A single-cell atlas of immunocytes in the spleen of a mouse model of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a disorder characterized by rare X-linked genetic immune deficiency with mutations in the Was gene, which is specifically expressed in hematopoietic cells. The spleen plays a major role in hematopoiesis and red blood cell clearance. However, to date, comprehensive analyses of the spleen in wild-type (WT) and WASp-deficient (WAS-KO) mice, especially at the transcriptome level, have not been reported. In this study, single-cell RNA sequencing (scRNA-seq) was adopted to identify various types of immune cells and investigate the mechanisms underlying immune deficiency. We identified 30 clusters and 10 major cell subtypes among 11,269 cells; these cell types included B cells, T cells, dendritic cells (DCs), natural killer (NK) cells, monocytes, macrophages, granulocytes, stem cells and erythrocytes. Moreover, we evaluated gene expression differences among cell subtypes, identified differentially expressed genes (DEGs), and performed enrichment analyses to identify the reasons for the dysfunction in these different cell populations in WAS. Furthermore, some key genes were identified based on a comparison of the DEGs in each cell type involved in specific and nonspecific immune responses, and further analysis showed that these key genes were previously undiscovered pathology-related genes in WAS-KO mice. In summary, we present a landscape of immune cells in the spleen of WAS-KO mice based on detailed data obtained at single-cell resolution. These unprecedented data revealed the transcriptional characteristics of specific and nonspecific immune cells, and the key genes were identified, laying a foundation for future studies of WAS, especially studies into novel and underexplored mechanisms that may improve gene therapies for WAS.

Anthraquinones as Potential Antibiofilm Agents Against Methicillin-Resistant Staphylococcus aureus.

Biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) are one of the contributing factors to recurrent nosocomial infection in humans. There is currently no specific treatment targeting on biofilms in clinical trials approved by FDA, and antibiotics remain the primary therapeutic strategy. In this study, two anthraquinone compounds isolated from a rare actinobacterial strain Kitasatospora albolonga R62, 3,8-dihydroxy-l-methylanthraquinon-2-carboxylic acid (1) and 3,6,8-trihydroxy-1-methylanthraquinone-2-carboxylic acid (2), together with their 10 commercial analogs 3-12 were evaluated for antibacterial and antibiofilm activities against MRSA, which led to the discovery of two potential antibiofilm anthraquinone compounds anthraquinone-2-carboxlic acid (6) and rhein (12). The structure-activity relationship analysis of these anthraquinones indicated that the hydroxyl group at the C-2 position of the anthraquinone skeleton played an important role in inhibiting biofilm formation at high concentrations, while the carboxyl group at the same C-2 position had a great influence on the antibacterial activity and biofilm eradication activity. The results of crystal violet and methyl thiazolyl tetrazolium staining assays, as well as scanning electron microscope and confocal scanning laser microscopy imaging of compounds 6 and 12 treatment groups showed that both compounds could disrupt preformed MRSA biofilms possibly by killing or dispersing biofilm cells. RNA-Seq was subsequently used for the preliminary elucidation of the mechanism of biofilm eradication, and the results showed upregulation of phosphate transport-related genes in the overlapping differentially expressed genes of both compound treatment groups. Herein, we propose that anthraquinone compounds 6 and 12 could be considered promising candidates for the development of antibiofilm agents.

Prevalent cytidylation and uridylation of precursor miRNAs in Arabidopsis.

A key step in microRNA biogenesis is the processing of a primary precursor RNA by the microprocessor into a precursor miRNA (pre-miRNA) intermediate. In plants, little is known about the processes that act on pre-miRNAs to influence miRNA biogenesis. Here, we performed 3' rapid amplification of complementary DNA ends sequencing to profile pre-miRNA 3' ends in Arabidopsis. 3' end heterogeneity was prevalent, and the three microprocessor components promoted 3' end precision. Extensive cytidylation and uridylation of precise and imprecise pre-miRNA 3' ends were uncovered. The nucleotidyl transferase HESO1 uridylated pre-miRNAs in vitro and was responsible for most pre-miRNA uridylation in vivo. HESO1, NTP6 and NTP7 contribute to pre-miRNA cytidylation. Tailing of pre-miRNAs tended to restore trimmed pre-miRNAs to their intact length to promote further processing. In addition, HESO1-mediated uridylation led to the degradation of certain imprecisely processed pre-miRNAs. Thus, we uncovered widespread cytidylation and uridylation of pre-miRNAs and demonstrated diverse functions of pre-miRNA tailing in plants.

The U-box family genes in Medicago truncatula: Key elements in response to salt, cold, and drought stresses.

The ubiquitination pathway regulates growth, development, and stress responses in plants, and the U-box protein family of ubiquitin ligases has important roles in this pathway. Here, 64 putative U-box proteins were identified in the Medicago truncatula genome. In addition to the conserved U-box motif, other functional domains, such as the ARM, kinase, KAP, and WD40 domains, were also detected. Phylogenetic analysis of the M. truncatula U-box proteins grouped them into six subfamilies, and chromosomal mapping and synteny analyses indicated that tandem and segmental duplications may have contributed to the expansion and evolution of the U-box gene family in this species. Using RNA-seq data from M. truncatula seedlings subjected to three different abiotic stresses, we identified 33 stress-inducible plant U-box genes (MtPUBs). Specifically, 25 salinity-, 15 drought-, and 16 cold-regulated MtPUBs were detected. Among them, MtPUB10, MtPUB17, MtPUB18, MtPUB35, MtPUB42, and MtPUB44 responded to all three stress conditions. Expression profiling by qRT-PCR was consistent with the RNA-seq data, and stress-related elements were identified in the promoter regions. The present findings strongly indicate that U-box proteins play critical roles in abiotic stress response in M. truncatula.

Identification and expression profiling of Oryza sativa nucleotidyl transferase protein (NTP) genes under various stress conditions.

Nucleotidyl transferase proteins (NTPs) modify the 3' ends of mature small RNAs, leading to their stabilization or degradation. The first two plant NTPs, HESO1 and URT1, were identified in Arabidopsis. These two NTPs act cooperatively to uridylate the 3' terminal nucleotide of specific miRNAs, leading to their degradation and thereby affecting the expression of genes regulated by these miRNAs. Little is known about NTPs in other plants. Here, we performed a comprehensive analysis of 13 putative NTP genes in Oryza sativa, a major crop in global food production. Phylogenetic analysis showed homology among the NTPs from diverse plant species. Analysis of cis-acting promoter elements at OsNTP loci identified several stress response elements, indicating the potential involvement of NTPs in plant stress responses. The promoter analysis results were validated by expression of the OsNTP genes under abiotic stress treatments, with some OsNTPs clearly induced by salt, drought or cold stress. Moreover, the RT-PCR data showed that the OsNTP genes were differentially expressed in different developmental stages and tissues. These findings suggest that NTPs, which are involved in small RNA metabolic pathways, might play roles in plant stress resistance.