Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

Evaluation of an Intensive Care Unit Sepsis Alert in Critically Ill Medical Patients.

BACKGROUND: Sepsis alerts commonly used for intensive care unit (ICU) patients can lead to alert fatigue because these patients generally meet 1 or more of the criteria for systemic inflammatory response syndrome. To identify ICU patients at greatest risk for sepsis-related consequences, an ICU-specific sepsis alert was implemented. OBJECTIVE: To evaluate an ICU sepsis alert based on modified criteria for systemic inflammatory response syndrome among critically ill medical patients. METHODS: This retrospective evaluation was conducted at a comprehensive tertiary referral center. Patients included were at least 18 years old, were admitted to the critical care medicine service, and had at least 1 sepsis alert between January 1 and February 29, 2020. The sepsis alert identified patients meeting at least 2 modified systemic inflammatory response syndrome criteria (white blood cell count, <4000/µL or >12 000/µL; body temperature, <36 °C or >38.3 °C; heart rate, >110/min; and respiratory rate, >21/min), with at least 1 of the 2 criteria being white blood cell count or body temperature. RESULTS: For 128 alerts evaluated, the positive predictive value was 72%. Of 713 patients who were admitted to the critical care medicine service and did not have a sepsis alert, 7 received a sepsis diagnosis. The ICU sepsis alert had a negative predictive value of 99%, sensitivity of 92.9%, and specificity of 95.1%. CONCLUSIONS: An ICU sepsis alert using modified systemic inflammatory response syndrome criteria was effective for identifying sepsis in critically ill medical patients.

Improving Initial Sepsis Management Through a Nurse-Driven Rapid Response Team Protocol.

BACKGROUND: Rapid identification and timely management of sepsis improve survival. Therefore, a bundled approach to care is recommended. LOCAL PROBLEM: In an acute care area of the study institution, a 2016 internal evaluation of 27 patients with sepsis showed a median time to first-dose antibiotic administration of 269 minutes, with no patients receiving antibiotics within the 60-minute target time. Additionally, only one-third of patients received appropriate fluid resuscitation (30-mL/kg bolus of intravenous crystalloids). Given poor bundle compliance, a nurse-driven rapid response team protocol for suspected sepsis was implemented. The purpose of this project was to assess the protocol's impact on the timeliness of treatment for sepsis. METHODS: This retrospective quality improvement evaluation involved patients aged 18 years or older for whom the suspected sepsis protocol was initiated during their acute care area admission. The evaluation focused on improvements in time to intravenous antibiotic administration and volume of fluid resuscitation compared with before protocol implementation. The protocol empowers the rapid response team to initiate sepsis management and includes pertinent laboratory tests, blood cultures, intravenous broad-spectrum antibiotic administration, and a crystalloid bolus (30 mL/kg) if indicated. RESULTS: A total of 32 patients were evaluated. Time to first-dose antibiotic administration was reduced by half (from 269 to 135 minutes). Eighteen patients met criteria for fluid resuscitation, with twice as many receiving appropriate fluid volumes compared with before protocol implementation. CONCLUSION: Implementation of the suspected sepsis protocol demonstrates the substantial role nurses have in optimizing patient care, especially in the timely treatment of sepsis.

Survey of the 2009 commercial optical biosensor literature.

We took a different approach to reviewing the commercial biosensor literature this year by inviting 22 biosensor users to serve as a review committee. They set the criteria for what to expect in a publication and ultimately decided to use a pass/fail system for selecting which papers to include in this year's reference list. Of the 1514 publications in 2009 that reported using commercially available optical biosensor technology, only 20% passed their cutoff. The most common criticism the reviewers had with the literature was that "the biosensor experiments could have been done better." They selected 10 papers to highlight good experimental technique, data presentation, and unique applications of the technology. This communal review process was educational for everyone involved and one we will not soon forget.

Biacore analysis with stabilized G-protein-coupled receptors.

Using stabilized forms of ß1 adrenergic and A2(A) adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.

Safety of a Nurse-Driven Standardized Potassium Replacement Protocol in Critically Ill Patients With Renal Insufficiency.

BACKGROUND: In critically ill patients, maintaining appropriate serum potassium concentrations requires careful supplementation to correct hypokalemia but avoid hyperkalemia. At the study institution, an institution-based, nurse-driven standardized electrolyte replacement protocol is used in critically ill patients with a serum creatinine concentration of 2 mg/dL or less. If the serum creatinine concentration is greater than 2 mg/dL, electrolyte replacement requires a physician order. OBJECTIVE: To determine if standardized potassium supplementation is safe in critically ill patients with renal insufficiency not requiring renal replacement therapy. METHODS: This study was an institutional review board-approved, single-center, retrospective evaluation of critically ill patients receiving intravenous potassium replacement per protocol. Patients were grouped according to serum creatinine concentration (≤ 2 mg/dL or > 2 mg/dL) at the time of replacement. The primary outcome was the incidence of hyperkalemia (potassium concentration ≥ 5 mEq/L) following potassium replacement. Secondary outcomes were the incidence of hyperkalemia, change in serum potassium concentration, and need for hyperkalemia treatment. Outcomes were analyzed using χ2 and t tests. RESULTS: Of 814 patients screened, 145 were included (99 with serum creatinine ≤ 2 mg/dL and 46 with serum creatinine > 2 mg/dL). The incidence of hyperkalemia was not different between groups (P = .57). Five patients experienced hyperkalemia; none received hyperkalemia treatment. Change in serum potassium was similar for patients in the 2 groups (P = .33). CONCLUSIONS: A standardized, nurse-driven electrolyte replacement protocol can be used safely in critically ill patients with renal insufficiency not requiring renal replacement therapy.

Grading the commercial optical biosensor literature-Class of 2008: 'The Mighty Binders'.

Optical biosensor technology continues to be the method of choice for label-free, real-time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high-school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind.

Kinetic analysis and fragment screening with Fujifilm AP-3000.

We evaluated the performance of Fujifilm's new AP-3000 surface plasmon resonance biosensor for kinetic analysis and fragment screening. Using carbonic anhydrase II as a model system, we characterized a set of 10 sulfonamide-based inhibitors that range in molecular mass from 98 to 341Da and approximately 10,000-fold in affinity (0.4mM to 20nM). Although the data collected from the AP-3000 were generally similar to those collected using a Biacore T100, the AP-3000's stop-flow analyte delivery system complicated the shapes of the association- and dissociation-phase binding responses. We illustrate how reasonable estimates of the kinetic rate constants can be extracted from AP-3000 data by limiting data analysis to only the regions of the responses collected during flow conditions. We also provide an example of the results obtained for a fragment-screening study with the AP-3000, which is the ideal application of this technology.

Biosensor-based fragment screening using FastStep injections.

We have developed a novel analyte injection method for the SensíQ Pioneer surface plasmon resonance-based biosensor referred to as "FastStep." By merging buffer and sample streams immediately prior to the reaction flow cells, the instrument is capable of automatically generating a two- or threefold dilution series (of seven or five concentrations, respectively) from a single analyte sample. Using sucrose injections, we demonstrate that the production of each concentration within the step gradient is highly reproducible. For kinetic studies, we developed analysis software that utilizes the sucrose responses to automatically define the concentration of analyte at any point during the association phase. To validate this new approach, we compared the results of standard and FastStep injections for ADP binding to a target kinase and a panel of compounds binding to carbonic anhydrase II. Finally, we illustrate how FastStep can be used in a primary screening mode to obtain a full concentration series of each compound in a fragment library.

Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies.

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.

A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Improved continuous-flow print head for micro-array deposition.

Limitations in depositing ligands using conventional micro-array pin spotting have hindered the application of surface plasmon resonance imaging (SPRi) technology. To address these challenges we introduce a modification to our continuous-flow micro-spotting technology that improves ligand deposition. Using Flexchip protein A/G and neutravidin capturing surfaces, we demonstrate that our new microfluidic spotter requires 1000 times less concentrated antibodies and biotinylated ligands than is required for pin spotting. By varying the deposition flow rate, we show that the design of our tip overlay flow cell is efficient at delivering sample to the substrate surface. Finally, contact time studies show that it is possible to capture antibodies and biotinylated ligands at concentrations of less than 0.1 ug/ml and 100 pM, respectively. These improvements in spotting technology will help to expand the applications of SPRi systems in areas such as antibody screening, carbohydrate arrays, and biomarker detection.

Spying on HIV with SPR.

The application of surface plasmon resonance (SPR)-based optical biosensors has contributed extensively to our understanding of functional aspects of HIV. SPR biosensors allow the analysis of real-time interactions of any biomolecule, be it protein, nucleic acid, lipid, carbohydrate or small molecule, without the need for intrinsic or extrinsic probes. As such, the technology has been used to analyze molecular interactions associated with every aspect of the viral life cycle, from basic studies of binding events occurring during docking, replication, budding and maturation to applied research related to vaccine and inhibitory drug development. Along the way, SPR biosensors have provided a unique and detailed view into the inner workings of HIV.

Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods.

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.

Why you should be using more SPR biosensor technology.

There are three ways of looking at surface plasmon resonance biosensor technology: bewilderment (I can not believe this sort of technology actually exists), love (without this technology I would cry), or hate (this technology does not work). Whether you love them or hate them, or just have never heard of them, it is time to take a fresh look at how SPR biosensors can help you get from point A to point B in your drug discovery efforts.:

Biophysical fragment screening of the ß1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design.

Biophysical fragment screening of a thermostabilized ß1-adrenergic receptor (ß1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein-ligand crystal structures of the ß1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized ß1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.

Fragment screening of stabilized G-protein-coupled receptors using biophysical methods.

Biophysical studies with G-protein-coupled receptors (GPCRs) are typically very challenging due to the poor stability of these receptors when solubilized from the cell membrane into detergent solutions. However, the stability of a GPCR can be greatly improved by introducing a number of point mutations into the protein sequence to give a stabilized receptor or StaR®. Here, we present the utility of StaRs for biophysical studies and the screening of fragment libraries. Two case studies are used to illustrate the methods: first, the screening of a library of fragments by surface plasmon resonance against the adenosine A(2A) receptor StaR, demonstrating how very small and weakly active xanthine fragments can be detected binding to the protein on chips; second, the screening and detection of fragment hits of a larger fragment library in an NMR format called TINS (target-immobilized NMR screening) against the ß(1) adrenergic StaR.

E2 interaction and dimerization in the crystal structure of TRAF6.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.

Survey of the year 2007 commercial optical biosensor literature.

In 2007, 1179 papers were published that involved the application of optical biosensors. Reported developments in instrument hardware, assay design, and immobilization chemistry continue to improve the technology's throughput, sensitivity, and utility. Compared to recent years, the widest range of platforms, both traditional format and array-based, were used. However, as in the past, we found a disappointingly low percentage of well-executed experiments and thoughtful data interpretation. We are alarmed by the high frequency of suboptimal data and over-interpreted results in the literature. Fortunately, learning to visually recognize good--and more importantly, bad--data is easy. Using examples from the literature, we outline several features of biosensor responses that indicate experimental artifacts versus actual binding events. Our goal is to have everyone, from benchtop scientists to project managers and manuscript reviewers, become astute judges of biosensor results using nothing more than their eyes.

High-affinity interaction between IKKbeta and NEMO.

The Ser/Thr-specific IkappaB kinase (IKK), which comprises IKKalpha or IKKbeta and the regulatory protein NEMO, is at the bottleneck for NF-kappaB activation. IKK activity relies on interaction between NEMO and IKKalpha or IKKbeta. A conserved region in the C-terminal tail of IKKbeta or IKKalpha (NEMO-binding domain, NBD, residues 734-745 of IKKbeta) is important for interaction with NEMO. Here we show that the NBD peptide of IKKbeta is not sufficient for interaction with NEMO. Instead, a longer region of the IKKbeta C-terminal region provides high affinity for NEMO. Quantitative measurements using surface plasmon resonance and isothermal titration calorimetry confirm the differential affinities of these interactions and provide insight into the kinetic and thermodynamic behaviors of the interactions. Biochemical characterization using multiangle light scattering (MALS) coupled with refractive index shows that the longer IKKbeta C-terminal region forms a 2:2 stoichiometirc complex with NEMO.