Origin of the Diversity in DNA Recognition Domains in Phasevarion Associated modA Genes of Pathogenic Neisseria and Haemophilus influenzae

Phase variable restriction-modification (R-M) systems have been identified in a range of pathogenic bacteria. In some it has been demonstrated that the random switching of the mod (DNA methyltransferase) gene mediates the coordinated expression of multiple genes and constitutes a phasevarion (phase variable regulon). ModA of Neisseria and Haemophilus influenzae contain a highly variable, DNA recognition domain (DRD) that defines the target sequence that is modified by methylation and is used to define modA alleles. 18 distinct modA alleles have been identified in H. influenzae and the pathogenic Neisseria. To determine the origin of DRD variability, the 18 modA DRDs were used to search the available databases for similar sequences. Significant matches were identified between several modA alleles and mod gene from distinct bacterial species, indicating one source of the DRD variability was via horizontal gene transfer. Comparison of DRD sequences revealed significant mosaicism, indicating exchange between the Neisseria and H. influenzae modA alleles. Regions of high inter- and intra-allele similarity indicate that some modA alleles had undergone recombination more frequently than others, generating further diversity. Furthermore, the DRD from some modA alleles, such as modA12, have been transferred en bloc to replace the DRD from different modA alleles

Major prospects for exploring canine vector borne diseases and novel intervention methods using 'omic technologies

Canine vector-borne diseases (CVBDs) are of major socioeconomic importance worldwide. Although many studies have provided insights into CVBDs, there has been limited exploration of fundamental molecular aspects of most pathogens, their vectors, pathogen-host relationships and disease and drug resistance using advanced, 'omic technologies. The aim of the present article is to take a prospective view of the impact that next-generation, 'omics technologies could have, with an emphasis on describing the principles of transcriptomic/genomic sequencing as well as bioinformatic technologies and their implications in both fundamental and applied areas of CVBD research. Tackling key biological questions employing these technologies will provide a 'systems biology' context and could lead to radically new intervention and management strategies against CVBDs

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

Is gonococcal disease preventable? The importance of understanding immunity and pathogenesis in vaccine development

Gonorrhea is a major, global public health problem for which there is no vaccine. The continuing emergence of antibiotic-resistant strains raises concerns that untreatable Neisseria gonorrhoeae may become widespread in the near future. Consequently, there is an urgent need for increased efforts towards the development of new anti-gonococcal therapeutics and vaccines, as well as suitable models for potential pre-clinical vaccine trials. Several current issues regarding gonorrhea are discussed herein, including the global burden of disease, the emergence of antibiotic-resistance, the status of vaccine development and, in particular, a focus on the model systems available to evaluate drug and vaccine candidates. Finally, alternative approaches to evaluate vaccine candidates are presented. Such approaches may provide valuable insights into the protective mechanisms, and correlates of protection, required to prevent gonococcal transmission, local infection and disease sequelae.Full Tex

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics I. Differential centrifugation fractionation B16F10 cells and use to study the intracellular fate of HPMA copolymer–doxorubicin

Polymer therapeutics are being designed for lysosomotropic, endosomotropic and transcellular drug delivery. Their appropriate intracellular routing is thus crucial for successful use. For example, polymer-anticancer drug conjugates susceptible to lysosomal enzyme degradation will never deliver their drug payload unless they encounter the appropriate activating enzymes. Many studies use confocal microscopy to monitor intracellular fate, but there is a pressing need for more quantitative methods able to define intracellular compartmentation over time. Only then will it be possible to optimise the next generation of polymer therapeutics for specific applications. The aim of this study was to establish a subcellular fractionation method for B16F10 murine melanoma cells and subsequently to use it to define the intracellular trafficking of N-(2-hydroxyproplylmethacrylamide) (HPMA) copolymer-bound doxorubicin (PK1). Free doxorubicin was used as a reference. The cell cracker method was used to achieve cell breakage and optimised to reproducibly achieve approximately 90% breakage efficiency. This ensured that subsequent subcellular fractionation experiments were representative for the whole cell population. To characterise the subcellular fractions obtained by differential centrifugation, DNA (nuclei), succinate dehydrogenase (mitochondria), N-acetyl-beta-glucosaminidase (lysosomes), alkaline phosphatase (plasma membrane) and lactate dehydrogenase (cytosol) were selected as markers and their assay was carefully validated. The relative specific activity (RSA) of the fractions obtained from B16F10 cells were: nuclei (2.2), mitochondria (4.1), lysosomes (3.7) and cytosol (2.5). When used to study the intracellular distribution at non-toxic concentrations of PK1 and doxorubicin, time-dependent accumulation of PK1 in lysosomes was evident and the expected nuclear localisation of free doxorubicin was seen. Live cell fluorescence microscopy and confocal co-localisation studies gave qualitative corroboration of these results, but by using this method, we were unable to accurately define organelle localisation. In conclusion, the B16F10 subcellular fractionation method developed here provides a useful tool to allow comparison of the intracellular trafficking of other polymer conjugates

Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring

Abstract SERS spectra excited at 785 nm of the bacteria Chlamydia trahomatis (elementary bodies, EB) and Neisseria gonorrheoae, the causative pathogens for the two most common sexually transmitted diseases (STD), chlamydia and gonorrhea, respectively, are reported. Although both are Gram-negative bacteria, the SERS signatures of C. trachomatis and N. gonorrheoae are completely different. N. gonorrheoae SERS spectra are due to the starvation induced nucleotide metabolites adenine and guanine, and the surface associated co-enzyme nicotinamide adenine dinucleotide and are very similar on Au and Ag although the spectrum appears more rapidly on Ag. The C. trachomatis SERS spectrum is dominated by the vibrational features of cell surface proteins. While features attributable to specific residues and the amide backbone characterize the C. trachomatis spectrum on Ag, the corresponding SERS spectrum on Au substrates displays vibrational characteristics of aggregated proteins. The prospects for the development of a SERS based platform for rapid (<one hour), low-cost bacterial STD diagnostics are promising based on these initial studies. Furthermore, this biomedical application demonstrates the potential for SERS to be a sensitive real time probe of the dynamics of biochemical activity in the cell wall and extracellular regions of microorganisms