Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms.

Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly's bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites' development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.

Non-destructive classification of unlabeled cells: Combining an automated benchtop magnetic resonance scanner and artificial intelligence.

In order to treat degenerative diseases, the importance of advanced therapy medicinal products has increased in recent years. The newly developed treatment strategies require a rethinking of the appropriate analytical methods. Current standards are missing the complete and sterile analysis of the product of interest to make the drug manufacturing effort worthwhile. They only consider partial areas of the sample or product while also irreversibly damaging the investigated specimen. Two-dimensional T1 / T2 MR relaxometry meets these requirements and is therefore a promising in-process control during the manufacturing and classification process of cell-based treatments. In this study a tabletop MR scanner was used to perform two-dimensional MR relaxometry. Throughput was increased by developing an automation platform based on a low-cost robotic arm, resulting in the acquisition of a large dataset of cell-based measurements. Two-dimensional inverse Laplace transformation was used for post-processing, followed by data classification performed with support vector machines (SVM) as well as optimized artificial neural networks (ANN). The trained networks were able to distinguish non-differentiated from differentiated MSCs with a prediction accuracy of 85%. To increase versatility, an ANN was trained on 354 independent, biological replicates distributed across ten different cell lines, resulting in a prediction accuracy of up to 98% depending on data composition. The present study provides a proof of principle for the application of T1 / T2 relaxometry as a non-destructive cell classification method. It does not require labeling of cells and can perform whole mount analysis of each sample. Since all measurements can be performed under sterile conditions, it can be used as an in-process control for cellular differentiation. This distinguishes it from other characterization techniques, as most are destructive or require some type of cell labeling. These advantages highlight the technique's potential for preclinical screening of patient-specific cell-based transplants and drugs.

New aziridine-based inhibitors of cathepsin L-like cysteine proteases with selectivity for the Leishmania cysteine protease LmCPB2.8.

In the present work a series of aziridine-2,3-dicarboxylate inhibitors of papain-like cysteine proteases was designed, synthesized and tested. The compounds displayed selectivity for the parasitic protozoon Leishmania mexicana cathepsin L-like cysteine protease LmCPB2.8. The computational methods of homology modelling and molecular docking predicted some significant differences in the S2 pocket of LmCPB2.8 and cruzain, a related enzyme from Trypanosoma cruzi. Due to the presence of Tyr209 in LmCPB2.8 rather than Glu208 in cruzain sterically demanding, lipophilic ester groups (inhibitor 7d, 9d, 12d and 14d) are predicted to occupy the S2 pocket of the Leishmania protease, but do not form favorable interactions in cruzain, which is in common with our experimental results. Further, inhibitor 18 bearing a free carboxylic acid attached to the aziridine moiety showed a time-dependent inhibition of LmCPB2.8 (Ki = 0.41 µM; k2nd = 190,569 M-1 min-1). Docking results suggested a strong ionic interaction with the positively charged His163 of the active site. Biological and theoretical data confirm that the novel selective aziridine-based inhibitors are promising candidates for further optimization as LmCPB2.8 inhibitors.