Easing the global burden of diarrhoeal disease: can synthetic biology help?

The Millennium Declaration committed the 193 member states of the United Nations to end poverty by 2015. Despite the efforts of the UN and World Health Organisation, and the G8 commitment to spend a fixed proportion of gross national income on overseas aid, more than 2.6 billion people still lack access to proper sanitation. The absence of effective public health strategies in developing countries results in significant health burdens following gastrointestinal infections. Diarrhoea associated with infections resulting from oral-faecal contamination is the second leading cause of death in children under 5 years of age, primarily in Africa and South Asia. Currently there are no appropriate vaccines that could be easily administered on a global scale to prevent these infections. Synthetic biology has the potential to contribute to development of such vaccines. Our work is directed at developing a range of multivalent oral vaccines against the most common diarrhoea-causing bacteria, e.g., Escherichia coli, Shigella and Salmonella. If synthetic biology is to avoid the suspicion and possible revulsion of the public, scientists need to demonstrate that this new field has something real to offer.     

Chromatographic profile of high boiling point organic acids in human urine

The profile of high boiling point organic acids in urine samples from both normal subjects and patients suspected of having some form of metabolic disorder has been determined by combined gas chromatography—mass spectrometry. Fifteen different compounds eluting after hippuric acid have been identified, including two, cinnamoylglycine and acetyltributylcitrate, which have not been recognised previously. Relative retention times and abbreviated mass spectra of the identified compounds are presented.     

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX₃δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β₂, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β₂ binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β₂. DNA synthesis activity can be restored by increased concentrations of β₂. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β₂ loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.     

Release of Dengue Virus Genome Induced by a Peptide Inhibitor

Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.     

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn²⁺) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn²⁺ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn²⁺-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn²⁺-limited Mtb in vivo. Prolonged Zn²⁺ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn²⁺ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn²⁺ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn²⁺-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn²⁺-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn²⁺ availability likely plays a key role during early interactions with host cells.     

A Comparison of Collection Techniques for Gene Expression Analysis of Human Oral Taste Tissue

Variability in human taste perception is associated with both genetic and environmental factors. The influence of taste receptor expression on this variability is unknown, in part, due to the difficulty in obtaining human oral tissue that enables quantitative expression measures of taste genes. In a comparison of six current techniques (Oragene RNeasy Kit, Isohelix swab, Livibrush cytobrush, tongue saliva, cheek saliva collection, and fungiform papillae biopsy), we identify the fungiform papillae biopsy is the optimal sampling technique to analyse human taste gene expression. The fungiform papillae biopsy resulted in the highest RNA integrity, enabling amplification of all the assessed taste receptor genes (TAS1R1, TAS1R2, TAS1R3, SCNN1A and CD36) and taste tissue marker genes (NCAM1, GNAT3 and PLCβ2). Furthermore, quantitative expression was observed in a subset of taste genes assessed from the saliva collection techniques (cheek saliva, tongue saliva and Oragene RNA kit). These saliva collection techniques may be useful as a non-invasive alternative sampling technique to the fungiform papillae biopsy. Identification of the fungiform papillae biopsy as the optimal collection method will facilitate further research into understanding the effect of gene expression on variability in human taste perception.