TRPM4 and PLCß3 contribute to normal behavioral responses to an array of sweeteners and carbohydrates but PLCß3 is not needed for taste-driven licking for glucose.

The peripheral taste system is more complex than previously thought. The novel taste-signaling proteins TRPM4 and PLCß3 appear to function in normal taste responding as part of Type II taste cell signaling or as part of a broadly responsive (BR) taste cell that can respond to some or all classes of tastants. This work begins to disentangle the roles of intracellular components found in Type II taste cells (TRPM5, TRPM4, and IP3R3) or the BR taste cells (PLCß3 and TRPM4) in driving behavioral responses to various saccharides and other sweeteners in brief-access taste tests. We found that TRPM4, TRPM5, TRPM4/5, and IP3R3 knockout (KO) mice show blunted or abolished responding to all stimuli compared with wild-type. IP3R3 KO mice did, however, lick more for glucose than fructose following extensive experience with the 2 sugars. PLCß3 KO mice were largely unresponsive to all stimuli except they showed normal concentration-dependent responding to glucose. The results show that key intracellular signaling proteins associated with Type II and BR taste cells are mutually required for taste-driven responses to a wide range of sweet and carbohydrate stimuli, except glucose. This confirms and extends a previous finding demonstrating that Type II and BR cells are both necessary for taste-driven licking to sucrose. Glucose appears to engage unique intracellular taste-signaling mechanisms, which remain to be fully elucidated.

Chronic Intermittent Sucrose Consumption Facilitates the Ability to Discriminate Opioid Receptor Blockade with Naltrexone in Rats.

The opioid antagonist naltrexone (NTX) decreases intake of preferred diets in rats at very low doses relative to doses needed to decrease intake of "bland" laboratory chow. In the absence of an opioid agonist, NTX is not discriminable using operant techniques. In the current study, we found that rats given intermittent access to a 25% sucrose solution learned to discriminate between various naltrexone doses and saline. None of the rats given only water learned to discriminate between naltrexone and saline. When access to the sucrose solution was discontinued for 14 days, the rats lost the ability to discriminate between NTX and saline. We also studied the changes of c-Fos IR in selected brain regions in rats treated with saline versus NTX that were drinking water or 25% sucrose. An injection of NTX or saline resulted in a significant drug, diet, and interaction effect in various brain regions associated with feeding behavior, particularly the amygdala, accumbens, and hypothalamic sites. Thus, we found that ingestion of a sucrose solution results in the ability of rats to reliably discriminate naltrexone administration. In addition, sucrose and naltrexone altered c-Fos IR in an interactive fashion in brain regions known to be involved in ingestion behavior.

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation.

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO2, formate, and H2 that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially Nε-acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, Nε-acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia.

Best Practices for Preparation of Staphylococcus aureus Metabolomics Samples.

Developments in mass spectrometry have made it possible to identify individual biomolecules in complex samples. This has led to advances in the detection and quantification of both extracellular and intracellular metabolites, such as amino acids, organic acids, fatty acids, nucleotides, and CoA-esters from growth media and cellular extracts. However, the reproducibility of metabolite data can be problematic if the concentrations and/or stability of metabolites fluctuate during culture harvesting and processing. Herein we describe a standardized and efficient collection protocol and best practices for preservation and harvesting of Staphylococcus aureus cellular and supernatant samples to improve reproducibility, reliability, and consistency in mass-spectrometry-based metabolite data sets.

Bacterial-like nitric oxide synthase in the haloalkaliphilic archaeon Natronomonas pharaonis.

Bacterial nitric oxide (NO) synthases (bNOS) play diverse and important roles in microbial physiology, stress resistance, and virulence. Although bacterial and mammalian NOS enzymes have been well-characterized, comparatively little is known about the prevalence and function of NOS enzymes in Archaea. Analysis of archaeal genomes revealed that highly conserved bNOS homologs were restricted to members of the Halobacteria. Of these, Natronomonas pharaonis NOS (npNOS) was chosen for further characterization. NO production was confirmed in heterologously expressed His-tagged npNOS by coupling nitrite production from N-hydroxy-L-arginine in an H2O2-supported reaction. Additionally, the nos gene was successfully targeted and disrupted to create a Nmn. pharaonis nos mutant by adapting an established Natrialba magadii transformation protocol. Genome re-sequencing of this mutant revealed an additional frameshift in a putative cation-acetate symporter gene, which could contribute to altered acetate metabolism in the nos mutant. Inactivation of Nmn. pharaonis nos was also associated with several phenotypes congruent with bacterial nos mutants (altered growth, increased oxygen consumption, increased pigment, increased UV susceptibility), suggesting that NOS function may be conserved between bacteria and archaea. These studies are the first to describe genetic inactivation and characterization of a Nmn. pharaonis gene and provides enhanced tools for probing its physiology.

Temperature Is Sufficient to Condition a Flavor Preference for a Cold-Paired Solution in Rats.

Increasing evidence suggests that stimulus temperature modifies taste signaling. However, understanding how temperature modifies taste-driven behavior is difficult to separate as we must first understand how temperature alone modifies behavior. Previous work has suggested that cold water is more rewarding and "satiating" than warm water, and water above orolingual temperature is avoided in brief-access testing. We explored the strength of cold water preference and warm water avoidance by asking: (1) if cold temperature alone was sufficient to condition a flavor preference and (2) if avoidance of warm stimuli is driven by novelty. We addressed these questions using custom-designed equipment that allows us to monitor and maintain solution temperatures. We conducted two-bottle preference tests, after pairing Kool-Aid flavors with 10 or 40 °C. Rats preferred the flavor paired with cold temperature, both while it was cold and for 1 day while solutions were presented at 22 °C. We then examined the role of novelty in avoidance of 40 °C. Rats were maintained on 10, 22, or 40 °C water in their home cage to increase familiarity with the temperatures. Rats were then subject to a series of brief-access taste tests to water or sucrose at 10 to 40 °C. Rats that had 40 °C experience licked more to 40 °C water, but not sucrose, during brief-access testing. In a series of two-bottle preference tests, rats maintained on 40 °C water had a decreased preference for 10 °C water when paired opposite 40 °C water. Together, these data contribute to our understanding of orosensory-driven behavior with water at different temperatures.

Altering salivary protein profile can decrease aversive oromotor responding to quinine in rats.

Bitter taste is often associated with toxins, but accepting some bitter foods, such as green vegetables, can be an important part of maintaining a healthy diet. It has previously been shown that animals exposed to quinine upregulate a set of salivary proteins (SPs), and those with upregulated SPs have increased rates of feeding on a quinine diet as well as increased brief-access licking to and higher detection thresholds for quinine. These studies suggest that SPs alter orosensory feedback; however, they rely on SPs upregulated by diet exposure and cannot control for the role of learning. Here, we use taste reactivity to determine if SPs can alter bitter taste in animals with no previous bitter diet experience. First, saliva with proteins stimulated by injections of isoproterenol and pilocarpine was collected from anesthetized rats; this "donor saliva" was analyzed for protein concentration and profile. Bitter-naïve rats were implanted with oral catheters and infused with taste stimuli dissolved in saliva that contained all of the SPs from the donors, saliva that was filtered of SPs, water, or artificial saliva. Their orofacial movements were recorded and quantified. We found that presence of quinine increased movements associated with aversive stimuli, but adding SPs to the infusion was sufficient to reduce aversive oromotor responding to quinine. The effect was dependent on the total protein concentration of the saliva, as protein concentration increased aversive responses decreased. Additionally, infusions of whole saliva altered aversive responding to quinine, but not other stimuli (citric acid, NaCl, sucrose). Our work suggests that effect of these SPs is specific and the presence of SPs is sufficient to decrease aversive orosensory feedback to bitter stimuli.

Impact of acute ethanol exposure on body temperatures in aged, adult and adolescent male rats.

The mean population age of the United States continues to increase, and data suggest that by the year 2060 the population of people over the age of 65 will more than double, providing a potentially massive strain on health care systems. Research demonstrates individuals 65 and older continue to consume ethanol, often at high levels. However, preclinical animal models are still being developed to understand how ethanol might interact with the aged population. The current experiments investigated differential body temperature responses in aged rats compared to adult rats and adolescent rats. Aged (19 months of age), adult (70 days of age), or adolescent (30 days of age) male Sprague Dawley rats were administered 1.0 g/kg, 2.0 g/kg, or 3.0 g/kg ethanol, intraperitoneally (i.p.), in a balanced Latin square design. Prior to ethanol administration, a core body temperature via an anal probe was obtained, and then repeatedly determined every 60 min following ethanol exposure for a total of 360 min. In addition, a blood sample was obtained from a tail nick 60, 180, and 300 min following the ethanol injection to investigate the relationship of ethanol levels and body temperature in the same animals. Aged rats had significantly greater reductions in body temperature compared to either adult or adolescent rats following both the 2.0 g/kg and 3.0 g/kg ethanol injection. Additionally, adolescent rats cleared ethanol significantly faster than aged or adult animals. These experiments suggest body temperature regulation in aged rats might be more sensitive to acute ethanol compared to adult rats or adolescent rats. Future studies are needed to identify the neurobiological effects underlying the differential sensitivity in aged rats to ethanol.

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids.

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Staphylococcus aureus Metabolism and Virulence.

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.

The impact of low to moderate chronic intermittent ethanol exposure on behavioral endpoints in aged, adult, and adolescent rats.

The average age of the population in the United States and other countries is increasing. Understanding the health consequences in the aged population is critical. Elderly individuals consume ethanol, often at elevated rates, and in some cases in a binge episode. The present study sought to investigate whether binge-like ethanol exposure in aged male rats produced differential health and behavioral effects compared to adult male and adolescent male rats. Subjects were exposed to either 1.0 g/kg or 2.0 g/kg ethanol every other day via intraperitoneal injection for 20 days, and tested on a variety of behavioral measures and body weight. Binge-like ethanol exposure produced differential effects on body weight between aged and adolescent and adult rats. In addition, aged rats had a significantly longer loss of righting reflex and demonstrated a trend toward tolerance following the 2.0-g/kg exposure. No significant effects on anxiety-like behavior as measured by open arm entries, depressive-like symptoms as measured by immobility in the forced swim test, or cognitive performance as measured by latency and path length in the Morris water maze were found. These results demonstrate that aged animals are differentially sensitive to the impact of chronic intermittent ethanol exposure in some, but not all behaviors. Future research is needed to understand the mechanisms of these differential effects.

Staphylococcus aureus nitric oxide synthase (saNOS) modulates aerobic respiratory metabolism and cell physiology.

Nitric oxide (NO) is generated from arginine and oxygen via NO synthase (NOS). Staphylococcus aureus NOS (saNOS) has previously been shown to affect virulence and resistance to exogenous oxidative stress, yet the exact mechanism is unknown. Herein, a previously undescribed role of saNOS in S. aureus aerobic physiology was reported. Specifically, aerobic S. aureus nos mutant cultures presented with elevated endogenous reactive oxygen species (ROS) and superoxide levels, as well as increased membrane potential, increased respiratory dehydrogenase activity and slightly elevated oxygen consumption. Elevated ROS levels in the nos mutant likely resulted from altered respiratory function, as inhibition of NADH dehydrogenase brought ROS levels back to wild-type levels. These results indicate that, in addition to its recently reported role in regulating the switch to nitrate-based respiration during low-oxygen growth, saNOS also plays a modulatory role during aerobic respiration. Multiple transcriptional changes were also observed in the nos mutant, including elevated expression of genes associated with oxidative/nitrosative stress, anaerobic respiration and lactate metabolism. Targeted metabolomics revealed decreased cellular lactate levels, and altered levels of TCA cycle intermediates, the latter of which may be related to decreased aconitase activity. Collectively, these findings demonstrate a key contribution of saNOS to S. aureus aerobic respiratory metabolism.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UNLABELLED: Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE: Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Vibrato Rate and Extent in College Music Majors: A Multicenter Study.

OBJECTIVES: To examine differences in vibrato rate and extent according to vowel, production type, gender, voice type, and vocal training. STUDY DESIGN: Cross-sectional. METHODS: Four collegiate voice teachers used a common protocol to gather data on habitual, best classical, and nonvibrato singing production of five vowels in 78 male and female vocal majors. Subject age, gender, voice type, academic degree program, number of years of training, and most frequent singing style were compared with mean vibrato rate and mean peak-to-peak vibrato extent for each vowel and for each production condition. RESULTS: The high versus low and female versus male comparisons in this study support results found in the literature. Both vibrato rate and vibrato extent were reduced when the singers sang nonvibrato as compared with their habitual and best classical production. CONCLUSIONS: The mechanisms for reducing vibrato rate and extent need further exploration.

Efficacy of selected treatments of HIV wasting: a systematic review and meta-analysis.

PURPOSE: To assess the efficacy of recombinant human growth hormone (rhGH), testosterone, and anabolic steroids in the treatment of HIV wasting. METHODS: A systematic review and meta-analysis of studies published in English since 1996 was conducted. Studies of anabolic steroids, testosterone, and rhGH in treatment of HIV wasting reporting the efficacy outcomes of body composition measures, work output, or health-related quality of life (QoL) were eligible. Meta-analyses were performed for mean pre-post change in lean body mass (LBM), the within-study mean difference in pre-post change for LBM, and of odds ratios for certain safety events. RESULTS: A total of 18 studies met inclusion criteria for this review. CONCLUSION: The 3 treatments for HIV wasting assessed--rhGH, testosterone, and anabolic steroids--all demonstrated significant efficacy in increasing LBM as compared with placebo. Although meta-analysis did not indicate any statistically significant differences between these agents in the degree of efficacy in this outcome, the Food and Drug Administration-approved dose of rhGH may have advantages over the other 2 therapies in terms of improvements in functional capacity and QoL.