when exposed to the defence compound SA (salicylic acid). Furthermore, immunoblot analysis revealed that the CML43 protein accumulates following treatment with SA. Collectively, our findings suggest that CML43 functions as a Ca(2+) sensor in root tips during both normal growth and plant immune response.

Structure and function of a hexameric cyanophycin synthetase 2.

Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide ß-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.

A cross sectional survey of the views of newly registered pharmacists in Great Britain on their potential prescribing role: a cautious approach.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Pharmacists, along with certain other health professionals, may train and practice as supplementary or independent prescribers. The implementation and sustainability of pharmacist prescribing services throughout Britain will require a sizeable workforce. However, a survey of GB pharmacists highlighted that only a minority has taken any action to investigate prescribing training. Newly registered pharmacists may be keen to explore extended clinical roles and their engagement is likely to be key to the future success of this initiative. WHAT THIS STUDY ADDS: Newly registered pharmacists are cautious in their approach to taking on prescribing training and roles. While almost all expressed interest in prescribing training, they acknowledged training needs in clinical examination, patient monitoring and medico-legal aspects of prescribing. Longitudinal research on a cohort of newly registered pharmacist prescribers is warranted, aiming to identify later prescribing training actions and subsequent impact on patient care. AIM To investigate newly registered pharmacists' awareness of pharmacist prescribing and views on potential future roles as prescribers. METHODS: A mailed questionnaire was sent to all 1658 pharmacists joining the Pharmacist Register in 2009. RESULTS: The response rate was 25.2% (n= 418). While most (86.4%) expressed interest in prescribing training, they acknowledged training needs in clinical examination, patient monitoring and medico-legal aspects of prescribing. Two thirds of respondents (66.3%) thought the current requirement of being registered as a pharmacist for 2 years prior to commencing prescribing training was appropriate. CONCLUSION: Newly registered pharmacists are cautious in their approach to taking on prescribing training and roles.

Structural and functional characterization of the monomeric U-box domain from E4B.

Substantial evidence has accumulated indicating a significant role for oligomerization in the function of E3 ubiquitin ligases. Among the many characterized E3 ligases, the yeast U-box protein Ufd2 and its mammalian homologue E4B appear to be unique in functioning as monomers. An E4B U-box domain construct (E4BU) has been subcloned, overexpressed in Escherichia coli, and purified, which enabled determination of a high-resolution NMR solution structure and detailed biophysical analysis. E4BU is a stable monomeric protein that folds into the same structure observed for other structurally characterized U-box domain homodimers. Multiple sequence alignment combined with comparative structural analysis reveals substitutions in the sequence that inhibit dimerization. The interaction between E4BU and the E2 conjugating enzyme UbcH5c has been mapped using NMR, and these data have been used to generate a structural model for the complex. The E2 binding site is found to be similar to that observed for dimeric U-box and RING domain E3 ligases. Despite the inability to dimerize, E4BU was found to be active in a standard autoubiquitination assay. The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization.

Dynamics in Thermotoga neapolitana adenylate kinase: 15N relaxation and hydrogen-deuterium exchange studies of a hyperthermophilic enzyme highly active at 30 degrees C.

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.

Critical role for a single leucine residue in leukemia induction by E2A-PBX1.

In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

Cross sectional survey of the Scottish general public's awareness of, views on, and attitudes toward nonmedical prescribing.

BACKGROUND: Nonmedical (ie, nonphysician) prescribing is a key development in the UK that has brought about many changes in prescribing policy and practice. Systematic research into the views of the general public toward such developments is limited. OBJECTIVE: To determine the awareness of, views on, and attitudes of members of the Scottish general public toward nonmedical prescribing, with an emphasis on pharmacist prescribing. METHODS: A questionnaire was mailed in November 2006 to a random sample of 5000 members of the general public in Scotland aged 18 and over, obtained from the UK electoral roll. The questionnaire contained items on awareness of nonmedical prescribing, levels of comfort with specific health professionals, and attitudes toward pharmacist prescribing. RESULTS: Response rate was 37.1%. More than half of the individuals who responded were taking prescribed drugs. Nine hundred and seventy-eight (56.6%) were aware that trained health professionals could write prescriptions for medicines previously only prescribed by physicians. Awareness was associated with: increasing age (p < 0.001), having a health professional in their immediate family (p < 0.001), self-rated general health (p < 0.005), and a higher education level (p < 0.01). In logistic regression, all factors were retained as independent predictors of awareness (p < 0.001). Comfort levels for nonmedical prescribing were highest for pharmacists (median 4, IQR 3-5 [1 = low, 5 = high]), closely followed by nurses, and lowest for radiographers (median 2, IQR 1-4) (p < 0.001). While more than half of the respondents supported pharmacists having a prescribing role, fewer felt that pharmacists should prescribe the same range of drugs as physicians. There were concerns about lack of privacy in a pharmacy, despite acknowledging its enhanced convenience. CONCLUSIONS: Our results indicate that more than half of the respondents were aware of nonmedical prescribing. A higher proportion was more comfortable with prescribing by pharmacists and nurses than with other healthcare professionals. Several issues relating to aspects of clinical governance were highlighted, specifically education and data handling.

ATXR5/6 Forms Alternative Protein Complexes with PCNA and the Nucleosome Core Particle.

The proliferating cell nuclear antigen (PCNA) is a sliding clamp associated with DNA polymerases and serves as a binding platform for the recruitment of regulatory proteins linked to DNA damage repair, cell cycle regulation, and epigenetic signaling. The histone H3 lysine-27 (H3K27) mono-methyltransferase Arabidopsis trithorax-related protein 5/6 (ATXR5/6) associates with PCNA, and this interaction has been proposed to act as a key determinant controlling the reestablishment of H3K27 mono-methylation following replication. In this study, we provide biochemical evidence showing that PCNA inhibits ATXR6 enzymatic activity. The structure of the ATXR6 PCNA-interacting peptide (PIP) in complex with PCNA indicates that a trio of hydrophobic residues contributes to the binding of the enzyme to the sliding clamp. Finally, despite the presence of three PIP binding clefts, only two molecules of ATXR6 bind to PCNA likely enabling the recruitment of a third protein to the sliding clamp. Collectively, these results rule out the model wherein PCNA-bound ATXR6 actively reestablishes H3K27 mono-methylation following DNA replication and provides insights into the role of ATXR6 PIP motif in its interaction with PCNA.

Self-Assembled N-Heterocyclic Carbene-Based Carboxymethylated Dextran Monolayers on Gold as a Tunable Platform for Designing Affinity-Capture Biosensor Surfaces.

Sensor surfaces play a predominant role in the development of optical biosensor technologies for the analysis of biomolecular interactions. Thiol-based self-assembled monolayers (SAMs) on gold have been widely used as linker layers for sensor surfaces. However, the degradation of the thiol-gold bond can limit the performance and durability of such surfaces, directly impacting their performance and cost-effectiveness. To this end, a new family of materials based on N-heterocyclic carbenes (NHCs) has emerged as an alternative for surface modification, capable of self-assembling onto a gold surface with higher affinity and superior stability as compared to the thiol-based systems. Here we demonstrate three applications of NHC SAMs supporting a dextran layer as a tunable platform for developing various affinity-capture biosensor surfaces. We describe the development and testing of NHC-based dextran biosensor surfaces modified with each of streptavidin, nitrilotriacetic acid, and recombinant Protein A. These affinity-capture sensor surfaces enable oriented binding of ligands for optimal performance in biomolecular assays. Together, the intrinsic high stability and flexible design of the NHC biosensing platforms show great promise and open up exciting possibilities for future biosensing applications.

Arabidopsis Calmodulin-Like Proteins, CML15 and CML16 Possess Biochemical Properties Distinct from Calmodulin and Show Non-overlapping Tissue Expression Patterns.

Calcium ions are used as ubiquitous, key second messengers in cells across eukaryotic taxa. In plants, calcium signal transduction is involved in a wide range of cellular processes from abiotic and biotic stress responses to development and growth. Calcium signals are detected by calcium sensor proteins, of which calmodulin (CaM), is the most evolutionarily conserved and well-studied. These sensors regulate downstream targets to propagate the information in signaling pathways. Plants possess a large family of calcium sensors related to CaM, termed CaM-like (CMLs), that are not found in animals and remain largely unstudied at the structural and functional level. Here, we investigated the biochemical properties and gene promoter activity of two closely related members of the Arabidopsis CML family, CML15 and CML16. Biochemical characterization of recombinant CML15 and CML16 indicated that they possess properties consistent with their predicted roles as calcium sensors. In the absence of calcium, CML15 and CML16 display greater intrinsic hydrophobicity than CaM. Both CMLs displayed calcium-dependent and magnesium-independent conformational changes that expose hydrophobic residues, but the degree of hydrophobic exposure was markedly less than that observed for CaM. Isothermal titration calorimetry indicated two and three calcium-binding sites for CML15 and CML16, respectively, with affinities expected to be within a physiological range. Both CML15 and CML16 bound calcium with high affinity in the presence of excess magnesium. Promoter-reporter analysis demonstrated that the CML16 promoter is active across a range of Arabidopsis tissues and developmental stages, whereas the CML15 promoter activity is very restricted and was observed only in floral tissues, specifically anthers and pollen. Collectively, our data indicate that these CMLs behave biochemically like calcium sensors but with properties distinct from CaM and likely have non-overlapping roles in floral development. We discuss our findings in the broader context of calcium sensors and signaling in Arabidopsis.

Carboxymethylated Dextran-Modified N-Heterocyclic Carbene Self-Assembled Monolayers on Gold for Use in Surface Plasmon Resonance Biosensing.

Surface chemistry is a key enabler for various biosensing applications. Biosensors based on surface plasmon resonance routinely employ thiol-based chemistry for the linker layer between gold-coated support surfaces and functional biosensor surfaces. However, there is a growing awareness that such sensor surfaces are prone to oxidation/degradation problems in the presence of oxygen, and previous efforts to improve the stability have shown limited advancements. As an alternative, recent studies employing N-heterocyclic carbene (NHC) self-assembled monolayers (SAMs) deposited on gold have shown significant promise in this area. Here, we describe a sensor surface employing an NHC SAM to couple a modified carboxymethylated dextran onto a gold surface. Such a dextran matrix is also used for affinity chromatography, and it is the most commonly employed matrix for commercial biosensor surfaces today. The performance reliability of the dextran-modified NHC chip to act as an alternative biosensing platform is compared with that of a thiol-based commercial chip in the proof-of-concept tests. The resultant NHC sensor surface shows a higher thermal stability compared to thiol analogues. Moreover, the plasma protein/drug and antibody/antigen interactions were validated on the NHC-based dextran chip and showed similar performance as compared to the thiol-based commercial chip. Ultimately, this study shows the strong potential applicability of chemical modifications to gold surfaces using NHC ligands for biosensing applications.

Prescription medications: beliefs, experiences, behavior, and adherence of sheltered housing residents.

BACKGROUND: Data on medication utilization among residents of sheltered housing complexes (SHCs) are limited. OBJECTIVE: To evaluate the extent of nonadherence to prescribed medications among residents of SHCs and identify factors associated with nonadherence. METHODS: A 58 item, pre-piloted questionnaire with validated items for patient self-assessment of medication risk; adherence; disability; health beliefs, experiences, and behavior; and general items on demographics, health, and medication use was mailed to 1137 SHC residents in Aberdeen, Scotland. Predictors of nonadherence (Morisky score >0) were identified using logistic regression. RESULTS: Of the 695 (61.1%) respondents, 645 (mean +/- SD age, 78.2 +/- 7.8 y) reported using prescribed drugs. Nonadherence was reported by 176 of 627 (28.1%) residents. The predictors of nonadherence were: disagreeing with the statement, "I ensure I have enough medicines so that I don't run out" (OR 0.48; 95% CI 0.30 to 0.77), agreeing with the statements, "I make changes in the recommended management to suit my lifestyle" (OR 1.32; 95% CI 1.09 to 1.60) and "I get confused about my medicines" (OR 1.39; 95% CI 1.10 to 1.77), younger age (OR 0.96; 95% CI 0.94 to 0.99), not getting help from someone to use the medications correctly (OR 2.20; 95% CI 1.21 to 3.99), and agreeing with the statement, "I am concerned about the side effects from my medicines" (OR 1.31; 95% CI 1.05 to 1.63). CONCLUSIONS: Over one-quarter of SHC residents were found to be nonadherent to prescribed drugs. Risk factors for nonadherence include younger age, confusion about drugs, lack of support for drug supply and administration, interference of treatment recommendations with lifestyle, and a perceived view of risks outweighing benefits in using recommended drug therapy.