Computational hemodynamic simulations of cerebral aneurysms have traditionally relied on stereotypical boundary conditions (such as blood flow velocity and blood pressure) derived from published values as patient-specific measurements are unavailable or difficult to collect. However, controversy persists over the necessity of incorporating such patient specific conditions into computational analyses. We perform simulations using both endovascular-derived patient-specific and typical literature-derived inflow and outflow boundary conditions. Detailed three-dimensional anatomical models of the cerebral vasculature are developed from rotational angiography data, and blood flow velocity and pressure are measured in situ by a dual-sensor pressure and velocity endovascular guidewire at multiple peri-aneurysmal locations in ten unruptured cerebral aneurysms. These measurements are used to define inflow and outflow boundary conditions for computational hemodynamic models of the aneurysms. The additional in situ measurements which are not prescribed in the simulation are then used to assess the accuracy of the simulated flow velocity and pressure drop. Simulated velocities using patient-specific boundary conditions show good agreement with the guidewire measurements at measurement locations inside the domain, with no bias in the agreement and a random scatter of ≈25%. Simulated velocities using the simplified, literature-derived values show a systematic bias and over-predicted velocity by ≈30% with a random scatter of ≈40%. Computational hemodynamics using endovascularly measured patient-specific boundary conditions have the potential to improve treatment predictions as they provide more accurate and precise results of the aneurysmal hemodynamics than those based on commonly accepted reference values for boundary conditions.
Hemodynamics; Cerebral Aneurysm; Patient-Specific Computational Modeling
Endogenously produced carbon monoxide (CO) is commonly believed to be a ubiquitous second messenger involved in a wide range of physiological and pathological responses. The major evidence supporting this concept is that CO is produced endogenously via heme oxygenase-catalyzed breakdown of heme and that experimental exposure to CO alters tissue function. However, it remains to be conclusively demonstrated that there are specific receptors for CO and that endogenous CO production is sufficient to alter tissue function. Unlike other signaling molecules, CO is not significantly metabolized, and it is removed from cells solely via rapid diffusion into blood, which serves as a near infinite sink. This non-metabolizable nature of CO renders the physiology of this gas uniquely susceptible to quantitative modeling. This review analyzes each of the steps involved in CO signaling: 1) the background CO partial pressure (PCO) and the blood and tissue CO binding; 2) the affinity of the putative CO receptors; 3) the rate of endogenous tissue CO production; and 4) the tissue PCO that results from the balance between this endogenous CO production and diffusion to the blood sink. Because existing data demonstrate that virtually all endogenous CO production results from the routine “housekeeping” turnover of heme, only a small fraction can play a signaling role. The novel aspect of the present report is to demonstrate via physiological modeling that this small fraction of CO production is seemingly insufficient to raise intracellular PCO to the levels required for the conventional, specific messenger receptor activation. It is concluded that the many physiological alterations observed with exogenous CO administration are probably produced by the non-specific CO inhibition of cytochrome C oxidase activity, with release of reactive oxygen species (ROS) and that this ROS signaling pathway is a potential effector mechanism for endogenously produced CO.
cytochrome; oxygenase; Krogh cylinder model; ROS; heme
Serum bilirubin measurements are commonly obtained for the evaluation of ill patients and to screen for liver disease in routine physical exams. An enormous research effort has identified the multiple mechanisms involved in the production and metabolism of conjugated (CB) and unconjugated bilirubin (UB). While the qualitative effects of these mechanisms are well understood, their expected quantitative influence on serum bilirubin homeostasis has received less attention. In this review, each of the steps involved in bilirubin production, metabolism, hepatic cell uptake, and excretion is quantitatively examined. We then attempt to predict the expected effect of normal and defective function on serum UB and CB levels in health and disease states including hemolysis, extra- and intrahepatic cholestasis, hepatocellular diseases (eg, cirrhosis, hepatitis), and various congenital defects in bilirubin conjugation and secretion (eg, Gilbert’s, Dubin–Johnson, Crigler–Najjar, Rotor syndromes). Novel aspects of this review include: 1) quantitative estimates of the free and total UB and CB in the plasma, hepatocyte, and bile; 2) detailed discussion of the important implications of the recently recognized role of the hepatic OATP transporters in the maintenance of CB homeostasis; 3) discussion of the differences between the standard diazo assay versus chromatographic measurement of CB and UB; 4) pharmacokinetic implications of the extremely high-affinity albumin binding of UB; 5) role of the enterohepatic circulation in physiologic jaundice of newborn and fasting hyperbilirubinemia; and 6) insights concerning the clinical interpretation of bilirubin measurements.
liver; conjugation; diazo; albumin; Rotor
An overview of applications of the deformable elastic network (DEN) refinement method is presented together with recommendations for its optimal usage.
Crystals of membrane proteins and protein complexes often diffract to low resolution owing to their intrinsic molecular flexibility, heterogeneity or the mosaic spread of micro-domains. At low resolution, the building and refinement of atomic models is a more challenging task. The deformable elastic network (DEN) refinement method developed previously has been instrumental in the determinion of several structures at low resolution. Here, DEN refinement is reviewed, recommendations for its optimal usage are provided and its limitations are discussed. Representative examples of the application of DEN refinement to challenging cases of refinement at low resolution are presented. These cases include soluble as well as membrane proteins determined at limiting resolutions ranging from 3 to 7 Å. Potential extensions of the DEN refinement technique and future perspectives for the interpretation of low-resolution crystal structures are also discussed.
deformable elastic network refinement; low resolution
Ribonucleic acid (RNA) molecules play important roles in a variety of biological processes. To properly function, RNA molecules usually have to fold to specific structures, and therefore understanding RNA structure is vital in comprehending how RNA functions. One approach to understanding and predicting biomolecular structure is to use knowledge-based potentials built from experimentally determined structures. These types of potentials have been shown to be effective for predicting both protein and RNA structures, but their utility is limited by their significantly rugged nature. This ruggedness (and hence the potential's usefulness) depends heavily on the choice of bin width to sort structural information (e.g. distances) but the appropriate bin width is not known a priori. To circumvent the binning problem, we compared knowledge-based potentials built from inter-atomic distances in RNA structures using different mixture models (Kernel Density Estimation, Expectation Minimization and Dirichlet Process). We show that the smooth knowledge-based potential built from Dirichlet process is successful in selecting native-like RNA models from different sets of structural decoys with comparable efficacy to a potential developed by spline-fitting — a commonly taken approach — to binned distance histograms. The less rugged nature of our potential suggests its applicability in diverse types of structural modeling.
Knowledge-based potential; mixture models; RNA structure
The protein density and arrangement of subunits of a complete, 31-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by cryo-electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs), and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.
TRiC is an important group II chaperonin that facilitates the folding of many eukaryotic proteins. The TRiC complex consists of two stacked rings, each comprised of eight paralogous subunits with a mutual sequence identity of 30–35%. Each subunit has unique functional roles that are manifested by corresponding sequence conservation. It is generally assumed that the subunit order within each ring is fixed, but this order is still uncertain. Here we address the problem of the intra-ring subunit order by combining two sources of information: evolutionary conservation and a structural hypothesis. Specifically, we identify residues in the TRiC subunits that are likely to be part of the intra-unit interface, based on homology modeling to the solved thermosome structure. Within this set of residues, we search for a subset that shows an evolutionary conservation pattern that is indicative of the subunit order key. This pattern shows considerable conservation across species, but large variation across the eight subunits. By this approach we were able to locate two parts of the interface where complementary interactions seem to favor certain pairing of subunits. This knowledge leads to restrictions on the 5,040 (=7!) possible subunits arrangements in the ring, and limits them to just 72. Although our findings give only partial understanding of the inter-subunit interactions that determine their order, we conclude that they are comprised of complementary charged, polar and hydrophobic interactions that occur in both the equatorial and middle domains of each subunit.
In eukaryotes, CCT is essential for the correct and efficient folding of many cytosolic proteins, most notably actin and tubulin. Structural studies of CCT have been hindered by the failure of standard crystallographic analysis to resolve its eight different subunit types at low resolutions. Here, we exhaustively assess the R-value fit of all possible CCT models to available crystallographic data of the closed and open forms with resolutions of 3.8 Å and 5.5 Å, respectively. This unbiased analysis finds the native subunit arrangements with overwhelming significance. The resulting structures provide independent crystallographic proof of the subunit arrangement of CCT, and map major asymmetrical features of the particle onto specific subunits. The actin and tubulin substrates both bind around subunit CCT6, which shows other structural anomalies. CCT is thus clearly partitioned, both functionally and evolutionary, into a substrate-binding side that is opposite to the ATP-hydrolyzing side.
Background and Purpose
Computational fluid dynamics modeling is useful in the study of the hemodynamic environment of cerebral aneurysms, but patient-specific measurements of boundary conditions, such as blood flow velocity and pressure, have not been previously applied to the study of flow-diverting stents. We integrated patient-specific intravascular blood flow velocity and pressure measurements into computational models of aneurysms before and after treatment with flow-diverting stents to determine stent effects on aneurysm hemodynamics.
Blood flow velocity and pressure were measured in peri-aneurysmal locations using an intravascular dual-sensor pressure and Doppler velocity guidewire before and after flow-diverting stent treatment of four unruptured cerebral aneurysms. These measurements defined inflow and outflow boundary conditions for computational models. Intra-aneurysmal flow rates, wall shear stress and wall shear stress gradient were calculated.
Measurements of inflow velocity and outflow pressure were successful in all four patients. Computational models incorporating these measurements demonstrated significant reductions in intra-aneurysmal wall shear stress and wall shear stress gradient, and a trend in reduced intra-aneurysmal blood flow.
Integration of intravascular dual-sensor guidewire measurements of blood flow velocity and blood pressure provided patient-specific computational models of cerebral aneurysms. Aneurysm treatment with flow-diverting stents reduces blood flow and hemodynamic shear stress in the aneurysm dome.
Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their “built-in lid” to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ~45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.
Lattice models of proteins have been extensively used to study protein thermodynamics, folding dynamics and evolution. Our study considers two different hydrophobic-polar models on the two-dimensional square lattice: the purely hydrophobic-polar (HP) model and a model where a compactness-favoring term is added. We exhaustively enumerate all the possible structures in our models and perform the study of their corresponding folds, HP arrangements in space and shapes. The two models considered differ greatly in their numbers of structures, folds, arrangements and shapes. Despite their differences both lattice models have distinctive protein-like features: (1) Shapes are compact in both models, especially when a compactness-favoring energy term is added. (2) The residue composition is independent of the chain length and is very close to 50% hydrophobic in both models, as we observe in real proteins. (3) Comparative modeling works well in both models, particularly in the more compact one. The fact that our models show protein-like features suggests that lattice models incorporate the fundamental physical principles of proteins. Our work supports the use of lattice models to study questions about proteins that require exactness and extensive calculations, such as protein design and evolution, which are often too complex and computationally demanding to be addressed with more detailed models.
lattice models; self-avoiding walk; residue composition; hydrophobicity; protein-like; protein universe
The multidisciplinary management of brain metastases has generated substantial controversy as treatment has diversified in recent years. Debate about the type, role, and timing of different diagnostic and therapeutic strategies has promoted rigorous scientific research into efficacy. However, much still remains unanswered in the treatment of this difficult disease process. This manuscript seeks to highlight some of the controversies identified in previous sections of this supplement, including prognosis, pathology, radiation and surgical treatment, neuroimaging, and the biochemical underpinnings of brain metastases. By recognizing what is yet unanswered, we hope to identify areas in which further research may yield promising results.
Brain metastases; biomarkers; chemotherapy; neuroimaging; stereotactic radiosurgery; whole-brain radiation therapy
In X-ray crystallography, molecular replacement and subsequent refinement is challenging at low resolution. We compared refinement methods using synchrotron diffraction data of photosystem I at 7.4 Å resolution, starting from different initial models with increasing deviations from the known high-resolution structure. Standard refinement spoiled the initial models moving them further away from the true structure and leading to high Rfree-values. In contrast, DEN-refinement improved even the most distant starting model as judged by Rfree, atomic root-mean-square differences to the true structure, significance of features not included in the initial model, and connectivity of electron density. The best protocol was DEN-refinement with initial segmented rigid-body refinement. For the most distant initial model, the fraction of atoms within 2 Å of the true structure improved from 24% to 60%. We also found a significant correlation between Rfree-values and the accuracy of the model, suggesting that Rfree is useful even at low resolution.
DEN refinement; membrane protein; low-resolution refinement; simulated annealing; free R value
SCOP is a hierarchical domain classification system for proteins of known structure. The superfamily level has a clear definition: Protein domains belong to the same superfamily if there is structural, functional and sequence evidence for a common evolutionary ancestor. Superfamilies are sub-classified into families, however, there is not such a clear basis for the family level groupings. Do SCOP families group together domains with sequence similarity, do they group domains with similar structure or by common function? It is these questions we answer, but most importantly, whether each family represents a distinct phylogenetic group within a superfamily.
Several phylogenetic trees were generated for each superfamily: one derived from a multiple sequence alignment, one based on structural distances, and the final two from presence/absence of GO terms or EC numbers assigned to domains. The topologies of the resulting trees and confidence values were compared to the SCOP family classification.
We show that SCOP family groupings are evolutionarily consistent to a very high degree with respect to classical sequence phylogenetics. The trees built from (automatically generated) structural distances correlate well, but are not always consistent with SCOP (hand annotated) groupings. Trees derived from functional data are less consistent with the family level than those from structure or sequence, though the majority still agree. Much of GO and EC annotation applies directly to one family or subset of the family; relatively few terms apply at the superfamily level. Maximum sequence diversity within a family is on average 22% but close to zero for superfamilies.
Human posttraumatic epilepsy (PTE) is highly heterogeneous, ranging from mild remitting to progressive disabling forms. PTE results in simple partial, complex partial, and secondarily generalized seizures with a wide spectrum of durations and semiologies. PTE variability is thought to depend on the heterogeneity of head injury and patient's age, gender, and genetic background. To better understand the role of these factors, we investigated the seizures resulting from calibrated fluid percussion injury (FPI) to adolescent male Sprague–Dawley rats with video electrocorticography. We show that PTE incidence and the frequency and severity of chronic seizures depend on the location and severity of FPI. The frontal neocortex was more prone to epileptogenesis than the parietal and occipital, generating earlier, longer, and more frequent partial seizures. A prominent limbic focus developed in most animals, regardless of parameters of injury. Remarkably, even with carefully controlled injury parameters, including type, severity, and location, the duration of posttraumatic apnea and the age and gender of outbred rats, there was great subject-to-subject variability in frequency, duration, and rate of progression of seizures, indicating that other factors, likely the subjects' genetic background and physiological states, have critical roles in determining the characteristics of PTE.
drug screening; electrocorticography; endophenotype; model; partial seizures; syndrome; trauma
Myeloid sarcoma is a rare extramedullary tumour consisting of immature myeloid cells. It can arise at any anatomical location and often develops in the bowel. This report describes a case of severe acute disseminated intravascular coagulation (DIC) with multi-organ failure occurring in a 57-year-old man with chronic myelomonocytic leukaemia during bowel resection for newly diagnosed adenocarcinoma of the sigmoid colon. Histopa thology however revealed a differentiating myeloid sarcoma encompassing a well-differentiated adenocarcinoma. This is the first documented case of acute DIC to be triggered following surgical manipulation of myeloid sarcoma.
myeloid sarcoma; disseminated intravascular coagulation; leukaemic transformation.
The KoBaMIN web server provides an online interface to a simple, consistent and computationally efficient protein structure refinement protocol based on minimization of a knowledge-based potential of mean force. The server can be used to refine either a single protein structure or an ensemble of proteins starting from their unrefined coordinates in PDB format. The refinement method is particularly fast and accurate due to the underlying knowledge-based potential derived from structures deposited in the PDB; as such, the energy function implicitly includes the effects of solvent and the crystal environment. Our server allows for an optional but recommended step that optimizes stereochemistry using the MESHI software. The KoBaMIN server also allows comparison of the refined structures with a provided reference structure to assess the changes brought about by the refinement protocol. The performance of KoBaMIN has been benchmarked widely on a large set of decoys, all models generated at the seventh worldwide experiments on critical assessment of techniques for protein structure prediction (CASP7) and it was also shown to produce top-ranking predictions in the refinement category at both CASP8 and CASP9, yielding consistently good results across a broad range of model quality values. The web server is fully functional and freely available at http://csb.stanford.edu/kobamin.
Prion proteins (PrP) are the infectious agent in transmissible spongiform encephalopathies (i.e. mad cow disease). To be infectious, prion proteins must undergo a conformational change involving a decrease of α-helical content along with an increase of β-strand structure. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two α-helices by one and two residues, as well as an extension of two β-strands by three residues each which could instigate the conformational change. The conformational change occurs in a region that is compatible with immunological studies, and it is observed more frequently in mutant prions which are prone to conversion, than in WT prions due to differences in their starting structures, which are amplified through normal modes. These findings are valuable for our comprehension of the conversion mechanism associated with the conformational change of prion proteins.
Prion proteins; normal modes; conversion mechanism; zipper; dynamics
Although the factors involved in cirrhotic ascites have been studied for a century, a number of observations are not understood, including the action of diuretics in the treatment of ascites and the ability of the plasma-ascitic albumin gradient to diagnose portal hypertension. This communication presents an explanation of ascites based solely on pathophysiological alterations within the peritoneal cavity. A quantitative model is described based on experimental vascular and intraperitoneal pressures, lymph flow, and peritoneal space compliance. The model's predictions accurately mimic clinical observations in ascites, including the magnitude and time course of changes observed following paracentesis or diuretic therapy.
Ascites; Cirrhosis; Portal hypertension; Wedge pressure
DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols.
Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle
Chaperonins are multisubunit entities that are composed of two stacked rings enclosing a central chamber for ATP-dependent protein folding. A series of cryo-EM structures of the eukaryotic group II chaperonin TRiC/CCT reveal the conformational changes during the ATPase cycle and provide insight into how the subunits cooperate to close the lid.
The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.
asymmetric intermediate; conformational cycle; cryo-EM; protein folding; TRiC/CCT
Protein structure refinement is an important but unsolved problem; it must be solved if we are to predict biological function that is very sensitive to structural details. Specifically, Critical Assessment of Techniques for Protein Structure Prediction (CASP) shows that the accuracy of predictions in the comparative modeling category is often worse than that of the template on which the homology model is based. Here we describe a refinement protocol that is able to consistently refine submitted predictions for all categories at CASP7. The protocol uses direct energy minimization of the knowledge-based potential of mean force that is based on the interaction statistics of 167 atom types (Summa and Levitt, Proc Natl Acad Sci USA 2007; 104:3177–3182). Our protocol is thus computationally very efficient; it only takes a few minutes of CPU time to run typical protein models (300 residues). We observe an average structural improvement of 1% in GDT_TS, for predictions that have low and medium homology to known PDB structures (Global Distance Test score or GDT_TS between 50 and 80%). We also observe a marked improvement in the stereochemistry of the models. The level of improvement varies amongst the various participants at CASP, but we see large improvements (>10% increase in GDT_TS) even for models predicted by the best performing groups at CASP7. In addition, our protocol consistently improved the best predicted models in the refinement category at CASP7 and CASP8. These improvements in structure and stereochemistry prove the usefulness of our computationally inexpensive, powerful and automatic refinement protocol.
Refinement; Comparative Modeling; CASP7; ENCAD; MESHI; Knowledge-based; Stereochemistry
The present article introduces a set of novel methods that facilitate the use of “natural moves” or arbitrary degrees of freedom that can give rise to collective rearrangements in the structure of biological macromolecules. While such “natural moves” may spoil the stereochemistry and even break the bonded chain at multiple locations, our new method restores the correct chain geometry by adjusting bond and torsion angles in an arbitrary defined molten zone. This is done by successive stages of partial closure that propagate the location of the chain break backwards along the chain. At the end of these stages, the size of the chain break is generally reduced so much that it can be repaired by adjusting the position of a single atom. Our chain closure method is efficient with a computational complexity of O(Nd), where Nd is the number of degrees of freedom used to repair the chain break. The new method facilitates the use of arbitrary degrees of freedom including the “natural” degrees of freedom inferred from analyzing experimental (X-ray crystallography and nuclear magnetic resonance [NMR]) structures of nucleic acids and proteins. In terms of its ability to generate large conformational moves and its effectiveness in locating low energy states, the new method is robust and computationally efficient.
chain closure algorithm; internal coordinates; Markov Chains; Monte Carlo Minimization; nucleic acids; proteins; stochastic optimization
Hydrogen sulfide (H2S) has long been associated with the gastrointestinal tract, especially the bacteria-derived H2S present in flatus. Along with evidence from other organ systems, the finding that gastrointestinal tissues are capable of endogenous production of H2S has led to the hypothesis that H2S is an endogenous gaseous signaling molecule. In this review, the criteria of gasotransmitters are reexamined, and evidence from the literature regarding H2S as a gaseous signaling molecule is discussed. H2S is produced enzymatically by gastrointestinal tissues, but evidence is lacking on whether H2S production is regulated. H2S causes well-defined physiologic effects in gastrointestinal tissues, but evidence for a receptor for H2S is lacking. H2S is inactivated through enzymatic oxidation, but evidence is lacking on whether manipulating H2S oxidation alters endogenous cell signaling. Remaining questions regarding the role of H2S as a gaseous signaling molecule in the gastrointestinal tract suggest that H2S currently remains a molecule in search of a physiologic function. Antioxid. Redox Signal. 12, 1135–1146.