MALDI-TOF Mass Spectrometry Analysis Research Articles

Synthesis and characterization of polyisobutylene telechelic prepolymers with epoxide functionality

Syntheses of novel polyisobutylene telechelic prepolymers with various types of epoxide functionality, including aliphatic glycidyl ether, phenyl glycidyl ether, non-glycidyl aliphatic epoxide, and cycloaliphatic epoxide have been developed. The polyisobutylene prepolymers with aliphatic and phenyl glydicyl ether functionalities were achieved by reaction of primary alcohol-terminated and phenol-terminated polyisobutylene, respectively, with epichlorohydrin. Exo-olefin epoxide-terminated polyisobutylene was synthesized by direct epoxidation of exo-olefin-terminated polyisobutylene with a strong oxidizing agent, meta-chloroperoxybenzoic acid. The cycloaliphatic epoxide-terminated polyisobutylene was obtained by nucleophilic substitution of primary bromide end-functional polyisobutylene with a pre-formed potassium cyclohex-3-enecarboxylate salt, followed by epoxidation with meta-chloroperoxybenzoic acid. 1H NMR and MALDI-TOF mass spectrometry analyses demonstrated that the functionality for all types of epoxide prepolymers was 2.0. Gel permeation chromatography (GPC) analysis showed that targeted molecular weight and narrow molecular weight distribution were achieved for these prepolymers and no polymer chain degradation or coupling occurred during post-polymerization modifications.

Subclinical mastitis in pastoralist dairy camel herds in Isiolo, Kenya: Prevalence, risk factors, and antimicrobial susceptibility

Mastitis is an important constraint to milk production in pastoralist camel (Camelus dromedarius) herds in Kenya. The objective of this study was to investigate the prevalence, risk factors, and bacterial panorama of subclinical mastitis (SCM) in pastoralist camel herds in Isiolo County, Kenya. Furthermore, antimicrobial susceptibility in udder pathogens was studied. A cross-sectional sample of 206 camels from 20 milking herds was screened using the California Mastitis Test (CMT), and quarter milk was subjected to bacterial culturing. Isolates were confirmed using MALDI-TOF mass spectrometry analysis, and antimicrobial susceptibility was determined using the broth microdilution method. Interviews focusing on herd management were conducted with camel owners. Subclinical mastitis, defined as a CMT score ≥ 3 (scale 1 to 5) and absence of clinical symptoms in the udder, were present in all visited herds. On the individual level, 46% of the camels had at least 1 quarter affected with SCM, and on the quarter level the prevalence was 26%. Intramammary infections (IMI) were common; out of 798 quarter milk samples, 33% yielded conclusive bacterial growth. The sensitivity and specificity of CMT for correctly identifying quarters with IMI were 82% and 92%, respectively. The most prevalent pathogen was Streptococcus agalactiae (72% of IMI-positive quarters), followed by non-aureus staphylococci (19%) and Staphylococcus aureus (13%). Antimicrobial susceptibility testing revealed that only a low proportion (4.9%) of Strep. agalactiae isolates was sensitive to tetracycline. For Staph. aureus, 59.1% of isolates exhibited sensitivity to penicillin. Skin lesions on the teats or udder were a risk factor for SCM. Increased age, parity, and stage of lactation were associated with increased risk of both SCM and IMI. Older camels with a blind teat or a previous history of mastitis were more likely to be infected with Strep. agalactiae. Hygiene routines for milking were largely absent in the observed herds, and knowledge of adequate milk handling was limited. The poor udder health is likely to depend on multiple factors, most prominently the within-herd maintenance of contagious udder pathogens, in combination with difficult sanitary conditions and lack of awareness among camel keepers. This study showed that in pastoralist camel herds around Isiolo town, SCM and IMI specifically caused by Strep. agalactiae are common udder health problems and are associated with increasing age, parity, and stage of lactation, and skin lesions on the teats and udder. Resistance to tetracycline in Strep. agalactiae was common. Control strategies specifically targeting SCM and adapted to pastorally managed camel herds need to be developed to reduce disease, combat antimicrobial resistance, and improve the livelihoods of pastoralists.

Short communication: Enterotoxigenic potential of coagulase-negative staphylococci isolated from bovine milk in Poland

Our objective was to assess the enterotoxigenic potential of coagulase-negative staphylococci (CNS) isolated from bovine milk in Poland. We analyzed CNS isolates collected from 133 bovine milk batches from dairy farms in the Western Pomerania district during 2 milking seasons. A total of 163 isolates were screened by multiplex/duplex PCR for the presence of 18 of 25 enterotoxin genes identified so far in Staphylococcus aureus. The CNS strains presumed to be potentially enterotoxigenic were identified at the species level based on MALDI-TOF mass spectrometry analysis. Based on the presence of an amplicon matching D, G, or O enterotoxin genes, we initially identified 32 of the 163 CNS isolates tested as potentially enterotoxigenic. However, only 8 of these strains were confirmed as such. All 8 of these CNS strains, identified as Staphylococcus haemolyticus, harbored the seg genes, a prerequisite for enterotoxin G production, but so far not connected with staphylococcal foodborne poisoning cases. None of the CNS bovine milk isolates tested was a potential producer of classical A to E staphylococcal enterotoxins. Results of our surveys revealed a low prevalence of enterotoxigenic CNS among the milk isolates from dairy farms in the Western Pomerania district, Poland, suggesting that they pose only a mild health risk in milk. In our opinion, confirmed formation of nonspecific amplicons leading to false-positive results excludes multiplex/duplex PCR as the sole method for assessing the enterotoxigenic potential of CNS.

Proofreading of single nucleotide insertion/deletion replication errors analyzed by MALDI-TOF mass spectrometry assay

Small nucleotide insertion/deletion (indel) errors are one of the common replication errors in DNA synthesis. The most frequent occurrence of indel error was thought to be due to repeated sequences being prone to slippage during DNA replication. Proofreading and DNA mismatch repair are important factors in indel error correction to maintain the high fidelity of genetic information transactions. We employed a MALDI-TOF mass spectrometry (MS) analysis to measure the efficiency of Klenow polymerase (KF) proofreading of indel errors. Herein, a non-labeled and non-radio-isotopic oligonucleotide primer is annealed to a template DNA forming a single nucleotide indel error and was proofread by KF in the presence of a combination of different deoxyribonucleotide triphosphates and/or dideoxyribonucleotide triphosphates. The proofreading products were identified by the KF modified mass change of the primer. We examined proofreading of DNAs containing indel errors at various positions of the primer-template junction. We found that indel errors located 1–5-nucleotides (nt) from the primer terminus can be proofread efficiently, while insertion/deletions at 6-nt from the 3’ end are partially corrected and extended. Indels located 7–9-nt from the primer terminus escape proofreading and are elongated by polymerase. The possible underlying mechanisms of these observations are discussed in the context of the polymerase and primer-template junction interactions via a structure analysis.

Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry.

MALDI-TOF mass spectrometry analyses revealed the oxidation of thiol-containing polymer chain-ends during sample preparation using THF as solvent. In these conditions, the extent of oxidation was hardly reproducible, and led to various types of oxidized compounds. Preparing the samples at the last minute using commercial THF stabilized with an antioxidant led to more reproducible results, with the least oxidation. However, it is demonstrated herein that thiol oxidation can be advantageously taken into profit to further ascertain the presence of the thiol at the polymer chain-end. To force thiol oxidation we used THF without any antioxidant stabilizer, thus more prone to form peroxides. Thiol-containing polymer chains can thereby be indirectly evidenced by the formation of oxidation products such as chain-chain disulfide bonds and sulfonic acid chains-ends. More importantly, in these oxidizing conditions and in the negative mode, sulfonic acid-terminated polymer chains can be more sensitively detected than thiol ones (the low pKa of sulfonic acids facilitating their anionization in MALDI source). In conclusion, performing MALDI-TOF mass spectrometry analyses in oxidizing conditions, as complement to regular analyses, was found to be very useful for the chain-end identification of different thiol-containing polymer chains.

Venom diversity in the Neotropical scorpion genus Tityus: Implications for antivenom design emerging from molecular and immunochemical analyses across endemic areas of scorpionism

Scorpions of the Neotropical genus Tityus are responsible for most severe envenomations in the Caribbean, South America, and Lower Central America (LCA). Although Tityus is taxonomically complex, contains high toxin polymorphism, and produces variable clinical manifestations, treatment is limited to antivenoms produced against species with restricted distributions. In this study, we explored the compositional and antigenic diversity of Tityus venoms to provide improved guidelines for the use of available antivenoms at a broader geographic scale. We used immunoblotting, competitive ELISA, and in vivo studies to compare reactivity against commercial antivenoms from Brazil, Venezuela, and Mexico, as well as MALDI-TOF mass spectrometry, cDNA sequencing, and phylogenetic analyses to assess venom sodium channel-active toxin (NaTx) content from medically important Tityus populations inhabiting Brazil, Colombia, Costa Rica, Ecuador, Panama, Trinidad and Tobago, and Venezuela. Additionally, we raised rabbit antibodies against Tityus venoms from LCA to test for cross-reactivity with congeneric species. The results suggest that Tityus spp. possess high venom antigenic diversity, underlying the existence of four toxinological regions in Tropical America, based on venom composition and immunochemical criteria: LCA/Colombia/Amazonia (Region I), Venezuela (Region II), southeast South America (Region III), and a fourth region encompassing species related to toxinologically divergent Tityus cerroazul. Importantly, our molecular and cross-reactivity results highlight the need for new antivenoms against species inhabiting Region I, where scorpions may produce venoms that are not significantly reactive against available antivenoms.

Susceptibility of chicken Lactobacillus bacteria to coccidiostats.

The aim of this study was to determine the susceptibility of Lactobacillus bacteria to selected coccidiostats. Seventy-five Lactobacillus isolates obtained from chickens were classified by MALDI-TOF mass spectrometry and 16S rDNA restriction analysis into seven species, among which L. salivarius (33%) and L. johnsonii (24%) were dominant. Susceptibility of lactobacilli to coccidiostats was determined by broth microdilution method. The ranges of minimum inhibitory concentrations (MICs) were 0.5–≥128 µg/ml for monensin, 0.125–8 µg/ml for salinomycin, ≤0.03–2 µg/ml for lasalocid A, and 4–16 µg/ml for robenidine. Coccidiostats in low concentrations inhibited in vitro growth of most lactobacilli and therefore there is a high probability that administration of this drugs to chickens would reduce the number of lactobacilli in the gut.

Organic-Inorganic Hybrid Materials for Room Temperature Light-Activated Sub-ppm NO Detection.

Nitric oxide (NO) is one of the main environmental pollutants and one of the biomarkers noninvasive diagnosis of respiratory diseases. Organic-inorganic hybrids based on heterocyclic Ru (II) complex and nanocrystalline semiconductor oxides SnO2 and In2O3 were studied as sensitive materials for NO detection at room temperature under periodic blue light (λmax = 470 nm) illumination. The semiconductor matrixes were obtained by chemical precipitation with subsequent thermal annealing and characterized by XRD, Raman spectroscopy, and single-point BET methods. The heterocyclic Ru (II) complex was synthesized for the first time and characterized by 1H NMR, 13C NMR, MALDI-TOF mass spectrometry and elemental analysis. The HOMO and LUMO energies of the Ru (II) complex are calculated from cyclic voltammetry data. The thermal stability of hybrids was investigated by thermogravimetric analysis (TGA)-MS analysis. The optical properties of Ru (II) complex, nanocrystalline oxides and hybrids were studied by UV-Vis spectroscopy in transmission and diffuse reflectance modes. DRIFT spectroscopy was performed to investigate the interaction between NO and the surface of the synthesized materials. Sensor measurements demonstrate that hybrid materials are able to detect NO at room temperature in the concentration range of 0.25–4.0 ppm with the detection limit of 69–88 ppb.

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18.

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.

Cysteamine-capped gold-copper nanoclusters for fluorometric determination and imaging of chromium(VI) and dopamine.

Highly emissive cysteamine-capped gold-copper bimetallic nanoclusters (CA-AuCu NCs) with a quantum yield of 18% were synthesized via one-pot anti-galvanic reduction. The CA-AuCu NCs were characterized by HR-TEM, XPS, FTIR, MALDI-TOF mass spectrometry, DLS, and zeta potential analyses. The NCs are shown to be viable fluorescent probes for Cr(VI) ions and dopamine (DA) via quenching of the blue fluorescence, typically measured at excitation/emission wavelengths of 350/436nm. During DA recognition, a dark brown color appears, which is distinguishable from that of Cr(VI) detection. The aggregation induced quenching due to electron transfer was demonstrated by photoluminescence, HR-TEM, FTIR, DLS, and zeta potential interrogations. In buffer of pH7, response is linear in the 0.2 ~ 100μM for Cr(VI) and from 0.4 ~ 250μM for DA. The respective detection limits are 80 and 135nM. The method was applied to the determination of both Cr(VI) and DA in (spiked) tap, lake and sea water, and in human urine samples. The low toxicity of CA-AuCu NCs was validated by the MTT assay, and their responses to Cr(VI) ions and DA was also proven by Raw 264.7 cell imaging. Graphical abstractCysteamine capped Au-Cu nanoclusters (CA-AuCu NCs) were synthesized via one-pot anti-galvanic reduction and utilized in sensing of Cr(VI) ions and dopamine (DA) with demonstrated real/urine and cell imaging applications.

Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Description of Klebsiella spallanzanii sp. nov. and of Klebsiella pasteurii sp. nov.

Klebsiella oxytoca causes opportunistic human infections and post-antibiotic haemorrhagic diarrhea. This Enterobacteriaceae species is genetically heterogeneous and is currently subdivided into seven phylogroups (Ko1 to Ko4 and Ko6 to Ko8). Here we investigated the taxonomic status of phylogroups Ko3 and Ko4. Genomic sequence-based phylogenetic analyses demonstrate that Ko3 and Ko4 formed well-defined sequence clusters related to, but distinct from, Klebsiella michiganensis (Ko1), K. oxytoca (Ko2), K. huaxiensis (Ko8), and K. grimontii (Ko6). The average nucleotide identity (ANI) of Ko3 and Ko4 were 90.7% with K. huaxiensis and 95.5% with K. grimontii, respectively. In addition, three strains of K. huaxiensis, a species so far described based on a single strain from a urinary tract infection patient in China, were isolated from cattle and human feces. Biochemical and MALDI-ToF mass spectrometry analysis allowed differentiating Ko3, Ko4, and Ko8 from the other K. oxytoca species. Based on these results, we propose the names Klebsiella spallanzanii for the Ko3 phylogroup, with SPARK_775_C1T (CIP 111695T and DSM 109531T) as type strain, and Klebsiella pasteurii for Ko4, with SPARK_836_C1T (CIP 111696T and DSM 109530T) as type strain. Strains of K. spallanzanii were isolated from human urine, cow feces, and farm surfaces, while strains of K. pasteurii were found in fecal carriage from humans, cows, and turtles.

Prevotella marseillensis sp. nov., a new bacterium isolated from a patient with recurrent Clostridium difficile infection.

Prevotella marseillensis strain Marseille-P8229T (= CSURP8229) is a new species isolated from a patient with recurrent Clostridium difficile infection. It is an anaerobic, non-motile, non-spore-forming Gram-negative coccobacillus isolated from the stool of patient with recurrent Clostridium difficile infection in Marseille. We present herein its phenotypic description together with MALDI-TOF mass spectrometry analysis and genome sequencing and comparison. The genome of P. marseillensis is 4.1607 Mbp long with 45.80 mol% of G+C content, and it contains 3078 protein-coding genes.

Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

Validation of the Bio-Response Solutions Human-28 Low-Temperature Alkaline Hydrolysis System.

High temperature alkaline hydrolysis (AH) is recognized as an alternative method for sterilization and disposition of animal carcasses and human remains. The aim of this study is to validate the low temperature (LT) AH process specific to its use in the Bio-Response Solutions, Inc. Human-28 LT System. A 313-lb pig was processed using the manufacturers recommended cycle parameters. Stainless steel sample vials containing spore suspensions of Geobacillus stearothermophilus were implanted into the pig's deep tissue to validate the efficacy of the process conditions. Spore suspensions of Bacillus thuringiensis were suspended in the vessel headspace to validate sterilization. The spore challenge was greater than the recommended 106 log used to determine sterilization. MALDI-TOF mass spectrometry analysis was used to validate the destruction of prion-sized particles in processed effluent. Complete inactivation of spores and digestion of animal tissue were achieved after processing in the Bio-Response Solutions Human-28 LT Alkaline Hydrolysis System. Complete inactivation of spores was achieved when exposed to heat in the animal carcass and headspace. No peptide fragments larger than 2500 Da were observed in the treatment effluent. The Bio-Response Solutions, Inc. Human-28 LT Alkaline Hydrolysis System was as effective as high-temperature alkaline hydrolysis for use on animal and human tissue. LT AH for tissue and bodies exceeded the sterility assurance level III of the US State and Territorial Association on Alternative Treatment Technologies and sterility requirements for animal biosafety level-3 and -4 facilities. LT AH process validated destruction of prion-sized particles.

Tamoxifen acts on Trypanosoma cruzi sphingolipid pathway triggering an apoptotic death process

This study shows the effects of tamoxifen, a known estrogen receptor antagonist used in the treatment of breast cancer, on the sphingolipid pathway of Trypanosoma cruzi, searching for potential chemotherapeutic targets. A dose-dependent epimastigote growth inhibition at increasing concentration of tamoxifen was determined. In blood trypomastigotes, treatment with 10 μM showed 90% lysis, while 86% inhibition of intracellular amastigote development was obtained using 50 μM. Lipid extracts from treated and non-treated metabolically labelled epimastigotes evidenced by thin layer chromatography different levels of sphingolipids and MALDI-TOF mass spectrometry analysis assured the identity of the labelled species. Comparison by HPLC-ESI mass spectrometry of lipids, notably exhibited a dramatic increase in the level of ceramide in tamoxifen-treated parasites and a restrained increase of ceramide-1P and sphingosine, indicating that the drug is acting on the enzymes involved in the final breakdown of ceramide. The ultrastructural analysis of treated parasites revealed characteristic morphology of cells undergoing an apoptotic-like death process. Flow cytometry confirmed cell death by an apoptotic-like machinery indicating that tamoxifen triggers this process by acting on the parasitic sphingolipid pathway.

Isolation of a Novel Complex Between Human NER Proteins XPC and XPA

Nucleotide excision repair (NER) is the primary mechanism to remove large covalent DNA lesions resulting from exposure to a wide variety of genotoxic agents. The human NER mechanism is dependent on the coordinated activity of over thirty proteins that build a series of protein machines to repair the damaged DNA. Two proteins, XPC and XPA, play critical roles in NER despite lacking any enzymatic activity. XPC serves as a generic sensor of damaged DNA, marking a site for repair and initiating assembly of the NER machinery. XPA is generally believed to act as a molecular scaffold upon which the NER machines are built. Previous studies have suggested a weak but direct physical interaction between XPC and XPA, and genetic evidence suggests this interaction may aid in recruitment of XPA to potential damage sites in cells. In this study, we identified a novel protein complex between human XPA and the globular transglutaminase-like domain of XPC (XPC-TGD). We employed a chemical cross-linking strategy to isolate protein complexes formed from highly purified recombinant proteins. Complexes were purified by SDS-PAGE and subjected to trypsin proteolysis. MALDI-TOF mass spectrometry analysis identified polypeptide fragments from both the XPA and XPC proteins in tryptic digests of isolated complex samples. These results suggest the XPA may interact with XPC by binding to the XPC-TGD during assembly of the NER machinery. Support or Funding Information Supported by The University of Virginia's College at Wise Fellowship in Natural Sciences (FINS) Undergraduate Research Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessing Molecular Epidemiology of Carbapenem-resistant Klebsiella pneumoniae (CR-KP) with MLST and MALDI-TOF in Central China

Carbapenem-resistant K. pneumoniae (CR-KP) posts significant public health challenge worldwide. The aim of this study is to assess clinical characteristics and molecular epidemiology of CR-KP infections with Multilocus sequence typing (MLST) and Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) in Central China. A total of 71 CR-KP isolates were recovered in a teaching hospital from October 2014 to December 2015. Among all CR-KP isolates, 73.2% (52) produced K. pneumoniae carbapenemases-2 (KPC-2). Eighteen ST types were identified by MLST, among these ST types, forty-seven isolates belonged to ST11 type, which was the predominant outbreak strain in China, and most ST11 isolates produced KPC-2. Eleven mass spectrometry (MS) types were identified by MALDI-TOF MS analysis, 53.5% isolates were MS4 and MS6, which matched with ST11 in MLST analysis. CR-KP infection was associated with increased medical cost and longer hospitalization. Therefore, we found that KPC-2-producing ST11 (MS4 and MS6) CR-KP isolates were the predominant clone identified by MLST and MALDI-TOF, and CR-KP infection was associated with increased hospital costs and longer hospitalization.

Polypeptides secreted from the columnar vesicles of the sea anemone Bunodosoma cangicum and their in vivo effects on Caenorhabditis elegans.

In this study we provide new evidence that the columnar vesicles of the sea anemone Bunodosoma cangicum are toxic in vivo and contain at least two active polypeptides, a neurotoxic and an apoptosis inducing polypeptide. Here we show that it is also an effective inducer of apoptosis in vivo in the nematode Caenorhabditis elegans. In addition, the anemone peptides rapidly paralyze C. elegans, and set in motion a sequence of events that result in the complete dissolution of the internal organs in adult animals within 60 min. Nematodes that survive the toxin treatment exhibit a decreased reproductive capacity. Interestingly, adult animals appear to be much more susceptible to the effects of the toxins than larval stages, suggesting possible developmentally dependent targets of the toxins. Here we also provide chemical characterization of the compounds through chromatographic analysis and mass spectrometry. Gel filtration chromatography coupled with reverse phase HPLC shows that our partially purified extract contains at least two principle components. Additionally, MALDI-TOF mass spectrometry analysis of our extract shows three principal compounds at 814.6, 2914.1, and 4360.3 m/z plus three other minor components or fragments. Mass spectrometry analysis also indicates the presence of three disulfide bridges. Which is in agreement with other characterizations of anemone venoms.

Protein carbonylation in human bronchial epithelial cells exposed to cigarette smoke extract.

Cigarette smoke is a well-established exogenous risk factor containing toxic reactive molecules able to induce oxidative stress, which in turn contributes to smoking-related diseases, including cardiovascular, pulmonary, and oral cavity diseases. We investigated the effects of cigarette smoke extract on human bronchial epithelial cells. Cells were exposed to various concentrations (2.5-5-10-20%) of cigarette smoke extract for 1, 3, and 24h. Carbonylation was assessed by 2,4-dinitrophenylhydrazine using both immunocytochemical and Western immunoblotting assays. Cigarette smoke induced increasing protein carbonylation in a concentration-dependent manner. The main carbonylated proteins were identified by means of two-dimensional electrophoresis coupled to MALDI-TOF mass spectrometry analysis and database search (redox proteomics). We demonstrated that exposure of bronchial cells to cigarette smoke extract induces carbonylation of a large number of proteins distributed throughout the cell. Proteins undergoing carbonylation are involved in primary metabolic processes, such as protein and lipid metabolism and metabolite and energy production as well as in fundamental cellular processes, such as cell cycle and chromosome segregation, thus confirming that reactive carbonyl species contained in cigarette smoke markedly alter cell homeostasis and functions.