Zoonotic and pandemic influenza A viruses: lessons from H5N1 and H2N2

Influenza A viruses can be directly transmitted from animals to humans (zoonotic viruses), cause worldwide outbreaks (pandemic viruses) or infect people annually (seasonal viruses). Influenza A viruses that are transmitted efficiently from one person to another have in common that they can spread via the air. We have studied zoonotic H5N1 and pandemic H2N2 influenza viruses with respect to their evolution over time, their potential to transmit via the air, and the virus properties that are required to be efficiently transmitted via the air. From 1997 onwards, H5N1 influenza viruses have occasionally infected humans resulting in severe illness and several deaths. To investigate if this new zoonotic virus had the capacity to spread via the air, we adapted this virus to ferrets, an animal model often used to study airborne transmission of influenza A viruses. We have shown that as little as five mutations in the genetic material of the virus, changing three virus properties, are sufficient for the virus to transmit via the air. Since influenza A viruses evolve constantly, our immune system cannot protect us from infections with new influenza viruses that might emerge in the future. H2N2 influenza virus has caused a pandemic in 1957 and caused seasonal outbreaks in humans until 1968. We have shown that the pandemic potential of H2H2 influenza viruses circulating in birds is low. Furthermore, we have investigated the genetic diversity of H2N2 influenza viruses over time and have determined the mutations that had an effect on recognition by the immune system

Relative Protein Lifetime Measurement in Plants Using Tandem Fluorescent Protein Timers

Targeted protein degradation plays a wide range of important roles in plant growth and development, but analyzing protein turnover in vivo is technically challenging. Until recently, there has been no straightforward methodology for quantifying protein dynamics at subcellular resolution during cellular transitions in plants. A tandem fluorescent protein timer (tFT) is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which allows estimation of relative protein age from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we describe approaches to use this technology to report relative protein lifetime in both transient and stable plant transformation systems. tFTs enable in vivo, real-time protein lifetime assessment within subcellular compartments and across tissues, permitting the analysis of protein degradation dynamics in response to stresses or developmental cues and in different genetic backgrounds

Competition-based model of pheromone component ratio detection in the moth

For some moth species, especially those closely interrelated and sympatric, recognizing a specific pheromone component concentration ratio is essential for males to successfully locate conspecific females. We propose and determine the properties of a minimalist competition-based feed-forward neuronal model capable of detecting a certain ratio of pheromone components independently of overall concentration. This model represents an elementary recognition unit for the ratio of binary mixtures which we propose is entirely contained in the macroglomerular complex (MGC) of the male moth. A set of such units, along with projection neurons (PNs), can provide the input to higher brain centres. We found that (1) accuracy is mainly achieved by maintaining a certain ratio of connection strengths between olfactory receptor neurons (ORN) and local neurons (LN), much less by properties of the interconnections between the competing LNs proper. An exception to this rule is that it is beneficial if connections between generalist LNs (i.e. excited by either pheromone component) and specialist LNs (i.e. excited by one component only) have the same strength as the reciprocal specialist to generalist connections. (2) successful ratio recognition is achieved using latency-to-first-spike in the LN populations which, in contrast to expectations with a population rate code, leads to a broadening of responses for higher overall concentrations consistent with experimental observations. (3) when longer durations of the competition between LNs were observed it did not lead to higher recognition accuracy

Internal Cholinergic Regulation of Learning and Recall in a Model of Olfactory Processing

In the olfactory system, cholinergic modulation has been associated with contrast modulation and changes in receptive fields in the olfactory bulb, as well the learning of odor associations in olfactory cortex. Computational modeling and behavioral studies suggest that cholinergic modulation could improve sensory processing and learning while preventing pro-active interference when task demands are high. However, how sensory inputs and/or learning regulate incoming modulation has not yet been elucidated. We here use a computational model of the olfactory bulb, piriform cortex (PC) and horizontal limb of the diagonal band of Broca (HDB) to explore how olfactory learning could regulate cholinergic inputs to the system in a closed feedback loop. In our model, the novelty of an odor is reflected in firing rates and sparseness of cortical neurons in response to that odor and these firing rates can directly regulate learning in the system by modifying cholinergic inputs to the system. In the model, cholinergic neurons reduce their firing in response to familiar odors—reducing plasticity in the PC, but increase their firing in response to novel odor—increasing PC plasticity. Recordings from HDB neurons in awake behaving rats reflect predictions from the model by showing that a subset of neurons decrease their firing as an odor becomes familiar

Cotranslational N-degron masking by acetylation promotes proteome stability in plants

N-terminal protein acetylation (NTA) is a prevalent protein modification essential for viability in animals and plants. The dominant executor of NTA is the ribosome tethered N-alpha-acetyltransferase A (NatA) complex. However, the impact of NatA on protein fate is still enigmatic. Here, we demonstrate that depletion of NatA activity leads to a 4-fold increase in global protein turnover via the ubiquitin-proteasome system in Arabidopsis. Surprisingly, a concomitant increase in translation, actioned via enhanced Target-of-Rapamycin activity, is also observed, implying that defective NTA triggers feedback mechanisms to maintain steady-state protein abundance. Quantitative analysis of the proteome, the translatome, and the ubiquitome reveals that NatA substrates account for the bulk of this enhanced turnover. A targeted analysis of NatA substrate stability uncovers that NTA absence triggers protein destabilization via a previously undescribed and widely conserved nonAc/N-degron in plants. Hence, the imprinting of the proteome with acetylation marks is essential for coordinating proteome stability. N-terminal protein acetylation is required for plant viability. Here the authors show that reducing N-terminal acetylation by NatA leads to an increase in global protein turnover that is facilitated by absent masking of a novel N-degro

The Molecular Basis for Antigenic Drift of Human A/H2N2 Influenza Viruses.

Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated between 1957 and 1968 was characterized. The antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin (HA) molecule, were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the 12-year period after introduction in humans was half that of A/H3N2 viruses, despite similar rates of genetic change.IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity, and the evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition (HI) titers of cell-cultured virus isolates and ferret postinfection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity, confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alphaanomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate

Tandem fluorescent protein timers for non-invasive relative protein lifetime measurement in plants

Targeted protein degradation is an important and pervasive regulatory mechanism in plants, required for perception and response to the environment as well as developmental signalling. Despite the significance of this process, relatively few studies have assessed plant protein turnover in a quantitative fashion. Tandem fluorescent protein timers (tFTs) offer a powerful approach for the assessment of in vivo protein turnover in distinct subcellular compartments of single or multiple cells. A tFT is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which enable protein age to be estimated from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we use short-lived auxin signalling proteins and model N-end rule pathway reporters to demonstrate the utility of tFTs for studying protein turnover in living plants. We present transient expression of tFTs as an efficient screen for relative protein lifetime, useful for testing the effects of mutations and different genetic backgrounds on protein stability, and demonstrate the potential for using stably expressed tFTs to study native protein dynamics with high temporal resolution in response to exogenous or endogenous stimuli

Identifying metabolites by integrating metabolome databases with mass spectrometry cheminformatics.

Novel metabolites distinct from canonical pathways can be identified through the integration of three cheminformatics tools: BinVestigate, which queries the BinBase gas chromatography-mass spectrometry (GC-MS) metabolome database to match unknowns with biological metadata across over 110,000 samples; MS-DIAL 2.0, a software tool for chromatographic deconvolution of high-resolution GC-MS or liquid chromatography-mass spectrometry (LC-MS); and MS-FINDER 2.0, a structure-elucidation program that uses a combination of 14 metabolome databases in addition to an enzyme promiscuity library. We showcase our workflow by annotating N-methyl-uridine monophosphate (UMP), lysomonogalactosyl-monopalmitin, N-methylalanine, and two propofol derivatives