Growth phase-dependent surface properties of Legionella pneumophila and their role in adhesion to stainless steel coated QCM-D sensors.

Legionella pneumophila cell surface hydrophobicity and charge are important determinants of their mobility and persistence in engineered water systems (EWS). These surface properties may differ depending on the growth phase of L. pneumophila resulting in variable adhesion and persistence within EWS. We describe the growth-dependent variations in L. pneumophila cell surface hydrophobicity and surface charge using the microbial adhesion to hydrocarbon assay and microelectrophoresis, respectively, and their role in cell adhesion to stainless steel using a quartz crystal microbalance with dissipation (QCM-D) monitoring instrument. We observed a steady increase in L. pneumophila hydrophobicity during their lifecycle in culture media. Cell surfaces of stationary phase L. pneumophila were significantly more hydrophobic than their lag and midexponential counterparts. No significant changes in L. pneumophila cell surface charge were noted. Morphology of L. pneumophila remained relatively constant throughout their lifecycle. In the QCM-D study, lag and exponential phase L. pneumophila weakly adhered to stainless steel surfaces resulting in viscoelastic layers. In contrast, stationary phase bacteria were tightly and irreversibly bound to the surfaces, forming rigid layers. Our results suggest that the stationary phase of L. pneumophila would highly favour their adhesion to plumbing surfaces and persistence in EWS.

Neuromodulation of orexin neurons reduces diet-induced adiposity.

BACKGROUND/OBJECTIVES: Low levels of orexin are associated with obesity and reduced physical activity in humans and animals. SUBJECTS/METHODS: Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) selectively activated orexin neurons in mouse lateral hypothalamus (LH) to measure effects on spontaneous physical activity (SPA). DREADD targeting was achieved by stereotaxic injection of AAV vectors into caudal lateral LH of heterozygous orexin-Cre or C57/B6J mice. In one set of studies, excitation of orexin neurons was examined (virus: AAV2-EF1a-DIO-hM3Dq-mCherry), and test sessions began 3-4 h after light cycle onset. In a study examining the inhibition of orexin neurons (virus: AAV2-hSyn-DIO-hM4Di-mCherry), testing began 15 min prior to dark cycle onset. Clozapine n-oxide (CNO; 1 or 5 mg/kg) or saline was injected intraperitoneally and time spent moving in open field chambers was recorded for 2 h. Follow-up studies in separate mouse cohorts quantified SPA in parallel with changes in energy expenditure (EE) and chow intake using indirect calorimetry chambers (SableSystem™). Following acclimation, testing sessions (saline and/or CNO) took place over the course of ~1 week, with injections administered every day. Changes in SPA, EE, chow intake, fecal boli, and body composition (EchoMRI™) were measured. Additional mice cohorts were fed a high-fat diet (HFD) and injected with CNO daily up to 10 days to assess the potential for orexin activation to prevent diet-induced obesity. RESULTS: Activation of orexin resulted in increases in SPA in male and female mice, and was accompanied by increases in energy expenditure without changes in overall chow intake. When orexin activation occurred in the context of high fat diet, weight gain and adiposity were significantly attenuated. SPA was decreased when DREADDs were used to inhibit orexin activity. CONCLUSION: These results demonstrate that orexin neurons play a critical role in mediating physical activity and suggest a novel therapeutic target for treating obesity.

Role of orexin-A in the ventrolateral preoptic area on components of total energy expenditure.

BACKGROUND: Identifying whether components of total energy expenditure (EE) are affected by orexin receptor (OXR1 and OXR2) stimulation or antagonism with dual orexin receptor antagonists (DORAs) has relevance for obesity treatment. Orexin receptor stimulation reduces weight gain by increasing total EE and EE during spontaneous physical activity (SPA). OBJECTIVE: The purpose of this study was to determine if a DORA (TCS-1102) in the ventrolateral preoptic area (VLPO) reduced orexin-A-induced arousal, SPA, total EE and EE during sleep, rest, wake and SPA and whether the DORA alone reduced total EE and its components. We hypothesized that: (1) a DORA would reduce orexin-A induced increases in arousal, SPA, components of total EE, reductions in sleep and the EE during sleep and (2) the DORA alone would reduce baseline (non-stimulated) SPA and total EE. SUBJECTS/METHODS: Sleep, wakefulness, SPA and EE were determined after microinjection of the DORA (TCS-1102) and orexin-A in the VLPO of male Sprague-Dawley rats with a unilateral cannula targeted towards the VLPO. Individual components of total EE were determined based on time-stamped data. RESULTS: The DORA reduced orexin-A-induced increases in arousal, SPA, total EE and EE during SPA, wake, rest and sleep 1 h post injection (P<0.05). Orexin-A significantly reduced sleep and significantly increased EE during sleep 1 h post injection (P<0.05). Furthermore, the DORA alone significantly reduced total EE, EE during sleep (NREM and REM) and resting EE 2 h post injection (P<0.05). CONCLUSIONS: These data suggest that orexin-A reduces weight gain by stimulating total EE through increases in EE during SPA, rest and sleep. Residual effects of the DORA alone include decreases in total EE and EE during sleep and rest, which may promote weight gain.

Role of orexin receptors in obesity: from cellular to behavioral evidence.

The orexin peptides and their two receptors are involved in multiple physiological processes, including energy homeostasis, arousal, stress and reward. Higher signaling of the orexin peptides at the orexin receptors (OXR) protects against obesity, but it is less clear how their activation in different brain regions contributes to this behavioral output. This review summarizes the evidence available for a role of central OXR in energy homeostasis and their contribution to obesity. A detailed analysis of anatomical, cellular and behavioral evidence shows that modulation of energy homeostasis by the OXR is largely dependent upon anatomical and cellular context. It also shows that obesity resistance provided by activation of the OXR is distributed across multiple brain sites with site-specific actions. We suggest that understanding the role of the OXR in the development of obesity requires considering both specific mechanisms within brain regions and interactions of orexinergic input between multiple sites.

A versatile high resolution objective for imaging quantum gases.

We present a high resolution objective lens made entirely from catalog singlets that has a numerical aperture of 0.36. It corrects for aberrations introduced by a glass window and has a long working distance of 35 mm, making it suitable for imaging objects within a vacuum system. This offers simple high resolution imaging for many in the quantum gas community. The objective achieves a resolution of 1.3 µm at the design wavelength of 780 nm, and a diffraction-limited field of view of 360 µm when imaging through a 5 mm thick window. Images of a resolution target and a pinhole show quantitative agreement with the simulated lens performance. The objective is suitable for diffraction-limited monochromatic imaging on the D2 line of all the alkalis by changing only the aperture diameter, retaining numerical apertures above 0.32. The design corrects for window thicknesses of up to 15 mm if the singlet spacings are modified.

Characteristics of three listeriaphages isolated from New Zealand seafood environments.

AIM: To isolate and characterize listeriaphages from seafood environments. METHODS AND  RESULTS: Listeriaphages (phages) isolated from seafood environments were distinguished by physical and biological techniques including restriction digestion of phage DNA. Three phages belonged to order Caudovirales and showed psychrotrophic characteristics. The phages had broad host ranges against 23 Listeria strains by productive infection or at least by adsorption. At 15 ± 1°C, adsorption rate constants of the three phages ranged from 8·93 × 10(-9) to 3·24 × 10(-11 ) ml min(-1) across different Listeria monocytogenes strains. In indicator hosts, the mean burst sizes of phages LiMN4L, LiMN4p and LiMN17 were c. 17, 17 and 11 plaque-forming units (PFU) per cell, respectively, at 15 ± 1°C. The respective latent periods were c. 270 min for phages LiMN4p and LiMN17, whereas for phage LiMN4L, it was c. 240 min. CONCLUSIONS: The three virulent psychrotrophic phages isolated from seafood-processing environments had broad host ranges and low productive replication. These characteristics suggest that the phages may be suitable as passive biocontrol agents against seafood-borne L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the isolation of autochthonous virulent listeriaphages from seafood-processing environments and information on single-step replication and adsorption characteristics of such listeriaphages.

Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef.

A number of outbreaks of Escherichia coli O157:H7 infections involving beef have been reported. Options for controlling bacterial pathogens in raw foods are limited, but one is to use bacteriophages (phages). We describe the isolation and characterisation of phage FAHEc1, which infects E. coli O157, and its ability to kill its host in vitro and on beef. The phage belonged to the family Myoviridae and lysed 28 of 30 E. coli O157 (:H7, :HNM and :H not specified) isolates, only one other non-O157 E. coli serotype (O162:H7), and none of the other 13 bacterial species tested. The phage did not contain stx1, stx2, eae or ehxA virulence genes as assessed by PCR. An approximate 4 log10 inactivation of E. coli O157:H7 occurred at 5 °C in the presence of phage FAHEc1 at >107 PFU/ml in broth in vitro. On thinly sliced beef pieces incubated at 37 °C, a > 2.7 log10 reduction occurred with 3.2 × 107 PFU/4 cm² meat piece. At lower phage concentrations (10³-104 PFU/4 cm² piece) phage replication occurred on beef at 37 °C. When the phage was applied to beef pieces under conditions simulating hot boning and conventional carcass cooling, inactivation of E. coli O157:H7 of approximately 2 log10 was measured under optimal conditions with phages applied at 3.2 × 107 PFU/4 cm² meat piece.

Spontaneous physical activity protects against fat mass gain.

OBJECTIVE: It is unclear whether elevated spontaneous physical activity (SPA, very low-intensity physical activity) positively influences body composition long term. We determined whether SPA and caloric intake were differentially related to the growth curve trajectories of body weight, fat mass (FM) and fat-free mass (FFM) between obesity resistant and Sprague-Dawley rats at specific age intervals. DESIGN AND SUBJECTS: Body composition, SPA and caloric intake were measured in selectively-bred obesity-resistant and out-bred Sprague-Dawley rats from 1 to 18 months. Data from development throughout maturation were analyzed by longitudinal growth curve modeling to determine the rate and acceleration of body weight, FM- and FFM-gain. RESULTS: Obesity-resistant rats had a lower rate of FM gain overall, a lower acceleration in body weight early in life, significantly greater SPA and lower cumulative caloric intake. Greater SPA in obesity-resistant rats was significantly associated with a lower rate of FM gain overall and lower acceleration in body weight early in life. Obesity resistant rats lost less FFM compared with Sprague-Dawley rats despite that obesity-resistant rats had a lower acceleration in FFM gain early in life. Obesity-resistant rats gained less FM and more FFM per gram body weight and were less energy efficient than Sprague-Dawley rats. Caloric intake was significantly and positively related to body weight, FM and FFM gain in both groups. Circadian patterns of caloric intake were group and age-dependent. Our data demonstrate that elevated and sustained SPA during development and over the lifespan are related to the reduced the rate of FM gain and may preserve FFM. CONCLUSION: These data support the idea that SPA level is a reproducible marker that reliably predicts propensity for obesity in rats, and that elevated levels of SPA maintained during the lifespan promote a lean phenotype.

Inhibition of Listeria monocytogenes by Enterococcus mundtii isolated from soil.

Two bacterial isolates with inhibitory activity against Listeria monocytogenes and Enterococcus faecalis were obtained from soil. Genotypic and phenotypic characterization identified them as Enterococcus mundtii, a species whose ability to compete with L. monocytogenes is relatively unexplored compared to other members of the genus. The thermal stability of the inhibitory factor and its sensitivity to proteolytic enzymes indicate that it is most likely a bacteriocin. Both isolates grew at comparable rates to L. monocytogenes at 5 °C and 10 °C in vitro. One isolate killed L. monocytogenes when it reached concentrations of 10(6)-10(8) CFU ml(-1). Minimum inocula of 10(6) and 10(5) CFU ml(-1) of E. mundtii were required to reduce and maintain L. monocytogenes concentrations beneath the level of detection at 5 °C and 10 °C, respectively. In situ experiments at 5 °C showed that E. mundtii inhibited the growth of L. monocytogenes on vacuum-packed cold smoked salmon during its four week shelf life. E. mundtii could, therefore, control the growth of L. monocytogenes at low temperatures, indicating a potential application in controlling this pathogen in chilled foods. To control growth of Listeria, the concentration of E. mundtii needs to be high, but it is possible that a purified bacteriocin could be used to achieve the same effect.

Isolation and characterization of phages infecting Bacillus cereus.

AIM: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. METHODS AND RESULTS: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single-step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform-resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log(10) CFU ml(-1) within 24 h at room temperature, when applied at a high concentration. CONCLUSIONS: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a 'cocktail' of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of biocontrol by phages of B. cereus in food.

Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage.

A bacteriophage (phage) that infected strains of the species Listeria monocytogenes as well as Listeria ivanovii and Listeria welshimeri, but not Listeria grayi or Listeria innocua, was isolated from sheep faeces. The phage had a contractile tail and an icosohedral head indicating that it was a myovirus, and was morphologically similar to phage A511. At 30 °C, phages added at 5.2 × 107 PFU ml⁻¹ prevented the growth in broth of L. monocytogenes present at approximately twice this concentration for 7 h, but re-growth occurred such that the concentration after 24 h incubation was similar in both control and phage-treated cultures. At the same temperature, but on the surface of vacuum-packed ready-to-eat chicken breast roll, there was an immediate 2.5 log10 CFU cm⁻² reduction in pathogen concentration following addition of phages and then re-growth. However, at a temperature reflecting that at which a chilled food might be held (5 °C), this re-growth was prevented over 21 days incubation. The data suggest a dose-dependent rapid reduction in pathogen concentration followed by no continued phage-mediated effect. These results, alongside other published data, indicate that a high concentration of phages per unit area is required to ensure significant inactivation of target pathogens on food surfaces.

Elevated sleep quality and orexin receptor mRNA in obesity-resistant rats.

OBJECTIVE: To determine if resistance to weight gain is associated with alterations in sleep-wake states and orexin receptor gene expression. DESIGN: Three-month-old obesity-susceptible Sprague-Dawley (SD) and obesity-resistant (OR) rats were fed standard rodent chow. Sleep-wake cycle was measured by radiotelemetry and orexin receptor profiles in sleep-wake regulatory areas of the brain were quantified by quantitative reverse transcriptase-PCR. SUBJECTS: Adult male obesity-susceptible SD and selectively bred OR rats. MEASUREMENTS: Body weight, food intake, energy efficiency, percent time spent in active wake (AW), quiet wake (QW), slow-wave sleep (SWS), rapid eye movement (REM) sleep, number and mean duration of sleep-wake episodes, number of stage transitions, SWS sleep delta power and orexin receptor mRNA levels were measured. RESULTS: OR rats weighed significantly less and had lower energy efficiency than SD rats. Food intake was not different between SD and OR rats. Time spent in QW was similar between groups, and therefore AW and QW were combined and are referred to as 'wakefulness'. OR rats spent significantly more time in wakefulness and less time in SWS compared with SD rats during the 24-h recording period. Relative to SD rats, OR rats had significantly fewer sleep-wake episodes and the duration of the episodes were prolonged, indicating less fragmented sleep. Furthermore, OR rats had fewer transitions between sleep stages, which indicates that OR rats were behaviorally more stable and had more consolidated sleep than obesity-susceptible SD rats. OR rats showed lower delta power during SWS, indicating a lower sleep drive. Our results showed greater orexin receptor gene expression in sleep regulatory brain areas in OR rats. CONCLUSION: These results show that prolonged wakefulness, better sleep quality, lower sleep drive and greater orexin signaling may confer protection against obesity.

A comparison of McGrath and Macintosh laryngoscopes in novice users: a manikin study.

Direct laryngoscopy using the Macintosh laryngoscope is a difficult skill to acquire. Videolaryngoscopy is a widely accepted airway management technique that may be easier for novices to learn. We compared the McGrath videolaryngoscope and Macintosh laryngoscope by studying the performance of 25 medical students with no previous experience of performing tracheal intubation using an easy intubation scenario in a manikin. The order of device use was randomised for each student. After brief instruction each participant performed eight tracheal intubations with one device and then eight tracheal intubations with the other laryngoscope. Novices achieved a higher overall rate of successful tracheal intubation, avoided oesophageal intubation and produced less dental trauma when using the McGrath. The view at laryngoscopy was significantly better with the McGrath. Intubation times were similar for both laryngoscopes and became shorter with practice. There was no difference in participants' rating of overall ease of use for each laryngoscope.

Neuropeptidergic mediators of spontaneous physical activity and non-exercise activity thermogenesis.

Lean individuals have high levels of spontaneous physical activity (SPA) and the energy expenditure derived from that activity, termed non-exercise activity thermogenesis or NEAT, appears to protect them from obesity. Conversely, obesity in different human populations is characterized by low levels of SPA and NEAT. Like in humans, elevated SPA in rats appears to protect against obesity: obesity-resistant rats have significantly greater SPA and NEAT than obesity-prone rats. We review the literature on brain mechanisms important in mediating SPA and NEAT. The focus is on neuropeptides, including cholecystokinin, corticotropin-releasing hormone (also known as corticotropin-releasing factor), neuromedin U, neuropeptide Y, leptin, agouti-related protein, orexin-A (also known as hypocretin-1), and ghrelin. We also review information regarding interactions between these neuropeptides and dopamine, a neurotransmitter important in mediating motor function. Finally, we present evidence that elevated signaling of pathways mediating SPA and NEAT may protect against weight gain and obesity.

Phage inactivation of foodborne pathogens on cooked and raw meat.

Phages infecting Salmonella Typhimurium PT160 and Campylobacter jejuni were added at a low or high (10 or 10(4)) multiplicity of infection (MOI) to either low or high (<100 or 10(4)cm(-2)) densities of host bacteria inoculated onto raw and cooked beef, and incubated at 5 and 24 degrees C to simulate refrigerated and room temperature storage. Counts of host bacteria were made throughout the incubation period, with phages being counted at the first and last sampling times. Host inactivation was variable and depended on the incubation conditions and food type. Significant host inactivations of the order of 2-3 log(10)cm(-2) at 5 degrees C and >5.9 log(10)cm(-2) at 24 degrees C were achieved compared to phage-free controls using the Salmonella phage under optimal conditions (high host cell density and MOI). These results alongside those already published indicate that phages may be useful in the control for foodborne pathogens.

Shedding new light on obesity. Appetite regulation.

Several genes involved in the regulation of appetite and energy metabolism have been cloned and characterized recently. Each seems to form part of the complex regulatory network, centred in the hypothalamus, that is responsible for striking a balance between food intake and energy expenditure.

Obesity: progress through genetic manipulation.

Transgenic mice have been produced that either lack or overproduce neuroregulatory substances implicated in the control of food intake and body weight. Are such mice useful models for understanding the underlying etiology of obesity in humans?

Regulation of proliferation of human colonic subepithelial myofibroblasts by mediators important in intestinal inflammation.

An increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring. We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts. Using primary cultures of these cells, we have shown increases in [3H]thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml). Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II. In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in [3H]thymidine incorporation. IL-1beta and platelet-derived growth factor together produced an increase in [3H]thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers. Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4. 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.

Effects of opioid antagonists naloxone and naltrexone on neuropeptide Y-induced feeding and brown fat thermogenesis in the rat. Neural site of action.

Neuropeptide Y administered intracerebroventricularly and into the paraventricular nucleus of the hypothalamus stimulates feeding and decreases brown adipose tissue thermogenesis. Although specific neuropeptide Y antagonists are not yet available, previous studies had shown that the opioid antagonist naloxone blocked neuropeptide Y-induced feeding when both drugs were injected intracerebroventricularly. We wanted to find out if naloxone injected into specific brain sites would block neuropeptide Y effects on feeding and brown fat thermogenesis. Rats were double injected in specific brain sites with neuropeptide Y and either naloxone or naltrexone (a congener of naloxone). Food intake and brown fat measures were assessed. Naloxone or naltrexone in the paraventricular nucleus weakly decreased paraventricular nucleus neuropeptide Y-induced feeding and did not affect neuropeptide Y-induced reductions in brown fat activity. Peripheral naloxone blocked intracerebroventricular neuropeptide Y-induced feeding and brown fat alterations. Fourth ventricular naloxone decreased paraventricular nucleus neuropeptide Y-induced feeding, and naltrexone given into the nucleus of the solitary tract blocked paraventricular nucleus neuropeptide Y-induced alterations in feeding and brown fat. These data indicate that neuropeptide Y in the paraventricular nucleus may act on feeding and brown fat thermogenesis through opioidergic pathways in the nucleus of the solitary tract.

Orexin activation counteracts decreases in nonexercise activity thermogenesis (NEAT) caused by high-fat diet.

Overweight and obesity result from an imbalance between caloric intake and energy expenditure, including expenditure from spontaneous physical activity (SPA). Changes in SPA and resulting changes in non-exercise activity thermogenesis (NEAT) likely interact with diet to influence risk for obesity. However, previous research on the relationship between diet, physical activity, and energy expenditure has been mixed. The neuropeptide orexin is a driver of SPA, and orexin neuron activity can be manipulated using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). We hypothesized that HFD decreases SPA and NEAT, and that DREADD-mediated activation of orexin neuron signaling would abolish this decrease and produce an increase in NEAT instead. To test these ideas, we characterized behaviors to determine the extent to which access to a high-fat diet (HFD) influences the proportion and probability of engaging in food intake and activity. We then measured NEAT following access to HFD and following a DREADD intervention targeting orexin neurons. Two cohorts of orexin-cre male mice were injected with an excitatory DREADD virus into the caudal hypothalamus, where orexin neurons are concentrated. Mice were then housed in continuous metabolic phenotyping cages (Sable Promethion). Food intake, indirect calorimetry, and SPA were automatically measured every second. For cohort 1 (n=8), animals were given access to chow, then switched to HFD. For cohort 2 (n=4/group), half of the animals were given access to HFD, the other access to chow. Then, among animals on HFD, orexin neurons were activated following injections of clozapine n-oxide (CNO). Mice on HFD spent significantly less time eating (p<0.01) and more time inactive compared to mice on chow (p<0.01). Following a meal, mice on HFD were significantly more likely to engage in periods of inactivity compared to those on chow (p<0.05). NEAT was decreased in animals on HFD, and was increased to the NEAT level of control animals following activation of orexin neurons with DREADDs. Food intake (kilocalories) was not significantly different between mice on chow and HFD, yet mice on chow expended more energy per unit of SPA, relative to that in mice consuming HFD. These results suggest that HFD consumption reduces SPA and NEAT, and increases inactivity following a meal. Together, the data suggest a change in the efficiency of energy expenditure based upon diet, such that SPA during HFD burns fewer calories compared to SPA on a standard chow diet.