Management of per- and polyfluoroalkyl substances (PFAS)-laden wastewater sludge in Maine: Perspectives on a wicked problem.

This article discusses the challenges and potential solutions for managing wastewater sludge that contains per- and polyfluoroalkyl substances (PFAS), using the experience in Maine as a guide toward addressing the issue nationally. Traditional wastewater treatment, designed to remove excess organic waste and nutrients, does not eliminate persistent toxic pollutants like PFAS, instead partitioning the chemicals between discharged effluent and the remaining solids in sludge. PFAS chemistry, the molecular size, the alkyl chain length, fluorine saturation, the charge of the head group, and the composition of the surrounding matrix influence PFAS partitioning between soil and water. Land application of sludge, incineration, and storage in a landfill are the traditional management options. Land application of Class B sludge on agricultural fields in Maine peaked in the 1990s, totaling over 2 × 106 cu yd over a 40-year period and has contaminated certain food crops and animal forage, posing a threat to the food supply and the environment. Additional Class A EQ (Exceptional Quality) composted sludge was also applied to Maine farmland. The State of Maine banned the land application of wastewater sludge in August 2022. Most sludge was sent to the state-owned Juniper Ridge Landfill, which accepted 94 270 tons of dewatered sludge in 2022, a 14% increase over 2019. Between 2019 and 2022, the sum of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) concentrations in sludge sent to the landfill ranged from 1.2 to 104.9 ng/g dw. In 2022, the landfill generated 71.6 × 106 l of leachate. The concentration of sum of six PFAS in the leachate increased sixfold between 2021 and 2022, reaching 2 441 ng/l. The retention of PFAS within solid-waste landfills and the potential for long-term release of PFAS through liners into groundwater require ongoing monitoring. Thermal treatment, incineration, or pyrolysis can theoretically mineralize PFAS at high temperatures, yet the strong C-F bond and reactivity of fluorine require extreme temperatures for complete mineralization. Future alternatives may include interim options such as preconditioning PFAS with nonpolar solvents prior to immobilization in landfills, removing PFAS from leachate, and interrupting the cycle of PFAS moving from landfill, via leachate, to wastewater treatment, and then back to the landfill via sludge. Long-term solutions may involve destructive technologies such as electron beam irradiation, electrochemical advanced oxidation, or hydrothermal liquefaction. The article highlights the need for innovative and sustainable solutions for managing PFAS-contaminated wastewater sludge.

Sex differences in choice-based thermal nociceptive tests in adult rats.

Interest in the role of sex as a biological variable has increased, including a mandate for the study of both sexes in NIH-funded research. As sex differences exist in both human chronic pain conditions and rodent models of nociception, it is critical to understand the impact of sex in nociceptive assays. Choice-based thermal nociceptive tests permit the study of avoidance responses to thermal stimuli compared to traditional nociceptive assays, which measure nocifensive reactions. However, to date no comparison of male and female responses to choice-based tests has been published. Herein, we examined the effect of sex on two choice-based thermal nociceptive tests, the thermal gradient test and the temperature place preference test, in adult rats. The activation of a 10 °C-to-47 °C thermal gradient results in an increase in time spent in the 10 °C zone in females, compared to a reduction in males. Additionally, in a temperature place preference test pairing a surface temperature of 22 °C with either 5 °C, 10 °C, 47 °C, or 50 °C, females appeared to have overall greater tolerance for non-ambient temperatures. Males spent less than 50% of their time in every non-22 °C zone, whereas in females this was only observed when testing 5 °C and 50 °C. Together, these results suggest that male rats show more avoidance behavior than females to both hot and cold non-ambient temperatures when given free access to multiple zones, including at milder temperatures than those typically used to evoke a nociceptive response in traditional hot and cold plate tests.

Morphine exposure alters Fos expression in a sex-, age-, and brain region-specific manner during adolescence.

Data in both humans and preclinical animal models clearly indicate drug exposure during adolescence, when the "reward" circuitry of the brain develops, increases the risk of substance use and other mental health disorders later in life. Human data indicate that different neural and behavioral sequelae can be observed in early versus late adolescence. However, most studies with rodent models examine a single adolescent age compared to a mature adult age, and often only in males. Herein, we sought to determine whether the acute response to the opioid morphine would also differ across adolescence, and by sex. By quantifying Fos positive cells, a proxy for neural activity, at different stages during adolescence (pre-, early, mid-, and late adolescence) and in multiple reward regions (prefrontal cortex, nucleus accumbens, caudate/putamen), we determined that the neural response to acute morphine is highly dependent on adolescent age, sex, and brain region. These data suggest that heterogeneity in the consequences of adolescent opioid exposure may be due to age- and sex-specific developmental profiles in individual reward processing regions. In future studies, it will be important to add age within adolescence as an independent variable for a holistic view of healthy or abnormal reward-related neural development.

A semi-automated brain atlas-based analysis pipeline for c-Fos immunohistochemical data.

BACKGROUND: The use of immunohistochemistry to quantify neural markers in various brain regions is a staple of neuroscience research. Numerous programs exist to automate quantification, but manual assignment of regions of interest (ROIs) within individual brain sections remains time consuming and can introduce interobserver variability. NEW METHOD: We have developed a novel open source FIJI-based immunohistochemical data analysis pipeline, Atlas-Based Analysis (ABA). ABA uses landmark-based image warping to adjust the experimental image to closely align with a published rat brain atlas. c-Fos positive cells are then quantified within predetermined ROI coordinates derived from the brain atlas. Image warping adjusts for natural variation in brain sections to ensure reliable alignment of ROIs for data analysis. This pipeline can be adapted for new atlases, landmarks, ROIs, and quantification measurements. RESULTS: ABA permits rapid quantification of immunoreactivity in multiple ROIs and produces results with high levels of interobserver consistency. COMPARISON WITH EXISTING METHODS: Compared to manual ROI designation, ABA reduces total analysis time by ∼70%. With correct use of landmarks for image warping, ABA produces similar results to manually drawn ROIs, results in no interobserver variability, and maintains c-Fos+ pixel dimensions. CONCLUSIONS: ABA reduces time to obtain reliable results when performing automated immunoreactivity quantification and allows multiple users to analyze data without compromising the reliability of data obtained.

Coagulation parameters following equine herpesvirus type 1 infection in horses.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) is the cause of respiratory disease, abortion storms, and outbreaks of herpesvirus myeloencephalopathy (EHM). Infection of the spinal cord is characterised by multifocal regions of virally infected vascular endothelium, associated with vasculitis, thrombosis and haemorrhage that result in ischaemia and organ dysfunction. However, the mechanism of thrombosis in affected horses is unknown. OBJECTIVES: To evaluate tissue factor (TF) procoagulant activity and thrombin-antithrombin complex (TAT) levels in horses following infection with EHV-1. STUDY DESIGN: In vitro and in vivo studies following experimental EHV-1 infection. METHODS: Horses were infected with EHV-1 and levels of peripheral blood mononuclear cell (PBMC)-associated TF activity; plasma and cerebrospinal fluid (CSF)-derived microvesicle (MV)-associated TF activity and TAT complexes in plasma were examined. RESULTS: EHV-1 infection increased PBMC TF procoagulant activity in vitro and in vivo. In infected horses, this increase was observed during the acute infection and was most marked at the onset and end of viraemia. However, no significant differences were observed between the horses that showed signs of EHM and the horses that did not develop EHM. Significant changes in MV-associated TF procoagulant activity and TAT complexes were not observed in infected horses. MAIN LIMITATIONS: A small number of horses typically exhibit clinical EHM following experimental infection. CONCLUSIONS: The results indicate that EHV-1 infection increases PBMC-associated TF procoagulant activity in vivo and in vitro. Additional in vivo studies are needed to better understand the role of TF-dependent coagulation during EHM pathogenesis in horses.

Spatial and temporal trends of mercury in the aquatic food web of the lower Penobscot River, Maine, USA, affected by a chlor-alkali plant.

Mercury (Hg) concentrations in aquatic biota, including fish and shellfish, were measured over the period 2006-2012 in the lower Penobscot River and upper estuary (Maine, USA). The Penobscot is a system contaminated with Hg by a chlor-alkali plant that operated from 1967 to 2000, discharging 6-12 tons of mercury into the river. Mercury levels in aquatic biota were highest at sites downstream of the chlor-alkali plant and spatial trends were similar to those of sediments. Mean total Hg concentrations in fish muscle (adjusted for size or age) in the most affected areas were 521 (480, 566; 95% CI) ng/g ww in American eels, 321 (261,395) in mummichog, 121 (104, 140) in rainbow smelt, 155 (142,169) in tomcod, 55.2 (42.7,71.4) in winter flounder, and 328 (259,413) in American lobster tail and 522 (488,557) ng/g dw in blue mussel. Levels exceeded the 50 ng/g ww considered protective for piscivorous predators and were of concern for human health, with American eels and American lobster exceeding Maine's mercury action level of 200 ng/g ww. Calculations of trophic position (using nitrogen isotopes) suggested that the spatial patterns observed in total Hg concentrations were not due to changes in feeding habits of the species. Fish feeding in benthic food webs, as defined by stomach content and stable carbon isotope analyses, showed no change in Hg concentrations over time. In contrast, declining trends in Hg were found in two species dependent on pelagic food webs. The absence of declines in Hg concentrations in the benthically-based food webs, despite the fact that most Hg was discharged into the system >40 years ago, is consistent with the long recovery predicted from dated sediment cores and from similar studies elsewhere.

Chronic liver injury drives non-traditional intrahepatic fibrin(ogen) crosslinking via tissue transglutaminase.

Essentials Fibrin clots are often implicated in the progression of liver fibrosis. Liver fibrosis was induced in transgenic mice with defects in clot formation or stabilization. Liver fibrosis and fibrin(ogen) deposition do not require fibrin polymerization or factor XIIIa. Fibrin(ogen) is an in vivo substrate of tissue transglutaminase in experimental liver fibrosis. SUMMARY: Background Intravascular fibrin clots and extravascular fibrin deposits are often implicated in the progression of liver fibrosis. However, evidence supporting a pathological role of fibrin in hepatic fibrosis is indirect and based largely on studies using anticoagulant drugs that inhibit activation of the coagulation protease thrombin, which has other downstream targets that promote fibrosis. Therefore, the goal of this study was to determine the precise role of fibrin deposits in experimental hepatic fibrosis. Methods Liver fibrosis was induced in mice expressing mutant fibrinogen insensitive to thrombin-mediated proteolysis (i.e. locked in the monomeric form), termed FibAEK mice, and factor XIII A2 subunit-deficient (FXIII-/- ) mice. Female wild-type mice, FXIII-/- mice and homozygous FibAEK mice were challenged with carbon tetrachloride (CCl4 ) twice weekly for 4 weeks or 6 weeks (1 mL kg-1 , intraperitoneal). Results Hepatic injury and fibrosis induced by CCl4 challenge were unaffected by FXIII deficiency or inhibition of thrombin-catalyzed fibrin polymer formation (in FibAEK mice). Surprisingly, hepatic deposition of crosslinked fibrin(ogen) was not reduced in CCl4 -challenged FXIII-/- mice or FibAEK mice as compared with wild-type mice. Rather, deposition of crosslinked hepatic fibrin(ogen) following CCl4 challenge was dramatically reduced in tissue transglutaminase-2 (TGM2)-deficient (TGM2-/- ) mice. However, the reduction in crosslinked fibrin(ogen) in TGM2-/- mice did not affect CCl4 -induced liver fibrosis. Conclusions These results indicate that neither traditional fibrin clots, formed by the thrombin-activated FXIII pathway nor atypical TGM2-crosslinked fibrin(ogen) contribute to experimental CCl4 -induced liver fibrosis. Collectively, the results indicate that liver fibrosis occurs independently of intrahepatic fibrin(ogen) deposition.

Distribution and biogeochemical controls on net methylmercury production in Penobscot River marshes and sediment.

The distribution of mercury and methylmercury (MeHg) in sediment, mudflats, and marsh soils of the Hg-contaminated tidal Penobscot River was investigated, along with biogeochemical controls on production. Average total Hg in surface samples (0-3 cm) ranged from 100 to 1200 ng/g; average MeHg ranged from 5 to 50 ng/g. MeHg was usually highest at or near the surface except in highly mobile mudflats. Although total Hg concentrations in the Penobscot are elevated, it is the accumulation of MeHg that stands out in comparison to other ecosystems. Surface soils in the large Mendall Marsh, about 17 km downstream from the contamination source, contained particularly high %MeHg (averaging 8%). In Mendall marsh soil porewaters, MeHg often accounted for more than half of total Hg. Salt marshes are areas of particular concern in the Penobscot River, for they are depositional environments for a Hg-contaminated mobile pool of river sediment, hot spots for net MeHg production, and sources of risk to marsh animals. We hypothesized that exceptionally low mercury partitioning between the solid and aqueous phases (with log Kd averaging ~4.5) drives high MeHg in Penobscot marshes. The co-occurrence of iron and sulfide in filtered soil porewaters, sometimes both above 100 µM, suggests the presence of nanoparticulate and/or colloidal metal sulfides. These colloids may be stabilized by high concentrations of aromatic and potentially sulfurized dissolved organic matter (DOM) in marsh soils. Thus, Hg in Penobscot marsh soils appears to be in a highly available for microbial methylation through the formation of DOM-associated HgS complexes. Additionally, low partitioning of MeHg to marsh soils suggests high MeHg bioavailability to animals. Overall, drivers of high MeHg in Penobscot marshes include elevated Hg in soils, low partitioning of Hg to solids, high Hg bioavailability for methylation, rapidly shifting redox conditions in surface marsh soils, and high rates of microbial activity.

Current and historical rates of input of mercury to the Penobscot River, Maine, from a chlor-alkali plant.

Mercury inputs by surface and ground water sources to Penobscot River from a defunct Hg-cell chlor-alkali plant were measured in 2009-10 and estimated for the entire period of operation of this facility. Over the measured interval (422 days) approximately 2.3 kg (5.4 g day-1) of mercury was discharged to the Penobscot River by the two surface streams that drain the site, with most of the combined loading (1.8 kg Hg, 78%) associated with a single storm with rainfall in excess of 100 mm. Groundwater seepage rates from the site, as estimated from both a radon tracer and seepage meter methods were in the range of 3 to 4 cm day-1 and, when combined with a best estimate of the area of groundwater discharge (11,000 m2) and average seepage/porewater mercury concentration (242 ng L-1, UCL95), yielded a loading of 0.11 g day-1 for site groundwater. None of the municipal or other industrial point sources of mercury to the river between Veazie and Bucksport, Maine exceeded 1 g day-1 individually, nor was the aggregate loading of all such sources >3 g day-1 (based on State of Maine data). Mercury loadings for the three largest tributaries downstream of Veazie Dam were estimated to contribute 4.2, 3.7 and 2.5 g day-1, respectively, to the Penobscot River. Based on sampling (total Hg ~ 2 to 4 ng L-1) and historical mean discharge data (340-460 m3 s-1), the Penobscot River upstream of the plant site contributes as much as 160 g day-1 to the downstream reach depending on river discharge. Estimates of historical (1967-2012) mercury loading using both generic emission factors and measured releases ranged from 2.6 to 27 MT while the mass of mercury found in downstream sediments amounted to 9 MT.

Elevated mercury in blood and feathers of breeding marsh birds along the contaminated lower Penobscot River, Maine, USA.

Mercury (Hg) concentrations in the blood and feathers of five species of migratory marsh birds, Nelson's sparrow (Ammodramus nelson subvirgatus), song sparrow (Melospiiza melodia), swamp sparrow (Melospiza geogiana), red-winged blackbird (Agelaius phoeniceus), and Virginia rail (Rallus limicola), breeding in marshes along the lower Penobscot River, Maine, far exceeded reference concentrations, exceeded concentrations associated with reproductive health, and are the highest Hg concentrations reported to date for several species. Blood Hg concentrations in adult Nelson's sparrows were greatest in 2007, at 6.6µg/gww (geometric mean) and in 2012, at 6.5µg/gww and greatest in red-winged blackbirds in 2012, 8.0µg/gww. Mercury in blood increased with residence time on the contaminated marshes at an estimated rate of 0.04 to 0.07µg/gww per day. Feather mercury concentrations in specific primary, secondary and tail feathers (P1, S2, R6) were strongly associated with exposure location at the time of feather formation. Geometric mean Hg concentrations in primary feathers (P1) reached 39.6µg/gfw in 2010 in Nelson's sparrows. The paper documents the dynamic nature of Hg concentrations in avian blood and feathers, an important consideration in contaminant study design, and the increased risk to marsh birds posed by Hg deposition from upstream sources.

Tidal fluxes of mercury and methylmercury for Mendall Marsh, Penobscot River estuary, Maine.

Tidal marshes are both important sites of in situ methylmercury production and can be landscape sources of methylmercury to adjacent estuarine systems. As part of a regional investigation of the Hg-contaminated Penobscot River and Bay system, the tidal fluxes of total suspended solids, total mercury and methylmercury into and out of a regionally important mesohaline fluvial marsh complex, Mendall Marsh, were intensively measured over several tidal cycles and at two spatial scales to assess the source-sink function of the marsh with respect to the Penobscot River. Over four tidal cycles on the South Marsh River, the main channel through which water enters and exits Mendall Marsh, the marsh was a consistent sink over typical 12-h tidal cycles for total suspended solids (8.2 to 41 g m-2), total Hg (9.2 to 47 µg m-2), total filter-passing Hg (0.4 to 1.1 µg m-2), and total methylmercury (0.2 to 1.4 µg m-2). The marsh's source-sink function was variable for filter-passing methylmercury, acting as a net source during a large spring tide that inundated much of the marsh area and that is likely to occur during approximately 17% of tidal cycles. Additional measurements on a small tidal channel draining approximately 1% of the larger marsh area supported findings at the larger scale, but differences in the flux magnitude of filter-passing fractions suggest a highly non-conservative transport of these fractions through the tidal channels. Overall the results of this investigation demonstrate that Mendall Marsh is not a significant source of mercury or methylmercury to the receiving aquatic systems (Penobscot River and Bay). While there is evidence of a small net export of filter-passing (<0.4 µm pore size) methylmercury under some tidal conditions, the mass involved represents <3% of the mass of filter-passing methylmercury carried by the Penobscot River.

Mercury in wintering American black ducks (Anas rubripes) downstream from a point-source on the lower Penobscot River, Maine, USA.

Waterfowl wintering along the lower Penobscot River, Maine continue to be exposed to elevated Hg concentrations from the HoltraChem chlor-alkali plant that operated along the river between 1967 and 2000. In American black ducks (Anas rubripes) total Hg in duck breast muscle increased with residence time on contaminated marshes, reaching means of 0.82±0.21µg/g ww (wet weight) by the end of the fall hunting season, and prompting Maine to issue a human consumption advisory on duck breast muscle. Methyl Hg comprised over 99% of the total Hg in breast muscle. The ratio of Hg concentrations in blood and muscle were strongly correlated and approached 1:1 after extended residence times. Primary feather (P1) total Hg concentrations averaged 2.2±1.3µg/g fw (fresh weight), verifying low Hg exposure during feather growth on distant breeding grounds the preceding summer. Mercury concentrations in black ducks, following winter residence along the lower Penobscot exceeded levels associated with reproductive toxicity. Carry-over of Hg to summer breeding grounds may limit the subsequent reproductive success of black ducks.

Assessing chronic fish health: An application to a case of an acute exposure to chemically treated crude oil.

Human alteration of marine ecosystems is substantial and growing. Yet, no adequate methodology exists that provides reliable predictions of how environmental degradation will affect these ecosystems at a relevant level of biological organization. The primary objective of this study was to develop a methodology to evaluate a fish's capacity to face a well-established environmental challenge, an exposure to chemically dispersed oil, and characterize the long-term consequences. Therefore, we applied high-throughput, non-lethal challenge tests to assess hypoxia tolerance, temperature susceptibility and maximal swimming speed as proxies for a fish's functional integrity. These whole animal challenge tests were implemented before (1 month) and after (1 month) juvenile European sea bass (Dicentrarchus labrax) had been acutely exposed (48h) to a mixture containing 0.08gL(-1) of weathered Arabian light crude oil plus 4% dispersant (Corexit© EC9500A), a realistic exposure concentration during an oil spill. In addition, experimental populations were then transferred into semi-natural tidal mesocosm ponds and correlates of Darwinian fitness (growth and survival) were monitored over a period of 4 months. Our results revealed that fish acutely exposed to chemically dispersed oil remained impaired in terms of their hypoxia tolerance and swimming performance, but not in temperature susceptibility for 1 month post-exposure. Nevertheless, these functional impairments had no subsequent ecological consequences under mildly selective environmental conditions since growth and survival were not impacted during the mesocosm pond study. Furthermore, the earlier effects on fish performance were presumably temporary because re-testing the fish 10 months post-exposure revealed no significant residual effects on hypoxia tolerance, temperature susceptibility and maximal swimming speed. We propose that the functional proxies and correlates of Darwinian fitness used here provide a useful assessment tool for fish health in the marine environment.

Dietary conjugated linoleic acid affects blood parameters, liver morphology and expression of selected hepatic genes in laying hens.

The objective of this research were to investigate the effect of a conjugated linoleic acid (CLA)-enriched diet on Isa Brown laying hen health status and to provide a comprehensive analysis of changes in blood parameters, liver morphology and selected hepatic gene expression. Hens were allocated to the control and experimental group (diet enriched with 0.75% CLA) for a total period of 4 m. At the end of the experiment half of the hens from each group were slaughtered for analyses. The remaining hens were transferred to an organic farm for the next 5 m and fed on the diet without CLA supplementation. The CLA-enriched diet resulted in significant changes in blood and serum parameters; specifically, haematocrit (HCT), mean corpuscular volume (MCV) and white blood cells (WBC) count were decreased compared to the control. The total cholesterol (TC) was not significantly affected while the triacylglycerol's (TG) concentration was elevated. The activity of alanine aminotransferase (ALT) was significantly increased in the CLA-supplemented group, while aspartate aminotransferase (AST) showed an increasing tendency. Liver biopsies showed pathological changes classified as non-alcoholic fatty liver disease (NAFLD). Additionally, the expression of hepatic genes involved in fatty acids synthesis (ME1, ACLY, ACC, FASN, SCD1), oxidation (CPT1α, PPARA), detoxification processes (Cytochrome P450, CYP, Flavin-containing monooxygenase, FMO3), oxidative stress (NOX4, XbP1) and inflammation (IL6, TNFα) were elevated. Cessation of CLA supplementation for 5 m of organic farming resulted in normalisation of blood and hepatic parameters to the levels observed in control hens. The results of this study indicate that dietary CLA triggers an integrated stress response in laying hens and activates mechanisms involved in liver detoxification.

Role of hemostatic factors in hepatic injury and disease: animal models de-liver.

Chronic liver damage is associated with unique changes in the hemostatic system. Patients with liver disease often show a precariously rebalanced hemostatic system, which is easily tipped towards bleeding or thrombotic complications by otherwise benign stimuli. In addition, some clinical studies have shown that hemostatic system components contribute to the progression of liver disease. There is a strong basic science foundation for clinical studies with this particular focus. Chronic and acute liver disease can be modeled in rodents and large animals with a variety of approaches, which span chronic exposure to toxic xenobiotics, diet-induced obesity, and surgical intervention. These experimental approaches have now provided strong evidence that, in addition to perturbations in hemostasis caused by liver disease, elements of the hemostatic system have powerful effects on the progression of experimental liver toxicity and disease. In this review, we cover the basis of the animal models that are most often utilized to assess the impact of the hemostatic system on liver disease, and highlight the role that coagulation proteases and their targets play in experimental liver toxicity and disease, emphasizing key similarities and differences between models. The need to characterize hemostatic changes in existing animal models and to develop novel animal models recapitulating the coagulopathy of chronic liver disease is highlighted. Finally, we emphasize the continued need to translate knowledge derived from highly applicable animal models to improve our understanding of the reciprocal interaction between liver disease and the hemostatic system in patients.

A novel quantitative method of pten expression assessment in tumor tissue.

Phosphatase and Tensin Homolog deleted on chromosome 10 (PTEN) gene is one of the most important tumor suppressor genes which is involved in the regulation of many signaling cascades (AKT/PKB and MAPK). Subtle changes in its activity lead to cancer susceptibility or aggressive tumor behaviour. Despite the diversity of mechanisms leading to PTEN inactivation, it is frequently associated with a decreased or complete loss of protein expression. About 20% decrease in PTEN expression could lead to the development of cancer. There have been no objective, quantitative methods of PTEN expression assessment that allow to measure the subtle variations of the protein concentration in a tissue-contextual manner. A new quantitative algorithm of immunostaining evaluation based on combination of color deconvolution and relative chromogen signal intensity was used in the study. The proposed algorithm was implemented in the popular ImageJ image analysis software and positively verified in cancer cell lines and tissue models as well as in the tissue samples of colorectal cancer (CRC) patients. The proposed quantitative method of PTEN expression assessment creates an alternative to currently available subjective methods and forms the basis for inter-case and inter-tissue comparisons. Using the algorithm it would be possible to identify three groups of patients with advanced colorectal cancer which could significantly differ in the overall survival. The research should be continued.

Coagulation-driven platelet activation reduces cholestatic liver injury and fibrosis in mice.

BACKGROUND: The coagulation cascade has been shown to participate in chronic liver injury and fibrosis, but the contribution of various thrombin targets, such as protease activated receptors (PARs) and fibrin(ogen), has not been fully described. Emerging evidence suggests that in some experimental settings of chronic liver injury, platelets can promote liver repair and inhibit liver fibrosis. However, the precise mechanisms linking coagulation and platelet function to hepatic tissue changes following injury remain poorly defined. OBJECTIVES: To determine the role of PAR-4, a key thrombin receptor on mouse platelets, and fibrin(ogen) engagement of the platelet αII b ß3 integrin (αIIb ß3 ) in a model of cholestatic liver injury and fibrosis. METHODS: Biliary and hepatic injury was characterized following 4 week administration of the bile duct toxicant α-naphthylisothiocyanate (ANIT) (0.025%) in PAR-4-deficient mice, mice expressing a mutant form of fibrin(ogen) incapable of binding integrin αII b ß3 (Fibγ(Δ5) ), and wild-type mice. RESULTS: Elevated plasma thrombin-antithrombin and serotonin levels, hepatic fibrin deposition, and platelet accumulation in liver accompanied hepatocellular injury and fibrosis in ANIT-treated wild-type mice. PAR-4 deficiency reduced plasma serotonin levels, increased serum bile acid concentration, and exacerbated ANIT-induced hepatocellular injury and peribiliary fibrosis. Compared with PAR-4-deficient mice, ANIT-treated Fibγ(Δ5) mice displayed more widespread hepatocellular necrosis accompanied by marked inflammation, robust fibroblast activation, and extensive liver fibrosis. CONCLUSIONS: Collectively, the results indicate that PAR-4 and fibrin-αII b ß3 integrin engagement, pathways coupling coagulation to platelet activation, each exert hepatoprotective effects during chronic cholestasis.

An evaluation of the levels of 25-hydroxyvitamin D3 and bone turnover markers in professional football players and in physically inactive men.

Vitamin D is synthesised in the skin during exposure to sunlight and its fundamental roles are the regulation of calcium and phosphate metabolism and bone mineralisation. The aim of our study was to evaluate serum levels of 25-hydroxyvitamin D3, PTH and bone turnover markers (P1NP, OC, beta-CTx, OC/beta-CTx) and the intake of calcium and vitamin D in Polish Professional Football League (Ekstraklasa) players and in young men with a low level of physical activity. Fifty healthy men aged 19 to 34 years were included in the study. We showed that 25(OH)D3 and P1NP levels and OC/beta-CTx were higher in the group of professional football players than in the group of physically inactive men. The daily vitamin D and calcium intake in the group of professional football players was also higher. We showed a significant relationship between 25(OH)D3 levels and body mass, body cell mass, total body water, fat-free mass, muscle mass, vitamin D and calcium intake. Optimum 25(OH)D3 levels were observed in a mere 16.7% of the football players and vitamin D deficiency was observed in the physically inactive men. The level of physical activity, body composition, calcium and vitamin D intake and the duration of exposure to sunlight may significantly affect serum levels of 25(OH)D3.