Small-molecule biosensors for high-throughput metabolic engineering

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. In this presentation we outline a versatile and high-throughput method to evolve and functionalize prokaryotic aTF ligand specificity and transfer functions in a eukaryote chassis, namely baker’s yeast Saccharomyces cerevisiae. From a single round of directed evolution of the aTF ligand-binding domain coupled with various toggled selection regimes, we robustly select aTF variants evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion of function from activation to repression. Importantly, by targeting only the ligand-binding domain, the evolved biosensors display DNA-binding affinities similar to parental aTFs and are functional when ported back into a non-native prokaryote chassis. The developed platform technology thus leverages aTF evolvability for the development of new biosensors with user-defined small-molecule specificities and transfer functions. Finally, the presentation will highlight examples on biosensor applications for high-throughput metabolic engineering. Please click Additional Files below to see the full abstract

Comprehensive and unbiased multiparameter high-throughput screening by compaRe finds effective and subtle drug responses in AML models

Large-scale multiparameter screening has become increasingly feasible and straightforward to perform thanks to developments in technologies such as high-content microscopy and high-throughput flow cytometry. The automated toolkits for analyzing similarities and differences between large numbers of tested conditions have not kept pace with these technological developments. Thus, effective analysis of multiparameter screening datasets becomes a bottleneck and a limiting factor in unbiased interpretation of results. Here we introduce compaRe, a toolkit for large-scale multiparameter data analysis, which integrates quality control, data bias correction, and data visualization methods with a mass-aware gridding algorithm-based similarity analysis providing a much faster and more robust analyses than existing methods. Using mass and flow cytometry data from acute myeloid leukemia and myelodysplastic syndrome patients, we show that compaRe can reveal interpatient heterogeneity and recognizable phenotypic profiles. By applying compaRe to high-throughput flow cytometry drug response data in AML models, we robustly identified multiple types of both deep and subtle phenotypic response patterns, highlighting how this analysis could be used for therapeutic discoveries. In conclusion, compaRe is a toolkit that uniquely allows for automated, rapid, and precise comparisons of large-scale multiparameter datasets, including high-throughput screens.Peer reviewe

Efficacy of SARS-CoV-2 Vaccination in Dialysis Patients: Epidemiological Analysis and Evaluation of the Clinical Progress

This study investigated the impact of the fourth COVID-19 pandemic wave on dialysis patients of Romagna territory, assessing the associations of vaccination status with infection risk, clinical severity and mortality. From November 2021 to February 2022, an epidemiological search was conducted on 829 patients under dialysis treatment for at least one month. The data were then analyzed with reference to the general population of the same area. A temporal comparison was also carried out with the previous pandemic waves (from March 2020 to October 2021). The epidemiological evolution over time in the dialysis population and in Romagna citizens replicated the global trend, as the peak of the fourth wave corresponded to the time of maximum diffusion of omicron variant (B.1.1.529). Of 771 prevalent dialysis patients at the beginning of the study, 109 (14.1%) contracted SARS-CoV-2 infection during the 4-month observation period. Vaccine adherence in the dialysis population of the reference area was above 95%. Compared to fully or partially vaccinated subjects, the unvaccinated ones showed a significantly higher proportion of infections (12.5% vs. 27.0% p = 0.0341), a more frequent need for hospitalization (22.2% vs. 50.0%) and a 3.3-fold increased mortality risk. These findings confirm the effectiveness of COVID-19 vaccines in keeping infectious risk under control and ameliorating clinical outcomes in immunocompromised patients

Production of steviol glycosides in recombinant hosts

Properties of nitrogen cluster plasmas produced by an intense, ultrashort laser pulse have been investigated numerically and experimentally. The classical dynamics simulations show that on increasing the cluster size a plasma with residual electron energy above 1 keV can be created due to collisional heating, which is considerably higher than the value obtained with a conventional low-density gas target. Experimentally, nitrogen gas jets created by two types of nozzles were irradiated with a laser pulse of 55 fs, up to 1.2× 1017 / cm2. A seeded gas jet consisting of nitrogen and helium was also employed to promote the production of large clusters. The influences of the shape of nozzle, the seeded gas, and the gas jet stagnation pressure on the properties of plasmas were examined by spectroscopic observations. K -shell emissions showed that for the gas jet using the conical nozzle the electrons underwent intense collisional heating within the large clusters, resulting in the production of highly charged ions. In contrast, the emissions observed with the capillary nozzle exhibited the characteristics of a cold plasma without suffering substantial electron heating, indicating the absence of large clusters. That is, the differences between the two types of nozzles in the efficiency of electron heating and subsequent residual energies after the passage of the laser pulse, which are strongly dependent upon the cluster size, drastically changed the properties of the produced plasmas. The reason that for the capillary gas jet the plasma density deduced from the recombination spectra was significantly higher than the value obtained using the conical nozzle is also given by the difference in residual electron energy

High-Resolution Scanning of Optimal Biosensor Reporter Promoters in Yeast.

Small-molecule binding allosteric transcription factors (aTFs) derived from bacteria enable real-time monitoring of metabolite abundances, high-throughput screening of genetic designs, and dynamic control of metabolism. Yet, engineering of reporter promoter designs of prokaryotic aTF biosensors in eukaryotic cells is complex. Here we investigate the impact of aTF binding site positions at single-nucleotide resolution in >300 reporter promoter designs in Saccharomyces cerevisiae. From this we identify biosensor output landscapes with transient and distinct aTF binding site position effects for aTF repressors and activators, respectively. Next, we present positions for tunable reporter promoter outputs enabling metabolite-responsive designs for a total of four repressor-type and three activator-type aTF biosensors with dynamic output ranges up to 8- and 26-fold, respectively. This study highlights aTF binding site positions in reporter promoters as key for successful biosensor engineering and that repressor-type aTF biosensors allows for more flexibility in terms of choice of binding site positioning compared to activator-type aTF biosensors