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     

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.     

Early kinetics of antibody response to inactivated influenza vaccine

The aim was to examine the rapidity of haemagglutination inhibiting (HI) antibody response induced by immunization with a current inactivated trivalent influenza vaccine. Five to six sequential serum samples collected in autumn 1992 from each of 68 vaccinees in three age groups were studied for HI antibodies to ten influenza virus strains representing vaccine and epidemic viruses. Geometric mean titres, response rates and protection rates are presented. Response rates of > 70% were overall, but not until two weeks after the vaccination. Significant two- and four-day post-vaccination antibody responses were detected only occasionally. In previously vaccinated persons, average antibody titres to some of the viruses decreased during the first days after the vaccination. In the subsequent samples, the titres remained lower than in persons who were not vaccinated against influenza in preceding years. Protection against influenza infection may be frequently developed not until two weeks after vaccination. This has relevance to prophylactic administration of amantadine and rimantadine when an influenza A outbreak is imminent and the vaccination is late.     

Die Chromosomenzahlen in den Leberzellen von Ratten verschiedenen Alters

Ohne ZusammenfassungMit Unterstützung der Deutschen Forschungsgemeinschaft.     

VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells

While caspases play an established role as intracellular executors of apoptosis, little is known about extracellular activities of this ubiquitously expressed family of proteases. We demonstrate here that recombinant caspase-3 retained enzymatic activity in various extracellular fluids. Experiments with cell lines, primary cells, and mice with fulminant CD95-triggered hepatitis showed that significant amounts of DEVD-aminofluoromethylcoumarine-cleaving activity, indicative of active effector caspases, were released into the medium/plasma during apoptosis. Furthermore, caspase activities were detected in liquor samples from human head trauma patients. These findings warrant closer investigation of DEVDase activity as a diagnostic marker, and of potential extracellular substrates for caspases.     

Platelet-associated tissue factor contributes to the collagen-triggered activation of blood coagulation

The extravascular localization of tissue factor (TF), the central initiator of coagulation, is thought to ensure that thrombus formation is prevented in the intact vessel. We observed that during a 5-min stimulation of human blood with collagen (type I), TF antigen appeared on the surface of platelets adhering to leukocytes. The rapidly presented intravascular TF was competent to start the coagulation cascade. The isolated platelets from healthy donors contained appreciable amounts of the TF protein, while no TF antigen was detected in the neutrophils and rapidly isolated monocytes. Direct interactions with the neutrophils and monocytes were apparently necessary to activate the platelet-associated TF. This was most likely mediated by inactivation of tissue factor pathway inhibitor through leukocyte elastase. In summary, the leukocyte-elicited activation of the platelet TF participates in the rapid initiation of coagulation by collagen.