Intervention to severe lower trachea obstruction supported by extracorporeal membrane oxygenation in a human immunodeficiency virus patient: A case report and literature review.

Here we reported a case, male, 33 years old, diagnosed with human immunodeficiency virus (HIV) infection 5 months ago, but he didn't take antiretroviral drugs regularly. He was admitted to intensive care unit emergently due to hypoxemia, hypercapnia, and hypotension. CT showed severe lower trachea obstruction caused by soft tissue. After rapid bedside assessment, the patient was considered to need endotracheal operation, but he couldn't tolerate intubation and mechanical ventilation. Extracorporeal membrane oxygenation (ECMO) was used. Hemodynamics improved significantly along with rehydration and low-dose vasoactive drugs. Subsequently, the patient underwent rigid bronchoscopy, airway tumor resection and Y-type silicone stent implantation. Postoperatively protective endotracheal intubation and mechanical ventilation was followed. ECMO was weaned off after the operation, and endotracheal cannula was removed 6 h later. The pathological examination of excisional tissue showed lung squamous cell carcinoma. Finally, the patient was discharged safely and went to local hospital for further treatment. From this case, we conclude that ECMO could play a key role for those who need endotracheal surgery while cannot endure conventional intubation and mechanical ventilation.

[Clinical and imaging features of acute histoplasmosis].

Objective: To investigate the clinical and imaging characteristics of acute histoplasmosis. Methods: The clinical and imaging data of 10 patients with acute histoplasmosis were studied. Their clinical and imaging characteristics were analyzed. All the patients returned from a South American republic in April 2019 and were treated at the Chongqing public health medical treatment center. Results: All the 10 patients were male, aged 30-56 years old, with an average age of 43.8 years old. Four of them were engaged in soil clearing, 2 in gas cutting, 2 in moving tools, and 2 in inspection. The disease in all the 10 patients was caused by inhaling a large amount of bacteria-bearing dust in a short time, with an incubation period of 9-13 days, and the main clinical manifestations were fever, insomnia, dizziness, headache, cough, poor appetite, rash and diarrhea. One patient's head CT showed extensive thickening and increased density of bilateral frontotemporal, parietal and occipital meninges, while the other 9 patients showed no obvious abnormalities. Chest CT findings were as follows: (1) Multiple nodular shadow: the chest CT findings of 4 patients were miliary nodular shadow with diffuse distribution in both lungs. Most of the nodules were less than 5 mm in diameter and distributed evenly or unevenly. CT findings of 6 cases showed scattered nodular shadows in both lungs, with diameters ranging from 2 to 15 mm, and obvious distribution in subpleural and inferior lobes of both lungs. (2) Consolidation shadow: in 2 cases, the size of the shadow was uneven and the density increased, mainly distributed in the subpleura and the lower lobe of both lungs. (3) Ground glass density shadow: mainly distributed around nodules, halo signs can be seen around some nodules. (4) Mediastinum and/or hilar lymph nodes were enlarged. (5) Pleural effusion: a small amount of pleural effusion was found in 4 cases. (6) Pericardial effusion in 3 cases. Abdominal CT showed splenomegaly in 8 cases and hepatomegaly in 1 case. Conclusions: Acute histoplasmosis has no specificity in clinical manifestations. However, there are still some features in CT manifestations, including multiple nodules in both lungs accompanied by halo, enlarged liver, spleen and mediastinal lymph nodes, and multiple serous cavity effusions.

Influence of lymph node dissection in patients undergoing radical nephrectomy for non-metastatic renal cell carcinoma: a systematic review and meta-analysis.

OBJECTIVE: Whether lymph node dissection (LND) should be performed concomitantly with radical nephrectomy (RN) for non-metastatic renal carcinoma has still been controversial recently. We conducted a meta-analysis assessing oncologic outcomes of radical nephrectomy with lymph node dissection (LND) and without lymph node dissection (non-LND) in non-metastatic renal cell carcinoma (NMRCC). PATIENTS AND METHODS: A systematic review was performed until April 2018 using a comprehensive search in PubMed, EMBASE, and Cochrane Library databases to identify eligible comparative studies. A formal meta-analysis was performed for studies comparing radical nephrectomy with LND and radical nephrectomy with non-LND for cT1-T4NxM0 tumors. Furthermore, a subgroup analysis for locally advanced renal cell carcinoma (cT3-T4NxM0) was conducted. RESULTS: Thirteen studies on patients with LND and non- LND were identified and included in the analysis. LND group did not have a significantly better survival than non-LND group for cT1-T4NxM0 tumors (HR 0.93, 95% CI 0.78-1.11, p=0.45), However, in the subgroup of locally advanced renal cell carcinoma (cT3-T4NxM0), it showed a significantly better OS rate in patients who had undergone LND compared to those without LND (HR 0.73, 95% CI 0.60-0.90; p=0.003). CONCLUSIONS: LND offers better cancer control and better long-term survival in locally advanced renal cell carcinomas (cT3-T4NxM0). This conclusion should be confirmed by a prospective randomized clinical trial.

Epigenetic silencing of microRNA-137 enhances ASCT2 expression and tumor glutamine metabolism.

Tumor cells must activate specific transporters to meet their increased glutamine metabolic demands. Relative to other glutamine transporters, the ASC family transporter 2 (ASCT2, also called SLC1A5) is profoundly elevated in a wide spectrum of human cancers to coordinate metabolic reprogramming and malignant transformation. Understanding the molecular mechanisms whereby tumor cells frequently upregulate this transporter is therefore vital to develop potential strategies for transporter-targeted therapies. Combining in-silico algorithms with systemic experimental screening, we herein identify the tumor suppressor microRNA, miR-137, as an essential regulator that targets ASCT2 and cancer cell glutamine metabolism. Metabolic analysis shows that miR-137 derepression, similar to ASCT2 inactivation, significantly inhibits glutamine consumption and TCA cycle anaplerosis. Mechanistically, methyl-CpG-binding protein 2 (MeCP2) and DNA methyltransferases (DNMTs) cooperate to promote active methylation of the miR-137 promoter and inhibit its transcription, conversely reactivating ASCT2 expression and glutamine metabolism. Moreover, expression between miR-137 and ASCT2 is inversely correlated in tumor specimens from multiple cancer types, and ectopic ASCT2 expression markedly rescued miR-137 suppression of tumorigenesis. These findings thus elucidate a previously unreported mechanism responsible for ASCT2 deregulation in human cancers and identify ASCT2 as a critical downstream effector of miR-137, revealing a molecular link between DNA methylation, microRNA and tumor metabolism.

Effect of polarization on HIV-1protease and fluoro-substituted inhibitors binding energies by large scale molecular dynamics simulations.

Molecular dynamics simulations in explicit water are carried out to study the binding of six inhibitors to HIV-1 protease (PR) for up to 700 ns using the standard AMBER force field and polarized protein-specific charge (PPC). PPC is derived from quantum mechanical calculation for protein in solution and therefore it includes electronic polarization effect. Our results show that in all six systems, the bridging water W301 drifts away from the binding pocket in AMBER simulation. However, it is very stable in all six complexes systems using PPC. Especially, intra-protease, protease-inhibitor hydrogen bonds are dynamic stabilized in MD simulation. The computed binding free energies of six complexes have a significantly linear correlation with those experiment values and the correlation coefficient is found to be 0.91 in PPC simulation. However, the result from AMBER simulation shows a weaker correlation with the correlation coefficient of -0.51 due to the lack of polarization effect. Detailed binding interactions of W301, inhibitors with PR are further analyzed and discussed. The present study provides important information to quantitative understanding the interaction mechanism of PR-inhibitor and PR-W301 and these data also emphasizes the importance of both the electronic polarization and the bridging water molecule in predicting precisely binding affinities.

Correlation between three glycometabolic-related hormones and muscle glycolysis, as well as meat quality, in three pig breeds.

BACKGROUND: The present study aimed to evaluate the correlations among muscle concentrations of three glycometabolic-related hormones (insulin, epinephrine and glucagon), muscle glycolysis and meat quality in representative muscles of either glycolytic or oxidative types. Moreover, the relative glycometabolic-related gene expression was measured. One Western crossbreed DLY (Duroc × (Landrace × Yorkshire)), one crossbreed with half-Chinese native-pig origin DL (Duroc × LiangShan) and one pure Chinese native pig TP (Tibetan pig) were used in the present study. RESULTS: Among the three breeds, DLY had the greatest glucagon and epinephrine (P < 0.01). Compared with DLY, TP and DL had lower lactic acid concentrations, showing lower glycolytic potentials (GP), greater ultimate pH values (P < 0.01) and lower relative expression levels of glycometabolic-related genes (GYS1, PRKAG3 and PKM2). Compared with the glycolytic muscle (musculus longissimus dorsi), oxidative muscle PM (musculus psoas major) had lower glucagon and epinephrine contents, lower GP and better meat quality. The concentration of glycometabolic-related hormones in the muscle had significant correlations with muscle glycolysis, meat pH and lightness. CONCLUSION: The results obtained in the present study imply that glucagon and epinephrine levels could be used to indicate early glycolytic metabolism during postmortem. These findings may be helpful in identifying pork with undesirable quality traits. 2016 Society of Chemical Industry.

DEPTOR is a direct NOTCH1 target that promotes cell proliferation and survival in T-cell leukemia.

Aberrant activation of NOTCH1 signaling plays a vital role in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Yet the molecular events downstream of NOTCH1 that drive T-cell leukemogenesis remain incompletely understood. Starting from genome-wide gene-expression profiling to seek important NOTCH1 transcriptional targets, we identified DEP-domain containing mTOR-interacting protein (DEPTOR), which was previously shown to be important in multiple myeloma but remains functionally unclear in other hematological malignancies. Mechanistically, we demonstrated NOTCH1 directly bound to and activated the human DEPTOR promoter in T-ALL cells. DEPTOR depletion abolished cellular proliferation, attenuated glycolytic metabolism and enhanced cell death, while ectopically expressed DEPTOR significantly promoted cell growth and glycolysis. We further showed that DEPTOR depletion inhibited while its overexpression enhanced AKT activation in T-ALL cells. Importantly, AKT inhibition completely abrogated DEPTOR-mediated cell growth advantages. Moreover, DEPTOR depletion in a human T-ALL xenograft model significantly delayed T-ALL onset and caused a substantial decrease of AKT activation in leukemic blasts. We thus reveal a novel mechanism involved in NOTCH1-driven leukemogenesis, identifying the transcriptional control of DEPTOR and its regulation of AKT as additional key elements of the leukemogenic program activated by NOTCH1.

Large-scale molecular dynamics simulation: Effect of polarization on thrombin-ligand binding energy.

Molecular dynamics (MD) simulations lasting 500 ns were performed in explicit water to investigate the effect of polarization on the binding of ligands to human α-thrombin based on the standard nonpolarizable AMBER force field and the quantum-derived polarized protein-specific charge (PPC). The PPC includes the electronic polarization effect of the thrombin-ligand complex, which is absent in the standard force field. A detailed analysis and comparison of the results of the MD simulation with experimental data provided strong evidence that intra-protein, protein-ligand hydrogen bonds and the root-mean-square deviation of backbone atoms were significantly stabilized through electronic polarization. Specifically, two critical hydrogen bonds between thrombin and the ligand were broken at approximately 190 ns when AMBER force field was used and the number of intra-protein backbone hydrogen bonds was higher under PPC than under AMBER. The thrombin-ligand binding energy was computed using the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method, and the results were consistent with the experimental value obtained using PPC. Because hydrogen bonds were unstable, it was failed to predict the binding affinity under the AMBER force field. Furthermore, the results of the present study revealed that differences in the binding free energy between AMBER and PPC almost comes from the electrostatic interaction. Thus, this study provides evidence that protein polarization is critical to accurately describe protein-ligand binding.

Metabolic targeting of oncogene MYC by selective activation of the proton-coupled monocarboxylate family of transporters.

Deregulation of the MYC oncogene produces Myc protein that regulates multiple aspects of cancer cell metabolism, contributing to the acquisition of building blocks essential for cancer cell growth and proliferation. Therefore, disabling Myc function represents an attractive therapeutic option for cancer treatment. However, pharmacological strategies capable of directly targeting Myc remain elusive. Here, we identified that 3-bromopyruvate (3-BrPA), a drug candidate that primarily inhibits glycolysis, preferentially induced massive cell death in human cancer cells overexpressing the MYC oncogene, in vitro and in vivo, without appreciable effects on those exhibiting low MYC levels. Importantly, pharmacological inhibition of glutamine metabolism synergistically potentiated the synthetic lethal targeting of MYC by 3-BrPA due in part to the metabolic disturbance caused by this combination. Mechanistically, we identified that the proton-coupled monocarboxylate transporter 1 (MCT1) and MCT2, which enable efficient 3-BrPA uptake by cancer cells, were selectively activated by Myc. Two regulatory mechanisms were involved: first, Myc directly activated MCT1 and MCT2 transcription by binding to specific recognition sites of both genes; second, Myc transcriptionally repressed miR29a and miR29c, resulting in enhanced expression of their target protein MCT1. Of note, expressions of MCT1 and MCT2 were each significantly elevated in MYCN-amplified neuroblastomas and C-MYC-overexpressing lymphomas than in tumors without MYC overexpression, correlating with poor prognosis and unfavorable patient survival. These results identify a novel mechanism by which Myc sensitizes cells to metabolic inhibitors and validate 3-BrPA as potential Myc-selective cancer therapeutics.

Electronic polarization stabilizes tertiary structure prediction of HP-36.

Molecular dynamic (MD) simulations with both implicit and explicit solvent models have been carried out to study the folding dynamics of HP-36 protein. Starting from the extended conformation, the secondary structure of all three helices in HP-36 was formed in about 50 ns and remained stable in the remaining simulation. However, the formation of the tertiary structure was difficult. Although some intermediates were close to the native structure, the overall conformation was not stable. Further analysis revealed that the large structure fluctuation of loop and hydrophobic core regions was devoted mostly to the instability of the structure during MD simulation. The backbone root-mean-square deviation (RMSD) of the loop and hydrophobic core regions showed strong correlation with the backbone RMSD of the whole protein. The free energy landscape indicated that the distribution of main chain torsions in loop and turn regions was far away from the native state. Starting from an intermediate structure extracted from the initial AMBER simulation, HP-36 was found to generally fold to the native state under the dynamically adjusted polarized protein-specific charge (DPPC) simulation, while the peptide did not fold into the native structure when AMBER force filed was used. The two best folded structures were extracted and taken into further simulations in water employing AMBER03 charge and DPPC for 25 ns. Result showed that introducing polarization effect into interacting potential could stabilize the near-native protein structure.

An implementation of hydrophobic force in implicit solvent molecular dynamics simulation for packed proteins.

MD simulations of five proteins in which helical chains are held together by hydrophobic packing were carried out to investigate the effect of hydrophobic force on simulated structures of these protein complexes in implicit generalized Born (GB) model. The simulation study employed three different methods to treat hydrophobic effect: the standard GB method that does not include explicit hydrophobic force, the LCPO method that includes explicit hydrophobic force based directly on solvent accessible surface area (SASA), and a proposed packing enforced GB (PEGB) method that includes explicit hydrophobic force based on the radius of gyration of the protein complex. Our simulation study showed that all five protein complexes were unpacked in the standard GB simulation (without explicit hydrophobic force). In the LCPO method, three of the five protein systems remained well packed during the simulation, indicating the need for an explicit hydrophobic force in GB model for these packed protein systems. However, two of the five systems were still unpacked during LCPO simulation. For comparison, all five protein systems remain well packed in simulation using the new PEGB method. Analysis shows that the failure of the LCPO method in two cases is related to the way that SASA changes during the unpacking process for these two systems. These examples showed that standard GB method without explicit hydrophobic force is not suitable for MD simulation of protein systems involving hydrophobic packing. A similar problem remains but to a much lesser extent in the LCPO method for some packed protein systems. The proposed PEGB method seems quite promising for MD simulation of large, multi-domain packed proteins in implicit solvent model.

Targeting IL-23 by employing a p40 peptide-based vaccine ameliorates murine allergic skin and airway inflammation.

BACKGROUND: Studies have found that the IL-23/Th17 pathway plays an important role in the pathogenesis of atopic dermatitis (AD) and severe and steroid-resistant asthma. Targeting IL-23/Th17 pathway with monoclonal antibodies (mAb) has been successful in the reduction of skin and airway inflammation in animal models. However, the mAb has a short half-life, requiring repeated administrations. For the long-term suppression of IL-23/Th17 pathway, we have previously developed an IL-23p40 peptide-based virus-like particle vaccine, which induces long-lasting autoantibodies to IL-23. OBJECTIVE: We sought to evaluate the effects of this IL-23p40 peptide-based vaccine on the down-regulation of allergic skin and airway inflammation in mice. METHODS: Mice were subcutaneously injected three times with the IL-23p40 vaccine, or the vaccine carrier protein or saline as controls. Two weeks later, mice were epicutaneously sensitized with ovalbumin four times at a 2-week interval. One week after the final sensitization, mice were nasally administrated with ovalbumin daily for 3 days. One day later, bronchoalveolar lavage fluids (BALF), sera, lung and skin tissues were obtained and analysed. RESULTS: Mice immunized with the vaccine produced high levels of IgG antibodies to IL-23, p40 and IL-12 that in vitro inhibited IL-23-dependent IL-17 production. The numbers of total cells, neutrophils, and eosinophils in BALF were significantly reduced in the vaccine group, compared with controls. The levels of IL-13, IL-5, IL-23 and, IL-17 in BALF and levels of serum ovalbumin-specific IgE, IgG1, and total IgE were also significantly decreased. Histological analysis showed less inflammation of the lung and skin tissues in the vaccine group, compared with controls. CONCLUSION AND CLINICAL RELEVANCE: Administration of an IL-23p40 peptide-based vaccine down-regulates allergic skin and airway inflammation, suggesting that this strategy may be a potential therapeutic approach in the treatment of AD and asthma.

Folding of a helix is critically stabilized by polarization of backbone hydrogen bonds: study in explicit water.

Multiple single-trajectory molecular dynamics (MD) simulation at room temperature (300 K) in explicit water was carried out to study the folding dynamics of an α-helix (PDB 2I9M ) using a polarized charge scheme that includes electronic polarization of backbone hydrogen bonds. Starting from an extended conformation, the 17-residue peptide was successfully folded into the native structure (α-helix) between 80 and 130 ns with a root-mean-square deviation of ~1.0 Å. Analysis of the time-dependent trajectories revealed that helix formation of the peptide started at the terminals and progressed toward the center of the peptide. For comparison, MD trajectories generated under various versions of standard AMBER force fields failed to show any significant or stable helix formation in our simulation. Our result shows clear evidence that the electronic polarization of backbone hydrogen bonds energetically stabilizes the helix formation and is critical to the stable folding of the short helix structure.

Folding of a helix at room temperature is critically aided by electrostatic polarization of intraprotein hydrogen bonds.

We report direct folding of a 17-residue helix protein (pdb:2I9M) by standard molecular dynamics simulation (single trajectory) at room temperature with implicit solvent. Starting from a fully extended structure, 2I9M successfully folds into the native conformation within 16 ns using adaptive hydrogen bond-specific charges to take into account the electrostatic polarization effect. Cluster analysis shows that conformations in the native state cluster have the highest population (78.4%) among all sampled conformations. Folding snapshots and the secondary structure analysis demonstrate that the folding of 2I9M begins at terminals and progresses toward the center. A plot of the free energy landscape indicates that there is no significant free energy barrier during folding, which explains the observed fast folding speed. For comparison, exactly the same molecular dynamics simulation but carried out under existing AMBER charges failed to fold 2I9M into native-like structures. The current study demonstrates that electrostatic polarization of intraprotein hydrogen bonding, which stabilizes the helix, is critical to the successful folding of 2I9m.

Intra-protein hydrogen bonding is dynamically stabilized by electronic polarization.

Molecular dynamics (MD) simulation has been carried out to study dynamical stability of intra-protein hydrogen bonds based on two set of atomic charges, the standard AMBER charge and the polarized protein-specific charge (PPC). The latter is derived from quantum mechanical calculation for protein in solution using a recently developed molecular fractionation with conjugate caps-Poisson-Boltzmann (MFCC-PB) approach and therefore includes electronic polarization effect of the protein at native structure. MD simulations are performed for a number of benchmark proteins containing helix and/or beta sheet secondary structures. The computational result shows that occupancy percentage of hydrogen bonds averaged over simulation time, as well as the number of hydrogen bonds as a function of simulation time, is consistently higher under PPC than AMBER charge. In particular, some intra-protein hydrogen bonds are found broken during MD simulation using AMBER charge but they are stable using PPC. The breaking of some intra-protein hydrogen bonds in AMBER simulation is responsible for deformation or denaturing of some local structures of proteins during MD simulation. The current study provides strong evidence that hydrogen bonding is dynamically more stable using PPC than AMBER charge, highlighting the stabilizing effect of electronic polarization on protein structure.

Clinical characteristics of pigment dispersion syndrome in Chinese patients.

PURPOSE: To report clinical findings and characteristics of pigment dispersion syndrome (PDS) in Chinese patients. METHODS: PDS suspects with any one of the following signs: corneal endothelial pigmentation, iris transillumination defects (ITDs), pigment granule dusting on anterior iris surface, posterior iris bowing, trabecular meshwork (TM) pigmentation, and lenticular or zonular pigmentation were evaluated for PDS at the glaucoma specialty clinic at Beijing Tongren Eye Centre. Diagnosis of PDS required at least two of the following signs: Krukenberg spindle, moderate-to-heavy TM pigmentation (>or=Scheie II) and any degree of lenticular and/or zonular pigmentation. RESULTS: Eighteen patients (12 males and six females) were identified as having PDS during a 1-year period, with mean age of 35.5+/-7.0 years (range, 22-49). All but two eyes from two patients had myopia of -0.5 D or greater, with mean spherical equivalent power of -5.20+/-5.80 D (range, -24.75+/-0.5). The average IOP at initial diagnosis was 33.7+/-10.5 mm Hg (range, 16-56). Fifteen patients (83.3%) were found to have pigmentary glaucoma at their initial diagnosis. All patients showed homogenous increased TM pigmentation as well as lenticular and/or zonular pigmentation. 61.1% of patients (11 of 18) had Krukenberg spindle. None of the patients exhibited spoke-like midperipheral ITDs except for trace-isolated transillumination in both eyes of the two patients. CONCLUSIONS: The most common clinical findings in Chinese PDS patients include homogeneous TM pigmentation and pigment granule dusting on lens zonules and/or posterior peripheral lens surface. ITDs are uncommon in Chinese patients with PDS.

Quantum study of HIV-1 protease-bridge water interaction.

We present a fully quantum mechanical calculation for binding interaction between HIV-1 protease (PR) and the water molecule W301 which bridges the flaps of the protease with the inhibitors of PR. The quantum calculation is made possible by applying a recently developed molecular fractionation with conjugate caps (MFCC) method which divides a protein molecule into capped amino acid-based fragments and their conjugate caps. These individual fragments are properly treated to preserve the chemical property of bonds that are cut. Ab initio methods at HF, B3LYP, and MP2 levels with a fixed basis set 6-31+G* have been employed in the present calculation. The MFCC calculation produces a quantum mechanical interaction "map" representing interactions between individual residues of PR and W301. This enables a detailed quantitative analysis on binding of W301 to specific residues of PR at quantum mechanical level.

Transient lower esophageal sphincter relaxations and reflux: mechanistic analysis using concurrent fluoroscopy and high-resolution manometry.

BACKGROUND & AIMS: The aim of this study was to perform a detailed analysis of the mechanics leading to esophagogastric junction (EGJ) opening during transient lower esophageal sphincter relaxations (tLESRs) using high-resolution manometry coupled with simultaneous fluoroscopy. METHODS: Six subjects without hiatus hernia had endoclips placed at the squamocolumnar junction and 10 cm proximal. A 36-channel solid-state manometric assembly was placed spanning from stomach to pharynx, and subjects were studied for 2 hours after a high-fat meal. An esophageal pH electrode also was placed and fluoroscopy was initiated at the onset of a tLESR. Axial clip movement was measured during replay of the videotaped fluoroscopy and was correlated with manometric data. RESULTS: Ninety-three tLESRs were recorded, 62 tLESRs of which had good fluoroscopic visualization. Seventy-eight tLESRs had manometric evidence of flow and the majority had evidence of a common cavity (88%), but few were detected by the pH electrode. Esophageal shortening and crural diaphragm inhibition always preceded EGJ opening and common cavity. A positive pressure gradient between the stomach and the EGJ lumen of 7.1 mm Hg (interquartile range, 4.1-9.1 mm Hg) preceded the EGJ opening. CONCLUSIONS: Key events leading to the EGJ opening during tLESRs were LES relaxation, crural diaphragm inhibition, esophageal shortening, and a positive pressure gradient between the stomach and the EGJ lumen. The manometric signature of opening was pressure equalization within the EGJ, but this only occasionally was associated with pH evidence of reflux. Future investigations will need to analyze how this delicately balanced anatomic-physiologic system is perturbed in subjects with reflux disease.