Dipolarophile-Controlled Regioselective 1,3-Dipolar Cycloaddition: A Switchable Divergent Access to Functionalized N-Fused Pyrrolidinyl Spirooxindoles.

N-fused pyrrolidinyl spirooxindole belongs to a class of privileged heterocyclic scaffolds and is prevalent in natural alkaloids and synthetic pharmaceutical molecules. To realize the switchable synthesis of divergent N-fused pyrrolidinyl spirooxindoles for further biological activity evaluation via a substrate-controlled strategy, a chemically sustainable, catalysis-free, and dipolarophile-controlled three-component 1,3-dipolar cycloaddition of isatin-derived azomethine ylides with diverse dipolarophiles is described in this work. A total of 40 functionalized N-fused pyrrolidinyl spirooxindoles were synthesized in 76-95% yields with excellent diastereoselectivities (up to >99:1 dr). The scaffolds of these products can be well-controlled by employing different 1,4-enedione derivatives as dipolarophiles in EtOH at room temperature. This study provides an efficient strategy to afford a spectrum of natural-like and potentially bioactive N-fused pyrrolidinyl spirooxindoles.

Novel organization of mitochondrial minicircles and guide RNAs in the zoonotic pathogen Trypanosoma lewisi.

Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.

The effect of normal human serum on the mouse trypanosome Trypanosoma musculi in vitro and in vivo.

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi. Both species belong to the subgenus Herpetosoma, and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro, and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei, but more sensitive than T. lewisi. This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi, our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients.

Investigation of blueberry juice fermentation by mixed probiotic strains: Regression modeling, machine learning optimization and comparison with fermentation by single strain in the phenolic and volatile profiles.

Three strains, including L. fermentum, L. plantarum and S. thermophilus, were combined to ferment blueberry juice. Through the sequential simplex lattice mixture design, regression modeling and genetic algorithm optimization, it was found that the combination of S. thermophilus with either L. fermentum or L. plantarum weakened the capacity of Lactobacillus strains to enrich phenolics, and the combinations of these strains had no synergistic effect of synthesizing lactic acid. The resulting optimal inoculation proportion to enrich phenolics was the mixed L. fermentum and L. plantarum at 0.5:0.5. After fermentation for 48 h, total phenolic, ferulic acid, rutin, and quercetin-3-rhamnoside of mixed fermented samples were 82.19 %, 15.22 %, 79.08 % and 98.59 % higher than the unfermented juice, and their contents were all highest among the fermented samples. Moreover, the samples fermented by mixed strains possessed higher amounts of 3-methyl-1-butanol, 2-methyl-1-propanol, 2-heptanone and 2-pentanone than samples fermented by L. fermentum, S. thermophilus and unfermented samples.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice.

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Fermentation of blueberry juices using autochthonous lactic acid bacteria isolated from fruit environment: Fermentation characteristics and evolution of phenolic profiles.

In this study, 4 Lactobacillus plantarum strains and 5 Lactobacillus fermentum strains adapting well to the unfavorable fruit system were isolated under different fruit environments. The fermentation ability of these autochthonous lactic acid bacteria (LAB) strains in blueberry juice, and the influence of microbial metabolism on juice composition were explored. After 48 h of fermentation, the viable cell counts exceeded 10.0 log CFU/mL, malic acid content decreased from 511.47 ± 10.50 mg/L to below 146.38 ± 3.79 mg/L, and lactic acid content increased from 0 mg/L to above 2184.90 ± 335.80 mg/L. Moreover, the metabolism of these strains exerted a profound influence on the phenolic composition of juice. Total phenolic content in blueberry juice increased by 6.1-81.2% under lactic acid fermentation, and the antioxidant capacity in vitro enhanced by at least 34.0%. Anthocyanin content showed a declining trend, while the profile of non-anthocyaninic phenolics exhibited complex changes. The increments of rutin, myricetin and gallic acid contents through 48 h lactic acid fermentation exceeded 136%, 71% and 38%, respectively. Instead, the contents of p-hydroxybenzoic acid and caffeic acid decreased with fermentation. Overall, Lactobacillus plantarum LSJ-TY-HYB-T9 and LSJ-TY-HYB-T7, and Lactobacillus fermentum LSJ-TY-HYB-C22 and LSJ-TY-HYB-L16 could be the suitable strains to produce fermented fruit juices, including blueberry in practical applications.

Fermentation of blueberry and blackberry juices using Lactobacillus plantarum, Streptococcus thermophilus and Bifidobacterium bifidum: Growth of probiotics, metabolism of phenolics, antioxidant capacity in vitro and sensory evaluation.

In this study, three potential probiotic strains were selected to ferment blueberry and blackberry juices. The viable cell counts of selected strains were increased by 0.4-0.7 log CFU/mL in berry juices environments after 48-h fermentation. Meanwhile, the contents of cyanindin-3-glucoside and peonidin-3-glucoside decreased over 30%. Heatmap presented an upgrade trend of syringic acid, ferulic acid, gallic acid and lactic acid during fermentation. However, the contents of p-coumaric acid, protocatechuic acid, chlorogenic acid, critic acid and malic acid showed downgrade trend. The metabolism of phenolics probably contributed to the enhancement of the ABTS radical scavenging activity (40%-60%) in fermented berry juices. Moreover, the three strains presented different capacities on changing the quality of berry juices according to the PCA and LDA analysis. The contents of individual organic acids had positive correlations with sensory quality, especially for sourness. Overall, probiotic fermentation could improve the sensory quality of berry juices.

Cell cycle and cleavage events during in vitro cultivation of bloodstream forms of Trypanosoma lewisi, a zoonotic pathogen.

Trypanosoma (Herpetosoma) lewisi is a globally distributed rat trypanosome, currently considered as a zoonotic pathogen; however, a detailed understanding of the morphological events occurring during the cell cycle is lacking. This study aimed to investigate the cell cycle morphology and cleavage events of Trypanosoma lewisi (T. lewisi) during in vitro cultivation. By establishing in vitro cultivation of T. lewisi at 37°C, various cell morphologies and stages could be observed. We have provided a quantitative analysis of the morphological events during T. lewisi proliferation. We confirmed a generation time of 12.14 ± 0.79 hours, which is similar to that in vivo (12.21 ± 0.14 hours). We also found that there are two distinct cell cycles, with a two-way transformation connection in the developmental status of this parasite, which was contrasted with the previous model of multiple division patterns seen in T. lewisi. We quantified the timing of cell cycle phases (G1n, 0.56 U; Sn, 0.14 U; G2n, 0.16 U; M, 0.06 U; C, 0.08 U; G1k, 0.65 U; Sk, 0.10 U; G2k, 0.17 U; D, 0.03 U; A, 0.05 U) and their morphological characteristics, particularly with respect to the position of kinetoplast(s) and nucleus/nuclei. Interestingly, we found that both nuclear synthesis initiation and segregation in T. lewisi occurred prior to kinetoplast, different to the order of replication found in Trypanosoma brucei and Trypanosoma cruzi, implicating a distinct cell cycle control mechanism in T. lewisi. We characterized the morphological events during the T. lewisi cell cycle and presented evidence to support the existence of two distinct cell cycles with two-way transformation between them. These results provide insights into the differentiation and evolution of this parasite and its related species.