A viral movement protein co-opts endoplasmic reticulum luminal-binding protein and calreticulin to promote intracellular movement.

Intracellular movement is an important step for the initial spread of virus in plants during infection. This process requires virus-encoded movement proteins (MPs) and their interaction with host factors. Despite the large number of known host factors involved in the movement of different viruses, little is known about host proteins that interact with one of the MPs encoded by potexviruses, the triple-gene-block protein 3 (TGBp3). The main obstacle lies in the relatively low expression level of potexviral TGBp3 in hosts and the weak or transient nature of interactions. Here, we used TurboID-based proximity labeling to identify the network of proteins directly or indirectly interacting with the TGBp3 of a potexvirus, Bamboo mosaic virus (BaMV). Endoplasmic reticulum (ER) luminal-binding protein 4 and calreticulin 3 of Nicotiana benthamiana (NbBiP4 and NbCRT3, respectively) associated with the functional TGBp3-containing BaMV movement complexes, but not the movement-defective mutant, TGBp3M. Fluorescent microscopy revealed that TGBp3 colocalizes with NbBiP4 or NbCRT3 and the complexes move together along ER networks to cell periphery in N. benthamiana. Loss- and gain-of-function experiments revealed that NbBiP4 or NbCRT3 is required for the efficient spread and accumulation of BaMV in infected leaves. In addition, overexpression of NbBiP4 or NbCRT3 enhanced the targeting of BaMV TGBp1 to plasmodesmata (PD), indicating that NbBiP4 and NbCRT3 interact with TGBp3 to promote the intracellular transport of virion cargo to PD that facilitates virus cell-to-cell movement. Our findings revealed additional roles for NbBiP4 and NbCRT3 in BaMV intracellular movement through ER networks or ER-derived vesicles to PD, which enhances the spread of BaMV in N. benthamiana.

Targeting histone deacetylase-3 blocked epithelial-mesenchymal plasticity and metastatic dissemination in gastric cancer.

BACKGROUND AND PURPOSE: Histone deacetylase (HDAC) inhibitors (HDIs) can modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs are attracted much attention in the field of drug discovery. This study aimed to discern the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression. EXPERIMENTAL APPROACH: Clinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signaling were analyzed using binding assays, differential scanning calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting, and Wnt/ß-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by PET/CT tomography. KEY RESULTS: HDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPß signaling, which could be reversed by the over-expression of plasmids of NFκBp65/CEBPß. Treatments with 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing inhibited Honokiol-inhibited NFκBp65/CEBPß activation. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/ß-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo. CONCLUSIONS AND IMPLICATIONS: Honokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked. Conceptual model showing the working hypothesis on the interaction among Honokiol, HDAC3, and ER stress in the peritoneal dissemination of gastric cancer. Honokiol targeting HDAC3 by ER stress cascade and mitigating the peritoneal spread of gastric cancer. Honokiol-induced ER stress-activated calpain activity targeted HDAC3 and blocked Tyr298 phosphorylation, subsequently blocked cooperating with EMT transcription factors and cancer progression. The present study provides evidence to demonstrate that HDAC3 is a positive regulator of EMT and metastatic growth of gastric cancer cells. The findings here imply that overexpressed HDAC3 is a potential therapeutic target for honokiol to reverse EMT and prevent gastric cancer migration, invasion, and metastatic dissemination. • Honokiol significantly abolished HDAC3 activity on catalytic tyrosine 298 residue site. In addition, Honokiol-induced ER stress markedly inhibited HDAC3 expression via inhibition of NFκBp65/CEBPß signaling. • HDAC3, which is a positive regulator of metastatic gastric cancer cell growth, can be significantly inhibited by Honokiol. • Opportunities for HDAC3 inhibition may be a potential therapeutic target for preventing gastric cancer metastatic dissemination.

Specificity Enhancement of Glutenase Bga1903 toward Celiac Disease-Eliciting Pro-Immunogenic Peptides via Active-Site Modification.

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease.

The Bph45 Gene Confers Resistance against Brown Planthopper in Rice by Reducing the Production of Limonene.

Brown planthopper (BPH), a monophagous phloem feeder, consumes a large amount of photoassimilates in rice and causes wilting. A near-isogenic line 'TNG71-Bph45' was developed from the Oryza sativa japonica variety 'Tainung 71 (TNG71) carrying a dominant BPH-resistance locus derived from Oryza nivara (IRGC 102165) near the centromere of chromosome 4. We compared the NIL (TNG71-Bph45) and the recurrent parent to explore how the Bph45 gene confers BPH resistance. We found that TNG71-Bph45 is less attractive to BPH at least partially because it produces less limonene. Chiral analysis revealed that the major form of limonene in both rice lines was the L-form. However, both L- and D-limonene attracted BPH when applied exogenously to TNG71-Bph45 rice. The transcript amounts of limonene synthase were significantly higher in TNG71 than in TNG71-Bph45 and were induced by BPH infestation only in the former. Introgression of the Bph45 gene into another japonica variety, Tainan 11, also resulted in a low limonene content. Moreover, several dominantly acting BPH resistance genes introduced into the BPH-sensitive IR24 line compromised its limonene-producing ability and concurrently decreased its attractiveness to BPH. These observations suggest that reducing limonene production may be a common resistance strategy against BPH in rice.

NbPsbO1 Interacts Specifically with the Bamboo Mosaic Virus (BaMV) Subgenomic RNA (sgRNA) Promoter and Is Required for Efficient BaMV sgRNA Transcription.

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in the infection cycle through the combined activities of viral replicase and host proteins. However, knowledge about host proteins involved in direct sgRNA promoter recognition is limited. Here, in the partially purified replicase complexes from Bamboo mosaic virus (BaMV)-infected tissue, we have identified the Nicotiana benthamiana photosystem II oxygen-evolving complex protein, NbPsbO1, which specifically interacted with the promoter of sgRNA but not that of genomic RNA (gRNA). Silencing of NbPsbO1 expression suppressed BaMV accumulation in N. benthamiana protoplasts without affecting viral gRNA replication. Overexpression of wild-type NbPsbO1 stimulated BaMV sgRNA accumulation. Fluorescent microscopy examination revealed that the fluorescence associated with NbPsbO1 was redistributed from chloroplast granal thylakoids to stroma in BaMV-infected cells. Overexpression of a mislocalized mutant of NbPsbO1, dTPPsbO1-T7, inhibited BaMV RNA accumulation in N. benthamiana, whereas overexpression of an NbPsbO1 derivative, sPsbO1-T7, designed to be targeted to chloroplast stroma, upregulated the sgRNA level. Furthermore, depletion of NbPsbO1 in BaMV RdRp preparation significantly inhibited sgRNA synthesis in vitro but exerted no effect on (+) or (-) gRNA synthesis, which indicates that NbPsbO1 is required for efficient sgRNA synthesis. These results reveal a novel role for NbPsbO1 in the selective enhancement of BaMV sgRNA transcription, most likely via direct interaction with the sgRNA promoter. IMPORTANCE Production of subgenomic RNAs (sgRNAs) for efficient translation of downstream viral proteins is one of the major strategies adapted for viruses that contain a multicistronic RNA genome. Both viral genomic RNA (gRNA) replication and sgRNA transcription rely on the combined activities of viral replicase and host proteins, which recognize promoter regions for the initiation of RNA synthesis. However, compared to the cis-acting elements involved in the regulation of sgRNA synthesis, the host factors involved in sgRNA promoter recognition mostly remain to be elucidated. Here, we found a chloroplast protein, NbPsbO1, which specifically interacts with Bamboo mosaic virus (BaMV) sgRNA promoter. We showed that NbPsbO1 is relocated to the BaMV replication site in BaMV-infected cells and demonstrated that NbPsbO1 is required for efficient BaMV sgRNA transcription but exerts no effect on gRNA replication. This study provides a new insight into the regulating mechanism of viral gRNA and sgRNA synthesis.

Application Potential of Bacterial Volatile Organic Compounds in the Control of Root-Knot Nematodes.

Plant-parasitic nematodes (PPNs) constitute the most damaging group of plant pathogens. Plant infections by root-knot nematodes (RKNs) alone could cause approximately 5% of global crop loss. Conventionally, chemical-based methods are used to control PPNs at the expense of the environment and human health. Accordingly, the development of eco-friendly and safer methods has been urged to supplement or replace chemical-based methods for the control of RKNs. Using microorganisms or their metabolites as biological control agents (BCAs) is a promising approach to controlling RKNs. Among the metabolites, volatile organic compounds (VOCs) have gained increasing attention because of their potential in the control of not only RKNs but also other plant pathogens, such as insects, fungi, and bacteria. This review discusses the biology of RKNs as well as the status of various control strategies. The discovery of VOCs emitted by bacteria from various environmental sources and their application potential as BCAs in controlling RKNs are specifically addressed.

Fumigant Activity of Bacterial Volatile Organic Compounds against the Nematodes Caenorhabditis elegans and Meloidogyne incognita.

Plant-parasitic nematodes infect a diversity of crops, resulting in severe economic losses in agriculture. Microbial volatile organic compounds (VOCs) are potential agents to control plant-parasitic nematodes and other pests. In this study, VOCs emitted by a dozen bacterial strains were analyzed using solid-phase microextraction followed by gas chromatography-mass spectrometry. Fumigant toxicity of selected VOCs, including dimethyl disulfide (DMDS), 2-butanone, 2-pentanone, 2-nonanone, 2-undecanone, anisole, 2,5-dimethylfuran, glyoxylic acid, and S-methyl thioacetate (MTA) was then tested against Caenorhabditis elegans. DMDS and MTA exhibited much stronger fumigant toxicity than the others. Probit analysis suggested that the values of LC50 were 8.57 and 1.43 µg/cm3 air for DMDS and MTA, respectively. MTA also showed stronger fumigant toxicity than DMDS against the root-knot nematode Meloidogyne incognita, suggesting the application potential of MTA.

The differential presence of human polyomaviruses, JCPyV and BKPyV, in prostate cancer and benign prostate hypertrophy tissues.

BACKGROUND: Studies have shown that human polyomavirus infection may be associated with various human cancers. We investigated the potential relationship between the prevalence of JCPyVor BKPyV and prostate cancer (PC) in patients from Taiwan. METHODS: Patients with PC and benign prostate hypertrophy (BPH; 76 and 30 patients, respectively) were recruited for this study. Paraffin-embedded tissues and clinical information of the patients were obtained. The tissue sections were used for viral DNA detection and immunohistochemistry analysis was performed for examining viral large T (LT) and VP1 proteins. Regression analysis was used to evaluate the relationship between the clinical characteristics of the patients and the risk of JCPyV/BKPyV infection. RESULTS: The prevalence of JCPyV/BKPyV DNA was different in PC and BPH tissues (27/76 [35.52%] and 2/30 [6.7%], respectively, p = 0.003)]. The LT and VP1 proteins were detected in 27 (35.52%) and 29 PC (38.2%) specimens, respectively, but neither protein was detected in BPH samples (p < 0.001). PC cells were more susceptible to JCPyV infection than BPH tissues [odds ratio (OR) 7.71, 95% CI: 1.71-34.09, p = 0.003). Patients with PC showing high levels of prostate-specific antigen and high Gleason scores were associated with a high risk of viral infection (ORs 1.1, 95% CI 1.000-1.003; p = 0.045 and ORs 6.18, 95% CI 1.26-30.33, p = 0.025, respectively). The expression of LT protein associated with the risk of PC increased 2923.39-fold (95% CI 51.19-166,963.62, p < 0.001). CONCLUSIONS: The findings indicate that JCPyV infection in PC cells may be associated with prostate cancer progression and prognosis.

Fungicidal Activity of Volatile Organic Compounds Emitted by Burkholderia gladioli Strain BBB-01.

A Burkholderia gladioli strain, named BBB-01, was isolated from rice shoots based on the confrontation plate assay activity against several plant pathogenic fungi. The genome of this bacterial strain consists of two circular chromosomes and one plasmid with 8,201,484 base pairs in total. Pangenome analysis of 23 B. gladioli strains suggests that B. gladioli BBB-01 has the closest evolutionary relationship to B. gladioli pv. gladioli and B. gladioli pv. agaricicola. B. gladioli BBB-01 emitted dimethyl disulfide and 2,5-dimethylfuran when it was cultivated in lysogeny broth and potato dextrose broth, respectively. Dimethyl disulfide is a well-known pesticide, while the bioactivity of 2,5-dimethylfuran has not been reported. In this study, the inhibition activity of the vapor of these two compounds was examined against phytopathogenic fungi, including Magnaporthe oryzae, Gibberella fujikuroi, Sarocladium oryzae, Phellinus noxius and Colletotrichumfructicola, and human pathogen Candida albicans. In general, 2,5-dimethylfuran is more potent than dimethyl disulfide in suppressing the growth of the tested fungi, suggesting that 2,5-dimethylfuran is a potential fumigant to control plant fungal disease.

Proliferating Cell Nuclear Antigen Suppresses RNA Replication of Bamboo Mosaic Virus through an Interaction with the Viral Genome.

Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail mosaic virus and Potato virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexviruses.IMPORTANCE Knowing the dynamic interplay between plant RNA viruses and their host is a basic step toward first understanding how the viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexviruses, including Bamboo mosaic virus, Foxtail mosaic virus, and Potato virus X Based on the tests on Bamboo mosaic virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexviruses in the rapidly dividing tissues of plants.

Increased inflammation in rheumatoid arthritis patients living where farm soils contain high levels of copper.

BACKGROUND/PURPOSE: Heavy metal pollution in farm soils is a problem in some parts of Taiwan. Copper can be a factor associated with increased disease activities of rheumatoid arthritis (RA). Thus, the aim of this study was to investigate whether copper pollution in farm soils is associated with worsened RA. METHODS: Clinical parameters from 122 RA patients were collected from a medical center in central Taiwan. Levels of heavy metals in the blood were measured using inductively coupled plasma mass spectrometry. Levels of copper in farm soils were retrieved from a national survey. These data were analyzed to find the factors related to RA disease activities. RESULTS: RA patients living where farm soils contained high levels of copper had increased white blood cell counts, erythrocyte sedimentation rate, and disease activity score 28, compared with patients living where copper levels were low. Among the nine types of heavy metal measured in the study, blood levels of copper and nickel correlated with erythrocyte sedimentation rate. CONCLUSION: Our cross-sectional data suggest a correlation between RA disease activity and the level of copper at township farm soils samples. Further longitudinal studies using more rigorous methodologies are warranted to examine whether this correlation is causal.

The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.

The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

Phosphorylation of coat protein by protein kinase CK2 regulates cell-to-cell movement of Bamboo mosaic virus through modulating RNA binding.

In this study, we investigated the fine regulation of cell-to-cell movement of Bamboo mosaic virus (BaMV). We report that the coat protein (CP) of BaMV is phosphorylated in planta at position serine 241 (S241), in a process involving Nicotiana benthamiana casein kinase 2α (NbCK2α). BaMV CP and NbCK2α colocalize at the plasmodesmata, suggesting that phosphorylation of BaMV may be involved in its movement. S241 was mutated to examine the effects of temporal and spatial dysregulation of phosphorylation on i) the interactions between CP and viral RNA and ii) the regulation of cell-to-cell movement. Replacement of S241 with alanine did not affect RNA binding affinity but moderately impaired cell-to-cell movement. A negative charge at position 241 reduced the ability of CP to bind RNA and severely interfered with cell-to-cell movement. Deletion of residues 240 to 242 increased the affinity of CP to viral RNA and dramatically impaired cell-to-cell movement. A threonine at position 241 changed the binding preference of CP toward genomic RNA and inhibited cell-to-cell movement. Together, these results reveal a fine regulatory mechanism for the cell-to-cell movement of BaMV, which involves the modulation of RNA binding affinity through appropriate phosphorylation of CP by NbCK2α.

Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR) of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione.

The gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from Rhodococcus equi USA-18, a cholesterol oxidase-producing strain formerly named Arthrobacter simplex USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in R. equi USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain that could accumulate up to 0.58 mg/ml 1,4-androstadiene-3,17-dione (ADD) in the culture medium when 0.2% cholesterol was used as the carbon source, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. In addition, this mutant also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC). Because both ADD and Δ1,4-BNC are important intermediates for the synthesis of steroid drugs, this mutant derived from R. equi USA-18 may deserve further investigation for its application potential.

A 24.7-kDa copper-containing oxidase, secreted by Thermobifida fusca, significantly increasing the xylanase/cellulase-catalyzed hydrolysis of sugarcane bagasse.

Thermobifida fusca is a moderately thermophilic soil bacterium belonging to Actinobacteria. It has been known for its capability to degrade plant cell wall polymers except lignin and pectin. To know whether it can produce enzymes to facilitate lignin degradation, the extracellular proteins bound to sugarcane bagasse were harvested and identified by liquid chromatography tandem mass spectrometry. Among the identified proteins, a putative copper-containing polyphenol oxidase of 241 amino acids, encoded by the locus Tfu_1114, was thought to presumably play a role in lignin degradation. This protein (Tfu1114) was thus expressed in E. coli and characterized. Similarly to common laccases, Tfu1114 is able to catalyze the oxidation reaction of phenolic and nonphenolic lignin related compounds such as 2,6-dimethoxyphenol and veratryl alcohol. More interestingly, it can significantly enhance the enzymatic hydrolysis of bagasse by xylanase and cellulase. Tfu1114 is stable against heat, with a half-life of 4.7 h at 90 °C, and organic solvents. It is sensitive to ethylenediaminetetraacetic acid and reducing agents but resistant to sodium azide, a potent inhibitor of laccases. Atomic absorption spectroscopy indicated that the ratio of copper to the protein monomer is 1, instead of 4, a feature of classical laccases. All these data suggest that Tfu1114 is a novel oxidase with laccase-like activity, potentially useful in biotechnology application.

Gallic acid provokes DNA damage and suppresses DNA repair gene expression in human prostate cancer PC-3 cells.

Our earlier studies have demonstrated that gallic acid (GA) induced cytotoxic effects including induction of apoptosis and DNA damage and inhibited the cell migration and invasion in human cancer cells. However, GA-affected DNA damage and repair gene expressions in human prostate cancer cells are still unclear. In this study, we investigated whether or not GA induces DNA damage and inhibits DNA repair gene expression in a human prostate cancer cell line (PC-3). The results from flow cytometric assay indicated that GA decreased the percentage of viable PC-3 cells in a dose- and time-dependent manner. PC-3 cells after exposure to different doses (50, 100, and 200 µM) of GA and various periods of time (12, 24, and 48 h) led to a longer DNA migration smear (comet tail) occurred based on the single cell gel electrophoresis (comet assay). These observations indicated that GA-induced DNA damage in PC-3 cells, which also confirmed by 4,6-diamidino-2-phenylindole dihydrochloride staining and DNA agarose gel electrophoresis. Alternatively, results from real-time polymerase chain reaction assay also indicated that GA inhibited ataxia telangiectasia mutated, ataxia-telangiectasia and Rad3-related, O6-methylguanine-DNA methyltransferase, DNA-dependent serine/threonine protein kinase, and p53 mRNA expressions in PC-3 cells. Taken together, the present study showed that GA caused DNA damage and inhibited DNA repair genes as well as both effects may be the critical factors for GA-inhibited growth of PC-3 cells in vitro.

Hydrolysis of Gluten-Derived Celiac Disease-Triggering Peptides across a Broad pH Range by RmuAP1: A Novel Aspartic Peptidase Isolated from Rhodotorula mucilaginosa.

An aspartate peptidase with proteolytic activity toward gluten was identified from an isolated red yeast Rhodotorula mucilaginosa strain. This peptidase consists of 425 amino acids, comprising an N-terminal signal peptide, a propeptide, and a C-terminal catalytic domain. The catalytic domain, termed RmuAP1CD, could be secreted by the recombinant oleaginous yeast Yarrowia lipolytica, whose genome contains the expression cassette for RmuAP1CD. RmuAP1CD exhibited optimum activity at pH 2.5 when acting on bovine serum albumin. Moreover, it facilitated the hydrolysis of gluten-derived immunogenic peptides (GIPs), which are responsible for triggering celiac disease symptoms, across a pH range of 3.0-6.0. The preferred cleavage sites are P-Q-Q-↓-P-Q in the 26-mer and P-Q-L-↓-P-Y in the 33-mer GIPs. Conversely, porcine pepsin cannot hydrolyze these two GIPs. The ability of RmuAP1CD to degrade GIPs under acidic conditions of the stomach indicates its potential as a viable oral enzyme therapy for celiac disease.

Harnessing microbial phylum-specific molecular markers for assessment of environmental estrogen degradation.

Steroidal estrogens are ubiquitous contaminants that have garnered attention worldwide due to their endocrine-disrupting and carcinogenic activities at sub-nanomolar concentrations. Microbial degradation is one of the main mechanisms through which estrogens can be removed from the environment. Numerous bacteria have been isolated and identified as estrogen degraders; however, little is known about their contribution to environmental estrogen removal. Here, our global metagenomic analysis indicated that estrogen degradation genes are widely distributed among bacteria, especially among aquatic actinobacterial and proteobacterial species. Thus, by using the Rhodococcus sp. strain B50 as the model organism, we identified three actinobacteria-specific estrogen degradation genes, namely aedGHJ, by performing gene disruption experiments and metabolite profile analysis. Among these genes, the product of aedJ was discovered to mediate the conjugation of coenzyme A with a unique actinobacterial C17 estrogenic metabolite, 5-oxo-4-norestrogenic acid. However, proteobacteria were found to exclusively adopt an α-oxoacid ferredoxin oxidoreductase (i.e., the product of edcC) to degrade a proteobacterial C18 estrogenic metabolite, namely 3-oxo-4,5-seco-estrogenic acid. We employed actinobacterial aedJ and proteobacterial edcC as specific biomarkers for quantitative polymerase chain reaction (qPCR) to elucidate the potential of microbes for estrogen biodegradation in contaminated ecosystems. The results indicated that aedJ was more abundant than edcC in most environmental samples. Our results greatly expand the understanding of environmental estrogen degradation. Moreover, our study suggests that qPCR-based functional assays are a simple, cost-effective, and rapid approach for holistically evaluating estrogen biodegradation in the environment.

The interaction between bamboo mosaic virus replication protein and coat protein is critical for virus movement in plant hosts.

Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.