Folate deficiency and DNA-methyltransferase inhibition modulate G-quadruplex frequency.

G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20nM folate medium compared with 2000nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza-2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by γH2AX immunofluorescence (r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level.

γH2AX responses in human buccal cells exposed to ionizing radiation.

DNA double strand breaks are induced by ionizing radiation (IR), leading to the phosphorylation of the core histone protein H2AX (termed γH2AX). The understanding of the γH2AX responses in irradiated human buccal cells is still very limited. We used visual scoring and laser scanning cytometry (LSC) methods to investigate γH2AX signaling following exposure of human buccal cells (from six individuals) to ionizing radiation at 0-4 Gy. The frequency of nuclei containing 15-30 γH2AX foci was significantly elevated 30 min post-IR exposure (by visual scoring). Concomitantly, there was a significant decrease in the frequency of cells without foci following exposure to IR. IR-induced γH2AX signal as determined by laser scanning cytometry (which included γH2AX integral and MaxPixel value) increased significantly in all individual's 2N nuclei 30 min post-IR and was similar for all three nuclear shapes identified. Individuals with the lowest baseline γH2AX integral (i.e., in nonirradiated cells) showed the greatest fold stimulation of γH2AX and significant dose-responses to IR doses of 1, 2, and 4 Gy. In 5 out of 6 individuals, the frequency of visually scored γH2AX in nuclei showed a strong correlation (up to r = 0.999) with LSC scored γH2AX integrals. The γH2AX response and subsequent decline varied between individuals but remained elevated above baseline levels 24 h post IR exposure. γH2AX response in irradiated human buccal cells has potential to be used as an index of baseline DNA damage in population studies. The variable response to IR exposure between individuals should be taken into consideration when using the γH2AX assay for radiation biodosimetry.

Altered cytological parameters in buccal cells from individuals with mild cognitive impairment and Alzheimer's disease.

Previous studies have shown that mild cognitive impairment (MCI) may be reflective of the early stages of more pronounced neurodegenerative disorders such as Alzheimer's disease (AD). There is a need for a minimally invasive and inexpensive diagnostic to identify those who exhibit cellular pathology indicative of MCI and AD risk so that they can be prioritized for primary preventative measures. The hypothesis was that a minimally invasive approach using cytological markers in isolated buccal mucosa cells can be used to identify individuals of both MCI and AD. An automated buccal cell assay was developed using laser scanning cytometry (LSC) to measure buccal cell type ratios, nuclear DNA content and shape, and neutral lipid content of buccal cells from clinically diagnosed AD (n = 13) and MCI (n = 13) patients prior to treatment compared to age- and gender-matched controls (n = 26). DNA content was significantly higher in all cell types in both MCI (P < 0.01) and AD (P < 0.05) compared with controls mainly due to an increase in >2N nuclei. Abnormal nuclear shape (circularity) was significantly increased in transitional cells in MCI (P < 0.001) and AD (P < 0.01) when compared to controls. In contrast, neutral lipid content (as measured by Oil red O "ORO" staining) of buccal cells was significantly lower in the MCI group (P < 0.05) compared with the control group. The ratio of DNA content/ORO in buccal basal cells for both MCI and AD was significantly higher compared to the control group, with ratios for MCI being approximately 2.8-fold greater (P < 0.01) and AD approximately 2.3-fold greater (P < 0.05) than the control group. Furthermore, there was a strong negative correlation between buccal cell DNA content and ORO content in the AD group (r(2) = 0.75, P < 0.0001) but not in MCI or controls. The changes in the buccal cell cytome observed in this study could prove useful as potential biomarkers in identifying individuals with an increased risk of developing MCI and eventually AD.

Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homologue (H2AvB).

The response of eukaryotic cells to ionising radiation (IR)-induced double-strand DNA breaks is highly conserved and involves a DNA repair mechanism characterised by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). Although the expression of an induced γH2AX variant has been detected in Drosophila melanogaster, the expression and radiation response of a γH2AX homologue has not been reported in economically important fruit flies. We use Bactrocera tryoni (Diptera: Tephritidae, Queensland fruit fly or 'Q-fly') to investigate this response with a view to developing molecular assays to detect/quantify exposure of fruit flies to IR and consequent DNA damage. Deep sequencing confirmed the presence of a H2AX homologue that we have termed H2AvB (i.e. variant Bactrocera) and has an identical sequence to a histone reported from the human disease vector Glossina morsitans. A linear dose-response of γH2AvB (0-400 Gy IR) was observed in whole Q-fly pupal lysates 24h post-IR and was detected at doses as low as 20 Gy. γH2AvB signal peaked at ~20min after IR exposure and at 24h post-IR the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by western blot or by laser scanning cytometry up to 17 days post-IR exposure in histone extracts or isolated nuclei from adult Q-flies (irradiated as pupae). We conclude that IR exposure in Q-fly leads to persistent γH2AvB signals (over a period of days) that can easily be detected by western blot or quantitative immunofluorescence techniques. These approaches have potential as the basis for assays for detection and quantification of prior IR exposure in pest fruit flies.

Identification of an in vitro interaction between an insect immune suppressor protein (CrV2) and G alpha proteins.

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gßγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gα(o) compared with rat Gα(i1). In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

How Healthy Are Non-Traditional Dietary Proteins? The Effect of Diverse Protein Foods on Biomarkers of Human Health.

Future food security for healthy populations requires the development of safe, sustainably-produced protein foods to complement traditional dietary protein sources. To meet this need, a broad range of non-traditional protein foods are under active investigation. The aim of this review was to evaluate their potential effects on human health and to identify knowledge gaps, potential risks, and research opportunities. Non-traditional protein sources included are algae, cereals/grains, fresh fruit and vegetables, insects, mycoprotein, nuts, oil seeds, and legumes. Human, animal, and in vitro data suggest that non-traditional protein foods have compelling beneficial effects on human health, complementing traditional proteins (meat/poultry, soy, eggs, dairy). Improvements in cardiovascular health, lipid metabolism, muscle synthesis, and glycaemic control were the most frequently reported improvements in health-related endpoints. The mechanisms of benefit may arise from their diverse range of minerals, macro- and micronutrients, dietary fibre, and bioactive factors. Many were also reported to have anti-inflammatory, antihypertensive, and antioxidant activity. Across all protein sources examined, there is a strong need for quality human data from randomized controlled intervention studies. Opportunity lies in further understanding the potential effects of non-traditional proteins on the gut microbiome, immunity, inflammatory conditions, DNA damage, cognition, and cellular ageing. Safety, sustainability, and evidence-based health research will be vital to the development of high-quality complementary protein foods that enhance human health at all life stages.

Salivary inflammatory biomarkers are predictive of mild cognitive impairment and Alzheimer's disease in a feasibility study.

Alzheimer's disease (AD) is an insidious disease. Its distinctive pathology forms over a considerable length of time without symptoms. There is a need to detect this disease, before even subtle changes occur in cognition. Hallmark AD biomarkers, tau and amyloid-ß, have shown promising results in CSF and blood. However, detecting early changes in these biomarkers and others will involve screening a wide group of healthy, asymptomatic individuals. Saliva is a feasible alternative. Sample collection is economical, non-invasive and saliva is an abundant source of proteins including tau and amyloid-ß. This work sought to extend an earlier promising untargeted mass spectrometry study in saliva from individuals with mild cognitive impairment (MCI) or AD with age- and gender-matched cognitively normal from the South Australian Neurodegenerative Disease cohort. Five proteins, with key roles in inflammation, were chosen from this study and measured by ELISA from individuals with AD (n = 16), MCI (n = 15) and cognitively normal (n = 29). The concentrations of Cystatin-C, Interleukin-1 receptor antagonist, Stratifin, Matrix metalloproteinase 9 and Haptoglobin proteins had altered abundance in saliva from AD and MCI, consistent with the earlier study. Receiver operating characteristic analysis showed that combinations of these proteins demonstrated excellent diagnostic accuracy for distinguishing both MCI (area under curve = 0.97) and AD (area under curve = 0.97) from cognitively normal. These results provide evidence for saliva being a valuable source of biomarkers for early detection of cognitive impairment in individuals on the AD continuum and potentially other neurodegenerative diseases.

Multi-Omics, an Integrated Approach to Identify Novel Blood Biomarkers of Alzheimer's Disease.

The metabolomic and proteomic basis of mild cognitive impairment (MCI) and Alzheimer's disease (AD) is poorly understood, and the relationships between systemic abnormalities in metabolism and AD/MCI pathogenesis is unclear. This study compared the metabolomic and proteomic signature of plasma from cognitively normal (CN) and dementia patients diagnosed with MCI or AD, to identify specific cellular pathways and new biomarkers altered with the progression of the disease. We analysed 80 plasma samples from individuals with MCI or AD, as well as age- and gender-matched CN individuals, by utilising mass spectrometry methods and data analyses that included combined pathway analysis and model predictions. Several proteins clearly identified AD from the MCI and CN groups and included plasma actins, mannan-binding lectin serine protease 1, serum amyloid A2, fibronectin and extracellular matrix protein 1 and Keratin 9. The integrated pathway analysis showed various metabolic pathways were affected in AD, such as the arginine, alanine, aspartate, glutamate and pyruvate metabolism pathways. Therefore, our multi-omics approach identified novel plasma biomarkers for the MCI and AD groups, identified changes in metabolic processes, and may form the basis of a biomarker panel for stratifying dementia participants in future clinical trials.

Laser scanning cytometry for automation of the micronucleus assay.

Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure.

Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability.

INTRODUCTION: Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. METHODS: An intestinal epithelial TLR4 conditional knockout (KO) mouse line (Tlr4ΔIEC, n = 6-8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (n = 5-7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. RESULTS: There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance-ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus Tlr4ΔIEC 20 ± 5.6 (Ω × cm2) p = 0.831, colon WT 30.8 ± 3.6 versus Tlr4ΔIEC 45.1 ± 9.5 p = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)-ZO-1: ileum-WT 1.49 ± 0.155 versus Tlr4ΔIEC 1.17 ± 0.07, p = 0.09; colon-WT 1.36 ± 0.26 versus Tlr4ΔIEC 1.12 ± 0.18 p = 0.47; occludin: ileum-WT 1.07 ± 0.12 versus Tlr4ΔIEC 0.95 ± 0.13, p = 0.53; colon-WT 1.26 ± 0.26 versus Tlr4ΔIEC 1.02 ± 0.16 p = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus Tlr4ΔIEC 0.09 ± 0.01 p = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus Tlr4ΔIEC 0.49 ± 0.08 p = 0.73. CONCLUSIONS: These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the Tlr4ΔIEC line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

The effect of zinc on human trophoblast proliferation and oxidative stress.

Adequate Zinc (Zn) intake is required to prevent multiple teratogenic effects however deviations from adequate Zn intake, including high maternal Zn status, have been linked to increased incidence of pregnancy complications, including those associated with inadequate placentation. Using placental trophoblast HTR8/SVneo cells and first trimester human placental explants (n = 12), we assessed the effects of varying Zn concentrations on trophoblast proliferation, viability, apoptosis and oxidative stress. Compared to physiologically normal Zn levels (20 µM), HTR-8/SVneo cell proliferation index was significantly lower in the presence of physiologically elevated (40 µM; P = .020) and supra-physiological (80 µM; P = .007) Zn. The latter was also associated with reduced proliferation (P = .004) and viability (P < .0001) in cultured placental explants, but not apoptosis. Reactive oxygen species production in HTR8/SVneo cultures was significantly higher in the presence of 80 µM Zn compared to all physiologically relevant levels. Oxidative stress, induced by an oxidizing agent menadione, was further exacerbated by high (80 µM) Zn. Zn did not affect lipid peroxidation in either HTR8/SVneo cells or placental explants or antioxidant defense mechanisms that included glutathione reductase and superoxide dismutase. Further study should focus on elucidating mechanisms behind impaired trophoblast proliferation and increased oxidative stress as a result of elevated Zn levels.

Salivaomics as a Potential Tool for Predicting Alzheimer's Disease During the Early Stages of Neurodegeneration.

BACKGROUND: The metabolomic and proteomic basis of mild cognitive impairment (MCI) and Alzheimer's disease (AD) is poorly understood and the relationships between systemic abnormalities in metabolism and AD/AMCI pathogenesis are unclear. OBJECTIVE: The aim of the study was to compare the metabolomic and proteomic signature of saliva from cognitively normal and patients diagnosed with MCI or AD, to identify specific cellular pathways altered with the progression of the disease. METHODS: We analyzed 80 saliva samples from individuals with MCI or AD as well as age- and gender-matched healthy controls. Saliva proteomic and metabolomic analyses were conducted utilizing mass spectrometry methods and data combined using pathway analysis. RESULTS: We found significant alterations in multiple cellular pathways, demonstrating that at the omics level, disease progression impacts numerous cellular processes. Multivariate statistics using SIMCA showed that partial least squares-data analysis could be used to provide separation of the three groups. CONCLUSION: This study found significant changes in metabolites and proteins from multiple cellular pathways in saliva. These changes were associated with AD, demonstrating that this approach might prove useful to identify new biomarkers based upon integration of multi-omics parameters.

Effect of Iodine and Selenium on Proliferation, Viability, and Oxidative Stress in HTR-8/SVneo Placental Cells.

Adequate maternal micronutrition is vital for placental formation, fetal growth, and development. Oxidative stress adversely affects placental development and function and an association between deficient placental development, oxidative stress, and micronutrient deficiency has been observed. Selenium and iodine are two essential micronutrients with antioxidant properties. Epidemiological studies have shown that poor micronutrient status in pregnant women is associated with a higher incidence of pregnancy complications. The aim of this study was to determine how selenium, iodine, and their combination impact oxidative stress in placental trophoblast cells. HTR8/SVneo extravillous trophoblasts were supplemented with a concentration range of organic and inorganic selenium, potassium iodide, or their combination for 24 h. Oxidative stress was then induced by treating cells with menadione or H2O2 for 24 h. Cell viability and lipid peroxidation as the biomarker of oxidative stress were assessed at 48 h. Both menadione and H2O2 reduced cell viability and increased lipid peroxidation (P < 0.05). Greater cell viability was found in selenium-supplemented cells when compared with vehicle treated cells (P < 0.05). Selenium and iodine supplementation separately or together were associated with lower lipid peroxidation compared with vehicle control (P < 0.05). Supplementation with the combination of selenium and iodine resulted in a greater reduction in lipid peroxidation compared with selenium or iodine alone (P < 0.05). Oxidative stress negatively impacts trophoblast cell survival and cellular integrity. Selenium and iodine protect placental trophoblasts against oxidative stress. Further research is warranted to investigate the molecular mechanisms by which selenium and iodine act in the human placenta.

Evaluation of GammaH2AX in Buccal Cells as a Molecular Biomarker of DNA Damage in Alzheimer's Disease in the AIBL Study of Ageing.

In response to double-stranded breaks (DSBs) in chromosomal DNA, H2AX (a member of histone H2A family) becomes phosphorylated to form γH2AX. Although increased levels of γH2AX have been reported in the neuronal nuclei of Alzheimer's disease (AD) patients, the understanding of γH2AX responses in buccal nuclei of individuals with mild cognitive impairment (MCI) and AD remain unexplored. In the current study, endogenous γH2AX was measured in buccal cell nuclei from MCI (n = 18) or AD (n = 16) patients and in healthy controls (n = 17) using laser scanning cytometry (LSC). The γH2AX level was significantly elevated in nuclei of the AD group compared to the MCI and control group, and there was a concomitant increase in P-trend for γH2AX from the control group through MCI to the AD group. Receiver-operating characteristic curves were carried out for different γH2AX parameters; γH2AX in nuclei resulted in the greatest area under the curve value of 0.7794 (p = 0.0062) with 75% sensitivity and 70% specificity for the identification of AD patients from control. In addition, nuclear circularity (a measure of irregular nuclear shape) was significantly higher in the buccal cell nuclei from the AD group compared with the MCI and control groups. Additionally, there was a positive correlation between the nuclear circularity and γH2AX signals. The results indicated that increased DNA damage is associated with AD.

G-protein coupled receptor array technologies: site directed immobilisation of liposomes containing the H1-histamine or M2-muscarinic receptors.

This paper describes a novel strategy to create a microarray of G-protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H(1)-histamine receptor and the M(2)-muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol-modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM-D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the Zepto-READER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self-sorting liposome array of GPCRs which would underpin a variety of future novel applications.

[35S]GTPgammaS binding in G protein-coupled receptor assays.

The [(35)S]GTPgammaS binding assay to measure G protein activation following agonist binding to G protein-coupled receptors (GPCRs) remains a powerful molecular technique to substantiate traditional pharmacological values of potency, efficacy, and affinity. The method described uses membrane preparations of the alpha(2A)-adrenergic receptor and purified G protein subunits expressed in Sf9 cells, reconstituted into a functional signaling system. This technology is generic and could be used with other GPCRs to demonstrate initial signaling events following receptor activation. Agonist-stimulated [(35)S]GTPgammaS binding is measured in a 96-well plate format using scintillation counting.

GPCR expression using baculovirus-infected Sf9 cells.

Expression of proteins in insect cells using recombinant baculoviruses has gained wide use in the G protein-coupled receptor (GPCR) community. This expression system produces high yields of functional receptor, is able to perform post-translational modifications, and is readily adaptable to large-scale culture. Here, we describe the generic methods for expressing a GPCR using baculovirus-infected insect cells, including the maintenance of insect cell culture. Data are presented for polyhedrin promoter-driven expression of a C-terminal 6 x histidine-tagged mammalian M(2) muscarinic receptor in Sf9 cells. Results demonstrate that expressed receptor could be detected and quantified using radiolabeled ligand binding, that expression was maximal at approximately 72 h post-infection, and that expression levels could be altered by addition of various ligands to cultures of infected insect cells.

Radioligand binding assays: application of [(125)I]angiotensin II receptor binding.

Angiotensin II (AngII) is an octapeptide hormone with a key role in blood pressure regulation. AngII increases blood pressure by stimulating G protein-coupled receptors in vascular smooth muscle. AngII receptors are therefore an important target in patients with high blood pressure. Strategies to lower high blood pressure (hypertension) include the use of drugs that compete for AngII at the angiotensin II Type 1 receptors (ATR) using ATR antagonists (e.g., irbesartan, valsartan, and losartan). This chapter will demonstrate the subtype specificity of ATR binding and we discuss some of the key experiments that are necessary in optimizing some of the parameters for GPCR screening. The latter protocols include saturation binding to determine K (d) and B (max), as well as competition/inhibition experiments to determine the IC(50) of binding. For these experiments we have used rat liver membranes which express ATR (type 1a) in relatively abundant amounts. Additionally, rat liver membrane preparations can be easily prepared in "bulk," frozen away for extended periods (up to 1 year) and used when necessary with no loss of receptor binding activity using the radiolabeled angiotensin II analogue, [(125)I][Sar(1),I le(8)]AngII.

Current State of Saliva Biomarkers for Aging and Alzheimer's Disease.

INTRODUCTION: Aging is the primary risk factor for major human pathologies, including cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases such as Alzheimer's Disease (AD). AD is a progressive degenerative disorder of the brain and is the most common form of dementia. METHODS: To-date no simple, inexpensive and minimally invasive procedure is available to confirm with certainty the early diagnosis of AD prior to the manifestations of symptoms characteristic of the disease. Therefore, if population screening of individuals is to be performed, easily accessible tissues would need to be used for a diagnostic test that would identify those who exhibit altered or aberrant aging profiles that may be indicative of AD risk, so that they can be prioritized for primary prevention. This need for minimally invasive tests could be achieved by targeting saliva, since it is now well recognized that many aging diseases including AD are associated with peripheral biomarkers that are not only restricted to pathology and biomarkers within the brain. RESULTS: Therefore, the aim of this review is to summarize some of the main findings of salivary biomarkers of aging and AD; including various proteins, metabolites, and alterations to DNA and miRNA. The future of healthy aging resides in innovative platforms, biosensors and point-of-care devices that can extract real time information on the health status of an individual. Those platforms may be achieved through the development and validation of novel biomarkers of health using saliva which, although being the least explored for biomedical purposes, has the distinct advantage that it can be self-collected in a non-invasive manner.