Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize (Zea mays L.).

BACKGROUND: Ubiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize (Zea mays L.). RESULTS: In this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response. CONCLUSIONS: In this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Description of AtCAX4 in Response to Abiotic Stress in Arabidopsis.

High-capacity tonoplast cation/H+ antiport in plants is partially mediated by a family of CAX transporters. Previous studies have reported that CAX activity is affected by an N-terminal autoinhibitory region. CAXs may be present as heterodimers in plant cells, and this phenomenon necessitates further study. In this study, we demonstrate that there is an interaction between CAX4 and CAX1 as determined by the use of a yeast two-hybrid system and a bimolecular fluorescence complementation assay. More specifically, the N-terminal of CAX4 interacts with CAX1. We further observed the over-expression and either a single or double mutant of CAX1 and CAX4 in response to abiotic stress in Arabidopsis. These results suggest that CAX1 and CAX4 can interact to form a heterodimer, and the N-terminal regions of CAX4 play important roles in vivo; this may provide a foundation for a deep study of CAX4 function in the future.

Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche.

Limited numbers of available hematopoietic stem cells (HSCs) limit the widespread use of HSC-based therapies. Expansion systems for functional heterogenous HSCs remain to be optimized. Here, we present a convenient strategy for human HSC expansion based on a biomimetic Microniche. After demonstrating the expansion of HSC from different sources, we find that our Microniche-based system expands the therapeutically attractive megakaryocyte-biased HSC. We demonstrate scalable HSC expansion by applying this strategy in a stirred bioreactor. Moreover, we identify that the functional human megakaryocyte-biased HSCs are enriched in the CD34+CD38-CD45RA-CD90+CD49f lowCD62L-CD133+ subpopulation. Specifically, the expansion of megakaryocyte-biased HSCs is supported by a biomimetic niche-like microenvironment, which generates a suitable cytokine milieu and supplies the appropriate physical scaffolding. Thus, beyond clarifying the existence and immuno-phenotype of human megakaryocyte-biased HSC, our study demonstrates a flexible human HSC expansion strategy that could help realize the strong clinical promise of HSC-based therapies.

Deciphering the Heterogeneity of Mitochondrial Functions During Hematopoietic Lineage Differentiation.

BACKGROUND: The heterogeneity of mitochondrial function is an important feature of hematopoietic cell lineage differentiation, but its stage wise contribution is not adequately studied. To establish a model to compare the lineage differentiation of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, the mitochondrial mass (MM), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitophagy level were analyzed. RESULTS AND DISCUSSION: HSCs had lower mitochondrial metabolic activity than committed progenitor populations, indicated by lower MM, MMP, and ROS and higher mitophagy. HPC1s shared more stem cell characteristics than HPC2s and committed progenitor populations in terms of mitochondrial number and function. The mitochondrial metabolism of mature blood cells had greater heterogeneity than hematopoietic stem and progenitor cells, with granulocytes being similar to monocytes. Moreover, HSCs exhibited heterogeneity in the selection of mitophagy-related PINK1/PARK2, BNIP3/NIX, and FUNDC1 pathways. Myeloid differentiation had greater morphological and functional heterogeneity of hematopoietic cells than lymphoid differentiation. Additionally, leukemia stem cells had higher aerobic metabolism and better stem cell function through elevated mitophagy than normal hematopoietic cells. ROS and MMP levels in differentiated leukemia cells were higher, but the level of mitophagy was lower than in differentiated hematopoietic cells. CONCLUSION: This study provides a complete set of methods and basic reference values for the systematic study of the mitochondrial metabolic function of different types of hematopoietic cells under physiological and pathological conditions. The findings contribute to the future research of tumor and aging based on mitochondrial metabolism.

Elucidating the mitochondrial function of murine lymphocyte subsets and the heterogeneity of the mitophagy pathway inherited from hematopoietic stem cells.

Background: Mitochondria are mainly involved in ATP production to meet the energy demands of cells. Researchers are increasingly recognizing the important role of mitochondria in the differentiation and activation of hematopoietic cells, but research on how mitochondrial metabolism influence different subsets of lymphocyte at different stages of differentiation and activation are yet to be carried out. In this work, the mitochondrial functions of lymphocytes were compared at different differentiation and activation stages and included CD8+ T lymphocytes, CD4+ T lymphocytes, B lymphocytes, NK cells as well as their subsets. For this purpose, a complete set of methods was used to comprehensively analyze mitophagy levels, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the mitochondrial mass (MM) of subsets of lymphocytes. It is expected that this will provide a complete set of standards, and drawing the mitochondrial metabolic map of lymphocyte subsets at different stages of differentiation and activation. Results and discussion: Of all lymphocytes, B cells had a relatively high mitochondrial metabolic activity which was evident from the higher levels of mitophagy, ROS, MMP and MM, and this reflected the highly heterogeneous nature of the mitochondrial metabolism in lymphocytes. Among the B cell subsets, pro-B cells had relatively higher levels of MM and MMP, while the mitochondrial metabolism level of mature B cells was relatively low. Similarly, among the subsets of CD4+ T cell, a relatively higher level of mitochondrial metabolism was noted for naive CD4+ T cells. Finally, from the CD8+ T cell subsets, CD8+ Tcm had relatively high levels of MM and MMP but relatively low ones for mitophagy, with effector T cells displaying the opposite characteristics. Meanwhile, the autophagy-related genes of lymphoid hematopoietic cells including hematopoietic stem cells, hematopoietic progenitor cells and lymphocyte subsets were analyzed, which preliminarily showed that these cells were heterogeneous in the selection of mitophagy related Pink1/Park2, BNIP3/NIX and FUNDC1 pathways. The results showed that compared with CD4+ T, CD8+ T and NK cells, B cells were more similar to long-term hematopoietic stem cell (LT-HSC) and short-term hematopoietic stem cell (ST-HSC) in terms of their participation in the Pink1/Park2 pathway, as well as the degree to which the characteristics of autophagy pathway were inherited from HSC. Compared with CLP and B cells, HSC are less involved in BNIP3/NIX pathway. Among the B cell subsets, pro-B cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with pre-B, immature B and immature B cells. Among CD4+ T cell subsets, nTreg cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with naive CD4+ T and memory CD4+ T cells. Among the CD8+ T cell subsets, compared with CLP and effector CD8+ T cells, CD8+ Tcm inherit the least characteristics of HSC in participating in Pink1/Park2 pathway. Meanwhile, CLP, naive CD4+ T and effector CD8+ T were more involved in BNIP3/NIX pathway than other lymphoid hematopoietic cells. Conclusion: This study is expected to provide a complete set of methods and basic reference values for future studies on the mitochondrial functions of lymphocyte subsets at different stages of differentiation and activation in physiological state, and also provides a standard and reference for the study of infection and immunity based on mitochondrial metabolism.

A mitophagy inhibitor targeting p62 attenuates the leukemia-initiation potential of acute myeloid leukemia cells.

There has been an increasing focus on the tumorigenic potential of leukemia initiating cells (LICs) in acute myeloid leukemia (AML). Despite the important role of selective autophagy in the life-long maintenance of hematopoietic stem cells (HSCs), cancer progression, and chemoresistance, the relationship between LICs and selective autophagy remains to be fully elucidated. Sequestosome 1 (SQSTM1), also known as p62, is a selective autophagy receptor for the degradation of ubiquitinated substrates, and its loss impairs leukemia progression in AML mouse models. In this study, we evaluated the underlying mechanisms of mitophagy in the survival of LICs with XRK3F2, a p62-ZZ inhibitor. We demonstrated that XRK3F2 selectively impaired LICs but spared normal HSCs in both mouse and patient-derived tumor xenograft (PDX) AML models. Mechanistically, we observed that XRK3F2 blocked mitophagy by inhibiting the binding of p62 with defective mitochondria. Our study not only evaluated the effectiveness and safety of XRK3F2 in LICs, but also demonstrated that mitophagy plays an indispensable role in the survival of LICs during AML development and progression, which can be impaired by blocking p62.

A Single-Cell Immunofluorescence Method for the Division Patterns Research of Mouse Bone Marrow-Derived Hematopoietic Stem Cells.

How hematopoietic stem cells (HSCs) maintain the balance of self-renewal and differentiation could be partially ascribed to asymmetric and symmetric division patterns. However, a simple and effective method to detect stem cell division patterns is lacking. In this study, we introduce a strategy to describe stem cells division patterns with high spatial resolution at the single-cell level. We show that the fate determinant, Numb, exhibits low expression levels in HSCs that increase upon the initiation of differentiation. Using this single-cell immunofluorescence technique, we found that HSCs mainly undergo symmetric self-renewal in the presence of only stem cell factor, but with the addition of trombopoietin this division pattern is transformed into a symmetric commitment dominant mode in vitro. In addition, our study indicated that the division pattern cannot be defined by cell size or the nuclear/cytoplasm ratio. These findings collectively demonstrate that this single-cell immunofluorescence technique provides a new biological strategy in stem cell division research, and can be more widely applied given its flexibility, easy operability, and inexpensiveness.

Treatment of artificial soybean wastewater anaerobic effluent in a continuous aerobic-anaerobic coupled (CAAC) process with excess sludge reduction.

In this study, treatment of artificial soybean wastewater anaerobic effluent was studied in a continuous aerobic-anaerobic coupled (CAAC) process. The focus was on COD and nitrogen removal as well as excess sludge reduction. During the continuous operation without reflux, the COD removal efficiency was 96.5% at the optimal hydraulic retention time (HRT) 1.3 days. When HRT was shortened to 1.0 day, reflux from anaerobic zone to moving bed biofilm reactor (MBBR) was introduced. The removal efficiencies of COD and TN were 94.4% and 76.0% at the optimal reflux ratio 30%, respectively. The sludge yield coefficient of CAAC was 0.1738, the simultaneous removal of COD and nitrogen with in situ sludge reduction could be achieved in this CAAC process. The sludge reduction mechanism was discussed by soluble components variation along the water flow.