The Interleukin-17 Gene of Herpesvirus Saimiri

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.     

A 4-Year Study of a Natural Tick-Borne Encephalitis Virus Focus in Hungary, 2010–2013

EcoHealth - A tick-borne encephalitis virus focus was identified in a former goat pasture that had been associated with a milk-borne encephalitis outbreak in 2007. Ticks and rodents were sampled...     

A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an in vivo high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.     

Sampling of intracellular metabolites for stationary and non-stationary C metabolic flux analysis in Escherichia coli

The analysis of metabolic intermediates is a rich source of isotopic information for ¹³C metabolic flux analysis (¹³C-MFA) and extends the range of its applications. The sampling of labeled metabolic intermediates is particularly important to obtain reliable isotopic information. The assessment of the different sampling procedures commonly used to generate such data, therefore, is crucial. In this work, we thoroughly evaluated several sampling procedures for stationary and non-stationary ¹³C-MFA using Escherichia coli. We first analyzed the efficiency of these procedures for quenching metabolism and found that procedures based on cold or boiling solvents are reliable, in contrast to fast filtration, which is not. We also showed that separating the cells from the broth is not necessary in isotopic stationary state conditions. On the other hand, we demonstrated that the presence of metabolic intermediates outside the cells strongly affects the transient isotopic data monitored during non-stationary ¹³C-labeling experiments. Meaningful isotopic data can be obtained by recovering intracellular labeled metabolites from pellets of cells centrifuged in cold solvent. We showed that if the intracellular pools are not separated from the extracellular ones, accurate flux maps can be established provided that the contribution of exogenous compounds is taken into account in the metabolic flux model.     

Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His²⁸⁴ in the active site replaced by Asn, Asp or Tyr showed up to 10⁴-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His²⁸⁴ in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.     

Population Genetic Consequences of the Allee Effect and the Role of Offspring-Number Variation

A strong demographic Allee effect in which the expected population growth rate is negative below a certain critical population size can cause high extinction probabilities in small introduced populations. But many species are repeatedly introduced to the same location and eventually one population may overcome the Allee effect by chance. With the help of stochastic models, we investigate how much genetic diversity such successful populations harbor on average and how this depends on offspring-number variation, an important source of stochastic variability in population size. We find that with increasing variability, the Allee effect increasingly promotes genetic diversity in successful populations. Successful Allee-effect populations with highly variable population dynamics escape rapidly from the region of small population sizes and do not linger around the critical population size. Therefore, they are exposed to relatively little genetic drift. It is also conceivable, however, that an Allee effect itself leads to an increase in offspring-number variation. In this case, successful populations with an Allee effect can exhibit less genetic diversity despite growing faster at small population sizes. Unlike in many classical population genetics models, the role of offspring-number variation for the population genetic consequences of the Allee effect cannot be accounted for by an effective-population-size correction. Thus, our results highlight the importance of detailed biological knowledge, in this case on the probability distribution of family sizes, when predicting the evolutionary potential of newly founded populations or when using genetic data to reconstruct their demographic history.     

Hydroxylamine as a thermal destabiliser of bacteriorhodopsin

The light-catalysed reaction of hydroxylamine (HA) with retinal is one of the basic features of bacteriorhodopsin (BR). Surprisingly, according to recent results, neither the photocycle and proton pumping of BR, nor the trans–cis isomerisation of retinal is prerequisite for photobleaching of BR in the presence of HA. How, then, is the accessibility of retinal to HA enhanced on illumination? We studied whether local thermal denaturation of BR, proposed recently, could provide an explanation for HA-promoted bleaching. According to our results, HA does not alter the absorption spectrum and the photocycle kinetics of BR substantially at room temperature, even at molar concentrations, but grossly affects the temperature of thermal denaturation. At pH 7, the presence of 0.5 M HA reduces the denaturation temperature from 100°C to as low as 72°C. The decrease is proportional to the logarithm of the HA concentration over more than three orders of magnitude, and even 0.5 mM HA has a significant effect. In addition, photobleaching becomes considerably faster with increasing temperature in the presence of HA, it takes a few seconds at 50–60°C. Our results suggest that photobleaching of BR in the presence of HA can be explained by overall destabilisation of the structure of the protein and local thermal denaturation that has already accounted for the photobleaching of the HA-free BR at elevated temperatures. These results further support the importance of thermooptic effects in protein photoreactions and identify HA as a thermal destabiliser of BR.