Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

Morphometric analysis of bite mark patterns caused by domestic dogs ( Canis lupus familiaris ) using dental wax registers / Análisis de patrones de huellas de mordedura producidos por perros domésticos ( Canis lupus familiaris ) utilizando registros en cera dental

Dog bites are a known public health problem involving physical, mental and emotional traumas. From a forensic point of view, it has been stated that their morphological characters, and the intercanine and interincisive measurements, could allow a taxonomic and specific identification of the implicated animal. The aim of this study was to differentiate and identify the biological profile of a potential aggressor dog by analysing eight morphometric bite patterns belonging to three different dog breeds. The data obtained were analyzed following three categories: a) breeds; b) sexes among breeds; and c) sex within breed. Significant differences were detected among the variables (p≤ 0.05), but only the maximum maxillary intercanines width (MaxCW) allowed a breed differentiation. The other variables allowed a differentiation between two breeds or one breed over the others. The principal components analysis (PCA) allowed visualizing the degree of dispersion and relationship among the scores. It showed three well-defined and separated breed groups, and different degrees of dispersion within and among breeds. The most important variable for such a differentiation was MaxCW. When considering sex among breeds for males, it showed a statistically significant difference, but only the diastema located between the third left mandibular, incisive and the left mandibular canine (C-I-ManL) allowed breed differentiation. For females, only MaxCW allowed a differentiation among breeds. The multivariate analysis permitted with a 95 % confidence interval, a breed and sex differentiation. Besides, the PCA models allowed classifying, identifying, separating and graphically showing the relationship among the variables. This made it possible to differentiate between breeds and sexes. Due to the large range of dog breeds around the world, this multivariate analysis could also help determining the dog's weight and size, narrowing down towards an approximate number of offending dogs, focussing on a certain kind of dog breed, and pinpointing any suspect dog.

Las mordeduras de perros son un conocido problema de salud pública que involucra traumas físicos, mentales y emocionales. En la faz forense, se ha establecido que características morfológicas, así como las medidas intercanina e interincisiva, permitirían una identificación taxonómica y especifica del animal involucrado. El objetivo fue diferenciar e identificar el perfil biológico de un potencial perro agresor analizando ocho patrones morfológicos de mordeduras pertenecientes a tres diferentes razas caninas. Los datos fueron analizados considerando tres categorías: a) razas; b) sexo entre razas; y c) sexo en cada raza. Se observaron diferencias entre las variables (p≤0,05), pero sólo la distancia máxima entre caninos maxilares (MaxCW), permitió una diferenciación entre razas. Las otras variables permitieron una diferenciación entre dos razas o de una raza sobre las otras. El análisis de componentes principales (PCA) permitió visualizar el grado de dispersión y la relación entre las puntuaciones (dentro y entre razas). Se observaron tres grupos (razas) bien definidos y separados con diferentes grados de dispersión dentro y entre razas. La variable más importante para tal diferenciación fue la MaxCW. Al considerar sexo (machos) entre razas, se encontró diferencia estadísticamente significativa, pero sólo la medida del diastema localizado entre el tercer incisivo y canino izquierdo mandibulares (C-I-ManL) permitió la diferenciación entre razas. Para hembras, sólo la MaxCW permitió una diferenciación entre razas. El análisis multivariante permitió, con un intervalo de confianza del 95 %, diferenciar raza y sexo. El modelo PCA permitió además clasificar, identificar, separar y mostrar gráficamente la relación entre las variables. Esto posibilitó diferenciar entre razas y sexos. Debido a la gran variedad de razas de perros en el mundo, este análisis multivariado permitiría estimar peso y tamaño del animal, indicando un número aproximado de perros atacantes, centrándose en ciertos tipos de raza, y localizar a cualquier cánido sospechoso.

A validated GC-MS method for ketamine and norketamine in hair and its use in authentic cases.

This work has shown that it is a valid method for determining ketamine, norketamine and amphetamines derivates in hair samples of forensic cases. This method was validated meeting the criteria of sensitivity and accuracy for detecting repeated consumption of ketamine in hair samples of forensic interest, according to the proposed cut-off for ketamine of 0.5 ng/mg. The detection of norketamine allowed discriminating between active uses and external contamination. The assessed method was applied for analyzing 1189 hair samples of judicial interest received in the INTCF along 15 months, obtaining 62 positive in ketamine consumption. This means a 5.2% of positivity. Ketamine consumers present a profile of young age (21-30 years old), polydrug use with consumption of synthetic substances preferably MDMA and, then, amphetamine. As consumer is collective, prone to consume new psychoactive substances, requires special attention due to they show a consumer profile with higher prevalence in MDMA than amphetamine, indicating that ketamine consumers belong to a subgroup with a different profile within the INTCF casuistry. The results of the exercises of the proficiency tests performed satisfactorily in all cases. In conclusion, it is a suitable method also to evaluate the chronic consumption of ketamine, in addition to amphetamines in the same method of analysis.

Efectividad del Decálogo de prevención cardiovascular en diabéticos / Effectiveness of a Decalogue for cardiovascular prevention in diabetics

INTRODUCCIÓN: Evaluar la efectividad en conocimiento, percepción de control y mejora del riesgo cardiovascular (RCV) mediante la entrega de un Decálogo visual. MATERIAL Y MÉTODOS: Ensayo clínico de intervención educativa, no farmacológico, con grupo control paralelo, aleatorizado, evaluado a ciegas, en pacientes diabéticos tipo 2, realizado en Atención Primaria de Salud. Ambos grupos recibieron una intervención educativa sobre RCV y su grado de control, y el de intervención además un Decálogo visual con los factores de RCV modificables que reflejaba visualmente su grado de control. Incluimos 50 pacientes en cada grupo para detectar una mejora del conocimiento multifactorial del RCV del 50%. Todos los pacientes recibieron un recuerdo telefónico a los 2 meses, evaluando enmascaradamente el conocimiento y la percepción de control del RCV, y al cabo de 6 meses se evaluó de nuevo presencialmente el conocimiento y el control real del RCV. RESULTADOS: Seleccionamos 51 hombres y 49 mujeres, de 62,9 años de edad media, 9,2 años de evolución y escaso nivel educativo (32% sin estudios y 52% estudios primarios). Basalmente, el conocimiento, la percepción y el control del RCV fueron del 55,0, 80,4 y 65,9%, respectivamente. A los 2 meses, el conocimiento aumentó en el grupo del Decálogo un 16,5% más que en el grupo de educación convencional (73,6% vs. 63,2%; p < 0,05); la percepción sobreestimada de control mejoró un 34,5% en el total de la muestra (p < 0,001) sin diferencias entre grupos, aunque mejoró más la concordancia en el grupo del Decálogo. A los 6 meses aumentó el conocimiento globalmente en un 25,6% (p < 0,001), desapareciendo las diferencias iniciales entre grupos. El control final del RCV aumentó globalmente y en el grupo del Decálogo en un 6,4% (p < 0,005) y un 9,4% (p < 0,001), respectivamente. El riesgo SCORE disminuyó significativamente de forma global y en ambos grupos, sin diferencias entre ambos. CONCLUSIONES: La intervención educativa mejora globalmente el conocimiento, la percepción y el control del RCV. El Decálogo de RCV incrementa rápidamente el conocimiento y disminuye la falsa percepción subjetiva de control del RCV, aunque el beneficio se equipara a los 6 meses si las intervenciones educativas persisten

INTRODUCTION: To assess the effects of a visual Decalogue aid on the degree of knowledge, control perception and improvement in cardiovascular risk factors (CVRF). MATERIAL AND METHODS: A Primary care randomised non-pharmacological trial of an educational intervention with a parallel control group, and blind evaluation in type 2 diabetic patients. Both groups received an educational intervention on the management of CVRF. The intervention group also received a visual Decalogue aid that showed the level of control patients have over the modifiable CVRF. A total of 50 patients were included in each group in order to identify an improvement of 50% in the multifactorial knowledge of CVRF. All patients received a reminder telephone call at 2 months, with masked evaluation of knowledge and CVRF control perception. In a 6 months visit the level of knowledge and real control of CVRF were re-evaluated. RESULTS: The study included 51 males and 49 females, with mean age of 62.9 years, a mean disease duration of 9.2 years, and low educational level. The level of knowledge, control perception, and real control at baseline was 55%, 80.4%, and 65.9%, respectively. After 2 months the level of knowledge in the Decalogue group increased by 16.5% more than in the conventional education group (73.6% vs. 63.2%; P < .05) and the overestimated control perception improved by 34.5% (P < .001) with no differences between groups, although concordance was better in the Decalogue group. At 6 months there was an overall increase 25.6% (P < .001) in the level of knowledge, with the previous difference between groups levelling off. The final CVRF control improved overall and in the Decalogue group by 6.4% (P < .005) and 9.4% (P < .001), respectively. The SCORE risk significantly decreased overall with no differences between groups. CONCLUSIONS: The educational intervention improves the overall level of knowledge, perception and control of CVRF. The CVRF Decalogue quickly increases the level of knowledge, and decreases the false subjective risk control perception. The benefit, however, becomes equal at 6 months with ongoing education interventions

[Effectiveness of a Decalogue for cardiovascular prevention in diabetics]. / Efectividad del Decálogo de prevención cardiovascular en diabéticos.

INTRODUCTION: To assess the effects of a visual Decalogue aid on the degree of knowledge, control perception and improvement in cardiovascular risk factors (CVRF). MATERIAL AND METHODS: A Primary care randomised non-pharmacological trial of an educational intervention with a parallel control group, and blind evaluation in type 2 diabetic patients. Both groups received an educational intervention on the management of CVRF. The intervention group also received a visual Decalogue aid that showed the level of control patients have over the modifiable CVRF. A total of 50 patients were included in each group in order to identify an improvement of 50% in the multifactorial knowledge of CVRF. All patients received a reminder telephone call at 2 months, with masked evaluation of knowledge and CVRF control perception. In a 6 months visit the level of knowledge and real control of CVRF were re-evaluated. RESULTS: The study included 51 males and 49 females, with mean age of 62.9 years, a mean disease duration of 9.2 years, and low educational level. The level of knowledge, control perception, and real control at baseline was 55%, 80.4%, and 65.9%, respectively. After 2 months the level of knowledge in the Decalogue group increased by 16.5% more than in the conventional education group (73.6% vs. 63.2%; P<.05) and the overestimated control perception improved by 34.5% (P<.001) with no differences between groups, although concordance was better in the Decalogue group. At 6 months there was an overall increase 25.6% (P<.001) in the level of knowledge, with the previous difference between groups levelling off. The final CVRF control improved overall and in the Decalogue group by 6.4% (P<.005) and 9.4% (P<.001), respectively. The SCORE risk significantly decreased overall with no differences between groups. CONCLUSIONS: The educational intervention improves the overall level of knowledge, perception and control of CVRF. The CVRF Decalogue quickly increases the level of knowledge, and decreases the false subjective risk control perception. The benefit, however, becomes equal at 6 months with ongoing education interventions.

Human Ultraweak Photon Emission: Key Analytical Aspects, Results and Future Trends - A Review.

Living systems emit what is called ultraweak photon emission (UPE). This visually undetectable phenomenon has only been studied in humans for the last 30 years, finding that UPE is a complex process depending on multitude factors. Considering previous literature, this review discusses the current trends in the analysis of in vivo UPE from human beings. To this aim, Analytical Approaches Employed for UPE Measurement section focuses on the analytical techniques employed (photomultipliers and charged coupled device cameras), summarizing analytical conditions and reporting figures of merit reached to date. Then, Human UPE Depending on External Factors and Human UPE Depending on Internal Factors sections address external and internal factors, which have proved to affect UPE, pointing out the important influence on oxidative processes outside and inside the body, and also highlighting some personal states of the individuals affecting UPE. Last section is devoted to give a general view on the goals and achieved up to date regarding UPE measurement, emphasizing some potential applications as well as recommendations which include: use of UPE spectra information together with UPE intensity, larger populations (≈50-100 subjects), further studies on internal states of individuals, and use of statistical tools.

Short wave infrared chemical imaging as future tool for analysing gunshot residues patterns in targets.

This work used chemical imaging in the short-wave infrared region for analysing gunshot residues (GSR) patterns in cotton fabric targets shot with conventional and non-toxic ammunition. It presents a non-destructive, non-toxic, highly visual and hiperspectral-based approach. The method was based on classical least squares regression, and was tested with the ammunition propellants and their standard components' spectra. The propellants' spectra were satisfactorily used (R2 >0.966, and CorrCoef >0.982) for identifying the GSR irrespective of the type of ammunition used for the shooting. In a more versatile approach, nitrocellulose, the main component in the ammunition propellants, resulted an excellent standard for identifying GSR patterns (R2>0.842, and CorrCoef >0.908). In this case, the propellants' stabilizers (diphenilamine and centralite), and its nitrated derivatives as well as dinitrotoluene, showed also high spectral activity. Therefore, they could be recommended as complementary standards for confirming the GSR identification. These findings establish the proof of concept for a science-based evidence useful to support expert reports and final court rulings. This approach for obtaining GSR patterns can be an excellent alternative to the current and traditional chemical methods, which are based in presumptive and invasive colour tests.

Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulin resistance: cause or consequence?

Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome and refers to a spectrum of disorders ranging from steatosis to steatohepatitis, a disease stage characterized by inflammation, fibrosis, cell death and insulin resistance (IR). Due to its association with obesity and IR the impact of NAFLD is growing worldwide. Consistent with the role of mitochondria in fatty acid (FA) metabolism, impaired mitochondrial function is thought to contribute to NAFLD and IR. Indeed, mitochondrial dysfunction and impaired mitochondrial respiratory chain have been described in patients with non-alcoholic steatohepatitis and skeletal muscle of obese patients. However, recent data have provided evidence that pharmacological and genetic models of mitochondrial impairment with reduced electron transport stimulate insulin sensitivity and protect against diet-induced obesity, hepatosteatosis and IR. These beneficial metabolic effects of impaired mitochondrial oxidative phosphorylation may be related not only to the reduction of reactive oxygen species production that regulate insulin signaling but also to decreased mitochondrial FA overload that generate specific metabolites derived from incomplete FA oxidation (FAO) in the TCA cycle. In line with the Randle cycle, reduced mitochondrial FAO rates may alleviate the repression on glucose metabolism in obesity. In addition, the redox paradox in insulin signaling and the delicate mitochondrial antioxidant balance in steatohepatitis add another level of complexity to the role of mitochondria in NAFLD and IR. Thus, better understanding the role of mitochondria in FA metabolism and glucose homeostasis may provide novel strategies for the treatment of NAFLD and IR.

Anionic markers for the forensic identification of Chemical Ignition Molotov Cocktail composition.

An improved version of the famous Molotov cocktail is the Chemical Ignition Molotov Cocktail (CIMC). This incendiary device contains chemical reagents that enable its self-ignition. The analysis of anions from CIMC residues by capillary electrophoresis (CE) allows the identification of the reagents used to produce the device, and provides forensic analysts with valuable information. Although, sulfate, chlorate, chloride, and perchlorate anions have been recently proposed in the literature as target anions to determine the CIMC composition, the identification of some of them could be controversial due to their presence in the environment. Therefore, the purpose of this study was to identify highly reliable anions capable of indicating the components used to prepare these self-initiated devices. The relationship among the detected anions in CIMC residues and the reagents employed in their elaboration is discussed. Some anions have been proposed as anionic markers of CIMC as incendiary devices. Additionally, the viability of different CIMC compositions was studied.

Characterization of carboxylate-terminated carbosilane dendrimers and their evaluation as nanoadditives in capillary electrophoresis for vegetable protein profiling.

Protein profiles are becoming an important tool to differentiate and classify varieties of several cultivars and to obtain a specific fingerprint for them. The use of protein profiles for these purposes needs to achieve high separation efficiencies to obtain a high number of well resolved peaks. In this work, carbosilane dendrimers with interior carbon-silicon bonds and negatively charged in the dendrimer surface with carboxylic acid as functional groups were employed as nanoadditives to separate soybean and olive seeds proteins. First, these dendrimers were characterized using CE to evaluate their possible impurities. A potentiometric titration was later carried out to determine their pK(a) values. Afterwards, the characterized dendrimers were used to improve the protein profiles obtained by EKC for vegetable proteins. Different dendrimer generations (G1, G2, and G3) and concentrations (0.01-1% m/v) were tested. The highest dendrimer generation G3 at 0.1% (m/v) allowed observing the best protein profiles for soybean and olive seeds. These results demonstrate that carboxylate-terminated carbosilane dendrimers are attractive nanoadditives in EKC for the effective separation of vegetable proteins.

Oxidative stress and altered mitochondrial function in neurodegenerative diseases: lessons from mouse models.

Oxidative stress has been consistently linked to ageing-related neurodegenerative diseases leading to the generation of lipid peroxides, carbonyl proteins and oxidative DNA damage in tissue samples from affected brains. Studies from mouse models that express disease-specific mutant proteins associated to the major neurodegenerative processes have underscored a critical role of mitochondria in the pathogenesis of these diseases. There is strong evidence that mitochondrial dysfunction is an early event in neurodegeneration. Mitochondria are the main cellular source of reactive oxygen species and key regulators of cell death. Moreover, mitochondria are highly dynamic organelles that divide, fuse and move along axons and dendrites to supply cellular energetic demands; therefore, impairment of any of these processes would directly impact on neuronal viability. Most of the disease-specific pathogenic mutant proteins have been shown to target mitochondria, promoting oxidative stress and the mitochondrial apoptotic pathway. In addition, disease-specific mutant proteins may also impair mitochondrial dynamics and recycling of damaged mitochondria via autophagy. Collectively, these data suggest that ROS-mediated defective mitochondria may accumulate during and contribute to disease progression. Strategies aimed to improve mitochondrial function or ROS scavenging may thus be of potential clinical relevance.

Estrés oxidativo mitocondrial en patología hepática / No disponible

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Brain mitochondrial alterations after chronic alcohol consumption

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The aim of this study was to demonstrate the existence of alterations in glutathione and cholesterol homeostasis in brain mitochondria from alcoholic rats. Glutathione concentration decreased, whereas oxidized glutathione and cholesterol contents increased in these organelles, suggesting the ethanol-induced generation of reactive oxygen species, and the impairment of mitochondrial uptake of glutathione, possibly due to the increase in cholesterol deposition. The release of apoptogenic proteins was increased after stimulating mitochondria from the brain of alcoholic rats with atractyloside. As a conclusion, chronic alcohol consumption might sensitize brain mitochondria to apoptotic stimuli, and promote the subsequent release of apoptotic proteins (AU)

Mitochondrial cholesterol in health and disease.

Cholesterol is a critical component of biological membranes, which not only plays an essential role in determining membrane physical properties, but also in the regulation of multiple signaling pathways. Cells satisfy their need for cholesterol either by uptake from nutrients and lipoproteins or de novo synthesis from acetyl-CoA. The latter process occurs in the endoplasmic reticulum, where transcription factors that regulate the expression of enzymes involved in the de novo cholesterol synthesis reside. Cholesterol is distributed to different membranes most prominently to plasma membrane, where it participates in the physical organization of specific membrane domains. Mitochondria, however, are considered cholesterol-poor organelles, and obtain their cholesterol load by the action of specialized proteins involved in its delivery from extramitochondrial sources and trafficking within mitochondrial membranes. Although mitochondrial cholesterol fulfills vital physiological functions, such as the synthesis of bile acids in the liver or the formation of steroid hormones in specialized tissues, recent evidence indicates that the accumulation of cholesterol in mitochondria may be a key step in disease progression, including steatohepatitis, carcinogenesis or Alzheimer disease.

Brain mitochondrial alterations after chronic alcohol consumption.

The aim of this study was to demonstrate the existence of alterations in glutathione and cholesterol homeostasis in brain mitochondria from alcoholic rats. Glutathione concentration decreased, whereas oxidized glutathione and cholesterol contents increased in these organelles, suggesting the ethanol-induced generation of reactive oxygen species, and the impairment of mitochondrial uptake of glutathione, possibly due to the increase in cholesterol deposition. The release of apoptogenic proteins was increased after stimulating mitochondria from the brain of alcoholic rats with atractyloside. As a conclusion, chronic alcohol consumption might sensitize brain mitochondria to apoptotic stimuli, and promote the subsequent release of apoptotic proteins.

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo.

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.