Crown ether dopant to reduce ion suppression and improve detection in the liquid microjunction surface sampling probe.

RATIONALE: Sodium and potassium are required in agar media for the growth of some microorganisms (e.g., marine bacteria). However, alkali cations are a significant source of contamination for mass spectrometry causing ion suppression and adduct formation. Conventionally, salts can be removed before mass spectrometric analysis with appropriate and often lengthy sample preparation. The direct mass spectrometric sampling of bacterial colonies grown on agar media seeks to minimize or eliminate sample preparation to improve workflow. However, this may exacerbate ion suppression and contamination since these metal cations will degrade spectral quality and limit the rapid profiling of microbial metabolites. Different approaches are needed to sequester sodium and potassium ions to minimize unwanted background interferences. Herein, we use crown ethers (CEs) in combination with a liquid microjunction surface sampling probe (LMJ-SSP) to directly sample the surface of the bacterial colonies from two marine bacteria species (Pseudoalteromonas rubra DSM6842 and Pseudoalteromonas tunicata DSM 14096). CEs (e.g., 18-crown-6 or 15-crown-5) are added to the carrier solvent of the LMJ-SSP, the chemical noise is reduced, and spectra are easier to interpret. METHODS: The liquid microjunction formed at the tip of LMJ-SSP was used to directly touch bacterial colonies on agar. The carrier solvent was either methanol (100%) or methanol: H2O (50:49.9%) with or without 0.01% CEs. Information-theoretic measures are employed to investigate qualitative changes between spectra before and after adding CEs. RESULTS: Our work demonstrates the capability of CEs to reduce background interferences within the direct profiling of bacterial colonies from agar plates. The data obtained from both P. rubra DSM6842 and P. tunicata DSM 14096 show that CEs can be used to mitigate the salty background and improve compound detection. CONCLUSION: Our approach can be implemented in natural product discovery using LMJ-SSP to allow fast and accurate detection of interesting/novel compounds.

Isolation and characterization of tambjamine MYP1, a macrocyclic tambjamine analogue from marine bacterium Pseudoalteromonas citrea.

Tambjamines are natural products that consist of a conserved bipyrrole core functionalized with different imines giving rise to many derivatives. The core structure of tambjamines allows ion coordination through the nitrogen atoms, which is a key aspect in many of their observed antimicrobial, anticancer, and antimalarial bioactivities. Minor variances in the compound structure have a considerable impact on the potency of these activities, so identifying new analogues is valuable for maximizing tambjamine biological potential. In this work, we describe the isolation and structure elucidation of the first naturally occurring macrocyclized tambjamine, tambjamine MYP1, from the marine microbe Pseudoalteromonas citrea. We also compare the apparent pK a of cyclic and linear tambjamine analogues and discuss how structural strain may effect the compound's ion coordination abilities.

Investigating the Biosynthesis of Natural Products from Marine Proteobacteria: A Survey of Molecules and Strategies.

The phylum proteobacteria contains a wide array of Gram-negative marine bacteria. With recent advances in genomic sequencing, genome analysis, and analytical chemistry techniques, a whole host of information is being revealed about the primary and secondary metabolism of marine proteobacteria. This has led to the discovery of a growing number of medically relevant natural products, including novel leads for the treatment of multidrug-resistant Staphylococcus aureus (MRSA) and cancer. Of equal interest, marine proteobacteria produce natural products whose structure and biosynthetic mechanisms differ from those of their terrestrial and actinobacterial counterparts. Notable features of secondary metabolites produced by marine proteobacteria include halogenation, sulfur-containing heterocycles, non-ribosomal peptides, and polyketides with unusual biosynthetic logic. As advances are made in the technology associated with functional genomics, such as computational sequence analysis, targeted DNA manipulation, and heterologous expression, it has become easier to probe the mechanisms for natural product biosynthesis. This review will focus on genomics driven approaches to understanding the biosynthetic mechanisms for natural products produced by marine proteobacteria.

The synthesis of active and stable diaminopimelate analogues of the lantibiotic peptide lactocin S.

Lantibiotic peptides are potent antimicrobial compounds produced by Gram-positive bacteria. They can be used in food preservation, and some also show potential for clinical applications. Unfortunately, some of these peptides can be susceptible to inactivation by oxidation of the sulfur-containing amino acid lanthionine, limiting their use. Here we describe the synthesis and testing of diaminopimelate analogues of the lantibiotic lactocin S. These analogues were designed to improve the oxidative stability of the peptide by replacing the sulfur in lanthionine with a methylene unit. Lanthionine was systematically replaced with diaminopimelate during solid-phase peptide synthesis to produce several analogues. One analogue, A-DAP lactocin S, was found to retain full biological activity in addition to displaying increased stability. This is the first time a synthetic lanthionine ring analogue of a lantibiotic has retained natural activity levels. This methodology is potentially very promising for use in producing more stable, medically relevant lantibiotics.

Fundamental functionality: recent developments in understanding the structure-activity relationships of lantibiotic peptides.

There has been great interest in the development of extremely potent antibacterial lantibiotic peptides for medicinal use and in food preservation. Of central importance to this endeavor is a strong understanding of which parts of the peptides are required for activity and which parts are expendable. Nisin, lacticin 481, nukacin ISK-1, mersacidin, lacticin 3147 and haloduracin represent many different types of lantibiotic peptides. In recent years, considerable advances toward understanding the structure-activity relationship of each of these lantibiotic systems have been achieved. This review will focus on the individual systems and the valuable information obtained from the many mutants produced.

Synthesis of the lantibiotic lactocin S using peptide cyclizations on solid phase.

Lactocin S is a lantibiotic peptide with potent antibacterial activity against a range of gram-positive bacteria. Because of challenges in obtaining sufficient quantities of this compound from natural sources, the stereochemistry of the lanthionine residues in lactocin S had not been confirmed. This report describes the chemical synthesis of lactocin S on chlorotrityl polystyrene resin in 10% overall yield using intramolecular cyclization to form the lanthionine rings and employing fragment coupling for the two N-terminal residues. This represents the first report of solid-supported synthesis of a naturally occurring lantibiotic. Comparison to lactocin S isolated from Lactobacillus sakei L45 using a combination of HPLC, MS/MS sequencing, bacterial testing, and chiral GC-MS analysis confirmed the initially proposed structure and the stereochemistry of the DL-lanthionine residues.

[Epidemiology of breast carcinoma]. / Epidemiologia del carcinoma della mammella.

With the aim of strengthening epidemiological knowledge on breast cancer, the authors analyze the geographical and temporal distribution of mortality and incidence of the neoplasy in Italy and other countries in the world; attention is focused on recent years and on the influence that the year of birth, the period and age of death have on Italian mortality trend. Analysis of the latest available data confirm the great variability in distribution of breast cancer in different countries of the world, with greater frequency in economically advanced western countries, urbanized and industrilized areas, white women and in higher socio-economic classes. The incidence of breast cancer in the world shows a continuous increases which is more evident and rapid in the countries and among the ethnic groups that were until now in lower risk categories. In Italy, during the period between 1950-88, there is a steady increase in the mortality rate, more evident in the first 20 years. The cumulative rates 25-49 years and 50-75 years demonstrates that the essential contribution to the trends is due to the mortality of elderly women. It is plausible that this trend is influenced by the increased survival, although a different cohort effect due to the exposure to different risk factors is possible. The cohort analysis of mortality shows a sharp cohort effect: the mortality increases in an evident way for all cohorts until those born in 1925, afterwhich we find a tendency towards reduction. The identification of specific reasons that are the basis of the observations can contribute to the knowledge of breast cancer eziology.