Denial of reproductive potential: a predictor of unperceived pregnancy in an Austrian neonaticide sample.

PURPOSE: This study aims to describe the phenomenon of unperceived pregnancy followed by neonaticide with a focus on the lack of awareness of reproductive potential in an Austrian sample. METHODS: An explorative comparative study of neonaticide cases with single and repeat perpetrators was conducted using nationwide register-based data from 1995 to 2017. A total number of 55 cases out of 66 were included in the analysis. A standardized coding sheet was used and calculations were performed. RESULTS: 48 women gave birth to 101 children, of which 55 were killed, 23 children lived out of home care and 23 lived with the perpetrator We found a higher fertility rate in both neonaticide perpetrators in the single (1,9) and the repeat group (4,25) in comparison to the general population (1,4). The use of contraception was only 31% among neonaticide perpetrators, deviating substantially from the general Austrian population age group (16-29yrs) which used contraception in 91%. The neonaticide perpetrators used an effective contraception method (pearl-index < 4) in only 2%, whereas 20% of the general population did so. The number of unperceived pregnancies was high in both groups (50/55) 91%. CONCLUSION: Future case reports and forensic evaluations should take reproductive behavior into account, as it may offer valuable insights into the events leading up to neonaticide. Our findings suggest that denial of reproductive potential often precedes unperceived pregnancies. In the Austrian cohort, women who experienced unperceived pregnancies resulting in unassisted births and subsequent neonaticide showed a low prevalence of contraceptive use. This is particularly noteworthy given that the primary motive for neonaticide is unwanted pregnancy.

Familicide: A Systematic Literature Review.

Familicides have received relatively little attention and are mostly discussed in studies with broader aims. Here, we reviewed 67 studies from 18 countries on familicides, in which an offender killed or attempted to kill their current or former spouse/intimate partner and one or more of their biological or stepchildren. We conducted a systematic literature search in PubMed, PsycINFO, and Google Scholar. Eight studies investigated familicide specifically, while the remaining reported on familicide cases as a subsample. We retrieved data on offenders' gender, age, and background as well as on victims and their relationship to the offender. We also retrieved data on contextual factors and offense characteristics (i.e., modus operandi, offense location, premeditation, and whether or not the offender had committed suicide). We also coded methodological aspects of the studies. Familicides were almost exclusively committed by men and about half of the familicide cases led to the suicide of the offender. Mental health problems, relationship problems, and financial difficulties were prevalent. Because few studies reported population base rates of the investigated characteristics, it is difficult to draw conclusions about specific risk factors. Future research should further investigate typologies of familicide and examine risk factors for different types of familicides.

A Global Screen for Assembly State Changes of the Mitotic Proteome by SEC-SWATH-MS.

Living systems integrate biochemical reactions that determine the functional state of each cell. Reactions are primarily mediated by proteins. In proteomic studies, these have been treated as independent entities, disregarding their higher-level organization into complexes that affects their activity and/or function and is thus of great interest for biological research. Here, we describe the implementation of an integrated technique to quantify cell-state-specific changes in the physical arrangement of protein complexes concurrently for thousands of proteins and hundreds of complexes. Applying this technique to a comparison of human cells in interphase and mitosis, we provide a systematic overview of mitotic proteome reorganization. The results recall key hallmarks of mitotic complex remodeling and suggest a model of nuclear pore complex disassembly, which we validate by orthogonal methods. To support the interpretation of quantitative SEC-SWATH-MS datasets, we extend the software CCprofiler and provide an interactive exploration tool, SECexplorer-cc.

Sensitive Quantitative Proteomics of Human Hematopoietic Stem and Progenitor Cells by Data-independent Acquisition Mass Spectrometry.

Physiological processes in multicellular organisms depend on the function and interactions of specialized cell types operating in context. Some of these cell types are rare and thus obtainable only in minute quantities. For example, tissue-specific stem and progenitor cells are numerically scarce, but functionally highly relevant, and fulfill critical roles in development, tissue maintenance, and disease. Whereas low numbers of cells are routinely analyzed by genomics and transcriptomics, corresponding proteomic analyses have so far not been possible due to methodological limitations. Here we describe a sensitive and robust quantitative technique based on data-independent acquisition mass spectrometry. We quantified the proteome of sets of 25,000 human hematopoietic stem/multipotent progenitor cells (HSC/MPP) and three committed progenitor cell subpopulations of the myeloid differentiation pathway (common myeloid progenitors, megakaryocyte-erythrocyte progenitors, and granulocyte-macrophage progenitors), isolated by fluorescence-activated cell sorting from five healthy donors. On average, 5,851 protein groups were identified per sample. A subset of 4,131 stringently filtered protein groups was quantitatively compared across the 20 samples, defining unique signatures for each subpopulation. A comparison of proteomic and transcriptomic profiles indicated HSC/MPP-specific divergent regulation of biochemical functions such as telomerase maintenance and quiescence-inducing enzymes, including isocitrate dehydrogenases. These are essential for maintaining stemness and were detected at proteome, but not transcriptome, level. The method is equally applicable to almost any rare cell type, including healthy and cancer stem cells or physiologically and pathologically infiltrating cell populations. It thus provides essential new information toward the detailed biochemical understanding of cell development and functionality in health and disease.

Repeated neonaticide: differences and similarities to single neonaticide events.

This study aims to identify differences between single and repeat perpetrators of filicide by using register-based data. The study used register-based, comprehensive, nationwide data from both Austria and Finland. The current study covers 23 perpetrators, 20 single and 3 repeat perpetrators, with a total of 28 victims. All victims had a maximum age of 24 h and all perpetrators were women. Every third victim of neonaticide was a victim of a repeat case. The repeat perpetrators were older; had a higher number of children over their lifespan, some of whom lived with them; were more likely to live within established family structures; had higher levels of education and employment; had a higher proportion of personality disorders; and were more likely to identify stress factors during pregnancy. One unexpected finding was low levels of awareness about pregnancy within the perpetrator's circle remain a risk factor, especially for repeat perpetrators. Arguably, the quality of interpersonal relationships these women have may be affected by their own mental health issues and life experience and vice versa.

Data-independent acquisition-based SWATH-MS for quantitative proteomics: a tutorial.

Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH-MS is a specific variant of data-independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH-MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH-MS data, a strategy based on peptide-centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH-MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH-MS data using peptide-centric scoring. Furthermore, concepts on how to improve SWATH-MS data acquisition, potential trade-offs of parameter settings and alternative data analysis strategies are discussed.

A hybrid retention time alignment algorithm for SWATH-MS data.

Recently, data-independent acquisition (DIA) MS has gained popularity as a qualitative-quantitative workflow for proteomics. One outstanding problem in the analysis of DIA-MS data is alignment of chromatographic retention times across multiple samples, which facilitates peptide identification and accurate quantification. Here, we present a novel hybrid (profile-based and feature-based) algorithm for LC-MS alignment and test it on sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) (a type of DIA) data. Our algorithm uses a profile-based dynamic time warping algorithm to obtain a coarse alignment and corrects large retention time shifts, and then uses a feature-based bipartite matching algorithm to match feature to feature at a fine scale. We evaluated our method by comparing our aligned feature pairs to peptide identification results of pseudo-MS2 spectra exported by DIA-Umpire, a recently reported tool for deconvoluting DIA-MS data. We proposed that our method can be used to align DIA-MS data prior to identification, and the alignment can be used to delete noise peaks or screen for differentially changed features. We found that a simple alignment-enabled denoising scheme can reduce the number of pseudo-MS2 spectra exported by DIA-Umpire by up to around 40%, while retaining a comparable number of identifications. Finally, we demonstrated the utility of our tool for accurate label-free relative quantification across multiple SWATH runs.

Molecular mechanism of regulation of the atypical protein kinase C by N-terminal domains and an allosteric small compound.

Protein kinases play important regulatory roles in cells and organisms. Therefore, they are subject to specific and tight mechanisms of regulation that ultimately converge on the catalytic domain and allow the kinases to be activated or inhibited only upon the appropriate stimuli. AGC protein kinases have a pocket in the catalytic domain, the PDK1-interacting fragment (PIF)-pocket, which is a key mediator of the activation. We show here that helix αC within the PIF-pocket of atypical protein kinase C (aPKC) is the target of the interaction with its inhibitory N-terminal domains. We also provide structural evidence that the small compound PS315 is an allosteric inhibitor that binds to the PIF-pocket of aPKC. PS315 exploits the physiological dynamics of helix αC for its binding and allosteric inhibition. The results will support research on allosteric mechanisms and selective drug development efforts against PKC isoforms.

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry.

Mass spectrometry has become a valuable method for studying structural dynamics of proteins in solution by measuring their backbone amide hydrogen/deuterium exchange (HDX) kinetics. In a typical exchange experiment one or more proteins are incubated in deuterated buffer at physiological conditions. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example, only 4% and 6% deuterium loss for fully deuterated ubiquitin and ß(2)-microglobulin were observed after 10 min of back-exchange. The practical value of our subzero-cooled setup for top-down fragmentation HDX analyses is demonstrated by electron-transfer dissociation of ubiquitin ions under carefully optimized mass spectrometric conditions where gas-phase hydrogen scrambling is negligible. Our results show that the known dynamic behavior of ubiquitin in solution is accurately reflected in the deuterium contents of the fragment ions.

Potential predictors in neonaticide: the impact of the circumstances of pregnancy.

This study aims to evaluate the psychosocial factors of neonaticide, especially the circumstances before delivery, the relationships of the pregnant women, and their social environment awareness of women's pregnancy. This nationwide study was register-based, comprising all known neonaticides in Austria and Finland between 1995 and 2005. Cases (n = 28) were obtained by screening the death certificates from coroner's departments and by analyzing them along with all further available reports. Few women (17.9 %, 5/28) admitted their pregnancy to others. Although most (16/28) offenders were in a relationship, the partner had knowledge of the pregnancy in only three cases. The main motive for negation of the pregnancy (named in 60.8 % of cases) was fear of abandonment/negative response from others. The fertility rate among the women was high, but half of those with children had lost the custody of them. In neonaticide, the lack of awareness surrounding offenders' pregnancy, as well as the awareness of social environment, is more relevant than any other social variable.

Allosteric regulation of protein kinase PKCζ by the N-terminal C1 domain and small compounds to the PIF-pocket.

Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by the N-terminal region cannot be explained by a simple pseudosubstrate inhibitory mechanism. Notably, we found that the C1 domain allosterically inhibits PKCζ activity and verified an allosteric communication between the PIF-pocket of atypical PKCs and the binding site of the C1 domain. Finally, we developed low-molecular-weight compounds that bind to the PIF-pocket and allosterically inhibit PKCζ activity. This work establishes a central role for the PIF-pocket on the regulation of PKCζ and allows us to envisage development of drugs targeting the PIF-pocket that can either activate or inhibit AGC kinases.

Gender differences in filicide offense characteristics--a comprehensive register-based study of child murder in two European countries.

OBJECTIVE: This study searched for gender differences in filicidal offense characteristics and associated variables. METHODS: In this bi-national register-based study all filicide perpetrators (75 mothers and 45 fathers) and their crimes in Austria and Finland 1995-2005 were examined for putative gender differences. The assessed variables were associated with the offense characteristics, the offenders' socioeconomic and criminal history, and related stressful events. RESULTS: Mothers had previously committed violent offenses less often than fathers (5% vs. 28%, p<0.001) and they were less often employed (27% vs. 49%, p<0.05). Mothers' victims were on average younger than those of fathers; median ages of the victims were 3.4 and 6.1 years, respectively (p<0.001). Fathers were more often intoxicated during the offense (11% vs. 42%, p<0.001) and also used shooting as the method of operation more often than mothers (5% vs. 27%, p<0.001). Mothers used drowning, criminal negligence, and poisoning more often than fathers. Fathers' motives were more impulsive in nature (13% vs. 41%, p<0.001). After the killing, mothers tried to get rid of the body more often than fathers (25% vs. 7%, p<0.05). CONCLUSIONS: Fathers who commit filicide may represent at least two subgroups, the one not unlike the common homicide offender; the other, the overloaded, working and suicidal father. Mothers may include several types of offenders, one of which is the neonaticide offender. More detailed descriptions and, therefore, more research are needed. PRACTICE IMPLICATIONS: Distressed parents and families need support and health care personnel, social work and other officials need to be alert to notice fatigued parents' signs of despair, especially when several stressful experiences amass. Straightforward enquiry to the situation and even practical and psychological help may be needed for enhanced protection of children. The role of employers should also be discussed in relation to the welfare of working parents.

Filicide in Austria and Finland--a register-based study on all filicide cases in Austria and Finland 1995-2005.

BACKGROUND: Filicide is the tragic crime of murdering one's own child. Previous research has found that the offending parents are commonly depressed and that suicide is often associated as an actual act or an intention. Yet, filicide is an underreported crime and previous studies have been strained with methodological problems. No comprehensive international studies on filicide have been presented in the literature until now. METHODS: This was a descriptive, comprehensive, register-based study of all filicides in Austria and Finland during 1995-2005. Filicide-suicide cases were also included. RESULTS: Most of the perpetrators were the biological mothers; in Austria 72%, in Finland 52%. Suicide followed filicide either as an attempt or a fulfilled act in 32% and 54% of the cases in Austria and Finland, respectively. Psychotic mood disorders were diagnosed for 10% of the living perpetrators in Austria, and 12% in Finland. Non-psychotic depression was diagnosed in 9% of surviving perpetrators in Austria, 35% in Finland. CONCLUSION: The data from the two countries demonstrated that filicide is such a multifaceted and rare phenomenon that national data from individual countries seldom offer sufficient scope for its thorough study. Further analyses are needed to produce a complete picture of filicide.

Confirmation of immuno-reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity-CIEF.

Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

Glycosylation analysis of glycoproteins and proteoglycans using capillary electrophoresis-mass spectrometry strategies.

This review highlights recent developments in glycosylation analysis by modern MS in combination with CE based preseparation. Focused on CE-MS strategies aimed for glycotyping, the review addresses the detailed glycoform analysis of glycoproteins, glycopeptides, and proteoglycans. Glycoform analysis in the context of modern glycoproteomics is briefly addressed, as well. CZE, CIEF, and frontal analysis CE approaches hyphenated to high-resolution multistage MS for the detailed analysis of protein-linked glycan structures are overviewed in a comprehensive way. Advantages and limitations of various methodological approaches and techniques as well as mass spectrometric instrumentation are discussed in the particular context of glycoanalysis.

Use of the arginine-specific butanedione/phenylboronic acid tag for analysis of peptides and protein digests using matrix-assisted laser desorption/ionization mass spectrometry.

We have applied an arginine-specific labeling technique to the study of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The reaction converts the guanidine group of the arginine side chain by reacting it with 2,3-butanedione and an arylboronic acid. Despite the general chemical lability of the tag under acidic conditions, it was possible to employ acidic matrices like alpha-cyano-4-hydroxycinnamic acid without adverse effects, using the thin-layer technique for preparation. After optimizing the method using arginine-containing model peptides--for which sensitivity down to the low fmol range was demonstrated--the procedure was applied to enzymatic digests of several model proteins in solution and to protein spots in gels obtained by two-dimensional electrophoretic separation of cell lysate samples. Information on the presence of arginine in peptides can be easily obtained from the mass spectra by the characteristic mass shift and the isotope pattern resulting from the incorporation of boron. This information might serve as a valuable additional search constraint for achieving a higher degree of confidence for protein identification by peptide mass fingerprinting.

Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function.

During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/kappa) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC-MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants--clipped and unclipped--elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.

Capillary zone electrophoresis of glycopeptides under controlled electroosmotic flow conditions coupled to electrospray and matrix-assisted laser desorption/ionization mass spectrometry.

Defined conditions of EOF along with different pH values of the BGE were compared for the purpose of analyzing glycopetides by CZE coupled to MS (CZE-MS). Hyphenation to MS involved ESI as well as MALDI, and single-stage and multistage MS were applied. Variation of the EOF was accomplished by selecting appropriate coatings for the capillary, namely hexadimethrine bromide (HDMB) and HDMB/dextran sulfate. A high and reproducible anodic and cathodic EOF, respectively, was obtained in both approaches, allowing the detection of analytes with net positive as well as negative charge in one single run. Thus, a fast and sensitive determination of the glycopeptides in a tryptic digest of antithrombin, chosen as a test sample, was achieved. Ionization suppression effects, a phenomenon typically observed with glycopeptides in MS analysis, were minimized thanks to separation from other peptides present. The high stability of the coatings permitted the generation of mass spectra without interfering peaks originating from the coating polymers. The high EOF generated by the coatings facilitated the maintenance of a stable spray when coupling to ESI-MS, and a stable CZE current when working with a sheath flow-assisted analyte deposition onto MALDI targets, respectively. In conclusion, CZE-MS could be demonstrated as a robust complementary method to capillary RP-HPLC-MS in combination with both soft-ionization techniques, ESI and MALDI, generally, and particularly in the context of glycopeptide analysis.

Standard additio--a way of improving quantification of banned azo dyes in leather.

An analytical procedure based on supercritical-fluid extraction and microwave-assisted extraction was applied on six different real leather samples for the determination of banned azo dyes. Determination of the dyes was performed indirectly by measuring their corresponding harmful aromatic amines, formed after reduction. A comparative study between external standard calibration and standard addition using both the dyes as well as the corresponding amines showed that the latter quantification method provided the highest accuracy.