A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway.

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization. In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites. The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.

Resident Kupffer cells and neutrophils drive liver toxicity in cancer immunotherapy.

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of TH1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-γ and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-γ were not toxic themselves but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways after anti-PD-1 and anti-CTLA-4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in TH1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity.

Can tumor cells take it all away?

Tumor cells utilize glucose to engage in aerobic glycolysis, fulfilling their metabolic demands for extensive proliferation. A recent study in Nature discovers that tumor-infiltrating myeloid cells exhibit a superior glucose uptake capacity over tumor cells, which present enhanced glutamine metabolism, suggesting that nutrient partitioning in the TME might be more complex than previously thought.

Tumor-Promoting Ly-6G+ SiglecFhigh Cells Are Mature and Long-Lived Neutrophils.

Myeloid cells co-expressing the markers CD11b, Ly-6G, and SiglecF can be found in large numbers in murine lung adenocarcinomas and accelerate cancer growth by fostering tumor cell invasion, angiogenesis, and immunosuppression; however, some of these cells' fundamental features remain unexplored. Here, we show that tumor-infiltrating CD11b+ Ly-6G+ SiglecFhigh cells are bona fide mature neutrophils and therefore differ from other myeloid cells, including SiglecFhigh eosinophils, SiglecFhigh macrophages, and CD11b+ Ly-6G+ myeloid-derived suppressor cells. We further show that SiglecFhigh neutrophils gradually accumulate in growing tumors, where they can live for several days; this lifespan is in marked contrast to that of their SiglecFlow counterparts and neutrophils in general, which live for several hours only. Together, these findings reveal distinct attributes for tumor-promoting SiglecFhigh neutrophils and help explain their deleterious accumulation in the tumor bed.

Path Following, Obstacle Detection and Obstacle Avoidance for Thrusted Underwater Snake Robots.

The use of unmanned underwater vehicles is steadily increasing for a variety of applications such as mapping, monitoring, inspection and intervention within several research fields and industries, e.g., oceanography, marine biology, military, and oil and gas. Particularly interesting types of unmanned underwater vehicles are bio-inspired robots such as underwater snake robots (USRs). Due to their flexible and slender body, these versatile robots are highly maneuverable and have better access capabilities than more conventional remotely operated vehicles (ROVs). Moreover, the long and slender body allows for energy-efficient transit over long distances similar to torpedo-shaped autonomous underwater vehicles (AUVs). In addition, USRs are capable of performing light intervention tasks, thereby providing intervention capabilities which exceed those of AUVs and inspection class ROVs. USRs may also propel themselves using energy-efficient motion patterns inspired by their biological counterparts. They can thereby increase the propulsion efficiency during transit and maneuvering, which is among the great challenges for autonomous underwater vehicles. In this paper, a control system for path following, and algorithms for obstacle detection and avoidance, are presented for a USR with thrusters attached at the tail module. The position of the obstacles is detected using a single camera in the head module of the USR and a developed computer vision algorithm. For the proposed control concept the robot joints are used for directional control while the thrusters are used for forward propulsion. The USR circumvents obstacles by following a circular path around them before converging back to the main straight line path when this is safe. Experimental results that validate the proposed methods are also presented.

Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor.

The introduction of a three-enzyme cascade (comprising a cyclohexanone monooxygenase (CHMO), an alcohol dehydrogenase (ADH) and a lipase (CAL-A)) for the production of oligo-ε-caprolactone provided self-sufficiency with respect to NADPH-cofactor regeneration and reduced inhibiting effects on the central CHMO enzyme. For further optimization of cofactor regeneration, now a co-expression of CHMO and ADH in E. coli using a Duet™ vector was performed. This led to higher conversion values of the substrate cyclohexanol in whole-cell biocatalysis compared to an expression of both enzymes from two separate plasmids. Furthermore, a more advantageous balance of expression levels between the partial cascade enzymes was achieved via engineering of the ribosome binding site. This contributed to an even faster cofactor regeneration rate.

Planar maneuvering control of underwater snake robots using virtual holonomic constraints.

This paper investigates the problem of planar maneuvering control for bio-inspired underwater snake robots that are exposed to unknown ocean currents. The control objective is to make a neutrally buoyant snake robot which is subject to hydrodynamic forces and ocean currents converge to a desired planar path and traverse the path with a desired velocity. The proposed feedback control strategy enforces virtual constraints which encode biologically inspired gaits on the snake robot configuration. The virtual constraints, parametrized by states of dynamic compensators, are used to regulate the orientation and forward speed of the snake robot. A two-state ocean current observer based on relative velocity sensors is proposed. It enables the robot to follow the path in the presence of unknown constant ocean currents. The efficacy of the proposed control algorithm for several biologically inspired gaits is verified both in simulations for different path geometries and in experiments.