The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.     

Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction

Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding “core” that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a K_d of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.     

Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.     

Stable isotopes as indicators of wastewater effects on the macroinvertebrates of urban rivers

Rivers in urban locations frequently receive contaminated wastewater and particulate waste either directly from storm overflows or from sewage treatment facilities. Although many urban streams are now recovering from wide-scale historic pollution, lower-level effects on water chemistry, nutrients and biotic composition are still widespread. We aimed to determine whether such effects could be detected using stable isotope ratios (??¹⁵N, ??¹³C and ??³⁴S) in macroinvertebrates alone or in conjunction with traditional biomonitoring. Macroinvertebrates were collected upstream and downstream of 11 different secondary wastewater treatment works (WwTW) in South Wales and the Welsh borders (United Kingdom). Overall, mean invertebrate ??¹⁵N signatures downstream of the WwTW were significantly enriched despite variation amongst sites. Moreover, changes between upstream and downstream macroinvertebrate ??¹⁵N values were highly correlated with patterns in macroinvertebrate community composition, increased total macroinvertebrate abundance, and reduced Shannon Diversity and other biomonitoring indices (% EPT, % shredders and ASPT scores). Changes in invertebrate ??¹⁵N values also paralleled the consented discharge volumes and population equivalents from each WwTW. In contrast, isotopic ratios of ??¹³C and ??³⁴S were unable to distinguish or quantify wastewater input into the rivers but differences were apparent amongst study streams. Overall, these results suggest that macroinvertebrate ??¹⁵N signatures can detect and quantify the effects of secondary sewage treatment inputs to riverine ecosystems. Moreover, the method potentially provides a sensitive means for tracing sewage-derived nutrients into food webs while inferring effects on aquatic communities where sewage-loads are subtle or confounded by other stressors.     

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ～90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.     

Screening Marine Fungi for Inhibitors of the C4 Plant Enzyme Pyruvate Phosphate Dikinase: Unguinol as a Potential Novel Herbicide Candidate

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C₄ plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 ± 0.8 μM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C₄ plant but no effect on a model C₃ plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.