The effect of various pH, ionic strength and temperature on papain hydrolysis of salivary film

Stimulated human whole saliva (WS) was used to study the dynamics of papain hydrolysis at defined pH, ionic strength and temperature with the view of reducing an acquired pellicle. A quartz crystal microbalance with dissipation (QCM-D) was used to monitor the changes in frequency due to enzyme hydrolysis of WS films and the hydrolytic parameters were calculated using an empirical model. The morphological and conformational changes of the salivary films before and after enzymatic hydrolysis were characterized by atomic force microscopy (AFM) imaging and grazing angle infrared spectroscopy (GA-FTIR) spectra, respectively. The characteristics of papain hydrolysis of WS films were pH-, ionic strength- and temperature-dependent. The WS films were partially removed by the action of enzyme, resulting thinner and smoother surfaces. The IR data suggested that hydrolysis-induced deformation did not occur onto the remnants salivary films. The processes of papain hydrolysis of WS films can be controlled by properly regulating pH, ionic strength and temperature.

Human milk xanthine oxidase and neonatal salivary nucleotide precursors generate hydrogen peroxide; a novel pathway with potential role in regulating oral microflora

Background: Xanthine oxidase (XO) is a complex molybdeno-flavoprotein occurring with high activity in the milk fat globule membrane (MFGM) in all mammalian milk and is involved in the final stage of degradation of purine nucleotides. It catalyzes the sequential oxidation of hypoxanthine to xanthine and uric acid, accompanied by production of hydrogen peroxide and superoxide anion. Human saliva has been extensively described for its composition of proteins, electrolytes, cortisol, melatonin and some metabolites such as amino acids, but little is known about nucleotide metabolites. Method: Saliva was collected with swabs from babies; at full-term 1-4 days, 6-weeks, 6-months and 12-months. Unstimulated fasting (morning) saliva samples were collected directly from 77 adults. Breast milk was collected from 24 new mothers. Saliva was extracted from swabs and ultra-filtered. Nucleotide metabolites were analyzed by RP-HPLC with UV-photodiode array and ESI-MS/MS. XO activity was measured as peroxide production from hypoxanthine. Bacterial inhibition over time was assessed using CFU/mL or OD. Results: Median concentrations (μmol/L) of salivary nucleobases and nucleosides for neonates/6-weeks/6-months/12-months/adult respectively were: uracil 5.3/0.8/1.4/0.7/0.8, hypoxanthine 27/7.0/1.1/0.8/2.0, xanthine 19/7.0/2.0/2.0/2.0, adenosine 12/7.0/0.9/0.8/0.1, inosine 11/5.0/0.3/0.4/0.2, guanosine 7.0/6.0/0.5/0.4/0.1, uridine 12/0.8/0.3/0.9/0.4. Deoxynucleosides and dihydropyrimidines concentrations were essentially negligible. XO activity (Vmax:mean ± SD) in breast milk was 8.9 ± 6.2 μmol/min/L and endogenous peroxide was 27 ± 12 μmol/L; mixing breast milk with neonate saliva generated ~40 μmol/L peroxide,which inhibited Staphylococcus aureus. Conclusions: Salivary metabolites, particularly xanthine/hypoxanthine, are high in neonates, transitioning to low adult levels between 6-weeks to 6-months (p < 0.001). Peroxide occurs in breast milk and is boosted during suckling as an antibacterial system.

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Saliva as an emerging biofluid for clinical diagnosis and applications of MEMS/NEMS in salivary diagnostics

Saliva as a biological fluid is gaining wider acceptance for diagnosing diseases. The growing interest in saliva as a biological fluid is due to its noninvasiveness, ease of use, cost-effectiveness, and multiple sample collection possibilities as well as minimal risk to health care professionals of contracting infectious organisms such as HIV and Hep B. However, the clinical translation of saliva is hampered by our lack of understanding of the biomolecular transportation from blood into saliva, the diurnal variations of biomolecules present in saliva, and relatively low levels of analytes (100th to a 1000th fold less than in blood). We provide information on the current status of salivary research, salivary diagnostics empowered by nanotechnology, and future prospects in this emerging field of saliva diagnostics.

Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.

Microstructural characterization of electron beam evaporated tungsten oxide films for gas sensing applications

Tungsten trioxide is one of the potential semiconducting materials used for sensing NH3, CO, CH4 and acetaldehyde gases. The current research aims at development, microstructural characterization and gas sensing properties of thin films of Tungsten trioxide (WO3). In this paper, we intend to present the microstructural characterization of these films as a function of post annealing heat treatment. Microstructural and elemental analysis of electron beam evaporated WO3 thin films and iron doped WO3 films (WO3:Fe) have been carried out using analytical techniques such as Transmission electron microscopy, Rutherford Backscattered Spectroscopy and XPS analysis. TEM analysis revealed that annealing at 300oC for 1 hour improves cyrstallinity of WO3 film. Both WO3 and WO3:Fe films had uniform thickness and the values corresponded to those measured during deposition. RBS results show a fairly high concentration of oxygen at the film surface as well as in the bulk for both films, which might be due to adsorption of oxygen from atmosphere or lattice oxygen vacancy inherent in WO3 structure. XPS results indicate that tungsten exists in 4d electronic state on the surface but at a depth of 10 nm, both 4d and 4f electronic states were observed. Atomic force microscopy reveals nanosize particles and porous structure of the film. This study shows e-beam evaporation technique produces nanoaparticles and porous WO3 films suitable for gas sensing applications and doping with iron decreases the porosity and particle size which can help improve the gas selectivity.

Electron beam evaporation of tungsten oxide films for gas sensors

Pure and Iron incorporated nanostructured Tungsten Oxide (WO3) thin films were investigated for gas sensing applications using noise spectroscopy. The WO3 sensor was able to detect lower concentrations (1 ppm-10 ppm) of NH3, CO, CH4 and Acetaldehyde gases at higher operating temperatures between 100oC to 250oC. The response of the WO3 sensor to NH3, CH4 and Acetaldehyde at lower temperatures (50oC-100oC) was significant when the sensor was photo-activated using blue-light emitting diode (Blue-LED). The WO3 with Fe (WO3:Fe) was found to show some response to Acetaldehyde gas only at relatively higher operating temperature (250oC) and gas concentration of 10 ppm.

Electron beam evaporation of tungsten oxide films for gas sensors

Pure and Iron incorporated nanostructured Tungsten Oxide (WO3) thin films were investigated for gas sensing applications using noise spectroscopy. The WO3 sensor was able to detect lower concentrations (1 ppm-10 ppm) of NH3, CO, CH4 and Acetaldehyde gases at operating temperatures between 100 degrees celcius to 250 degrees celcius. The iron doped Tungsten Oxide sensor (WO3:Fe) showed some response to Acetaldehyde gas at relatively higher operating temperature (250 degrees celcius) and gas concentration of 10 ppm. The sensitivity of the WO3 sensor towards NH3, CH4 and Acetaldehyde at lower operating temperatures (50 degrees celcius - 100 degrees celcius) was significant when the sensor was photo-activated using blue-light emitting diode (Blue-LED). From the results, photo-activated WO3 thin film that operates at room temperature appeared to be a promising gas sensor. The overall results indicated that the WO3 sensor exhibited reproducibility for the detection of various gases and the WO3:Fe indicated some response towards Acetaldehyde gas.

Effect of acupressure on thirst in hemodialysis patients

Background: Thirst and dry mouth are common among hemodialysis (HD) patients. This paper reports a study to evaluate the impact of an acupressure program on HD patients’ thirst and salivary flow rates. Methods: The acupressure program included placebo, followed by true acupressure each applied for 4 weeks. Twenty-eight patients (mean age 57.6, SD = 16.13 years) first received a sticker as placebo acupressure at two acupoints CV23 and TE17 three times a week for 4 weeks, and then received true acupressure in the same area for the next 4 weeks. Salivary flow rate and thirst intensity were measured at baseline, during and after treatment completion for both the placebo and true acupressure program. Results: The true acupressure program was associated with significantly increased salivary flow rate (0.09 ± 0.08 ml/min at baseline to 0.12 ± 0.08 ml/min after treatments completion, p = 0.04). The mean thirst intensity also improved from 4.21 ± 2.66 at baseline to 2.43 ± 2.32 (p = 0.008) after treatment completion in HD patients. There was no statistically significant difference in pre-post program salivary flow rate; however, significant improvement in thirst intensity scores was observed (p = 0.009) in the placebo acupressure program. Conclusion: This study provides preliminary evidence that acupressure may be effective in improving salivary flow rates and thirst intensity.

The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18–30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.

Evolutionary genetics and human assisted movement of a globally invasive pest (Russian wheat aphid : Diuraphis noxia)

This PhD study has examined the population genetics of the Russian wheat aphid (RWA, Diuraphis noxia), one of the world’s most invasive agricultural pests, throughout its native and introduced global range. Firstly, this study investigated the geographic distribution of genetic diversity within and among RWA populations in western China. Analysis of mitochondrial data from 18 sites provided evidence for the long-term existence and expansion of RWAs in western China. The results refute the hypothesis that RWA is an exotic species only present in China since 1975. The estimated date of RWA expansion throughout western China coincides with the debut of wheat domestication and cultivation practices in western Asia in the Holocene. It is concluded that western China represents the limit of the far eastern native range of this species. Analysis of microsatellite data indicated high contemporary gene flow among northern populations in western China, while clear geographic isolation between northern and southern populations was identified across the Tianshan mountain range and extensive desert regions. Secondly, this study analyzed the worldwide pathway of invasion using both microsatellite and endosymbiont genetic data. Individual RWAs were obtained from native populations in Central Asia and the Middle East and invasive populations in Africa and the Americas. Results indicated two pathways of RWA invasion from 1) Syria in the Middle East to North Africa and 2) Turkey to South Africa, Mexico and then North and South America. Very little clone diversity was identified among invasive populations suggesting that a limited founder event occurred together with predominantly asexual reproduction and rapid population expansion. The most likely explanation for the rapid spread (within two years) from South Africa to the New World is by human movement, probably as a result of the transfer of wheat breeding material. Furthermore, the mitochondrial data revealed the presence of a universal haplotype and it is proposed that this haplotype is representative of a wheat associated super-clone that has gained dominance worldwide as a result of the widespread planting of domesticated wheat. Finally, this study examined salivary gland gene diversity to determine whether a functional basis for RWA invasiveness could be identified. Peroxidase DNA sequence data were obtained for a selection of worldwide RWA samples. Results demonstrated that most native populations were polymorphic while invasive populations were monomorphic, supporting previous conclusions relating to demographic founder effects in invasive populations. Purifying selection most likely explains the existence of a universal allele present in Middle Eastern populations, while balancing selection was evident in East Asian populations. Selection acting on the peroxidase gene may provide an allele-dependent advantage linked to the successful establishment of RWAs on wheat, and ultimately their invasion potential. In conclusion, this study is the most comprehensive molecular genetic investigation of RWA population genetics undertaken to date and provides significant insights into the source and pathway of global invasion and the potential existence of a wheat-adapted genotype that has colonised major wheat growing countries worldwide except for Australia. This research has major biosecurity implications for Australia’s grain industry.

Variability in odour reception in the peripheral sensory system of Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

Mixtures of single odours were used to explore the receptor response profile across individual antennae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Seven odours were tested including floral and green-leaf volatiles: phenyl acetaldehyde, benzaldehyde, β-caryophyllene, limonene, α-pinene, 1-hexanol, 3Z-hexenyl acetate. Electroantennograms of responses to paired mixtures of odours showed that there was considerable variation in receptor tuning across the receptor field between individuals. Data from some moth antennae showed no additivity, which indicated a restricted receptor profile. Results from other moth antennae to the same odour mixtures showed a range of partial additivity. This indicated that a wider array of receptor types was present in these moths, with a greater percentage of the receptors tuned exclusively to each odour. Peripheral receptor fields show variation in the spectrum of response within a population (of moths) when exposed to high doses of plant volatiles. This may be related to recorded variation in host choice within moth populations as reported by other authors.