The efficacy of the analgesic GlyT2 inhibitor, ORG25543, is determined by two connected allosteric sites.

Glycine Transporter 2 (GlyT2) inhibitors have shown considerable potential as analgesics for the treatment of neuropathic pain but also display considerable side effects. One potential source of side effects is irreversible inhibition. In this study, we have characterized the mechanism of ORG25543 inhibition of GlyT2 by first considering three potential ligand binding sites on GlyT2-the substrate site, the vestibule allosteric site and the lipid allosteric site. The three sites were tested using a combination of molecular dynamics simulations and analysis of the inhibition of glycine transport of a series point mutated GlyT2 using electrophysiological methods. We demonstrate that the lipid allosteric site on GlyT2 is the most likely binding site for ORG25543. We also demonstrate that cholesterol derived from the cell membrane can form specific interactions with inhibitor-bound transporters to form an allosteric network of regulatory sites. These observations will guide the future design of GlyT2 inhibitors with the objective of minimising on-target side effects and improving the therapeutic window for the treatment of patients suffering from neuropathic pain.

Stereoselective Single Step Cyclization to Give Belt-Functionalized Pillar[6]arenes.

Two macrocycles were synthesized through cyclization reactions of secondary benzylic alcohols, giving pillar[6]arenes with a methyl substituent at each belt position. These macrocycles form stereoselectively with only the rtctct isomer with alternating up and down orientations of the belt methyl groups definitively identified. Isolated yields were modest (7 and 9%), but the macrocycles are prepared in a single step from either a commercially available alcohol or a very readily prepared precursor. X-ray crystal structures of the macrocycles indicate they have a capsule-like structure, which is far from the conventional pillar shape. Density functional theory calculations reveal that the energy barrier required to obtain the pillar conformation is significantly higher for these belt-functionalized macrocycles than for conventional belt-unfunctionalized pillar[6]arenes.

Outcomes of patients with Barrett's oesophagus with low-grade dysplasia undergoing endoscopic surveillance in a tertiary centre: a retrospective cohort study.

BACKGROUND AND AIM: Barrett's oesophagus predisposes individuals to oesophageal adenocarcinoma (OAC), with the risk of progression to malignancy increasing with the degree of dysplasia, categorized as either low-grade dysplasia (LGD) or high-grade dysplasia (HGD). The reported incidence of progression to OAC in LGD ranges from 0.02% to 11.43% per annum. In patients with LGD, Australian guidelines recommend 6-monthly endoscopic surveillance. We aimed to describe the surveillance practices within a tertiary centre, and to determine the predictive value of surveillance as well as other risk factors for progression. METHODS: Endoscopy and pathology databases were searched over a 10-year period to collate all cases of Barrett's oesophagus with LGD. Medical records were reviewed to document patient factors and endoscopic and histologic details. Because follow-up times varied greatly, survival analysis techniques were employed. RESULTS: Fifty-nine patients were found to have LGD. Thirteen patients (22.0%) progressed to either HGD or OAC (10 (16.9%) and three (5.1%) respectively); the annual incidence rates of progression to HGD/OAC and OAC were 5.5% and 1.1% respectively. All patients who developed OAC had non-guideline-adherent surveillance. A Cox model found only two predictors of progression: (i) guideline-adherent surveillance, performed in 16 (27.1%), detected progression to HGD/OAC four times earlier than non-guideline-adherent surveillance (95% confidence interval (CI) = 1.3-12.3; P = 0.016). (ii) The detection of visible lesions at exit endoscopy independently predicted progression (hazard ratio = 6.5; 95% CI = 1.9-22.8; P = 0.003). CONCLUSION: Barrett's oesophagus with LGD poses a significant risk of progression to HGD/OAC. Guideline-recommended surveillance is effective, but is difficult to adhere to. Clinical predictors for those who are more likely to progress are yet to be defined.

Amphetamine-like Deferiprone and Clioquinol Derivatives as Iron Chelating Agents.

The accumulation of iron in dopaminergic neurons can cause oxidative stress and dopaminergic neuron degeneration. Iron chelation therapy may reduce dopaminergic neurodegeneration, but chelators should be targeted towards dopaminergic cells. In this work, two series of compounds based on 8-hydroxyquinoline and deferiprone, iron chelators that have amphetamine-like structures, have been designed, synthesized and characterized. Each of these compounds chelated iron ions in aqueous solution. The hydroxyquinoline-based compounds exhibited stronger iron-binding constants than those of the deferiprone derivatives. The hydroxyquinoline-based compounds also exhibited greater free radical scavenging activities compared to the deferiprone derivatives. Molecular dynamics simulations showed that the hydroxyquinoline-based compounds generally bound well within human dopamine transporter cavities. Thus, these compounds are excellent candidates for future exploration as drugs against diseases that are affected by iron-induced dopaminergic neuron damage, such as Parkinson's disease.

Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA-protein complexes.

A set of >300 nonredundant high-resolution RNA-protein complexes were rigorously searched for π-contacts between an amino acid side chain (W, H, F, Y, R, E and D) and an RNA nucleobase (denoted π-π interaction) or ribose moiety (denoted sugar-π). The resulting dataset of >1500 RNA-protein π-contacts were visually inspected and classified based on the interaction type, and amino acids and RNA components involved. More than 80% of structures searched contained at least one RNA-protein π-interaction, with π-π contacts making up 59% of the identified interactions. RNA-protein π-π and sugar-π contacts exhibit a range in the RNA and protein components involved, relative monomer orientations and quantum mechanically predicted binding energies. Interestingly, π-π and sugar-π interactions occur more frequently with RNA (4.8 contacts/structure) than DNA (2.6). Moreover, the maximum stability is greater for RNA-protein contacts than DNA-protein interactions. In addition to highlighting distinct differences between RNA and DNA-protein binding, this work has generated the largest dataset of RNA-protein π-interactions to date, thereby underscoring that RNA-protein π-contacts are ubiquitous in nature, and key to the stability and function of RNA-protein complexes.

Lipopolysaccharide Pretreatment Prevents Medullary Vascular Congestion following Renal Ischemia by Limiting Early Reperfusion of the Medullary Circulation.

BACKGROUND: Vascular congestion of the renal medulla-trapped red blood cells in the medullary microvasculature-is a hallmark finding at autopsy in patients with ischemic acute tubular necrosis. Despite this, the pathogenesis of vascular congestion is not well defined. METHODS: In this study, to investigate the pathogenesis of vascular congestion and its role in promoting renal injury, we assessed renal vascular congestion and tubular injury after ischemia reperfusion in rats pretreated with low-dose LPS or saline (control). We used laser Doppler flowmetry to determine whether pretreatment with low-dose LPS prevented vascular congestion by altering renal hemodynamics during reperfusion. RESULTS: We found that vascular congestion originated during the ischemic period in the renal venous circulation. In control animals, the return of blood flow was followed by the development of congestion in the capillary plexus of the outer medulla and severe tubular injury early in reperfusion. Laser Doppler flowmetry indicated that blood flow returned rapidly to the medulla, several minutes before recovery of full cortical perfusion. In contrast, LPS pretreatment prevented both the formation of medullary congestion and its associated tubular injury. Laser Doppler flowmetry in LPS-pretreated rats suggested that limiting early reperfusion of the medulla facilitated this protective effect, because it allowed cortical perfusion to recover and clear congestion from the large cortical veins, which also drain the medulla. CONCLUSIONS: Blockage of the renal venous vessels and a mismatch in the timing of cortical and medullary reperfusion results in congestion of the outer medulla's capillary plexus and promotes early tubular injury after renal ischemia. These findings indicate that hemodynamics during reperfusion contribute to the renal medulla's susceptibility to ischemic injury.

The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity.

The role of lipids in modulating membrane protein function is an emerging and rapidly growing area of research. The rational design of lipids that target membrane proteins for the treatment of pathological conditions is a novel extension in this field and provides a step forward in our understanding of membrane transporters. Bioactive lipids show considerable promise as analgesics for the treatment of chronic pain and bind to a high-affinity allosteric-binding site on the human glycine transporter 2 (GlyT2 or SLC6A5). Here, we use a combination of medicinal chemistry, electrophysiology, and computational modeling to develop a rational structure-activity relationship for lipid inhibitors and demonstrate the key role of the lipid tail interactions for GlyT2 inhibition. Specifically, we examine how lipid inhibitor head group stereochemistry, tail length, and double-bond position promote enhanced inhibition. Overall, the l-stereoisomer is generally a better inhibitor than the d-stereoisomer, longer tail length correlates with greater potency, and the position of the double bond influences the activity of the inhibitor. We propose that the binding of the lipid inhibitor deep into the allosteric-binding pocket is critical for inhibition. Furthermore, this provides insight into the mechanism of inhibition of GlyT2 and highlights how lipids can modulate the activity of membrane proteins by binding to cavities between helices. The principles identified in this work have broader implications for the development of a larger class of compounds that could target SLC6 transporters for disease treatment.

Multiscale computational investigations of the translesion synthesis bypass of tobacco-derived DNA adducts: critical insights that complement experimental biochemical studies.

Among the numerous agents that damage DNA, tobacco products remain one of the most lethal and result in the most diverse set of DNA lesions. This perspective aims to provide an overview of computational work conducted to complement experimental biochemical studies on the mutagenicity of adducts derived from the most potent tobacco carcinogen, namely 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosaminoketone or NNK). Lesions ranging from the smallest methylated thymine derivatives to the larger, flexible pyridyloxobutyl (POB) guanine adducts are considered. Insights are obtained from density functional theory (DFT) calculations and molecular dynamics (MD) simulations into the damaged nucleobase and nucleoside structures, the accommodation of the lesions in the active site of key human polymerases, the intrinsic base pairing potentials of the adducts, and dNTP incorporation opposite the lesions. Overall, the computational data provide atomic level information that can rationalize the differential mutagenic properties of tobacco-derived lesions and uncover important insights into the impact of adduct size, nucleobase, position, and chemical composition of the bulky moiety.

High resolution crystal structure of a KRAS promoter G-quadruplex reveals a dimer with extensive poly-A π-stacking interactions for small-molecule recognition.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.

Pointless spatial modeling.

The analysis of area-level aggregated summary data is common in many disciplines including epidemiology and the social sciences. Typically, Markov random field spatial models have been employed to acknowledge spatial dependence and allow data-driven smoothing. In the context of an irregular set of areas, these models always have an ad hoc element with respect to the definition of a neighborhood scheme. In this article, we exploit recent theoretical and computational advances to carry out modeling at the continuous spatial level, which induces a spatial model for the discrete areas. This approach also allows reconstruction of the continuous underlying surface, but the interpretation of such surfaces is delicate since it depends on the quality, extent and configuration of the observed data. We focus on models based on stochastic partial differential equations. We also consider the interesting case in which the aggregate data are supplemented with point data. We carry out Bayesian inference and, in the language of generalized linear mixed models, if the link is linear, an efficient implementation of the model is available via integrated nested Laplace approximations. For nonlinear links, we present two approaches: a fully Bayesian implementation using a Hamiltonian Monte Carlo algorithm and an empirical Bayes implementation, that is much faster and is based on Laplace approximations. We examine the properties of the approach using simulation, and then apply the model to the classic Scottish lip cancer data.

Structural Rationalization for the Nonmutagenic and Mutagenic Bypass of the Tobacco-Derived O4-4-(3-Pyridyl)-4-oxobut-1-yl-thymine Lesion by Human Polymerase η: A Multiscale Computational Study.

Tobacco-derived pyridyloxobutyl (POB) DNA adducts are unique due to the large size and flexibility of the alkyl chain connecting the pyridyl ring to the nucleobase. Recent experimental work suggests that the O4-4-(3-pyridyl)-4-oxobut-1-yl-T (O4-POB-T) lesion can undergo both nonmutagenic (dATP) and mutagenic (dGTP) insertion by the translesion synthesis (TLS) polymerase (pol) η in human cells. Interestingly, the mutagenic rate for O4-POB-T replication is reduced compared to that for the smaller O4-methylthymine (O4-Me-T) lesion, and O4-POB-T yields a different mutagenic profile than the O2-POB-T variant (dTTP insertion). The present work uses a combination of density functional theory calculations and molecular dynamics simulations to probe the impact of the size and flexibility of O4-POB-T on pol η replication outcomes. Due to changes in the Watson-Crick binding face upon damage of canonical T, O4-POB-T does not form favorable hydrogen-bonding interactions with A. Nevertheless, dATP is positioned for insertion in the pol η active site by a water chain to the template strand, which suggests a pol η replication pathway similar to that for abasic sites. Although a favorable O4-POB-T:G mispair forms in the pol η active site and DNA duplexes, the inherent dynamical nature of O4-POB-T periodically disrupts interstrand hydrogen bonding that would otherwise facilitate dGTP insertion and stabilize damaged DNA duplexes. In addition to explaining the origin of the experimentally reported pol η outcomes associated with O4-POB-T replication, comparison to structural data for the O4-Me-T and O2-POB-T adducts highlights an emerging common pathway for the nonmutagenic replication of thymine alkylated lesions by pol η, yet underscores the broader impacts of bulky moiety size, flexibility, and position on the associated mutagenic outcomes.

Child mortality estimation incorporating summary birth history data.

The United Nations' Sustainable Development Goal 3.2 aims to reduce under-five child mortality to 25 deaths per 1000 live births by 2030. Child mortality tends to be concentrated in developing regions where information needed to assess achievement of this goal often comes from surveys and censuses. In both, women are asked about their birth histories, but with varying degrees of detail. Full birth history (FBH) data contain the reported dates of births and deaths of every surveyed mother's children. In contrast, summary birth history (SBH) data contain only the total number of children born and total number of children who died for each mother. Specialized methods are needed to accommodate this type of data into analyses of child mortality trends. We develop a data augmentation scheme within a Bayesian framework where for SBH data, birth and death dates are introduced as auxiliary variables. Since we specify a full probability model for the data, many of the well-known biases that exist in this data can be accommodated, along with space-time smoothing on the underlying mortality rates. We illustrate our approach in a simulation, showing robustness to model misspecification and that uncertainty is reduced when incorporating SBH data over simply analyzing all available FBH data. We also apply our approach to data from the Central region of Malawi and compare with the well-known Brass method.

The role of plasmalogens, Forssman lipids, and sphingolipid hydroxylation in modulating the biophysical properties of the epithelial plasma membrane.

A coarse-grain model of the epithelial plasma membrane was developed from high-resolution lipidomic data and simulated using the MARTINI force field to characterize its biophysical properties. Plasmalogen lipids, Forssman glycosphingolipids, and hydroxylated Forssman glycosphingolipids and sphingomyelin were systematically added to determine their structural effects. Plasmalogen lipids have a minimal effect on the overall biophysical properties of the epithelial plasma membrane. In line with the hypothesized role of Forssman lipids in the epithelial apical membrane, the introduction of Forssman lipids initiates the formation of glycosphingolipid-rich nanoscale lipid domains, which also include phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol (CHOL). This decreases the lateral diffusion in the extracellular leaflet, as well as the area per lipid of domain forming lipids, most notably PE. Finally, hydroxylation of the Forssman glycosphingolipids and sphingomyelin further modulates the lateral organization of the membrane. Through comparison to the previously studied average and neuronal plasma membranes, the impact of membrane lipid composition on membrane properties was characterized. Overall, this study furthers our understanding of the biophysical properties of complex membranes and the impact of lipid diversity in modulating membrane properties.

Uncovering a unique approach for damaged DNA replication: A computational investigation of a mutagenic tobacco-derived thymine lesion.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent nicotine carcinogen that leads to many DNA lesions, the most persistent being the O2-[4-oxo-4-(3-pyridyl)butyl]thymine adduct (POB-T). Although the experimental mutagenic profile for the minor groove POB-T lesion has been previously reported, the findings are puzzling in terms of the human polymerases involved. Specifically, while pol κ typically replicates minor groove adducts, in vivo studies indicate pol η replicates POB-T despite being known for processing major groove adducts. Our multiscale modeling approach reveals that the canonical (anti) glycosidic orientation of POB-T can fit in the pol κ active site, but only a unique (syn) POB-T conformation is accommodated by pol η. These distinct binding orientations rationalize the differential in vitro mutagenic spectra based on the preferential stabilization of dGTP and dTTP opposite the lesion for pol κ and η, respectively. Overall, by uncovering the first evidence for the replication of a damaged pyrimidine in the syn glycosidic orientation, the current work provides the insight necessary to clarify a discrepancy in the DNA replication literature, expand the biological role of the critical human pol η, and understand the mutational signature in human cancers associated with tobacco exposure.

Understanding the Link between Lipid Diversity and the Biophysical Properties of the Neuronal Plasma Membrane.

Cell membranes contain incredible diversity in the chemical structures of their individual lipid species and the ratios in which these lipids are combined to make membranes. Nevertheless, our current understanding of how each of these components affects the properties of the cell membrane remains elusive, in part due to the difficulties in studying the dynamics of membranes at high spatiotemporal resolution. In this work, we use coarse-grained molecular dynamics simulations to investigate how individual lipid species contribute to the biophysical properties of the neuronal plasma membrane. We progress through eight membranes of increasing chemical complexity, ranging from a simple POPC/CHOL membrane to a previously published neuronal plasma membrane [Ingólfsson, H. I., et al. (2017) Biophys. J. 113 (10), 2271-2280] containing 49 distinct lipid species. Our results show how subtle chemical changes can affect the properties of the membrane and highlight the lipid species that give the neuronal plasma membrane its unique biophysical properties. This work has potential far-reaching implications for furthering our understanding of cell membranes.

Oral NaHCO3 Activates a Splenic Anti-Inflammatory Pathway: Evidence That Cholinergic Signals Are Transmitted via Mesothelial Cells.

We tested the hypothesis that oral NaHCO3 intake stimulates splenic anti-inflammatory pathways. Following oral NaHCO3 loading, macrophage polarization was shifted from predominantly M1 (inflammatory) to M2 (regulatory) phenotypes, and FOXP3+CD4+ T-lymphocytes increased in the spleen, blood, and kidneys of rats. Similar anti-inflammatory changes in macrophage polarization were observed in the blood of human subjects following NaHCO3 ingestion. Surprisingly, we found that gentle manipulation to visualize the spleen at midline during surgical laparotomy (sham splenectomy) was sufficient to abolish the response in rats and resulted in hypertrophy/hyperplasia of the capsular mesothelial cells. Thin collagenous connections lined by mesothelial cells were found to connect to the capsular mesothelium. Mesothelial cells in these connections stained positive for the pan-neuronal marker PGP9.5 and acetylcholine esterase and contained many ultrastructural elements, which visually resembled neuronal structures. Both disruption of the fragile mesothelial connections or transection of the vagal nerves resulted in the loss of capsular mesothelial acetylcholine esterase staining and reduced splenic mass. Our data indicate that oral NaHCO3 activates a splenic anti-inflammatory pathway and provides evidence that the signals that mediate this response are transmitted to the spleen via a novel neuronal-like function of mesothelial cells.

Computational insights into the mutagenicity of two tobacco-derived carcinogenic DNA lesions.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent carcinogen found in all tobacco products that leads to a variety of DNA lesions in cells, including O6-[4-oxo-4-(3-pyridyl)butyl]guanine (POB-G) and O6-[4-hydroxy-4-(3-pyridyl)butyl]guanine (PHB-G), which differ by only a single substituent in the bulky moiety. This work uses a multiscale computational approach to shed light on the intrinsic conformational and base-pairing preferences of POB-G and PHB-G, and the corresponding properties in DNA and the polymerase η active site. Our calculations reveal that both lesions form stable pairs with C and T, with the T pairs being the least distorted relative to canonical DNA. This rationalizes the experimentally reported mutational profile for POB-G and validates our computational model. The same approach predicts that PHB-G is more mutagenic than POB-G due to a difference in the bulky moiety hydrogen-bonding pattern, which increases the stability of the PHB-G:T pair. The mutagenicity of PHB-G is likely further increased by stabilization of an intercalated DNA conformation that is associated with deletion mutations. This work thereby uncovers structural explanations for the reported mutagenicity of POB-G, provides the first clues regarding the mutagenicity of PHB-G and complements a growing body of literature highlighting that subtle chemical changes can affect the biological outcomes of DNA adducts.

DNA base sequence effects on bulky lesion-induced conformational heterogeneity during DNA replication.

4-Aminobiphenyl (ABP) and its structure analog 2-aminofluorene (AF) are well-known carcinogens. In the present work, an unusual sequence effect in the 5'-CTTCTG1G2TCCTCATTC-3' DNA duplex is reported for ABP- and AF-modified G. Specifically, the ABP modification at G1 resulted in a mixture of 67% major groove B-type (B) and 33% stacked (S) conformers, while at the ABP modification at G2 exclusively resulted in the B-conformer. The AF modification at G1 and G2 lead to 25%:75% and 83%:17% B:S population ratios, respectively. These differences in preferred conformation are due to an interplay between stabilizing (hydrogen bonding and stacking that is enhanced by lesion planarity) and destabilizing (solvent exposure) forces at the lesion site. Furthermore, while the B-conformer is a thermodynamic stabilizer and the S-conformer is a destabilizer in duplex settings, the situation is reversed at the single strands/double strands (ss/ds) junction. Specifically, the twisted biphenyl is a better stacker at the ss/ds junction than the coplanar AF. Therefore, the ABP modification leads to a stronger strand binding affinity of the ss/ds junction than the AF modification. Overall, the current work provides conformational insights into the role of sequence and lesion effects in modulating DNA replication.

Computational Insight into the Differential Mutagenic Patterns of O-Methylthymine Lesions.

Differential mutagenic patterns were recently reported for O-methylated thymine lesions, which indicate that O4-methylthymine (O4-Me-T) frequently leads to G misinsertions, whereas O2-methylthymine (O2-Me-T) is primarily nonmutagenic. The reasons for these differences are unclear since both lesions similarly alter the Watson-Crick binding face of T. To rationalize these replication outcomes at a molecular level, this work uses density functional theory calculations and molecular dynamics simulations to probe the lesion base-pairing properties as well as lesion accommodation by human polymerase η (pol η) and post-extension DNA duplexes. O4-Me-T forms two strong hydrogen bonds with an opposing G in the active site of pol η, which rationalizes the observed lesion mutagenicity. Nevertheless, dATP insertion opposite O4-Me-T can proceed through water-mediated hydrogen bonding, which is similar to the pathway previously proposed for pol η bypass of abasic sites and other T alkylation lesions. In contrast, the position of O2-Me-T in the pol η active site is dynamic due to the presence of the aberrant methyl group on the minor groove side of DNA. In fact, the experimental replication outcomes can only be rationalized when the syn glycosidic orientation of O2-Me-T is considered, which stabilizes the pre-insertion complex by placing the damage in the polymerase open pocket on the major groove side of DNA. Although dATP insertion can occur opposite syn-O2-Me-T through a water-mediated pathway similar to O4-Me-T replication, rotation about the glycosidic bond precludes a stable pol η ternary complex corresponding to dGTP insertion, which correlates with the reported nonmutagenic bypass of O2-Me-T. In addition to providing structural insights into the differential mutagenicity of methylated T adducts, our data highlight an emerging theme in the literature for the replication of pyrimidine alkylation products in noncanonical glycosidic orientations and sets the stage for future work on the replication of other alkylated lesions by TLS polymerases.

A basic solution to activate the cholinergic anti-inflammatory pathway via the mesothelium?

Much research now indicates that vagal nerve stimulation results in a systemic reduction in inflammatory cytokine production and an increase in anti-inflammatory cell populations that originates from the spleen. Termed the 'cholinergic anti-inflammatory pathway', therapeutic activation of this innate physiological response holds enormous promise for the treatment of inflammatory disease. Much controversy remains however, regarding the underlying physiological pathways mediating this response. This controversy is anchored in the fact that the vagal nerve itself does not innervate the spleen. Recent research from our own laboratory indicating that oral intake of sodium bicarbonate stimulates splenic anti-inflammatory pathways, and that this effect may require transmission of signals to the spleen through the mesothelium, provide new insight into the physiological pathways mediating the cholinergic anti-inflammatory pathway. In this review, we examine proposed models of the cholinergic anti-inflammatory pathway and attempt to frame our recent results in relation to these hypotheses. Following this discussion, we then provide an alternative model of the cholinergic anti-inflammatory pathway which is consistent both with our recent findings and the published literature. We then discuss experimental approaches that may be useful to delineate these hypotheses. We believe the outcome of these experiments will be critical in identifying the most appropriate methods to harness the therapeutic potential of the cholinergic anti-inflammatory pathway for the treatment of disease and may also shed light on the etiology of other pathologies, such as idiopathic fibrosis.