Analysis of Pesticide Residues on Fruit Using Swab Spray Ionization Mass Spectrometry.

The vast quantity and high variety of pesticides globally used in agriculture entails considerable risks for the environment and requires ensuring the safety of food products. Therefore, powerful analytical tools are needed to acquire qualitative and quantitative data for monitoring pesticide residues. The development of ambient ionization mass spectrometry methods in the past two decades has demonstrated numerous ways to generate ions under atmospheric conditions and simultaneously to reduce the need for extended sample preparation and circumvent chromatographic separation prior to mass analysis. Swab spray ionization enables the generation of ions directly from swabs via the application of high voltage and solvent flow. In this study, swab sampling of fruit surfaces and subsequent ionization directly from the swab in a modified electrospray ion source was employed for the screening and quantitation of pesticide residues. Aspects regarding sample collection, sampling efficacy on different surfaces, and swab background are discussed. The effect of solvent composition on pesticide-sodium adduct formation and the suppression of ionization by the background matrix have been investigated. Furthermore, a novel approach for the quantitation of pesticide residues based on depletion curve areas is presented. It is demonstrated that swab spray ionization is an effective and quick method for spectral library-based identification and the quantitative analysis of polar contact pesticide residues on food.

Challenges in the Analysis of Gangliosides by LC-MS.

Gangliosides are a family of conjugates consisting of a polar sialoglycan head group and a hydrophobic ceramide tail. Gangliosides are of high abundance in neuronal tissues and are involved in numerous biological processes, such as cell-cell recognition, adhesion, and signal transduction. Alteration of the ganglioside profile is associated with various neurodegenerative diseases and there is indication that gangliosides are involved in the pathogenesis of Parkinson's and Huntington's disease. The development of refined methods for the analysis of gangliosides by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) has supported research with qualitative and quantitative data. However, the amphiphilic character of gangliosides renders their separation and mass spectrometric analysis challenging. In this article, the strengths of hydrophilic interaction liquid chromatography (HILIC) for baseline separation of gangliosides, including two structural isomers, and their structural characterization by tandem mass spectrometry are demonstrated. The importance of ion source parameter optimization is highlighted to prevent misleading ganglioside transformation due to in-source dissociation.

Haplotype structure defines effects of common DPYD variants c.85T > C (rs1801265) and c.496A > G (rs2297595) on dihydropyrimidine dehydrogenase activity: Implication for 5-fluorouracil toxicity.

AIMS: The aim of this study was to identify risk variants and haplotypes that impair dihydropyrimidine dehydrogenase (DPD) activity and are, therefore, candidate risk variants for severe toxicity to 5-fluorouracil (5-FU) chemotherapy. METHODS: Plasma dihydrouracil/uracil (UH2 /U) ratios were measured as a population marker for DPD activity in a total of 1382 subjects from 4 independent studies. Genotype and haplotype correlations with UH2 /U ratios were assessed. RESULTS: Significantly lower UH2 /U ratios (panova < 2 × 10-16 ) were observed in carriers of the 4 well-studied 5-FU toxicity risk variants with mean differences (MD) of -43.7% for DPYD c.1905 + 1G > A (rs3918290), -46.0% for DPYD c.1679T > G (rs55886062), -37.1%, for DPYD c.2846A > T (rs67376798), and -13.2% for DPYD c.1129-5923C > G (rs75017182). An additional variant, DPYD c.496A > G (rs2297595), was also associated with lower UH2 /U ratios (P < .0001, MD: -12.6%). A haplotype analysis was performed for variants in linkage disequilibrium with c.496A > G, which consisted of the common variant c.85T > C (rs1801265) and the risk variant c.1129-5923C > G. Both haplotypes carrying c.496A > G were associated with decreased UH2 /U ratios (H3, P = .003, MD: -9.6%; H5, P = .002, MD: -16.9%). A haplotype carrying only the variant c.85T > C (H2) was associated with elevated ratios (P = .004, MD: +8.6%). CONCLUSIONS: Based on our data, DPYD-c.496A > G is a strong candidate risk allele for 5-FU toxicity. Our data suggest that DPYD-c.85T > C might be protective; however, the deleterious impacts of the linked alleles c.496A > G and c.1129-5923C > G likely limit this effect in patients. The possible protective effect of c.85T > C and linkage disequilibrium with c.496A > G and c.1129-5923C > G may have hampered prior association studies and should be considered in future clinical studies.

Mass Spectrometric Evaluation of ß-Cyclodextrins as Potential Hosts for Titanocene Dichloride.

Bent metallocene dichlorides (Cp2MCl2, M = Ti, Mo, Nb, …) have found interest as anti-cancer drugs in order to overcome the drawbacks associated with platinum-based therapeutics. However, they suffer from poor hydrolytic stability at physiological pH. A promising approach to improve their hydrolytic stability is the formation of host-guest complexes with macrocyclic structures, such as cyclodextrins. In this work, we utilized nanoelectrospray ionization tandem mass spectrometry to probe the interaction of titanocene dichloride with ß-cyclodextrin. Unlike the non-covalent binding of phenylalanine and oxaliplatin to ß-cyclodextrin, the mixture of titanocene and ß-cyclodextrin led to signals assigned as [ßCD + Cp2Ti-H]+, indicating a covalent character of the interaction. This finding is supported by titanated cyclodextrin fragment ions occurring from collisional activation. Employing di- and trimethylated ß-cyclodextrins as hosts enabled the elucidation of the influence of the cyclodextrin hydroxy groups on the interaction with guest structures. Masking of the hydroxy groups was found to impair the covalent interaction and enabling the encapsulation of the guest structure within the hydrophobic cavity of the cyclodextrin. Findings are further supported by breakdown curves obtained by gas-phase dissociation of the various complexes.

Identification of a precursor processing protease from the spider Cupiennius salei essential for venom neurotoxin maturation.

Spider venom neurotoxins and cytolytic peptides are expressed as elongated precursor peptides, which are post-translationally processed by proteases to yield the active mature peptides. The recognition motifs for these processing proteases, first published more than 10 years ago, include the processing quadruplet motif (PQM) and the inverted processing quadruplet motif (iPQM). However, the identification of the relevant proteases was still pending. Here we describe the purification of a neurotoxin precursor processing protease from the venom of the spider Cupiennius salei The chymotrypsin-like serine protease is a 28-kDa heterodimer with optimum activity at venom's pH of 6.0. We designed multiple synthetic peptides mimicking the predicted cleavage sites of neurotoxin precursors. Using these peptides as substrates, we confirm the biochemical activity of the protease in propeptide removal from neurotoxin precursors by cleavage C-terminal of the PQM. Furthermore, the PQM protease also cleaves the iPQM relevant for heterodimerization of a subgroup of neurotoxins. An involvement in the maturing of cytolytic peptides is very likely, due to high similarity of present protease recognition motifs. Finally, bioinformatics analysis, identifying sequences of homolog proteins from 18 spiders of 9 families, demonstrate the wide distribution and importance of the isolated enzyme for spiders. In summary, we establish the first example of a PQM protease, essential for maturing of spider venom neurotoxins. In the future, the here described protease may be established as a powerful tool for production strategies of recombinant toxic peptides, adapted to the maturing of spider venom toxins.

Ammonium Salts as a Source of Small Molecules Observed with High-Resolution Electron-Impact Ionization Mass Spectrometry.

Recent high-resolution in situ mass spectrometry at comet 67P/Churyumov-Gerasimenko visited by European Space Agency's Rosetta spacecraft raised the question, if sublimating ammonium salts can unequivocally be detected in the cometary coma. In laboratory experiments with the twin model of the space instrument, two prototypic ammonium salts NH4B, namely, ammonium chloride (B = Cl-) and ammonium formate (B = HCOO-) (as well as methodologically relevant isotopologues), were allowed to sublimate in vacuum while mass spectra were collected. High-resolution electron-impact ionization mass spectrometry provides an outstanding experimental tool to investigate the complex physicochemical processes occurring during the sublimation of ammonium salts. Sublimation of ammonium chloride led to the observation of the ammonium cation NH4+ and the chloramide molecule NH2Cl in the neutral gas mode of the instrument. These observations could be jointly interpreted as indirect evidence for the existence of a neutral gaseous parent species (either as the molecular complex NH3···HB or the double-ionic species NH4+···B-). However, the qualitative fragmentation pattern we present for 13C15N-ammonium formate suggests an alternative route of NH4+ production within the ionization region of the instrument, namely, by protonation/hydrogenation. Besides NH4+, other species were observed that were formed in protonation/hydrogenation reactions. Moreover, together with the two major species from the decomposition of the salt, ammonia and formic acid, three minor species also contributed to the fragmentation pattern: HCN/HNC, HOCN/HNCO, and CH3NO. Like chloramide, formamide (CH3NO) also is a secondary species probably formed in a pseudo-intramolecular chemical reaction while ammonia and the respective acid are in a state of association. HCN/HNC and HOCN/HNCO are ternary products coming out of formamide decomposition reactions. We discuss our experimental findings, summarized in a tentative chemical reaction network, in light of the available theoretical literature and highlight their relevance for the interpretation of in situ measurements in space research.

Characterization of nucleic acids by tandem mass spectrometry - The second decade (2004-2013): From DNA to RNA and modified sequences.

Nucleic acids play key roles in the storage and processing of genetic information, as well as in the regulation of cellular processes. Consequently, they represent attractive targets for drugs against gene-related diseases. On the other hand, synthetic oligonucleotide analogues have found application as chemotherapeutic agents targeting cellular DNA and RNA. The development of effective nucleic acid-based chemotherapeutic strategies requires adequate analytical techniques capable of providing detailed information about the nucleotide sequences, the presence of structural modifications, the formation of higher-order structures, as well as the interaction of nucleic acids with other cellular components and chemotherapeutic agents. Due to the impressive technical and methodological developments of the past years, tandem mass spectrometry has evolved to one of the most powerful tools supporting research related to nucleic acids. This review covers the literature of the past decade devoted to the tandem mass spectrometric investigation of nucleic acids, with the main focus on the fundamental mechanistic aspects governing the gas-phase dissociation of DNA, RNA, modified oligonucleotide analogues, and their adducts with metal ions. Additionally, recent findings on the elucidation of nucleic acid higher-order structures by tandem mass spectrometry are reviewed. © 2014 Wiley Periodicals, Inc., Mass Spec Rev 35:483-523, 2016.

Phosphatidylethanol (PEth) detected in blood for 3 to 12 days after single consumption of alcohol-a drinking study with 16 volunteers.

In most studies, the alcohol marker phosphatidylethanol (PEth) was used to differentiate social drinking from alcohol abuse. This study investigates PEth's potential in abstinence monitoring by performing a drinking study to assess the detection window of PEth after ingesting a defined amount of alcohol. After 2 weeks of abstinence, 16 volunteers ingested a single dose of alcohol, leading to an estimated blood alcohol concentration (BAC) of 1 g/kg. In the week after drinking, blood and urine samples were taken daily; in the second week, samples were taken every other day. PEth 16:0/18:1 and 16:0/18:2 were analyzed in blood by online-SPE-LC-MS/MS. Ethyl glucuronide and ethyl sulfate were determined in urine for abstinence monitoring. Prior to start of drinking, PEth 16:0/18:1 exceeded 30 ng/mL in blood samples of five volunteers despite the requested abstinence period. Positive PEth values resulted from drinking events prior to this abstinence period. After the start of drinking, maximum BACs were reached after 2 h with a mean of 0.80 ± 0.13 g/kg (range: 0.61-1.11 g/kg). PEth 16:0/18:1 increased within 8 h to maximum concentrations (mean: 88.8 ± 47.0 ng/mL, range: 37.2-208 ng/mL). After this event, PEth was detectable for 3 to 12 days with a mean half-life time of approximately 3 days. PEth has a potential in abstinence monitoring, since PEth could be detected for up to 12 days after a single drinking event. Further investigations are necessary, to establish cut-off levels for PEth as diagnostic marker for the determination of drinking habits like abstinence, social drinking, or risky alcohol consumption.

Transition Metal-based Anticancer Drugs Targeting Nucleic Acids: A Tandem Mass Spectrometric Investigation.

The search for effective drugs against cisplatin-resistant tumors resulted in a large number of organometallic compounds that are evaluated for their antiproliferative activity. Among the most promising candidates are bent metallocenes based on various transition metal ions and ligands. The elucidation of structural features and the characterization of the interaction of a drug candidate with its target require accurate and sensitive analytical tools. Tandem mass spectrometry is applied to the investigation of the adduct sites and binding patterns of metallodrugs bound to single-stranded oligonucleotides and higher-order nucleic acids. Results reveal the binding specificities of the different metallodrugs and demonstrate the influence they exert on the dissociation pathways of the adducts in the gas-phase.

Determination of fatty acid ethyl esters in dried blood spots by LC-MS/MS as markers for ethanol intake: application in a drinking study.

The forensic utility of fatty acid ethyl esters (FAEEs) in dried blood spots (DBS) as short-term confirmatory markers for ethanol intake was examined. An LC-MS/MS method for the determination of FAEEs in DBS was developed and validated to investigate FAEE formation and elimination in a drinking study, whereby eight subjects ingested 0.66-0.84 g/kg alcohol to reach blood alcohol concentrations (BAC) of 0.8 g/kg. Blood was taken every 1.5-2 h, BAC was determined, and dried blood spots were prepared, with 50 µL of blood, for the determination of FAEEs. Lower limits of quantitation (LLOQ) were between 15 and 37 ng/mL for the four major FAEEs. Validation data are presented in detail. In the drinking study, ethyl palmitate and ethyl oleate proved to be the two most suitable markers for FAEE determination. Maximum FAEE concentrations were reached in samples taken 2 or 4 h after the start of drinking. The following mean peak concentrations (c̅(max)) were reached: ethyl myristate 14 ± 4 ng/mL, ethyl palmitate 144 ± 35 ng/mL, ethyl oleate 125 ± 55 ng/mL, ethyl stearate 71 ± 21 ng/mL, total FAEEs 344 ± 91 ng/mL. Detectability of FAEEs was found to be on the same time scale as BAC. In liquid blood samples containing ethanol, FAEE concentrations increase post-sampling. This study shows that the use of DBS fixation prevents additional FAEE formation in blood samples containing ethanol. Positive FAEE results obtained by DBS analysis can be used as evidence for the presence of ethanol in the original blood sample.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency.

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 µg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Elucidation of nucleic acid-drug interactions by tandem mass spectrometry.

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid-drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double-stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle.

Isolation and characterization of synthesis intermediates and side products in hexahydrocannabiphorol.

After the Swiss ban of hexahydrocannabinol (HHC) in March 2023, other semisynthetic dibenzopyran cannabinoids emerged on the Swiss gray market. Hexahydrocannabiphorol (HHCP) was the most prominent of them due to its potent cannabimimetic effects, as anecdotal reports from recreational users suggest. In October 2023, a class wide ban of dibenzopyran cannabinoids was introduced in Switzerland to prevent new similar substances from entering the drug market. Various vendors in online shops claim that HHCP is made from CBD, even though they possess different alkyl chain lengths. An HHCP sample was analyzed by gas chromatography coupled to mass spectrometry (GC-MS), showing that a mixture of molecules with the same or a similar molecular mass as HHCP was present. Six different substances could be isolated from this sample using column chromatography. Four phenols ((9R)-HHCP, iso-HHCP, cis-HHCP, and abn-HHCP) and two ketones (possible intermediates to (9R)-HHCP and abn-HHCP) were identified by various nuclear magnetic resonance spectroscopy (NMR) techniques. (9S)-HHCP was obtained in an impure fraction. In addition, a fraction was obtained that showed characteristic molecular and fragment ions consistent with bisalkylated products from the synthesis of similar compounds. The presence of abnormal cannabinoids (abn-HHCP) and bisalkylated cannabinoids is a confirmation that this sample was produced purely synthetically as initially suspected, as these compounds have not been reported in Cannabis. Chiral derivatization of the phenols with Mosher acid chlorides showed that only iso-HHCP was present as a scalemic mixture, indicating a good stereocontrol of this synthetic procedure.

Comparative evaluation of the My5-FU™ immunoassay and LC-MS/MS in monitoring the 5-fluorouracil plasma levels in cancer patients.

BACKGROUND: Chemotherapies of solid tumors commonly include 5-fluorouracil (5-FU). With standard doses of 5-FU, substantial inter-patient variability has been observed in exposure levels and treatment response. Recently, improved outcomes in colorectal cancer patients due to pharmacokinetically guided 5-FU dosing were reported. We aimed at establishing a rapid and sensitive method for monitoring 5-FU plasma levels in cancer patients in our routine clinical practice. METHODS: Performance of the Saladax My5-FU™ immunoassay was evaluated on the Roche Cobas® Integra 800 analyzer. Subsequently, 5-FU concentrations of 247 clinical plasma samples obtained with this assay were compared to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and other commonly used clinical analyzers (Olympus AU400, Roche Cobas c6000, and Thermo Fisher CDx90). RESULTS: The My-FU assay was successfully validated on the Cobas Integra 800 analyzer in terms of linearity, precision, accuracy, recovery, interference, sample carryover, and dilution integrity. Method comparison between the Cobas Integra 800 and LC-MS/MS revealed a proportional bias of 7% towards higher values measured with the My5-FU assay. However, when the Cobas Integra 800 was compared to three other clinical analyzers in addition to LC-MS/MS including 50 samples representing the typical clinical range of 5-FU plasma concentrations, only a small proportional bias (≤1.6%) and a constant bias below the limit of detection was observed. CONCLUSIONS: The My5-FU assay demonstrated robust and highly comparable performance on different analyzers. Therefore, the assay is suitable for monitoring 5-FU plasma levels in routine clinical practice and may contribute to improved efficacy and safety of commonly used 5-FU-based chemotherapies.

LC-MS/MS method for simultaneous analysis of uracil, 5,6-dihydrouracil, 5-fluorouracil and 5-fluoro-5,6-dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients.

The chemotherapeutic drug 5-fluorouracil (5-FU) is widely used for treating solid tumors. Response to 5-FU treatment is variable with 10-30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6-dihydrouracil (UH(2) ), and analogously, 5-FU into 5-fluoro-5,6-dihydrouracil (5-FUH(2) ). Combined quantification of U and UH(2) with 5-FU and 5-FUH(2) may provide a pre-therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of U, UH(2) , 5-FU and 5-FUH(2) in human plasma. Samples were prepared by liquid-liquid extraction with 10:1 ethyl acetate-2-propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 µL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01-10 µm for U, 0.1-10 µm for UH(2) , 0.1-75 µm for 5-FU and 0.75-75 µm for 5-FUH(2) , covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5-FU-treated colorectal cancer patients. The present method merges the analysis of 5-FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5-FU-based chemotherapy.

Identification of human hexahydrocannabinol metabolites in urine.

Hexahydrocannabinol (HHC) is a cannabinoid that has been known since 1940 but has only recently found its way into recreational use as a psychoactive drug. HHC has been used as a legal alternative to tetrahydrocannabinol (THC) in many countries, but first countries already placed it under their narcotic substances law. Our aim was to evaluate a reliable analytical method for the proof of HHC consumption by LC-MS/MS and GC-MS. We identified the two epimers of HHC and metabolites after HHC consumption by two volunteers (inhalation by use of a vaporizer and oral intake). LC-HR-MS/MS, LC-MS/MS and GC-MS with literature data (EI-MS spectra of derivatives) and reference compounds - as far as commercially available - were used for metabolite identification. Phase-II-metabolites (glucuronides) of HHC and OH-HHC were found in urine samples with LC-HR-MS/MS and LC-MS/MS. The main metabolite was tentatively identified with GC-MS as 4'OH-HHC (stereochemistry on C9 and C4' unknown). Another major side-chain hydroxylated metabolite found by LC-MS/MS could not be unambiguously identified. Both epimers of 11-OH-HHC were found in considerable amounts in urine. (8R, 9R)-8-OH-HHC was identified as a minor metabolite with GC-MS and LC-MS/MS. While (9S)-HHC was found in urine after oral intake and inhalation of HHC, the more psychoactive epimer (9R)-HHC was only found in urine after inhalation. Several other minor metabolites were detected but not structurally identified. We found that after oral or inhalative consumption the urinary main metabolites of a diastereomeric mixture of HHC are different from the respective, major Δ9-THC metabolites (11-OH-Δ9-THC and 11-nor-9-carboxy-Δ9-THC). Although a sensitive LC-MS/MS and GC-SIM-MS method were set-up for the reference compounds (9R)-11-nor-9-carboxy-HHC and (9S)-11-nor-9-carboxy-HHC, these oxidation products were not detected in urine with these techniques. To further increase sensitivity, a GC-MS/MS method was developed, and the 11-nor-9-carboxy metabolites of HHC were confirmed to be present as minor metabolites.

Structural and biochemical characterization of native and recombinant single insulin-like growth factor-binding domain protein (SIBD-1) from the Central American hunting spider Cupiennius salei (Ctenidae).

Cupiennius salei single insulin-like growth factor binding domain protein (SIBD-1) is an 8.6 kDa Cys-, Pro-, and Gly-rich protein, discovered in the hemocytes of the Central American hunting spider Cupiennius salei. SIBD-1 exhibits high sequence similarity to the N-terminal domain of the insulin-like growth factor-binding protein superfamily and has been reported to play an important role in the spider's immune system. Here, the recombinant expression and the elucidation of the three-dimensional structure of recombinant SIBD-1 and the characterization of the sugar moiety at Thr2 of native SIBD-1 is described in detail.

Development and in vivo validation of phospholipid-based depots for the sustained release of bupivacaine.

By direct deposition of the drug at the local site of action, injectable depot formulations - intended for treatment of a local disease or for local intervention - are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to zwitterionic ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP.

Furan-oxidation-triggered inducible DNA cross-linking: acyclic versus cyclic furan-containing building blocks--on the benefit of restoring the cyclic sugar backbone.

Oligodeoxynucleotides incorporating a reactive functionality can cause irreversible cross-linking to the target sequence and have been widely studied for their potential in inhibition of gene expression or development of diagnostic probes for gene analysis. Reactive oligonucleotides further show potential in a supramolecular context for the construction of nanometer-sized DNA-based objects. Inspired by the cytochrome P450 catalyzed transformation of furan into a reactive enal species, we recently introduced a furan-oxidation-based methodology for cross-linking of nucleic acids. Previous experiments using a simple acyclic building block equipped with a furan moiety for incorporation into oligodeoxynucleotides have shown that cross-linking occurs in a very fast and efficient way and that substantial amounts of stable, site-selectively cross-linked species can be isolated. Given the destabilization of duplexes observed upon introduction of the initially designed furan-modified building block into DNA duplexes, we explore here the potential benefits of two new building blocks featuring an extended aromatic system and a restored cyclic backbone. Thorough experimental analysis of cross-linking reactions in a series of contexts, combined with theoretical calculations, permit structural characterization of the formed species and allow assessment of the origin of the enhanced cross-link selectivity. Our experiments clearly show that the modular nature of the furan-modified building blocks used in the current cross-linking strategy allow for fine tuning of both yield and selectivity of the interstrand cross-linking reaction.

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei.

Three novel glycine-rich peptides, named ctenidin 1-3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mass.