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Aeromonas hydrophila is a Gram-negative bacterium that is a critical causative agent of infections in fish and is occasionally responsible for human infections following contact with contaminated w...

The current approaches remain insufficient for measuring chicken egg spoilage or present analytical limitations. This study aimed to complement the existing analyses and identify novel markers using liquid chromatography-high resolution mass spectrometry-based foodomics strategies. In the discovery set, comparative untargeted metabolomics was utilized to identify marker candidates in microbially inoculated chicken eggs. Markers were annotated by spectral matching with authentic standards, experimental libraries, or in silico fragmentation. In the validation set, targeted metabolomics was employed to verify the markers in stored chicken eggs from five farms. Statistical differences at a p-value < 0.001 revealed increases in lactic and 3-hydroxybutyric acids and decreases in phosphocholine, LPE(O-18:1), LPC(16:0), and LPC(18:0) in stored eggs. Receiver operating characteristic curve analysis of the six combined markers yielded an AUC of 0.956 and a sensitivity and specificity of ∼90%. Four phospholipids were highlighted as a novel class of spoilage markers. Our findings may contribute to further industrial implementation, benefiting the quality assurance and food safety of poultry egg production.

Natural astaxanthin is widely used as a food and cosmetics additive because of its multiple biological activities. However, astaxanthin produced by Haematococcus pluvialis is generally esterified, and its activity is far less than that of free astaxanthin. Hydrolysis of astaxanthin esters to free astaxanthin by enzymes can overcome the drawbacks of chemical saponification methods. In this paper, a slug-flow microchannel reactor was constructed and tested in enzymatic hydrolysis of astaxanthin esters. The reactor consists of a “T” slug-flow generator, a stainless-steel microchannel, two constant-flow pumps, and a temperature controller. The reactor has the advantages of simple configuration and easy scale-up, and is suitable for two-phase biochemical reactions. Using the microchannel reactor, astaxanthin esters in H. pluvialis oil were efficiently hydrolyzed to free astaxanthin by lipase from Aspergillus niger. After hydrolysis, the content of free astaxanthin in H. pluvialis oil was 18.8 mg/L, 7.83-times higher than that before hydrolysis (2.13 mg/L). The hydrolysis rate reached 75.4 %. These results indicate that the microchannel reactor can be useful for the production of free astaxanthin from its esters.

For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion-pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate multimolecular (or secondary) association of unit PICs for non-covalent (or chemical crosslinking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm-sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be ~2,500 by using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at ~10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of non-covalently stabilized nanovesicles for the codelivery of two varying biomacromolecule payloads for ensuring their cooperative biological activity.

BACKGROUND The aim of this study is to investigate the application of two lactic acid bacteria and dry condensed molasses fermentation solubles (CMS) in the making and preservation of corn silage. Forage corn materials are divided into eight treatment groups as follows: (i) control, (ii) B2 (Lactobacillus plantarum B2, 1 × 109 cfu kg-1 ), (iii) LAS (Lactobacillus buchneri 40788, 3 × 108 cfu kg-1 ), (iv) B2 + LAS, (v) CMS (35 g kg-1 , fresh weight), (vi) B2 + CMS, (vii) LAS + CMS and (viii) B2 + LAS + CMS. The silage composition and aerobic stability are determined after ensiling for 90 days. Furthermore, the digestibility of the silage product and gas production are evaluated using a trotro digestion procedure. RESULTS The assay results indicate that the CMS supplementation and B2 inoculation significantly increased lactic acid concentration (P

Mitochondria are well known to serve as the powerhouse for cells and also the initiator for some vital signaling pathways. A variety of diseases are discovered to be associated with the abnormalities of mitochondria, including cancers. Thus, targeting mitochondria and their metabolisms are recognized to be promising for cancer therapy. In recent years, great efforts have been devoted to developing mitochondria-targeted pharmaceuticals, including small molecular drugs, peptides, proteins, and genes, with several molecular drugs and peptides enrolled in clinical trials. Along with the advances of nanotechnology, self-assembled peptide-nanomaterials that integrate the biomarker-targeting, stimuli-response, self-assembly, and therapeutic effect, have been attracted increasing interest in the fields of biotechnology and nanomedicine. Particularly, in situ mitochondria-targeted self-assembling peptides that can assemble on the surface or inside mitochondria have opened another dimension for the mitochondria-targeted cancer therapy. Here, we highlight the recent progress of mitochondria-targeted peptide-nanomaterials, especially those in situ self-assembly systems in mitochondria, and their applications in cancer treatments.

This case series reported a group of patients with Guillain–Barré syndrome (GBS) and their plasma cytokine changes before and after immunotherapy. We aimed to understand GBS's pathogenesis and pathophysiology through observing the interval differences of the representative cytokines, which were the thymus and activation regulated chemokine (TARC) for T-cell chemotaxis, CD40 ligand (CD40L) for cosimulation of B and T cells, activated complement component C5/C5a, and brain-derived neurotrophic factor (BDNF) for survival and regenerative responses to nerve injuries. The fluorescence magnetic bead-based multiplexing immunoassay simultaneously quantified the five cytokines in a single sample. From June 2018 to December 2019, we enrolled five GBS patients who had completed before–after blood cytokine measurements. One patient was diagnosed with paraneoplastic GBS and excluded from the following cytokine analysis. The BDNF level decreased consistently in all the patients and made it a potential biomarker for the acute stage of GBS. Interval changes of the other four cytokines were relatively inconsistent and possibly related to interindividual differences in the immune response to GBS triggers, types of GBS variants, and classes of antiganglioside antibodies. In summary, utilizing the multiplexing immunoassay helps in understanding the complex immune mechanisms of GBS and the variation of immune responses in GBS subtypes; this method is feasible for identifying potential biomarkers of GBS.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spatzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

N6-methyladenosine (m6A) is the most abundant modification of eukaryotic mRNA. Although m6A has been demonstrated to affect almost all aspects of RNA metabolism, its global contribution to the post-transcriptional balancing of translational efficiency remains elusive in plants. In this study, we performed a parallel analysis of the transcriptome-wide mRNA m6A distribution and polysome profiling in two maize (Zea mays) inbred lines to assess the global correlation of m6A modification with translational status. m6A sites are widely distributed in thousands of protein-coding genes, confined to a consensus motif and primarily enriched in the 3′ untranslated regions, and highly coordinated with alternative polyadenylation usage, suggesting a role of m6A modification in regulating alternative polyadenylation site choice. More importantly, we identified that the m6A modification shows multifaceted correlations with the translational status depending on its strength and genic location. Moreover, we observed a substantial intraspecies variation in m6A modification, and this natural variation was shown to be partly driven by gene-specific expression and alternative splicing. Together, these findings provide an invaluable resource for ascertaining transcripts that are subject to m6A modification in maize and pave the way to a better understanding of natural m6A variation in mediating gene expression regulation.

Genistein is known to have multiple biological activities and has great potential for use as a preventative medicine and in disease treatment. Genistein can be extracted from plants, but also can be obtained from its glycoside form, sophoricoside, which is more abundant in some plants. Biotransformation by unpurified microbial enzymes has the advantage of low cost and is a preferred method for production of natural compounds. This study isolated a strain of Rhizopus oryzae that could produce β-glucosidase, which efficiently hydrolyzes sophoricoside into genistein, from an enrichment culture of the dried fruits of Sophora japonica. After the composition of enzyme-producing medium and biotransformation conditions were optimized, a genistein yield of 85.6% was obtained after 24 h in a shake-flask biotransformation at pH 7.0 using an initial substrate concentration of 1 g/L. The developed process provides an alternative method for production of genistein, and would be suitable for scale-up production in the pharmaceutical industry.

Pre-mRNA splicing is an important step for gene expression regulation. Yeast Bud13p (bud-site selection protein 13) regulates the budding pattern and pre-mRNA splicing in yeast cells; however, no Bud13p homologs have been identified in plants. Here, we isolated two mutants that carry T-DNA insertions at the At1g31870 locus and shows early embryo lethality and seed abortion. At1g31870 encodes an Arabidopsis homolog of yeast Bud13p, AtBUD13. Although AtBUD13 homologs are widely distributed in eukaryotic organisms, phylogenetic analysis revealed that their protein domain organization is more complex in multicellular species. AtBUD13 is expressed throughout plant development including embryogenesis and AtBUD13 proteins is localized in the nucleus in Arabidopsis. RNA-seq analysis revealed that AtBUD13 mutation predominantly results in the intron retention, especially for shorter introns (≤100 bases). Within this group of genes, we identified 52 genes involved in embryogenesis, out of which 22 are involved in nucleic acid metabolism. Our results demonstrate that AtBUD13 plays critical roles in early embryo development by effecting pre-mRNA splicing.

In mammalians and yeast, the splicing factor U2AF65/Mud2p functions in precursor messenger RNA (pre-mRNA) processing. Arabidopsis AtU2AF65b encodes a putative U2AF65 but its specific functions in plants are unknown. This paper examines the function of AtU2AF65b as a negative regulator of flowering time in Arabidopsis. We investigated the expression and function of AtU2AF65b in abscisic acid (ABA)-regulated flowering as well as the transcript abundance and pre-mRNA splicing of flowering-related genes in the knock-out mutants of AtU2AF65b. The atu2af65b mutants show early-flowering phenotype under both long-day and short-day conditions. The transcript accumulation of the flowering repressor gene FLOWERING LOCUS C (FLC) is reduced in the shoot apex of atu2af65b, due to both increased intron retention and reduced transcription activation. Reduced transcription of FLC results, at least partially, from the abnormal splicing and reduced transcript abundance of ABSCISIC ACID-INSENSITIVE 5 (ABI5), which encodes an activator of FLC in ABA-regulated flowering signaling. Additionally, the expression of AtU2AF65b is promoted by ABA. Transition to flowering and splicing of FLC and ABI5 in the atu2af65b mutants are compromised during ABA-induced flowering. ABA-responsive AtU2AF65b functions in the pre-mRNA splicing of FLC and ABI5 in shoot apex, whereby AtU2AF65b is involved in ABA-mediated flowering transition in Arabidopsis.

Abscisic acid (ABA) is an important phytohormone regulating plant growth, development and stress responses. A multitude of key factors implicated in ABA signaling have been identified; however, the regulation network of these factors needs for further information. AtS40.4, a plant-specific DUF584 domain-containing protein, was identified previously as a senescence regulator inArabidopsis. In this study, our finding showed that AtS40.4 was negatively involved in ABA signaling during seed germination and early seedling growth.AtS40.4was highly expressed in seeds and seedlings, and the expression level was promoted by ABA. AtS40.4 was localized both in the nucleus and the cytoplasm. Moreover, the subcellular localization pattern of AtS40.4 was affected by ABA. The knockdown mutants ofAtS40.4exhibited an increased sensitivity to ABA, whereas the overexpression ofAtS40.4decreased the ABA response during seed germination and seedling growth ofArabidopsis. Furthermore, AtS40.4 was involved in ABRE-dependent ABA signaling and influenced the expression levels ofABA INSENTIVE(ABI)1-5andSnRK2.6. Further genetic evidence demonstrated thatAtS40.4functioned upstream ofABI4.These findings support the notion that AtS40.4 is a novel negative regulator of the ABA response network during seed germination and early seedling growth.

Magnesium (Mg) is an abundant and important cation in cells. Plants rely on Mg transporters to take up Mg from the soil, and then Mg is transported to anthers and other organs. Here, we showed that MGT6+/− plants display reduced fertility, while mgt6 plants are fertile. MGT6 is expressed in the anther at the early stages. Pollen mitosis and intine formation are impaired in aborted pollen grains (PGs) of MGT6+/− plants, which is similar to the defective pollen observed in mgt5 and mgt9 mutants. These results suggest that Mg deficiency leads to pollen abortion in MGT6+/− plants. Our data showed that mgt6 organs including buds develop significantly slower and mgt6 stamens accumulate a higher level of Mg, compared with wild-type (WT) and MGT6+/− plants. These results indicate that slower bud development allows mgt6 to accumulate sufficient amounts of Mg in the pollen, explaining why mgt6 is fertile. Furthermore, we found that mgt6 can restore fertility of mgt5, which has been reported to be male sterile due to defects in Mg transport from the tapetum to microspores and that an additional Mg supply can restore its fertility. Interestingly, mgt5 fertility is recovered when grown under short photoperiod conditions, which is a well-known factor regulating plant fertility. Taken together, these results demonstrate that slow development is a general mechanism to restore mgts fertility, which allows other redundant magnesium transporter (MGT) members to transport sufficient Mg for pollen formation.

BackgroundExtranodal natural killer/T-cell lymphoma (ENKTL) is a rare and extremely malignant tumor. The systemic inflammation score (SIS), which is based on the pretreatment level of lymphocyte-to-monocyte ratio (LMR) and serum albumin (Alb), has been shown to be of prognostic value in a number of cancers. We integrate several other pretreatment serum inflammatory indicators, including the neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), serum C-reactive protein (CRP) and albumin (Alb) level, to establish a modified systemic inflammatory scoring system to predict clinical outcomes of ENKTL.MethodsA total of 184 patients with newly diagnosed ENKTL was retrospectively investigated. Systemic inflammatory indexes, including NLR, LMR, CRP, and Alb level were reviewed. Receiver operating characteristic (ROC) curve analysis was carried out to obtain the optimal cut-off value. The associations between cutoff values and overall survival (OS) were analyzed by Kaplan–Meier curves and Cox proportional models.ResultsThe median age of patients was 44.0 years, ranging from 15 to 82 years. There were 129 (70.1%) male patient. About 57.1% of patients had stage III or IV disease. The optimal cut-off values of NLR and LMR in predicting OS were 3.1 and 2.4, respectively. The clinical standard of CRP and Alb levels at 10 and 40 mg/L, respectively, were chosen as the optimal cut-off values. By multivariate analysis, hemophilic syndrome (hazard ratio [HR]: 10.540, 95% confidence interval [CI]: 3.440–32.291, P < 0.001), advanced Ann Arbor stages (III–IV) (HR: 4.606, 95% CI: 1.661–12.774, P = 0.003), paranasal sinus invasion (HR: 2.323, 95% CI: 1.069–5.047, P = 0.033), NLR ≥ 3.1 (HR: 3.019, 95% CI: 1.317–6.923, P = 0.009), Alb level of

Cordyceps militaris has been widely studied for its various pharmacological activities such as antitumor, anti-inflammation, and immune regulation. The binding of an allergen to IgE-sensitized mast cells in nasal mucosa triggers allergic rhinitis. We found that oral administration of 300 mg/kg of the ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruiting bodies significantly alleviated the symptoms of ovalbumin-induced allergic rhinitis in mice, including sneeze/scratch, mast cell activation, eosinophil infiltration, and Syk activation. The treatment of ethanol extract significantly suppressed the release of β-hexosaminidase (a degranulation marker) and mRNA expression levels of various cytokines, including IL-3, IL-10, and IL-13 in activated RBL2H3 cells. The ethanol extract and β-sitostenone, which was purified from the extract, could respectively reduce the Ca2+ ion mobilization in activated RBL-2H3 cells. Furthermore, results collected from western immunoblotting demonstrated that ethanol extract significantly retarded Ca2+ ion mobilization-initiated signaling cascade, which provoked the expression of various allergic cytokines. Also, the extract incubation interfered with P38 as well as NF-kB activation and Nrf-2 translocation. Our study suggested that ethanol extract possessed some natural constituents which could inhibit immediate degranulation and de novo synthesis of allergic cytokines via inhibition of Ca2+ ion mobilization in mast cells in the nasal mucosa of allergic rhinitis mice.

Hormones play a crucial role both in plants and animals. As we all known, hormones work through specific receptors in different organisms. Therefore, the hormone interaction between plant and animal is an interesting question worth discussing. Recently, food safety has become a common topic concerned by consumers. A widely discussed rumor such as “Fruit bulking agent is some human reproductive hormones” leads us to consider whether animal hormones did regulate plant growth. Based on the large amount of previous research, we demonstrated that animal hormones did affect plant, such as steroid hormones for livestock feed could regulate physiological processes in plant including flowering, senescence and stress resistance after excreted into the environment. Here, we summarize the previous studies and systematically discuss the effects of animal hormones on plant growth and development.

Coastal wetlands along migratory flyways are crucial in supporting staging or wintering waterbirds, yet they are often targeted for wind energy development. Potential conflicts are likely to be strong in densely populated East and Southeast Asia, where many bird species along the flyway are endangered, and wind energy projects are just flourishing. We investigated waterbird abundance and flight behavior at a coastal wind farm at the mid of the East Asian-Australasian Flyway. For shorebirds roosting in the aquacultural ponds, the abundance showed no significant change in the study area compared with the control area across all development stages of the wind farm. For egrets breeding in the mangroves, fewer Cattle Egrets Bubulcus ibis were observed in the year of wind farm construction and the first year of wind farm operation, then the number recovered afterwards. Since the operation of the wind farm, birds avoided crossing closely spaced (200 m) turbines while travelling through widely spaced (500 m) ones more frequently. Shorebirds, egrets, and landbirds flew lower when turbines were present, reducing the overlap of their flight height with the swept zone. Our study suggests that coastal wind farms are not necessarily a great threat to waterbirds. Yet environmentally sound planning and rigorous monitoring are crucial in minimizing potential impacts.

IAV regulates inflammatory signaling via glucose metabolism., In this study, we demonstrated an essential function of the hexosamine biosynthesis pathway (HBP)–associated O-linked β-N-acetylglucosamine (O-GlcNAc) signaling in influenza A virus (IAV)–induced cytokine storm. O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, mediated IAV-induced cytokine production. Upon investigating the mechanisms driving this event, we determined that IAV induced OGT to bind to interferon regulatory factor–5 (IRF5), leading to O-GlcNAcylation of IRF5 on serine-430. O-GlcNAcylation of IRF5 is required for K63-linked ubiquitination of IRF5 and subsequent cytokine production. Analysis of clinical samples revealed that IRF5 is O-GlcNAcylated, and higher levels of proinflammatory cytokines correlated with higher levels of blood glucose in IAV-infected patients. We identified a molecular mechanism by which HBP-mediated O-GlcNAcylation regulates IRF5 function during IAV infection, highlighting the importance of glucose metabolism in IAV-induced cytokine storm.

Histone deacetylases (HDACs) are important for global gene expression and contribute to numerous physiological events. Deacetylase Rpd3 in yeast and its conserved homolog HDAC1 in mammals oppositely regulate autophagy; however, how Rpd3/HDAC1 is regulated to mediate autophagy remains unclear. Here, we showed autophagy occurrence in silkworm (Bombyx mori) required BmRpd3, wherein steroid hormone 20-hydroxyecdysone (20E) signaling regulated its protein level and nuclear localization negatively. Inhibition of MTOR led to dephosphorylation and nucleo-cytoplasmic translocation of BmRpd3/HsHDAC1. Besides, cholesterol, 20E, and 27-hydroxycholesterol could all induce massive dephosphorylation and cytoplasmic localization of BmRpd3/HsHDAC1, and thus autophagy by affecting MTORC1 activity. In addition, three phosphorylation sites (Ser392, Ser421, and Ser423) identified in BmRpd3 were conserved in HsHDAC1. Single or triple phosphorylation-site mutation attenuated the phosphorylation levels of BmRpd3/HsHDAC1, leading to their cytoplasmic localization and autophagy activation. In general, cholesterol derivatives, especially hydroxylated cholesterol, caused dephosphorylation and nucleo-cytoplasmic shuttling of BmRpd3/HsHDAC1 through inhibition of MTOR signaling to facilitate autophagy in B. mori and mammals. These findings improve our understandings of BmRpd3/HsHDAC1-mediated autophagy induced by cholesterol derivatives and shed light on their potential as a therapeutic target for neurodegenerative diseases and autophagy-related studies. Abbreviations: 20E: 20-hydroxyecdysone; 27-OH: 27-hydroxycholesterol; ACTB: actin beta; AMPK: AMP-activated protein kinase; Atg: autophagy-related; BmSqstm1: Bombyx sequestosome 1; CQ: chloroquine; HDAC: histone deacetylase; LMNB: Lamin B1; MTOR: mechanistic target of rapamycin kinase; PE: phosphatidylethanolamine; SQSTM1/p62: sequestosome 1; TUBA1A: tubulin alpha 1a.

Background and aims Major vault protein (MVP) is up-regulated during infections with hepatitis B virus (HBV) and hepatitis C virus (HCV). Here, we found that MVP deficiency inhibited hepatocellular carcinoma (HCC) development induced by diethylnitrosamine, hepatitis B X protein, and HCV core. Approach and results Forced MVP expression was sufficient to induce HCC in mice. Mechanistic studies demonstrate that the ubiquitin ligase human double minute 2 (HDM2) forms mutual exclusive complexes either with interferon regulatory factor 2 (IRF2) or with p53. In the presence of MVP, HDM2 is liberated from IRF2, leading to the ubiquitination of the tumor suppressor p53. Mouse xenograft models showed that HBV and HCV promote carcinogenesis through MVP induction, resulting in a loss of p53 mediated by HDM2. Analyses of clinical samples from chronic hepatitis B, liver cirrhosis, and HCC revealed that MVP up-regulation correlates with several hallmarks of malignancy and associates with poor overall survival. Conclusions Taken together, through the sequestration of IRF2, MVP promotes an HDM2-dependent loss of p53 that promotes HCC development.

ObjectiveTo investigate the possible human leukocyte antigen (HLA) association of both myelin oligodendrocyte glycoprotein (MOG-IgG)-associated diseases (MOGAD) and aquaporin-4 antibody (AQP4-IgG)-positive neuromyelitis optica spectrum disorders (NMOSDs) in the Dutch population with European ancestry to clarify similarities or differences in the immunogenetic background of both diseases.MethodsBlood samples from patients in the Dutch national MS/NMOSD expert clinic were tested for MOG-IgG and AQP4-IgG using a cell-based assay. HLA Class I and II genotyping was performed in 43 MOG-IgG–seropositive and 42 AQP4-IgG–seropositive Dutch patients with European ancestry and compared with those of 5,604 Dutch healthy blood donors.ResultsNo significant HLA association was found in MOG-IgG–seropositive patients. The AQP4-IgG–seropositive patients had a significant higher frequency of HLA-A*01 (61.9% vs 33.7%, OR 3.16, 95% CI, 1.707–5.863, p after correction [pc] = 0.0045), HLA-B*08 (61.9% vs 25.6%, OR 4.66, 95% CI, 2.513–8.643, pc < 0.0001), and HLA-DRB1*03 (51.2% vs 27.6%, OR 2.75, 95% CI, 1.495–5.042, pc = 0.0199) compared with controls.ConclusionsThe present study demonstrates differences in the immunogenetic background of MOGAD and AQP4-IgG–positive NMOSD. The strong positive association with HLA-A*01, -B*08, and -DRB1*03 is suggestive of a role of this haplotype in the etiology of AQP4-IgG–positive NMOSD in patients with European ancestry, whereas in MOGAD no evidence was found for any HLA association in these disorders.

Specifically blocking more than one oncogenic pathway simultaneously in a cancer cell with a combination of different drugs is the mainstay of the majority of cancer treatments. Being able to do this via two targeted pathways without inducing side effects through a general mechanism, such as chemotherapy, could bring benefit to patients. In this work we describe a new dual inhibitor of the JAK-STAT and HDAC pathways through designing and developing two types of molecule based on the JAK2 selective inhibitor XL019 and the pan-HDAC inhibitor, vorinostat. Both series of compounds had examples with low nanomolar JAK2 and HDAC1/6 inhibition. In some cases good HDAC1 selectivity was achieved while retaining HDAC6 activity. The observed potency is explained through molecular docking studies of all three enzymes. One example, 69c had 16–25 fold selectivity against the three other JAK-family proteins JAK1, JAK3 and TYK2. A number of compounds had sub-micromolar potencies against a panel of 4 solid tumor cell lines and 4 hematological cell lines with the most potent compound, 45h, having a cellular IC50 of 70 nM against the multiple myeloma cell line KMS-12-BM. Evidence of both JAK and HDAC pathway inhibition is presented in Hela cells showing that both pathways are modulated. Evidence of apoptosis with two compounds in 4 sold tumor cell lines is also presented.

Flowering is a critical stage of plant development and is closely correlated with seed production and crop yield. Flowering transition is regulated by complex genetic networks in response to endogenous and environmental signals. FLOWERING LOCUS C (FLC) is a central repressor in the flowering transition of Arabidopsis thaliana. The regulation of FLC expression is well studied at transcriptional and post-transcriptional levels. A subset of antisense transcripts from FLC locus, collectively termed cold-induced long antisense intragenic RNAs (COOLAIR), repress FLC expression under cold exposure. Recent studies have provided important insights into the alternative splicing of COOLAIR and FLC sense transcripts in response to developmental and environmental cues. Herein, at the 20th anniversary of FLC functional identification, we summarise new research advances in the alternative splicing of FLC sense and antisense transcripts that regulates flowering.

Triple-negative breast cancer (TNBC), which lacks estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, is closely related to basal-like breast cancer. Previously, we and others report that cyclin E/cyclin-dependent kinase 2 (CDK2) phosphorylates enhancer of zeste homolog 2 (EZH2) at T416 (pT416-EZH2). Here, we show that transgenic expression of phospho-mimicking EZH2 mutant EZH2T416D in mammary glands leads to tumors with TNBC phenotype. Coexpression of EZH2T416D in mammary epithelia of HER2/Neu transgenic mice reprograms HER2-driven luminal tumors into basal-like tumors. Pharmacological inhibition of CDK2 or EZH2 allows re-expression of ERα and converts TNBC to luminal ERα-positive, rendering TNBC cells targetable by tamoxifen. Furthermore, the combination of either CDK2 or EZH2 inhibitor with tamoxifen effectively suppresses tumor growth and markedly improves the survival of the mice bearing TNBC tumors, suggesting that the mechanism-based combination therapy may be an alternative approach to treat TNBC., EZH2 phosphorylation by CDK2 promotes progression of triple-negative breast cancer (TNBC). Here, the authors show that this signaling axis downregulates ERα, and thus combinatorial blockade of CDK2 and EZH2 sensitizes TNBC cells to tamoxifen.

Strains of Bifidobacterium animalis subsp. lactis (BAL) are the most commonly added probiotic strains in infant formulas on the Chinese market. In this study, four genotyping methods, 16S rRNA sequencing, multilocus sequence typing, pulsed-field gel electrophoresis and single nucleotide polymorphism-insertion deletion polymorphism were used to determine the 30 isolates from 20 commercial products at the species and strain levels. A combination of the label analysis, quantification and identification results showed that the label claims from the 20 samples had an overall accuracy of 80%. This study was the first to use molecular techniques to identify bifidobacteria strains in probiotic infant formulas on the Chinese market. The results demonstrated that the combination of genotyping methods can effectively identify commonly used commercial BAL strains with similar genetic backgrounds and highlight the importance of accurate strain identification by manufacturers to ensure product legitimacy and to avoid potential health risks.

Objective Kalanchoe hybrida (Crassulaceae) is naturalized throughout all the island of Taiwan in China. The preliminary bioassay-guided fractionation of the crude extract of K. hybrida exhibited that the chloroform and n-butanol fractions possessed potent cytotoxicity against MCF-7, NCI-H460, and SF-268 tumor cell lines at 50 µg/mL concentration. Therefore, K. hybrida was selected as a target and the chemical constituents from the chloroform and n-butanol fractions of the crude extracts of K. hybrida were identified. The potential constituents were examined for their cytotoxicity against the tumor cell lines. Methods A combination of conventional chromatographic techniques was performed on the crude extract of K. hybrida. The chemical structures of the purified constituents were identified on the basis of spectroscopic and spectrometric analysis. Results The purification results had led to the characterization of totally 37 compounds. The isolated compounds 1, 2, and 4–12 were examined for their cytotoxicity in vitro, and bufadienolides 4–8 and flavonol glycoside 11 displayed significant cytotoxicity towards all the tested tumor cell lines among these tested compounds. Conclusion The results indicated that these principles should be responsible for the bioactivity of corresponding partial fractions. The potential constituents could be further investigated to explore the new natural lead drugs.

Background To investigate and compare the cytotoxicity of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or foetal bovine serum (FBS). Methods The cultured human retina pigment epithelium ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG and TB. The cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial membrane potential (Δψ) and mitochondrial mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined. Results Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5 mg/mL for ICG but no cytotoxicity for BBG and TB at the concentrations used. Cell growth was arrested at a concentration 1 mg/mL when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout. Conclusions As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

The populations of many waterbird species along the East Asian-Australasian Flyway (EAAF) are declining, mainly due to the loss of natural wetland. Meanwhile, artificial wetlands, such as aquaculture ponds, saltpans and rice paddies, have increased in area. Artificial wetlands typically host fewer species and fewer individuals of waterbirds compared to natural ones, thus the conservation importance of artificial wetlands in general appears secondary. However, over large areas of densely populated East and Southeast Asia, few coastal lands above the high tide line remain natural. Waterbirds may utilize the natural intertidal zone at low tide, but are forced to feed or roost in artificial environments when tidal flats are submerged. In such landscapes, coastal artificial wetlands are not only supplementary or alternative to natural ones, but provide crucial habitats for waterbirds at high tide. To investigate the use of artificial wetlands by waterbirds in such a landscape, we studied supratidal wa...

Importance: Myelin oligodendrocyte glycoprotein antibodies (MOG-Abs) are consistently identified in a range of demyelinating disorders in adults and children. Current therapeutic strategies are largely center specific, and no treatments have been formally evaluated. Objective: To examine the clinical phenotypes, treatment responses, and outcomes of children with relapsing MOG-Ab-associated disease. Design, Setting, and Participants: This study prospectively collected demographic, clinical, and radiologic data from 102 patients from 8 countries of the EU Paediatric Demyelinating Disease Consortium from January 1, 2014, through December 31, 2016. Patients were treated according to local protocols. Main Outcomes and Measures: Annualized relapse rates (ARRs) and Expanded Disability Status Scale (EDSS) scores before and during treatment with disease-modifying drugs (DMDs). Results: A total of 102 children were identified (median [range] age, 7.0 [1.5-7.9] years; male to female ratio, 1.0:1.8; white to other race/ethnicity ratio, 3.6:1.0). Original diagnoses were neuromyelitis optica spectrum disorder (44 patients [43.1%]), acute disseminated encephalomyelitis followed by optic neuritis (20 [19.6%]), multiphasic disseminated encephalomyelitis (20 [19.6%]), and relapsing optic neuritis (18 [17.6%]). In all, 464 demyelinating events were reported. Treated patients had more relapses (median, 3.0; range, 1.0-17.0) than untreated patients (median, 1.0; range 1.0-7.0) (P = .009) and higher EDSS scores (median, 1.5; interquartile range, 0-2.5) than untreated patients (median, 1.0; interquartile range, 0-1.5) (P Conclusions and Relevance: Although commonly used to treat patients with multiple sclerosis, DMDs were not associated with clinical improvement in children with MOG-Ab-associated disease, whereas azathioprine, mycophenolate mofetil, rituximab, and particularly intravenous immunoglobulins were associated with a reduction in relapse frequency. A correct diagnosis of relapsing MOG-Ab-associated disorders is therefore important to optimize immune treatment.

Previous studies demonstrated that nuclear EGFR is associated with malignancy and resistance to tyrosine kinase inhibitor (TKI). To decipher the cellular compartmentalized functions and underlying mechanism of EGFR, we generated EGFR mutants at nuclear translocation signal (NLS) and nuclear export signal (NES). The results showed that EGFR NLS mutant with decreased nuclear translocation sensitized the cancer cells to TKI and impaired invasiveness and stemness of the cancer cells. In contrast, EGFR NES mutant with increased nuclear accumulation promoted aggressiveness of the cancer cells including TKI resistance. Next, we generated genetically engineered mouse model (GEMM) to validate the resulting phenotypes of the EGFR mutants. To this end, CRISPR-Cas9-mediated genome editing approach was used for generating EGFR mutant knock-in mouse, which harbors an internalization-deficient mutation (EGFRLL/AA), pro-nuclear (NES) mutation (EGFRL749P(S)) and the dual mutations. Upon EGF stimulation nuclear EGFR (nEGFR) accumulation in the hepatocytes from EGFRL749P heterozygous mice was increased. Conversely, a significant reduction of nEGFR was observed in the hepatocyte from EGFTRLL/AA homozygous mice. We found that germline expression of EGFRL749P(S) gave rise to deficiency in breeding. However, EGFRLL/AA mutant mice displayed no gross abnormal even in homozygous offspring, suggesting that depletion of nEGFR has no significant phenotype. We failed to generate homozygotes of EGFR NES mutant mice due to embryonic lethality. Of note, the female and male founders and F1 EGFRL798P(S) mice developed tumors with huge spleen. The IHC staining results indicated that the infiltrating lymphocytes in liver, stomach and kidney were B cells. The latency of the B cell lymphomagenesis was from 17 to 23 months, and tumor incidence of F1 offspring of EGFRL749P(S) knock-in mice was 10 of 15 (66.7%). These findings indicated that single allele mutation in EGFR NES domain is capable of driving lymphomagenesis, suggesting that the EGFR NES mutant possesses a strong oncogenic activity. Surprisingly, by searching database of EGFR mutation in human tumors (Cosmic database https://cancer.sanger.ac.uk/cosmic ), we noticed that among the somatic mutations in EGFR gene the gene amplification represents only small part of the gene alternations in human cancers, and EGFR L747P and L747S mutation occur in the lung cancer patients with intrinsic TKI resistance. In addition, the majority of human B lymphocyte originated tumors harboring EGFR NES mutation are also heterozygotes. Cosmic database shows that somatic mutations at the EGFR NES region occur in the patients with plasma cell lymphoma. Taken together, our findings suggest that nEGFR contributes to malignant potential of the tumors and EGFR with NES mutation is a tumor driver. Citation Format: Lei Nie, Junwei Hou, Yu-Yi Chu, Ying-Nai Wnag, Li-Chuan Chan, Longfei Huo, Paul Chiao, Shao-Chun Wang, Mien-Chie Hung, Michael Wang. Compartmentalized functions of epidermal growth factor receptor (EGFR) in tumorigenesis and malignant phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 120.

We previously performed long non-coding RNA (lncRNA) expression microarray analyses to identify novel indicators for gastric cancer (GC) metastasis and prognosis in which we identified lncRNA XLOC_010235 (XLOC) as a candidate RNA. However, XLOC_010235 molecular mechanism of action remains unclear. Gain and loss of function approaches were used to investigate the biological role of XLOC in vitro. The effects of XLOC on cell viability were assessed by CCK-8 proliferation assays. Real-time PCR, western-blot and immunofluorescence were used to evaluate the mRNA and protein expression of Snail and multiple EMT related molecules. The positive XLOC/Snail1 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis. Ectopic expression of XLOC facilitate cell viability, migration and invasion, leading to the acceleration of metastasis, while depletion of XLOC expression hindered cell migration and invasion. Moreover, over-expression of XLOC was found to play a important role in epithelial-to-mesenchymal transition (EMT) through the regulation of E-cadherin, N-cadherin and Vimentin expression, in which transcriptional factor Snail1 was involved. These results advance our understanding of the role of lncRNA XLOC_010235 as a active regulator of EMT by associating with Snail1, which may help in the development of new therapeutics.

Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20-hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain-A/B with DmCalpain-A/B, BmCalpain-C with DmCalpain-C, and BmCalpain-7 with HsCalpain-7. Then, the expression profiles of BmCalpains were analyzed by quantitative real-time polymerase chain reaction, and results showed that expression of BmCalpain-A/B, BmCalpain-C and BmCalpain-7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase-1. Moreover, the apoptosis-associated cleavage of BmATG6 in Bm-12 cells was significantly enhanced when BmCalpain-A/B and BmCalpain-7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain-A/B, -C and -7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis-associated cleavage of BmATG6.

For systemic delivery of small interfering RNA (siRNA) to solid tumors, we developed an actively-targeted unimer polyion complex-assembled gold nanoparticle (uPIC-AuNP) by a two-step assembling process. First is the monodispersed uPIC formation from the single molecules of therapeutic siRNA and the block catiomer, cyclic RGD (cRGD) peptide-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (cRGD-PEG-PLL-LA). Second is the surface decoration of a 20nm-sized AuNP with uPICs. The cRGD-installed uPIC-AuNPs (cRGD-uPIC-AuNP) provided the targetability for selective binding to the cancer and cancer-related endothelial cellular surface, while regulating their size

Flowering transition is regulated by complex genetic networks in response to endogenous and environmental signals. Pre-mRNA splicing is an essential step for the post-transcriptional regulation of gene expression. Alternative splicing of key flowering genes has been investigated in detail over the past decade. However, few splicing factors have been identified as being involved in flowering transition. Human heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) consists of two subunits, U2AF35 and U2AF65, and functions in 3' splice site recognition in mRNA splicing. Recent studies reveal that Arabidopsis U2AF65a/b and U2AF35a/b play important roles in the splicing of key flowering genes. We summarize recent advances in research on splicing-regulated flowering transition by focusing on the role of Arabidopsis U2AF in the splicing of key flowering-related genes at ambient temperature and in the abscisic acid signaling pathways.

A biomimetic platelet based on assembling peptides initiates artificial coagulation for tumor therapy., Platelets play a critical role in the regulation of coagulation, one of the essential processes in life, attracting great attention. However, mimicking platelets for in vivo artificial coagulation is still a great challenge due to the complexity of the process. Here, we design platelet-like nanoparticles (pNPs) based on self-assembled peptides that initiate coagulation and form clots in blood vessels. The pNPs first bind specifically to a membrane glycoprotein (i.e., CD105) overexpressed on angiogenetic endothelial cells in the tumor site and simultaneously transform into activated platelet-like nanofibers (apNFs) through ligand-receptor interactions. Next, the apNFs expose more binding sites and recruit and activate additional pNPs, forming artificial clots in both phantom and animal models. The pNPs are proven to be safe in mice without systemic coagulation. The self-assembling peptides mimic platelets and achieve artificial coagulation in vivo, thus providing a promising therapeutic strategy for tumors.

Vacuolar-type H + -adenosine triphosphatases (V-ATPases) are indispensable for lysosome acidification and participate in autophagic processes. The steroid hormone 20-hydroxyecdysone (20E) predominantly induces autophagy and regulates insect larval molting and metamorphosis; however, the specific mechanism of lysosome acidification regulation by 20E remains unclear. Here, we showed that the developmental profiles of Bombyx V-ATPases were in accordance with autophagy occurrence and lysosome acidification in the fat body during larval-pupal metamorphosis. BmV-ATPase-A and BmV-ATPase-B were required for lysosome acidification and autophagic flux. Both 20E treatment and starvation were able to induce lysosome acidification. Furthermore, BmV-ATPase transcription was induced by 20E treatment and reduced by RNAi targeting the 20E receptor BmUsp. On the one hand, 20E upregulated the transcription of BmV-ATPases through inducing Bombyx transcription factor EB (TFEB) and its nuclear translocation; on the other hand, 20E inhibited mTOR signaling to induce the transcription and assembly of BmV-ATPase subunits. Overall, 20E induces lysosome acidification by upregulating the transcription and assembly of V-ATPase subunits via activating BmTFEB and cooperating with nutrient signaling. These findings improve our understanding of the regulatory mechanisms underlying lysosome acidification and autophagic flux in Bombyx mori.

In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.

During insect larval-pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph-decreased protein bands (55-100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin-dependent endocytosis predominantly blocked the transportation of 55-100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ∼90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph-specific proteins were mainly involved in material transportation, while fat body-specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin-dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3′-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3′ part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3′ NTR (300 nt) showing 91.8–98.9% and 71.4–72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) H. T. Hsu et al. Phytopathology 90:615, 2000. (3) E. W. Kitajima. Acta. Hortic. 234:451, 1988.

Lisianthus (Eustoma exaltatum (L.) Salisb. ex G. Don subsp. russellianum (Hook.) Kartesz) is an economically important ornamental crop in Taiwan. Over the past decade, nine viruses have been identified or detected in lisianthus including: Bean yellow mosaic virus (BYMV), Lisianthus necrosis virus (LNV) (2), Cucumber mosaic virus (CMV) (1), Turnip mosaic virus (TuMV), Tomato spotted wilt virus (TSWV), Broad bean wilt virus (BBWV), Tomato mosaic virus (ToMV), Pepper veinal mottle virus (PVMV), and Ageratum yellow vein virus (AYVV) (4). In May 2007 (late period of growing season) in central Taiwan, systemic necrotic spots, which are similar to that caused by LNV (2), were found on approximately 20% of the lisianthus plants. Spherical virus particles, approximately 32 nm in diameter, were found in the crude sap of infected lisianthus collected from the fields. However, the diseased samples did not react with antisera against domestic lisianthus-infecting spherical viruses, LNV (2) and CMV (1). A virus culture was isolated via mechanical inoculation on Chenopodium quinoa and serologically identified as Carnation mottle virus (CarMV) by ELISA, western blotting, and immunoelectron microscopy using antiserum against the CarMV zantedeschia strain (3). The virus induced necrotic local lesions on the inoculated leaves of C. quinoa, C. amaranticolor, Gomphrena globosa, Cucurbita moschata, Phaseolus angularis, P. vulgaris, and Vigna unguiculata. Lisianthus was previously reported as a local lesion host for CarMV (3). In current studies with 8 of 10 lisianthus plants, the newly isolated virus induced necrotic local lesions on inoculated leaves 20 days post inoculation (dpi). However, systemic necrotic lesions on noninoculated upper leaves, as were observed in the fields, appeared 120 dpi on inoculated plants, indicating that CarMV induces systemic infection in lisianthus during late growth stages. Noninoculated plants did not develop symptoms. Complementary DNA fragments of viral genomic RNA were amplified with a specific primer of the coat protein gene (3) and sets of degenerate primer for CarMV. The amplified cDNA fragments were cloned and sequenced. The full-length sequence was submitted as GenBank Accession No. FJ843021. The genomic RNA consists of 4,003 nucleotides and has an identical genome organization to that reported for members of the genus Carmovirus. The nucleotide sequence of the full-length genome shares more than 95% identity to isolates of CarMV (GenBank Accession Nos. AF192772, AJ304989, AJ811998, NC_001265, and X02986), and the nucleotide and deduced amino acid sequence of coat protein shares more than 98% identity with that of CarMV-TW (AY383566) (3), CarMV-FO25 (EF622206), CarMV-Italy-Ca1 (EF622207), and CarMV-Netherland Ca2 (EF622210). To our knowledge, this is the first report of natural infection of CarMV in lisianthus in Taiwan. References: (1) C. C. Chen and C. C. Hu, Plant Prot. Bull. 41:179, 1999. (2) C. C. Chen et al. Plant Dis. 84:506, 2000. (3) C. C. Chen et al. Plant Dis. 87:1539, 2003. (4) Y. H. Cheng et al. J. Taiwan Agric. Res. 58:196, 2009.

Phytoplasmas have been reported to cause various disorders in papaya (Carica papaya L.), including dieback, mosaic, and yellow crinkle in Australia, Nivun Haamir dieback in Israel, and bunchy top-like disease in Cuba (1). Papaya is an economically important crop in Taiwan, and therefore, is monitored for viral infections. In 2005, papaya plants showing chlorosis, yellows and shriveling of leaves, dieback and lateral growth of branches, bending of apical branches, latexosis of fruits, and brown necrosis in phloem tissues were observed in southern Taiwan. Examination by an electron microscope revealed the presence of pleomorphic phytoplasma cells in sieve tubes of the phloem of petioles and leaf veins of diseased plants. Total DNA was extracted individually from at least three diseased plants at each location with a commercial DNA preparation kit (Axygen Scientific, Union City, CA) and used for amplification of the phytoplasma 16S rRNA gene in PCR with universal primer pairs P1 and Tint (3). The full-length 16S rRNA gene has been amplified and cloned. Sequence analysis revealed that the fragment was 1,581 bp long (GenBank Accession No. AJ919994) and shared 99.6% sequence identity with that of the ‘Candidatus Phytoplasma solani’ reference strain (GenBank Accession No. AF248959). A virtual restriction fragment length polymorphism analysis of the 16S rDNA sequence amplified from the R16F2n/R16R2 primers (2) was performed with iPhyClassifier (4) and pDRAW32. In silico restriction analysis identified the studied papaya phytoplasma as a subgroup 16SrXII-A strain. The sequence had 97 to 98% sequence identity with papaya phytoplasmas of the 16SrXII group in Australia (GenBank Accession No. Y10095), Israel (GenBank Accession No. AY903951), and Cuba (GenBank Accession No. AY725234). The disease incidence was 30 to 35% during the 2006 to 2010 growing seasons, and field surveys indicated that the disease has spread to central Taiwan with sporadic occurrence in recent years. To our knowledge, this is the first report of phytoplasma associated with papaya yellows in Taiwan. References: (1) Y. Arocha et al. Int. J. Syst. Evol. Microbiol. 55:2451, 2005. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Desert rose (Adenium obesum (Forssk.) Roem. & Schult, family Apocynaceae) is native to southeastern Africa, and is a perennial potted ornamental with colorful flowers that are popular in Taiwan. Symptoms of mosaic and chlorotic ringspots and line patterns on leaves were observed in July 2010, on all eight plants in a private garden in Potzu, Chiayi, Taiwan. Spherical virus particles with a diameter of approximately 28 nm were observed in crude sap prepared from symptomatic leaves. Virus culture was established by successive local lesion isolation in Chenopodium quinoa and was maintained in the systemic host Nicotiana tabacum van Hicks. The virus was mechanically transmissible to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). Observed symptoms included local lesions on inoculated leaves of C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, N. benthamiana, N. glutinosa, and N. rustica. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Serological tests using ELISA assays and western blotting indicated that the virus reacted positively to a rabbit antiserum prepared to CMV (4). Amplicons of an expected size (1.1 kb) were obtained in reverse transcription-PCR with primers specific to the 3′-half of CMV RNA 3 (3) using total RNA extracted from infected desert rose and N. tabacum. The amplified cDNA fragment was cloned and sequenced (GenBank Accession No. AB667971). Nucleotide sequences of the coat protein open reading frame (CP ORF) (657 nt) had 92 to 96% and 76 to 77% sequence identity to those of CMV in subgroups I (GenBank Accession Nos. NC_001440, D00385, M57602, D28780, and AB008777) and II (GenBank Accession Nos. L15336, AF127976, AF198103, and M21464), respectively. Desert roses infected by Tomato spotted wilt virus (TSWV) (1) and CMV (2) have been reported previously. In spite of the plants showing mosaic symptoms similar to that caused by CMV (2) and chlorotic ringspots and line patterns caused by TSWV (1), only CMV was detected in and isolated from these infected desert roses. However, the possibility of mixed infection of CMV and other viruses were not excluded in this research. To our knowledge, this is the first report of CMV infection in desert rose plants occurring in Taiwan. References: (1) S. Adkins and C. A. Baker. Plant Dis. 89:526, 2005. (2) C. A. Baker et al. Plant Dis. 87:1007, 2003. (3) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (4) Y. K. Chen and C. C. Yang. Plant Dis. 89:529, 2005.

Garland chrysanthemum (Chrysanthemum coronarium) and lettuce (Lactuca sativa) are winter vegetables in Taiwan. Turnip mosaic virus (TuMV) and Lettuce mosaic virus (LMV) were potyviruses commonly isolated from garland chrysanthemum and lettuce, respectively (1). Symptoms of mosaic and deformation in leaves and stunting of plants have been observed in both Compositae crops in the fields since 2007 in the Chiayi area and with an increasing incidence in recent years (26 and 33% in garland chrysanthemum and lettuce, respectively). Filamentous virus particles (approximately 780 × 13 nm) in the crude sap and pinwheel inclusions in infected cells can be observed in the preparations of both diseased hosts with electron microscopy. However, TuMV, LMV, and other potyviruses (Bean yellow mosaic virus, Papaya ringspot virus, and Zucchini yellow mosaic virus) were not detectable in diseased samples by ELISA and western blotting tests, indicating a new potyvirus infection. Virus cultures were isolated from infected garland chrysanthemum and lettuce separately via mechanical inoculations in Chenopodium quinoa. Each isolate was mechanically inoculated to their original host individually and all caused symptoms similar to that observed in the field, indicating their pathogenicity to their original host. A cDNA fragment consisting of partial nuclear inclusion (NIb) and coat protein (CP) genes were amplified with potyvirus degenerate primers (forward: 5′-GGBAAYAATAGTGGNCAACC and reverse: 5′-GGGGAGGTGCCGTTCTCDATRCACCA) and was found to share 90% nucleotide sequence identity to that of Bidens mottle virus (BiMoV; GenBank Accession No. AF538686). The sequences of the CP gene and 3′ untranslated region (3′-UTR) of tested viruses were further amplified with a specific primer of BiMoV CP and oligo-dT in reverse transcription-PCR. The amplified fragments were cloned, sequenced, and the combined cDNA sequences were deposited in GenBank (Accession No. AB491763 for isolate garland chrysanthemum and Accession No. AB491764 for isolate lettuce). Sequence analysis showed that both cloned sequences shared more than 97% nucleotide similarity to that of BiMoV. The amino acid sequence of the CP of both isolates shared a 99.3% identity and a 98.9 to 99.3% identity to that of other BiMoV isolates deposited in GenBank. BiMoV was first described to be infecting lettuce and Cichorium endivia in the United States (4) and was first reported in sunflower and calendula in Taiwan recently (2,3). To our knowledge, this is the first report of the occurrence of BiMoV in garland chrysanthemum and lettuce in Taiwan. References: (1) Y. K. Chen et al. Plant Pathol. Bull. 5:55,1996. (2) C.-H. Huang and F.-J. Jan. Plant Dis. 95:362, 2011. (3) J. Y. Liao et al. Arch. Virol. 154:723, 2009. (4) F. Youssef et al. Arch. Virol 153:227, 2008.

Macroptilium atropurpureum (siratro plants) is a perennial wild legume plant introduced to Taiwan as a forage crop in 1961 (3) and has become a naturalized weed found all over the island. In 2010, siratro plants with virus-like symptoms of mosaic and leaf deformation were observed on the campus of Da-Yeh University in central Taiwan. Flexuous virus-like particles about 750 × 12 nm were observed in the crude sap extracted from symptomatic leaves with a transmission electron microscope. Crude sap was mechanically inoculated to Chenopodium quinoa and local lesions can be observed on inoculated leaves 4 to 5 days after inoculation. Virus was purified from the leaves of inoculated C. quinoa with modified protocols of Gonsalves and Ishii (2). The virus coat protein (CP) consisted of a single major peptide with relative molecular weight of approximately 33 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral RNA extracted from the purified virus was used as a template and was primed with several primer sets corresponding to potyviruses and carlaviruses in reverse transcription-PCR to amplify possible corresponding cDNA fragments. After several attempts, a cDNA fragment of about 1,300 bp could be amplified with the degenerated primer set of BCMNV-F (5′CCDTGGACDGTWGGVATGAC3′) and BCMNV-R (5′CACCAHACCATRAARCCATTCAT3′), which were designed on the basis of the conserved region of the nuclear inclusion b (NIb) and CP genes of some potyviruses including Bean common mosaic necrosis virus, Soybean mosaic virus, Blackeye cowpea mosaic virus, East Asian passiflora virus, and Passion fruit woodiness virus. BLAST analyses showed the amplicon was highly homologous to that of Passiflora virus Y (PaVY). Together with oligo dT, a specific primer (5′GATGACACTCAAATGGCTG3′) corresponding to PaVY CP was used to amplify the cDNA fragment of the most 3′ region of the viral RNA (about 800 bp). The assembled cDNA fragment of 1,958 bp (Accession No. AB679294) contains a partial NIb gene (877 nt), a complete CP gene (819 nt), and the 3′ noncoding region (262 nt). The CP gene shared sequence identities of 89.4 to 98.9% and 92.7 to 98.9% in nucleotide and amino acid, respectively, to that of documented PaVY isolates. PaVY has also been found to be infecting Vigna trilobata, Rhynchosia minima, Clitoria ternatea, and Passiflora foetida in Australia (1). Here we present the first report to our knowledge of PaVY and its infection of siratro (M. atropurpureum) in Taiwan. Additional work is needed to investigate the spread of PaVY and its interaction with other legume plants in Taiwan. References: (1) B. A. Coutts et al. Arch. Virol. 156:1757, 2011. (2) D. Gonsalves and M. Ishii. Phytopathology 70:1028, 1980. (3) Y. Y. Lai et al. J. Taiwan Livestock Res. 42:19, 2009.

Lipoteichoic acid (LTA), a major component of the cell wall of Staphylococcus aureus (S. aureus), is not generally considered as an ideal vaccine candidate since it is a thymus-independent antigen. In this study, we screened a 12-mer phage peptide library and identified a series of peptide sequences that can mimic the epitope of LTA. A tetra-branched multiple antigenic peptide, named MAP2-3, comprising one of the positive peptide sequences (GHKEDRQWCQHS), was synthesized. Immunization with MAP2-3 induced LTA-specific IgG antibodies, prolonged the survival time, and decreased the bacterial burden in organs of mice infected with S. aureus. Moreover, passive immunization with polyclonal anti-MAP2-3 sera reduced bacterial load in organs of mice with bacteremia, alleviated acute lung injury in mice with pneumonia, and decreased the size of lesions in mice with skin infection. The number of LTA-specific antibody-secreting cells in the spleen of MAP2-3 immunized mice were significantly higher than that in the control mice. In summary, as a surrogate of LTA, vaccination with MAP2-3 elicited humoral immune response and protected mice from S. aureus infection. This study provides a new option to design vaccines against S. aureus.

Peptidoglycan recognition proteins (PGRPs) are members of an important class of pattern recognition receptors in insects that can specifically recognize peptidoglycan (PGN) in bacterial cell walls and participate in immune regulation and bacterial clearance. Although the role of PGRPs in regulating the innate immune response in Drosophila melanogaster has been studied, little is known regarding PGRPs in Lepidoptera species. In this study, five short (S)-type Bombyx mori PGRPs (BmPGRPs) were cloned, expressed, and evaluated for their function in innate immunity. B. mori larvae that were injected with the gram-positive bacterium Bacillus megaterium or the gram-negative bacterium Escherichia coli exhibited a rapid and significant upregulation in S-type BmPGRP expression. The results showed that the five evaluated BmPGRPs have significant agglutination activity toward E. coli and B. megaterium and more notable amidase activity toward meso-diaminopimelic acid peptidoglycan (DAP-PGN). Furthermore, only in the presence of BmPGRP-S5 did B. mori larval hemocytes exhibit significant phagocytosis against E. coli and B. megaterium.