The Alberta Telestewardship Network: Building a platform to enable capacity building in antimicrobial stewardship-results of an initial pilot study.

Background: Resources to improve antimicrobial stewardship (AS) are limited, but a telestewardship platform can enable capacity building and scalability. The Alberta Telestewardship Network (ATeleNet) was designed to focus on outreach across the province of Alberta, Canada, and facilitate AS activities. Methods: Outreach occurred virtually between pharmacists and physicians in hospital and long-term care settings throughout Alberta via secure, enterprise video conferencing software on both desktop and mobile devices. We used a quantitative questionnaire adapted from the telehealth usability questionnaire to capture the health provider's experience during each session. The questionnaire consisted of 39 questions, and a 5-point Likert scale was used to assess the degree of agreement and collate responses into a descriptive analysis. Results: A total of 33 pilot consultations were completed between July 6, 2020 and December 15, 2021. The majority (22, 85%) of respondents agreed that video conference-based virtual sessions are an acceptable means to provide health care and that they were able to express themselves effectively to other health care professionals (23, 88%). Respondents agreed the system was simple to use (23, 96%), and that they could become productive quickly using the system (23, 88%). Overall, 24 (92%) respondents were satisfied or very satisfied with the virtual care platform. Conclusions: We implemented and evaluated a telehealth consultation and collaborative care service between AS providers at multiple centres. AHS has since prioritized similar workflows, including access to specialists in acute care, as part of their virtual health strategy. Evaluation results will be shared with provincial stakeholders for further strategic planning and deployment.

Historique: Les ressources pour améliorer la gérance antimicrobienne (GA) sont limitées, mais une plateforme de télégérance peut favoriser le renforcement des capacités et l'échelonnabilité. L'Alberta Telestewardship Network (réseau de télégérance de l'Alberta, ou ATeleNet) a été conçu pour mettre l'accent sur le rayonnement dans la province de l'Alberta, au Canada et pour faciliter les activités de GA. Méthodologie: Le rayonnement s'est produit virtuellement entre des pharmaciens et des médecins d'établissements hospitaliers et d'établissements de soins de longue durée de l'Alberta par logiciel de visioconférence sécurisé sur des ordinateurs de bureau et des appareils mobiles. Les chercheurs ont utilisé un questionnaire quantitatif adapté du questionnaire sur la convivialité de la télésanté pour saisir l'expérience du dispensateur de soins lors de chaque séance. Le questionnaire était composé de 39 questions, et une échelle de Likert de cinq points a permis d'évaluer le degré d'entente et de recueillir les réponses dans une analyse descriptive. Résultats: Au total, les chercheurs ont effectué 33 consultations pilotes entre le 6 juillet 2020 et le 15 décembre 2021. La majorité des répondants (n = 22, 85 %) ont convenu que les séances en visioconférence représentaient un moyen acceptable de fournir des soins de santé et leur permettaient de s'exprimer avec efficacité auprès des autres professionnels de la santé (n = 23, 88 %). Les répondants ont indiqué que le système était facile à utiliser (n = 23, 96 %), et qu'ils pouvaient vite devenir productifs (n = 23, 88 %). Dans l'ensemble, 24 répondants (92 %) étaient satisfaits ou très satisfaits de la plateforme de soins virtuels. Conclusions: Les chercheurs ont lancé et évalué une consultation en télésanté et un service de soins coopératifs entre fournisseurs de GA de multiples centres. Depuis, les Services de santé de l'Alberta ont priorisé des processus de travail semblables dans leur stratégie de santé virtuelle, y compris pour l'accès à des spécialistes en soins aigus. Les résultats de l'évaluation seront transmis à des intervenants provinciaux en vue d'une planification et d'un déploiement stratégiques.

In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells.

Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.

NAD+/NADH redox alterations reconfigure metabolism and rejuvenate senescent human mesenchymal stem cells in vitro.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease.

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.

Metabolism in Human Mesenchymal Stromal Cells: A Missing Link Between hMSC Biomanufacturing and Therapy?

Human mesenchymal stem cells (hMSCs) are the most commonly-tested adult stem cells in cell therapy. While the initial focus for hMSC clinical applications was to exploit their multi-potentiality for cell replacement therapies, it is now apparent that hMSCs empower tissue repair primarily by secretion of immuno-regulatory and pro-regenerative factors. A growing trend in hMSC clinical trials is the use of allogenic and culture-expanded cells because they are well-characterized and can be produced in large scale from specific donors to compensate for the donor pathological condition(s). However, donor morbidity and large-scale expansion are known to alter hMSC secretory profile and reduce therapeutic potency, which are significant barriers in hMSC clinical translation. Therefore, understanding the regulatory mechanisms underpinning hMSC phenotypic and functional property is crucial for developing novel engineering protocols that maximize yield while preserving therapeutic potency. hMSC are heterogenous at the level of primary metabolism and that energy metabolism plays important roles in regulating hMSC functional properties. This review focuses on energy metabolism in regulating hMSC immunomodulatory properties and its implication in hMSC sourcing and biomanufacturing. The specific characteristics of hMSC metabolism will be discussed with a focus on hMSC metabolic plasticity and donor- and culture-induced changes in immunomodulatory properties. Potential strategies of modulating hMSC metabolism to enhance their immunomodulation and therapeutic efficacy in preclinical models will be reviewed.

Commitment to Aerobic Glycolysis Sustains Immunosuppression of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue repair in part by coordinating multiple components of the host immune system in response to environmental stimuli. Recent studies have shown that hMSCs are metabolically heterogeneous and actively reconfigure metabolism to support the biochemical demands of tissue repair. However, how hMSCs regulate their energy metabolism to support their immunomodulatory properties is largely unknown. This study investigates hMSC metabolic reconfiguration during immune activation and provides evidence that the hMSC metabolic state significantly influences their immunomodulatory properties. Specifically, hMSC immune polarization by interferon-gamma (IFN-γ) treatment leads to remodeling of hMSC metabolic pathways toward glycolysis, which is required to sustain the secretion of immunosuppressive factors. IFN-γ exposure also inhibited mitochondrial electron transport activity, and the accumulation of mitochondrial reactive oxygen species plays an important signaling role in this metabolic reconfiguration. The results also show that activation of the Akt/mTOR signaling pathway is required for metabolic reconfiguration during immune polarization and that interruption of these metabolic changes alters the immune response in IFN-γ licensed hMSCs. The results demonstrate the potential of altering hMSC metabolism to enhance their immunomodulatory properties and therapeutic efficacy in various diseases. Stem Cells Translational Medicine 2019;8:93-106.

Use of MRI, metabolomic, and genomic biomarkers to identify mechanisms of chemoresistance in glioma.

Gliomas are the most common form of central nervous system tumor. The most prevalent form, glioblastoma multiforme, is also the most deadly with mean survival times that are less than 15 months. Therapies are severely limited by the ability of these tumors to develop resistance to both radiation and chemotherapy. Thus, new tools are needed to identify and monitor chemoresistance before and after the initiation of therapy and to maximize the initial treatment plan by identifying patterns of chemoresistance prior to the start of therapy. Here we show how magnetic resonance imaging, particularly sodium imaging, metabolomics, and genomics have all emerged as potential approaches toward the identification of biomarkers of chemoresistance. This work also illustrates how use of these tools together represents a particularly promising approach to understanding mechanisms of chemoresistance and the development individualized treatment strategies for patients.

Metabolic Reconfiguration Supports Reacquisition of Primitive Phenotype in Human Mesenchymal Stem Cell Aggregates.

Spontaneous aggregation and the associated enhancement of stemness have been observed in many anchorage dependent cells. Recently, aggregation of human mesenchymal stem cells (hMSCs) in nonadherent culture has been shown to reverse expansion-induced heterogeneity and loss of stemness and reprogram the hMSC to reacquire their primitive phenotype, a phenomenon that can significantly enhance therapeutic applications of hMSC. The objective of this study was to investigate the mechanistic basis underlying the connection between multicellular aggregation and stemness enhancement in hMSC by testing the hypothesis that cellular events induced during three-dimensional aggregation on nonadherent substratum induces changes in mitochondrial metabolism that promote the expression of stem cell genes Oct4, Sox2, and Nanog. Our results show that aggregation changes mitochondrial morphology and reduces mitochondrial membrane potential, resulting in a metabolic reconfiguration characterized by increased glycolytic and anaplerotic flux, and activation of autophagy. We further demonstrate that interrupting mitochondrial respiration in two-dimensional planar culture with small molecule inhibitors partially recapitulates the aggregation-mediated enhancement in stem cell properties, whereas enhancement of mitochondrial oxidative phosphorylation in the aggregated state reduces the aggregation-induced upregulation of Oct4, Sox2, and Nanog. Our findings demonstrate that aggregation-induced metabolic reconfiguration plays a central role in reacquisition of primitive hMSC phenotypic properties. Stem Cells 2017;35:398-410.

Density-Dependent Metabolic Heterogeneity in Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are intrinsically heterogeneous and comprise subpopulations that differ in their proliferation, multi-potency, and functional properties, which are commonly demonstrated by culturing hMSCs at different plating densities. The objective of this study was to investigate the metabolic profiles of different subpopulations of hMSC by testing the hypothesis that the clonogenic hMSC subpopulation, which is selectively enriched in clonal density (CD) and low density (LD) culture (10 and 100 cells per square centimeter, respectively), possesses a metabolic phenotype that differs from that of hMSC in medium- or high-density (MD: 1,000 and HD: 3,000 cells per square centimeter, respectively). Cells at CD and LD conditions exhibited elevated expression of CD146 and colony forming unit-fibroblast compared with cells at MD- or HD. Global metabolic profiles revealed by gas chromatography-mass spectrometry of cell extracts showed clear distinction between LD and HD cultures, and density-dependent differences in coupling of glycolysis to the TCA cycle. Metabolic inhibitors revealed density-dependent differences in glycolysis versus oxidative phosphorylation (OXPHOS) for ATP generation, in glutamine metabolism, in the dependence on the pentose phosphate pathway for maintaining cellular redox state, and sensitivity to exogenous reactive oxygen species. We also show that active OXPHOS is not required for proliferation in LD culture but that OXPHOS activity increases senescence in HD culture. Together, the results revealed heterogeneity in hMSC culture exists at the level of primary metabolism. The unique metabolic characteristics of the clonogenic subpopulation suggest a novel approach for optimizing in vitro expansion of hMSCs.

Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

Gas chromatography-mass spectrometry analysis of human mesenchymal stem cell metabolism during proliferation and osteogenic differentiation under different oxygen tensions.

Bone marrow derived human mesenchymal stem cells (hMSC) are the primary cell type in bone tissue engineering, and their life span during osteogenic differentiation is associated with changes in oxygen tension. As a ubiquitous regulator of cellular metabolic activity, oxygen tension influences the profiles of metabolites in the entire metabolic network and plays an important role in hMSC survival, function, and osteogenic differentiation. In the current study, we hypothesize that hMSC have a metabolic phenotype that supports growth in low oxygen environments and that this phenotype changes upon differentiation, leading to differential responses to oxygen tension. We developed a gas chromatography-mass spectrometry (GC-MS) based metabolic profiling approach to analyze the metabolic fate of (13)C-glucose in glycolysis and the tricarboxylic acid cycle (TCA) in undifferentiated hMSC and hMSC-derived osteoblasts (hMSC-OS) in response to perturbation in oxygen tension; specifically we compared changes induced by culture under 20% vs. 2% O2. The isotope enrichments in the metabolites were calculated and used to infer activities of specific metabolic enzymes and the associated pathways. The results revealed contrasting metabolic profiles for hMSC and the hMSC-OS in both 20% and 2% O2 states, with the most significant differences involving coupling of glycolysis to the TCA cycle, glutaminolysis, and the malate-aspartate shuttle. The results have important implications in defining the optimal culture conditions for hMSC expansion and osteogenic differentiation.

Linear segmentation algorithm for detecting layer boundary with lidar.

The automatic detection of aerosol- and cloud-layer boundary (base and top) is important in atmospheric lidar data processing, because the boundary information is not only useful for environment and climate studies, but can also be used as input for further data processing. Previous methods have demonstrated limitations in defining the base and top, window-size setting, and have neglected the in-layer attenuation. To overcome these limitations, we present a new layer detection scheme for up-looking lidars based on linear segmentation with a reasonable threshold setting, boundary selecting, and false positive removing strategies. Preliminary results from both real and simulated data show that this algorithm cannot only detect the layer-base as accurate as the simple multi-scale method, but can also detect the layer-top more accurately than that of the simple multi-scale method. Our algorithm can be directly applied to uncalibrated data without requiring any additional measurements or window size selections.

Zn(II) stimulation of Fe(II)-activated repression in the iron-dependent repressor from Mycobacterium tuberculosis.

Thermodynamic measurements of Fe(II) binding and activation of repressor function in the iron-dependent repressor from Mycobacterium tuberculosis (IdeR) are reported. IdeR, a member of the diphtheria toxin repressor family of proteins, regulates iron homeostasis and contributes to the virulence response in M. tuberculosis. Although iron is the physiological ligand, this is the first detailed analysis of iron binding and activation in this protein. The results showed that IdeR binds 2 equiv of Fe(II) with dissociation constants that differ by a factor of 25. The high- and low-affinity iron binding sites were assigned to physical binding sites I and II, respectively, using metal binding site mutants. IdeR was also found to contain a high-affinity Zn(II) binding site that was assigned to physical metal binding site II through the use of binding site mutants and metal competition assays. Fe(II) binding was modestly weaker in the presence of Zn(II), but the coupled metal binding-DNA binding affinity was significantly stronger, requiring 30-fold less Fe(II) to activate DNA binding compared to Fe(II) alone. Together, these results suggest that IdeR is a mixed-metal repressor, where Zn(II) acts as a structural metal and Fe(II) acts to trigger the physiologically relevant promoter binding. This new model for IdeR activation provides a better understanding of IdeR and the biology of iron homeostasis in M. tuberculosis.

A conserved tandem cyclophilin-binding site in hepatitis C virus nonstructural protein 5A regulates Alisporivir susceptibility.

Cyclophilin A (CyPA) and its peptidyl-prolyl isomerase (PPIase) activity play an essential role in hepatitis C virus (HCV) replication, and mounting evidence indicates that nonstructural protein 5A (NS5A) is the major target of CyPA. However, neither a consensus CyPA-binding motif nor specific proline substrates that regulate CyPA dependence and sensitivity to cyclophilin inhibitors (CPIs) have been defined to date. We systematically characterized all proline residues in NS5A domain II, low-complexity sequence II (LCS-II), and domain III with both biochemical binding and functional replication assays. A tandem cyclophilin-binding site spanning domain II and LCS-II was identified. The first site contains a consensus sequence motif of AØPXW (where Ø is a hydrophobic residue) that is highly conserved in the majority of the genotypes of HCV (six of seven; the remaining genotype has VØPXW). The second tandem site contains a similar motif, and the ØP sequence is again conserved in six of the seven genotypes. Consistent with the similarity of their sequences, peptides representing the two binding motifs competed for CyPA binding in a spot-binding assay and induced similar chemical shifts when bound to the active site of CyPA. The two prolines (P310 and P341 of Japanese fulminant hepatitis 1 [JFH-1]) contained in these motifs, as well as a conserved tryptophan in the spacer region, were required for CyPA binding, HCV replication, and CPI resistance. Together, these data provide a high-resolution mapping of proline residues important for CyPA binding and identify critical amino acids modulating HCV susceptibility to the clinical CPI Alisporivir.

A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach.

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells.

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-¹³C-glucose and ¹5N-glutamate as labeled precursors. This study suggests that uniformly ¹5N,¹³C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Analysis of the dynamics of assembly and structural impact for a histidine tagged FGF1-1.5 nm Au nanoparticle bioconjugate.

Whether assembling proteins onto nanoscale, mesoscopic, or macroscropic material surfaces, maintaining a protein's structure and function when conjugated to a surface is complicated by the high propensity for electrostatic or hydrophobic surface interactions and the possibility of direct metal coordination of protein functional groups. In this study, the assembly of a 1.5 nm CAAKA passivated gold nanoparticle (AuNP) onto FGF1 (human acidic fibroblast growth factor) using an amino terminal His(6) tag is analyzed. The impact of structure and time-dependent changes in the structural elements in FGF1and FGF1-heparin in the presence of the AuNP is probed by a molecular beacon fluorescence assay, circular dichroism, and NMR spectroscopy. Analysis of the results indicates that a time-dependent evolution of the protein structure without loss of FGF1 heparin binding occurs following the formation of the initial FGF1-AuNP complex. The time-dependent changes are believed to reflect protein sampling of the AuNP surface to minimize the free energy of the AuNP-FGF1 complex without impacting FGF1 function.

Single peptide assembly onto a 1.5 nm Au surface via a histidine tag.

Nanoparticle surfaces functionalized with proteins or other biomolecules provide a mechanism for interfacing the unique properties of nanomaterials with biological samples. In most of these studies, the biomolecule is conjugated to a gold nanoparticles (AuNP) surface through the thiol group of native or introduced cysteine residues. Here we demonstrate the direct attachment of a hexa-histidine tagged (His(6)) peptide to a 1.5 nm AuNP. Binding occurs via a specific interaction between the Ne of the His imidazole, forming a 1:1 stoichiometric complex. Given the widespread use of histidine tags in producing recombinant proteins, this approach promises to expand the applications of AuNP in biological applications.

Error prevention as developed in airlines.

The airline industry is a high-risk endeavor. Tens of thousands of flights depart each day carrying millions of passengers with the potential for catastrophic consequences. To manage and mitigate this risk, airline operators, labor unions, and the Federal Aviation Administration have developed a partnership approach to improving safety. This partnership includes cooperative programs such as the Aviation Safety Action Partnership and the Flight Operational Quality Assurance. It also involves concentrating on the key aspects of aircraft maintenance reliability and employee training. This report discusses recent enhancements within the airline industry in the areas of proactive safety programs and the move toward safety management systems that will drive improvements in the future.

Backbone dynamics in an intramolecular prolylpeptide-SH3 complex from the diphtheria toxin repressor, DtxR.

The diphtheria toxin repressor contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment and stabilizes the inactive state of the repressor. Upon activation of diphtheria toxin repressor (DtxR) by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. Here we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined using NMR spectroscopy and molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times, while residues in the loops connecting beta-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment exhibited low-order parameters with internal rotational correlation times on the order of 0.6 ns-1 ns. Further analysis showed that the SH3 domain was rich in millisecond time scale motions while the Pr segment exhibited motions on the 100 mus time scale. Molecular dynamics simulations indicated structural rearrangements that may contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding<-->unbinding equilibrium. The results here provide new insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity.