Quantitative cell release from plant tissues for single-cell genomics

ABSTRACTSingle-cell RNA-sequencing (scRNA-seq) can provide invaluable insight into cell development, cell type identification, and plant evolution. However, the resilience of the cell wall makes it difficult to dissociate plant tissues and release individual cells. Here, we show that plant tissues can be rapidly and quantitatively dissociated if the tissues are fixed prior to enzymatic digestion. Fixation enables digestion at high temperatures at which enzymatic activity is optimal and stabilizes the plant cell cytoplasm, rendering cells resistant to mechanical shear force. This protocol was applied to maize anthers and provided suitable single-cell expression data for the identification of the tapetum, endothecium, meiocytes, and epidermis, while providing putative marker genes and gene ontology information for the identification of unknown cell types. This approach also preserves morphology of the isolated cells, permitting many cell types to be identified without staining. Our fixation-based protocol can be applied to a range of plant species and tissues with minimal optimization.

Effects of 2′-Fucosyllactose-Based Encapsulation on Probiotic Properties in Streptococcus thermophilus

Streptococcus thermophilus is widely used in dairy fermentation as a starter culture for yogurt and cheese production. Many strains are endowed with potential probiotic properties; however, since they might not survive in adequate amounts after transit through the human gastrointestinal tract, it is advisable to improve cell survivability during this passage. The present study evaluates the use of 2′-fucosyllactose, a prebiotic molecule from human milk, compared with other known molecules, such as gelatin and inulin, to form alginate-based microcapsules to fulfill these requirements. Such microcapsules, obtained by the extrusion technique, were evaluated in terms of encapsulation efficiency, storage stability, gastrointestinal condition resistance, and cell release kinetics. Results reveal that microcapsules made using 2′-fucosyllactose and those with inulin resulted in the most efficient structure to protect S. thermophilus strain TH982 under simulated gastrointestinal conditions (less than 0.45 log CFU/g decrease for both agents). In addition, a prompt and abundant release of encapsulated cells was detected after only 30 min from microcapsules made with sodium alginate plus 2′-fucosyllactose in simulated gastrointestinal fluid (more than 90% of the cells). These encouraging results represent the first report on the effects of 2′-fucosyllactose used as a co-encapsulating agent.

Pollen-Pistil Interactions as Reproductive Barriers

Pollen-pistil interactions serve as important prezygotic reproductive barriers that play a critical role in mate selection in plants. Here, we highlight recent progress toward understanding the molecular basis of pollen-pistil interactions as reproductive isolating barriers. These barriers can be active systems of pollen rejection, or they can result from a mismatch of required male and female factors. In some cases, the barriers are mechanistically linked to self-incompatibility systems, while others represent completely independent processes. Pollen-pistil reproductive barriers can act as soon as pollen is deposited on a stigma, where penetration of heterospecific pollen tubes is blocked by the stigma papillae. As pollen tubes extend, the female transmitting tissue can selectively limit growth by producing cell wall–modifying enzymes and cytotoxins that interact with the growing pollen tube. At ovules, differential pollen tube attraction and inhibition of sperm cell release can act as barriers to heterospecific pollen tubes.

Factors of VEGF, IGF, and TGF-β1 families in omentum tissues to mark premetastatic niches in ovarian cancer.

e17535 Background: Analysis of the nature of cancer cell release and interaction with the microenvironment determines the fundamental basis of ovarian cancer pathogenesis, which is necessary for the development of new methods for peritoneal dissemination treatment and prevention. The purpose of the study was to analyze levels of VEGF-A, VEGFR1, VEGF-C, VEGFR3, TGF-β, IGF-1, IGF-2 and IGFBP3 in omental tissues (O) with and without metastasis (Mts) in ovarian cancer patients. Methods: Samples of O tissues were obtained from 51 ovarian cancer patients aged 60±1.9 years (G2-G3 serous cystadenocarcinoma – AC, Т2-3NxM0-1). 17 non-cancer patients of similar age were controls (C). Levels of VEGF-A, VEGFR1, VEGF-C, VEGFR3, IGF-1, IGF-2, IGFBP3 and TGF-β1 were measured in Mts and O tissues by standard ELISA test systems. Results: VEGF-A levels in O of AC patients were 2.6 times higher than in C. In Mts, VEGF-A and VEGFR1 exceeded the levels in C (by 6.3 and 3.1 times) and O (by 2.5 and 2.8 times, respectively). VEGF-C in O was lower than in C by 5.5 times, while VEGFR3 was 2.2 times higher. In Mts, VEGF-C and VEGFR3 exceeded the levels in C (by 7.8 and 3.6 times) and O (by 43.3 and 1.6 times, respectively, p<0.05). Levels of IGF-2 and IGFBP3 in O were similar to the levels in C, while IGF-1 was twice higher. In Mts, IGF-1, IGF-2 and IGFBP3 exceeded the levels in C (by 4.9, 2.8 and 3.4 times) and O (by 2.5, 2.2 and 3.6 times, respectively). Levels of TGF-β1 were higher in O by 2.9 times and in Mts – by 2.3 times. Conclusions: The interaction between VEGF-A, IGF-1, and TGF-β can serve as a regulator of the metabolic state of the “soil” for tumor dissemination, marking the premetastatic niche and providing mesenchymal-epithelial transition of circulating tumor cells.

Vibrio cholerae biofilm dispersal regulator causes cell release from matrix through type IV pilus retraction

The extracellular matrix is a defining feature of bacterial biofilms and provides structural stability to the community by binding cells to the surface and to each other. Transitions between bacterial biofilm initiation, growth, and dispersion require different regulatory programs, all of which result in modifications to the extracellular matrix composition, abundance, or functionality. However, the mechanisms by which individual cells in biofilms disengage from the matrix to enable their departure during biofilm dispersal are unclear. Here, we investigated active biofilm dispersal of Vibrio cholerae during nutrient starvation, resulting in the discovery of the conserved Vibrio biofilm dispersal regulator VbdR. We show that VbdR triggers biofilm dispersal by controlling cellular release from the biofilm matrix, which is achieved by inducing the retraction of the mannose-sensitive hemagglutinin (MSHA) type IV pili and the expression of a matrix protease IvaP. We further show that MSHA pili have numerous binding partners in the matrix and that the joint effect of MSHA pilus retraction and IvaP activity is necessary and sufficient for causing biofilm dispersal. These results highlight the crucial role of type IV pilus dynamics during biofilm dispersal and provide a new target for controlling V. cholerae biofilm abundance through the induction and manipulation of biofilm dispersal.

Chromochloris zofingiensis (Chlorophyceae) Divides by Consecutive Multiple Fission Cell-Cycle under Batch and Continuous Cultivation

Several green algae can divide by multiple fission and spontaneously synchronize their cell cycle with the available light regime. The yields that can be obtained from a microalgal culture are directly affected by cell cycle events. Chromochloris zofingiensis is considered as one of the most promising microalgae for biotechnological applications due to its fast growth and the flexible trophic capabilities. It is intensively investigated in the context of bio-commodities production (carotenoids, storage lipids); however, the pattern of cell-cycle events under common cultivation strategies was not yet characterized for C. zofingiensis. In this study, we have employed fluorescence microscopy to characterize the basic cell-cycle dynamics under batch and continuous modes of phototrophic C. zofingiensis cultivation. Staining with SYBR green—applied in DMSO solution—enabled, for the first time, the clear and simple visualization of polynuclear stages in this microalga. Accordingly, we concluded that C. zofingiensis divides by a consecutive pattern of multiple fission, whereby it spontaneously synchronizes growth and cell division according to the available illumination regime. In high-light continuous culture or low-light batch culture, C. zofingiensis cell-cycle was completed within several light-dark (L/D) cycles (14 h/10 h); however, cell divisions were synchronized with the dark periods only in the high-light continuous culture. In both modes of cultivation, daughter cell release was mainly facilitated by division of 8 and 16-polynuclear cells. The results of this study are of both fundamental and applied science significance and are also important for the development of an efficient nuclear transformation system for C. zofingiensis.