Recommendations and quality criteria for micronucleus studies with humans.

Micronucleus (MN) analyses in peripheral blood lymphocytes and exfoliated cells from different organs (mouth, nose, bladder and cervix) are at present the most widely used approaches to detect damage of genetic material in humans. MN are extranuclear DNA-containing bodies, which can be identified microscopically. They reflect structural and numerical chromosomal aberrations and are formed as a consequence of exposure to occupational, environmental and lifestyle genotoxins. They are also induced as a consequence of inadequate intake of certain trace elements and vitamins. High MN rates are associated with increased risk of cancer and a range of non-cancer diseases in humans. Furthermore, evidence is accumulating that measurements of MN could be a useful tool for the diagnosis and prognosis of different forms of cancer and other diseases (inflammation, infections, metabolic disorders) and for the assessment of the therapeutic success of medical treatments. Recent reviews of the current state of knowledge suggest that many clinical studies have methodological shortcomings. This could lead to controversial findings and limits their usefulness in defining the impact of exposure concentrations of hazardous chemicals, for the judgment of remediation strategies, for the diagnosis of diseases and for the identification of protective or harmful dietary constituents. This article describes important quality criteria for human MN studies and contains recommendations for acceptable study designs. Important parameters that need more attention include sufficiently large group sizes, adequate duration of intervention studies, the exclusion of confounding factors which may affect the results (sex, age, body mass index, nutrition, etc.), the evaluation of appropriate cell numbers per sample according to established scoring criteria as well as the use of proper stains and adequate statistical analyses.

Impact of infections, preneoplasia and cancer on micronucleus formation in urothelial and cervical cells: A systematic review.

Approximately 165,000 and 311,000 individuals die annually from urothelial (UC) and cervical (CC) cancer. The therapeutic success of these cancers depends strongly on their early detection and could be improved by use of additional diagnostic tools. We evaluated the current knowledge of the use of micronucleus (MN) assays (which detect structural and numerical chromosomal aberrations) with urine- (UDC) and cervix-derived (CDC) cells for the identification of humans with increased risks and for the diagnosis of UC and CC. Several findings indicate that MN rates in UDC are higher in individuals with inflammation and schistosomiasis that are associated with increased prevalence of UC; furthermore, higher MN rates were also found in CDC in women with HPV, Candidiasis and Trichomonas infections which increase the risks for CC. Only few studies were published on MN rates in UDS in patients with UC, two concern the detection of recurrent bladder tumors. Strong correlations were found in individuals with abnormal CC cells that are scored in Pap tests and histopathological abnormalities. In total, 16 studies were published which concerned these topics. MN rates increased in the order: inflammation < ASC-US/ASC-H < LSIL < HSIL < CC. It is evident that MNi numbers increase with the risk to develop CC and with the degree of malignant transformation. Overall, the evaluation of the literature indicates that MNi are useful additional biomarkers for the prognosis and detection of CC and possibly also for UC. In regard to the diagnosis/surveillance of UC, further investigations are needed to draw firm conclusions, but the currently available data are promising. In general, further standardization of the assays is needed (i.e. definition of optimal cell numbers and of suitable stains as well as elucidation of the usefulness of parameters reflecting cytotoxicity and mitotic activity) before MN trials can be implemented in routine screening.

An automated method for dynamic red blood cell aggregate detection in microfluidic flow.

OBJECTIVE: Red blood cell (RBC) aggregation is a unique phenomenon that occurs when red blood cells are subjected to low shear rates. Little is known about the sizes, shapes and behaviour of aggregates flowing in healthy humans. However, excessive aggregation has been shown to be an indication of pathological conditions. Therefore, characterizing RBC aggregates is important to medical research. The objective of this study was to develop a reliable technique based on image processing to assess and characterize human RBC aggregation subjected to controlled and measurable shear rates in a two-fluid flow microfluidic shearing system. APPROACH: Images of RBC suspensions at [Formula: see text], [Formula: see text] and [Formula: see text] entrained by a phosphate buffered saline solution in a PDMS microchannel were captured with a high speed camera. An algorithm for processing the RBC aggregate images is presented and validated (1) on a sample of known diameter hollow glass microspheres and (2) by comparing RBC aggregate size results with those of an ImageJ image processing technique and those obtained by manual detection by two independent researchers. MAIN RESULTS: The proposed image processing algorithm provides a very good agreement with the manufacturer data for the glass microspheres. It also performs well on the RBC suspension images, with errors of 2-4 [Formula: see text] with respect to the manual results. SIGNIFICANCE: The proposed automated method for RBC aggregate detection is found to be reliable and fairly accurate and will serve researchers and, perhaps in the future, clinicians to assess healthy and pathological RBC aggregation under flowing conditions.

Prenatal omega-3 fatty acid supplementation does not affect offspring telomere length and F2-isoprostanes at 12 years: A double blind, randomized controlled trial.

BACKGROUND: Oxidative stress and nutritional deficiency may influence the excessive shortening of the telomeric ends of chromosomes. It is known that stress exposure in intrauterine life can produce variations in telomere length (TL), thereby potentially setting up a long-term trajectory for disease susceptibility. OBJECTIVE: To assess the effect of omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) supplementation during pregnancy on telomere length and oxidative stress in offspring at birth and 12 years of age (12y). DESIGN: In a double-blind, placebo-controlled, parallel-group study, 98 pregnant atopic women were randomised to 4g/day of n-3 LCPUFA or control (olive oil [OO]), from 20 weeks gestation until delivery. Telomere length as a marker of cell senescence and plasma and urinary F2-isoprostanes as a marker of oxidative stress were measured in the offspring at birth and 12y. RESULTS: Maternal n-3 LCPUFA supplementation did not influence offspring telomere length at birth or at 12y with no changes over time. Telomere length was not associated with F2-isoprostanes or erythrocyte total n-3 fatty acids. Supplementation significantly reduced cord plasma F2-isoprostanes (P<0.001), with a difference in the change over time between groups (P=0.05). However, the differences were no longer apparent at 12y. Between-group differences for urinary F2-isoprostanes at birth and at 12y were non-significant with no changes over time. CONCLUSIONS: This study does not support the hypothesis that n-3 LCPUFA during pregnancy provides sustained effects on postnatal oxidative stress and telomere length as observed in the offspring.

Wound swab use and misuse at a regional general hospital.

OBJECTIVE: Guidelines for swab use at our centre cover lower-limb wounds, ulcers and postoperative wound infections but not all types of wound. The objective of this study was to assess current practices in wound management at Mater Dei Hospital and to identify areas for improvement. METHOD: Wound swabs received at the microbiology department between February and April 2013 from adult inpatients departments were included. Wound swabs from the ophthalmology and paediatric departments were excluded. Patient comorbidities, detailed wound descriptions, acknowledgement of and documentation of culture and sensitivity results, and antibiotic changes during treatment were collected. Indictors of infection including white cell counts (WCCs) and C-reactive protein (CRP) were recorded. RESULTS: The study included 134 patients. Diabetes mellitus (61.9%, n=83) was the most common underlying comorbidity. Postoperative wounds were the most common type of wounds swabbed (34.3%). The wound swab characteristics were not fully documented in 27 patients (20.1%). The CRP results were not recorded in 39.6% and WCCs were not taken in 10.4% of patients. Wound swab results were not acknowledged in the medical notes of 76% of cases. CONCLUSION: Wound swabs that were not indicated, lack of documentation and untimely acknowledgement of results were evident. This suggests that a significant proportion of wound swabs may not have been justified and had no impact on wound management. Our study clearly underlines the need for a more comprehensive guideline. DECLARATION OF INTEREST: There was no sponsorship of this study. The authors have no conflict of interest to declare.

Design of a microfluidic system for red blood cell aggregation investigation.

The purpose of this paper is to design a microfluidic apparatus capable of providing controlled flow conditions suitable for red blood cell (RBC) aggregation analysis. The linear velocity engendered from the controlled flow provides constant shear rates used to qualitatively analyze RBC aggregates. The design of the apparatus is based on numerical and experimental work. The numerical work consists of 3D numerical simulations performed using a research computational fluid dynamics (CFD) solver, Nek5000, while the experiments are conducted using a microparticle image velocimetry system. A Newtonian model is tested numerically and experimentally, then blood is tested experimentally under several conditions (hematocrit, shear rate, and fluid suspension) to be compared to the simulation results. We find that using a velocity ratio of 4 between the two Newtonian fluids, the layer corresponding to blood expands to fill 35% of the channel thickness where the constant shear rate is achieved. For blood experiments, the velocity profile in the blood layer is approximately linear, resulting in the desired controlled conditions for the study of RBC aggregation under several flow scenarios.

Elevated plasma magnesium and calcium may be associated with shorter telomeres in older South Australian women.

Telomeres are structures that cap the ends of chromosomes. The integrity of the telomere structure and its DNA hexamer (TTAGGG)n repeat sequence is critical for protecting the ends of chromosomes from degradation and in maintaining overall chromosomal stability. Currently, there are limited data on the influence that nutrition has on telomere length. Recent studies have suggested that micronutrients may influence telomere length. Here we examined the relationship between telomere length in lymphocytes and plasma calcium, magnesium, selenium and zinc status in a healthy cohort of younger and older adults. We report a negative association between telomere length and both plasma calcium and magnesium levels, (r=-0.47, P=0.03 and r=-0.61, P=0.001 respectively), in older females; Intriguingly Ca/Mg ratio was positively associated with telomere length (r=0.55, P=0.007). These relationships were not observed in the younger adults, nor in the older males. In conclusion, our study provides preliminary evidence suggesting that levels of plasma magnesium and calcium may impact on telomere length in lymphocytes in older women.

Porosity and root dentine to material interface assessment of calcium silicate-based root-end filling materials.

OBJECTIVES: The aim of this study was to evaluate the porosity and assess the root dentine to material interface of four root-end filling materials based on tricalcium silicate cement using two microscopy techniques. METHODS: The porosity of Bioaggregate, Biodentine, a prototype radiopacified tricalcium silicate cement (TCS-20-Zr) and intermediate restorative material (IRM) was evaluated after immersion for 28 days in Hank's balanced salt solution (HBSS) using mercury intrusion porosimetry. The root dentine to material interface of the cements when used as root-end filling materials in extracted human teeth was assessed after 28 days of dry storage and immersion in HBSS using a confocal microscope together with fluorescent tracers and also a field emission gun scanning electron microscope. RESULTS: Biodentine and IRM exhibited the lowest level or degree of porosity. The confocal microscopy used in conjunction to fluorescent tracers demonstrated that dry storage resulted in gaps at the root dentine to material interface and also cracks in the material with Biodentine being the most affected. Zinc was shown to be present in root dentine adjacent to the IRM restorations. CONCLUSIONS: Dry storage of Biodentine resulted in changes in the material microstructure and cracks at the root dentine to Biodentine interface. Furthermore, the gaps resulting from material shrinkage allowed the passage of the fluorescent microspheres thus indicating that these gaps are significant and can potentially allow the passage of micro-organisms.

Automatic nuclear bud detection using ellipse fitting, moving sticks or top-hat transformation.

Micronucleus assays are extensively used by biologists to assess genotoxicity and to monitor human exposure to genotoxic materials. As recent studies suggested that nuclear buds can be a new source of micronuclei formed in interphase, the quantification of nuclear buds, which are micronucleus like objects that are attached to the nuclei in interphase, in normal and control group is needed. Three automatic nuclear bud detection algorithms fit for different situations are proposed in this paper. One is based on ellipse fitting, one is based on a stick model and the other is based on the top-hat transform. Comparison of the three methods is also given in this paper. Experimental results showed that the proposed algorithms are all effective and efficient for nuclear bud detection.

Contact angle study of blood dilutions on common microchip materials.

Biocompatible polymers are commonly used to fabricate microfluidic channels for the study of biological flows such as blood microflows. The most common of these materials is polydimethylsiloxane (PDMS) which is very hydrophobic. Oxygenated plasma is advocated to treat the PDMS with reported decreases in contact angle i.e. increase the hydrophilicity of the material in order to make the liquid flow easily. All contact angle studies have been reported with water. Here the contact angles of blood suspensions, in saline and native plasma, are compared to each other and water on common microfluidic chip materials. The hydrophilic effect of plasma-treatment on PDMS is not found to be as significant with blood suspensions as it is with water. Red blood cells suspended in native plasma are found to have a greater contact angle than those suspended in saline.

Acute rhabdomyolysis caused by combination therapy with atorvastatin and warfarin.

Atorvastatin and warfarin are commonly prescribed in combination. Acute rhabdomyolysis is a rare but recognized side effect of atorvastatin occurring within a few weeks of initiation. This article presents a case of a 69-year-old man, on stable atorvastatin therapy, who developed acute rhabdomyolysis following initiation of warfarin. Rising international normalized ratio is a well-recognized feature of interaction between warfarin and various statins (fluvastatin, lovastatin and simva-statin). There has only been one previous similar case of acute rhabdomyolysis following the commencement of warfarin, reported in a patient on stable simvastatin therapy. To the authors' knowledge, no similar case has been reported with atorvastatin.

Use of haemoglobin A1c to detect impaired fasting glucose or Type 2 diabetes in a United Kingdom community based population.

AIMS: To evaluate the diagnostic accuracy of haemoglobin A1c (HbA1c) in screening for impaired fasting glucose and Type 2 diabetes (T2DM). METHODS: We screened 3904 adults aged 45-70 (mean age 58.6 [standard deviation (SD) 6.9] years, mean body mass index (BMI) 29.9 [SD 4.7]kg/m(2)), with fasting plasma glucose (FPG) and HbA1c as part of a large diabetes prevention programme. We assessed the diagnostic accuracy of HbA1c for predicting impaired fasting glucose (IFG), (defined either as FPG 5.6-6.9 mmol/l, or 6.1-6.9 mmol/l), and T2DM (FPG ≥ 7.0 mmol/l). RESULTS: The prevalences of IFG were 13.8% (FPG 5.6-6.9 mmol/l) and 4.5% (FPG 6.1-6.9 mmol/l) and of T2DM was 2.1%. Using FPG 5.6-6.9 mmol/l as the IFG reference standard, HbA1c of 39-47 mmol/mol (5.7-6.4%) was 63% sensitive and 81% specific, and HbA1c 43-47 mmol/mol (6.1-6.4%) was 21% sensitive and 98% specific, in diagnosing IFG. HbA1c ≥ 48 mmol/mol (6.5%) was 61% sensitive and 99% specific in diagnosing T2DM. Having HbA1c 39-47 mmol/mol (5.7-6.4%), male sex, and body mass index >29.5 together increased the odds of IFG 6.5-fold (95% confidence interval (CI) 5.5-7.8) compared to the pre-test odds. CONCLUSION: Defining 'pre-diabetes' at a lower HbA1c threshold of 39 mmol/mol (5.7%) instead of 47 mmol/mol (6.1%) increases its sensitivity in diagnosing IFG, but current American Diabetes Association definitions of 'pre-diabetes' based on HbA1c would fail to detect almost 40% of people currently classified as IFG. This has implications for current and future diabetes prevention programmes, for vascular risk management, and for clinical advice given to people with 'pre-diabetes' based on fasting glucose data.

Transcriptomic network analysis of micronuclei-related genes: a case study.

Mechanistically relevant information on responses of humans to xenobiotic exposure in relation to chemically induced biological effects, such as micronuclei (MN) formation can be obtained through large-scale transcriptomics studies. Network analysis may enhance the analysis and visualisation of such data. Therefore, this study aimed to develop a 'MN formation' network based on a priori knowledge, by using the pathway tool MetaCore. The gene network contained 27 genes and three gene complexes that are related to processes involved in MN formation, e.g. spindle assembly checkpoint, cell cycle checkpoint and aneuploidy. The MN-related gene network was tested against a transcriptomics case study associated with MN measurements. In this case study, transcriptomic data from children and adults differentially exposed to ambient air pollution in the Czech Republic were analysed and visualised on the network. Six genes from the network, i.e. BAX, DMNT1, PCNA, HIC1, p21 and CDC20, were retrieved. Based on these six genes and in combination with p53 and IL-6, a dedicated network was created. This dedicated network is possibly suited for the development of a reporter gene assay that could be used to screen populations complementary to the current MN test assay. In conclusion, we have shown that network analysis of transcriptomics data in relation to the formation of MN is possible and provides a novel mechanistic hypothesis by indicating which genes are regulated and influence others.

Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells.

Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

Increased lymphocyte micronucleus frequency in early pregnancy is associated prospectively with pre-eclampsia and/or intrauterine growth restriction.

Genome stability is essential for normal foetal growth and development. To date, genome stability in human lymphocytes has not been studied in relation to late pregnancy diseases, such as pre-eclampsia (PE) and intrauterine growth restriction (IUGR), which can be life-threatening to mother and baby and together affect >10% of pregnancies. We performed a prospective cohort study investigating the association of maternal chromosomal damage in mid-pregnancy (20 weeks gestation) with pregnancy outcomes. Chromosome damage was measured using the cytokinesis-block micronucleus cytome (CBMNcyt) assay in peripheral blood lymphocytes. The odds ratio for PE and/or IUGR in a mixed cohort of low- and high-risk pregnancies (N = 136) and a cohort of only high-risk pregnancies (N = 91) was 15.97 (P = 0.001) and 17.85 (P = 0.007), respectively, if the frequency of lymphocytes with micronuclei (MN) at 20 weeks gestation was greater than the mean + 2 SDs of the cohort. These results suggest that the presence of lymphocyte MN is significantly increased in women who develop PE and/or IUGR before the clinical signs or symptoms appear relative to women with normal pregnancy outcomes. The CBMNcyt assay may provide a new approach for the early detection of women at risk of developing these late pregnancy diseases and for biomonitoring the efficacy of interventions to reduce DNA damage, which may in turn ameliorate pregnancy outcome.

Flow through a defective mechanical heart valve: a steady flow analysis.

Approximately 250,000 valve replacement operations occur annually around the world and more than two thirds of these operations use mechanical heart valves (MHV). These valves are subject to complications such: pannus and/or thrombus formation. Another potential complication is a malfunction in one of the valve leaflets. Although the occurrence of such malfunctions is low, they are life-threatening events that require emergency surgery. It is, therefore, important to develop parameters that will allow an early non-invasive diagnosis of such valve malfunction. In the present study, we performed numerical simulations of the flow through a defective mechanical valve under several flow and malfunction severity conditions. Our results show that the flow upstream and downstream of the defective valve is highly influenced by malfunction severity and this resulted in a misleading improvement in the correlation between simulated Doppler echocardiographic and catheter transvalvular pressure gradients. In this study, we were also able to propose and test two potential non-invasive parameters, using Doppler echocardiography and phase contrast magnetic resonance imaging, for an early detection of mechanical heart valve malfunction. Finally, we showed that valve malfunction has a significant impact on platelet activation and therefore on thrombus formation.

Investigations into the relationship between hemodynamics and vascular alterations in an established arteriovenous fistula.

Arteriovenous fistula are specific vessels created by a vascular operation in order to provide sufficient blood access for extracorporeal circulation in hemodialysis. They are subject to numerous pathologies that may be caused by hemodynamic effects. To better understand these effects, a specific patient's arteriovenous fistula was reconstructed from computed tomography angiography. Computational fluid dynamics software made it possible to solve fluid mechanics equations under physiological conditions. An accurate map of unsteady velocity profiles and wall shear stress was drawn up. The computed velocity profiles were successfully confronted with Echo Doppler investigation. Selected regions with or without calcification, the end stage of wall alteration, were examined in terms of the mechanical constraints generated by blood flow. In contrast with other authors, we did not observe any association between calcification and areas of oscillating shear stress. Nevertheless, a statistical analysis of the whole vessel envelop and specific sites of calcification suggested a potential association between calcification and high temporal wall shear stress gradients.

Moderate acute intake of de-alcoholized red wine, but not alcohol, is protective against radiation-induced DNA damage ex vivo -- results of a comparative in vivo intervention study in younger men.

Moderate intake of wine is associated with reduced risk of cardiovascular disease and possibly cancer however it remains unclear whether the potential health benefits of wine intake are due to alcohol or the non-alcoholic fraction of wine. We therefore tested the hypothesis that the non-alcoholic fraction of wine protects against genome damage induced by oxidative stress in a crossover intervention study involving six young adult males aged 21-26 years. The participants adhered to a low plant phenolic compound diet for 48 h prior to consuming 300 mL of complete red wine, de-alcoholized red wine or ethanol on separate occasions 1 week apart. Blood samples were collected 0.5, 1.0 and 2.0 h after beverage consumption. Baseline and radiation-induced genome damage was measured using the cytokinesis-block micronucleus assay and total plasma catechin concentration was measured. Consumption of de-alcoholized red wine significantly decreased the gamma radiation-induced DNA damage at 1 and 2 h post-consumption by 20%. In contrast alcohol tended to increase radiation-induced genome damage and complete wine protected against radiation-induced genome damage relative to alcohol. The observed effects were only weakly correlated with the concentration of total plasma catechin (R=-0.23). These preliminary data suggest that only the non-alcoholic fraction of red wine protects DNA from oxidative damage but this effect cannot be explained solely by plasma catechin.