Structure-Controllable and Mass-Produced Glycopolymersomes as a Template of the Carbohydrate@Ag Nanobiohybrid with Inherent Antibacteria and Biofilm Eradication.

Glycopolymer-supported silver nanoparticles (AgNPs) have demonstrated a promising alternative to antibiotics for the treatment of multidrug-resistant bacteria-infected diseases. In this contribution, we report a class of biohybrid glycopolymersome-supported AgNPs, which are capable of effectively killing multidrug-resistant bacteria and disrupting related biofilms. First of all, glycopolymersomes with controllable structures were massively fabricated through reversible addition-fragmentation chain transfer (RAFT) polymerization-induced self-assembly (PISA) in an aqueous solution driven by complementary hydrogen bonding interaction between the pyridine and amide groups of N-(2-methylpyridine)-acrylamide (MPA) monomers. Subsequently, Ag+ captured by glycopolymersomes through the coordination between pyridine-N and Ag+ was reduced into AgNPs stabilized by glycopolymersomes upon addition of the NaBH4 reducing agent, leading to the formation of the glycopolymersome@AgNPs biohybrid. As a result, they showed a wide-spectrum and enhanced removal of multidrug-resistant bacteria and biofilms compared to naked AgNPs due to the easier adhesion onto the bacterial surface and diffusion into biofilms through the specific protein-carbohydrate recognition. Moreover, the in vivo results revealed that the obtained biohybrid glycopolymersomes not only demonstrated an effective treatment for inhibiting the cariogenic bacteria but also were able to repair the demineralization of caries via accumulating Ca2+ through the recognition between carbohydrates and Ca2+. Furthermore, glycopolymersomes@AgNPs showed quite low in vitro hemolysis and cytotoxicity and almost negligible acute toxicity in vivo. Overall, this type of biohybrid glycopolymersome@AgNPs nanomaterial provides a new avenue for enhanced antibacterial and antibiofilm activities and the effective treatment of oral microbial-infected diseases.

Metalloestrogens exposure and risk of gestational diabetes mellitus: Evidence emerging from the systematic review and meta-analysis.

BACKGROUND: Metalloestrogens are metals and metalloid elements with estrogenic activity found everywhere. Their impact on human health is becoming more apparent as human activities increase. OBJECTIVE: Our aim is to conduct a comprehensive systematic review and meta-analysis of observational studies exploring the correlation between metalloestrogens (specifically As, Sb, Cr, Cd, Cu, Se, Hg) and Gestational Diabetes Mellitus (GDM). METHODS: PubMed, Web of Science, and Embase were searched to examine the link between metalloestrogens (As, Sb, Cr, Cd, Cu, Se, and Hg) and GDM until December 2023. Risk estimates were derived using random effects models. Subgroup analyses were conducted based on study countries, exposure sample, exposure assessment method, and detection methods. Sensitivity analyses and adjustments for publication bias were carried out to assess the strength of the findings. RESULTS: Out of the 389 articles identified initially, 350 met our criteria and 33 were included in the meta-analysis, involving 141,175 subjects (9450 cases, 131,725 controls). Arsenic, antimony, and copper exposure exhibited a potential increase in GDM risk to some extent (As: OR = 1.28, 95 % CI [1.08, 1.52]; Sb: OR = 1.73, 95 % CI [1.13, 2.65]; Cu: OR = 1.29, 95 % CI [1.02, 1.63]), although there is a high degree of heterogeneity (As: Q = 52.93, p < 0.05, I2 = 64.1 %; Sb: Q = 31.40, p < 0.05, I2 = 80.9 %; Cu: Q = 21.14, p < 0.05, I2 = 71.6 %). Conversely, selenium, cadmium, chromium, and mercury exposure did not exhibit any association with the risk of GDM in our study. DISCUSSION: Our research indicates that the existence of harmful metalloestrogens in the surroundings has a notable effect on the likelihood of GDM. Hence, we stress the significance of environmental elements in the development of GDM and the pressing need for relevant policies and measures.

The influence of dexmedetomidine added to ropivacaine for transversus abdominis plane block on perioperative neurocognitive disorders after radical colorectal cancer surgery: randomized, double-blind, controlled trial.

OBJECTIVE: Perioperative Neurocognitive Disorders (PND) is a common neurological complication after radical colorectal cancer surgery, which increases adverse outcomes. So, our objective is to explore the influence of dexmedetomidine added to ropivacaine for transversus abdominis plane block (TAPB) on perioperative neurocognitive disorders, and to provide a new way to reduce the incidence of PND. METHODS: One hundred and eighty patients submitted to radical laparoscopic colorectal cancer surgery were randomly divided into Control group and Dex group. Ultrasound guided TAPB was performed after anesthesia induction: 0.5% ropivacaine 20 ml was injected into each transversus abdominis plane in Control group, 0.5% ropivacaine + 1 µg/kg dexmedetomidine (amounting to 20 ml) in Dex group. We observed the incidence of PND within 30 days after surgery. RESULTS: One hundred and sixty-nine cases were finally analyzed, including 84 cases in Control group and 85 cases in Dex group. Compared with Control group, there was no significant difference in terms of the incidence of PND on the 3rd day and the 7th day (P > 0.05), but the incidence significantly decreased at the 6th hour, at the 24th hour and on the 30th day after surgery (P < 0.05) in Dex group. CONCLUSION: Dexmedetomidine added to ropivacaine for TAPB can reduce the incidence of PND in the first 24 h after surgery and on the 30th postoperative day, which may be related to reduce the consumption of general anesthetics and provide satisfactory postoperative analgesia. TRIAL REGISTRATION: 29 /05/ 2021, ChiCTR2100046876.

Proposal for a hybrid clock system consisting of passive and active optical clocks and a fully stabilized microcomb.

Optical atomic clocks produce highly stable frequency standards and frequency combs bridge clock frequencies with hundreds of terahertz difference. In this paper, we propose a hybrid clock scheme, where a light source pumps an active optical clock through a microresonator-based nonlinear third harmonic process, serves as a passive optical clock via indirectly locking its frequency to an atomic transition, and drives a chip-scale microcomb whose mode spacing is stabilized using the active optical clock. The operation of the whole hybrid system is investigated through simulation analysis. The numerical results show: (i) The short-term frequency stability of the passive optical clock follows an Allan deviation of σy(τ) = 9.3 × 10-14τ-1/2 with the averaging time τ, limited by the population fluctuations of interrogated atoms. (ii) The frequency stability of the active optical clock reaches σy(τ) = 6.2 × 10-15τ-1/2, which is close to the quantum noise limit. (iii) The mode spacing of the stabilized microcomb has a shot-noise-limited Allan deviation of σy(τ) = 1.9 × 10-11τ-1/2. Our hybrid scheme may be realized using recently developed technologies in (micro)photonics and atomic physics, paving the way towards on-chip optical frequency comparison, synthesis, and synchronization.

Adenosine Triphosphate-Responsive Glyconanorods through Self-Assembly of ß-Cyclodextrin-Based Glycoconjugates for Targeted and Effective Bacterial Sensing and Killing.

Polymer-based nanomaterials have exhibited promising alternative avenues to combat the globe challenge of multidrug-resistant bacterial infection. However, most of the reported polymeric nanomaterials have facially linear amphiphilic structures with positive net charges, which may lead to nonspecific binding, high hemolysis, and uncontrollable self-organization, limiting their practical applications. In this contribution, we report a one-dimensional glyconanorod (GNR) through self-assembly of well-defined ß-cyclodextrin-based glycoconjugates (RMan) featuring hydrophobic carbon-based chains and amide rhodamines with an adenosine triphosphate (ATP)-recognition site and targeted and hydrophilic mannoses and positively net-charged ethylene amine groups. The GNRs show superior targeting sensing and killing for Gram-negative Escherichia coli (E. coli) dominantly through the multivalent recognition between mannoses on the nanorod and the lectin on the surface of E. coli. Moreover, red fluorescence was light on due to the hydrogen bonding between amide rhodamine and ATP. Benefiting from the designs, the GNRs are capable of possessing a higher therapeutic index and of encapsulating other antibiotics. They exhibit an enhanced effect against E. coli strains. Intriguingly, the GNRs displayed a more reduced hemolysis effect and lower cytotoxicity compared to that of ethylene glyco-modified nanorods. These results reveal that the glyconanomaterials not only feature superior and targeted bacterial sensing and antibacterial activity, but also better biocompatibility compared with the widely used PEG-covered nanomaterials. Furthermore, the in vivo studies demonstrate that the targeted and ATP-responsive GNRs complexed with antibiotics showed better treatment using a mouse model of abdominal sepsis following intraperitoneal E. coli infection. The present work describes a targeted and effective sensing and antibacterial platform based on glycoconjugates that have potential applications for the treatment of infections caused by pathogenic microorganisms.

Dynamic Glycopeptide Dendrimers: Synthesis and Their Controllable Self-Assembly into Varied Glyco-Nanostructures for the Biomimicry of Glycans.

A library of 14 dynamic glycopeptide amphiphilic dendrimers composed of 14 hydrophilic and bioactive saccharides (seven kinds) as dendrons and 7 hydrophobic peptides (di- and tetrapeptides) as arms with ß-cyclodextrin (CD) as a core were facially designed and synthesized in several steps. Fourteen saccharides were first conjugated to the C-2 and C-3 positions of CD, forming glycodendrons. Subsequently, seven oligopeptide arms were introduced at the C-6 positions of a CD moiety by an acylhydrazone dynamic covalent bond, resulting in unique Janus amphiphilic glycopeptide dendrimers with precise and varied molecular structures. The kinds of hydrophilic parts of saccharides and hydrophobic parts of peptides were easily varied to prepare a series of amphiphilic Janus glycopeptide dendrimers. Intriguingly, these obtained amphiphilic glycopeptide dendrimers showcased very different self-assembly behaviors from the traditional amphiphilic linear block-copolymers and self-assembled into different glyco-nanostructures with controllable morphologies including glycospheres, worm-like micelles, and fibers depending upon the repeat unit ratio of saccharides and phenylalanine. Both glycodendrons and glycopeptide assemblies displayed strong and specific recognitions with C-type mannose-specific lectin. Moreover, these glycopeptide nanomaterials can encapsulate exemplary hydrophobic molecules such as Nile red (NR). The dye-loaded glycopeptide nanostructures showed a pH-controllable release behavior around the physiological and acidic tumor environment. Furthermore, cell experiments demonstrated that such glyco-nanostructures can further facilitate the functions of a model drug of the pyridone agent to reduce the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin -1beta (IL-1ß) in the primary peritoneal macrophages via encapsulating drugs. Considering all the abovementioned advantages including unique and precise structures, bioactivity, targeting, and controllable cargo release, we believe that these findings can not only enrich the library of glycopeptides but also provide a new avenue to the fabrication of smart and structure-controllable glyco-nanomaterials which hold great potential biological applications such as targeted delivery and release of therapeutic and bioactive molecules.

The G-protein coupled receptor GPRK contributes to fungal development and full virulence in Metarhizium robertsii.

G-protein-coupled receptor K (GPRK), which is a class VI fungal G-protein-coupled receptor (GPCR), plays a critical role in plant immunity against pathogens by mediating the endocytic pathway, influencing metabolism in response to environmental signals, and regulating asexual reproduction and pathogenic development. However, the function of these proteins in entomopathogenic fungi has rarely been investigated. Accordingly, we characterized MrGPRK, a GPCR in the entomopathogenic fungus Metarhizium robertsii containing a C-terminal seven-transmembrane and a conserved regulator of G protein signaling domain, and found that it localized to endosomes. Mutant phenotype assays showed that a ΔMrGprk strain displayed increased defects in radial growth (~28%) and decreased conidial production (~80%) compared with a wild-type strain. Decreased conidiation rates coincided well with the repression of conidiation-related regulatory genes, including three key conidial transcription factors: brlA, abaA, and wetA. MrGprk deficiency impaired full virulence (both topical and injectable inoculations). Further analysis demonstrated that deleting fungal MrGprk decreased the rates of appressorium formation and suppressed the transcription of several genes contributing to appressorial turgor pressure, cuticle penetration, and pH regulation. Additionally, the ΔMrGprk strain showed higher cyclic (cAMP) levels, suggesting that this GPCR is critical for cAMP signal transduction. In summary, MrGPRK was found to contribute to vegetative growth, conidial production, and full virulence of M. robertsii. These findings are conducive to a better understanding of the roles of GPCRs in the development and pathogenicity of entomopathogenic fungi.

TSARM-UDP: An Efficient Time Series Association Rules Mining Algorithm Based on Up-to-Date Patterns.

In many industrial domains, there is a significant interest in obtaining temporal relationships among multiple variables in time-series data, given that such relationships play an auxiliary role in decision making. However, when transactions occur frequently only for a period of time, it is difficult for a traditional time-series association rules mining algorithm (TSARM) to identify this kind of relationship. In this paper, we propose a new TSARM framework and a novel algorithm named TSARM-UDP. A TSARM mining framework is used to mine time-series association rules (TSARs) and an up-to-date pattern (UDP) is applied to discover rare patterns that only appear in a period of time. Based on the up-to-date pattern mining, the proposed TSAR-UDP method could extract temporal relationship rules with better generality. The rules can be widely used in the process industry, the stock market, etc. Experiments are then performed on the public stock data and real blast furnace data to verify the effectiveness of the proposed algorithm. We compare our algorithm with three state-of-the-art algorithms, and the experimental results show that our algorithm can provide greater efficiency and interpretability in TSARs and that it has good prospects.

[A simplified technique for reconstruction of vesicourethral support in laparoscopic radical prostatectomy improves immediate continence].

OBJECTIVE: To investigate the application of a simplified technique for reconstruction of vesicourethral support (RVUS) in laparoscopic radical prostatectomy (LRP). METHODS: From January 2017 to August 2019, 122 patients with localized prostate cancer underwent extraperitoneal LRP, 65 with RVUS (the RVUS group) and 57 without RVUS (the non-RVUS group). We compared the operation time, intraoperative blood loss, rate of pelvic lymph node dissection, neurovascular bundle sparing, incidence of urethrovesical anastomotic urinary leakage (UVAUL), postoperative urinary continence, postoperative hospital stay, intraperitoneal drainage tube removal time, and urethral catheter removal time between the two groups of patients. RESULTS: No statistically significant differences were observed between the two groups in the operation time, intraoperative blood loss, rate of pelvic lymph node dissection, neurovascular bundle sparing, or urethral catheter removal time (P > 0.05). The incidence rate of UVAUL was lower in the non-RVUS than in the RVUS group (8.8% vs 0%, P < 0.05), and so were the rates of postoperative urinary continence immediate after (0% vs 32.3%, P < 0.05) and at 1 month (38.6% vs 56.9%, P < 0.05), 3 months (59.6% vs 80%, P < 0.05), 6 months (78.9% vs 84.6%, P > 0.05) and 12 months after catheter removal (87.7% vs 92.3%, P > 0.05). The postoperative hospital stay was dramatically longer in the non-RVUS than in the RVUS group (ï¼»9.1 ± 4.3ï¼½ vs ï¼»6.7 ± 1.8ï¼½ d, P < 0.01) and so was the intraperitoneal drainage tube removal time (ï¼»6.9 ± 4.5ï¼½ vs ï¼»4.8 ± 1.5ï¼½ d, P < 0.01). CONCLUSIONS: The simplified technique for reconstruction of vesicourethral support in laparoscopic radical prostatectomy improves early urinary continence, especially immediate continence, decreases the incidence rate of urethrovesical anastomotic urinary leakage, and shortens the intraperitoneal drainage tube removal time and postoperative hospital stay.?

MiR-429 regulates blood-spinal cord barrier permeability by targeting Krüppel-like factor 6.

The blood-spinal cord barrier (BSCB) is an effective, tightly-connected tissue that reduces secondary spinal cord injury (SCI) by decreasing blood cell infiltration, inflammation, and neuronal cell death during primary SCI. However, the methods and molecular mechanisms of BSCB openness remain elusive. In the present study, we found that microRNA429 (miR-429) plays a vital role in the opening of the blood-spinal cord. Inhibiting the expression of miR-429 (antagomiR-429) resulted in increased expression levels of the tight junction (TJ) proteins, ZO-1, occludin, and claudin-5, in the BSCB and reduced BSCB permeability. Moreover, overexpression of miR-429 (agomiR-429) had the opposite effect. Krüppel-like factor 6 (KLF6) is a transcription factor of the zinc-finger family. Using RT-qPCR and western blotting, we found that miR-429 can negatively regulate the expression of the KLF6. Co-transfection of KLF6 and miR-429 demonstrated that miR-429 negatively regulates KLF6 to mediate TJ protein expression and BSCB permeability. Based on these results, we suggest that KLF6 may be a downstream target of miR-429, mediating TJ protein expression to regulate the BSCB.

Characteristics of the urinary microbiome in kidney stone patients with hypertension.

BACKGROUND: Kidney stone disease (KSD) is more common in individuals with hypertension (HTN) than in individuals with normotension (NTN). Urinary dysbiosis is associated with urinary tract disease and systemic diseases. However, the role of the urinary microbiome in KSD complicated with HTN remains unclear. METHODS: This study investigated the relationship between the pelvis urinary microbiome and blood pressure (BP) in patients with KSD co-occurring with HTN (KSD-HTN) and healthy controls (HC) by conducting 16S rRNA gene sequencing of bacteria in urine samples. The urine samples were collected (after bladder disinfection) from 50 patients with unilateral kidney calcium stones and NTN (n = 12), prehypertension (pHTN; n = 11), or HTN (n = 27), along with 12 HCs. RESULTS: Principal coordinates analysis showed that there were significant differences in the urinary microbiomes not only between KSD patients and HCs but also between KSD-pHTN or KSD-HTN patients and KSD-NTN patients. Gardnerella dominated in HCs, Staphylococcus dominated in KSD-NTN patients and Sphingomonas dominated in both KSD-pHTN and KSD-HTN patients. The abundance of several genera including Acidovorax, Gardnerella and Lactobacillus was correlated with BP. Adherens junction and nitrogen and nucleotide metabolism pathways, among others, were associated with changes in BP. CONCLUSIONS: The findings suggest that patients with KSD complicated with HTN have a unique urinary microbiome profile and that changes in the microbiome may reflect disease progression and may be useful to monitor response to treatments.

DNM1, a Dynamin-Related Protein That Contributes to Endocytosis and Peroxisome Fission, Is Required for the Vegetative Growth, Sporulation, and Virulence of Metarhizium robertsii.

Although dynamins and dynamin-related proteins (DRPs), a large GTPase superfamily, are involved in the budding of transport vesicles and division of organelles in eukaryotic cells, the function of these proteins in entomopathogenic fungi has not been reported to date. Here, DNM1, a DRP in Metarhizium robertsii, was characterized using gene disruption and complementation strategies. Mutant phenotype assays showed that the ΔDnm1 strain displayed increased defects in radial growth (∼24%) and conidial production (∼42%) compared to those of the wild type (WT), and reduced conidiation levels were accompanied by the repression of several key conidiation-related genes, including flbA, wetA, and flbD Additionally, mutant bioassays revealed that disruption of Dnm1 impaired the virulence (both topical inoculation and injection) of M. robertsii in the insect Galleria mellonella Further analysis demonstrated that deleting Dnm1 in fungi suppressed the transcriptional levels of several virulence genes in the insect hemocoel. Moreover, we found that DNM1 colocalized with peroxisomes and mitochondria. Importantly, disruption of Dnm1 abolished normal fungal endocytosis, resulting in significantly decreased numbers of, as well as morphological changes in, peroxisomes. These findings indicate that deletion of Dnm1 causes significant changes in the vegetative growth, sporulation, and virulence of M. robertsii due to changes in cell function and peroxisomes.IMPORTANCEDnm1 was found to be involved in fungal development and virulence, mediated peroxisomal fission, and normal endocytosis. This finding provides new insights into the cellular processes and pathogenicity in entomopathogenic fungi.

Genome-wide study of saprotrophy-related genes in the basal fungus Conidiobolus heterosporus.

Conidiobolus spp. are important saprophytic basal fungi. However, to date, no genomic-level data for decaying plant materials in the genus Conidiobolus has been reported. Here, we report that the 33.4-Mb genome of Conidiobolus heterosporus encodes 10,857 predicted genes. Conidiobolus heterosporus harbors 394 CAZyme-encoding genes belonging to 4 major modules but does not encode a polysaccharide lyase (PL). Many carbohydrate esterases (CEs) belonging to the family CE12 play crucial roles as pectin acetylesterases, and 14 genes were upregulated in the IM (fungus grown on inducing medium) among 17 expressed CE12 family genes. In addition, most of the genes in the GH132 CAZyme family showed a greater than 5-fold increase in expression in the IM compared with that in the wild type. Furthermore, 122 P450-encoding genes grouped into 11 families were detected in the fungal genome, most of which belonged to the CYP547 family (36 genes) followed by CYP548 (27 genes) and CYP5856 (25 genes). Interestingly, members of the families CYP5014 and CYP5136 were identified, the first time such enzymes have been described in a fungus. Our findings provide new insights into the genomics and genomic features of the saprophytic basal fungus C. heterosporus.Key Points• Genome of the saprobiotic basal fungus C. heterosporus was sequenced and analyzed.• 394 CAZymes but no PL family genes were found and expression levels were determined.• CE12 and GH132 proteins may play roles in the pectin and plant material degradation.• A large number of P450s but few P450 families existed in the fungus.

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice.

Rice (Oryza sativa) responds to various abiotic stresses during growth. Plant-specific NAM, ATAF1/2, and CUC2 (NAC) transcription factors (TFs) play an important role in controlling numerous vital growth and developmental processes. To date, 170 NAC TFs have been reported in rice, but their roles remain largely unknown. Herein, we discovered that the TF OsNAC006 is constitutively expressed in rice, and regulated by H2O2, cold, heat, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), NaCl, and polyethylene glycol (PEG) 6000 treatments. Furthermore, knockout of OsNAC006 using the CRISPR-Cas9 system resulted in drought and heat sensitivity. RNA sequencing (RNA-seq) transcriptome analysis revealed that OsNAC006 regulates the expression of genes mainly involved in response to stimuli, oxidoreductase activity, cofactor binding, and membrane-related pathways. Our findings elucidate the important role of OsNAC006 in drought responses, and provide valuable information for genetic manipulation to enhance stress tolerance in future plant breeding programs.

MrSVP, a secreted virulence-associated protein, contributes to thermotolerance and virulence of the entomopathogenic fungus Metarhizium robertsii.

BACKGROUND: Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies. RESULTS: The gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock-related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT50) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type-and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection-revealed that MrSVP is required for virulence in the insect haemocoel. CONCLUSIONS: Overall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection.

An easily-performed high-throughput method for plant genomic DNA extraction.

Genomic DNA isolation is a crucial technique for researchers studying plant molecular biology. A current widely-used protocol for DNA extraction needs a pestle and mortal for each sample and consumes a large amount of liquid nitrogen in grinding the samples. Most high-throughput methods depend on expensive machines for tissue homogenization. Here we developed a CTAB-based DNA extraction method using 2.0 mL microcentrifuge tubes for sample processing. This protocol has the advantages that it is suitable for a variety of plants, easily-performed without special equipment, and high-throughput; it effectively avoids sample cross-contamination, and is inexpensive, rapid and safe.

Complete genome sequence of a novel partitivirus from the entomogenous fungus Beauveria bassiana in China.

In this study, we report a novel double-stranded RNA (dsRNA) virus, Beauveria bassiana partitivirus 3 (BbPV-3), derived from the entomogenous fungus Beauveria bassiana isolate RCEF5853 from China. The genome of BbPV-3, whose sequence was determined by metagenomic sequencing, RT-PCR, and RACE cloning, comprises two dsRNA genome segments that are 1,856 and 1,719 bp long. The first segment contains a single ORF (ORF-1) encoding a 584-amino-acid-long protein (66.05 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif. The second segment also has a single ORF (ORF-2) encoding a 500-amino-acid-long coat protein (CP) (55.9 kDa). The CP and RdRp sequences showed highest identity of 43.4% and 60.2%, respectively, to those of Colletotrichum eremochloae partitivirus 1. Phylogenetic analysis of the RdRp domain of the polyprotein revealed that BbPV-3 grouped together with the members of the genus Epsilonpartitivirus. Hence, we proposed that Beauveria bassiana partitivirus 3 is a novel member of the proposed genus Epsilonpartitivirus.

The Highly Conserved Barley Powdery Mildew Effector BEC1019 Confers Susceptibility to Biotrophic and Necrotrophic Pathogens in Wheat.

Effector proteins secreted by plant pathogens play important roles in promoting colonization. Blumeria effector candidate (BEC) 1019, a highly conserved metalloprotease of Blumeria graminis f. sp. hordei (Bgh), is essential for fungal haustorium formation, and silencing BEC1019 significantly reduces Bgh virulence. In this study, we found that BEC1019 homologs in B. graminis f. sp. tritici (Bgt) and Gaeumannomyces graminis var. tritici (Ggt) have complete sequence identity with those in Bgh, prompting us to investigate their functions. Transcript levels of BEC1019 were abundantly induced concomitant with haustorium formation in Bgt and necrosis development in Ggt-infected plants. BEC1019 overexpression considerably increased wheat susceptibility to Bgt and Ggt, whereas silencing this gene using host-induced gene silencing significantly enhanced wheat resistance to Bgt and Ggt, which was associated with hydrogen peroxide accumulation, cell death, and pathogenesis-related gene expression. Additionally, we found that the full and partial sequences of BEC1019 can trigger cell death in Nicotiana benthamiana leaves. These results indicate that Bgt and Ggt can utilize BEC1019 as a virulence effector to promote plant colonization, and thus these genes represent promising new targets in breeding wheat cultivars with broad-spectrum resistance.

Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants.

RNA interference (RNAi), based on hairpin RNA (hpRNA) expression, plays an important role in functional analysis of plant genes. Traditional methods for making RNAi constructs usually involve multiple time-consuming cloning steps. We have developed a Gateway-compatible binary vector for RNAi-mediated gene knockdown in plants from pCAMBIA2301 and pHANNIBAL vectors. The new plant RNAi binary vector, named pCAMBIA2301-GW-RNAi, has two inverted repeated Gateway cassettes driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter. This enables site-specific recombination at two sites by one Gateway LR reaction without restriction enzymes and ligases. The pCAMBIA2301-GW-RNAi vector's effectiveness was evaluated by Agrobacterium-mediated transient co-expression assays of overexpression and silencing constructs of HvCEBiP in Nicotiana benthamiana followed by western blot analysis. Obtained results show that the developed RNAi vector successfully knocked down 35S-driven expression of HvCEBiP, as expression levels of the encoded HvCEBiP protein were significantly reduced.

MiR-429 improved the hypoxia tolerance of human amniotic cells by targeting HIF-1α.

MicroRNA-429(miR-429) plays an important role in mesenchymal stem cells. Hypoxia-inducible factor 1α (HIF-1α) is a nuclear transcription factor that regulates the proliferation, apoptosis and tolerance to hypoxia of mesenchymal stem cells. HIF-1α is also a target gene of miR-429. We investigated whether miR-429 plays a role in hypoxia tolerance with HIF-1α in human amniotic mesenchymal stem cells (hAMSCs). The expression of miR-429 was increased by hypoxia in hAMSCs. miR-429 expression resulted in decreased HIF-1α protein level, but little effect on HIF-1α mRNA. While overexpression of HIF-1α increased the survival rate and exhibited anti-apoptosis effects in hAMSCs under hypoxia, co-expression of miR-429 reduced survival and increased apoptosis. However, miR-429 silencing with HIF-1α overexpression stimulated cell survival and reduced apoptosis. Co-expression of HIF-1α and miR-429 reduced VEGF and Bcl-2 proteins and increased Bax and C-Caspase-3 levels in hAMSCs under hypoxia compared with cells expressing only HIF-1α; cells with HIF-1α overexpression and miR-429 silencing showed the opposite effects. These results indicate that HIF-1α and angomiR-429 reciprocally antagonized each other, while HIF-1α and antagomiR-429 interacted with each other to regulate survival and apoptosis in hAMSCs under hypoxia. miR-429 increased VEGF and Bcl-2 protein levels and decreased Bax and cleaved Caspase-3 protein levels by promoting the synthesis of HIF-1α. These results indicate that miR-429 negatively regulates the survival and anti-apoptosis ability of hAMSCs by mediating HIF-1α expression and improves the ability of hAMSCs to tolerate hypoxia.