New aspects of dental implants and DNA technology in human identification.

Missing, ineligible or delayed reference data to establish conventional dental or DNA identification are common scenarios in forensic practice. Therefore, it is worthwhile to explore new avenues that facilitate human identification. Due to the recent remarkable evolution in the prosthetic dental restorations based on dental implants and the emergence of novel DNA technologies utilized to infer the biological profile, the identification process has become easier than ever before. We report on a characteristic case, which highlights the particular importance of dental implants and DNA approaches in the prospective investigations for human identification. The aim of this publication is to focus on the possibility of identifying the batch numbers, even if they were not engraved in dental implants, making antemortem dental records of dental implants more easily accessible to establish a comparative dental identification. In addition, the reported case presents the supplementary data yielded through estimating the epigenetic age using DNA methylation as well as the biogeographical origin using Y-Haplotype and mitochondrial DNA analyses. Our results demonstrate that expanded oral implant investigations that also include implants extraction and comprehensive microscopic measurements can lead to identifying their batch numbers despite the numerous number of implants systems manufactured and distributed worldwide. Data saved by dental implant manufacturers can be very supportive and represent additional reference data for dental identification, when antemortem dental records are still missing. Furthermore, DNA methylation and mitochondrial DNA analyses can support the progress of investigation.

Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.

Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.

[Carbon monoxide poisoning by a heating system]. / Kohlenmonoxidvergiftungen durch Heizungsanlagen.

A case of accidental carbon monoxide poisoning in several occupants of two neighboring residential buildings in Hamburg-Harburg (Germany) caused by a defective gas central heating system is described. Because of leaks in one of the residential buildings and the directly adjacent wall of the neighboring house, the gas could spread and accumulated in both residential buildings, which resulted in a highly dangerous situation. Exposure to the toxic gas caused mild to severe intoxication in 15 persons. Three victims died still at the site of the accident. Measures to protect the occupants were taken only with a great delay. As symptoms were unspecific, it was not realized that the various alarms given by persons involved in the accident were related to the same cause. In order to take appropriate measures in time it is indispensible to recognize, assess and check potential risks, which can be done by using carbon monoxide warning devices and performing immediate COHb measurements with special pulse oximeters on site. Moreover, the COHb content in the blood should be routinely determined in all patients admitted to an emergency department with unspecific symptoms.

Substrate-induced radical formation in 4-hydroxybutyryl coenzyme A dehydratase from Clostridium aminobutyricum.

4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S](2+) cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose.

A flexible, interactive software tool for fitting the parameters of neuronal models.

The construction of biologically relevant neuronal models as well as model-based analysis of experimental data often requires the simultaneous fitting of multiple model parameters, so that the behavior of the model in a certain paradigm matches (as closely as possible) the corresponding output of a real neuron according to some predefined criterion. Although the task of model optimization is often computationally hard, and the quality of the results depends heavily on technical issues such as the appropriate choice (and implementation) of cost functions and optimization algorithms, no existing program provides access to the best available methods while also guiding the user through the process effectively. Our software, called Optimizer, implements a modular and extensible framework for the optimization of neuronal models, and also features a graphical interface which makes it easy for even non-expert users to handle many commonly occurring scenarios. Meanwhile, educated users can extend the capabilities of the program and customize it according to their needs with relatively little effort. Optimizer has been developed in Python, takes advantage of open-source Python modules for nonlinear optimization, and interfaces directly with the NEURON simulator to run the models. Other simulators are supported through an external interface. We have tested the program on several different types of problems of varying complexity, using different model classes. As targets, we used simulated traces from the same or a more complex model class, as well as experimental data. We successfully used Optimizer to determine passive parameters and conductance densities in compartmental models, and to fit simple (adaptive exponential integrate-and-fire) neuronal models to complex biological data. Our detailed comparisons show that Optimizer can handle a wider range of problems, and delivers equally good or better performance than any other existing neuronal model fitting tool.

[Suicide among physicians--a current analysis for the City of Hamburg]. / Suizide von Ärztinnen und Ärzten--eine aktuelle Analyse für Hamburg.

According to the literature, physicians have an increased risk of committing suicide, which can partly be explained by occupational stress. A retrospective analysis of the deaths investigated by the Hamburg office of Criminal Investigation and documents of the Hamburg Institute of Forensic Medicine for the years 2001 to 2013 showed that the average suicide rate among physicians is not significantly higher than that of the total population, it is 0.021% for physicians, 0.023% for dentists and 0.013% for the general public; thus the difference is not statistically significant (p: 0.57 for physicians and 0.74 for dentists). Drug intoxication has been found to be the most common method of suicide committed by physicians. However, these results must be interpreted with caution in view of the fact that a detailed evaluation of data was often not possible, especially because information as to the profession of the deceased and the motive for the suicide were missing or insufficiently documented.

Experimental study of hydrogen bonding potentially stabilizing the 5'-deoxyadenosyl radical from coenzyme†B12.

Coenzyme B(12) can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co-C bond homolysis of coenzyme B(12) to cob(II)alamin and the 5'-deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19-H to the 3'-O of the ribose moiety of the incipient 5'-deoxyadenosyl radical, which is stabilized by 30 kJ mol(-1) (B. Durbeej et al., Chem. Eur. J. 2009, 15, 8578-8585). The diastereoisomers (R)- and (S)-2,3-dihydroxypropylcobalamin were used as models for coenzyme B(12). A downfield shift of the NMR signal for the C19-H proton was observed for the (R)-isomer (δ=4.45 versus 4.01 ppm for the (S)-isomer) and can be ascribed to an intramolecular hydrogen bond between the C19-H and the oxygen of CHOH. Crystal structures of (R)- and (S)-2,3-dihydroxypropylcobalamin showed C19-H⋅⋅⋅O distances of 3.214(7) Š(R-isomer) and 3.281(11) Š(S-isomer), which suggest weak hydrogen-bond interactions (-ΔG<6 kJ mol(-1)) between the CHOH of the dihydroxypropyl ligand and the C19-H. Exchange of the C19-H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3-11. No exchange was found for any of the cobalt oxidation states. 3',5'-Dideoxyadenosylcobalamin, but not the 2',5'-isomer, was found to act as a coenzyme for glutamate mutase, with a 15-fold lower k(cat)/K(M) than 5'-deoxyadenosylcobalamin. This indicates that stabilization of the 5'-deoxyadenosyl radical by a hydrogen bond that involves the C19-H and the 3'-OH group of the cofactor is, at most, 7 kJ mol(-1) (-ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2'-OH and 3'-OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19-H and the 3'-O of the ribose moiety of the 5'-deoxyadenosyl group is concluded not to exceed 6 kJ mol(-1) (-ΔG).

CalpB modulates border cell migration in Drosophila egg chambers.

BACKGROUND: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. RESULTS: We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), ß-PS integrin (mys) and talin (rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-head(R367A), a mutant form which is not able to bind ß-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. CONCLUSIONS: The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.

Enzyme catalyzed radical dehydrations of hydroxy acids.

BACKGROUND: The steadily increasing field of radical biochemistry is dominated by the large family of S-adenosylmethionine dependent enzymes, the so-called radical SAM enzymes, of which several new members are discovered every year. Here we report on 2- and 4-hydroxyacyl-CoA dehydratases which apply a very different method of radical generation. In these enzymes ketyl radicals are formed by one-electron reduction or oxidation and are recycled after each turnover without further energy input. Earlier reviews on 2-hydroxyacyl-CoA dehydratases were published in 2004 [J. Kim, M. Hetzel, C.D. Boiangiu, W. Buckel, FEMS Microbiol. Rev. 28 (2004) 455-468. W. Buckel, M. Hetzel, J. Kim, Curr. Opin. Chem. Biol. 8 (2004) 462-467.] SCOPE OF REVIEW: The review focuses on four types of 2-hydroxyacyl-CoA dehydratases that are involved in the fermentation of amino acids by anaerobic bacteria, especially clostridia. These enzymes require activation by one-electron transfer from an iron-sulfur protein driven by hydrolysis of ATP. The review further describes the proposed mechanism that is highlighted by the identification of the allylic ketyl radical intermediate and the elucidation of the crystal structure of 2-hydroxyisocapryloyl-CoA dehydratase. With 4-hydroxybutyryl-CoA dehydratase the crystal structure, the complete stereochemistry and the function of several conserved residues around the active site could be identified. Finally potential biotechnological applications of the radical dehydratases are presented. GENERAL SIGNIFICANCE: The action of the activator as an 'Archerase' shooting electrons into difficultly reducible acceptors becomes an emerging principle in anaerobic metabolism. The dehydratases may provide useful tools in biotechnology. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

Novel approach for improved assessment of phenotypic and functional characteristics of BKV-specific T-cell immunity.

BACKGROUND: BKV-associated nephropathy represents a serious complication of the posttransplant period in kidney transplant recipients. Monitoring BKV-specific immunity is of a special importance for estimation of clinical course in patients with BKV reactivation. Our recent data demonstrated that all five BKV antigens are immunogenic and elicit T-cell responses varying within patients. Therefore, all five BKV proteins should be evaluated for the assessment of BKV-specific immunity. However, analysis of five proteins performed separately is time- and cost-intensive and requires large amount of blood. METHODS: Using novel approach of a mixture of overlapping peptide pools encompassing all five BKV antigens (viral protein [VP] 1, VP2, VP3, large tumor antigen, and small tumor antigen) and multiparameter flow cytometry, we evaluate BKV-specific T cells in patients with a previous/present severe long-lasting or transient BKV reactivation. Patients without BKV reactivation were used as control. RESULTS: In this study, we show that using mixture of overlapping peptide pool results in the magnitude of CD4- and CD8-positive BKV-specific T-cell response, which is significantly higher compared with any frequencies detected by previously used single BKV antigen stimulation. Of interest, patients with a history of rapid BKV clearance had significantly higher frequency of multifunctional interferon gamma-γ/interleukin (IL)-2/tumor necrosis factor-α and IL-2/tumor necrosis factor-α CD4-positive T cells, suggesting protective potential of polyfunctional T cells. Furthermore, we did not find IL-17-producing BKV-specific memory T cells in patients recovered from BKV reactivation. CONCLUSIONS: Here, we established a fast and sensitive approach allowing the most comprehensive assessment of the total BKV immunity performed to date and offer a new platform for further prospective studies.

Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.

BACKGROUND: Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase. METHODOLOGY/PRINCIPAL FINDINGS: Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,ß-imido-dUTP did not show any effect on Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization. CONCLUSIONS/SIGNIFICANCE: Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase.

BK-VP3 as a new target of cellular immunity in BK virus infection.

BACKGROUND: Polyomavirus BK virus (BKV) infection represents a serious complication leading to BKV-associated nephropathy (BKVAN) and subsequent kidney graft loss in up to 10% of transplant patients. Cellular immunity is known to play a crucial role in the control of BKV replication. However, the knowledge on the BKV-T-cell response is limited: only two (VP1 and large T antigen) of six known BKV proteins were evaluated for their antigenicity so far. METHODS: By using 10-color flow cytometry and newly created overlapping peptide pools of five BKV antigens (VP1, VP2, VP3, large T antigen, and small t antigen), we performed cross-sectional phenotypic and functional analysis of BKV-specific T cells in kidney transplant patients with a history of BKVAN. Patients with clinically unapparent BKV infection (history of transient/no BKV reactivation) were used as control group. RESULTS: Our data demonstrate for the first time the antigenic properties of all five evaluated proteins with VP3 as a new important target of cellular immunity. Further, we found a correlation between the severity of the previous BKV infection and the magnitude of memory CD4+ T-cell response. Thus, compared with the control group, patients with a history of BKVAN demonstrated significantly higher frequencies of interferon-γ- and interleukin-2-producing effector memory CD4+ T cells. In the control group, more patients with detectable interferon-γ+/interleukin-2+/tumor necrosis factor+ triple producers were found, suggesting possibly a protective function of these multifunctional T cells. CONCLUSIONS: In conclusion, our study results suggest an implementation of new targets for monitoring of BKV immunity. Further studies are required to evaluate the protective function of the found BKV-specific T-cell subsets.

Conservation of male-specific expression of novel phosphoprotein phosphatases in Drosophila.

In the genome of Drosophila melanogaster, there are 19 phosphoprotein phosphatase (PPP) catalytic subunit coding genes. Seven of the novel members of the gene family turned out to be Drosophila-specific. The expression and evolution of these genes was investigated in the present study. CG11597 is a recently evolved gene that is expressed during all stages of morphogenesis in D. melanogaster. In contrast, the transcription of PpD5, PpD6, Pp1-Y1, and Pp1-Y2 genes is restricted to the pupa and imago developmental stages and to the testis of the males, just as that of the previously characterized PpY-55A and PpN58A. With the exception of the Y-localized Pp1-Y1 and Pp1-Y2, the testis-specific phosphatase genes are expressed in X/0 males, while none of them are expressed in XX/Y females. The mRNA of PpD5, Pp1-Y1, and PpY-55A were detected in the developing cysts by in situ hybridization, in contrast with the PpD6 transcript that was found in the distal ends of elongating spermatids. The latter localization suggests post-meiotic expression. The comparison of PPP genes in five Drosophila species revealed that the sequence of the six testis-specific phosphatases changed more rapidly than that of the housekeeping phosphatases. Our results support the "faster male" hypothesis. On the other hand, the male-biased expression of the six genes remained conserved during evolution despite the fact that Pp1-Y1, Pp1-Y2, and PpD6 moved from autosomes to the Y chromosome. Interestingly, the PpD6 gene was found to be Y-linked only in Drosophila ananassae.

Regulation of calpain B from Drosophila melanogaster by phosphorylation.

Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies.

Folding transitions in calpain activator peptides studied by solution NMR spectroscopy.

Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca(2+) sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an alpha-helical conformation in 50% CD(3)OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca(2+) induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain.

Cold stability of intrinsically disordered proteins.

Contrary to globular proteins, intrinsically disordered proteins (IDPs) lack a folded structure and they do not lose solubility at elevated temperatures. Although this should also be true at low temperatures, cold stability of IDPs has not been addressed in any scientific work so far. As direct characterization of cold-denaturation is difficult, we approached the problem through a freezing-induced loss-of-function model of globular-disordered functional protein pairs (m-calpain-calpastatin, tubulin-Map2c, Hsp90-ERD14). Our results affirm that in contrast with globular proteins IDPs are resistant to cold treatment. The theoretical and functional aspects of this observation are discussed.

Identifying calpain substrates in intact S2 cells of Drosophila.

Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca(2+) and ionomycin added), C, inactivated (additions as in B+specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A>B<

Synthetic calpain activator boosts neuronal excitability without extra Ca2+.

Earlier we have shown that an equimolar mixture of calpastatin subdomains A and C (19 amino acids each) strongly activates m-calpain in vitro. In the present work we developed a membrane-permeable activator system, by conjugating an oligo-arginine tail to both peptides. We tested calpain activation as well as synaptic excitability on rat brain slices ex vivo. In hippocampal slices both basic excitability and long-term synaptic efficacy were significantly increased upon treatment with the activator. We propose that the activator peptide conjugates can be used with any mammalian cell, to specifically challenge the calpain system apparently without raising cytoplasmic Ca2+. Such an effector may be a useful tool in dissecting intracellular mechanisms involving the calpain system.

Calcium-induced tripartite binding of intrinsically disordered calpastatin to its cognate enzyme, calpain.

The activity of calpain is controlled by the free intracellular calcium level and by the protein's intrinsically disordered endogenous inhibitor, calpastatin, mediated by short conserved segments: subdomains A-C. The exact binding mode of calpastatin to the enzyme has until now been unclear. Our NMR data of the 141 amino acid long inhibitor, with and without calcium and calpain, have revealed structural changes and a tripartite binding mode, in which the disordered inhibitor wraps around, and contacts, the enzyme at three points, facilitated by flexible linkers. This unprecedented binding mode permits a unique combination of specificity, speed and binding strength in regulation.