Transreplication Preference of the Tomato Leaf Curl Joydebpur Virus for a Noncognate Betasatellite through Iteron Resemblance on Nicotiana bethamiana.

Pepper plants (Capsicum annuum) with severe leaf curl symptoms were collected in 2013 from Bangalore, Karnataka, India. The detection results showed a co-infection between the tomato leaf curl Joydebpur virus (ToLCJoV) and tomato leaf curl Bangladesh betasatellite (ToLCBDB) through the sequencing analysis of PCR amplicons. To pinpoint the molecular mechanism of this uncommon combination, infectious clones of ToLCJoV and two different betasatellites-ToLCBDB and tomato leaf curl Joydebpur betasatellite (ToLCJoB)-were constructed and tested for their infectivity in Nicotiana benthamiana. Together, we conducted various combined agroinoculation studies to compare the interaction of ToLCJoV with non-cognate and cognate betasatellites. The natural non-cognate interaction between ToLCJoV and ToLCBDB showed severe symptoms compared to the mild symptoms of a cognate combination (ToLCJoV × ToLCJoB) in infected plants. A sequence comparison among betasatellites and their helper virus wasperformed and the iteron resemblances in ToLCBDB as well as ToLCJoB clones were processed. Mutant betasatellites that comprised iteron modifications revealed that changes in iteron sequences could disturb the transreplication process between betasatellites and their helper virus. Our study might provide an important consideration for determining the efficiency of transreplication activity between betasatellites and their helper virus.

M13KO7 bacteriophage enables Potato Virus Y detection.

IMPORTANCE: In this study, we confirmed the binding of M13KO7 to Potato virus Y (PVY) using enzyme-linked immunosorbent assay. M13KO7 is a "bald" bacteriophage in which no recombinant antibody is displayed. M13KO7 is easy to propagate by using Escherichia coli, making this method more reasonable in economic perspective. Based on this study, we suggest that M13KO7 detection system has applicability as a novel biological tool for the detection of PVY.

Coat protein is responsible for tomato leaf curl New Delhi virus pathogenicity in tomato.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite Begomovirus belonging to the family Geminiviridae, causes severe damage to many economically important crops worldwide. In the present study, pathogenicity of Asian (ToLCNDV-In from Pakistan) and Mediterranean isolates (ToLCNDV-ES from Italy) were examined using infectious clones in tomato plants. Only ToLCNDV-In could infect the three tomato cultivars, whereas ToLCNDV-ES could not. Genome-exchange of the two ToLCNDVs revealed the ToLCNDV DNA-A segment as the main factor for ToLCNDV infectivity in tomato. In addition, serial clones with chimeric ToLCNDV-In A and ToLCNDV-ES A genome segments were generated to identify the region determining viral infectivity in tomatoes. A chimeric clone carrying the ToLCNDV-In coat protein (CP) exhibited pathogenic adaptation in tomatoes, indicating that the CP of ToLCNDV is essential for its infectivity. Analyses of infectious clones carrying a single amino acid substitution revealed that amino acid at position 143 of the CP is critical for ToLCNDV infectivity in tomatoes. To better understand the molecular basis whereby CP function in pathogenicity, a yeast two-hybrid screen of a tomato cDNA library was performed using CPs as bait. The hybrid results showed different interactions between the two CPs and Ring finger protein 44-like in the tomato genome. The relative expression levels of upstream and downstream genes and Ring finger 44-like genes were measured using quantitative reverse transcription PCR (RT-qPCR) and compared to those of control plants. This is the first study to compare the biological features of the two ToLCNDV strains related to viral pathogenicity in the same host plant. Our results provide a foundation for elucidating the molecular mechanisms underlying ToLCNDV infection in tomatoes.

Insights into the Differential Composition of Stem-Loop Structures of Nanoviruses and Their Impacts.

Multipartite viruses package their genomic segments independently and mainly infect plants; few of them target animals. Nanoviridae is a family of multipartite single-stranded DNA (ssDNA) plant viruses that individually encapsidate ssDNAs of ~1 kb and transmit them through aphids without replication in aphid vectors, thereby causing important diseases in host plants, mainly leguminous crops. All of these components constitute an open reading frame to perform a specific role in nanovirus infection. All segments contain conserved inverted repeat sequences, potentially forming a stem-loop structure and a conserved nonanucleotide, TAGTATTAC, within a common region. This study investigated the variations in the stem-loop structure of nanovirus segments and their impact using molecular dynamics (MD) simulations and wet lab approaches. Although the accuracy of MD simulations is limited by force field approximations and simulation time scale, explicit solvent MD simulations were successfully used to analyze the important aspects of the stem-loop structure. This study involves the mutants' design, based on the variations in the stem-loop region and construction of infectious clones, followed by their inoculation and expression analysis, based on nanosecond dynamics of the stem-loop structure. The original stem-loop structures showed more conformational stability than mutant stem-loop structures. The mutant structures were expected to alter the neck region of the stem-loop by adding and switching nucleotides. Changes in conformational stability are suggested expression variations of the stem-loop structures found in host plants with nanovirus infection. However, our results can be a starting point for further structural and functional analysis of nanovirus infection. IMPORTANCE Nanoviruses comprise multiple segments, each with a single open reading frame to perform a specific function and an intergenic region with a conserved stem-loop region. The genome expression of a nanovirus has been an intriguing area but is still poorly understood. We attempted to investigate the variations in the stem-loop structure of nanovirus segments and their impact on viral expression. Our results show that the stem-loop composition is essential in controlling the virus segments' expression level.

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana.

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus Mastrevirus (family Geminiviridae) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into Nicotiana benthamiana using three Agrobacterium tumefaciens strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The A. tumefaciens strain LBA4404 was the most effective at transferring the SPSMV-1 genome to N. benthamiana. We confirmed the viral replication in N. benthamiana samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

Transcriptional Analysis of the Differences between ToLCNDV-India and ToLCNDV-ES Leading to Contrary Symptom Development in Cucumber.

Tomato leaf curl New Delhi virus-ES (ToLCNDV-ES), a high threat to cucurbits in the Mediterranean Basin, is listed as a different strain from the Asian ToLCNDV isolates. In this study, the infectivity of two clones previously isolated from Italy and Pakistan were compared in cucumbers, which resulted in the opposite symptom appearance. The swapping subgenome was processed; however, the mechanisms related to the disease phenotype remain unclear. To identify the disease-associated genes that could contribute to symptom development under the two ToLCNDV infections, the transcriptomes of ToLCNDV-infected and mock-inoculated cucumber plants were compared 21 days postinoculation. The number of differentially expressed genes in ToLCNDV-India-infected plants was 10 times higher than in ToLCNDV-ES-infected samples. The gene ontology (GO) and pathway enrichment were analyzed using the Cucurbits Genomics Database. The flavonoid pathway-related genes were upregulated in ToLCNDV-ES, but some were downregulated in ToLCNDV-India infection, suggesting their role in resistance to the two ToLCNDV infections. The relative expression levels of the selected candidate genes were validated by qRT-PCR under two ToLCNDV-infected conditions. Our results reveal the different infectivity of the two ToLCNDVs in cucumber and also provide primary information based on RNA-seq for further analysis related to different ToLCNDV infections.

Interspecies Recombination-Led Speciation of a Novel Geminivirus in Pakistan.

Recombination between isolates of different virus species has been known to be one of the sources of speciation. Weeds serve as mixing vessels for begomoviruses, infecting a wide range of economically important plants, thereby facilitating recombination. Chenopodium album is an economically important weed spread worldwide. Here, we present the molecular characterization of a novel recombinant begomovirus identified from C. album in Lahore, Pakistan. The complete DNA- A genome of the virus associated with the leaf distortion occurred in the infected C. album plants was cloned and sequenced. DNA sequence analysis showed that the nucleotide sequence of the virus shared 93% identity with those of the rose leaf curl virus and the duranta leaf curl virus. Interestingly, this newly identified virus is composed of open reading frames (ORFs) from different origins. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the sequences. The infectious clone of the newly detected virus was found to be fully infectious in C. album and Nicotiana benthamiana as the viral DNA was successfully reconstituted from systemically infected tissues of inoculated plants, thus fulfilling Koch's postulates. Our study reveals a new speciation of an emergent ssDNA plant virus associated with C. album through recombination and therefore, proposed the tentative name 'Chenopodium leaf distortion virus' (CLDV).

Different Infectivity of Mediterranean and Southern Asian Tomato Leaf Curl New Delhi Virus Isolates in Cucurbit Crops.

Tomato leaf curl New Delhi virus (ToLCNDV) became an alerting virus in Europe from 2017 to 2020 because of its significant damage to Cucurbitaceae cultivation. Until now, just some cucurbit crops including sponge gourd, melon, pumpkin, and cucumber were reported to be resistant to ToLCNDV, but no commercial cultivars are available. In this study, a new isolate of ToLCNDV was identified in Pakistan and analyzed together with ToLCNDV-ES which was previously isolated in Italy. Furthermore, infectious clones of two ToLCNDV isolates were constructed and agroinoculated into different cucurbit crops to verify their infectivity. Results showed that both isolates exhibited severe infection on all tested cucurbit (>70%) except watermelon. Thus, those cultivars may be good candidates in the first step of screening genetic resources for resistance on both Southeast Asian and Mediterranean ToLCNDV isolates. Additional, comparison pathogenicity of different geographical ToLCNDV isolates will be aided to understand viral characterization as such knowledge could facilitate breeding resistance to this virus.

Fast and Sensitive Real-Time PCR Detection of Major Antiviral-Drug Resistance Mutations in Chronic Hepatitis B Patients by Use of a Predesigned Panel of Locked-Nucleic-Acid TaqMan Probes.

We developed a novel real-time PCR assay that simultaneously evaluates 11 major nucleos(t)ide antiviral (NA) drug resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine [LMV] resistance), N/H238A/T (adefovir [ADF] resistance), which are circulating in Vietnam; and T184G/L, S202I, and M250V (entecavir [ETV] resistance) and A194T (tenofovir resistance), which have been recently reported in several studies across the globe. We detected drug-resistant mt in hepatitis B virus (HBV) samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low-HBV DNA population of ≥5 × 103 IU/ml and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV and ADF resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (children, 42.3%; adults, 36.7%) and rtN238T/A (children, 15.4%; adults, 16.3%) being the most frequent mt in these populations. Multiple-point mt, including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I, were identified in only two children, resulting in LMV-ADF resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in nonhospitalized patients who need their results within half a day, before starting treatment.

Clinical and Pathogenic Characteristics of Lower Respiratory Tract Infection Treated at the Vietnam National Children's Hospital.

Lower respiratory tract infections are commonly caused by viruses and cause significant morbidity and mortality among children. Early identification of the pathological agent causing these infections is essential to avoid unnecessary antibiotic use and improve patient management. Multiplex PCR techniques were recently developed to detect multiple viral pathogens using a single PCR reaction. In this study, we identify viral pathogens in children with respiratory infections. We collected 194 nasopharyngeal aspirates from infants (2-24 months old) with lower respiratory tract infections treated at the Vietnam National Children's Hospital between November 2014 and June 2015 and assessed the presence of 16 virus types and subtypes by multiplex PCR using the xTAG Respiratory Viral Panel (RVP) assay. Overall, 73.7% of the samples were positive for at least one virus, and 24.2% corresponded to infections with multiple viruses. The most common viruses were respiratory syncytial virus and enterovirus/rhinovirus. These viruses were more frequent among younger patients (2-5 months old) and caused symptoms similar to those of bronchiolitis and pneumonia. The most common clinical manifestation caused by respiratory tract infection was bronchiolitis. Elevated neutrophils levels were associated with adenovirus infection. Our results showed that the xTAG Respiratory Viral Panel (RVP) can effectively detect multiple viruses causing respiratory infections in children and that the nasopharyngeal aspirates are a good sample choice to detect respiratory viruses in children. Applying this approach in the clinical setting would improve patient management and allow early diagnosis, thus avoiding the unnecessary use of antibiotics.

The predominance of seafood allergy in Vietnamese adults: Results from the first population-based questionnaire survey.

BACKGROUND: Food allergy (FA) is a serious, costly and growing health problem worldwide. FA occurs in both children and adults; however, there is a paucity of information on FA prevalence and its clinical features in the adult population, especially in Asia. We sought to assess the prevalence of FAs in Vietnamese adults and the distribution of offending food items among different regions throughout Vietnam. METHODS: A nationwide, cross-sectional, population-based survey was conducted among University students aged 16-50 years. We used a structured, anonymous questionnaire, which was modified from recently published FA epidemiologic studies and based on European Academy of Allergy and Clinical Immunology (EAACI) guidelines, to collect data on FA prevalence, clinical presentations, and implicated food groups. Statistical analysis was performed to generate the prevalence of self-reported and doctor-diagnosed FA and to examine the association of key environmental factors and FA incidence in this population. RESULTS: Of the 14,500 surveys distributed, a total of 9,039 responses were returned, resulting in a response rate of 62.4%. Among participants who reported food-induced adverse reactions, 48.0% have repeated reactions. 18.0% of the participants perceived FA symptoms, but less than half of them sought medical services for confirmation (37.9%). Stratifying for true FA symptoms, the prevalence of self-reported FA was 11.8% and of doctor-diagnosed FA, 4.6%. The most common doctor-diagnosed FA was to crustacean (3.0%; 95% CI, 2.6-3.3), followed by fish (1.6%; 95% CI, 1.3-1.8), mollusk (1.3%; 95% CI, 1.0-1.5) and beef (1.0%; 95% CI, 0.8-1.2). The prevalence of doctor-diagnosed FA differed among participants living in urban (6.5%) and rural regions (4.9%) (P < 0.001). Atopic family history was the strongest predictor for FA (odds ratio 8.0; 95% CI, 6.2-10.4). CONCLUSIONS: Seafood allergy among adults is predominant in Vietnam, followed by beef, milk, and egg, while peanut, soy, and tree nut allergy are much less common. Populations in rural regions have considerably less FA; however, the protective environmental factors have yet to be identified.

The threat of seed-transmissible pepper yellow leaf curl Indonesia virus in chili pepper.

Recently, chili pepper (Capsicum annuum) plants in Indonesia have been devastated by a notorious bipartite begomovirus infection named Pepper yellow leaf curl Indonesia virus (PepYLCIV), which causes a distinct decrease in chili pepper production. Pepper yellow diseases have been known since early 2000; however, the spread of this virus thus far is distressing. These diseases can reduce chili yields by 20-100% in Indonesia. As previously known, begomovirus can be transmitted through whitefly to several host plants from the families Solanaceae, Compositae, and Leguminosae. In the field, a single plant was observed with severe symptoms of pepper yellow leaf curl disease, while other plants in the same field were asymptomatic and healthy. The observation leads to the possibility that the virus can be transmitted from previously infected chili pepper plants through seeds, as begomovirus transmission through seeds has been reported before. This study was conducted using seeds from chili peppers infected with viruses from different places in Indonesia. Whole seeds, embryos, and seedlings from PepYLCIV infected seeds were investigated in this study by performing viral genome DNA extraction, uracil DNA glycosylase-PCR, and sequencing analysis. Results revealed that both DNA-A and DNA-B of PepYLCIV in seeds and embryos of infected chili pepper plants were detected. The results also showed that 25-67% of PepYLCIV DNA-A and 50-100% of DNA-B were detected from seedlings grown from infected chili pepper seed collected from different location, thus confirming PepYLCIV as a seed-transmissible virus in chili pepper plants.

Newly emerged enterovirus-A71 C4 sublineage may be more virulent than B5 in the 2015-2016 hand-foot-and-mouth disease outbreak in northern Vietnam.

Enterovirus-A71 (EV-A71) is a common cause of hand-foot-and-mouth disease (HFMD) and, rarely, causes severe neurological disease. This study aimed to elucidate the epidemiological and genetic characteristics and virulence of EV-A71 strains isolated from children diagnosed with HFMD. Rectal and throat swabs were collected from 488 children with HFMD in Hanoi, Vietnam, in 2015-2016. From 391 EV-positive patients, 15 EVs, including coxsackievirus A6 (CV-A6; 47.1%) and EV-A71 (32.5%, n = 127), were identified. Of the 127 EV-A71 strains, 117 (92.1%) were the B5 subgenotype and 10 (7.9%) were the C4 subgenotype. A whole-genome analysis of EV-A71 strains showed that seven of the eight C4a strains isolated in 2016 formed a new lineage, including two possible recombinants between EV-A71 C4 and CV-A8. The proportion of inpatients among C4-infected children was higher than among B5-infected children (80.0% vs. 27.4%; P = 0.002). The virulence of EV-A71 strains was examined in human scavenger receptor class B2 (hSCARB2)-transgenic mice, and EV-A71 C4 strains exhibited higher mortality than B5 strains (80.0% vs. 30.0%, P = 0.0001). Thus, a new EV-A71 C4a-lineage, including two possible recombinants between EV-A71 C4 and CV-A8, appeared in 2016 in Vietnam. The EV-A71 C4 subgenotype may be more virulent than the B5 subgenotype.

A draft genome of the striped catfish, Pangasianodon hypophthalmus, for comparative analysis of genes relevant to development and a resource for aquaculture improvement.

BACKGROUND: The striped catfish, Pangasianodon hypophthalmus, is a freshwater and benthopelagic fish common in the Mekong River delta. Catfish constitute a valuable source of dietary protein. Therefore, they are cultured worldwide, and P. hypophthalmus is a food staple in the Mekong area. However, genetic information about the culture stock, is unavailable for breeding improvement, although genetics of the channel catfish, Ictalurus punctatus, has been reported. To acquire genome sequence data as a useful resource for marker-assisted breeding, we decoded a draft genome of P. hypophthalmus and performed comparative analyses. RESULTS: Using the Illumina platform, we obtained both nuclear and mitochondrial DNA sequences. Molecular phylogeny using the mitochondrial genome confirmed that P. hypophthalmus is a member of the family Pangasiidae and is nested within a clade including the families Cranoglanididae and Ictaluridae. The nuclear genome was estimated at approximately 700 Mb, assembled into 568 scaffolds with an N50 of 14.29 Mbp, and was estimated to contain ~ 28,600 protein-coding genes, comparable to those of channel catfish and zebrafish. Interestingly, zebrafish produce gadusol, but genes for biosynthesis of this sunscreen compound have been lost from catfish genomes. The differences in gene contents between these two catfishes were found in genes for vitamin D-binding protein and cytosolic phospholipase A2, which have lost only in channel catfish. The Hox cluster in catfish genomes comprised seven paralogous groups, similar to that of zebrafish, and comparative analysis clarified catfish lineage-specific losses of A5a, B10a, and A11a. Genes for insulin-like growth factor (IGF) signaling were conserved between the two catfish genomes. In addition to identification of MHC class I and sex determination-related gene loci, the hypothetical chromosomes by comparison with the channel catfish demonstrated the usefulness of the striped catfish genome as a marker resource. CONCLUSIONS: We developed genomic resources for the striped catfish. Possible conservation of genes for development and marker candidates were confirmed by comparing the assembled genome to that of a model fish, Danio rerio, and to channel catfish. Since the catfish genomic constituent resembles that of zebrafish, it is likely that zebrafish data for gene functions is applicable to striped catfish as well.

Prevalence of food allergy in Vietnam: comparison of web-based with traditional paper-based survey.

BACKGROUND: Web-based surveys (WBS) are increasingly applied in epidemiological studies as an appealing alternative to traditional survey methods. Rapid data collection, reduced expenditure and ease of access to large populations are some of the clear advantages of online surveys. However, WBS are still subject to limitations in terms of sample size, response rate and other additional biases compared to traditional survey methods. In the present study, we seek to validate data on food allergy (FA) in two independent sample populations collected from a WBS, and compare it to a paper-based survey (PBS). METHODS: Data collected from two survey modes were compared by hypothesis testing for independent sample population. The WBS included 1185 respondents, while the PBS included 9039 respondents. RESULTS: Overall, the data from the WBS were comparable to the PBS conducted over the same period of time in Vietnamese adults. There were no effects of different survey modes on the lifetime prevalence of doctor-diagnosed FA (5.7%; P = 0.7795, ß = 0.05) and IgE-mediated FA (5.8%; P = 0.9590, ß = 0.05). Both surveys showed the dominance of seafood allergy in this population (up to 2.6%), followed by beef allergy. Close correlation was seen in the patterns of FAs and different clinical symptoms. The contribution of family history of allergic diseases and place of residence to FA were confirmed in both surveys. CONCLUSIONS: The consistency of the WBS results with the PBS indicates a promising application of online surveys as an economic and validated model for future epidemiological studies, specifically in developing countries.

Improvement of a P450-Based Recombinant Escherichia coli Whole-Cell System for the Production of Oxygenated Sesquiterpene Derivatives.

Sesquiterpenes are common constituents of essential oil in plants. Their oxygenated derivatives often possess desirable flavor, fragrance, and pharmaceutical properties. Recently, the CYP264B1-based recombinant Escherichia coli whole-cell system has been constructed for the oxidation of sesquiterpenes. However, limiting factors of this system related to the high volatility of substrates and the suitability of the P450 redox partner need to be addressed. In this work, the improvement of the system was implemented with (+)-α-longipinene as a model substrate. By using 2-hydroxypropyl-ß-cyclodextrin and an alternative ferredoxin reductase, the conversion of (+)-α-longipinene was improved 77.1%. Applying the optimized conditions, the yields of the main products were 54.2, 34.2, and 47.2 mg L-1, corresponding to efficiencies of 82.1, 51.8, and 71.5% for the conversion of (+)-α-longipinene, (-)-isolongifolene, and α-humulene, respectively, at a 200 mL scale. These products were characterized as 12-hydroxy-α-longipinene, isolongifolene-9-one, and 5-hydroxy-α-humulene, respectively, by nuclear magnetic resonance spectroscopy.

New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56.

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non-activated C-H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)-nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.

Characterization of the gene cluster CYP264B1-geoA from Sorangium cellulosum So ce56: biosynthesis of (+)-eremophilene and its hydroxylation.

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1-geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X-ray diffraction were carried out by employing an Escherichia coli whole-cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)-eremophilene synthase. The very tight binding of (+)-eremophilene (∼0.40 µM), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1-geoA gene cluster is required for the biosynthesis of (+)-eremophilene derivatives.

Calbindin-D9k as a sensitive molecular biomarker for evaluating the synergistic impact of estrogenic chemicals on GH3 rat pituitary cells.

Various endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), alkylphenols [4-nonylphenol (NP) and 4-tert octylphenol (OP)] and isobutylparaben (IBP) are a constant concern due to their widespread distribution. It has been reported that some combinations of hormone-disrupting chemicals are much more powerful than any of the chemicals alone. In this study, we measured the expression of an estrogenic biomarker gene, calbindin-D9k (CaBP-9k), and progesterone receptor (PR) to evaluate the individual or combined estrogenic activity of BPA, NP, OP and IBP in GH3 rat pituitary cells. Most doses of the individual compounds and all the doses of the combined chemicals significantly increased CaBP-9k and PR mRNA and protein expression compared to the vehicle (except for PR expression after treatment with OP and NP at 10-7 M). Of note, high doses (10-6 and 10-5 M) of the EDC combinations increased the translational and transcriptional levels of CaBP-9k by 1.3- to 2.4-fold compared to each individual equivalent concentrations of EDCs. To determine whether the increased CaBP-9k gene expression was induced via intracellular estrogen receptor (ER), we blocked ER signaling using fulvestrant, an ER antagonist. The results showed that fulvestrant significantly reversed the CaBP-9k and PR upregulation following treatment with individual EDCs or their combinations. Taken together, we conclude that combinations of BPA, NP, OP and IBP in GH3 rat pituitary cells have synergistic estrogenic activities mediated by ER signaling. In addition, the expression of the CaBP-9k gene may be used as a biomarker to assess the synergistic effects of EDCs in vitro.

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase.

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and ß-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and ß-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-ß-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/ß-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.