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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.

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.

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

An early cellular response to DNA double-strand breaks is the phosphorylation of histone H2AX to form γH2AX. Although increased levels of γH2AX have been reported in neuronal nuclei of Alzheimer’s disease (AD) patients, γH2AX responses in the lymphocytes of individuals with mild cognitive impairment (MCI) and AD remain unexplored. In this study, the endogenous γH2AX level was measured, using laser scanning cytometry (LSC) and visual scoring, in lymphocyte nuclei from MCI (n = 18), or AD (n = 20) patients and healthy controls (n = 40). Levels were significantly elevated in nuclei of the AD group compared to the MCI and control groups, and there was a concomitant increase, with a significant trend, from the control group through MCI to the AD group. A significant negative correlation was seen between γH2AX and the mini mental state examination (MMSE) score, when the analysis included all subjects. Receiver Operation Characteristic curves were carried out for different γH2AX parameters; visually scored percent cells containing overlapping γH2AX foci displayed the best area under the curve value of 0.9081 with 85% sensitivity and 92% specificity for the identification of AD patients versus control. Plasma homocysteine, creatinine, and chitinase-3-like protein 1 (CHI3L1) were positively correlated with lymphocyte γH2AX signals, while glomerular filtration rate (GFR) was negatively correlated. Finally, there was a diminished γH2AX response to X-rays in lymphocytes of the MCI and AD groups compared to the control group. Our results indicate that lymphocyte γH2AX levels are a potential marker for identifying individuals at increased risk of developing AD. Prospective studies with normal healthy individuals are needed to test whether there is indeed a link between γH2AX levels and AD risk.

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n = 5) and healthy individuals (n = 5) were cultured in RPMI medium under condition of folate deficiency (20 nM) or sufficiency (200 nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and γH2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p 0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20 nM folate media also showed significant increases in G4 (p 0.001) and γH2AX (p 0.01) signals compared with the same cells grown in 200 nM folate. Control cells grown in 20 nM folate also showed a significant reduction in DNA methylation levels (P 0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.

The placenta is an important organ in pregnancy, however, very little is understood about placental development at a molecular level. This includes the role of epigenetic mechanisms and how they change throughout gestation. DNA methylation studies in this organ are complicated by the different cell types that make up the placenta, each with their own unique transcriptome and epigenome. Placental dysfunction is often associated with pregnancy complications such as preeclampsia (PE). Aberrant DNA methylation in the placenta has been identified in pregnancy complications. We used immunohistochemistry (IHC) and immunofluorescence (IF) to localize 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in placenta tissue from first and second trimester as well as uncomplicated term and PE samples. IHC analysis of whole placental tissues showed 5-mC increased across gestation. When cytotrophoblasts (CTB) and syncytiotrophoblasts (STB) were isolated and assessed using IF, both 5-mC and 5-hmC increased in term CTBs compared to first/second-trimester samples. Staining intensity of 5-hmC was higher in first/second trimester STBs compared to CTBs (P = 0.0011). Finally, IHC staining of term tissue from PE and uncomplicated pregnancies revealed higher 5-mC staining intensity in placentas from PE pregnancies (P = 0.028). Our study has shown increased 5-mC and 5-hmC staining intensities across gestation and differed between two trophoblast populations. Differences in DNA methylation profiles between placental cell types may be indicative of different functions and requires further study to elucidate what changes accompany placental pathologies.

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.

Guanine-quadruplexes (G4) are highly stable DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 are known to be associated with DNA damage and we propose that G4 are involved in the ageing disorder mild cognitive impairment (MCI). Lymphocytes were obtained from healthy controls and individuals with MCI. The intensity and frequency of G4 foci as well as γH2AX (a marker of DNA damage) intensity were measured by quantitative immunofluorescence and were correlated with cognitive function and cytokinesis-block micronucleus cytome markers of DNA damage. γH2AX intensity as well as G4 frequency and intensity were significantly elevated in MCI lymphocytes compared to controls. The combined biomarker panel was tested in a predictive statistical model, which improved the demarcation of MCI from controls with 80.3% accuracy. The results obtained from this pilot study showed for the first time that G4 levels are increased with cognitive impairment and thus, may be involved in the early development of Alzheimer's disease possibly via an association with chromosomal DNA damage and DNA double strand breaks.

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.