Affinity-matured antibody with a disulfide bond in H-CDR3 loop.

Affinity maturation increases antigen-binding affinity and specificity of antibodies by somatic hypermutation. Various monoclonal antibodies against (4-hydroxy-3-nitrophenyl)acetyl (NP) were obtained during affinity maturation. Among them, highly matured anti-NP antibodies, such as E11 and E3, possess Cys96H and Cys100H in the complementarity-determining region 3 of the heavy chain, which would form a disulfide bond. In this study, we evaluated the effects of disulfide bonds on antigen binding by generating single-chain Fv (scFv) antibodies of E11 and its mutants, E11_C96KH/C100EH and E11_C96KH/C100QH, and determined their antigen-binding thermodynamics and kinetics. The binding affinities of the Cys mutants were lower than that of E11 scFv, indicating that the disulfide bond contributed to antigen binding, especially for stable complex formation. This was also supported by the decreased affinity of E11 scFv in the presence of a reducing agent. The crystal structures of NP-free and NP-bound E11 scFvs were determined at high resolution, showing the existence of a disulfide bond between Cys96H and Cys100H, and the antigen recognition mechanism, which could be compared with those of other anti-NP antibodies, such as germline-type N1G9 and matured-type C6, as reported previously. These structures could explain the molecular basis of changes in antigen-binding affinity and thermal stability in the absence or presence of antigens. Small-angle X-ray scattering further showed a local conformational change in E11 scFv upon antigen binding in solution.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

Ligand-Dependent Intramolecular Motion of Native Nicotinic Acetylcholine Receptors Determined in Living Myotube Cells via Diffracted X-ray Tracking.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in signal transduction at the neuromuscular junction (NMJ). Movement of the nAChR extracellular domain following agonist binding induces conformational changes in the extracellular domain, which in turn affects the transmembrane domain and opens the ion channel. It is known that the surrounding environment, such as the presence of specific lipids and proteins, affects nAChR function. Diffracted X-ray tracking (DXT) facilitates measurement of the intermolecular motions of receptors on the cell membranes of living cells, including all the components involved in receptor function. In this study, the intramolecular motion of the extracellular domain of native nAChR proteins in living myotube cells was analyzed using DXT for the first time. We revealed that the motion of the extracellular domain in the presence of an agonist (e.g., carbamylcholine, CCh) was restricted by an antagonist (i.e., alpha-bungarotoxin, BGT).

Comparison of the Molecular Motility of Tubulin Dimeric Isoforms: Molecular Dynamics Simulations and Diffracted X-ray Tracking Study.

Tubulin has been recently reported to form a large family consisting of various gene isoforms; however, the differences in the molecular features of tubulin dimers composed of a combination of these isoforms remain unknown. Therefore, we attempted to elucidate the physical differences in the molecular motility of these tubulin dimers using the method of measurable pico-meter-scale molecular motility, diffracted X-ray tracking (DXT) analysis, regarding characteristic tubulin dimers, including neuronal TUBB3 and ubiquitous TUBB5. We first conducted a DXT analysis of neuronal (TUBB3-TUBA1A) and ubiquitous (TUBB5-TUBA1B) tubulin dimers and found that the molecular motility around the vertical axis of the neuronal tubulin dimer was lower than that of the ubiquitous tubulin dimer. The results of molecular dynamics (MD) simulation suggest that the difference in motility between the neuronal and ubiquitous tubulin dimers was probably caused by a change in the major contact of Gln245 in the T7 loop of TUBB from Glu11 in TUBA to Val353 in TUBB. The present study is the first report of a novel phenomenon in which the pico-meter-scale molecular motility between neuronal and ubiquitous tubulin dimers is different.

A prospective randomized study to compare standard versus intensive training strategies on long-term improvement in critical care ultrasonography proficiency.

BACKGROUND: Critical care ultrasonography (CCUS) has become a daily diagnostic tool for intensivists. While the effective training measures for ultrasound novices are discussed widely, the best curriculum for the novices to retain a long-term proficiency is yet to be determined. METHODS: Critical care medicine fellows who underwent an introductory CCUS workshop were randomly allocated into the standard training (ST) or the intensive training (IT) group. The IT group received an 8-h training besides the standardized fellowship education that the ST group received. Participant improvement in CCUS proficiency tests (maximum score, 200) after a 6-month training intervention was compared between the groups. CCUS examinations performed in patient care were observed over 2 years. RESULTS: Twenty-one fellows were allocated into the ST (n = 10) or the IT (n = 11) group. No statistically significant difference was observed in the median (interquartile range [IQR]) improvement in CCUS proficiency tests between the ST group and the IT group: 18 (3.8-38) versus 31 (21-46) (P = .09). Median (IQR) test scores were significantly higher in postintervention than preintervention for both groups: ST, 103 (87-116) versus 124 (111-143) (P = .02), and IT, 100 (87-113) versus 143 (121-149) (P < .01). Participating fellows performed 226 examinations over the 2 years of observation. CONCLUSIONS: Fellows improved their CCUS proficiency significantly after 6-month training intervention. However, an additional 8-h training did not provide further benefits.

Epinephrine dosing interval and neurological outcome in out-of-hospital cardiac arrest.

OBJECTIVE: Current guidelines for cardiopulmonary resuscitation (CPR) recommend that standard-dose epinephrine be administered every 3-5 minutes during cardiac arrest. However, there is a knowledge gap regarding the optimal epinephrine dosing interval. This study aimed to examine the association between epinephrine dosing intervals and outcomes after out-of-hospital cardiac arrest (OHCA). METHODS: This was a nationwide population-based observational study using data from a Japanese government-led registry of OHCA, including patients who experienced OHCA in Japan from 2011 to 2017. We defined the epinephrine dosing interval as the time interval between the first epinephrine administration and return of spontaneous circulation in the prehospital setting, divided by the total number of epinephrine doses. The primary outcome was 1-month neurologically favorable survival. RESULTS: A total of 10,965 patients (mean (SD) age, 75.8 (14.3) years; 59.8% male) were included. The median epinephrine dosing interval was 3.5 minutes (IQR, 2.5-4.5; mean (SD), 3.6 (1.8)). Only approximately half of the patients received epinephrine administration with a standard dosing interval, as recommended in the current CPR guidelines. After multivariable adjustment, compared with the standard dosing interval, neither shorter nor longer epinephrine dosing intervals were associated with neurologically favorable survival after OHCA (Short vs Standard: adjusted OR 0.87 [95%CI 0.66-1.15]; and Long vs Standard: adjusted OR 1.08 [95%CI 0.76-1.55]). Similar associations were observed in propensity score-matched analyses. CONCLUSIONS: The epinephrine dosing interval was not associated with 1-month neurologically favorable survival after OHCA. Our findings do not deny the recommended epinephrine dosing interval in the current CPR guidelines.

Diffracted X-ray Tracking Method for Measuring Intramolecular Dynamics of Membrane Proteins.

Membrane proteins change their conformations in response to chemical and physical stimuli and transmit extracellular signals inside cells. Several approaches have been developed for solving the structures of proteins. However, few techniques can monitor real-time protein dynamics. The diffracted X-ray tracking method (DXT) is an X-ray-based single-molecule technique that monitors the internal motion of biomolecules in an aqueous solution. DXT analyzes trajectories of Laue spots generated from the attached gold nanocrystals with a two-dimensional axis by tilting (θ) and twisting (χ). Furthermore, high-intensity X-rays from synchrotron radiation facilities enable measurements with microsecond-timescale and picometer-spatial-scale intramolecular information. The technique has been applied to various membrane proteins due to its superior spatiotemporal resolution. In this review, we introduce basic principles of DXT, reviewing its recent and extended applications to membrane proteins and living cells, respectively.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

Diffracted X-ray blinking measurements of interleukin 15 receptors in the inner/outer membrane of living NK cells.

Interleukin 15 receptor (IL-15R) is a transmembrane signalling protein consisting of 3 subsets: α, ß (IL-15Rß), and γ (γc). IL-2 and IL-15 share the signalling domains IL-15Rß and γc, although they bind to intrinsic α-subsets and non-signalling domains. Additionally, IL-2 and IL-15 play different roles; therefore, there have been many observations of the dynamic behaviours of IL-15R, which are linked to physiological functions. For more practical discrimination between IL-2 and IL-15, a study was designed and carried out in which α-subsets were removed and a cytoplasmic inhibitor was applied to create a simplified environment in which secondary signalling molecules were reduced. We also applied a new measurement method, diffracted X-ray blinking (DXB), to achieve higher accuracy (<0.01 Å). The dynamics of IL-2 binding (confined motion, max range = 0.71 Å) and IL-15 binding (normal motion) in live natural killer cells were different. We also confirmed. that DXB was a suitable method to quantitatively evaluate the transmembrane protein dynamics of inner/outer live cell membranes by labeling the extracellular domain since the measurements were dependent on the cytosolic environment.

Assessing Competence in Critical Care Echocardiography: Development and Initial Results of an Examination and Certification Processes.

OBJECTIVES: To describe the development and initial results of an examination and certification process assessing competence in critical care echocardiography. DESIGN: A test writing committee of content experts from eight professional societies invested in critical care echocardiography was convened, with the Executive Director representing the National Board of Echocardiography. Using an examination content outline, the writing committee was assigned topics relevant to their areas of expertise. The examination items underwent extensive review, editing, and discussion in several face-to-face meetings supervised by National Board of Medical Examiners editors and psychometricians. A separate certification committee was tasked with establishing criteria required to achieve National Board of Echocardiography certification in critical care echocardiography through detailed review of required supporting material submitted by candidates seeking to fulfill these criteria. SETTING: The writing committee met twice a year in person at the National Board of Medical Examiner office in Philadelphia, PA. SUBJECTS: Physicians enrolled in the examination of Special Competence in Critical Care Electrocardiography (CCEeXAM). MEASUREMENTS AND MAIN RESULTS: A total of 524 physicians sat for the examination, and 426 (81.3%) achieved a passing score. Of the examinees, 41% were anesthesiology trained, 33.2% had pulmonary/critical care background, and the majority had graduated training within the 10 years (91.6%). Most candidates work full-time at an academic hospital (46.9%). CONCLUSIONS: The CCEeXAM is designed to assess a knowledge base that is shared with echocardiologists in addition to that which is unique to critical care. The National Board of Echocardiography certification establishes that the physician has achieved the ability to independently perform and interpret critical care echocardiography at a standard recognized by critical care professional societies encompassing a wide spectrum of backgrounds. The interest shown and the success achieved on the CCEeXAM by practitioners of critical care echocardiography support the standards set by the National Board of Echocardiography for testamur status and certification in this imaging specialty area.

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors.

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 µs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the "ionic lock" between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.

X-ray-based living-cell motion analysis of individual serotonin receptors.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins, which transmit extracellular signals inside cells via activating G proteins. GPCRs are involved in a wide variety of physiological functions, such as signal sensing, immune system processes, and neurotransmission. Although the structures and functions of GPCRs have been well studied, little has been known about their real-time dynamics on live cells. In this study, we used Diffracted X-ray Tracking (DXT) and Diffracted X-ray Blinking (DXB) techniques for analysis. These methods are very precise single-molecular analytical techniques that elucidate protein dynamics by analyzing the diffraction spots from the gold nanocrystals labeled on the protein surface. DXT tracks diffraction spot movements, whereas DXB analyzes continuation of signals by calculating the autocorrelation function of each pixel from the recorded data. Serotonin receptor subtype 2A (5-HT2A receptors) were transiently expressed on HEK 293 cells, and the gold nanocrystals were attached to the N-terminally introduced FLAG-tag via anti-FLAG antibodies. Fast- and mid-range motions were recorded by DXT with 100µs and 1.25 ms/frame rate, respectively. Slow-range motion was obtained using the DXB method with 100 ms/frame rate. An agonist interestingly suppressed the fluctuations of 5-HT2A receptors at the microsecond-ranged fast measurement. On the contrary, the motion was enhanced by the agonist in the hundred-millisecond-ranged slow time scale. These dual-natured data may suggest that we succeeded in extracting different modes of receptor's motion on live cells; microsecond ranged fluctuation on the cell membrane, and millisecond-ranged dynamic movement comprising interactions with intracellular signaling molecules.

Mechanical Stabilization of Deoxyribonucleic Acid Solid Films Based on Hydrated Ionic Liquid.

Solid films of deoxyribonucleic acid (DNA) containing a hydrated ionic liquid, choline dihydrogen phosphate (CDP), were prepared by a solvent-casting method. Thermal properties, aggregation structure, thermal molecular motion, and tensile properties of CDP-containing DNA films were examined by thermogravimetry (TG), wide-angle X-ray diffraction (WAXD) measurement, dynamic mechanical analysis (DMA), and tensile tests, respectively. The water retentivity of the films at room temperature was much improved with CDP. The packing density of DNA helical chains clearly depended on the amount of CDP in the film. A small amount of CDP contributed to the suppression of the BI → BII conformational transition and the cooperative motion of the DNA duplex in the film. The tensile properties of the film drastically changed in the presence of CDP. When the amount of hydrated CDP in the film increased, the mechanical response of the film changed from glassy-like to rubbery-like via a semicrystalline-like state. The above results make it clear that CDP plays two major roles as a water absorber and plasticizer in the DNA film. Thus, it can be concluded that the use of an ionic liquid as an additive significantly increases the possibility of using a DNA solid film as a structural material.

Computerized data mining analysis of keywords as indicators of the concepts in AHA-BLS guideline updates.

INTRODUCTION: Cardiopulmonary resuscitation (CPR) guidelines have been updated every 5 years since 2000. Significant changes have been made in each update, and every time a guideline is changed, the instructors of each country that ratify the American Heart Association (AHA) must review the contents of the revised guideline to understand the changes made in the concept of CPR. The purpose of this study was to use a computerized data mining method to identify and characterize the changes in the key concepts of the AHA-Basic Life Support (BLS) updates between 2000 and 2015. METHODS: We analyzed the guidelines of the AHA-BLS provider manual of 2000, 2005, 2010, and 2015 using a computerized data mining method and attempted to identify the changes in keywords along with changes in the guideline. RESULTS: In particular, the 2000 guideline has focused on the detailed BLS technique of an individual health care provider, whereas the 2005 and 2010 guidelines have focused on changing the ratio of chest compressions and breathing and changing the BLS sequence, respectively. In the most recent 2015 guideline, the CPR team was the central topic. We observed that as the guidelines were updated over the years, keywords related to CPR and automated external defibrillators (AED) associated with co-occurrence network continued to appear. CONCLUSIONS: Analysis revealed that keywords related to CPR and AED associated with the co-occurrence network continued to appear. We believe that the results of this study will ultimately contribute to optimizing AHA's educational strategies for health care providers.

DNA-binding induced conformational change of c-Myb R2R3 analyzed using diffracted X-ray tracking.

Previous structural analyses have shown that R2R3, the minimum unit of the DNA-binding domain of the transcriptional factor c-Myb, is largely flexible in solution, and changes to a more rigid structure upon DNA binding. In this study, we evaluated the structural dynamics using the diffracted X-ray tracking method, in correlation with DNA-binding abilities under different salt conditions, and compared them with the previous results. The resultant curve of the mean square angular displacements (MSD) clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.

Importance of Proteasome Gene Expression during Model Dough Fermentation after Preservation of Baker's Yeast Cells by Freezing.

Freeze-thaw stress causes various types of cellular damage, survival and/or proliferation defects, and metabolic alterations. However, the mechanisms underlying how cells cope with freeze-thaw stress are poorly understood. Here, model dough fermentations using two baker's yeast strains, 45 and YF, of Saccharomyces cerevisiae were compared after 2 weeks of cell preservation in a refrigerator or freezer. YF exhibited slow fermentation after exposure to freeze-thaw stress due to low cell viability. A DNA microarray analysis of the YF cells during fermentation revealed that the genes involved in oxidative phosphorylation were relatively strongly expressed, suggesting a decrease in the glycolytic capacity. Furthermore, we found that mRNA levels of the genes that encode the components of the proteasome complex were commonly low, and ubiquitinated proteins were accumulated by freeze-thaw stress in the YF strain. In the cells with a laboratory strain background, treatment with the proteasome inhibitor MG132 or the deletion of each transcriptional activator gene for the proteasome genes (RPN4, PDR1, or PDR3) led to marked impairment of model dough fermentation using the frozen cells. Based on these data, proteasomal degradation of freeze-thaw-damaged proteins may guarantee high cell viability and fermentation performance. We also found that the freeze-thaw stress-sensitive YF strain was heterozygous at the PDR3 locus, and one of the alleles (A148T/A229V/H336R/L541P) was shown to possess a dominant negative phenotype of slow fermentation. Removal of such responsible mutations could improve the freeze-thaw stress tolerance and the fermentation performance of baker's yeast strains, as well as other industrial S. cerevisiae strains.IMPORTANCE The development of freezing technology has enabled the long-term preservation and long-distance transport of foods and other agricultural products. Fresh yeast, however, is usually not frozen because the fermentation performance and/or the viability of individual cells is severely affected after thawing. Here, we demonstrate that proteasomal degradation of ubiquitinated proteins is an essential process in the freeze-thaw stress responses of S. cerevisiae Upstream transcriptional activator genes for the proteasome components are responsible for the fermentation performance after freezing preservation. Thus, this study provides a potential linkage between freeze-thaw stress inputs and the transcriptional regulatory network that might be functionally conserved in higher eukaryotes. Elucidation of the molecular targets of freeze-thaw stress will contribute to advances in cryobiology, such as freezing preservation of human cells, tissues, and embryos for medical purposes and breeding of industrial microorganisms and agricultural crops that adapt well to low temperatures.

When to incorporate point-of-care ultrasound (POCUS) into the initial assessment of acutely ill patients: a pilot crossover study to compare 2 POCUS-assisted simulation protocols.

BACKGROUND: The purpose of this study was to determine the ideal timing for providers to perform point-of-care ultrasound (POCUS) with the least increase in workload. METHODS: We conducted a pilot crossover study to compare 2 POCUS-assisted evaluation protocols for acutely ill patients: sequential (physical examination followed by POCUS) vs parallel (POCUS at the time of physical examination). Participants were randomly assigned to 2 groups according to which POCUS-assisted protocol (sequential vs parallel) was used during simulated scenarios. Subsequently, the groups were crossed over to complete assessment by using the other POCUS-assisted protocol in the same patient scenarios. Providers' workloads, measured with the National Aeronautics and Space Administration Task Load Index (NASA-TLX) and time to complete patient evaluation, were compared between the 2 protocols. RESULTS: Seven providers completed 14 assessments (7 sequential and 7 parallel). The median (IQR) total NASA-TLX score was 30 (30-50) in the sequential and 55 (50-65) in the parallel protocol (P = .03), which suggests a significantly lower workload in the sequential protocol. When individual components of the NASA-TLX score were evaluated, mental demand and frustration level were significantly lower in the sequential than in the parallel protocol (40 [IQR, 30-60] vs 50 [IQR, 40-70]; P = .03 and 25 [IQR, 20-35] vs 60 [IQR, 45-85]; P = .02, respectively). The time needed to complete the assessment was similar between the sequential and parallel protocols (8.7 [IQR, 6-9] minutes vs 10.1 [IQR, 7-11] minutes, respectively; P = .30). CONCLUSIONS: A sequential POCUS-assisted protocol posed less workload to POCUS operators than the parallel protocol.

Reprint of: Pathologic manifestations of Immunoglobulin(Ig)G4-related lung disease.

Immunoglobulin(Ig)G4-related disease (IgG4-RD) is a fibroinflammatory condition that can affect virtually any organ and usually presents as tumefactive lesions involving multiple sites. Characteristic histopathology of IgG4-RD consists of dense lymphoplasmacytic infiltrate, fibrosis (often in storiform pattern), and obliterative phlebitis, accompanied by tissue infiltration of IgG4-positive plasma cells with or without elevation of serum IgG4 level. Despite a general similarity in the morphologic manifestations of IgG4-RD, site-specific unique morphologic features have been described in some organs including the lung. Compared with other sites, pulmonary involvement by IgG4-RD has been recognized more recently, and lung biopsy interpretation for this condition is often challenging, as both a relative paucity of pathognomonic features and a plethora of overlapping findings with other fibroinflammatory processes of the lung. This review is focused on the morphologic spectrum of IgG4-related lung disease documented in the current literature and on the pertinent issues in the differential diagnoses with other conditions encountered in the lung.

Time-Resolved Measurement of the ATP-Dependent Motion of the Group II Chaperonin by Diffracted Electron Tracking.

Previously, we demonstrated the ATP-dependent dynamics of a group II chaperonin at the single-molecule level by diffracted X-ray tracking (DXT). The disadvantage of DXT is that it requires a strong X-ray source and also perfect gold nano-crystals. To resolve this problem, we developed diffracted electron tracking (DET). Electron beams have scattering cross-sections that are approximately 1000 times larger than those of X-rays. Thus, DET enables us to perform super-accurate measurements of the time-resolved 3D motion of proteins labeled with commercially available gold nanorods using a scanning electron microscope. In this study, we compared DXT and DET using the group II chaperonin from Methanococcus maripaludis (MmCpn) as a model protein. In DET, the samples are prepared in an environmental cell (EC). To reduce the electron beam-induced protein damage, we immobilized MmCpn on the bottom of the EC to expose gold nanorods close to the carbon thin film. The sample setup worked well, and the motions of gold nanorods were clearly traced. Compared with the results of DXT, the mobility in DET was significantly higher, which is probably due to the difference in the method for immobilization. In DET, MmCpn was immobilized on a film of triacetyl cellulose. Whereas proteins are directly attached on the surface of solid support in DXT. Therefore, MmCpn could move relatively freely in DET. DET will be a state-of-the-art technology for analyzing protein dynamics.

Focused cardiac ultrasound in the early resuscitation of severe sepsis and septic shock: a prospective pilot study.

PURPOSE: Point-of-care ultrasonography has been increasingly used in the care of critically ill patients; however, reports on its use during active resuscitation are limited. The aim of this study was to investigate the true impact of focused cardiac ultrasound (FCU) during the management of sepsis with early (6-h) resuscitation. METHODS: A prospective pilot observational study was conducted at an academic medical center from March 2011 through July 2012. Patients undergoing resuscitation for severe sepsis or septic shock were prospectively enrolled at medical and combined medical-surgical intensive care units. Patients underwent a 10-min FCU examination when echocardiography was not part of their care plan. FCU was performed by sonographers and interpreted by cardiologists to minimize risks of inadequate image acquisition and misinterpretation. Intensivists completed surveys on their diagnostic and therapeutic plans before and after receiving FCU information. RESULTS: Of the 30 patients enrolled, 18 (60%) were male and the median age was 61 years [interquartile range (IQR) 50-71 years]. Median central venous oxygen saturation and lactate levels were 59.6% (IQR 53.1-66.2%) and 2.7 mmol/L (IQR 1.2-4.1 mmol/L), respectively. Clinical assessment by intensivists before FCU commonly failed to correctly estimate ventricular function; specifically, left ventricular in 12 patients [40%, 95% confidence interval (CI) 25-58%] and right ventricular function in 15 patients (50%, 95% CI 33-67%). Intensivists' therapeutic plans changed in eight cases (27%, 95% CI 14-44%) after FCU information became available. The most common changes were fluid management and imaging tests. Intensivists' confidence in their therapeutic plans improved for 11 patients (37%, 95% CI 22-55%). CONCLUSION: FCU is a valuable examination tool during early resuscitation of severe sepsis and septic shock.