Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic MARKers (LANDMark): Baseline findings

Purpose:Over the past decade, corneal nerve morphology and corneal sensation threshold have been explored as potential surrogate markers for the evaluation of diabetic neuropathy. We present the baseline findings of a Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic Markers (LANDMark). Methods:The LANDMark Study is a 5-year, two-site, natural history (observational) study of individuals with Type 1 diabetes stratified into those with (T1W) and without (T1WO) neuropathy according to the Toronto criteria, and control subjects. All study participants undergo detailed annual assessment of neuropathy including corneal nerve parameters measured using corneal confocal microscopy and corneal sensitivity measured using non-contact corneal esthesiometry. Results:396 eligible individuals (208 in Brisbane and 188 in Manchester) were assessed: 76 T1W, 166 T1WO and 154 controls. Corneal sensation threshold (mbars) was significantly higher in T1W (1.0 ± 1.1) than T1WO (0.7 ± 0.7) and controls (0.6 ± 0.4) (P=0.002); post-hoc analysis (PHA) revealed no difference between T1WO and controls (Tukey HSD, P=0.502). Corneal nerve fiber length (mm/mm2) (CNFL) was lower in T1W (13.8 ± 6.4) than T1WO (19.1 ± 5.8) and controls (23.2 ± 6.3) (P<0.001); PHA revealed CNFL to be lower in T1W than T1WO, and lower in both of these groups than controls (P<0.001). Corneal nerve branch density (branches/mm2) (CNBD) was significantly lower in T1W (40 ± 32) than T1WO (62 ± 37) and controls (83 ± 46) (P<0.001); PHA showed CNBD was lower in T1W than T1WO, and lower in both groups than controls (P<0.001). Alcohol and cigarette consumption did not differ between groups, although age, BMI, BP, waist circumference, HbA1c, albumin-creatinine ratio, and cholesterol were slightly greater in T1W than T1WO (p<0.05). Some site differences were observed. Conclusions:The LANDMark baseline findings confirm that corneal sensitivity and corneal nerve morphometry can detect differences in neuropathy status in individuals with Type 1 diabetes and healthy controls. Corneal nerve morphology is significantly abnormal even in diabetic patients ‘without neuropathy’ compared to control participants. Results of the longitudinal trial will assess the capability of these tests for monitoring change in these parameters over time as potential surrogate markers for neuropathy.

Exploring Retinal Markers of Diabetic Neuropathy

Purpose : To investigate the application of retinal nerve fibre layer (RNFL) thickness as a marker for severity of diabetic peripheral neuropathy (DPN) in people with Type 2 diabetes. Methods : This was a cross-sectional study whereby 61 participants (mean age 61 [41-75 years], mean duration of diabetes 14 [1-40 years], 70% male) with Type 2 diabetes and DPN underwent optical coherence tomography (OCT) scans. Global and 4 quadrant (TSNI) RNFL thicknesses were measured at 3.45mm around the optic nerve head of one eye. Neuropathy disability score (NDS) was used to assess the severity of DPN on a 0 to 10 scale. Participants were divided into three age-matched groups representing mild (NDS=3-5), moderate (NDS=6-8) and severe (NDS=9-10) neuropathy. Two regression models were fitted for statistical analysis: 1) NDS scores as co-variate for global and quadrant RNFL thicknesses, 2) NDS groups as a factor for global RNFL thickness only. Results : Mean (SD) RNFL thickness (µm) was 103(9) for mild neuropathy (n=34), 101(10) for moderate neuropathy (n=16) and 95(13) in the group with severe neuropathy (n=11). Global RNFL thickness and NDS scores were statistically significantly related (b=-1.20, p=0.048). When neuropathy was assessed across groups, a trend of thinner mean RNFL thickness was observed with increasing severity of neuropathy; however, this result was not statistically significant (F=2.86, p=0.065). TSNI quadrant analysis showed that mean RNFL thickness reduction in the inferior quadrant was 2.55 µm per 1 unit increase in NDS score (p=0.005). However, the regression coefficients were not statistically significant for RNFL thickness in the superior (b=-1.0, p=0.271), temporal (b=-0.90, p=0.238) and nasal (b=-0.99, p=0.205) quadrants. Conclusions : RNFL thickness was reduced with increasing severity of DPN and the effect was most evident in the inferior quadrant. Measuring RNFL thickness using OCT may prove to be a useful, non-invasive technique for identifying severity of DPN and may also provide additional insight into common mechanisms for peripheral neuropathy and RNFL damage.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

DNA adduction by the potent carcinogen aflatoxin B1 : mechanistic studies

Aflatoxin B1, a potently carcinogenic fungal metabolite, is converted to the biologically active form by chemical oxidation using dimethyldioxirane and enzymatically by cytochrome P450 mixed-function oxidases. Both processes give rise to mixtures of the exo- and endo-8,9-epoxides. Methanolysis studies reveal exclusive trans opening of both epoxides under neutral conditions in CH3OH and CH3OH/H2O mixtures; an SN2 mechanism is postulated. Under acidic conditions, the exo isomer gives mixtures of trans and cis solvolysis products, suggesting that the reaction is, at least in part, SN1; the endo isomer gives only the trans product. The exo isomer reacts with DNA by attack of the nitrogen atom at the 7 position of guanine on C8 of the epoxide to give the trans adduct; the endo epoxide fails to form an adduct at this or any other site in DNA. The exo isomer is strongly mutagenic in a base-pair reversion assay employing Salmonella typhimurium; the endo isomer is essentially nonmutagenic. Aflatoxin B1 and its derivatives intercalate in DNA. These results are consistent with a mechanism in which intercalation of the exo epoxide optimally orients the epoxide for an SN2 reaction with guanine but intercalation of the endo isomer places the epoxide in an orientation which precludes reaction. Thus, while the exo epoxide is a potent mutagen, the endo epoxide fails to react with DNA.

DNA binding study of isophorone in rats and mice

Following isophorone exposure, in a 2-year study with F344 rats and B6C3F1 mice performed under the National Toxicology Program (NTP), an elevated incidence of tumors was observed in male rats (kidney tumors) and male mice (liver tumors). Female rats and mice showed no elevation of tumor rates by isophorone (NTP 1986).

Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

Methods for extracting genomic DNA from whole blood samples : current perspectives

Deoxyribonucleic acid (DNA) extraction has considerably evolved since it was initially performed back in 1869. It is the first step required for many of the available downstream applications used in the field of molecular biology. Whole blood samples are one of the main sources used to obtain DNA, and there are many different protocols available to perform nucleic acid extraction on such samples. These methods vary from very basic manual protocols to more sophisticated methods included in automated DNA extraction protocols. Based on the wide range of available options, it would be ideal to determine the ones that perform best in terms of cost-effectiveness and time efficiency. We have reviewed DNA extraction history and the most commonly used methods for DNA extraction from whole blood samples, highlighting their individual advantages and disadvantages. We also searched current scientific literature to find studies comparing different nucleic acid extraction methods, to determine the best available choice. Based on our research, we have determined that there is not enough scientific evidence to support one particular DNA extraction method from whole blood samples. Choosing a suitable method is still a process that requires consideration of many different factors, and more research is needed to validate choices made at facilities around the world.

Detection of DNA mutations using novel SERS (Surface-Enhanced Raman Spectroscopy) diagnostic platform

This article describes the detection of DNA mutations using novel Au-Ag coated GaN substrate as SERS (surface-enhanced Raman spectroscopy) diagnostic platform. Oligonucleotide sequences corresponding to the BCR-ABL (breakpoint cluster region-Abelson) gene responsible for development of chronic myelogenous leukemia were used as a model system to demonstrate the discrimination between the wild type and Met244Val mutations. The thiolated ssDNA (single-strand DNA) was immobilized on the SERS-active surface and then hybridized to a labeled target sequence from solution. An intense SERS signal of the reporter molecule MGITC was detected from the complementary target due to formation of double helix. The SERS signal was either not observed, or decreased dramatically for a negative control sample consisting of labeled DNA that was not complementary to the DNA probe. The results indicate that our SERS substrate offers an opportunity for the development of novel diagnostic assays.

Regulatory focus moderates the relationship between task control and physiological and psychological markers of stress : a work simulation study

This experiment examined whether trait regulatory focus moderates the effects of task control on stress reactions during a demanding work simulation. Regulatory focus describes two ways in which individuals self-regulate toward desired goals: promotion and prevention. As highly promotion-focused individuals are oriented toward growth and challenge, it was expected that they would show better adaptation to demanding work under high task control. In contrast, as highly prevention-focused individuals are oriented toward safety and responsibility they were expected to show better adaptation under low task control. Participants (N = 110) completed a measure of trait regulatory focus and then three trials of a demanding inbox activity under either low, neutral, or high task control. Heart rate variability (HRV), affective reactions (anxiety & task dissatisfaction), and task performance were measured at each trial. As predicted, highly promotion-focused individuals found high (compared to neutral) task control stress-buffering for performance. Moreover, highly prevention-focused individuals found high (compared to low) task control stress-exacerbating for dissatisfaction. In addition, highly prevention-focused individuals found low task control stress-buffering for dissatisfaction, performance, and HRV. However, these effects of low task control for highly prevention-focused individuals depended on their promotion focus.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.