Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.     

Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2^?/? mice were exposed to short term application of CCl₄, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl₄ intoxication showed increased liver damage in LCN2^?/? mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2^?/? mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4 weeks bile duct ligation or 8 weeks CCl₄ application), LCN2^?/? mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.     

Hindcasting Historical Breeding Conditions for an Endangered Salamander in Ephemeral Wetlands of the Southeastern USA: Implications of Climate Change

The hydroperiod of ephemeral wetlands is often the most important characteristic determining amphibian breeding success, especially for species with long development times. In mesic and wet pine flatwoods of the southeastern United States, ephemeral wetlands were a common landscape feature. Reticulated flatwoods salamanders (Ambystoma bishopi), a federally endangered species, depend exclusively on ephemeral wetlands and require at least 11 weeks to successfully metamorphose into terrestrial adults. We empirically modeled hydroperiod of 17 A. bishopi breeding wetlands by combining downscaled historical climate-model data with a recent 9-year record (2006–2014) of observed water levels. Empirical models were subsequently used to reconstruct wetland hydrologic conditions from 1896–2014 using the downscaled historical climate datasets. Reconstructed hydroperiods for the 17 wetlands were highly variable through time but were frequently unfavorable for A. bishopi reproduction (e.g., only 61% of years, using a conservative estimate of development time [12 weeks], were conducive to larval development and metamorphosis). Using change-point analysis, we identified significant shifts in average hydroperiod over the last century in all 17 wetlands. Mean hydroperiods were shorter in recent years than at any other point since 1896, and thus less suitable for A. bishopi reproduction. We suggest that climate change will continue to impact the reproductive success of flatwoods salamanders and other ephemeral wetland breeders by reducing the number of years these wetlands have suitable hydroperiods. Consequently, we emphasize the importance of conservation and management for mitigating other forms of habitat degradation, especially maintenance of high quality breeding sites where reproduction can occur during appropriate environmental conditions.     

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing

Introduction

Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing.

Methods

We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99^th percentile and 176 lean adults (BMI ≤ 15^th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults.

Results and Conclusion

We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (p_TDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.     

Domestication and crop evolution of wheat andbarley: Genes,genomics, and future directions

Wheat and barley are two of the founder crops of the agricultural revolution that took place 10,000 years ago in the Fertile Crescent and both crops remain among the world's most important crops. Domestication of these crops from their wild ancestors required the evolution of traits useful to humans, rather than survival in their natural environment. Of these traits, grain retention and threshability, yield improvement, changes to photoperiod sensitivity and nutritional value are most pronounced between wild and domesticated forms. Knowledge about the geographical origins of these crops and the genes responsible for domestication traits largely pre-dates the era of nextgeneration sequencing, although sequencing will lead to new insights. Molecular markers were initially used to calculate distance(relatedness), genetic diversity and to generate genetic maps which were useful in cloning major domestication genes. Both crops are characterized by large,complex genomes which were long thought to be beyond the scope of whole-genome sequencing. However, advances in sequencing technologies have improved the state of genomic resources for both wheat and barley. The availability of reference genomes for wheat and some of its progenitors,as well as for barley, sets the stage for answering unresolved questions in domestication genomics of wheat and barley.     

Modeling population dynamics in a microbial consortium under control of a synthetic pheromone‐mediated communication system

Microbial consortia can be used to catalyze complex biotransformations. Tools to control the behavior of these consortia in a technical environment are currently lacking. In the present study, a synthetic biology approach was used to build a model consortium of two Saccharomyces cerevisiae strains where growth and expression of the fluorescent marker protein EGFP by the receiver strain is controlled by the concentration of α‐factor pheromone, which is produced by the emitter strain. We have developed a quantitative experimental and theoretical framework to describe population dynamics in the model consortium. We measured biomass growth and metabolite production in controlled bioreactor experiments, and used flow cytometry to monitor changes of the subpopulations and protein expression under different cultivation conditions. This dataset was used to parameterize a segregated mathematical model, which took into account fundamental growth processes, pheromone‐induced growth arrest and EGFP production, as well as pheromone desensitization after extended exposure. The model was able to predict the growth dynamics of single‐strain cultures and the consortium quantitatively and provides a basis for using this approach in actual biotransformations.     

Helicobacter pylori dampens gut epithelial self-renewal by inhibiting apoptosis, a bacterial strategy to enhance colonization of the stomach

Colonization of the gastric pits in the stomach by Helicobacter pylori (Hp) is a major risk factor for gastritis, gastric ulcers, and cancer. Normally, rapid self-renewal of gut epithelia, which occurs by a balance of progenitor proliferation and pit cell apoptosis, serves as a host defense mechanism to limit bacterial colonization. To investigate how Hp overcomes this host defense, we use the Mongolian gerbil model of Hp infection. Apoptotic loss of pit cells induced by a proapoptotic agent is suppressed by Hp. The ability of Hp to suppress apoptosis contributed to pit hyperplasia and persistent bacterial colonization of the stomach. Infection with WT Hp but not with a mutant in the virulence effector cagA increased levels of the prosurvival factor phospho-ERK and antiapoptotic protein MCL1 in the gastric pits. Thus, CagA activates host cell survival and antiapoptotic pathways to overcome self-renewal of the gastric epithelium and help sustain Hp infection.     

Assessing enzyme activities using stable isotope labeling and mass spectrometry

Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.