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Titanium dioxide nanotubes (TNTs) have been extensively researched for their enhanced biomedical characteristics, including biocompatibility, cell apposition, and growth morphology. However, there is a significant concern in metallic medical devices owing to micro-movements between implants and bone in aggressive environments due to wear and corrosion synergies, leading to adverse body responses and implant failure. In this study, TNTs on Commercially Pure titanium (C.P. Ti) with three different thickness layers were grown to correlate their corrosion and wear behavior. Initially amorphous after anodization, TNTs underwent annealing at 450 °C for 2 h to transform the distorted, into a crystalline structure. The characterization included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX), X-ray Photoelectron Spectroscopy (XPS), and contact profilometry. Potentiodynamic polarization (PD) and tribocorrosion tests were conducted in Simulated Body Fluid Electrolyte (SBF). SEM micrographs showed TNT thickness layers of 314, 691, and 5280 nm, and X-ray Diffraction showed anatase transformation post-annealing. Results confirmed a significant influence of TNT thickness layers on corrosion and tribocorrosion properties, with higher current density obtained from PD for thicker TNTs layers, associated with surface fluorine content. During tribocorrosion, annealed samples demonstrated lower corrosion tendency, potential drop, and coefficient of friction. Morphology of worn surfaces and volumetric wear rate varied with TNT thickness layer and thermal treatment.

Given the tubal origin of high-grade serous ovarian cancer (HGSC), we sought to investigate intrauterine lavage (IUL) as a novel method of biomarker detection. IUL and serum samples were collected from patients with HGSC or benign pathology. Although CA-125 and HE4 concentrations were significantly higher in IUL samples compared to serum, they were similar between IUL samples from patients with HGSC vs benign conditions. In contrast, CA-125 and HE4 serum concentrations differed between HGSC and benign pathology (P =.002 for both). IUL and tumor samples from patients with HGSC were subjected to targeted panel sequencing and droplet digital PCR (ddPCR). Tumor mutations were found in 75 % of matched IUL samples. Serum CA-125 and HE4 biomarker levels allowed for better differentiation of HGSC and benign pathology compared to IUL samples. We believe using IUL for early detection of HGSC requires optimization, and current strategies should focus on prevention until early detection strategies improve.

Summary: Background: Previous findings from the Swissped RECOVERY trial showed that patients with Pediatric Inflammatory Multisystem Syndrome–Temporally Associated with SARS-CoV-2 (PIMS-TS) who were randomly assigned to intravenous immunoglobulins or methylprednisolone have a comparable length of hospital stay. Here, we report the 6-month follow-up outcomes of cardiac pathologies and normalisation of clinical or laboratory signs of inflammation from this study population. Methods: This pre-planned follow-up of patients with PIMS-TS included the Swissped RECOVERY Trial reports on the 6-month outcomes of the cohort after randomisation, with a focus on cardiac, haematological, and biochemical findings. The trial was an investigator-initiated randomised multicentre open-label two-arm trial in children and adolescents hospitalised with PIMS-TS at ten hospitals in Switzerland. Cardiological assessments and laboratory analyses were prospectively collected in the intention-to-treat analysis on pre-defined intervals after hospital discharge. Differences between randomised arms were investigated using Chi-square test for categorical and Wilcoxon test for continuous variables. The trial is registered with the Swiss National Clinical Trials Portal (SNCTP000004720) and ClinicalTrials.gov (NCT04826588). Findings: Between May 21, 2021 and April 15, 2022, 75 patients with a median age of 9.1 years (IQR 6.2–12.2) were included in the intention-to-treat population (37 in the methylprednisolone group and 38 in the intravenous immunoglobulin group). During follow-up, the incidence of abnormal left ventricular systolic function, coronary artery aneurysms (CAA), and other signs of inflammation were comparable in both groups. However, we detected cardiac abnormalities with low incidence and a mild degree grade of pathology. CAAs were observed in 2/38 children (5.3%) in the IVIG group and 1/37 children (2.7%) in the methylprednisolone group at 6-month follow-up (difference proportion 0.75; 95% confidence interval (CI) −0.05 to 1.0; p = 0.39). Interpretation: Methylprednisolone alone may be an acceptable first-line treatment as left ventricular systolic dysfunction and clinical/laboratory evidence for inflammation quickly resolved in all children. However, our findings need further confirmation through larger studies as our sample size is likely to be of insufficient power to address rare clinically relevant adverse outcomes. Funding: NOMIS, Vontobel, and Gaydoul Foundation.

Summary: Background: Urinary biomarkers may improve the prediction of chronic kidney disease (CKD) progression. Yet, data reporting the applicability of most commercial biomarker assays to the detection of their target analyte in urine together with an evaluation of their predictive performance are scarce. Methods: 30 commercial assays (ELISA) were tested for their ability to quantify the target analyte in urine using strict (FDA-approved) validation criteria. In an exploratory analysis, LASSO (Least Absolute Shrinkage and Selection Operator) logistic regression analysis was used to identify potentially complementary biomarkers predicting fast CKD progression, determined as the 51CrEDTA clearance-based measured glomerular filtration rate (mGFR) decline (>10% per year) in a subsample of 229 CKD patients (mean age, 61 years; 66% men; baseline mGFR, 38 mL/min) from the NephroTest prospective cohort. Findings: Among the 30 assays, directed against 24 candidate biomarkers, encompassing different pathophysiological mechanisms of CKD progression, 16 assays fulfilled the FDA-approved criteria. LASSO logistic regressions identified a combination of five biomarkers including CCL2, EGF, KIM1, NGAL, and TGF-α that improved the prediction of fast mGFR decline compared to the kidney failure risk equation variables alone: age, gender, mGFR, and albuminuria. Mean area under the curves (AUC) estimated from 100 re-samples was higher in the model with than without these biomarkers, 0.722 (95% confidence interval 0.652–0.795) vs. 0.682 (0.614–0.748), respectively. Fully-adjusted odds-ratios (95% confidence interval) for fast progression were 1.87 (1.22, 2.98), 1.86 (1.23, 2.89), 0.43 (0.25, 0.70), 1.10 (0.71, 1.83), 0.55 (0.33, 0.89), and 2.99 (1.89, 5.01) for albumin, CCL2, EGF, KIM1, NGAL, and TGF-α, respectively. Interpretation: This study provides a rigorous validation of multiple assays for relevant urinary biomarkers of CKD progression which combination may improve the prediction of CKD progression. Funding: This work was supported by Institut National de la Santé et de la Recherche Médicale, Université de Paris, Assistance Publique Hôpitaux de Paris, Agence Nationale de la Recherche, MSDAVENIR, Pharma Research and Early Development Roche Laboratories (Basel, Switzerland), and Institut Roche de Recherche et Médecine Translationnelle (Paris, France).

The public water system management in Mexico has recently been criticized due to severe problems with the availability of drinkable water. Significant differences exist between Mexican regions regarding the availability of renewable water and water system distribution. Moreover, water governance in the country is inefficient due to the lack of transparency in the water policies. Therefore, the article aims to investigate whether there is a direct relationship between water system management efficiency and water system improvement efficiency and how the efficiency reflects the size of the population and the availability of renewable water in each state. For this, a two-stage DEA model is constructed to evaluate the water system management efficiency in Mexico. The results indicate high average water management efficiency but very low efficiency in the water system improvements. Furthermore, immense differences in the water system efficiency can be observed with respect to the characteristics and adopted policies.

Introduction: Chronic kidney disease (CKD) exposes to an increased incidence of fragility fractures. International guidelines recommend performing bone mineral density (BMD) if the results will impact treatment decisions. It remains unknown where bone loss occurs and what would preclude the longitudinal loss in patients with CKD. Here, we aimed to investigate factors influencing BMD and to analyze the longitudinal BMD changes. Methods: In the NephroTest cohort, we measured BMD at the femoral neck, total hip, lumbar spine, and proximal radius, together with circulating biomarkers and standardized measured glomerular filtration rate (mGFR) by 51Cr-EDTA in a subset of patients with CKD stage 1 to 5 followed during 4.3 ± 2.0 years. A linear mixed model explored the longitudinal bone loss and the relationship of associated factors with BMD changes. A total of 858 patients (mean age 58.9 ± 15.2 years) had at least 1 and 477 had at least 2 BMD measures. Results: At baseline, cross-sectional analysis showed a significantly lower BMD at femoral neck and total hip and a significant higher serum parathyroid hormone (PTH) along with CKD stages. Baseline age, gender, tobacco, low body mass index (BMI), and high PTH levels were significantly associated with low BMD. Longitudinal analysis during the mean 4.3 years revealed a significant bone loss at the radius only. BMD changes at the femoral neck were associated with BMI, but not CKD stages or basal PTH levels. Conclusions: CKD is associated with low BMD and high PTH in the cross-sectional analysis. Longitudinal bone loss occurred at the proximal radius after 4.3 years.

Introduction: Glomerular filtration rate (GFR) is measured from the late plasma disappearance curve of an exogenous tracer, after correction for the early decay—corresponding to the distribution of the tracer—using various equations. These equations display the highest discrepancies in the GFR range above 90 ml/min per 1.73 m2, and their respective performances against a reference, urinary GFR measurement are unclear. Methods: In patients with mGFR >90 ml/min per 1.73 m2 from 6 different cohorts, we compared GFR obtained from the plasma clearance of iohexol or 51Cr-ethylenediamine tetraacetic acid (EDTA), after correction using Chantler (C), Bröchner-Mortensen (BM), Fleming (F), Jodal-Bröchner-Mortensen (JBM), and Ng (N) equations, with urinary clearance of the same tracers or inulin. Results: In 438 participants (median age 41 [39–42] years, 43% women), the median urinary clearance was 100.8 (94.7–112.6) ml/min per 1.73 m2. Plasma clearances using the correction equations were 105.7 (96.8–119.2), 102.4 (95.2–112.9), 100.7 (93.6–111.1), 102.6 (95.2–113.4), and 106.0 (98.2–117.6) ml/min per 1.73 m2 for C, BM, F, JBM, and N, respectively. Concordance correlation coefficients between plasma and urinary clearances were poor for all equations. Compared with urinary clearances, BM, F, and JBM displayed the best accuracy within 10% (73%, 72%, and 71%, respectively, vs. 63% and 66% for C and N), whereas BM and JBM had the lowest median biases. Accuracy of all equations was especially low in the hyperfiltration range (urinary clearance >130 ml/min per 1.73 m2). Conclusion: The BM and JBM equations displayed the best overall performances to correct for the early disappearance curve. Results of these equations should be interpreted with caution, especially in the highest GFR range.

The spatial distribution of activities and agents within cities, conceptualised as an urban function, profoundly affects how different areas are perceived and lived. This dataset introduces the concept of functional signatures - contiguous areas of a similar urban function delineated based on enclosed tessellation cells (ETC) - and applies it to the area of Great Britain. ETCs are granular spatial units, which capture function based on interpolations from open data inputs stretching from remote sensing to land use, census and points of interest data. The spatial extent of each signature type is defined by grouping ETCs using cluster analysis, based on similarity between their functional profiles, inferred by the data linked to each cell. This approach results in a dataset that reflects urban function as a composite of aspects, rather than a singular use, and is built up from granular spatial units. Furthermore, the underlying data are sourced from available open data products, which together with a method and code fully available, yields a fully reproducible pipeline and makes our dataset and open data product.Both the final classification composed of 17 types of functional signatures and the underlying data collected on the level of enclosed tessellation cells are included in the release and described in this report.

A 21-year-old woman was hospitalized due to coronavirus disease 2019 (COVID-19)-associated respiratory and hepatic impairment concomitant with severe hemolytic anemia. Upon diagnosis of secondary hemophagocytic lymphohistiocytosis, immunosuppression with anakinra and steroids was started, leading to a hepatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and viremia. Subsequent liver biopsy revealed virus particles in hepatocytes by electron microscopy and SARS-CoV-2 virus could be isolated and cultured. Immunosuppression was stopped and convalescent donor plasma given. In the differential diagnosis, an acute crisis of Wilson’s disease was raised by laboratory and genetic testing. This case highlights the complexity of balancing immunosuppression to control hyperinflammation versus systemic SARS-CoV-2 dissemination.

Summary: Background: Erythrocytosis is a hematological disorder usually related to hematopoietic stem cell somatic mutations. However, unexplained erythrocytosis remains frequent. In this study, we evaluated the involvement of IgA1, a regulator of erythropoiesis also implicated in IgA nephropathy (IgAN) pathophysiology, in unexplained polycythemia/erythrocytosis (PE) of IgAN patients. Methods: IgAN-PE patients’ serum was collected, analyzed and used to study IgA1 effect on proliferation and differentiation of erythroid progenitors. Hematological parameters of transgenic mice for human alpha1 heavy chain were studied. Multicentric observational cohorts of chronic kidney disease (CKD) patients, including both native kidney diseases and renal transplants, were studied to analyze patient hemoglobin levels. Findings: We retrospectively identified 6 patients with IgAN and unexplained PE. In large CKD cohorts, IgAN was associated with PE in 3.5% of patients (p

Objectives: Higher education institutions all over the world struggled to balance the need for infection control and educational requirements, as they prepared to reopen after the first wave of the COVID-19 pandemic. A particularly difficult choice was whether to offer for in-person or online teaching. Norwegian universities and university colleges opted for a hybrid model when they reopened for the autumn semester, with some students being offered more in-person teaching than others. We seized this opportunity to study the association between different teaching modalities and COVID-19 risk, quality of life (subjective well-being), and teaching satisfaction. Study design: Prospective, observational cohort study. Methods: We recruited students in higher education institutions in Norway who we surveyed biweekly from September to December in 2020. Results: 26 754 students from 14 higher education institutions provided data to our analyses. We found that two weeks of in-person teaching was negatively associated with COVID-19 risk compared to online teaching, but the difference was very uncertain (−22% relative difference; 95% CI -77%–33%). Quality of life was positively associated with in-person teaching (3%; 95% CI 2%–4%), as was teaching satisfaction (10%; 95% CI 8%–11%). Conclusion: The association between COVID-19 infection and teaching modality was highly uncertain. Shifting from in-person to online teaching seems to have a negative impact on the well-being of students in higher education.

This work presents an example of the design and manufacture capabilities that 3D printing can introduce in catalysis. A multi-purpose catalyst, with fast heat and mass transfer and low-pressure drop has been designed and manufactured by 3D printing. The novelty of the methodology is the combination of advanced techniques for accurate control on the micropore-level allied with a generic framework for the design of macropore and structural levels. The ability to design ordered macroporous should be combined with adequate and controllable implantation of surface functionalities. With this combination of advanced techniques for macro and micro-pore control, it is possible to produce catalysts that unlock traditional trade-off compromises between diffusion, pressure drop and heat transfer.To demonstrate this novel methodology, we have designed and 3D printed a cubic iso-reticular foam in AlSi10Mg. After producing the support, its entire internal area was anodized to high-surface alumina followed by Pt deposition. We have verified the reproducibility of this technique by manufacturing a catalyst for a demonstrator with 8 m length. The test reaction was oxidation of NO to NO2 with the main aim to accelerate this reaction for additional recovery of energy in the production of nitric acid. Keywords: 3D printing, Catalyst, Anodization, Process intensification, Diffusion, Heat transfer

Reducing protein intake is recommended for slowing chronic kidney disease (CKD) progression, but assessment of its true effectiveness is sparse. Methods: Using the Maroni formula, we assessed dietary protein intake (DPI) from 24-hour urinary urea excretion in 1594 patients (67% men and 33% women) with CKD, 784 of whom also had 7-day food records. Cause-specific hazard ratios (HRs) and 95% confidence intervals for the competing risks of DPI-associated end-stage renal disease (ESRD) or death were estimated in 1412 patients with baseline glomerular filtration rate ≥15 ml/min per 1.73 m2, measured by 51Cr-EDTA renal clearance (mGFR). Results: Overall, mean DPI estimated from urea excretion was 1.09 ± 0.30 g/kg of body weight per day (range = 0.34−2.76); 20% of patients had values > 1.3 g/kg per day, and 1.9% had values < 0.6 g/kg per day. Urea excretion and food records produced similar estimates of mean DPI. The lower the mGFR, the lower the mean DPI. Over a median follow-up of 5.6 years, there were 319 ESRD events and 189 pre-ESRD deaths. After adjusting for relevant covariates, each 0.1 g/kg daily higher baseline urea excretion−based DPI or food record−based DPI was associated with an HR for ESRD of 1.05 (95% confidence interval 1.01−1.10) or 1.09 (95% confidence interval 1.04−1.14), respectively. HRs were stronger in patients with baseline mGFR < 30 ml/min per 1.73 m2. There was no association with mortality. The mean age of the patients was 59 ± 15 years, and mean body mass index was 26.6 ± 5.2 kg/m2. Conclusion: In this prospective observational study, the lower the baseline DPI, the slower the progression toward ESRD. Most importantly, the absence of threshold for the relation between DPI and ESRD risk indicates that there is no optimal DPI in the range observed in this cohort.

The data presented in this article are related to the research article entitled “The Diagnostic Value of Rescaled Renal Biomarkers Serum Creatinine and Serum Cystatin C and their Relation with Measured Glomerular Filtration Rate” (Pottel et al. (2017) [1]). Data are presented demonstrating the rationale for the normalization or rescaling of serum cystatin C, equivalent to the rescaling of serum creatinine. Rescaling biomarkers brings them to a notionally common scale with reference interval [0.67–1.33]. This article illustrates the correlation between rescaled biomarkers serum creatinine and serum cystatin C by plotting them in a 2-dimensional graph. The diagnostic value in terms of sensitivity and specificity with measured Glomerular Filtration Rate as the reference method is calculated per age-decade for both rescaled biomarkers. Finally, the interchangeability between detecting impaired kidney function from renal biomarkers and from the Full Age Spectrum FAS-estimating GFR-equation and measured GFR using a fixed and an age-dependent threshold is shown.

Sarcolemma-based focal adhesions (costameres) are a central hub for the cytoskeletal anchoring of myofibrils and mechano-regulated signaling. Here we report the time course of alterations in focal adhesion-associated signaling and fiber composition in rat soleus muscle after Achilles tenotomy. The report includes data from tenotomized muscles and contralateral mock controls to expose whether muscle degeneration after tenotomy is due to the transection of the Achilles tendon, or circumjacent surgical manipulations of the tendon. With respect to the interpretation of the data regarding mechanistic implications of costamere-associated processes for surgical repair of the detached muscle-tendon complex the reader is referred to the accompanying research article ‘Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction’ Ferrié et al., 2019.

Microplastics (MPs) and benzo[a]pyrene (B[a]P) are prevalent environmental pollutants. Numerous studies have extensively reported their individual adverse effects on organisms. However, the combined effects and mechanisms of exposure in mammals remain unknown. Thus, this study aims to investigate the potential effects of oral administration of 0.5μm polystyrene (PS) MPs (1 mg/mL or 5 mg/mL), B[a]P (1 mg/mL or 5 mg/mL) and combined (1 mg/mL or 5 mg/mL) on 64 male SD rats by gavage method over 6-weeks. The results demonstrate that the liver histopathological examination showed that the liver lobules in the combined (5 mg/kg) group had blurred and loose boundaries, liver cord morphological disorders, and significant steatosis. The levels of AST, ALT, TC, and TG in the combined dose groups were significantly higher than those in the other groups, the combined (5 mg/kg) group had the lowest levels of antioxidant enzymes and the highest levels of oxidants. The expression of Nrf2 was lowest and the expression of P38, NF-κB, and TNF-α was highest in the combined (5 mg/kg) group. In conclusion, these findings indicate that the combination of PSMPs and B[a]P can cause the highest levels of oxidative stress and elicit markedly enhanced toxic effects, which cause severe liver damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microplastics (MPs) are ubiquitous contaminants that have become an emerging pollutant of concern, potentially threatening human health and ecosystem environments. Although current detection methods can accurately identify various types of MPs, it remains necessary to develop non-destructive and rapid methods to meet growing demands for detection. Herein, we combine a hyperspectral unmixing method and machine learning to analyse Raman imaging data of environmental MPs. Five MPs types including poly(butylene adipate-co-terephthalate) (PBAT), poly(butylene succinate) (PBS), p-polyethylene (PE), polystyrene (PS) and polypropylene (PP) were visualized and identified. Individual or mixed pure or aged MPs along with environmental samples were analysed by Raman imaging. Alternating volume maximization (AVmax) combined with unconstrained least squares (UCLS) method estimated end members and abundance maps of each of the MPs in the samples. Pearson correlation coefficients (r) were used as the evaluation index; the results showed that there is a high similarity between the raw spectra and the average spectra calculated by AVmax. This indicates that Raman imaging based on machine learning and hyperspectral unmixing is a novel imaging analysis method that can directly identify and visualize MPs in the environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Attenuated Total Reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy is an emerging technology in the medical field. Blood D-dimer was initially studied as a marker of the activation of coagulation and fibrinolysis. It is mainly used as a potential diagnosis screening test for pulmonary embolism or deep vein thrombosis but was recently associated with COVID-19 severity. This study aimed to evaluate the use of ATR-FTIR spectroscopy with machine learning to classify plasma D-dimer concentrations. The plasma ATR-FTIR spectra from 100 patients were studied through principal component analysis (PCA) and two supervised approaches: genetic algorithm with linear discriminant analysis (GA-LDA) and partial least squares with linear discriminant (PLS-DA). The spectra were truncated to the fingerprint region (1800-1000 cm -1 ). The GA-LDA method effectively classified patients according to D-dimer cutoff (≤0.5 μg/mL and >0.5 μg/mL) with 87.5 % specificity and 100 % sensitivity on the training set, and 85.7 % specificity, and 95.6 % sensitivity on the test set. Thus, we demonstrate that ATR-FTIR spectroscopy might be an important additional tool for classifying patients according to D-dimer values. ATR-FTIR spectral analyses associated with clinical evidence can contribute to a faster and more accurate medical diagnosis, reduce patient morbidity, and save resources and demand for professionals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The co-authors Maneesh N. Singh and Francis L. de Martin have old positions and shareholdings in Biocel UK Ltd. and its subsidiary companies., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Breast cancer is the most common malignant tumor in women worldwide, and environmental pollutants are considered to be risk factors. Currently, most studies into benzo[a]pyrene (B[a]P)-induced breast cancer focus on biological effects such as proliferation, invasion, and metastasis, DNA damage, estrogen receptor (ER)-related molecular mechanisms, oxidative damage, and other metabolic pathways. This study aims to provide insights into the role of B[a]P in breast cancer development through RNA-seq and bioinformatics analysis and construction of a competing endogenous RNA (ceRNA) regulatory network. By analyzing RNA-seq results, we identified 144 differentially-expressed circRNAs, 69 differentially-expressed lncRNAs, 20 differentially-expressed miRNAs, and 212 differentially-expressed mRNAs. Following on, we analyzed the gene ontology (GO) and KEGG enrichment functions of the differentially-expressed RNAs. In addition, the protein-protein interaction (PPI) network was mapped for differentially-expressed mRNAs. Subsequently, we constructed ceRNA networks, one of which consisted of 45 dysregulated circRNAs, 11 miRNAs, and 9 mRNAs, and a second consisted of 40 lncRNAs, 11 miRNAs, and 9 mRNAs. Finally, 6 circRNAs, 4 lncRNAs, 1 miRNA, and 4 mRNAs were randomly selected for quantitative real-time PCR verification. PCR results were further verified by Western blotting assays. These results show that the expression level of differentially-expressed RNA was consistent with the sequencing data, and the Western blotting results were highly consistent with the PCR results, confirming that the sequencing result was very reliable. This study systematically explores the ceRNA atlas of differentially-expressed genes related to B[a]P exposure in breast cancer cells, providing new insights into mechanisms of environmental pollutants in breast cancer., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Meningiomas remains a clinical dilemma. They are the commonest "benign" types of brain tumours and, although being typically benign, they are divided into three WHO grades categories (I, II and III) which are associated with the tumour growth rate and likelihood of recurrence. Recurrence depends on extend of surgery as well as histopathological diagnosis. There is a marked variation amongst surgeons in the follow-up arrangements for their patients even within the same unit which has a significant clinical, and financial implication. Knowing the tumour grade rapidly is an important factor to predict surgical outcomes and adequate patient treatment. Clinical follow up sometimes is haphazard and not based on clear evidence. Spectrochemical techniques are a powerful tool for cancer diagnostics. Raman hyperspectral imaging is able to generate spatially-distributed spectrochemical signatures with great sensitivity. Using this technique, 95 brain tissue samples (66 meningiomas WHO grade I, 24 meningiomas WHO grade II and 5 meningiomas that reoccurred) were analysed in order to discriminate grade I and grade II samples. Newly-developed three-dimensional discriminant analysis algorithms were used to process the hyperspectral imaging data in a 3D fashion. Three-dimensional principal component analysis quadratic discriminant analysis (3D-PCA-QDA) was able to distinguish grade I and grade II meningioma samples with 96% test accuracy (100% sensitivity and 95% specificity). This technique is here shown to be a high-throughput, reagent-free, non-destructive, and can give accurate predictive information regarding the meningioma tumour grade, hence, having enormous clinical potential with regards to being developed for intra-operative real-time assessment of disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microplastics (MPs) contamination is ubiquitous in environmental matrices worldwide. Moreover these pollutants can be ingested by organisms and transported to organs via the circulatory system. Although efficient methods for the analysis of MPs derived from environment matrices and organisms' tissue samples have been developed after special sample pre-treatment, there remains a need for an optimised approach allowing direct identification and visualisation these MPs in real environmental matrices and organismal samples. Herein, we firstly used a multivariate curve resolution-alternating least squares (MCR-ALS) analysis of Raman hyperspectral imaging data to direct identification and visualisation of MPs in a complex serum background. Four common MPs types including polyethylene (PE), polystyrene (PS), polypropylene (PP) and polyethylene terephthalate (PET) were identified and visualised either individually or in mixtures within spiked samples at an 8-μm spatial resolution. Moreover, Raman imaging based on MCR-ALS was successfully applied in fish faeces biological samples and environmental sand samples for in situ MPs identification directly without washing or removal of organic matter. The current results demonstrate Raman imaging based on MCR-ALS as a novel imaging approach for direct identification and visualisation of MPs, through extraction of MPs' chemical spectra within a complicated biological or environmental background whilst eliminating overlapping Raman bands and fluorescence interference., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The present study aimed to investigate whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy coupled with multivariate analysis could be applied to discriminate and classify among breast tumour molecular subtypes based on the unique spectral "fingerprints" of their biochemical composition. The different breast cancer tissues and normal breast tissues were collected and identified by pathology and ATR-FTIR spectroscopy respectively. The study indicates that the levels of the lipid-to-protein, nucleic acid-to-lipid, phosphate-to-carbohydrate and their secondary structure ratio, including RNA-to-DNA, Amide I-to-Amide II, and RNA-to-lipid ratios were significantly altered among the molecular subtype of breast tumour compared with normal breast tissues, which helps explain the changes in the biochemical structure of different molecular phenotypes of breast cancer. Tentatively-assigned characteristic peak ratios of infrared (IR) spectra reflect the changes of the macromolecule structure in different issues to a great extent and can be used as a potential biomarker to predict the molecular subtype of breast tumour. The present study acts as the first case study to show the successful application of IR spectroscopy in classifying subtypes of breast cancer with biochemical alterations. Therefore, the present study is likely to help to provide a new diagnostic approach for the accurate diagnosis of breast tumours and differential molecular subtypes and has the potential to be used for further intraoperative management., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Compliance with ethical standards Conflict of interest FLM is a shareholder in Biocel UK Ltd, a company seeking to develop spectrochemical tools and algorithms for commercial gain. All remaining authors declare that they have no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Uncertainty estimation provides a quantitative value of the predictive performance of a classification model based on its misclassification probability. Low misclassification probabilities are associated with a low degree of uncertainty, indicating high trustworthiness; while high misclassification probabilities are associated with a high degree of uncertainty, indicating a high susceptibility to generate incorrect classification. Herein, misclassification probability estimations based on uncertainty estimation by bootstrap were developed for classification models using discriminant analysis [linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA)] and support vector machines (SVM). Principal component analysis (PCA) was used as variable reduction technique prior classification. Four spectral datasets were tested (1 simulated and 3 real applications) for binary and ternary classifications. Models with lower misclassification probabilities were more stable when the spectra were perturbed with white Gaussian noise, indicating better robustness. Thus, misclassification probability can be used as an additional figure of merit to assess model robustness, providing a reliable metric to evaluate the predictive performance of a classifier., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although it is well acknowledged that the anti-androgenic phthalate diesters can be readily hydrolysed into their monoester counterparts, their metabolites' toxicology remains obscure. Herein, we tested the hypothesis that hydrolysis of one of the two ester bonds can mediate phthalate diesters' potential endocrine effects in MLTC-1 Leydig cells, in line with their ability to disrupt androgen secretion in humans. Five diesters (DMP, DEP, DBP, DBzP and DEHP) and five monoesters (MMP, MEP, MBP, MBzP and MEHP) phthalates as mixtures or individually were applied to cell lines to investigate differences in phthalates' hydrolysis associated with varying side-chain structures and steroidogenic effects. Short-chain diesters DMP, DEP and DBP are more readily hydrolysed compared to the long-chain DEHP, while aromatic alkyl chain DBzP cannot be metabolized completely in vitro. When the hydrolysis processes are interrupted, the diester phthalates' steroidogenic effects can be influenced via regulating related steroidogenic pathway genes. With 10-100 μM treatment exposures, androgenic effects were observed only with DMP or DEP but not for MMP or MEP; while the phthalate diesters DBP, DBzP or DEHP generally exhibited more complex steroidogenic effects than their corresponding monoester counterparts (i.e., biphasic androgen and anti-androgen effects for diesters but monotonic androgen effects for monoesters were observed). DBP elicited hydrolysis-related steroidogenic modulation, in which the anti-androgenic effects of diester DBP reversed into the androgenic effects of monoester MBP at 100 μM. Phthalate metabolites appear to exert different effects at an endocrine level compared to parent compounds, and deeper insights into how the hydrolytic process is related to this alternating toxicity would improve our understanding of a risk assessment for these widespread contaminants in male reproduction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Much effort is currently being placed into developing new blood tests for cancer diagnosis in the hope of moving cancer diagnosis earlier and by less invasive means than current techniques, e.g., biopsy. Current methods are expected to diagnose and begin treatment of cancer within 62 days of patient presentation, though due to high volume and pressures within the NHS in the UK any technique that can reduce time to diagnosis would allow reduction in the time to treat for patients. The use of vibrational spectroscopy, notably infrared (IR) spectroscopy, has been under investigation for many years with varying success. This technique holds promise as is would combine a generally well accepted test (a blood test) with analysis that is reagent free and cheap to run. It has been demonstrated that, when asked simple clinical questions (i.e., cancer vs. no cancer), results from spectroscopic studies are promising. However, in order to become a clinically useful tool, it is important that the test differentiates a variety of cancer types from healthy patients. This study has analysed plasma samples with attenuated total reflection Fourier-transform IR spectroscopy (ATR-FTIR), to establish if the technique is able to distinguish normal from primary or metastatic brain tumours. We have shown that when asked specific questions, i.e., high-grade glioma vs. low-grade glioma, the results show a significantly high accuracy (100%). Crucially, when combined with meningiomas and metastatic lesions, the accuracy remains high (88-100%) with only minimal overlap between the two metastatic adenocarcinoma groups. Therefore in a clinical setting, this novel technique demonstrates potential benefit when used in conjuction with existing diagnostic methods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Robust diagnosis of ovarian cancer is crucial to improve patient outcomes. The lack of a single and accurate diagnostic approach necessitates the advent of novel methods in the field. In the present study, two spectroscopic techniques, Raman and surface-enhanced Raman spectroscopy (SERS) using silver nanoparticles, have been employed to identify signatures linked to cancer in blood. Blood plasma samples were collected from 27 patients with ovarian cancer and 28 with benign gynecological conditions, the majority of which had a prolapse. Early ovarian cancer cases were also included in the cohort (n = 17). The derived information was processed to account for differences between cancerous and healthy individuals and a support vector machine (SVM) algorithm was applied for classification. A subgroup analysis using CA-125 levels was also conducted to rule out that the observed segregation was due to CA-125 differences between patients and controls. Both techniques provided satisfactory diagnostic accuracy for the detection of ovarian cancer, with spontaneous Raman achieving 94% sensitivity and 96% specificity and SERS 87% sensitivity and 89% specificity. For early ovarian cancer, Raman achieved sensitivity and specificity of 93% and 97%, respectively, while SERS had 80% sensitivity and 94% specificity. Five spectral biomarkers were detected by both techniques and could be utilised as a panel of markers indicating carcinogenesis. CA-125 levels did not seem to undermine the high classification accuracies. This minimally invasive test may provide an alternative diagnostic and screening tool for ovarian cancer that is superior to other established blood-based biomarkers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The estrogenic chemical 4-nonylphenol (NP) is known to impair testicular devolopment and spermatogenesis in rodents. The objective of this study was to explore the effects of NP on autophagy induction and AMPK-mTOR signaling pathway in Sertoli cells (SCs), which are the "nursemaid cells" for meiosis of spermatocytes. In this study we exposed 7-week-old male rats to NP by intra-peritoneal injection at 0, 20, 50 or 100mg/kg body weight/2days for 20 consecutive days. Our results showed that exposure to NP dose-dependently induces the formation of autophagosomes in SCs, increases the expression of Beclin-1, the conversion of LC3-I to LC3-II and the mRNA expression of Atg3, Atg5, Atg7 and Atg12 in testis, and these effects are concomitant with the activation of AMPK, and the suppression of TSC2-mTOR-p70S6K/4EBP1 signaling cascade in testis. Furthermore, 10μM Compound C or AMPKα1 siRNA pre-treatment effectively attenuated autophagy and reversed AMPK-mTOR-p70S6K/4EBP1 signaling in NP-treated SCs. Co-treatment with 1mM AICAR remarkably strengthened NP-induced autophagy and mTOR inhibition in SCs. Together, these data suggest that NP stimulates Sertoli cell autophagy and inhibits mTOR-p70S6K/4EBP1 activity through AMPK activation, which is the potential mechanism responsible for the regulation of testis function and differentiation following NP exposure., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Epidemiological studies implicate environmental risk factors and epigenetic alterations in the multistage process of hepatocellular carcinoma (HCC) development. However, associations between environmental factors and DNA methylation of tumour suppressor genes (TSGs) in HCC development remain ambiguous. Understanding how possible interactions influence risk may provide insights into the complexity of hepato-carcinogenesis. For this study, blood samples were collected from HCC patients (n=90) and healthy volunteers (n=99) from Xiamen (China) and data for selected environmental risk factors [e.g., benzo[a]pyrene (B[a]P), hepatitis B or C virus (HBV or HCV) infection, smoking and alcohol consumption] were recorded; factors identified as significantly higher (P<0.05) amongst case subjects compared to controls were identified. In order to assess associations for epigenetic alterations and HCC risk factors, serum DNA methylation of TSGs was quantified using high-resolution melting (HRM) analysis. Our results clearly indicate elevated methylation patterns for detoxification gene [glutathione-S-transferase Pi (GSTP)] promoter regions in cases compared to control subjects. Additionally, GSTP promoter hypermethylation and B[a]P diol epoxide-albumin (BPDE-Alb) were positively correlated with HCC incidence. Our epidemiological and in vitro cell model studies indicated that GSTP promoter DNA methylation regulates this gene's expression. Moreover, GSTP also plays an important role in B[a]P detoxification and potential protective role against B[a]P-induced liver cell toxicity and hepato-carcinogenesis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Exposure to chemicals such as benzo[a]pyrene (B[a]P) can generate intracellular toxic mechanisms. Fourier-transform infrared (FTIR) spectroscopy is a novel approach that allows the non-destructive analysis of underlying chemical bond alterations in patho-physiological processes. This study set out to examine whether B[a]P-induced whole cell alterations could be distinguished from effects on nuclei of exposed cells. Using attenuated total reflection FTIR (ATR-FTIR) spectroscopy, alterations in nuclei isolated from B[a]P-treated MCF-7 cells concentrated either in G0/G1- or S-phase were observed. B[a]P-induced effects in whole-cells included alterations to lipids, DNA and protein spectral regions. Absorbance areas for protein and DNA/RNA regions in B[a]P-treated whole cells differed significantly (P<0.0001) from vehicle controls and these observations correlated with alterations noted in isolated nuclei. Our findings provide evidence that FTIR spectroscopy has the ability to identify specific chemical-induced alterations., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Arsenic exposure has been implicated to alter DNA methylation process in vitro and in vivo, but it remains obscure whether it disrupts DNA demethylation process, which is pivotal for epigenetic regulation. The objective of this descriptive study was to investigate the relationship between arsenic exposure and 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) alterations in various organs. In this study, we exposed male Sprague-Dawley rats to sodium arsenite (0.5, 2 or 10 ppm) via drinking water for 8 weeks. Spleen accumulated 2- to 3-fold higher arsenic levels than liver and heart. Lower arsenic levels were observed in the kidney, pancreas and lung. No significant arsenic-induced global 5mC alterations were observed in the majority of investigated organs. However, arsenic induced organ-specific alterations of 5hmC and/or 5hmC/5mC in some investigated organs, i.e. lung, heart, kidney, pancreas and spleen. Our observations suggest that 5hmC is a more sensitive biomarker of arsenic-induced impacts on epigenetic processes than 5mC. Moreover, demethylation via hydroxylation of 5mC appears to play a central role in the toxic mechanism of arsenic., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Perfluorooctanoic acid (PFOA) is one of the most commonly used perfluorinated compounds, and exposure to it has been associated with a number of adverse health effects. However, the molecular mechanisms involved in PFOA toxicity are still not well characterized. In the present study, flow cytometry analysis revealed that PFOA induced oxidative stress, cell cycle arrest and apoptosis in human non-tumor hepatic cells (L-02). Furthermore, we investigated the alterations in protein profile within L-02 cells exposed to PFOA, aiming to explore the mechanisms underlying PFOA hepatotoxicity on the proteome level. Of the 28 proteins showing significant differential expression in response to PFOA, 24 were down-regulated and 4 were up-regulated. This proteomic study proposed that the inhibition of some proteins, including GRP78, HSP27, CTSD and hnRNPC may be involved in the activation of p53, which consequently triggered the apoptotic process in L-02 cells. Induction of apoptosis via the p53-dependent mitochondrial pathway is further suggested as one of the key toxicological events occurring in L-02 cells under PFOA stress. We hope these data will shed new light on the molecular mechanisms responsible for PFOA-mediated toxicity in human liver cells, and from such studies useful biomarkers indicative of PFOA exposure could be developed., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Risk assessment and remediation of contaminated land is inherently dependent on the contaminants present and their availability for interaction with soil biota. An ever-growing body of evidence suggests that current regulatory procedures over-estimate the 'true' fraction available to biota. Thus, a procedure that predicts the 'bioavailable fraction' would be useful for predicting 'actual' exposure limits and provide a more relevant basis for risk assessment. The aim of this paper is to address several important questions: "How should bioavailability be defined?" "What factors affect bioavailability measurement?" "To what extent have existing protocols measured bioavailability?" "What is actually measured by chemical techniques purported to determine bioavailability?" We offer two definitions (namely 'bioavailability' and 'bioaccessibility') and review commonly employed chemical extraction techniques to measure putative bioavailability. Relative advantages and disadvantages of the techniques are highlighted to elucidate underlying factors for the wide range of conclusions observed in the literature. Although the concept of bioavailability is implicit to contaminated land risk assessment and remediation, explicit reference to and use of adjustment factors is rare amongst regulatory bodies and remediators. Use of chemical determinants for bioavailability, applicable within current legislation and due consideration to inherent variability, are proposed and barriers to their implementation discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

CYP1B1 is a key P450 enzyme involved in the metabolism of exogenous and endogenous substrates and plays a key role in hormone-induced carcinogenesis. Risk factors for ovarian cancer are related to hormonal exposure and reproduction, and polymorphisms within genes involved in metabolism of oestrogen and certain xenobiotics may influence the risk of developing ovarian cancer. Current meta-analysis evaluated four CYP1B1 polymorphisms (Leu432Val, Arg48Gly, Ala119Ser and Asn453Ser) for their association with ovarian cancer risk. A search of the MEDLINE bibliographic database for the period up to April 2012 identified five relevant studies. With regards to Leu432Val polymorphism, all of the five studies were eligible (1199 cases and 2596 controls) for analysis, while for Arg48Gly (799 cases and 1169 controls), Ala119Ser (799 cases and 1172 controls) and Asn453Ser (361cases and 1577 controls) only two studies were eligible for analysis. Fixed-effect models were used to estimate pooled odds ratios (OR) with 95% confidence intervals (95% CI) and chi-square based Q-test was used to test for heterogeneity. The pooled OR (95% CI) for CYP1B1&#95;Leu432Val polymorphism were 1.1 (0.84-1.31) for heterozygous subjects and 0.82 (0.57-1.17) for homozygous Val subjects. In a recessive model, homozygous carriers of Leu432Val showed a weak trend towards reduced risk as compared to 'wild type' and heterozygous carriers (OR 0.8, 95% CI; 0.66-0.99); however, this association was of limited significance. Regarding Arg48Gly, the pooled OR (95% CI) were 1.06 (0.89-1.27) for heterozygous and 0.98 (1.72-1.33) for homozygous Gly subjects. With respect to Ala119Ser and Asn453Ser, the pooled OR were 1.06 (0.87-1.29) and 1.24 (0.94-1.63) for heterozygous and 1.1 (0.8-1.52) and 1.09 (0.5-2.34) for homozygous respectively. In conclusion, this meta-analysis suggests that CYP1B1 polymorphisms are not associated with ovarian cancer risk. Studies evaluating CYP1B1&#95;Leu432Val polymorphism are required to further elucidate the risk of ovarian cancer with this polymorphism. Additionally, studies amongst Asian and African subjects are required to estimate race-specific effects., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Chemical contaminants, such as benzo[a]pyrene (B[a]P), may modulate transcriptional responses in cells via the activation of aryl hydrocarbon receptor (AhR) or through responses to DNA damage following adduct formation. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be employed in a non-destructive fashion to interrogate the biochemical signature of cells via generation of infrared (IR) spectra. By applying to generated spectral datasets subsequent computational approaches such as principal component analysis plus linear discriminant analysis (PCA-LDA), derived data reduction is achieved to facilitate the visualization of wavenumber-related alterations in target cells. Discriminating spectral variables might be associated with lipid or glycogen content, conformational protein changes and phosphorylation, and structural alterations in DNA/RNA. Using this approach, we investigated the dose-related effects of B[a]P in MCF-7 cells concentrated in S- or G₀/G₁-phase. Our findings identified that in PCA-LDA scores plots a clear segregation of IR spectra was evident, with the major spectral alterations associated with DNA/RNA, secondary protein structure and lipid. Dose-related effects were observed and even with exposures as low as 10⁻⁹ M B[a]P, significant (P ≤ 0.001) separation of B[a]P-treated vs. vehicle control cells was noted. ATR-FTIR spectroscopy with computational analysis is a novel approach to identify the effects of environmental contaminants in target cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Perfluorooctanoic acid (PFOA) is one of the most commonly used perfluorinated compounds. Being a persistent environmental pollutant, it can accumulate in human tissues via various exposure routes. PFOA may interfere in a toxic fashion on the immune system, liver, development, and endocrine systems. In utero human exposure had been associated with cord serum global DNA hypomethylation. In light of this, we investigated possible PFOA-induced DNA methylation alterations in L02 cells in order to shed light into its epigenetic-mediated mechanisms of toxicity in human liver. L02 cells were exposed to 5, 10, 25, 50 or 100 mg/L PFOA for 72h. Global DNA methylation levels were determined by LC/ESI-MS, glutathione-S-transferase Pi (GSTP) gene promoter DNA methylation was investigated by methylation-specific polymerase chain reaction (PCR) with bisulfite sequencing, and consequent mRNA expression levels were measured with quantitative real-time reverse transcriptase PCR. A dose-related increase of GSTP promoter methylation at the transcription factor specificity protein 1 (SP1) binding site was observed. However, PFOA did not significantly influence global DNA methylation; nor did it markedly alter the promoter gene methylation of p16 (cyclin-dependent kinase inhibitor 2A), ERα (estrogen receptor α) or PRB (progesterone receptor B). In addition, PFOA significantly elevated mRNA transcript levels of DNMT3A (which mediates de novo DNA methylation), Acox (lipid metabolism) and p16 (cell apoptosis). Considering the role of GSTP in detoxification, aberrant methylation may be pivotal in PFOA-mediated toxicity response via the inhibition of SP1 binding to GSTP promoter., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Human papillomavirus (HPV) is a sexually-transmitted infection associated with cervical cancer. Of over 100 HPV types identified, 13 are high-risk oncogenic. In unvaccinated women worldwide, the incidence of cervical cancer from HPV16 and HPV18 will remain. Cervical cytology can be graded from normal (atypia-free) to low-grade to high-grade. Infrared (IR) spectroscopy is a non-destructive technique that allows the acquisition of a biochemical-cell fingerprint based on vibrational states of chemical bonds., Methods: Exfoliative cervical cytology specimens (n=147) were retrieved, graded by a cytologist and HPV-tested/genotyped using hybrid capture 2 and the Roche HPV Linear Array. Additionally, the spectral signatures of cervical cell lines C33A, HeLa and SiHa were examined. After washing, cellular material was transferred to low-E glass slides and interrogated using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. Given the complex nature of the dataset consisting of thousands of variables (wavenumbers), we used multivariate analysis for data reduction and information retrieval. Principal component analysis coupled with linear discriminant analysis (PCA-LDA) generated a visual representation of the data (scores plot) and, identification of the wavenumbers and consequent biochemical entities responsible for segregation (loadings plot)., Results: Immortalised cell lines were readily distinguishable from each other. It was difficult to segregate categories of cytology associated with HPV infection types. However, in low-grade cytology infected with high-risk oncogenic HPV16 or HPV18, it was possible to segregate women based on whether they were aged 20-29years vs. 30-39years., Conclusions: Our findings suggest a spectral phenotype in exfoliative cervical cytology associated with transient vs. persistent HPV infection., (Copyright 2010 Elsevier B.V. All rights reserved.)

Markers of gastrointestinal (GI) stem cells remain elusive. We employed synchrotron Fourier-transform infrared (FTIR) microspectroscopy to derive mid-infrared (IR) spectra along the length of human GI crypts. Tissue sections (10-μm thick) were floated onto BaF2 windows and image maps were acquired of small intestine and large bowel crypts in transmission mode with an aperture of ≤10 μm×10 μm. Counting upwards in a step-size (≤10 μm) fashion from the crypt base, IR spectra were extracted from the image maps and each spectrum corresponding to a particular location was identified. Spectra were analyzed using principal component analysis plus linear discriminant analysis. Compared to putative crypt base columnar/Paneth cells, those assigned as label-retaining cells were chemically more similar to putative large bowel stem cells and, the small intestine transit-amplifying cells were closest to large bowel transit-amplifying cells; interestingly, the base of small intestine crypts was the most chemically-distinct. This study suggests that in the complex cell lineage of human GI crypts, chemical similarities as revealed by FTIR microspectroscopy between regions putatively assigned as stem cell, transit-amplifying and terminally-differentiated facilitates identification of cell function.

Primary Syrian hamster embryo (SHE) cells might be used to assess morphological transformation following treatment with chemical carcinogens. We employed attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to interrogate SHE colonies, as complex biomolecules absorb in the mid-infrared (IR; lambda=2-20microm) giving vibrational spectra associated with structure and function. Early-passage SHE cells were cultured (pH 6.7) in the presence or absence of benzo[a]pyrene (B[a]P; 5.0microg/ml). Unstained colonies were applied to an ATR crystal, and vibrational spectra were obtained in the ATR mode using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment. These were individually baseline-corrected and normalised. Spectra were then analysed using principal component analysis (PCA) plus linear discriminant analysis (LDA). PCA was used to reduce the dataset dimensions before LDA was employed to reveal clustering. This determined whether wavenumber-absorbance relationships expressed as single points (scores) in 'hyperspace' might on the basis of multivariate distance reveal biophysical differences associated with morphologies in vehicle control (non-transformed or transformed) or carcinogen-treated (non-transformed or transformed) cells. Retrospectively designated SHE colonies (following staining and microscopic analysis) clustered according to whether they were vehicle control (non-transformed), B[a]P-treated (non-transformed) or transformed (control and B[a]P-treated). Scores plots pointed to a B[a]P-treated phenotype and derived loadings plots highlighted distinguishing markers in control transformed vs. B[a]P-treated transformed; these were mostly associated with Amide I, Amide II and phosphate stretching (asymmetric and symmetric) vibrations. Combined application of ATR-FTIR spectroscopy and unsupervised (PCA)/supervised (LDA) may be a novel approach to scoring SHE colonies for morphological transformation.

Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues (n=18) classified as benign (n=6), non-tamoxifen-associated carcinoma (n=6) and tamoxifen-associated carcinoma (n=6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol-O-methyltransferase (COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

Tamoxifen remains a frontline treatment for hormone-responsive breast cancer despite its use being associated with a 2-7-fold elevated risk of developing endometrial carcinoma. Several groups have investigated whether tamoxifen induces DNA-damaging (genotoxic) versus non-genotoxic mechanisms. Some studies point to the presence of tamoxifen-DNA adducts while others suggest otherwise. In many of these studies, the histological sub-type has not been considered; as type 1 carcinomas are associated with PTEN and KRAS2 mutations whereas type 2 carcinomas exhibit TP53 and ERBB-2 mutations, the absence of this information makes comparisons between such independent investigations difficult. An examination of the sub-types of endometrial carcinoma points to histological and mechanistic distinctions between sporadic and tamoxifen-associated disease; this could suggest differing aetiologies. On this basis, we propose a dual mechanism of action highlighted by the different patterns of endometrial carcinoma sub-types. Tamoxifen may initially be pro-oestrogenic in the endometrium giving rise to elevated type 1 endometrioid carcinoma occurrence whereas after long-term use, there is an increase of type 2 disease or malignant mixed mullerian tumours associated with a hormone-independent mechanism of action. Despite these associated risk factors, and the introduction of new selective oestrogen receptor modulators (SERMs), we suggest that the organ-specific pleiotrophic effects of tamoxifen mean that this effective therapeutic agent for breast cancer will continue to have significant usage. The focus of future research should concentrate on the different aetiologies of tamoxifen-associated endometrial carcinomas while efforts continue to develop future SERMs.

Although cervical cancer screening in the UK has led to reductions in the incidence of invasive disease, this programme remains flawed. We set out to examine the potential of infrared (IR) microspectroscopy to allow the profiling of cellular biochemical constituents associated with disease progression. Attenuated total reflection-Fourier Transform IR (ATR) microspectroscopy was employed to interrogate spectral differences between samples of exfoliative cervical cytology collected into liquid based cytology (LBC). These were histologically characterised as normal (n = 5), low-grade (n = 5), high-grade (n = 5) or severe dyskaryosis (? carcinoma) (n = 5). Examination of resultant spectra was coupled with principal component analysis (PCA) and subsequent linear discriminant analysis (LDA). The interrogation of LBC samples using ATR microspectroscopy with PCA-LDA facilitated the discrimination of different categories of exfoliative cytology and allowed the identification of potential biomarkers of abnormality; these occurred prominently in the IR spectral region 1200 cm(-1) - 950 cm(-1) consisting of carbohydrates, phosphate, and glycogen. Shifts in the centroids of amide I (approximately 1650 cm(-1)) and II (approximately 1530 cm(-1)) absorbance bands, indicating conformational changes to the secondary structure of intracellular proteins and associated with increasing disease progression, were also noted. This work demonstrates the potential of ATR microspectroscopy coupled with multivariate analysis to be an objective alternative to routine cytology.

Background: Neutral endopeptidase (NEP) degrades atrial natriuretic peptide (ANP) that via cyclic guanosine monophosphate (cGMP) is natriuretic and aldosterone-inhibiting. We hypothesized that chronic oral NEP inhibition (NEPI), initiated in early experimental congestive heart failure (CHF), would delay onset of decreases in sodium excretion during the progression of CHF and, in the severe phase, suppress aldosterone activation and reduce the magnitude of sodium retention. We also hypothesized that chronic NEPI during progressive CHF (PCHF) would improve the natriuretic response to acute volume expansion., Methods: In a novel canine model that progresses over 38 days from early to moderate and finally severe CHF, we defined the actions of chronic NEPI (candoxatril, 10 mg/kg, orally, twice a day) upon cardiorenal and neurohumoral function as well as the clinical well being of treated and untreated dogs in CHF., Results: From baseline through the moderate phase of CHF, NEPI maintained sodium excretion. In contrast, in moderate CHF, sodium excretion was reduced compared to the early phase in the controls. In severe CHF, sodium excretion was higher with NEPI compared to control. Chronic NEPI also resulted in lower plasma aldosterone as compared to controls. In severe CHF, the natriuretic response to acute saline volume expansion was enhanced with oral NEPI as compared to control., Conclusion: This study supports the conclusion that chronic oral NEPI delays the onset of reduction in sodium excretion during the transition from early to severe CHF in this model of PCHF. This therapeutic strategy also improved the natriuretic response to acute volume expansion in severe CHF while enhancing ANP and suppressing aldosterone activation. Thus, these studies demonstrated a selective renal and adrenal action of chronic NEPI in heart failure indicating a therapeutic potential.

Dietary factors are important in the aetiology of human cancer and carcinogens, mostly heterocyclic aromatic amines, have been isolated from cooked proteinaceous foodstuffs. Whilst such carcinogens have induced tumours in rodent bioassays, the dosages required were much higher than estimates of human exposure levels. We have examined the possibility that genotoxins, which were not extractable prior to enzymic digestion, may be released from cooked beef by proteolysis. Dichloromethane and/or a solid-phase tandem extraction procedure were used with aqueous homogenates of pan-fried or uncooked beef, both before and after proteolysis (proteinase K). Genotoxicity was measured using the alkaline single cell-gel electrophoresis ('Comet') assay in MCL-5 cells and mutagenicity in Salmonella typhimurium strains TA1538 or YG1019. Proteolysis released significant amounts of DNA-damaging material that was not extractible prior to enzymic digestion, suggesting that human exposures to diet-derived genotoxins may have been underestimated.

Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins. Extracts of human milk from UK-resident women (n=7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays. Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9. A seventh extract, tested only in the absence of S9, was inactive. Extracts were either active or inactive in at least three of the four tests applied. Four extracts were active or inactive in all four tests. The results suggest that human milk could be used as a resource for investigations of the as-yet-unidentified transforming agents previously detected in mammary lipid.

Exfoliated cells, isolated from breast milk samples donated by UK-resident women (n=15), were incubated, either immediately or after culture for 7 days, with one of a series of genotoxins, either in the presence or absence of the DNA-repair inhibitors, hydroxyurea (HU), and cytosine arabinoside (ara-C). The numbers of DNA single-strand breaks induced were then assessed as comet tail length (CTL) (microm) using the alkaline single cell-gel electrophoresis ('Comet') assay; cell viability was measured by trypan blue exclusion. The heterocyclic aromatic amines (HAAs) (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (0.4 mM), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) (1.67 mM), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) (1.77 mM)), a polycyclic aromatic hydrocarbon (benzo[a]pyrene (B[a]P) (0.36 mM)), a nitro-polycyclic aromatic hydrocarbon (1-nitropyrene (1-NP) (1.84 mM)) and aromatic amines (o-toluidine (0.85 mM), p-chloroaniline (0. 71 mM)) each induced statistically significant (P<0.0001, Mann-Whitney test) increases in median CTLs in breast milk cells from all the donors examined when incubated (30 min, 37 degrees C) in the presence of HU/ara-C. In some cases, these compounds were also active in the absence of the repair inhibitors. There were marked variations in comet formation between donors and between genotoxins. Cell culture appeared to increase the epithelial cell proportion and cultured cells retained their ability to activate genotoxins. The results suggest that breast milk is a valuable source of human mammary cells for the study of the metabolic activation of possible carcinogens.

We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7, 12-dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked comet formation even in the presence of HU/ara-C. Aniline and o-toluidine produced significant levels of comet formation in the absence of HU/ara-C, but in their presence comet formation was markedly increased. Benzene, lindane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride induced comet formation in the absence of HU/ara-C, but, with the exception of cisplatin, their presence enhanced comet formation. Neither sucrose nor DES elicited comet formation under the conditions used in this study. Many more agents need to be tested in order to determine how well the comet assay using MCL-5 cells (or modified versions of it) can distinguish genotoxins from non-genotoxins.