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.

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.

Infant birth outcomes are associated with DNA damage biomarkers as measured by the cytokinesis block micronucleus cytome assay: the DADHI study.

Accumulation of DNA damage in the first 1000 days may increase risk of accelerated ageing and degenerative diseases in adult life such as cancers. The extent of DNA damage in infants and the correlation of maternal factors during pregnancy with neonate birth outcomes and DNA damage is not known in infants born in Australia. Therefore, we performed a prospective cohort study to collect data on DNA damage in lymphocytes of Australian infants (aged 0, 3 and 6 months), using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay. The study also explored correlation of CBMN-Cyt biomarkers with infant birth outcomes and maternal anthropometric and lifestyle variables. Peripheral blood lymphocytes were isolated from the infants at birth (cord blood) (n = 82), 3 months (n = 64) and 6 months (n = 53) after birth. DNA damage biomarkers measured ex vivo in binucleated lymphocytes (BNC) included: micronuclei (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUD). Apoptotic and necrotic lymphocytes were also scored and nuclear division index (NDI) was measured using the frequency of mono-, bi- and multinucleated lymphocyte. MN and NBUD were also scored in mononucleated lymphocytes (MNC). The mean (± SD) frequency of MN, NPB and NBUD in BNCs at birth was 2.0 (± 1.2), 5.8 (± 3.7) and 11.1 (± 5.7) per 1000 BNC, respectively, and tended to decrease significantly at 3 months (P < 0.01, P < 0.0001, P < 0.001, respectively) and 6 months (P < 0.05, P < 0.0001, P < 0.0001, respectively) after birth relative to cord blood when compared with the same cohort of infants (n = 48 at birth, 48 at 3 months and 39 at 6 months). None of the CBMN cytome biomarkers measured at birth was associated with maternal smoking status, alcohol and folic acid intake during pregnancy. The mean gestation age correlated positively with MN (r = 0.38, P = 0.006), NPB (r = 0.30, P = 0.03) and negatively with NDI (r = -0.29, P = 0.03). Infant birth weight associated positively with MN, NPB and NBUD in cord blood (r = 0.24, P = 0.08; r = 0.32, P = 0.02; r = 0.28, P = 0.04, respectively), birth length associated positively with NPB (r = 0.32, P = 0.02) and NBUD (r = 0.27, P = 0.04) while head circumference associated negatively with apoptotic cells (r = -0.27, P = 0.06). APGAR score at 1 and 5 min after birth associated positively with NDI at birth (r = 0.3, P = 0.05, r = 0.28, P = 0.06, respectively). Mother's weight and body mass index (BMI) recorded at the time of recruitment associated positively with NPB (r = 0.38, P = 0.006, r = 0.32, P = 0.02, respectively) and negatively with APGAR score at 5 min (r = -0.25, P = 0.07). The significant positive associations of infant birth weight and length and maternal BMI with CBMN-Cyt biomarkers suggest the possibility of a genotoxic effect of metabolic processes that promote excessive growth and high BMI.

Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international inter-laboratory scoring exercise.

The buccal micronucleus cytome (BMNcyt) assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and follow-up of precancerous oral lesions. The main limitation of the assay is the large variability observed in the baseline values of micronuclei (MNi) and other nuclear anomalies mainly related to different scoring criteria. The aim of this international collaborative study, involving laboratories with different level of experience, was to evaluate the inter- and intra-laboratory variations in the BMNcyt parameters, using recently implemented guidelines, in scoring cells from the same pooled samples obtained from healthy subjects (control group) and from cancer patients undergoing radiotherapy (treated group). The results indicate that all laboratories correctly discriminated samples from the two groups by a significant increase of micronucleus (MN) and nuclear bud (NBUD) frequencies and differentiated binucleated (BN) cells, associated with the exposure to ionizing radiation. The experience of the laboratories was shown to play an important role in the identification of the different cell types and nuclear anomalies. MN frequency in differentiated mononucleated (MONO) and BN cells showed the greatest consistency among the laboratories and low variability was also detected in the frequencies of MONO and BN cells. A larger variability was observed in classifying the different cell types, indicating the subjectivity in the interpretation of some of the scoring criteria while reproducibility of the results between scoring sessions was very good. An inter-laboratory calibration exercise is strongly recommended before starting studies with BMNcyt assay involving multiple research centers.

High Content, Multi-Parameter Analyses in Buccal Cells to Identify Alzheimer's Disease.

Alzheimer's disease (AD) is a degenerative brain disorder and is the most common form of dementia. Minimally invasive approaches are required that combine biomarkers to identify individuals who are at risk of developing mild cognitive impairment (MCI) and AD, to appropriately target clinical trials for therapeutic discovery as well as lifestyle strategies aimed at prevention. Buccal mucosa cells from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing cohort (n=60) were investigated for cytological markers that could be used to identify both MCI and AD individuals. Visual scoring of the buccal cytome demonstrated a significantly lower frequency of basal and karyorrhectic cells in the MCI group compared with controls. A high content, automated assay was developed using laser scanning cytometry to simultaneously measure cell types, nuclear DNA content and aneuploidy, neutral lipid content, putative Tau and amyloid-ß (Aß) in buccal cells. DNA content, aneuploidy, neutral lipids and Tau were similar in all groups. However, there was significantly lower Tau protein in both basal and karyolytic buccal cell types compared with differentiated buccal cells. Aß, as measured by frequency of cells containing Aß signal, as well as area and integral of Aß signal, was significantly higher in the AD group compared with the control group. Buccal cell Aß was correlated with mini-mental state examination (MMSE) scores (r = -0.436, P=0.001) and several blood-based biomarkers. Combining newly identified biomarkers from buccal cells with those already established may offer a potential route for more specific biomarker panels which may substantially increase the likelihood of better predictive markers for earlier diagnosis of AD.

Buccal micronucleus cytome assay: results of an intra- and inter-laboratory scoring comparison.

The buccal micronucleus cytome (BMCyt) assay is a minimally invasive approach for measuring DNA damage, cell proliferation, cell differentiation and cell death in exfoliated buccal cells. The main limitation for its use is the lack of knowledge about inter- and intra-laboratory variability in scoring micronuclei and other end points included in the cytome approach. In order to identify the main sources of variability across the BMCyt biomarkers, a scoring exercise was carried out between three experienced laboratories using the same set of slides and an identical set of detailed scoring criteria and associated images for the different end points. Single batches of slides were prepared from pooled samples of four groups of subjects characterised by different frequencies of cell types and micronuclei, namely Down syndrome patients, head and neck cancer patients undergoing radiotherapy and two age- and gender-matched control groups. A good agreement among the laboratories in the identification of normal differentiated cells and of micronuclei was obtained. A 3-fold and 20-fold increase in the frequency of micronucleated cells and micronuclei in differentiated cells of Down syndrome patients and in cancer patients, respectively, compared to matched controls, was a consistent result in the three laboratories. The scores of other cell types and nuclear anomalies, such as basal, binucleated, condensed chromatin and karyorrhectic cells showed significant disagreement between and within laboratories indicating that their evaluation using the current visual scoring protocol does not yield robust results for these parameters. The guidelines for BMCyt assay application could be improved by combining the anomalies associated with cell death (condensed chromatin and karyorrhectic cells) in a single category and by defining more stringent criteria in classifying basal cell, binucleated cells and buds.

DNA melting and genotoxicity induced by silver nanoparticles and graphene.

We have revealed a connection between DNA-nanoparticle (NP) binding and in vitro DNA damage induced by citrate- and branched polyethylenimine-coated silver nanoparticles (c-AgNPs and b-AgNPs) as well as graphene oxide (GO) nanosheets. All three types of nanostructures triggered an early onset of DNA melting, where the extent of the melting point shift depends upon both the type and concentration of the NPs. Specifically, at a DNA/NP weight ratio of 1.1/1, the melting temperature of lambda DNA dropped from 94 °C down to 76 °C, 60 °C, and room temperature for GO, c-AgNPs and b-AgNPs, respectively. Consistently, dynamic light scattering revealed that the largest changes in DNA hydrodynamic size were also associated with the binding of b-AgNPs. Upon introduction to cells, b-AgNPs also exhibited the highest cytotoxicity, at the half-maximal inhibitory (IC50) concentrations of 3.2, 2.9, and 5.2 mg/L for B and T-lymphocyte cell lines and primary lymphocytes, compared to the values of 13.4, 12.2, and 12.5 mg/L for c-AgNPs and 331, 251, and 120 mg/L for GO nanosheets, respectively. At cytotoxic concentrations, all NPs elicited elevated genotoxicities via the increased number of micronuclei in the lymphocyte cells. However, b-AgNPs also induced micronuclei at subtoxic concentrations starting from 0.1 mg/L, likely due to their stronger cellular adhesion and internalization, as well as their subsequent interference with normal DNA synthesis or chromosome segregation during the cell cycle. This study facilitates our understanding of the effects of NP chemical composition, surface charge, and morphology on DNA stability and genotoxicity, with implications ranging from nanotoxicology to nanobiotechnology and nanomedicine.

Workers exposed to wood dust have an increased micronucleus frequency in nasal and buccal cells: results from a pilot study.

Wood dust is recognised as a human carcinogen, based on the strong association of wood dust exposure and the elevated risk of malignant tumours of the nasal cavity and paranasal sinuses [sino-nasal cancer (SNC)]. The study aimed to assess genetic damage in workers exposed to wood dust using biomarkers in both buccal and nasal cells that reflect genome instability events, cellular proliferation and cell death frequencies. Nasal and buccal epithelial cells were collected from 31 parquet layers, installers, carpenters and furniture workers (exposed group) and 19 non-exposed workers located in Switzerland. Micronucleus (MN) frequencies were scored in nasal and buccal cells collected among woodworkers. Other nuclear anomalies in buccal cells were measured through the use of the buccal micronucleus cytome assay. MN frequencies in nasal and buccal cells were significantly higher in the exposed group compared to the non-exposed group; odds ratio for nasal cells 3.1 [95% confidence interval (CI) 1.8-5.1] and buccal cells 1.8 (95% CI 1.3-2.4). The exposed group had higher frequencies of cells with nuclear buds, karyorrhectic, pyknotic, karyolytic cells and a decrease in the frequency of basal, binucleated and condensed cells compared to the non-exposed group. Our study confirms that woodworkers have an elevated risk for chromosomal instability in cells of the aerodigestive tract. The MN assay in nasal cells may become a relevant biomonitoring tool in the future for early detection of SNC risk. Future studies should seek to standardise the protocol for MN frequency in nasal cells similar to that for MN in buccal cells.

Sorted cell microarrays as platforms for high-content informational bioassays.

We report on surface-engineered microarrays that provide in situ cell sorting, localization, and immobilization of various subsets of human primary lymphocytes, followed by an on-chip bioassay for ionizing-radiation-induced cytogenetic damage. The microarray format eliminates the necessity of separating cell sub-populations by alternative means (such as fluorescence- or magnetic-activated cell sorting) prior to performing informational bioassays. To exemplify the potential of this on-chip cytometry approach, we have integrated the cytokinesis-block micronucleus cytome (CBMNcyt) assay with the microarray platform for analysis of the chromosome damage profile of specific subsets of human peripheral lymphocytes. Microarray results were compared with data obtained from the traditional CBMNcyt assay on heterogeneous lymphocyte populations, and with flow cytometry data. Our results suggest that cytogenetic damage caused by ionizing radiation is not uniformly distributed across all lymphocytes subsets, but rather concentrated in specific subsets. The salient features of our approach are that it requires very small volumes of reagents, allows sorting of lymphocyte subsets in situ, increases parallelism of cell assays and is amenable to high content microscopy analysis. The on-chip cytometry format opens new vistas for advanced cell-based assays, potentially bringing to light important information which remains hidden with conventional assays and hence engendering new discoveries in cell biology.

Cell microarrays for the screening of factors that allow the enrichment of bovine testicular cells.

Cell microarrays can serve as high-throughput platforms for the screening of a diverse range of biologically active factors and biomaterials that can induce desired cellular responses such as attachment, proliferation, or differentiation. Here, we demonstrate that surface-engineered microarrays can be used for the screening and identification of factors that allow the enrichment and isolation of rare cells from tissue-derived heterogeneous cell populations. In particular, we have focused on the enrichment of bovine testicular cells including type A spermatogonia and Sertoli cells. Microarray slides were coated with a copolymer synthesized from poly(ethylene glycol) methacrylate and glycidyl methacrylate to enable both the prevention of cell attachment between printed spots and the covalent anchoring of various factors such as antibodies, lectins, growth factors, extracellular matrix proteins, and synthetic macromolecules on printed spots. Microarrays were incubated with mixed cell populations from freshly isolated bovine testicular tissue. Overall, cell attachment was evaluated using CellTracker staining, whereas differential attachment of testicular cells was determined by immunohistochemistry staining with Plzf and vimentin antibodies as markers for type A spermatogonia and Sertoli cells, respectively. The results indicate that various surface immobilized factors, but in particular Dolichos biflorus lectin, allowed the enrichment of Plzf positive cells. Furthermore, Pisum sativum lectin, concanavalin A, collagen type IV, and vitronectin were identified as suitable negative selection factors. To our best knowledge, this work is the first to demonstrate the utility of surface engineered cell-based microarrays for the identification of factors that allow the selective capture of rare cells from tissue isolated heterogeneous mixtures.