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We report the conformational analysis by [sup 1]H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations at 300K of peptoid analogs of the cyclic hexapeptide c-[Phe[sup 11]-Pro[sup 6]-Phe[sup 7]-D-Trp[sup 8]-Lys[sup 9]-Thr[sup 10]]. The analogs c-[Phe[sup 11]-Nasp[sup 6]-Phe[sup 7]-D-Trp[sup 8]-Lys[sup 9 ]-Thr[sup 10]] (1), c-[Phe[sup 11]-Ndab[sup 6]-Phe[sup 7]-D-Trp[sup 8]-Lys[sup 9 ]-Thr[sup 10]] (2) and c-[Phe[sup 11]-Nlys[sup 6]-Phe[sup 7]-D-Trp[sup 8]-Lys[sup 9 ]-Thr[sup 10]] (3) where Nasp denotes N-(2-carboxyethyl) glycine, Ndab N-(2-aminoethyl) glycine and Nlys N-(4-aminobutyl) glycine are subject to conformational studies. The results of free and restrained molecular dynamics simulations at 300K are reported and give insight into the conformational behaviour of these analogs. The compounds show two sets of nuclear magnetic resonance signals corresponding to the cis and trans orientations of the peptide bond between residues 11 and 6. The backbone conformation of the cis isomers that we believe are the bioactive isomers of the three compounds are very similar to each other while there are larger variations amongst the trans isomers. The binding data to the isolated receptors show that the introduction of the Nlys residue in analog 3 leads to an enhancement of binding potency to the hsst5 receptor compared with analog 2 while maintaining identical binding potency to the hsst2 receptor. The Nasp[sup 6] analog 1 binds weakly to the hsst2 and is essentially inactive towards the other receptors. Comparison of the conformations and binding activities of these three analogs indicates that the Nlys residue extends sufficiently far to allow binding to a negatively charged binding domain on the hsst5 receptor. According to this model, the Ndab analog 2 cannot extend far enough to allow for binding to the receptor pocket. The loss of activity observed for the Nasp[sup 6] compound 1 indicates that the presence of a negatively charged residue in position 6 is unfavorable for binding to the hsst receptors. [ABSTRACT FROM AUTHOR]

Twelve patients with intra-abdominal or mediastinal abscesses were treated by percutaneous drainage. Three abscesses were subphrenic, three were adjacent to enteric leaks, two were intrahepatic, two were pancreatic pseudocysts, one was a pancreatic abscess extending to the lesser sac, and one was an infected adrenal hematoma. All 12 lesions were entered percutaneously using fluoroscopic guidance without traumatizing the adjacent normal tissue. Localization was frequently aided by computed tomography. Various catheters were positioned using basic angiographic techniques. Following drainage all patients had a favorable clinical response. Seven of the 12 patients required no surgical management. Careful radiologic follow-up and frequent changing of catheters was necessary in six of the patients. Two patients benefited from the addition of auxiliary drains. Five of the 12 patients were electively operated upon because of incomplete drainage of the abscess cavity. Causes of failure were: persistent anastomotic leak (two patients), sequestered, loculated extension of abscess cavity (two patients), or necrotic, viscous hepatic tissue requiring removal at laparotomy (one patient). [ABSTRACT FROM AUTHOR]

Based on evidence of an increased rate of respiratory infections in sudden infant death (SID) infants as well as the observation of familial occurrence, we analysed in a retrospective study class II and class III genes of the major histocompatibility complex in 40 cases of SID by Southern blot analysis of DNA obtained post mortem from tissue samples. In 24 cases, the parents were interviewed and confirmatory human lymphocyte antigen (HLA) and DNA typing was carried out. Using HLA-DRß and-DQß probes, no evidence of an abnormal HLA-DR frequency distribution in SID infants was detected ( P=0.97). Using DNA probes for the tandemly arranged complement C4 and steroid 21-hydroxylase genes, an increased number of C4B gene deletions in SID cases was found. The increase in C4 gene deletions was significant ( P=0.0125) in infants with recurrent infections. These data indicate a possible role of partial C4 deficiency as a genetically predisposing risk factor in SID. [ABSTRACT FROM AUTHOR]

Abstract: Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradational processes can affect PCR-based Short Tandem Repeat (STR) analysis. Degradation of DNA is observed to vary among different tissues and with time. Therefore, the stability of DNA in Achilles tendon samples is compared to that in muscle and kidney specimens with a variety of postmortem histories. Tissue samples from 28 autopsy cases, including 15 decomposed corpses and a control group of 13 nondecayed corpses were analysed. DNA was isolated using the All-tissue DNA Kit (GEN-IAL, Troisdorf, Germany), quantified by spectrophotometric measurement, amplified by the multiplex PCR genRES MPX-2 (Serac, Bad Homburg, Germany), and analysed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Quantitative analysis of nondecomposed tissues revealed that the recovery of DNA was highest in kidney followed by muscle, whereas Achilles tendon tissue was the poorest source of isolated DNA. Only small amounts of DNA were present in both kidney and muscle samples from decomposed corpses. However, from decayed Achilles tendon samples twice as much DNA as from nondecayed samples could be isolated on average. These results suggest DNA to be better protected in Achilles tendons. Moreover, postmortem changes in Achilles tendons may even improve DNA isolation. [Copyright &y& Elsevier]

Elimination of microbes in the root canal system is crucial for achieving long-term success in endodontic treatment. Further efforts in study design and standardization are needed in order to improve the validity and comparability of in vitro results on endodontic disinfection procedures, in turn improving clinical outcomes. This study optimizes two models at all steps: tooth selection, pretreatment, inoculation method (by growth or centrifugation), and confocal laser scanning microscopy (CLSM)-guided imaging of LIVE/DEAD-stained specimens. Individual anatomical conditions lead to substantial differences in penetration depth. Sclerosis grading (SCG), a classification system introduced in this study, provides information about the sclerosis status of the dentine and is helpful for careful, specific, and comparable tooth selection in in vitro studies. Sonically activated EDTA for the pretreatment of roots, inoculation of Enterococcus faecalis in an overflow model, 3-4 weeks of incubation, as well as polishing of dentine slices before staining, led to advances in the visualization of bacterial penetration and irrigation depths. In contrast, NaOCl pretreatment negatively affected performance reproducibility and should be avoided in any pretreatment. Nonsclerotized teeth (SCG0) can be used for microbial semilunar-shaped inoculation by centrifugation as a "quick-and-dirty" model for initial orientation. In conclusion, CLSM-guided imaging for quantifying endodontic infection/disinfection is a very powerful method after the fine-tuning of materials and methods.

Aims: Prevention of heart failure relies on the early identification and control of risk factors. We aimed to identify the frequency and characteristics of individuals at risk of heart failure in the general population., Methods and Results: We report cross-sectional data from the prospective Characteristics and Course of Heart Failure Stages A-B and Determinants of Progression (STAAB) cohort study investigating a representative sample of residents of Würzburg, Germany. Sampling was stratified 1:1 for sex and 10:27:27:27:10 for age groups of 30-39/40-49/50-59/60-69/70-79 years. Heart failure precursor stages were defined according to American College of Cardiology/American Heart Association: stage A (risk factors for heart failure), stage B (asymptomatic cardiac dysfunction). The main results were internally validated in the second half of the participants. The derivation sample comprised 2473 participants (51% women) with a distribution of 10%/28%/25%/27%/10% in respective age groups. Stages A and B were prevalent in 42% and 17% of subjects, respectively. Of stage B subjects, 31% had no risk factor qualifying for stage A (group 'B-not-A'). Compared to individuals in stage B with A criteria, B-not-A were younger, more often women, and had left ventricular dilation as the predominant B qualifying criterion (all P < 0.001). These results were confirmed in the validation sample (n = 2492)., Conclusion: We identified a hitherto undescribed group of asymptomatic individuals with cardiac dysfunction predisposing to heart failure, who lacked established heart failure risk factors and therefore would have been missed by conventional primary prevention. Further studies need to replicate this finding in independent cohorts and characterise their genetic and -omic profile and the inception of clinically overt heart failure in subjects of group B-not-A., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Purpose: To present a case of unusual corneal infection early in the course of peripheral ulcerative keratitis in a patient with severe vitamin A deficiency., Method: Single observational case report in urban USA., Case Presentation: An alcoholic patient with pancreatitis, chronic diarrhea, and vitamin A deficiency presented with a marginal corneal ulcer from which two bacteria of the family Micrococcaceae were cultured and identified by genome sequence analysis, namely Kocuria palustris and Rothia mucilaginosa. Soon after, severe bilateral peripheral ulcerative keratitis developed, later accompanied by eyelid cellulitis of one lid. These conditions improved with antibiotics, treatment of the underlying gastrointestinal conditions, and treatment of the vitamin deficiency., Conclusion: Susceptibility to keratitis with unusual bacteria of the Micrococcaceae family can occur in the setting of alcoholism-related gastrointestinal disease with severe vitamin A deficiency. To our knowledge, K. palustris is a species not previously identified in any human disease, and the Kocuria genus has not previously been reported as a participant in eye infection. Documented cases of R. mucilaginosa in ocular disease are rare. These unusual infections heralded the onset of severe marginal corneal melts.

Phosphatidylethanol (PEth), which is formed extrahepatically by the action of phospholipase D on phosphatidylcholine in the presence of ethanol, has been suggested as a promising marker of alcohol misuse. Analysis of dried blood spots (DBS) is particularly advantageous for the determination of delicate analytes such as PEth. Therefore, measurement of PEth species (18:1/18:1, 16:0/18:1) in DBS versus whole blood was performed to ascertain whether respective results are directly comparable. Samples were obtained from subjects (n = 40) undergoing alcohol detoxification treatment. Analysis involved liquid-liquid extraction from both, DBS and whole blood (100 μL, respectively), with phosphatidylpropanol as the internal standard. Extracts were subjected to LC gradient separation using multiple reaction monitoring of deprotonated molecules. Results from measurements of corresponding DBS and whole blood specimens were compared by estimating the respective mean values and by a Bland and Altman analysis. Concentrations of PEth 18:1/18:1 ranged from 46.1 to 3,360 ng/mL in whole blood (mean, 461.7 ng/mL) and from 35.8 to 3,360 ng/mL in DBS (mean, 457.6 ng/mL); for PEth 16:0/18:1, concentrations were from 900 to 213,000 ng/mL (mean, 23,375 ng/mL) and 922-213,000 ng/mL (mean, 23,470 ng/mL) in blood and DBS, respectively. Estimated mean differences were -4.3 ng/mL for PEth 18:1/18:1 and 95.8 ng/mL for PEth 16:0/18:1. The Bland-Altman plot of both PEth species showed that the variation around the mean difference was similar all through the range of measured values and that all differences except one were within the limits of agreement. It could be shown that the determination of PEth species in DBS is as reliable as in whole blood samples. This assay may facilitate monitoring of alcohol misuse.

Analysis of dried blood spots is an increasingly accepted method in therapeutic drug monitoring, whereas its application by analogy to forensic samples has not been studied in detail. Therefore, we investigated whether determination of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolite 3,4-methylendioxyamphetamine (MDA) from dried blood spots (DBS) is as reliable as that from whole blood specimens. Analysis was performed by liquid chromatography-tandem mass spectrometry following liquid-liquid extraction of blood and corresponding DBS samples from 20 volunteers participating in a controlled driving experiment under the influence of MDMA. The assay was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection (LLOD) and quantitation, extraction efficiency and the within-run and between-run assay imprecision for both whole blood and DBS. The LLODs were 2.0 and 1.6 ng/mL for MDMA in whole blood and DBS, respectively, using a volume of 100 μL. LLODs of MDA were determined to be 0.25 ng/mL in whole blood specimens and 0.12 ng/mL in DBS. Extraction efficiency and imprecision did not differ significantly between the two methods for both MDMA and MDA. The mean concentration ratio of corresponding whole blood and DBS samples, t-test, and the Bland-Altman difference plot were used to test hypothesis of equality. Statistical analyses revealed that methods did not significantly differ for MDMA or MDA. Thus, DBS analysis has potential as a precise and inexpensive alternative to whole blood analysis of MDMA.

This paper compares the blood-to-serum distribution (B/S ratio) of 3,4-methylenedioxymethamphetamine (MDMA) and its major metabolite 3,4-methylenedioxyamphetamine (MDA). B/S ratios were determined by liquid chromatography-tandem mass spectrometry analysis following liquid-liquid extraction as a function of the hematocrit value (experimental specimens) and in blood and corresponding serum samples (n = 63) from 16 healthy volunteers participating in a controlled driving experiment (authentic specimens). A regression analysis to calculate the B/S ratio was performed followed by an analysis of covariances (ANCOVA). A linear relationship between the hematocrit value and the B/S ratio of both MDMA and MDA could be established from the experimental data. For MDMA, the regressions provided mean B/S ratios of 1.22 and 1.26 for the experimental setting and the authentic samples, respectively. For MDA, the analysis determined slopes of 1.15 and 1.27 for the experimental setting and field study, respectively. ANCOVA revealed that the method of determination (experimental vs. authentic specimens) did not influence the resulting slopes. A conversion factor of 0.80 may give an adequate estimate to derive the serum concentration for MDMA if only the concentration in whole blood is known, whereas such a definitive factor could not be established for MDA because of its very low levels in authentic samples.

The use of dried blood spots (DBS) which has successfully been introduced in neonatal metabolic screening is an appropriate method to reduce virus infection risk to a minimum, facilitating regular mailing and handling of samples in the laboratory. Injection diacetylmorphine use is notably associated with a prevalence of infection and a risk of transmission of blood-borne viruses. The aim of the present study was to establish a method to determine morphine and 6-acetylmorphine (6-AM) as accurately and sensitively from DBS as from whole blood. Analysis by liquid chromatography/tandem mass spectrometry was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection and quantification, and the within-run and between-run assay imprecision for both whole blood and DBS after liquid/liquid extraction. DBS drying time and elution were optimized, and extraction efficiency from DBS was compared with that of blood and of a solution of the pure compounds. Short-term stability of morphine and 6-AM was determined at -20 degrees C, 4 degrees C, and 40 degrees C up to 7 days from both whole blood and DBS. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating 50 authentic samples. The lower limit of detection was 0.4 ng of morphine per spot and 0.8 ng of 6-AM per spot using a DBS blood volume of 100 microL and was 0.3 and 0.7 ng/100 microL whole blood for morphine and 6-AM, respectively. Recovery rates of the analytes from DBS did not differ from those from whole blood and were > or =55% for 6-AM and > or =25% for morphine, and the within- and between-run coefficients of variation were always < or =9%. 6-AM degraded rapidly at both 4 degrees C and 40 degrees C in whole blood; however, it seemed to be much more stable in DBS. Significant correlations between real whole-blood samples and DBS were obtained. The DBS assay has potential as a precise and inexpensive option for the determination of morphine and 6-AM in small blood samples. Further, the DBS matrix proved to excellently stabilize 6-AM.

The postmortem diagnosis of shaken baby syndrome, a severe form of child abuse, may be difficult, especially when no other visible signs of significant trauma are obvious. An important finding in shaken baby syndrome is subdural haemorrhage, typically originating from ruptured cerebral bridging veins. Since these are difficult to detect at autopsy, we have developed a special postmortem computed tomographic (PMCT) method to demonstrate the intracranial vein system in infants. This method is minimally invasive and can be carried out conveniently and quickly on clinical computed tomography (CT) systems. Firstly, a precontrast CT is made of the infant's head, to document the original state. Secondly, contrast fluid is injected manually via fontanel puncture into the superior sagittal sinus, followed by a repeat CT scan. This allows the depiction of even very small vessels of the deep and superficial cerebral veins, especially the bridging veins, without damaging them. Ruptures appear as extravasation of contrast medium, which helps to locate them at autopsy and examine them histologically, whenever necessary.

Objective: The aim of the present study was to estimate the drug interaction potential of psychtropic medication on buprenorphine (BUP) N-dealkylation using cDNA-expressed cytochrome P450 (CYP) enzymes., Methods: BUP was incubated with psychotropic drugs and cDNA-expressed CYP 3A4 and CYP 2C8 enzymes. Seven substances were screened for their inhibition potency. To check for a mechanism-based component in inhibition, all substances were tested with and without preincubation, respectively. Norbuprenorphine (NBUP) concentrations were determined by liquid chromatography/tandem mass spectrometry, following liquid/liquid extraction., Results: Midazolam and zolpidem demonstrated greatest inhibition in screening experiments. As expected, IC(50) values without preincubation were higher than those after 30-min preincubation, with zolpidem 113.1 microM and midazolam 20.25 microM. Following a 30-min preincubation period in the absence of the probe substrate BUP, the apparent IC(50) values for zolpidem and midazolam were 20.17 microM and 3.5 microM., Conclusion: Both midazolam and zolpidem showed a distinct inhibitory potency towards NBUP formation by CYP 3A4, implicating a decreased conversion of BUP. When preincubated, the inhibitory potency was increased, which strongly suggests a metabolically activated component in inhibition.

Contrary to the traditional dogma of being a relatively invariable and quiescent organ lacking the capability to regenerate, there is now widespread evidence that the human brain harbors multipotent neural stem cells, possibly throughout senescence. These cells can divide and give rise to neuroectodermal progeny in vivo and are now regarded as powerful prospective candidates for repairing or enhancing the functional capability of neural tissue in trauma or diseases associated with degeneration or malperfusion. Hopes primarily rest upon techniques to either recruit endogenous stem cells or to utilize exogenous donor-derived material for transplantation. In the search for suitable human cell sources, embryonic, fetal, and adult stem cells appear highly controversial, as they are accompanied by various still-unresolved moral and legal challenges. Fascinatingly, however, recent reports indicate the successful isolation and expansion of viable neural stem cells from the rodent and human brain within a considerable postmortem interval, suggesting that postmortem neural stem cells could potentially become an acceptable alternative cellular resource. This article will provide a brief overview about neural stem cells, their prominent features, and prospects for a cellular therapy, and will furthermore illuminate the cells in particular with respect to their newly discovered postmortem provenience, their advantage as a potential cell source, and several unfolding forensic considerations. Also, important ethical, social, and legal implications arising from this hitherto unpracticed cellular harvest of brain tissue from the deceased are outlined.

An ingestion of an unknown quantity of mirtazapine in a suicide attempt leading to death is described. Sertraline and amitriptyline have been co-ingested. Because mirtazapine is reported to be relatively safe in overdose, body fluids and tissues were investigated for both mirtazapine and desmethylmirtazapine by high-pressure liquid chromatography/tandem mass spectrometry following liquid-liquid extraction. The limit of detection was sufficiently low to also apply the assay in pharmacokinetic studies. The levels of amitriptyline and nortriptyline were very low (38 and 19 ng/mL femoral venous blood) and the amount of sertraline in blood taken from the femoral vein (880 ng/mL) was considerably lower than those seen in overdosage. Accumulation of mirtazapine and N-desmethylmirtazapine was evident in fluids and tissues involved in enterohepatic circulation and excretion. The concentration determined in a brain sample suggests a contribution of the metabolite to the drug's pharmacodynamic activity. Based on literature data, significant adverse or synergistic effects among the drugs detected as well as adverse reactions such as a serotonin reaction appeared less probable. Mirtazapine exhibits alpha(1)-antagonistic properties on the cardiac-vascular system and may cause hyponatraemia. In the face of the cardiac findings at autopsy and the lack of an apparent cause of death, these effects of mirtazapine may have initiated a process leading to death.

Background: Dyslipidaemia, inflammation and oxidative stress are prominent risk factors that potentially cause vascular disease in haemodialysis patients. Dialysis modalities affect uraemic dyslipidaemia, possibly by modifying oxidative stress, but the effects of dialyser flux and membrane material on atherogenic remnant particles and oxidized low-density lipoproteins (LDL) are unknown., Methods: We performed a randomized crossover study in 36 patients on haemodialysis to analyse the effect of dialyser flux and membrane material on plasma lipids, apolipoproteins and markers of inflammation and oxidative stress. Stable patients on low-flux dialysis with polysulphone for >/=6 weeks were assigned to high-flux polysulphone or high-flux modified cellulose with similar dialyser surface area and permeability characteristics and crossed over twice every 6 weeks., Results: Thirty patients completed the study per protocol. Treatments with high-flux polysulphone and modified cellulose lowered serum triglyceride (by 20% and 10%, respectively; P<0.05) and remnant-like particle cholesterol by 32% (P<0.001) and 11% (NS) after the first 6 weeks of treatment. Oxidized LDL decreased significantly with high-flux polysulphone, but not with modified cellulose. Apolipoproteins CII and CIII were reduced, whereas the ratio CII/CIII was increased (all P<0.05). Acute-phase proteins and LDL and high-density lipoprotein cholesterol remained unaffected., Conclusions: This randomized crossover study demonstrates a potent effect of high-flux haemodialysis on uraemic dyslipidaemia. Polysulphone membrane material showed superiority on oxidatively modified LDL, an indicator of oxidative stress in haemodialysis patients.

The immune response in the central nervous system (CNS) is under tight control of regulatory mechanisms, resulting in the establishment of immune privilege. CNS injury induces an acute inflammatory reaction, composed mainly of invading leukocytes and activated microglial cells/macrophages. The generation of this robust immune response requires binding of receptors such as CD14, a pattern recognition receptor of the immune system. CD14, a surface molecule of monocytic cells, is up-regulated after monocyte stimulation and is involved in cellular activation. To examine CD14 expression in human brain lesions we investigated sections of brains obtained at autopsy from 25 cases following closed traumatic brain injury (TBI) and 5 control brains by immunohistochemistry. Detection of CD14 in controls demonstrated constitutive expression by perivascular cells, but not in parenchymal microglial cells, equivalent to known expression pattern of ED2 in rats. Following TBI, numbers of CD14(+) cells in perivascular spaces and in the brain parenchyma increased in parallel within 1-2 days, both at the lesion and in adjacent perilesional areas. The number of CD14(+) cells in perivascular spaces and in the brain parenchyma reached maximum levels within 4-8 days and remained elevated until weeks after trauma. In contrast to activated parenchymal microglia/macrophages, resting parenchymal microglial cells lacked CD14. Thus, early CD14 expression constitutes an essential part of the acute inflammatory CNS response following trauma.