Revisiting of Carex sect. Confertiflorae s.l. (Cyperaceae): New data from molecular and morphological evidence and first insights on Carex biogeography in East Asia

Carex sect. Confertiflorae s.l. is a medium-sized species group (ca. 40 species) with its center of diversity in E Asia (China and Japan). According to morphological traits, the section has been proposed to split into two sections (sects. Confertiflorae sensu Ohwi and Molliculae Ohwi) up to five different ones (sects. Confertiflorae s.s., Molliculae, Dispalatae Ohwi, Ischnostachyae Ohwi, and Alliiformes Akiyama). Recent phylogenetic reconstructions showed Confertiflorae s.l. not to be monophyletic, as species traditionally considered part of it were found to belong to other clades, whereas species traditionally ascribed to other sections were nested within it. In this study, we investigated the phylogenetic structure, morphological affinities, and biogeographic history of sect. Confertiflorae s.l. We employed a taxon-based approach to explore the morphological affinities of the species considered in sect. Confertiflorae and compared the micromorphology of the nutlets of almost all the taxa using SEM. We included 40 samples representing 31 species/subspecies of sect. Confertiflorae s.l. and used two nuclear (ETS and ITS) and three plastid (trnL-F, matK, and rpl32-trnL ^UAG) molecular markers to reconstruct the phylogeny of the group. The phylogenetic analyses confirmed the polyphyly of sect. Confertiflorae s.l., whose representatives were found within five distinct clades. From these, two clades, sect. Confertiflorae and sect. Molliculae, were found to be closely related and contained the majority of the species. The composition of the two clades agreed with the morphological structure of the group, and we confirmed an exclusive combination of features (namely color of basal sheaths, presence of bract sheath, peduncle of lowest spike, inflorescence sex distribution, shape of pistillate glume apex, and color and veins of utricle, among others) characterizing each of the two clades. The origin of the two clades was found to be in the early Pliocene; however, the majority of the diversification events within each clade took place during the Pleistocene. This illustrates that although Asia has been regarded as having little potential ecological space for Carex to diversify due to its climate stability, groups of sedges sub-endemic from that area may have a fairly recent origin related to glaciations. We proposed the rearrangement of sect. Confertiflorae as previously conceived as three independent sections: the monotypic Alliiformes, sect. Molliculae, and sect. Paludosae.     

Three‐dimensional reconstruction and comparison of vacuolar membranes in response to viral infection

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three‐dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV‐A^C), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck‐like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV‐A^C auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.     

Effective methods for isolation and purification of extracellular vesicles from plants

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.     

Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system

Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.     

Relationship of the lung microbiome with PD-L1 expression and immunotherapy response in lung cancer

IL-35 subunit EBI3 is up-regulated in pulmonary fibrosis tissues. In this study, we investigated the pathological role of EBI3 in pulmonary fibrosis and dissected the underlying molecular mechanism. Bleomycin-induced pulmonary fibrosis mouse model was established, and samples were performed gene expression analyses through RNAseq, qRT-PCR and Western blot. Wild type and EBI3 knockout mice were exposed to bleomycin to investigate the pathological role of IL-35, via lung function and gene expression analyses. Primary lung epithelial cells were used to dissect the regulatory mechanism of EBI3 on STAT1/STAT4 and STAT3. IL-35 was elevated in both human and mouse with pulmonary fibrosis. EBI3 knockdown aggravated the symptoms of pulmonary fibrosis in mice. EBI3 deficiency enhanced the expressions of fibrotic and extracellular matrix-associated genes. Mechanistically, IL-35 activated STAT1 and STAT4, which in turn suppressed DNA enrichment of STAT3 and inhibited the fibrosis process. IL-35 might be one of the potential therapeutic targets for bleomycin-induced pulmonary fibrosis.     

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell’s exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.     

Regulation of the innate immune cells during pregnancy: An immune checkpoint perspective

The foetus can be regarded as a half-allograft implanted into the maternal body. In a successful pregnancy, the mother does not reject the foetus because of the immune tolerance mechanism at the maternal-foetal interface. The innate immune cells are a large part of the decidual leukocytes contributing significantly to a successful pregnancy. Although the contributions have been recognized, their role in human pregnancy has not been completely elucidated. Additionally, the accumulated evidence demonstrates that the immune checkpoint molecules expressed on the immune cells are co-inhibitory receptors regulating their activation and biological function. Therefore, it is critical to understand the immune microenvironment and explore the function of the innate immune cells during pregnancy. This review summarizes the classic immune checkpoints such as PD-1, CTLA-4 and some novel molecules recently identified, including TIM-3, CD200, TIGIT and the Siglecs family on the decidual and peripheral innate immune cells during pregnancy. Furthermore, it emphasizes the role of the immune checkpoint molecules in pregnancy-associated complications and reproductive immunotherapy.