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KLHL-12 is a substrate specific adapter protein for a Cul3-Ring ligase complex. It is a member of the Kelch β-propeller domain subclass of Cullin-Ring substrate recognition domains. This E3 ubiquitin ligase complex has many activities, including acting as a negative regulator of the Wnt signaling pathway by mediating ubiquitination and subsequent proteolysis of Dvl3/Dsh3. KLHL-12 is also known to mediate the polyubiquitination of the dopamine D4 receptor (D4.2), the ubiquitination of KHSRP, a protein that is involved in IRES translation, and also the ubiquitination of Sec31, which is involved in endoplasmic reticulum-Golgi transport by regulating the size of COPII coats. Earlier studies broadly defined the substrate binding regions for D4.2 and Dvl3/Dsh3 to KLHL-12. We tested several peptides from these regions and succeeded in identifying a short peptide that bound to KLHL-12 with low micromolar affinity. To better understand the sequence specificity of this peptide, we used alanine substitutions to map the important residues and obtained an X-ray structure of this peptide bound to KLHL-12. This structure and our peptide affinity measurements suggest a sequence motif for peptides that bind to the top face of KLHL-12. Understanding this binding site on KLHL-12 may contribute to efforts to find small molecule ligands that can either directly inhibit the degradation of substrate proteins or be used in targeted protein degradation strategies using PROTACs.

Layered inoculation can achieve rapid start-up and promote methanation performance of anaerobic digesters. Daily specific methane yield (SMY) rapidly increased to 2.93 mL/g VS/d during 0-13 days, and cumulative SMY reached 212 mL/g VS in the solid-state anaerobic co-digestion (SS-AcoD) of pig manure and corn straw. Data were collected at macro-, micro-, and genetic-levels of each substrate layer. The results showed that layered inoculation could improve volatile fatty acids utilization and prevent adverse effects of high total ammonium nitrogen concentrations. Layered inoculation accelerated hydrolysis, acidification, and methanogenesis of substrates, as evidenced by the efficient inoculation of Bacteroidetes, Anaerolineales, Methanosphaerula, and Methanothrix, which were primarily from inocula. The various stages of SS-AcoD were synergistically initiated during the first 13 days, and acetoclastic pathway was boosted. These results further explain why layered inoculation is an efficient method for improving methanation performance of SS-AcoD and achieving efficient utilization of organic solid waste.

Based on dynamic analysis of rolling bearings, the nonlinear dynamic differential equations of a cylindrical roller bearing with a trilobe-raceway were established and solved by the GSTIFF (gear stiff) integer algorithm with a variable step. The influences of structural parameters and the tolerance of the trilobe-raceway, working conditions of the bearing, and the outer ring installation method on cage slip characteristics were investigated. The results show that: (i) The cage slip ratio and bearing rating life of a cylindrical roller bearing with a trilobe-raceway would reduce when the low-radius (radius of the outer raceway contour at the lowest point) and D-value (difference value between the high and low points of the outer raceway contour) decrease, and the former (low-radius) contributes more significantly. (ii) The cage slip ratio of a cylindrical roller bearing with a trilobe-raceway rises with the increase of the bearing speed, and decreases with the increase of the radial force; the variation range increases with the increase of the low-radius. (iii) When the installation angle of the outer ring increases in a period, the cage slip ratio remains unchanged while the bearing rating life rises up a little. Therefore, when installing a cylindrical roller bearing with a trilobe-raceway, the location of the maximum radius shall be under that of the radial force to improve the bearing rating life. (iv) With the increase of the roundness of the base circle where the radius of the lowest points of the trilobe-raceway contour locates, the cage slip ratio rises gradually and the bearing rating life decreases. Keywords: Bearing rating life, Cage slip ratio, Dynamics, Raceway contour, Trilobe-raceway

Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix–helix interactions within the membrane, enabling the ability to probe the activation of the integrins α(IIb)β(3) and α(v)β(3). Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α(5)β(1) transmembrane (TM) domains, targeting the Ala–X(3)–Gly motif within α(5). Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α(5)β(1) but not α(V)β(3) in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α(5) TM domain peptide in detergent micelles to a similar degree as a β(1) TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin β(1) TM domain, which itself has a Gly–X(3)–Gly motif. The second peptide associated modestly with both α(5) and β(1) constructs, with slight preference for α(5). Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α(5)β(1) that can serve as useful reagents for probing integrin biology.

The Notch signaling pathway is critical in development, neuronal maintenance, and hematopoiesis. An obligate step in the activation of this pathway is cleavage of its transmembrane (TM) domain by γ-secretase. While the soluble domains have been extensively studied, little has been done to characterize its TM and flanking juxtamembrane (JM) segments. Here, we present the results of nuclear magnetic resonance (NMR) studies of the human Notch1 TM/JM domain. The TM domain is largely α-helical. While the flanking JM segments do not adopt regular secondary structure, they interact with the membrane surface, suggesting membrane interactions may play a role in modulating its cleavage by γ-secretase and subsequent NOTCH signaling function.

Our recent study has shown that cellular junctions in myelin and in the epi-/perineruium that encase nerve fibers regulate the permeability of the peripheral nerves. This permeability may affect propagation of the action potential. Direct interactions between the PDZ₁ domain of zonula occludens (ZO₁ or ZO₂) and the C-termini of claudins are known to be crucial for the formation of tight junctions. Using the purified PDZ₁ domain of ZO₂ and a variety of C-terminal mutants of peripheral nerve claudins (claudin-1, claudin-2, claudin-3, claudin-5 in epi-/perineurium; claudin-19 in myelin), we have utilized NMR spectroscopy to determine specific roles of the 3 C-terminal claudin residues (position -2, -1, 0) for their interactions with PDZ₁ of ZO₂. In contrast to the canonical model that emphasizes the importance of residues at the -2 and 0 positions, our results demonstrate that, for peripheral nerve claudins, the residue at position -1 plays a critical role in association with PDZ₁, while the side-chain of residue 0 plays a significant but lesser role. Surprisingly, claudin-19, the most abundant claudin in myelin, exhibited no binding to ZO₂. These findings reveal that the binding mechanism of claudin/ZO in epi-/perineurium is distinct from the canonical interactions between non-ZO PDZ-containing proteins with their ligands. This observation provides the molecular basis for a strategy to develop drugs that target tight junctions in the epi-/perineurium of peripheral nerves.

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is D-galactose-α(1→6)-D-glucose. Light scattering showed the β-DDMB micelle to be roughly 30kD smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than for DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22 (PMP22). β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

The Notch transmembrane domain exhibits significant structural differences from that of the amyloid precursor protein., γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-β precursor C99 protein as potential Alzheimer’s disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.

C99 (also known as β-CTF) is the 99 residue transmembrane C-terminal domain (residues 672-770) of the amyloid precursor protein and is the immediate precursor of the amyloid-β (Aβ) polypeptides. To test the dependence of the C99 structure on the composition of the host model membranes, NMR studies of C99 were conducted both in anionic lyso-myristoylphosphatidylglycerol (LMPG) micelles and in a series of five zwitterionic bicelle compositions involving phosphatidylcholine and sphingomyelin in which the acyl chain lengths of these lipid components varied from 14 to 24 carbons. Some of these mixtures are reported for the first time in this work and should be of broad utility in membrane protein research. The site-specific backbone (15)N and (1)H chemical shifts for C99 in LMPG and in all five bicelle mixtures were seen to be remarkably similar, indicating little dependence of the backbone structure of C99 on the composition of the host model membrane. However, the length of the transmembrane span was seen to vary in a manner that alters the positioning of the γ-secretase cleavage sites with respect to the center of the bilayer. This observation may contribute to the known dependency of the Aβ42-to-Aβ40 production ratio on both membrane thickness and the length of the C99 transmembrane domain.

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins, are heterodimeric transmembrane receptors consisting of α and β subunits and critical integrins in the kidney collecting system express the β1subunit. The β1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and link them to the actin cytoskeleton. We defined the role of talin binding to the β1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the β1 integrin subunit NPxY motif.

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 80s. “Without receivers fitted and kept in order, the air may tingle and thrill with the message, but it will not reach my spirit and consciousness.” Mary Slessor, Calabar, circa 1910

Integrins are transmembrane receptors composed of α and β subunits. Although most integrins contain β1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbβ3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted β3 TM/CT that leads to activation when disrupted. We show significant structural differences between β1 and β3 TM/CT in bicelles. Moreover, the ‘snorkeling’ lysine at the TM/CT interface of β subunits, previously proposed to regulate αIIbβ3 activation by ion pairing with nearby lipids, plays opposite roles in β1 and β3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbβ3 is seen between various α subunits and β1 TM/CTs. The αIIbβ3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins. DOI: http://dx.doi.org/10.7554/eLife.18633.001, eLife digest Proteins called integrins span the membranes of most human cells, and help our cells to interact with their surroundings, enabling them to organise, communicate and to form a variety of structures. Cells in different parts of the body typically produce different integrins so that they can specifically connect with other cells and proteins in their local environment. There are many different kinds of integrin proteins found in cell membranes and they consist of one alpha and one beta subunit. Different integrin pairs can have different effects based on their environment and the other molecules that they encounter. Much of the research into how integrins work has involved one specific integrin found in platelets – cells in blood that aid clotting and wound repair. Yet, it is unknown if all integrins actually operate in the same way as the platelet integrin. Lu, Mathew, Chen et al. studied the part of integrins that are located inside cells (referred to as the cytoplasmic tail) and the part that crosses the membrane (the transmembrane domain). Three-dimensional structures of these parts of the proteins showed that they varied between different beta integrin proteins. Further experiments revealed that the strength of the association between different alpha and beta integrins also varied. Finally Lu, Mathew, Chen et al. demonstrated that components shared by several beta integrins actually have different purposes in different contexts. The diversity of structures and interactions within the group of integrin proteins suggests that integrins are likely to behave very differently in different cells. This means that platelet integrins cannot be used to fully understand the activity of all other types of integrin. More work is now needed to understand how the differences between integrins affect the roles that they fulfil and the molecules that they interact with. A deeper understanding of the differences between integrins could ultimately shape the development of strategies to specifically target them to treat a range of diseases – such as cancer and diseases in which there is a build-up of fibrous connective tissue. DOI: http://dx.doi.org/10.7554/eLife.18633.002

Integrins are heterodimeric trans-membrane receptor proteins that mediate the interaction of cells with extracellular matrix proteins (ECM). They mediate various growth factor dependent cell-signaling pathways during the development of fibrosis that is characteristic of all forms of chronic kidney diseases. Although integrin β1 is the most abundant integrin subunit in kidney and can form complexes with 12 different α subunits, integrin β3 is the best studied integrin β subunit and serves as the canonical model for integrin function based on the high sequence homology between the trans-membrane and cytosolic domains of integrin β subunits. A conserved lysine residue towards the C-terminus of integrin β3 subunit is reported to be important for regulating the activation of integrin αIIbβ3 complexes; however the functional importance of this lysine is unknown in β1 integrins. We investigated the role of this lysine residue in integrin β1-dependent kidney collecting duct cell function. We expressed the mutant protein where the lysine is mutated to glutamic acid in collecting duct cells null for integrin β1. Collecting duct cells expressing mutant protein had decreased the adhesion of cells to collagen IV mediated by integrin α1β1 by 80% (0.95 vs 0.18). This mutation also decreased the ability of IMCD cells adhesion to collagen I mediated by integrin α2β1 by 82% (0.78 vs 0.15). In contrast to earlier reports in integrin β3, this mutation did not significantly alter the amount of active integrin β1 on the cell surface as estimated by FACS analysis; however we did observe a decrease in conformation specific antibody binding on cells adhered to collagen (0.70 vs 0.30). We also investigated the role of this lysine residue in complex formation of purified integrin β1 with integrin α1 and α2 TM/CT domains in phospholipid bicelles using fluorescence anisotropy. The dissociation constant for binding was estimated to be >3.2 mol and mutation of lysine residue did not significantly alter their binding ability. This contrasted with integrin αIIb β3 where we found fourfold decrease in binding ability (Kd 0.09 ± 0.03 mol and 0.33 ± 0.05 mol). Our data clearly suggest that conserved transmembrane lysine in both integrin β3 and integrin β1 regulate cell functions by distinct mechanisms.

Loss of β1 integrin expression inhibits renal collecting-system development. Two highly conserved NPXY motifs in the distal β1 tail regulate integrin function by associating with phosphtyrosine binding (PTB) proteins, such as talin and kindlin. Here, we define the roles of these two tyrosines in collecting-system development and delineate the structural determinants of the distal β1 tail using nuclear magnetic resonance (NMR). Mice carrying alanine mutat ions have moderate renal collecting-system developmental abnormalities relative to β1-null mice. Phenylalanine mutations did not affect renal collecting-system development but increased susceptibility to renal injury. NMR spectra in bicelles showed the distal β1 tail is disordered and does not interact with the model membrane surface. Alanine or phenylalanine mutations did not alter β1 structure or interactions between β and β1 subunit transmembrane/cytoplasmic domains; however, they did decrease talin and kindlin binding. Thus, these studies highlight the fact that the functional roles of the NPXY motifs are organ dependent. Moreover, the β1 cytoplasmic tail, in the context of the adjacent transmembrane domain in bicelles, is significantly different from the more ordered, membrane-associated β3 integrin tail. Finally, tyrosine mutations of β1 NPXY motifs induce phenotypes by disrupting their interactions with critical integrin binding proteins like talins and kindlins. © 2012, American Society for Microbiology.

The configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein were determined by measuring differential and trans-hydrogen-bond H/D isotope effects by using the isotopomer-selective (IS)-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl (13)C' shifts, which are defined as Delta(2)Delta(13)C'(ND) = (2)Delta(13)C'(ND(E))-(2)Delta(13)C'(ND(Z)), provide a reliable means for the configuration assignment for side-chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values. Second and more importantly, the significant variations in the differential one-bond isotope effects on (15)N chemical shifts, which are defined as Delta(1)Delta(15)N(D) = (1)Delta(15)N(D(E))-(1)Delta(15)N(D(Z)) can be correlated with hydrogen-bonding interactions, particularly those involving charged acceptors. The differential one-bond isotope effects are additive, with major contributions from intrinsic differential conjugative interactions between the E and Z configurations, H-bonding interactions, and charge effects. Furthermore, the pattern of trans-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks that involve bifurcated hydrogen-bonds. Third, the correlations between Delta(1)Delta(15)N(D) and hydrogen-bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side-chain amides. Rotamer correction by differential isotope effects is not only robust, but also simple and can be applied to large proteins.

Caveolins mediate the formation of caveolae, which are small omega-shaped membrane invaginations involved in a variety of cellular processes. There are three caveolin isoforms, the third of which (Cav3) is expressed in smooth and skeletal muscles. Mutations in Cav3 cause a variety of human muscular diseases. In this work, we characterize the secondary structure, dynamics, and topology of the monomeric form of the full-length lipidated protein. Cav3 consists of a series of membrane-embedded or surface-associated helical elements connected by extramembrane connecting loops or disordered domains. Our results also reveal that the N-terminal domain undergoes a large scale pH-mediated topological rearrangement between soluble and membrane-anchored forms. Considering that roughly one-third of pathogenic mutations in Cav3 influence charged residues located in this domain, we hypothesize that this transition is likely to be relevant to the molecular basis of Cav3-linked diseases. These results provide insight into the structure of Cav3 and set the stage for mechanistic investigations of the effects of pathogenic mutations.

Our recent study has shown that cellular junctions in myelin and in the epi-/perineruium that encase nerve fibers regulate the permeability of the peripheral nerves. This permeability may affect propagation of the action potential. Direct interactions between the PDZ1 domain of zonula occludens (ZO1 or ZO2) and the C-termini of claudins are known to be crucial for the formation of tight junctions. Using the purified PDZ1 domain of ZO2 and a variety of C-terminal mutants of peripheral nerve claudins (claudin-1, claudin-2, claudin-3, claudin-5 in epi-/perineurium; claudin-19 in myelin), we have utilized NMR spectroscopy to determine specific roles of the 3 C-terminal claudin residues (position -2, -1, 0) for their interactions with PDZ1 of ZO2. In contrast to the canonical model that emphasizes the importance of residues at the -2 and 0 positions, our results demonstrate that, for peripheral nerve claudins, the residue at position -1 plays a critical role in association with PDZ1, while the side-chain of residue 0 plays a significant but lesser role. Surprisingly, claudin-19, the most abundant claudin in myelin, exhibited no binding to ZO2. These findings reveal that the binding mechanism of claudin/ZO in epi-/perineurium is distinct from the canonical interactions between non-ZO PDZ-containing proteins with their ligands. This observation provides the molecular basis for a strategy to develop drugs that target tight junctions in the epi-/perineurium of peripheral nerves.

Background: Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires. Results: To understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-A resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands β 3a ndβ4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product. Conclusions: The EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.

Bilayered detergent-lipid assemblies known as bicelles have been widely used as model membranes in structural biological studies and are being explored for wider applications, including pharmaceutical use. Most studies to date have involved the use of concentrated bicelle mixtures, such that little is known about the capacity of bicellar mixtures to be diluted without unwanted transitions to non-isotropic phases. Here, different detergent/lipid mixtures have been explored, leading to the identification of two different families of bicelles for which it is possible to lower the total amphiphile (detergent+lipid) concentration to

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), which follows an ordered bi-bi kinetic mechanism with ATP binding to the enzyme first. HPPK undergoes dramatic conformational changes during its catalytic cycle as revealed by X-ray crystallography, and the conformational changes are essential for the enzymatic catalysis as shown by site-directed mutagenesis and biochemical and crystallographic analysis of the mutants. However, the dynamic properties of the enzyme have not been measured experimentally. Here, we report a (15)N NMR relaxation study of the dynamic properties of Escherichia coli HPPK from the apo form to the binary substrate complex with MgATP (represented by MgAMPCPP, an ATP analogue) to the Michaelis complex (ternary substrate complex) with MgATP (represented by MgAMPCPP) and HP (represented by 7,7-dimethyl-6-hydroxypterin, an HP analogue). The results show that the binding of the nucleotide to HPPK does not cause major changes in the dynamic properties of the enzyme. Whereas enzymes are often more rigid when bound to the ligand or the substrate, the internal mobility of HPPK is not reduced and is even moderately increased in the binary complex, particularly in the catalytic loops. The internal mobility of the catalytic loops is significantly quenched upon the formation of the ternary complex, but some mobility remains. The enhanced motions in the catalytic loops of the binary substrate complex may be required for the assembling of the ternary complex. On the other hand, some degrees of mobility in the catalytic loops of the ternary complex may be required for the optimal stabilization of the transition state, which may need the instantaneous adjustment and alignment of the side-chain positions of catalytic residues. Such dynamic behaviors may be characteristic of bisubstrate enzymes.

We introduce a novel technique for continuous (i.e., automatic) monitoring of cardiac output (CO) and left atrial pressure (LAP) by mathematical analysis of a pulmonary artery pressure (PAP) waveform. To obtain an initial evaluation of the technique, we applied it to PAP waveforms obtained from nine critically ill patients and compared the resulting CO and LAP estimates with standard operator- dependent thermodilution and pulmonary capillary wedge pressure measurements, respectively. We report that the technique achieved an overall CO error of 17.2% and an overall LAP error of 15.8%. With further testing, the technique may ultimately be employed so as to permit, for the first time, continuous CO and LAP monitoring in critically ill patients. I. INTRODUCTION ARDIAC output (CO) represents the total blood flow rate in the circulation, while left atrial pressure (LAP) generally indicates the blood pressure attained in the left ventricle during the cardiac filling phase. CO and LAP are two of the most important quantities to be able to monitor in critically ill patients, as they facilitate the diagnosis, monitoring, and treatment of various disease processes such as left ventricular failure, mitral valve disease, and shock of any cause. For example, a decrease in CO while LAP is rising would indicate that the patient is in left ventricular failure, whereas a decrease in CO while LAP is falling may indicate that the patient is going into hypovolemic shock. The standard methods for monitoring CO and LAP in critically ill patients both involve the use of the balloon- tipped, flow-directed pulmonary artery catheter (1)(2). CO is specifically estimated according to the thermodilution method. This method involves injecting a bolus of cold saline in the right atrium, measuring temperature downstream in the pulmonary artery, and computing the average CO based on conservation laws. LAP is estimated through the pulmonary capillary wedge pressure (PCWP) method. This method involves advancing the catheter into a branch of the pulmonary artery, inflating the balloon, and measuring the resulting steady-state PCWP. In theory, PCWP should nearly equal LAP, since flow has ceased

The maximum left ventricular elastance (Esupmax/supinfiv/inf) is a valuable index of ventricular contractility. However, conventional methods for its measurements are too invasive for routine use. We propose a non-invasive technique for tracking changes in Esupmax/supinfiv/infby mathematically determining the dynamic coupling between beat-to-beat fluctuations in instantaneous lung volume and arterial blood pressure measured during random-interval breathing. The mathematical technique also provides an estimate of the dominant time constant of the ventilatory system (), which is equal to the product of the airway resistance and lung compliance. We then describe a theoretical evaluation of the technique with respect to realistic beat-to-beat variability generated by a cardiovascular simulator. Our results show that the technique is able to detect actual changes in the Esupmax/supinfiv/infand values of the simulator. With successful experimental testing, the technique may ultimately be employed to help guide therapy in heart failure patients.

Hydrogen bonds play critical roles in protein structure, stability, and function. Conventionally, hydrogen bonds are mainly determined by X-ray crystallography and NOE-based NMR spectroscopy in indirect manners. In recent years, it was demonstrated that hydrogen bonds can be directly detected through NMR measurements of trans-hydrogen-bond scalar coupling constants. Here we report across hydrogen-bond protium/deuterium isotope effects in a 35 kDA protein observed with the isotopomer-selective TROSY NMR technique (Liu et al. J. Biomol. NMR 2006, 36, 205−214; Liu et al. J. Magn. Reson. 2007, 186, 319−326) and show that such isotope effects can be used to detect a most common type of bifurcated hydrogen bonds, in which a heavy atom, usually oxygen, is involved in two hydrogen bonds, including a pair of bifurcated hydrogen bonds involving a bound water molecule.