Both humans and animals seek primary rewards in the environment, even when such rewards do not correspond to current physiological needs. An example of this is a dissociation between food-seeking behaviour and metabolic needs, a notoriously difficult-to-treat symptom of eating disorders. Feeding relies on distinct cell groups in the hypothalamus, the activity of which also changes in anticipation of feeding onset. The hypothalamus receives strong descending inputs from the lateral septum, which is connected, in turn, with cortical networks, but cognitive regulation of feeding-related behaviours is not yet understood. Cortical cognitive processing involves gamma oscillations, which support memory, attention, cognitive flexibility and sensory responses. These functions contribute crucially to feeding behaviour by unknown neural mechanisms. Here we show that coordinated gamma (30-90 Hz) oscillations in the lateral hypothalamus and upstream brain regions organize food-seeking behaviour in mice. Gamma-rhythmic input to the lateral hypothalamus from somatostatin-positive lateral septum cells evokes food approach without affecting food intake. Inhibitory inputs from the lateral septum enable separate signalling by lateral hypothalamus neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. Upstream, medial prefrontal cortical projections provide gamma-rhythmic inputs to the lateral septum; these inputs are causally associated with improved performance in a food-rewarded learning task. Overall, our work identifies a top-down pathway that uses gamma synchronization to guide the activity of subcortical networks and to regulate feeding behaviour by dynamic reorganization of functional cell groups in the hypothalamus.

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. Key points • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins.

The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural, structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-free protein synthesis in a fast and efficient manner. The inhibitory effect of the non-structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for the validation of available antibodies. The cytotoxic envelope protein was characterized in electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates the potential of eukaryotic cell-free systems as a rapid response mechanism for the synthesis, functional characterization and antibody validation against a viral pathogen.

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA’s active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin’s mechanism of action.

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.