Unraveling the surface marker signature of cell-derived vesicles via proteome analysis and nanoparticle flow cytometry.

The cell-derived vesicles (CDVs) obtained using a proprietary extrusion process are the foundation of BioDrone platform technology. With superior productivity and versatility, this technology has garnered increasing attention in broad applications, particularly as a drug delivery vehicle. Previously, we showed that CDVs exhibited varying levels of expression for tetraspanin and organelle membrane markers while revealing no discernible differences in physical characteristics compared to naturally produced extracellular vesicles (EVs). To further understand and utilize the therapeutic potentials of CDVs, a more comprehensive study of membrane protein profiles is necessary. In addition, it is crucial to validate that the CDVs produced from extrusion are indeed intact lipid vesicles rather than other impurities. Here, we produced multiple batches of CDVs and EVs from HEK293 cells. CDVs and EVs were subjected to the same purification processes for subsequent proteome and particle analyses. The proteome analyses revealed unique proteome signatures between CDVs, EVs, and parental cells. Extensive proteome analyses identified the nine most prominent membrane markers that are abundant in CDVs compared to cells and EVs. Subsequent western blotting and nanoparticle flow cytometry analyses confirmed that CD63, lysosome-associated membrane glycoprotein 1 (LAMP1), and nicastrin (NCSTN) are highly enriched in CDVs, whereas CD81, CD9, and prostaglandin F2 receptor negative regulator (PTGFRN) are more abundant in EVs. This highlights the unique membrane composition and marker signature of CDVs that are distinct from EVs. Lastly, we demonstrated that more than 90% of the CDVs are genuine lipid vesicles by combining two different classes of vesicle labeling dyes and detergents to disrupt lipid membranes. This indicates that our proprietary extrusion technology is highly compatible with other well-characterized EV production methods. The robust CDV markers identified in this study will also facilitate the engineering of CDVs to achieve enhanced therapeutic effects or tissue-selective cargo delivery.

Seroprevalence of Measles IgG Antibodies in Married Immigrant Women from Multicultural Families in Korea.

BACKGROUND: Although an effective vaccine has been available, measles still causes mast morbidity and mortality world widely. In Korea, a small number of measles cases have been reported through exposure to imported cases among young people with vaccine-induced measles immunity. Recently due to international migration including marriage, marriage migrants were the second-largest group of foreign population in Korea. Our study was carried out to obtain positive rate of measles antibody among married immigrant women from 12 countries in 10 Gun-Counties and 6 Cities, Korea. MATERIALS AND METHODS: A total of 547 blood samples were collected from maternal multicultural members from 12 countries. The measles-specific IgG antibody was measured by ELISA (Enzyme-linked immunosorbent assay; Enzygnost® Anti-measles virus/IgG, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). We performed a simple logistic regression to test whether the measles antibody seroprevalence differed by participant age, location, or country of birth and then calculated the likelihood ratio statistics to determine whether measles antibody seroprevalence differed by country of birth. RESULTS: Overall positive measles seroprevalence was 75.3% (95% confidence interval: 71.7 - 78.9). Participants aged 20 - 24 years, 25 - 29 years, and 30 - 63 years has respective seropositivities of 52.5%, 55.3%, and 82.7%. In this study, the geometric mean titers of participants aged 21 - 29 years were slightly lower than those of participants aged over 30 years, which were 1,372 mIU/ml and 2,261 mIU/ml, respectively (average of total participants: 2,027 mIU/ml). CONCLUSION: The study provides detailed information about seroimmunity of the married immigrant population in Korea, which is important for measles elimination. Since the 1980s, most vaccine-preventable diseases including measles have been well-controlled. Nevertheless, sporadic measles outbreaks are still reported. Thus, special attention should be paid to the possible importation of infectious diseases such as measles by immigration.

Inhibition of Arabidopsis thaliana CIN-like TCP transcription factors by Agrobacterium T-DNA-encoded 6B proteins.

Agrobacterium T-DNA-encoded 6B proteins cause remarkable growth effects in plants. Nicotiana otophora carries two cellular T-DNAs with three slightly divergent 6b genes (TE-1-6b-L, TE-1-6b-R and TE-2-6b) originating from a natural transformation event. In Arabidopsis thaliana, expression of 2×35S:TE-2-6b, but not 2×35S:TE-1-6b-L or 2×35S:TE-1-6b-R, led to plants with crinkly leaves, which strongly resembled mutants of the miR319a/TCP module. This module is composed of MIR319A and five CIN-like TCP (TEOSINTHE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR) genes (TCP2, TCP3, TCP4, TCP10 and TCP24) targeted by miR319a. The CIN-like TCP genes encode transcription factors and are required for cell division arrest at leaf margins during development. MIR319A overexpression causes excessive growth and crinkly leaves. TE-2-6b plants did not show increased miR319a levels, but the mRNA levels of the TCP4 target gene LOX2 were decreased, as in jaw-D plants. Co-expression of green fluorescent protein (GFP)-tagged TCPs with native or red fluorescent protein (RFP)-tagged TE-6B proteins led to an increase in TCP protein levels and formation of numerous cytoplasmic dots containing 6B and TCP proteins. Yeast double-hybrid experiments confirmed 6B/TCP binding and showed that TE-1-6B-L and TE-1-6B-R bind a smaller set of TCP proteins than TE-2-6B. A single nucleotide mutation in TE-1-6B-R enlarged its TCP-binding repertoire to that of TE-2-6B and caused a crinkly phenotype in Arabidopsis. Deletion analysis showed that TE-2-6B targets the TCP4 DNA-binding domain and directly interferes with transcriptional activation. Taken together, these results provide detailed insights into the mechanism of action of the N. otophora TE-encoded 6b genes.

Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates.

In the model plant Arabidopsis thaliana, four Dicer-like proteins (DCL1-4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII-like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4-dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4-dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold-back double-stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR-dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.

New DRB complexes for new DRB functions in plants.

Double-stranded RNA binding (DRB) proteins are generally considered as promoting cofactors of Dicer or Dicer-like (DCL) proteins that ensure efficient and precise production of small RNAs, the sequence-specificity guide of RNA silencing processes in both plants and animals. However, the characterization of a new clade of DRB proteins in Arabidopsis has recently challenged this view by showing that DRBs can also act as potent inhibitors of DCL processing. This is achieved through sequestration of a specific class of small RNA precursors, the endogenous inverted-repeat (endoIR) dsRNAs, thereby selectively preventing production of their associated small RNAs, the endoIR-siRNAs. Here, we concisely summarize the main findings obtained from the characterization of these new DRB proteins and discuss how the existence of such complexes can support a potential, yet still elusive, biological function of plant endoIR-siRNAs.

A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis.

BRI1-Associated Receptor Kinase 1 (BAK1) is a leucine-rich repeat serine/threonine receptor-like kinase (LRR-RLK) that is involved in multiple developmental pathways, such as brassinosteroid (BR) signaling, plant immunity and cell death control in plants. Because the roundish and compact rosette leaves of bak1 mutant plants are characteristic phenotypes for deficient BR signaling, we screened genetic suppressors of bak1 according to changes in leaf shape to identify new components that may be involved in BAK1-mediated BR signaling using the activation-tagging method. Here, we report bak1-SUP1, which exhibited longer and narrower rosette leaves and an increased BR sensitivity compared with those of bak1. Analyses of the T-DNA insertional site and the gene expression that was affected by the T-DNA insertion revealed that a microRNA, namely, miR172, over-accumulates in bak1-SUP1. Detailed phenotypic analyses of bak1-SUP1 and a single mutant in which the bak1 mutation was segregated out (miR172-D) revealed that the overexpression of miR172 promotes leaf length elongation in adult plants and increases the root and hypocotyl growth during the seedling stage compared with that of wild type plants. Taken together with its increased BR sensitivity, these results suggest that miR172 regulates vegetative growth patterns by modulating BR sensitivity as well as by the previously identified developmental phase transition.

A novel Arabidopsis MYB-like transcription factor, MYBH, regulates hypocotyl elongation by enhancing auxin accumulation.

Critical responses to developmental or environmental stimuli are mediated by different transcription factors, including members of the ERF, bZIP, MYB, MYC, and WRKY families. Of these, MYB genes play roles in many developmental processes. The overexpression of one MYB gene, MYBH, significantly increased hypocotyl elongation in Arabidopsis thaliana plants grown in the light, and the expression of this gene increased markedly in the dark. The MYBH protein contains a conserved motif, R/KLFGV, which was implicated in transcriptional repression. Interestingly, the gibberellin biosynthesis inhibitor paclobutrazol blocked the increase in hypocotyl elongation in seedlings that overexpressed MYBH. Moreover, the function of MYBH was dependent on phytochrome-interacting factor (PIF) proteins. Taken together, these results suggest that hypocotyl elongation is regulated by a delicate and efficient mechanism in which MYBH expression is triggered by challenging environmental conditions such as darkness, leading to an increase in PIF accumulation and subsequent enhanced auxin biosynthesis. These results indicate that MYBH is one of the molecular components that regulate hypocotyl elongation in response to darkness.

STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis.

MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1 (STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesis-defective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1 is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level.

Arabidopsis serine decarboxylase mutants implicate the roles of ethanolamine in plant growth and development.

Ethanolamine is important for synthesis of choline, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in plants. The latter two phospholipids are the major phospholipids in eukaryotic membranes. In plants, ethanolamine is mainly synthesized directly from serine by serine decarboxylase. Serine decarboxylase is unique to plants and was previously shown to have highly specific activity to l-serine. While serine decarboxylase was biochemically characterized, its functions and importance in plants were not biologically elucidated due to the lack of serine decarboxylase mutants. Here we characterized an Arabidopsis mutant defective in serine decarboxylase, named atsdc-1 (Arabidopsis thaliana serine decarboxylase-1). The atsdc-1 mutants showed necrotic lesions in leaves, multiple inflorescences, sterility in flower, and early flowering in short day conditions. These defects were rescued by ethanolamine application to atsdc-1, suggesting the roles of ethanolamine as well as serine decarboxylase in plant development. In addition, molecular analysis of serine decarboxylase suggests that Arabidopsis serine decarboxylase is cytosol-localized and expressed in all tissue.

A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress.

In response to environmental challenges, plant cells activate several signaling pathways that trigger the expression of transcription factors. Arabidopsis MYB60 was reported to be involved in stomatal regulation under drought conditions. Here, two splice variants of the MYB60 gene are shown to play a crucial role in stomatal movement. This role was demonstrated by over-expressing each variant, resulting in enhanced sensitivity to water deficit stress. The MYB60 splice variants, despite the fact that one of which lacks the first two exons encoding the first MYB DNA binding domain, both localize to the nucleus and promote guard cell deflation in response to water deficit. Moreover, MYB60 expression is increased in response to a low level of ABA and decreased in response to high level of ABA. At initial stage of drought stress, the plant system may modulate the root growth behavior by regulating MYB60 expression, thus promotes root growth for increased water uptake. In contrast, severe drought stress inhibits the expression of the MYB60 gene, resulting in stomatal closure and root growth inhibition. Taken together, these data indicate that MYB60 plays a dual role in abiotic stress responses in Arabidopsis through its involvement in stomatal regulation and root growth.

The ethylene signaling pathway has a negative impact on sucrose-induced anthocyanin accumulation in Arabidopsis.

In an attempt to understand the complex regulatory mechanisms underlying sucrose-induced flavonoid biosynthesis, we examined several Arabidopsis mutants with altered anthocyanin accumulation. We determined that disruption of ethylene signaling results in a dramatic increase in sucrose-induced anthocyanin accumulation. Furthermore, we investigated why the ein2-1 (ethylene insensitive) Arabidopsis mutant accumulates higher levels of anthocyanin in response to sucrose than wild-type Arabidopsis. An increased level of PAP1 transcript in the ein2-1 mutant appears to be the main factor responsible for the increased accumulation of anthocyanin in response to sucrose. Therefore, our results indicate that the ethylene signaling pathway plays a negative role in sucrose-induced anthocyanin accumulation. We believe that the explanation for this observation may be related to the initiation of the senescence program in plants.

Altered ARA2 (RABA1a) expression in Arabidopsis reveals the involvement of a Rab/YPT family member in auxin-mediated responses.

Ras super family proteins serve as molecular switches regulating many different cellular processes. However, given the large number of family members, sequence information has provided little insight into the function of individual proteins. This study examined phenotypic alterations in an Arabidopsis ara2 mutant, in which a Ras super family member-encoding gene is disrupted by a T-DNA insertion. Although one mutant line (Salk_013811) was hypersensitive to auxin, its T-DNA insertion was in the 5'-UTR of ARA2. Thus, we examined a true ARA2 knock-out mutant (Salk_077747) which contains an insertion in the first exon of ARA2. We found that ARA2 expression is responsive to auxin and at low concentrations, ara2 mutant plants exhibit increased numbers of lateral roots. ARA2 overexpression causes plants to exhibit hypersensitivity to auxin, due to altered expression of auxin-responsive genes, and these plants exhibited reduced numbers of lateral roots. A GFP-ARA2 fusion protein localized to the endosomes, suggesting that ARA2 may play a role in vesicle trafficking of components involved in polar auxin transport. Taken together, these results show that ARA2 is an essential component of a pathway that couples auxin signaling to plant growth and development.

Arabidopsis hot2 encodes an endochitinase-like protein that is essential for tolerance to heat, salt and drought stresses.

The Arabidopsis hot2 mutant was originally identified based on its lack of thermotolerance, but pleiotropic abnormal phenotypes are also exhibited under normal conditions, including semi-dwarfism, ethylene overproduction and aberrant cell shape with incomplete cell walls. Here we present additional characterization of the hot2 mutant, and the map-based cloning of HOT2. Mutants of hot2 had an aberrant tolerance to salt and drought stresses, and accumulated high levels of Na(+) in cells under either normal or NaCl stress conditions. Expression of the stress-inducible COR15A and KIN1 gene in hot2 mutants in response to increased NaCl concentrations was normal. HOT2 encoded a chitinase-like protein (AtCTL1) that has not previously been shown to be involved in tolerance to salt stress. Ten-day-old seedlings of wild-type plants exhibited constitutive expression of the AtCTL1 transcript, the level of which was unaffected by treatment with NaCl, mannitol or mild heat. These observations provide genetic evidence that a chitinase-like protein prevents the overaccumulation of Na(+) ions, thereby contributing to the salt tolerance in Arabidopsis. A possible role for this chitinase-like protein in Arabidopsis tolerance to abiotic stress is discussed.