Application of Micro-Computed Tomography for the Estimation of the Post-Mortem Interval of Human Skeletal Remains.

It is challenging to estimate the post-mortem interval (PMI) of skeletal remains within a forensic context. As a result of their interactions with the environment, bones undergo several chemical and physical changes after death. So far, multiple methods have been used to follow up on post-mortem changes. There is, however, no definitive way to estimate the PMI of skeletal remains. This research aimed to propose a methodology capable of estimating the PMI using micro-computed tomography measurements of 104 human skeletal remains with PMIs between one day and 2000 years. The present study indicates that micro-computed tomography could be considered an objective and precise method of PMI evaluation in forensic medicine. The measured parameters show a significant difference regarding the PMI for Cort Porosity p < 0.001, BV/TV p > 0.001, Mean1 p > 0.001 and Mean2 p > 0.005. Using a machine learning approach, the neural network showed an accuracy of 99% for distinguishing between samples with a PMI of less than 100 years and archaeological samples.

Nerve at risk: anatomical variations of the left recurrent laryngeal nerve and implications for thoracic surgeons.

OBJECTIVES: Recurrent laryngeal nerve (RLN) injury during thoracic surgery may result in life-threatening postoperative complications including recurrent aspiration and pneumonia. Anatomical details of the intrathoracic course are scarce. However, only an in-depth understanding of the anatomy will help reduce nerve injury. The aim of this study was to assess the anatomic variations of the intrathoracic left RLN. METHODS: Left-sided vagal nerves and RLN were dissected in 100 consecutive Caucasian cadavers during routine autopsy. Anatomical details were documented. Available demographic data were assessed for possible correlations. RESULTS: All nerves were identified during dissection. Variant courses were classified in 3 different groups according to the level at which the RLN separated from the vagal nerve: above the aortic arch, level with the aortic arch and below the aortic arch. We found 11% of RLN separating above the aortic arch and crossing the aortic arch at a considerable distance to the vagal nerve. In 48% of the RLN, the nerve split off when it was level with the aortic arch, and 41% of the RLN leave the vagal nerve in a perpendicular direction below the aortic arch. All nerves crossed the ligamentum arteriosum on the posterior side. No gender-specific differences were observed. CONCLUSIONS: Mediastinal lymph node dissection in left-sided lung cancer patients puts the RLN at risk. With more detailed anatomical knowledge about its course, it is possible to avoid risking the nerve. Visualization will help protect the nerve.

Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains.

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm-1 to 2800 cm-1. Raman spectra illustrated a similar picture with less ν2PO43-at 450 cm-1 and ν4PO43- from 590 cm-1 to 584 cm-1, amide III at 1272 cm-1 and protein CH2 deformation at 1446 cm-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains.

Deregulated cell death and lymphocyte homeostasis cause premature lethality in mice lacking the BH3-only proteins Bim and Bmf.

BH3 domain-only proteins (BH3-only) proteins are members of the Bcl-2 family that play crucial roles in embryogenesis and the maintenance of tissue homeostasis by triggering apoptotic cell death. The BH3-only protein Bim is critical for developmental apoptosis of lymphocytes, securing establishment of tolerance and for the termination of immune responses. Bim is believed to act in concert with other BH3-only proteins or members of the tumor necrosis factor receptor family in getting rid of unwanted cells. Bmf, a related BH3-only protein, was shown to play a role in B-cell homeostasis and to mediate cell death in response to certain apoptotic triggers, including glucocorticoid, histone deacetylase inhibitors, and overexpression of the c-Myc proto-oncogene. Here we show that Bim and Bmf have overlapping functions during mouse development and coregulate lymphocyte homeostasis and apoptosis in a nonredundant manner. Double deficiency of Bim and Bmf caused more B lymphadenopathy than loss of either BH3-only protein alone, and this was associated with autoimmune glomerulonephritis and a range of malignancies in aged mice. Thus, our results demonstrate that Bim and Bmf act in concert to prevent autoimmunity and malignant disease, strengthening the rational for the development of BH3-only protein mimicking therapeutics for the treatment of such disorders.

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Anti-apoptotic Bcl-2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro-apoptotic antagonists, i.e. 'BH3-only' proteins, in this cell compartment. Based on the analysis of cytokine deprivation-induced changes in mRNA expression levels of Bcl-2 family proteins, we determined the consequences of BH3-only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution. HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio-depleted recipients. Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

Challenging small human hepatocytes with opiates: further characterization of a novel prototype bioartificial liver.

Bioartificial liver (BAL) systems can take over liver functions in patients undergoing liver failure until transplantation. Recently, a novel prototype rotary BAL has been developed using small human hepatocytes (SH). This study investigated the metabolism of opiates morphine and methadone in the BAL and their influence on the basic cell culture parameters, viability, and growth of SH. Opiates may be present in patients due to pain therapy, anticancer treatment, or drug abuse. Cells were cultivated in the BAL for a total of 12 days and exposed twice to 100 microg/L of morphine or methadone. Morphine and methadone concentrations were analyzed using gas chromatography with a mass spectrometry detector. Further, the production of albumin, lactate dehydrogenase release, lactate release, urea production, and glucose consumption were measured. Cell viability and growth were determined by confocal microscopy. Cytochrome P 3A4 and uridindiphosphat (UDP) glucuronosyl transferase 2B7 in SH were analyzed by western blot. The mean cell density during treatment was 5.5 +/- 0.7 x 10(6) cells/mL (n = 6) and was not altered significantly by the opiates. Cell viability stayed above 90%. Morphine was not reduced by SH and was a stress factor as determined by decreased metabolic activity. On the other hand, SH metabolized methadone showing first-order kinetics: the first-order rate constant k = 0,019, half-life t(1/2) = 36 h. Methadone metabolism led to decreased urea and albumin production. The expression of cytochrome P 3A4, mainly responsible for methadone metabolism, was proved in SH. The prototype BAL is basically suited to support liver functions, provided patients receive therapy with methadone.