Cell-cell communication in Gram-negative bacteria

Over the last decade or so, a wealth of research has established that bacteria communicate with one another using small molecules. These signals enable the individuals in a population to coordinate their behaviour. In the case of pathogens, this behaviour may include decisions such as when to attack a host organism or form a biofilm. Consequently, such signalling systems are excellent targets for the development of new antibacterial therapies. In this review, we assess how Gram-negative bacteria use small molecules for cell-cell communication, and discuss the main approaches that have been developed to interfere with it.     

Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.     

KiSS-1: a likely candidate for the photoperiodic control of reproduction in seasonal breeders

In seasonal species, photoperiod exerts tight regulation of reproduction to ensure that birth occurs at the most favorable time of yr. A distinct photoneuroendocrine circuit composed of the retina, suprachiasmatic nucleus (SCN) of the hypothalamus, and pineal gland transduces daylength into a rhythmic secretion of melatonin. The duration of the night-time rise of this hormone conveys daylength information to the organism. Melatonin is known to mediate the control of seasonal reproduction, but how it modulates sexual activity is far from understood. Recent data indicate that the product of the KiSS-1 gene is a potent stimulator of the hypothalamic-pituitary-gonadal axis and may play, together with its receptor GPR54, a central role in the neuroendocrine regulation of gonadotropin secretion. This article briefly reviews these findings and presents arguments that KiSS-1 could take part in the seasonal control of reproduction.     

Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors

This study used a culture-independent molecular approach to investigate the archaeal community composition of thermophilic bioleaching reactors. Two culture samples, MTC-A and MTC-B, grown with different concentrations of chalcopyrite (CuFeS₂), a copper sulfidic ore, at a temperature of 78°C and pH 1.6 were studied. Phylogenetic analysis of the 16S rRNA genes revealed that both cultures consisted of Archaea belonging to the Sulfolobales . The 16S rRNA gene clone library of MTC-A grown with 4% (w/v) chalcopyrite was dominated by a unique phylotype related to Sulfolobus shibatae (69% of total clones). The remaining clones were affiliated with Stygiolobus azoricus (11%), Metallosphaera sp. J1 (8%), Acidianus infernus (2%), and a novel phylotype related to Sulfurisphaera ohwakuensis (10%). In contrast, the clones from MTC-B grown with 12% (w/v) chalcopyrite did not appear to contain Sulfolobus shibatae -like organisms. Instead the bioleaching consortium was dominated by clones related to Sulfurisphaera ohwakuensis (73.9% of total clones). The remaining microorganisms detected were similar to those found in MTC-A.     

Designed optimization of a single-step extraction of fucose-containing sulfated polysaccharides from Sargassum sp.

Fucose-containing sulfated polysaccharides can be extracted from the brown seaweed, Sargassum sp. It has been reported that fucose-rich sulfated polysaccharides from brown seaweeds exert different beneficial biological activities including anti-inflammatory, anticoagulant, and anti-viral effects. Classical extraction of fucose-containing sulfated polysaccharides from brown seaweed species typically involves extended, multiple-step, hot acid, or CaCl₂ treatments, each step lasting several hours. In this work, we systematically examined the influence of acid concentration (HCl), time, and temperature on the yield of fucose-containing sulfated polysaccharides (FCSPs) in statistically designed two-step and single-step multifactorial extraction experiments. All extraction factors had significant effects on the fucose-containing sulfated polysaccharides yield, with the temperature and time exerting positive effects, and the acid concentration having a negative effect. The model defined an optimized single-step FCSPs extraction procedure for Sargassum sp. (a brown seaweed). A maximal fucose-containing sulfated polysaccharides yield of ～7% of the Sargassum sp. dry matter was achieved by the optimal extraction procedure of: 0.03?M HCl, 90°C, 4?h. HPAEC-PAD analysis confirmed that fucose, galactose, and glucuronic acid were the major constituents of the polysaccharides obtained by the optimized method. Lower polysaccharide yield, but relatively higher fucose content was obtained with shorter extraction time. The data also revealed that classical multi-step extraction with acid ≥0.2?M HCl at elevated temperature and extended time had a detrimental effect on the FCSPs yield as this treatment apparently disrupted the structural integrity of the polymer and evidently caused degradation of the carbohydrate chains built up of fucose residues.     

Phylogenetic analysis of four nuclear protein-encoding genes largely corroborates the traditional classification of Bivalvia (Mollusca)

Revived interest in molluscan phylogeny has resulted in a torrent of molecular sequence data from phylogenetic, mitogenomic, and phylogenomic studies. Despite recent progress, basal relationships of the class Bivalvia remain contentious, owing to conflicting morphological and molecular hypotheses. Marked incongruity of phylogenetic signal in datasets heavily represented by nuclear ribosomal genes versus mitochondrial genes has also impeded consensus on the type of molecular data best suited for investigating bivalve relationships. To arbitrate conflicting phylogenetic hypotheses, we evaluated the utility of four nuclear protein-encoding genes-ATP synthase β, elongation factor-1α, myosin heavy chain type II, and RNA polymerase II-for resolving the basal relationships of Bivalvia. We sampled all five major lineages of bivalves (Archiheterodonta, Euheterodonta [including Anomalodesmata], Palaeoheterodonta, Protobranchia, and Pteriomorphia) and inferred relationships using maximum likelihood and Bayesian approaches. To investigate the robustness of the phylogenetic signal embedded in the data, we implemented additional datasets wherein length variability and/or third codon positions were eliminated. Results obtained include (a) the clade (Nuculanida+Opponobranchia), i.e., the traditionally defined Protobranchia; (b) the monophyly of Pteriomorphia; (c) the clade (Archiheterodonta+Palaeoheterodonta); (d) the monophyly of the traditionally defined Euheterodonta (including Anomalodesmata); and (e) the monophyly of Heteroconchia, i.e., (Palaeoheterodonta+Archiheterodonta+Euheterodonta). The stability of the basal tree topology to dataset manipulation is indicative of signal robustness in these four genes. The inferred tree topology corresponds closely to those obtained by datasets dominated by nuclear ribosomal genes (18S rRNA and 28S rRNA), controverting recent taxonomic actions based solely upon mitochondrial gene phylogenies.     

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.     

Inhibition of the proteolytic activity of pregnancy-associated plasma protein-A by targeting substrate exosite binding

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.