A new approach to cultural scripts of trauma sequelae assessment: The sample case of Switzerland

Background The novel concept of cultural scripts of trauma sequelae captures culture-specific expressions of posttraumatic distress (e.g., cognitive, emotional, interpersonal, psychosomatic changes) and their temporal associations. Cultural scripts of trauma sequelae complement pan-cultural (etic) diagnoses, such as posttraumatic stress disorder (PTSD) and Complex PTSD, as well as the cultural syndromes concept. Objective This study aimed to develop the cultural scripts of trauma inventory (CSTI) for German-speaking Switzerland and to explore temporal associations of script elements. Method Five semi-structured focus groups were conducted with psychotraumatologists (n = 8) and Swiss trauma survivors (n = 7). The interview schedule included open questions about different domains of potential posttraumatic changes (emotions, cognitions, worldviews, interpersonal relationships, body-related experiences, behavior, and growth). Data were analyzed using qualitative content analysis. Results The Swiss CSTI includes 57 emic elements that represent salient trauma sequelae (30 conformed with a theoretically derived item pool, 27 were newly phrased). Temporal script associations were visualized in a network, whereby self-deprecation, the urge to function and overcompensate, and the urge to hide and endure suffering had the highest number of connections. Conclusion While many posttraumatic changes identified in the present work seem to mirror pan-cultural phenomena represented in the Complex PTSD concept (e.g., self-deprecation), others (e.g., urge to function and perform, urge to hide and endure suffering) may be prominently related to Swiss culture with its value orientations. Knowledge about cultural scripts of trauma sequelae may provide a culture-specific framework that can help to understand individual experiences of distress and enable mental health practitioners to administer culturally sensitive interventions. Pending further validation, the Swiss CSTI bears the potential to advance culture-sensitive assessment of trauma sequelae

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO-), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO- ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3 -)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3 - loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO- and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs. Graphical abstract

Binding of Antitumor Metallocenes to Nucleic Acids

Cancer is a severe disease that is responsible for the annual death of millions of people worldwide. For decades, the most prominent approach to inhibit the rapid growth of cancer cells has been the administration of platinum-based anticancer drugs, such as cisplatin and its newer generation derivatives. These agents bind to nuclear DNA and thus, interfere with cell replication. However, the drugs suffer from severe side effects. The search for potent chemotherapeutic agents that represent an alternative to platinum-related compounds led to the synthesis of numerous DNA-binding metallodrugs. Metallocenes and their derivatives are a class of such antitumor complexes that target nucleic acids. For the investigation of their interaction with DNA, powerful and reliable analytical methods are needed. High-resolution tandem mass spectrometry constitutes a versatile technique for the structural characterization of nucleic acid-drug complexes and for the localization of the preferred binding sites. In the first part of this thesis the investigation of the binding of metallocenes to deoxydinucleoside monophosphates is reported. The binding was found to essentially depend on the type of transition metal, following Pearsons' hard and soft Lewis acids and bases concept. The metallocenes based on the hard Lewis acids titanium and vanadium were found to primarily bind to the phosphate oxygen, while the softer molybdenum additionally interacts with the nucleobases. No interaction with DNA was found for niobocene, which indicates a different mechanism of action. Studies on hexanucleotides revealed that hard Lewis acids bind to phosphate groups in a nucleobase- and sequencedependent manner. Since the phosphate group adjacent to a thymine exhibits the highest acidity, it constitutes the preferred binding site for metallocenes comprising a hard Lewis acid coordination center. While cationic adducts dissociated via backbone cleavage, thus, enabling to localize favored binding sites, adduct anions released both cylcopentadienyl ligands upon collision-induced dissociation (CID) and only minor backbone scission occurred. The loss of the ligands was found to be accompanied by electron transfer from one cyclopentadienyl ligand to the remaining metallated adduct. To gain deeper insight into this unusual electron transfer process, the adducts were analyzed by electron transfer dissociation (ETD) and by ETD in combination with CID (ETcaD). Results revealed the nucleobase rather than the metal center as preferred site for electron transfer. In summary, the presented work represents the first mass spectrometry-based elucidation of the interaction of anticancer metallocenes with their DNA target and thereby contributes to a deeper understanding of their unique binding characteristics

Tandem Mass Spectrometric Investigation of Titanocene and Cisplatin Adducts

Metallocene dichlorides (Cp2MCl2, Cp=cyclopentadienyl ligand, M=Ti, V, Nb, Mo) are metal-based compounds, which were found to be highly effective against several cancer cell lines. Evidence for the accumulation of the transition metal in the cell nucleus suggests DNA as one of the major targets. However, the characteristics of the interaction between different types of metallocenes and nucleic acids remain largely unknown. The progress of this therapeutic approach comprises the development of cyclopentadienyl-modified metallocenes and precise knowledge of their interaction with the target. Tandem mass spectrometry is the ideal method to investigate the formation of metallodrug adducts and to elucidate nucleobase selectivity and binding. In this study, four different metallocenes were incubated with eight dinucleotide monophosphates (AT, TA, CG, GC, CT, TC, AG, GA) to elucidate the favored binding sites. ESI-MS experiments emphasized that adduct formation is primarily determined by the type of transition metal attached. While only titanocene and molybdenocene yield adducts including the two Cp ligands, vanadocene exhibits adducts that underwent extensive ligand exchange, and no niobocene adducts were detected at pH 7. ESI-MS/MS data of the adducts revealed that the nucleobase is involved in the binding of the metallocenes and that the type (pyrimidine or purine) and the position (3' or 5') of the nucleobase essentially determine the binding motif and the gas-phase fragmentation

Tandem Mass Spectrometry of Titanocene-Oligonucleotide Adducts

Antitumor titanocene dichloride (Cp2TiCl2, Cp = cyclopentadienyl) and its derivatives are considered very promising in chemotherapy, attributable to their high activity in cancer cell studies and their low toxicity against healthy tissue. Though the precise mechanism of action has not been elucidated yet, the accumulation of the transition metal in the nucleus points towards DNA as one of the primary targets. Different analytical techniques and computational studies evidenced the interaction of metallocenes with oligonucleotides, but the exact ligand composition of the formed adducts and the underlying binding specificities remain unknown. Mass spectrometry constitutes an ideal tool to illuminate the ligand stoichiometries of the formed adduct. The ability to select and collisionally activate a certain metallocene-oligonucleotide complex further allows the specific localization of preferred binding sites. In this study, positive nanoESI-MS/MS experiments were conducted on titanocene-nucleic acid adducts. Binding preferences were examined by competition experiments on DNA and RNA hexamers. Detailed elucidation of the binding pattern was performed by tandem mass spectrometric approaches comprising collisional activation and electron transfer. Furthermore, the influence of the transition metal coordination center on the gas-phase dissociation of the olignucleotides is discussed

The interaction of transition metal-based anticancer compounds with nucleic acids

The discovery of the antitumor activity of cisplatin represented a milestone in the transition metal-based chemotherapy. Despite the great success of cisplatin against a variety of cancer types, severe side effects and resistances (aquired or intrinsic) necessitated the development of alternative anticancer agents. Metallocene dichlorides (Cp2MCl2, with Cp=cyclopentadienyl, M=Ti, V, Nb, Mo) and their derivatives are a class of metallodrugs with promising anticancer characteristics. Their activity against cisplatin-resistant cell lines combined with their general low toxicity motivated the synthesis of novel Cp-functionalized metallocenes. The antitumor activity of certain metallocenes (e.g., titanocene) is reported to arise from the interaction with nuclear DNA. However, the precise mechanisms of action are not fully elucidated yet and appear to be rather diverse. Building on previous ESI-MS/MS experiments with dinucleoside monophosphates, this study investigates the binding preferences and binding sites of titanocene with DNA and RNA hexanucelotides. The influence of the sugar moiety, the types of nucleobases, and the nucleobase sequences on the adduct formation was elucidated by means of competition experiments. To localize the exact binding sites in MS/MS experiments, the precursors were dissociated by different activation techniques (CID, HCD, and ETD). The effect of the titanium coordination center on the gas-phase dissociation of the nucleic acids is discussed

Interaction of Antitumor Metallocenes with Nucleic Acids

Introduction: Metallocenes Cp2M2+ (with M = Ti, V, Nb, Mo; Cp = cyclopentadienyl and derivatives) represent a promising class of antitumor agents. Evidence was found for the accumulation of the transition metals in the nucleus, thus implying DNA as a primary target. However, the characteristics of the interaction between different types of metallocenes and nucleic acids remain largely unknown. The development of novel metallodrugs requires the identification of the active compounds as well as precise knowledge of their interaction with the target. In the presented study, tandem mass spectrometry is applied to elucidate the patterns and specificities of metallocenes binding to nucleic acids. Methods: Solutions of metallocene dichlorides (M=Ti, V, Nb, Mo, Hf, and W) were incubated with deoxyoligonucleotides of varying length and nucleotide composition. The resulting adducts were subsequently analyzed by ESI-MS and ESI-MS/MS (CID) in positive and negative ion mode on a LTQ Orbitrap XL instrument. Preliminary Data or Plenary Speakers Abstract: Adduct formation was found to depend on the type of transition metal and the pH during incubation. ESI-MS data gave evidence for the formation of adducts that retained both cyclopentadienyl ligands as well as adducts that underwent ligand exchange (Cp- to OH-). The adducts were found to be remarkably stable upon CID and sets of fragment ions including the transition metal coordination center could be detected. Tandem mass spectrometric analysis of DNA adducts with first-row transition metals (Ti, V) gave evidence for the consecutive loss of the two Cp ligands. It is hypothesized that the adducts undergo an intramolecular rearrangement involving an adjacent phosphate group, which results in the ejection of the neutral ligands and a strengthened interaction with the oligonucleotide. This hypothesis is further supported by MS3 data, which revealed that product ions including the transition metal exhibit a minimum of three phosphate groups, thus reflecting the pivotal role of the phosphate linker in the fragmentation process. Second- and third-row transition metals (Nb, Mo, W) displayed a considerably altered behavior upon CID with backbone cleavage preferred over the loss of the ligands. Our data reveals that the nucleobase must be involved in the binding of the metallocenes, as base loss fragments carrying the intact metallocene were observed. Novel Aspect: The elucidation of the binding motifs of metallocenes to nucleic acids is fundamental to the development of novel anticancer drugs

Elucidation of the binding sites in antitumor metallocene - dinucleoside monophosphate adducts

Antitumor metallocenes [Cp2M]2+ (with Cp = cyclopentadienyl and M = Ti, V, Mo) and their Cp-functionalized analogues were found to be highly effective against tumor cell lines, even those that are resistant against cisplatin [(NH3)2Pt]2+ [1, 2]. Although the accumulation of the transition metals in the nucleus [3] points towards DNA as a major target, the interaction of metallocenes with nucleic acids has not been elucidated yet. Mass spectrometry represents the ideal method for the fast and reliable investigation of the formed metallodrug-adducts and allows the selective study of the adduct of interest. The presented tandem mass spectrometric study compares the adduct formation and the subsequent dissociation pathways upon CID with the dinucleoside monophosphate d(TpA) and four transition metals

Characterization of metallocene-oligonucleotide adducts with different activation techniques

Introduction: Bent metallocenes [Cp2M]2+ (with Cp = cyclopentadienyl and derivatives) based on the transition metals Ti, V, Nb, and Mo are a promising class of antitumor compounds that are active against cisplatin sensitive or resistant cancer cell lines. The anticancer activity of these metallodrugs is mainly based on the interaction with nuclear DNA. Building on previous experiments on dinucleoside monophosphates, we investigated the suitability of the Pearson concept to predict the preferred binding sites in DNA and RNA oligonucleotides. Furthermore, we aim at the elucidation of the dissociation mechanisms of oligonucleotide-metallocene adducts. Methods: Unmodified and modified oligonucleotides were incubated with solutions of metallocene dichlorides in an equimolar ratio. The adducts were investigated in the positive ion mode and subsequently fragmented by collision-induced dissociation (CID) and electron transfer dissociation (ETD) on a LTQ Orbitrap Velos instrument. Preliminary Data: The incubation of the metallocenes with DNA hexamers resulted in the formation of adducts with titanium, molybdenum, and vanadium, whereas no adducts were obtained with niobium. ESI-MS experiments in the positive ion mode revealed the formation of adducts that either retained both cyclopentadienyl ligands (titanocene and molybdenocene) or underwent ligand exchange (vanadocene). Reaction of DNA with vanadocene resulted in the release of both cyclopentadienyl ligands and retention of a single hydroxo ligand. Collision induced dissociation of metallocene-DNA hexamer adducts revealed a significantly altered dissiciation behavior compared to their unadducted counterparts, resulting in an increased number of fragment ions. Our data on adducts with molybdenum give proof for binding to the nucleobases, yielding product ions of the type (B+Cp2Mo). On the other hand, the metal centers titanium and vanadium were found to preferably bind to the deprotonated phosphate oxygen atoms. Even for metallocenes that primarily target the deprotonated phosphate groups, a steering effect of the nucleobase sequence was detected. This was reflected by the data on titanocene adducts, which revealed interaction of the metal center with the phosphate linkers in proximity to thymine nucleobases. Oligonucleotides with modified backbone served as targets to further investigate the detailed binding patterns. Furthermore, alternative activation methods, such as electron transfer, were evaluated for the characterization of the metallodrug adducts. Novel Aspect: Data demonstrate the unique binding patterns of metallocenes to oligonucleotides and reveal the specific fragmentation pathways of the metallodrug adducts

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry