Visualizing regulatory interactions in metabolic networks

Background  

Direct visualization of data sets in the context of biochemical network drawings is one of the most appealing approaches in the field of data evaluation within systems biology. One important type of information that is very helpful in interpreting and understanding metabolic networks has been overlooked so far. Here we focus on the representation of this type of information given by the strength of regulatory interactions between metabolite pools and reaction steps.     

Usefulness of multi-parameter opiates-amphetamines-cocainics analysis in hair of drug users for the evaluation of an abuse profile by means of LC-APCI-MS-MS

The report presents a segmental hair analysis for the retrospective multi-parameter evaluation of drugs of abuse including opioids, cocainics and amphetamines. The analysis was carried out with the use of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS). The authors have evaluated the differences in the contents of particular opiates in the hair as related to the origin of a sample taken from Polish drug users taking "Polish heroin", and also from heroin abusers from Western European countries taking "Western heroin". The results indicate distinct differences in the 6-MAM concentration values in the Polish and foreigners, suggesting that the foreigners take products containing high concentrations of heroin and the Polish take the poppy product "compote" characterized by its variable and low heroin content. An additional argument for a different abuse profile in the Polish and Western drug users is found in the presence of cocaine detected in hair samples originating from the latter, while cocaine is much less frequently detected in Polish drug users.     

SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo

The detection and analysis of protein-protein interactions is one of the central tasks of proteomics in the postgenomic era. For this purpose, we present a procedure, the Strep-protein interaction experiment (SPINE) that combines the advantages of the Strep-tag protein purification system with those of reversible in vivo protein crosslinking by formaldehyde. Using two Bacillus subtilis regulator proteins, we demonstrate that this method is well suited to isolate protein complexes with high purity and virtually no background. Plasmids allowing the high-level expression of proteins carrying an N- or C-terminal Strep-tag in B. subtilis were constructed.     

The redox-switch domain of Hsp33 functions as dual stress sensor

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.     

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3^?/?) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1β (IL-1β), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3^?/? mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.     

Algae sediment dynamics are mediated by herbivorous fishes on a nearshore coral reef

Coral Reefs - Epilithic algae are a ubiquitous component of coral reefs. Components of the epilithic algal matrix (EAM) can have a significant influence on coral settlement and benthic feeding by...     

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.Subject terms: Environmental microbiology, Sequencing, Microbiology