Use of an oral stable isotope label to confirm variation in red blood cell mean age that influences HbA1c interpretation.

HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC ) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral (15) N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15-20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R(2) = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c.

The effect of repeated microwave irradiation on the dimensional stability of a specific acrylic denture resin.

PURPOSE: To determine the dimensional stability of a poly(methyl methacrylate) (PMMA) acrylic resin when subjected to multiple sessions of repeated microwave irradiation at power settings of 700 and 420 W. MATERIALS AND METHODS: Twenty standardized denture bases were fabricated using a PMMA resin. Points of measurement were marked on each denture base with a standardized template, and the distances between points were recorded using a digital microscope. The denture bases were randomly placed into two experimental groups of 10 bases each. Individual denture bases were placed into a glass beaker containing 200 ml of room temperature deionized water and then exposed to either 700 or 420 W of microwave radiation for 3 minutes. The denture bases were allowed to cool to room temperature, and measurements between points were recorded. This process was carried out for two microwave periods with measurements being completed after each period. The data were then analyzed for any significant changes in distances between points using a Student's t-test. RESULTS: All denture bases experienced 1.0 to 2.0 mm or approximately 3% linear dimensional change after each period of microwaving. Results were significant with all t-tests having values of p < 0.05. CONCLUSION: This report showed that the denture bases deformed significantly under experimental conditions at either 700 W for 3 minutes in 200 ml of water or 420 W for 3 minutes in 200 ml of water.

Quantitative liver function tests improve the prediction of clinical outcomes in chronic hepatitis C: results from the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis Trial.

UNLABELLED: Risk for future clinical outcomes is proportional to the severity of liver disease in patients with chronic hepatitis C virus (HCV). We measured disease severity by quantitative liver function tests (QLFTs) to determine cutoffs for QLFTs that identified patients who were at low and high risk for a clinical outcome. Two hundred and twenty-seven participants in the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis (HALT-C) Trial underwent baseline QLFTs and were followed for a median of 5.5 years for clinical outcomes. QLFTs were repeated in 196 patients at month 24 and in 165 patients at month 48. Caffeine elimination rate (k(elim)), antipyrine (AP) clearance (Cl), MEGX concentration, methionine breath test (MBT), galactose elimination capacity (GEC), dual cholate (CA) clearances and shunt, perfused hepatic mass (PHM), and liver and spleen volumes (by single-photon emission computed tomography) were measured. Baseline QLFTs were significantly worse (P = 0.0017 to P < 0.0001) and spleen volumes were larger (P < 0.0001) in the 54 patients who subsequently experienced clinical outcomes. QLFT cutoffs that characterized patients as "low" and "high risk" for clinical outcome yielded hazard ratios ranging from 2.21 (95% confidence interval [CI]: 1.29-3.78) for GEC to 6.52 (95% CI: 3.63-11.71) for CA clearance after oral administration (Cl(oral)). QLFTs independently predicted outcome in models with Ishak fibrosis score, platelet count, and standard laboratory tests. In serial studies, patients with high-risk results for CA Cl(oral) or PHM had a nearly 15-fold increase in risk for clinical outcome. Less than 5% of patients with "low risk" QLFTs experienced a clinical outcome. CONCLUSION: QLFTs independently predict risk for future clinical outcomes. By improving risk assessment, QLFTs could enhance the noninvasive monitoring, counseling, and management of patients with chronic HCV.

Comparison of Metabolic Response to Colonic Fermentation in Lean Youth vs Youth With Obesity.

Importance: Pediatric obesity is a growing health care burden. Understanding how the metabolic phenotype of youth with obesity may modify the effect of intestinal fermentation on human metabolism is key to designing early intervention. Objective: To assess whether adiposity and insulin resistance in youth may be associated with colonic fermentation of dietary fibers and its production of acetate, gut-derived hormone secretion, and adipose tissue lipolysis. Design, Setting, and Participants: Cross-sectional study of youths aged 15 to 22 years with body mass index in the 25th to 75th percentile or higher than the 85th percentile for age and sex throughout the New Haven County community in Connecticut. Recruitment, studies, and data collection occurred from June 2018 to September 2021. Youths were assigned to a lean, obese insulin sensitive (OIS), or obese insulin resistant (OIR) group. Data were analyzed from April 2022 to September 2022. Exposure: Participants consumed 20 g of lactulose during a continuous 10-hour sodium d3-acetate intravenous infusion to measure the rate of appearance of acetate in plasma. Main Outcomes and Measures: Plasma was obtained hourly to measure acetate turnover, peptide tyrosine tyrosine (PYY), ghrelin, active glucagon-like peptide 1 (GLP-1), and free fatty acids (FFA). Results: A total of 44 youths participated in the study (median [IQR] age, 17.5 [16.0-19.3] years; 25 [56.8%] were female; 23 [52.3%] were White). Consequent to lactulose ingestion, there was a reduction of plasma FFA, an improvement of adipose tissue insulin sensitivity index, an increase in colonic acetate synthesis, and an anorexigenic response characterized by an increased plasma concentration of PYY and active GLP-1 and a reduction of ghrelin in the subgroups. Compared with the lean and OIS groups, the OIR group showed a less marked median (IQR) rate of acetate appearance (OIR: 2.00 [-0.86 to 2.69] µmol × kg-1 × min-1; lean: 5.69 [3.04 to 9.77] µmol × kg-1 × min-1; lean vs OIR P = .004; OIS: 2.63 [1.22 to 4.52] µmol × kg-1 × min-1; OIS vs OIR P = .09), a blunted median (IQR) improvement of adipose insulin sensitivity index (OIR: 0.043 [ 0.006 to 0.155]; lean: 0.277 [0.220 to 0.446]; lean vs OIR P = .002; OIS: 0.340 [0.048 to 0.491]; OIS vs OIR P = .08), and a reduced median (IQR) PYY response (OIR: 25.4 [14.8 to 36.4] pg/mL; lean: 51.3 [31.6 to 83.3] pg/mL; lean vs OIR P = .002; OIS: 54.3 [39.3 to 77.2] pg/mL; OIS vs OIR P = .011). Conclusions and Relevance: In this cross-sectional study, lean, OIS, and OIR youth demonstrated different associations between colonic fermentation of indigestible dietary carbohydrates and the metabolic response, with OIR youth showing minimal metabolic modifications as compared with the other 2 groups. Trial Registration: ClinicalTrials.gov Identifier: NCT03454828.

A state-dependent salt-bridge interaction exists across the ß/α intersubunit interface of the GABAA receptor.

The GABA(A) receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (ß(2)Asp163 and α(1)Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC(50-GABA), decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that ß(2)Asp163 and α(1)Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.

Three arginines in the GABAA receptor binding pocket have distinct roles in the formation and stability of agonist- versus antagonist-bound complexes.

Binding of the agonist GABA to the GABA(A) receptor causes channel gating, whereas competitive antagonists that bind at the same site do not. The details of ligand binding are not well understood, including which residues interact directly with ligands, maintain the structure of the binding pocket, or transduce the action of binding into opening of the ion channel gate. Recent work suggests that the amine group of the GABA molecule may form a cation-π bond with residues in a highly conserved "aromatic box" within the binding pocket. Although interactions with the carboxyl group of GABA remain unknown, three positively charged arginines (α(1)Arg67, α(1)Arg132, and ß(2)Arg207) just outside of the aromatic box are likely candidates. To explore their roles in ligand binding, we individually mutated these arginines to alanine and measured the effects on microscopic ligand binding/unbinding rates and channel gating. The mutations α(1)R67A or ß(2)R207A slowed agonist binding and sped unbinding with little effect on gating, demonstrating that these arginines are critical for both formation and stability of the agonist-bound complex. In addition, α(1)R67A sped binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide (SR-95531), indicating that this arginine poses a barrier to formation of the antagonist-bound complex. In contrast, ß(2)R207A and α(1)R132A sped antagonist unbinding, indicating that these arginines stabilize the antagonist-bound state. α(1)R132A also conferred a new long-lived open state, indicating that this arginine influences the channel gate. Thus, each of these arginines plays a unique role in determining interactions with agonists versus antagonists and with the channel gate.

A tight coupling between ß2Y97 and ß2F200 of the GABA(A) receptor mediates GABA binding.

The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/ß interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α1R67, α1R120, α1R132, and ß2R207) and two aromatic residues (ß2Y97 and ß2F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α1R67 nor α1R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α1R132 and both aromatic residues. Significant functional coupling between ß2R207 and both ß2Y97 and ß2F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, ß2Y97 and ß2F200, and for the first time provided direct evidence for the involvement of ß2Y97 and ß2F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

An epilepsy-related region in the GABA(A) receptor mediates long-distance effects on GABA and benzodiazepine binding sites.

The GABA(A) receptor mutation gamma(2)R43Q causes absence epilepsy in humans. Homology modeling suggests that gamma(2)Arg43, gamma(2)Glu178, and beta(2)Arg117 participate in a salt-bridge network linking the gamma(2) and beta(2) subunits. Here we show that several mutations at these locations exert similar long-distance effects on other intersubunit interfaces involved in GABA and benzodiazepine binding. These mutations alter GABA-evoked receptor kinetics by slowing deactivation, enhancing desensitization, or both. Kinetic modeling and nonstationary noise analysis for gamma(2)R43Q reveal that these effects are due to slowed GABA unbinding and slowed recovery from desensitization. Both gamma(2)R43Q and beta(2)R117K also speed diazepam dissociation from the receptor's benzodiazepine binding interface, as assayed by the rate of decay of diazepam-induced potentiation of GABA-evoked currents. These data demonstrate that gamma(2)Arg43 and beta(2)Arg117 similarly regulate the stability of both the GABA and benzodiazepine binding sites at the distant beta/alpha and alpha/gamma intersubunit interfaces, respectively. A simple explanation for these results is that gamma(2)Arg43 and beta(2)Arg117 participate in interactions between the gamma(2) and beta(2) subunits, disruptions of which alter the neighboring intersubunit binding sites in a similar fashion. In addition, gamma(2)Arg43 and gamma(2)Glu178 regulate desensitization, probably mediated within the transmembrane domains near the pore. Therefore, mutations at the gamma/beta intersubunit interface have specific long-distance effects that are propagated widely throughout the GABA(A) receptor protein.

A super-agonist of growth hormone-releasing hormone causes rapid improvement of nutritional status in patients with chronic kidney disease.

Chronic kidney disease is frequently associated with protein-energy wasting related to chronic inflammation and a resistance to anabolic hormones such as insulin and growth hormone (GH). In this study, we determined whether a new GH-releasing hormone super-agonist (AKL-0707) improved the anabolism and nutritional status of nondialyzed patients with stage 4-5 chronic kidney disease randomized to twice daily injections of the super-agonist or placebo. After 28 days, this treatment significantly increased 24-h GH secretion by almost 400%, without altering the frequency or rhythmicity of secretory bursts or fractional pulsatile GH release, and doubled the serum insulin-like growth factor-1 level. There was a significant change in the Subjective Global Assessment from 'mildly to moderately malnourished' to 'well-nourished' in 6 of 9 patients receiving AKL-0707 but in none of 10 placebo-treated patients. By dual-energy X-ray absorptiometry, both the mean fat-free mass and the body mineral content increased, but fat mass decreased, all significantly. In the AKL-0707-treated group, both serum urea and normalized protein equivalent of nitrogen appearance significantly decreased with no change in dietary protein intake, indicating a protein anabolic effect of treatment. Thus, our study shows that stimulation of endogenous GH secretion by AKL-0707 overcomes uremic catabolism of patients with advanced chronic kidney disease.

Low-wind/high particulate matter episodes in the Calexico/Mexicali region.

The U.S. Environmental Protection Agency (EPA) currently classifies Imperial County, CA, as a nonattainment area for PM10 (particulate matter [PM] < or = 10 microm in diameter), and this region suffers from high rates of chronic bronchitis and childhood asthma. Although high annual and daily average PM levels can have negative health and economic effects, recent studies have identified an association between adverse health effects and short-term PM spikes of tens of micrograms per cubic meter. This study identified PM episodes in Calexico/Mexicali that involve PM concentration spikes with concentrations up to 10 times greater than those reported to cause adverse health effects. These episodes appear to be relatively common during the winter months, are associated with wind speeds below 2 m/sec and stable boundary level heights below 500 m, and can comprise a large portion of the 24-hr PM levels. The organic composition of the PM10 samples collected during the low-wind/ high-PM episodes differed from that collected at other times. However, a preliminary source attribution identified only one significant difference between the source classes: agricultural burning accounted for 6.7% of organic-fraction PM10 for low-wind/high-PM episodes versus 0.25% at other times. This preliminary source attribution also revealed that motor vehicles were the most important relative contributor to organic PM10.

An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans.

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

Whole Body Protein Turnover and Net Protein Balance After Pediatric Thoracic Surgery: A Noninvasive Single-Dose 15 N Glycine Stable Isotope Protocol With End-Product Enrichment.

BACKGROUND: We used the 15 N glycine urinary end-product enrichment technique to quantify whole body protein turnover following thoracic surgery. MATERIALS AND METHODS: A single dose of 15 N glycine (2 mg/kg) was administered orally on postoperative day 1 to children (1-18 years) following thoracic surgery. 15 N enrichment of ammonia and urea was measured in mixed urine after 12 and 24 hours, respectively, and protein synthesis, breakdown, and net balance determined. Nitrogen balance (dietary intake minus urinary excretion) was calculated. Urinary 3-methylhistidine:creatinine ratio was measured as a marker of skeletal muscle protein breakdown. RESULTS: We enrolled 19 subjects-median (interquartile range): age, 13.8 years (12.2-15.1); weight, 49.2 kg (38.4-60.8)-who underwent thoracotomy (n = 12) or thoracoscopic (n = 7) surgery. Protein synthesis and breakdown by 15 N enrichment were 7.1 (5.5-9) and 7.1 (5.6-9) g·kg-1 ·d-1 with ammonia (12 hours) as the end product, and 5.8 (3.8-6.7) and 6.7 (4.5-7.6) with urea (24 hours), respectively. Net protein balance by the 15 N glycine and urinary urea nitrogen methods were -0.34 (-0.47, -0.3) and -0.48 (-0.65, -0.28) g·kg-1 ·d-1 , respectively (rs = 0.828, P < .001). Postoperative change in 3-methylhistidine:creatinine ratio did not correlate significantly with protein breakdown or balance. CONCLUSION: The single-dose oral administration of 15 N glycine stable isotope with measurement of urinary end-product enrichment is a feasible and noninvasive method to investigate whole body protein turnover in children. After major surgery, children manifest increased protein turnover and net negative balance due to increased protein breakdown.

Comparison of the 13C-urea blood test to histology and rapid urease testing in the diagnosis of Helicobacter pylori infection in children.

OBJECTIVE: Helicobacter pylori infection is commonly investigated in children with abdominal pain. The definitive means of diagnosing infection, histology, requires endoscopy and sedation, making it invasive and expensive. Our objective was to compare histology against a less invasive and safer method, the 13C-urea blood test. PATIENTS AND METHODS: Forty children with abdominal pain undergoing upper endoscopy were randomized into either of 2 dosages of 13C-urea. Several biopsies were taken for histology and rapid urease testing. After endoscopy, each child ingested a randomly assigned dosage of either 75 mg or 125 mg 13C-urea, and blood was withdrawn 30 min later. RESULTS: Irrespective of the dosage of 13C-urea, the 13C-urea blood test performed with high accuracy (89%) when compared against either histology or rapid urease testing. The sensitivity and specificity of the blood test was 83% and 91%, respectively. When the smaller dosage of 13C-urea was used, the accuracy of the blood test was 100% compared with histology. There were no adverse events related to using either dosage of 13C-urea. CONCLUSIONS: The 13C-urea blood test may be comparable with histology in diagnosing H. pylori infection in children, and the smaller dosage of 13C-urea does not adversely affect blood test performance. The 13C-urea blood test is well tolerated in children.

Evaluation of postinjury hepatocyte function by central amino acid clearance.

It has been demonstrated by other investigators that central plasma clearance of amino acids accurately predicts hepatocyte function in patients with liver disease and correlates with clinical outcome. This methodology has not heretofore been studied in the trauma patient. It is our hypothesis that central amino acid clearance in trauma patients is more reflective of hepatocyte function than traditional liver function tests. We examined the plasma amino acid clearance rates using L-[1-13C]phenylalanine. Clearance rates were compared to standard liver function tests (LFTs) and the sensitivity and predictability of the technique were determined. The study was conducted on uninjured control subjects and in seriously injured patients, both with and without significant liver injuries. Compared to baseline values in the control group, initial phenylalanine breath scores were reduced in the injured, but exceeded control levels at 7 days postinjury. These changes were statistically significant. There was no difference between those with and without liver trauma. LFTs showed inconsistent and conflicting results. Thus, central amino acid clearance measured by L-[1-13C]phenylalanine oxidation is depressed immediately following injury but reaches supranormal levels at 7 days postinjury. Compared to LFTs, amino acid clearance suggests initial hepatocyte suppression followed by hyperactivity and is a more accurate determinant of hepatocyte function.

Kinetics and spontaneous open probability conferred by the epsilon subunit of the GABAA receptor.

GABAA receptors mediate synaptic and extrasynaptic inhibition. Native receptors consist of alpha and beta subunits, which are required for function, and another "modulatory" subunit, for example, gamma, delta, or epsilon. Of these, the epsilon subunit has the most restricted distribution, confers resistance to neurosteroid and anesthetic modulation, and causes spontaneous channel opening. Little is known, however, about how epsilon affects receptor kinetics, which in turn shape responses to both ambient and synaptic GABA exposure. Here, we expressed human alpha2beta1, alpha2beta1gamma2, or alpha2beta1epsilon subunit combinations in human embryonic kidney 293 cells and used rapid solution exchange to study receptor kinetics in outside-out patches. The epsilon subunit greatly slowed deactivation and recovery after brief GABA pulses. During long, saturating GABA pulses, the rate of desensitization was slower for alpha2beta1epsilon and alpha2beta1gamma2 than for alpha2beta1. However, in alpha2beta1epsilon, the final extent of desensitization was large compared with that of alpha2beta1gamma2. Responses in alpha2beta1epsilon, but not the others, were often followed by an "overshoot" above the baseline, suggesting that a fraction of channels are spontaneously open and are transiently silenced by receptor activation and subsequent desensitization. The baseline current and associated noise were reduced by picrotoxin, revealing that epsilon-containing channels are open approximately 4% of the time in the absence of GABA. These results suggest that, if epsilon-containing receptors are expressed at synapses, the synaptic currents would be long-lasting but may rundown quickly under high-frequency activation. In addition, silencing of spontaneous openings by desensitization raises the possibility that tonic inhibition mediated by epsilon-containing receptors may be regulated by phasic inhibition.

Role of Gut Microbiota and Short Chain Fatty Acids in Modulating Energy Harvest and Fat Partitioning in Youth.

OBJECTIVE: We aimed at determining the relationship of the gut microbiota and short chain fatty acids with obesity and fat partitioning and at testing potential differences in the ability of gut microbiota to ferment equal amounts of carbohydrates (CHO) between lean and obese youth. RESEARCH DESIGN AND METHODS: We analyzed the gut microbiota of 84 youth in whom body fat distribution was measured by fast-magnetic resonance imaging, de novo lipogenesis (DNL) quantitated using deuterated water, and the capability of gut flora to ferment CHO was assessed by 13C-fructose treatment in vitro. RESULTS: A significant association was found between the Firmicutes to Bacteroidetes ratio, and the abundance of Bacteroidetes and Actinobacteria with body mass index, visceral and SC fat (all P < .05). Plasma acetate, propionate, and butyrate were associated with body mass index and visceral and SC fat (all P < .05) and with hepatic DNL (P = .01, P = .09, P = .04, respectively). Moreover, the rate of CHO fermentation from the gut flora was higher in obese than in lean subjects (P = .018). CONCLUSIONS: These data demonstrate that obese youth show a different gut flora composition than lean and that short chain fatty acids are associated with body fat partitioning and DNL. Also, the gut microbiota of obese youth have a higher capability than the gut flora of lean to oxidize CHO.

An arginine involved in GABA binding and unbinding but not gating of the GABA(A) receptor.

GABA(A) receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose-response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function. Here, we focused on a residue (beta2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of beta2-R207, we expressed alpha1beta2 and alpha1beta2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because beta2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Palmitoleic acid is elevated in fatty liver disease and reflects hepatic lipogenesis.

BACKGROUND: Biochemical evidence has linked the coordinate control of fatty acid (FA) synthesis with the activity of stearoyl-CoA desaturase-1 (SCD1). The ratio of 16:1n-7 to 16:0 [SCD116] in plasma triacylglycerol FA has been used as an index to reflect liver SCD116 activity and has been proposed as a biomarker of FA synthesis, although this use has not been validated by comparison with isotopically measured de novo lipogenesis (DNL(Meas)). OBJECTIVE: We investigated plasma lipid 16:1n-7 and FA indexes of elongation and desaturation in relation to lipogenesis. DESIGN: In this cross-sectional investigation of metabolism, 24 overweight adults, who were likely to have elevated DNL, consumed D2O for 10 d and had liver fat (LF) measured by magnetic resonance spectroscopy. Very-low-density lipoprotein (VLDL)-triacylglycerols and plasma free FA [nonesterified fatty acids (NEFAs)] were analyzed by using gas chromatography for the FA composition (molar percentage) and gas chromatography-mass spectrometry and gas chromatography-combustion isotope ratio mass spectrometry for deuterium enrichment. RESULTS: In all subjects, VLDL-triacylglycerol 16:1n-7 was significantly (P < 0.01) related to DNL(Meas) (r = 0.56), liver fat (r = 0.53), and adipose insulin resistance (r = 0.56); similar positive relations were shown with the SCD116 index, and the pattern in NEFAs echoed that of VLDL-triacylglycerols. Compared with subjects with low LF (3.1 ± 2.7%; n = 11), subjects with high LF (18.4 ± 3.6%; n = 13) exhibited a 45% higher VLDL-triacylglycerol 16:1n-7 molar percentage (P < 0.01), 16% of subjects had lower 18:2n-6 (P = 0.01), and 27% of subjects had higher DNL as assessed by using a published DNL index (ratio of 16:0 to 18:2n-6; P = 0.03), which was isotopically confirmed by DNL(Meas) (increased 2.5-fold; P < 0.01). Compared with 16:0 in the diet, the low amount of dietary 16:1n-7 in VLDL-triacylglycerols corresponded to a stronger signal of elevated DNL. CONCLUSION: The current data provide support for the use of the VLDL-triacylglycerol 16:1n-7 molar percentage as a biomarker for elevated liver fat when isotope use is not feasible; however, larger-scale confirmatory studies are needed.