Estudio mediante técnicas moleculares del tanatomicrobioma intestinal en la estimación del intervalo post mortem temprano empleando un modelo de ratón / Early post mortem interval estimation in a mouse model using molecular analyses of the gut thanatomicrobiome

Introducción: en la actualidad, la falta de métodos cuantitativos fiables ha llevado a distintas líneas de investigación a buscar un modelo que prediga el intervalo post mortem (IPM). El tanatomicrobioma, presente desde el momento de la muerte, parece sufrir cambios predecibles y que siguen una correlación con el IPM.Materiales y métodosse ha analizado experimentalmente el comportamiento del tanatomicrobioma en la región del intestino delgado posterior y del colon ascendente durante las primeras 24 h de descomposición en Mus musculus. Para ello, se ha llevado a cabo una aproximación molecular basada en el análisis del gen ribosomal 16S (ARNr 16S) mediante electroforesis en gel con gradiente desnaturalizante (DGGE) y, seguidamente un análisis de la alfa y beta diversidad.Resultadoslos resultados basados en el análisis de los índices de diversidad ecológica reflejaron cambios estadísticamente significativos antes de las 12 h, y un descenso de la diversidad a partir de esas 12 h postmortem, siendo este estadísticamente significativo en las 2 regiones intestinales analizadas. Por otro lado, el estudio comparativo de las comunidades microbianas mostró que cambian estructurada y diferenciablemente desde el momento de la muerte, alejándose en similitud de las mostradas en vida (IPM 0 h).Discusiónestos resultados coinciden con el descenso de la diversidad sugerido a largo plazo por distintos autores. Sin embargo, en las condiciones del estudio, se ha visto que este descenso no se inicia hasta las 12 h. Conclusión como conclusión, se han podido establecer, según los cambios en la diversidad bacteriana, fases de la dinámica bacteriana durante la descomposición que podrían ayudar a mejorar los modelos de correlación microbiana para la estimación del IPM. (AU)

Introduction: Currently, the lack of reliable quantitative methods has led different research lines to find a model that predicts the postmortem interval (PMI). The thanatomicrobiome, present from the moment of death, has been shown to change in predictable ways, allowing a correlation with PMI.Materials and methodsIn this study, the shifts of the thanatomicrobiome in the region of the posterior small intestine and the ascending colon in Mus musculus during the first 24 hrs of decomposition have been analyzed experimentally. For this purpose, a molecular approach based on the analysis of the 16S ribosomal gene (16S rRNA) and a denaturing gradient gel electrophoresis (DGGE) was adopted, followed by analyses of the ecological diversity indices Alpha and beta diversity.ResultsThe results based on the analysis of the ecological diversity indices reflected statistically significant changes before 12 hrs, and a decrease in diversity after 12 hrs postmortem, this being statistically significant in the two intestinal regions analyzed. Moreover, the comparative study of microbial communities indicated distinct and structured changes from the moment of death, with shifts in the degree of similarity from the composition detected in life (PMI 0 hrs).DiscussionThese results agree with other studies demonstrating a decrease in microbial diversity. However, under the conditions of the study, this decrease does not begin until 12 hrs after death. Conclusions: In conclusion, by examining the dynamics of bacterial diversity our study has identified phases during decomposition that could help to improve microbial correlation models for PMI estimation. (AU)

Revisión sobre las nuevas perspectivas de datación cadavérica desde el necrobioma / Review of cadaveric dating methods and new perspectives from the necrobiome

Hoy en día existen numerosas estrategias desde un punto de vista científico que ayudan a esclarecer los casos forenses, entre ellas la datación cadavérica. La ausencia de métodos fiables cuantitativos para estimar el intervalo post mortem explica el incremento de nuevas líneas de investigación prometedoras con dicha finalidad. Tras la aparición de las nuevas técnicas de secuenciación masiva y bioinformáticas, surge también el estudio del necrobioma como un área novedosa y poco estudiada dentro de las ciencias forenses, que se ha llegado a denominar «microbiología forense». En esta revisión se realiza un breve recorrido por las técnicas y procedimientos existentes de datación cadavérica, centrándose en la utilidad del tanatomicrobioma, o conjunto de microorganismos presentes en el momento de la muerte, que podría ser un método prometedor para la estimación del intervalo post mortem en el futuro. (AU)

Nowadays there are numerous scientific strategies that helping to clarify forensic cases, including time since death. The absence of reliable quantitative methods to estimate the post-mortem interval explains the increase in promising new lines of research for this purpose. After the appearance of the new techniques of massive sequencing and bioinformatics, also arises the study of the necrobiome through a new and little studied area within the forensic sciences, Forensic Microbiology. In this review, a tour of the existing techniques and procedures of cadaveric dating is made, which includes new cutting-edge techniques in different areas of knowledge and also mentions the utilities of Forensic Microbiology, where the thanatomicrobiome, present from the moment of death, according to recent studies, points to be a promising method for estimating the post-mortem interval in the future. (AU)

Body Composition as a Mediator between Cardiorespiratory Fitness and Bone Mass during Growth.

INTRODUCTION AND PURPOSE: To examine the effect of cardiorespiratory fitness (CRF) and muscle power output (MPO) on bone mass of prepubertal and pubertal children using lean mass (LM) and percentage of fat mass (%FM) as mediator variables. The hypothesis was that both LM and %FM would be independent mediators of the relationships during the sexual maturation period. METHODS: We analyzed 200 children (88 boys and 112 girls [11.5 ± 2.0 yr]). Body composition was analyzed by bone densitometry, and indirect calorimetry and cycle ergometer were used to calculate V˙O2peak (mL·kg·min) and MPO (W) during an incremental exercise test. Sample was divided by pubertal status. RESULTS: In the prepubertal group, LM and %FM acted independently as mediators in the relationship between bone mass and CRF or MPO (22%-25% for LM and 37%-50% for %FM, respectively). In pubertal children, LM acted as mediator at 37%. CONCLUSIONS: Although the independent mediator role of LM and %FM in the associations between CRF or MPO and bone mass was present during the prepubertal stage, only LM remain its mediator role in these associations during the postpubertal period. Therefore, with growth and sexual maturation, the full effect of LM seems to increase, whereas the influence of %FM seems to disappear.

Functional characterization of MODY2 mutations highlights the importance of the fine-tuning of glucokinase and its role in glucose sensing.

Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s(-1) vs 47.86±2.78 s(-1)) is balanced by an increased glucose affinity (S(0.5) = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing.

Identification of the first de novo PAR1 deletion downstream of SHOX in an individual diagnosed with Léri-Weill dyschondrosteosis (LWD).

Léri-Weill dyschondrosteosis (LWD, MIM 127300), is a dominantly inherited skeletal dysplasia with disproportionate short stature, mesomelic limb shortening, and the characteristic Madelung deformity. Two regions of the pseudoautosomal region 1 (PAR1) have been shown to be involved in LWD, SHOX (short-stature homeobox-containing gene) and the downstream enhancer region. We report our genetic findings of a young girl clinically diagnosed with LWD. We analyzed the proband and her family using MLPA and microsatellite analysis. We identified a deletion, 726-866 kb in size, of the downstream SHOX enhancer region in the proband. Neither parent carried the deletion. Microsatellite analysis showed that the deleted allele was of paternal origin. The mutation is more likely to have arisen from a de novo event but paternal gonadal mosaicism cannot be excluded. In conclusion, we report the clinical and molecular details of the first case of a de novo deletion of the downstream PAR1 region in an LWD individual. De novo deletions of SHOX and the downstream enhancer region must be therefore considered in cases of isolated LWD.

Lipid and lipoprotein concentrations at age 4. Association with neonatal and parental levels.

BACKGROUND AND OBJECTIVE: To study the influence of diet, anthropometrical measurements and neonatal and parental lipoprotein variables on lipoprotein concentrations at age 4. SUBJECTS AND METHOD: 18 neonates with normal serum lipoprotein values (group 1), 19 neonates with high total cholesterol (TC) levels (group 2) and 21 neonates with normal TC but altered levels in other lipids, apolipoproteins, lipoproteins or ratios (group 3) were selected for a follow-up study. Body weight, body mass index (BMI) and the suitability of diet at age 4 for coronary heart disease prevention were evaluated. Multivariable stepwise linear regression analyses were performed for each lipid or lipoprotein parameter at age 4 considering group at birth, diet, neonatal and parental BMI, lipid or lipoprotein parameters. RESULTS: A large percentage of 4 year-olds had high low density lipoproteins-cholesterol (LDLc) and low high density lipoproteins-cholesterol (HDLc) and followed an unsuitable diet. Prevalence of altered lipoprotein variables, except for TC/HDLc, was similar in the 3 groups. Correlations of birth versus 4 year levels were significant (p = 0.021-0.0001) for all parameters except triglycerides, TC and LDLc. However, all tracking correlations were not significant in group 3. In the multiple regression study, parental and neonatal parameters were retained as explicative variables in many of the models but diet was not retained in any of them. Maternal concentrations were always more explicative than paternal or neonatal ones. Models for TC/HDLc and LDLc/HDLc were the most explicative (both, R2 > 0.578; p < 0.0001). CONCLUSIONS: Lipoprotein variables at age 4 were more closely associated with progenitors' and neonatal lipoprotein values than BMI or diet.

Lipid and lipoprotein concentrations at age 4. Association with neonatal and parental levels

Fundamento y objetivo: Estudiar la influencia de la dieta, de los parámetros antropométricos y de las características lipoproteínicas al nacimiento y de los padres sobre la concentración de lipoproteínas a los 4 años de edad. Sujetos y método: Se seleccionó para participar en un estudio de seguimiento a los 4 años a 18 niños con un perfil lipoproteico normal al nacer (grupo 1), a otros 19 con concentraciones elevadas de colesterol total (CT) al nacer (grupo 2) y a 21 con CT normal pero valores anormales al nacer de triglicéridos, apolipoproteínas, lipoproteínas y cocientes lipoproteínicos (grupo 3). Se evaluaron el peso corporal, el índice de masa corporal (IMC) y la adecuación de la dieta a los 4 años desde el punto de vista cardiovascular. Se realizó un análisis de regresión lineal múltiple por pasos para explicar la concentración a los 4 años, considerando la dieta, el IMC y los parámetros lipoproteínicos de ambos padres y de los recién nacidos. Resultados: A los 4 años un gran porcentaje de niños tenía concentraciones elevadas de colesterol unido a lipoproteínas de baja densidad (cLDL), cifras bajas de colesterol unido a lipoproteínas de alta densidad (cHDL) y una dieta incorrecta, rica en colesterol y ácidos grasos saturados. La prevalencia de concentraciones lipoproteínicas alteradas fue similar en los 3 grupos, excepto para el cociente CT/cHDL. Las correlaciones entre las variables al nacimiento y a los 4 años fueron todas significativas (p = 0,021-0,0001), salvo para los triglicéridos, el CT y el cLDL. En el grupo 3 se perdía la significación en todas las variables. En las regresiones múltiples las variables maternas, paternas y de los recién nacidos aparecieron como variables explicativas en un gran número de modelos. Las concentraciones maternas fueron más explicativas que las paternas y las de los neonatos. Los modelos más explicativos fueron para CT/cHDL y cLDL/cHDL (ambos, R2 > 0,578; p < 0,0001). Conclusiones: A los 4 años, las variables lipoproteínicas, más que el IMC o la dieta, se asocian a los valores lipoproteínicos de los progenitores y los de los niños al nacer

Background and objective: To study the influence of diet, anthropometrical measurements and neonatal and parental lipoprotein variables on lipoprotein concentrations at age 4. Subjects and method: 18 neonates with normal serum lipoprotein values (group 1), 19 neonates with high total cholesterol (TC) levels (group 2) and 21 neonates with normal TC but altered levels in other lipids, apolipoproteins, lipoproteins or ratios (group 3) were selected for a follow-up study. Body weight, body mass index (BMI) and the suitability of diet at age 4 for coronary heart disease prevention were evaluated. Multivariable stepwise linear regression analyses were performed for each lipid or lipoprotein parameter at age 4 considering group at birth, diet, neonatal and parental BMI, lipid or lipoprotein parameters. Results: A large percentage of 4 year-olds had high low density lipoproteins-cholesterol (LDLc) and low high density lipoproteins-cholesterol (HDLc) and followed an unsuitable diet. Prevalence of altered lipoprotein variables, except for TC/HDLc, was similar in the 3 groups. Correlations of birth versus 4 year levels were significant (p = 0.021-0.0001) for all parameters except triglycerides, TC and LDLc. However, all tracking correlations were not significant in group 3. In the multiple regression study, parental and neonatal parameters were retained as explicative variables in many of the models but diet was not retained in any of them. Maternal concentrations were always more explicative than paternal or neonatal ones. Models for TC/HDLc and LDLc/HDLc were the most explicative (both, R2 > 0.578; p < 0.0001). Conclusions: Lipoprotein variables at age 4 were more closely associated with progenitors' and neonatal lipoprotein values than BMI or diet

High density lipoprotein-cholesterol changes in children with high cholesterol levels at birth.

UNLABELLED: The predictive value of serum lipoprotein concentrations at birth for the same parameters later in life is under debate. A group of 20 children displaying high total cholesterol (TC) levels at birth (group 2) were compared at age 4 years with 18 control children who had presented a normal lipoprotein profile at birth (group 1). There was a significant correlation between TC, low density lipoprotein-cholesterol, high density lipoprotein (HDL)-cholesterol, and apolipoprotein (Apo) A-I levels at age 4 years and at birth. The increases in TC and HDL-cholesterol levels from birth to age 4 years were significantly lower (P < 0.05, P < 0.01, respectively) in group 2 than in the control group and inversely correlated with the concentrations of these parameters at birth. The increases in HDL-cholesterol and Apo A-I levels were higher in males while those of triacylglycerol and Apo B were higher in females (P < 0.05). However, the increases in TC and HDL-cholesterol were higher in controls (P< 0.05). Diets of children of both groups were similar regarding the energy contribution of saturated, monounsaturated and polyunsaturated fatty acids, although children from group 2 ate less fish and omega-3 fatty acids (P < 0.05). CONCLUSION: the present data suggest for the first time that when high density lipoprotein-cholesterol levels are high at birth, those levels increase less during the first four years of life. Moreover, low density lipoprotein-cholesterol increased about five times as much as high density lipoprotein-cholesterol did in controls and about 15 times as much as in the children with high cholesterol at birth.