ALBERT

Description: Peroxisome proliferator-activated receptor β/ (PPARβ/) is a lipid ligand-inducible transcription factor with established metabolic functions, whereas its anti-inflammatory function is poorly understood. To address this issue, we determined the global PPARβ/-regulated signaling network in human monocyte-derived macrophages. Besides cell type-independent, canonical target genes with metabolic and immune regulatory functions we identified a large number of inflammation-associated NFB and STAT1 target genes that are repressed by agonists. Accordingly, PPARβ/ agonists inhibited the expression of multiple pro-inflammatory mediators and induced an anti-inflammatory, IL-4-like morphological phenotype. Surprisingly, bioinformatic analyses also identified immune stimulatory effects. Consistent with this prediction, PPARβ/ agonists enhanced macrophage survival under hypoxic stress and stimulated CD8 + T cell activation, concomitantly with the repression of immune suppressive target genes and their encoded products CD274 (PD-1 ligand), CD32B (inhibitory Fc receptor IIB) and indoleamine 2,3-dioxygenase 1 (IDO-1), as well as a diminished release of the immune suppressive IDO-1 metabolite kynurenine. Comparison with published data revealed a significant overlap of the PPARβ/ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines. Our findings indicate that PPARβ/ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/ in immune regulation.

Description: Numerous regional plate reorganizations and the coeval ages of the Hawaiian Emperor bend (HEB) and Louisville bend of 50–47 Ma have been interpreted as a possible global tectonic plate reorganization at ~chron 21 (47.9 Ma). Yet for a truly global event we would expect a contemporaneous change in Africa absolute plate motion (APM) reflected by physical evidence distributed on the Africa Plate. This evidence has been postulated to take the form of the Réunion-Mascarene bend which exhibits many HEB-like features, such as a large angular change close to ~chron 21. However, the Réunion hotspot trail has recently been interpreted as a sequence of continental fragments with incidental hotspot volcanism. Here we show that the alternative Réunion-Mascarene Plateau trail can also satisfy the age progressions and geometry of other hotspot trails on the Africa Plate. The implied motion, suggesting a pivoting of Africa from 67 to 50 Ma, could explain the apparent bifurcation of the Tristan hotspot chain, the age reversals seen along the Walvis Ridge, the sharp curve of the Canary trail, and the diffuse nature of the St. Helena chain. To test this hypothesis further we made a new Africa APM model that extends back to ~80 Ma using a modified version of the Hybrid Polygonal Finite Rotation Method. This method uses seamount chains and their associated hotspots as geometric constraints for the model, and seamount age dates to determine APM through time. While this model successfully explains many of the volcanic features, it implies an unrealistically fast global lithospheric net rotation, as well as improbable APM trajectories for many other plates, including the Americas, Eurasia and Australia. We contrast this speculative model with a more conventional model in which the Mascarene Plateau is excluded in favour of the Chagos-Laccadive Ridge rotated into the Africa reference frame. This second model implies more realistic net lithospheric rotation and far-field APMs, but fails to explain key details of the Atlantic Ocean volcanic chains. Both models predict a Canary plume influence beneath the Madeiras. Neither model, when projected via the global plate circuit into the Pacific, predicts any significant change in plate motion around chron 21. Consequently, Africa APM models do not appear to provide independent support for a chron 21 global reorganization.

Description: Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org . Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

Description: We construct a model of remanence for the oceans, combine it with a model of induced magnetization for the whole Earth from a previous study, compute the predicted lithospheric geomagnetic field and compare the result with a model, MF7, that is based on satellite data. Remanence is computed by assigning magnetizations to the oceanic lithosphere acquired at the location and time of formation. The magnetizing field is assumed to be an axial dipole that switches polarity with the reversal time scale. The magnetization evolves with time by decay of thermal remanence and acquisition of chemical remanence. The direction of remanence is calculated by Euler rotation of the original geomagnetic field direction with respect to an absolute reference frame, significantly improving previous results which did not include realistic oceanic magnetization computed this way. Remanence only accounts for 24 per cent of the energy of the oceanic magnetization, the induced magnetization being dominant, increasing slightly to 30 per cent of the part of the magnetization responsible for generating geomagnetic anomalies and 39 per cent of the Lowes energy of the geomagnetic anomalies. This is because our model of oceanic crust and lithosphere is fairly uniform, and a uniform layer magnetized by a magnetic field of internal origin produces no external field. The largest anomalies are produced by oceanic lithosphere magnetized during the Cretaceous Normal Superchron. Away from ridges and magnetic quiet zones the prediction fails to match the MF7 values; these are also generally, but not always, somewhat smaller than the observations. This may indicate that the magnetization estimates are too small, in which case the most likely error is in the poorly-known magnetization deep in the crust or upper mantle, or it may indicate some other source such as locally underplated continental lithosphere or anomalous oceanic crust, or even small-scale core fields.

Description: Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli , usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.

Description: Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Red, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.

Description: Nephronophthisis (NPH) is a genetically heterogenous kidney disease and represents the most common genetic cause for end-stage renal disease in children. It is caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs) which localize to primary cilia or centrosomes, classifying this disease as a ‘ciliopathy’. Recently, it has been shown that NPHP4 acts as a potent negative regulator of mammalian Hippo signalling by interacting with the Lats protein kinase and controlling the phosphorylation of the oncogenic transcriptional activator TAZ. Here, we demonstrate that NPHP9, another NPH family member, also controls TAZ activity by a distinct mechanism. NPHP9, which is also called NEK8, directly interacted with TAZ and induced nuclear translocation of the TAZ/NPHP9 protein complex. Binding of NPHP9 to TAZ was enhanced in a TAZ mutant that lost its ability to bind 14-3-3, suggesting that 14-3-3 and NPHP9 may compete for TAZ binding, with 14-3-3 favouring cytoplasmic retention and NPHP9 mediating nuclear delivery. Consistently, co-expression of NPHP4, which inhibits TAZ phosphorylation at the 14-3-3 binding site through the inhibition of Lats kinase activity, induced efficient nuclear delivery of the TAZ/NPHP9 protein pair. Consistent with a role for TAZ in controlling proliferation and tumorigenesis, the downregulation of NPHP9 inhibited the TAZ-dependent proliferation of hippo-responsive normal epithelial and also breast cancer cells. As NPHP9 has been shown to be upregulated in breast cancer, these data do not only support a critical role for TAZ/hippo signalling in the pathogenesis of NPH but may also imply a possible role for NPHP9 in TAZ-mediated tumorigenesis.