Influence of human gut microbiota on the metabolic fate of glucosinolates

Glucosinolates (GSLs) are secondary metabolites predominantly found in cruciferous vegetables such as broccoli, brussel sprout, cabbage and cauliflower which upon chopping and chewing will release the indigenous plant myrosinase enzyme that catalyzes the hydrolysis of GSLs. This hydrolysis releases a range of breakdown products including isothiocyanates (ITCs), which have been implicated in the cancer-protective effects of cruciferous vegetables. Certain human gut bacteria are able to metabolize GSLs and produce ITCs for human health benefits. In this work, six GSL-metabolizing bacterial strains were isolated from human faecal sample and identified. Most bacteria were capable of producing both nitriles (NITs) and ITCs from the metabolism of GSLs however Enterococcus sp. C213 and Enterococcus faecium KT4S13 produced only NITs. Enterococcus casseliflavus NCCP-53, Escherichia coli O83:H1 NRG 857C and Lactobacillus agilis R16 were able to metabolize different types (allyl, aromatic, methylthioalkyl, methylsulfinylalkyl and indolyl) of GSLs differently over 24 h of in vitro anaerobic fermentations. For all GSLs, ITC production seemed to peak between 4 and 8 h of incubation and then declined due to the inherent instability of ITCs in culture broths and buffers. In contrast, NIT productions gradually increased over time and remained relatively constant. The total percentage products from each GSL metabolism in all three bacteria never reached 100%. Interestingly, E. coli O83:H1 NRG 857C produced methylthioalkyl ITCs and NITs from methylsulfinylalkyl GSLs while E. casseliflavus NCCP-53 produced only methylsulfinylalkyl ITCs from the same GSLs. This difference was due to reductase activity in E. coli O83:H1 NRG 857C intact cells and cell-free extracts that biotransforms the sulfoxide groups of methylsulfinylalkyl GSLs to the sulfide groups. The reductase enzyme is yet to be identified at the gene and protein level, however it has been characterized using cell-free extracts in this work. This reductase is inducible by GSLs, oxygen-independent and requires Mg2+ ion and NADP(H) as co-factors for its activity with optimum pH and temperature at pH 7.0 and 37˚C, respectively. Arylsulfatase activity was also detected in this bacterium. The corresponding recombinant SUL2 enzyme (57 kDa) of E. coli O83:H1 NRG 857C expressed in BL21(DE3) exhibited arylsulfatase activity by desulfating synthetic p-nitrocatachol sulfate substrate with optimum pH and temperature at pH 6.0 and 30˚C, respectively. In addition, GSL-sulfatase activity was detected in crude extracts by being able to desulfate different intact GSLs to produce desulfo-glucosinolates (DS-GSLs) with less efficiency in comparison with the commercially available snail sulfatase from Helix pomatia. The catalytic efficiency of recombinant SUL2 enzyme for GSLs in descending order is as follows; sinigrin > glucoerucin > gluconasturtiin > glucoiberin. The DS-GSLs (except DS-glucoraphanin) then act as substrates for the recombinant GH3 enzyme defived from E. casseliflavus NCCP-53 to produce the corresponding NIT products in NB broths and the buffer with the presence of 5 mM Fe2+ ions. This enzyme (79 kDa) showed β-O-glucosidase activity for p-nitrophenyl β-D-glucopyranoside with optimum pH and temperature at pH 7.0 and 37˚C, respectively. NIT productions only occurred from the metabolism of intact GSLs in bacterial culture broths, but not in the buffers unless 5 mM Fe2+ ions are present as co-factors. Putative bacterial GSL-degrading enzymes responsible for ITC and NIT productions from GSL metabolisms are inducible by GSL in resting cells experiments. By using two-dimensional gel electrophoresis (2-DE) and liquid chromatography mass spectrometry (LC-MS/MS) for the comparative analysis between proteins obtained from cultures of L. agilis R16 and E. coli O83:H1 NRG 857C with and without GSL supplementation, upregulated/distinct proteins that may be involved in the metabolism of GSLs by these bacteria were identified. These proteins belong to (sugar) transport system, carbohydrate metabolism especially kinases and oxidoreduction process. To date, bacterial GSL-degrading enzyme is yet to be identified.Open Acces

CHARACTERIZATIONS OF METHYLTRANSFERASE-LIKE PROTEIN 2 IN RNA MODIFICATION

Ph.DPHD IN CANCER BIOLOG

Application of Soil Bacteria as Bioinoculants to Promote Growth of Cowpea (Vigna unguiculata)

This work aimed to evaluate the capacity of soil bacteria as bioinoculants (biofertilizers) to promote cowpea (Vigna unguiculata) growth. Three pure bacterial cultures namely Acinetobacter pittii PT1.3.4 (AP), Achromobacter sp.C2.23 (AS), and Achromobacter xylosoxidans N3.4 (AX) were used as bioinoculants to enhance germination and development of cowpea seeds. Pre-decide formulations of single or mixed cultures were prepared, soaked with cowpea seeds, and cultivated on agar in a growth chamber for 7 days at 25°C. Shoot and root length were measured and percentage germination was determined. Similarly, bacterial formulations were prepared in talcum powder and were used as bioinoculants to adhere to cowpea seeds. The inoculated seeds were cultivated in pots for 28 days for the shoot and root length, fresh and dry weight, and percentage germination. Among the tested various formulations, treatment has A. pittii (AP) displayed the highest shoot length (14.67 cm) and fresh weight (0.58 g/plant) of cowpea under laboratory conditions after seven days of inoculation. Similarly, cowpea plants treated with A. pittii (AP) also have the tallest shoots (14.25 cm) under natural conditions after 7 days of inoculation, while the highest root length (10.5 cm) and fresh weight (1.57 g/plant) were recorded from the treatment of Achromobacter sp. (AS). Further, the results of the study also revealed that soil bacteria can survive for one month in talcum powder at 4°C and room temperature storage. These bioinoculants can be used for agricultural application by local farmers to mitigate the cost of chemicals that cause environmental concerns to promote sustainable agriculture in Thailand

Taxonomic placement of Paphiopedilum rungsuriyanum (Cypripedioideae; Orchidaceae) based on morphological, cytological and molecular analyses

Additional file 9: Table S4. The comparison of main significant traits between subgenera and sections of Paphiopedilum by Gorniak et al. (2014) and the present study

Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes

Published online: 4 February 2017The cuticle forms a hydrophobic waxy layer that covers plant organs and provides protection from biotic and abiotic stresses. Transcription of genes responsible for cuticle formation is regulated by several types of transcription factors (TFs). Five orthologous to WAX PRODUCTION (WXP1 and WXP2) genes from Medicago truncatula were isolated from a cDNA library prepared from flag leaves and spikes of drought tolerant wheat (Triticum aestivum, breeding line RAC875) and designated TaWXP-like (TaWXPL) genes. Tissue-specific and drought-responsive expression of TaWXPL1D and TaWXPL2B was investigated by quantitative RT-PCR in two Australian wheat genotypes, RAC875 and Kukri, with contrasting glaucousness and drought tolerance. Rapid dehydration and/or slowly developing cyclic drought induced specific expression patterns of WXPL genes in flag leaves of the two cultivars RAC875 and Kukri. TaWXPL1D and TaWXPL2B proteins acted as transcriptional activators in yeast and in wheat cell cultures, and conserved sequences in their activation domains were localised at their C-termini. The involvement of wheat WXPL TFs in regulation of cuticle biosynthesis was confirmed by transient expression in wheat cells, using the promoters of wheat genes encoding two cuticle biosynthetic enzymes, the 3-ketoacyl-CoA-synthetase and the cytochrome P450 monooxygenase. Using the yeast 1-hybrid (Y1H) assay we also demonstrated the differential binding preferences of TaWXPL1D and TaWXPL2B towards three stress-related DNA cis-elements. Protein structural determinants underlying binding selectivity were revealed using comparative 3D molecular modelling of AP2 domains in complex with cis-elements. A scheme is proposed, which links the roles of WXPL and cuticle-related MYB TFs in regulation of genes responsible for the synthesis of cuticle components.Huihui Bi, Sukanya Luang, Yuan Li, Natalia Bazanova, Nikolai Borisjuk, Maria Hrmova, Sergiy Lopat

Cytotoxic effects of Saccharomyces cerevisiae TC6 and Lactobacillus brevis TBRC 3003 isolated from Thai fermented foods

Purpose: To determine the cytotoxic effect, anti-colony formation effect and antimigratory effect of Saccharomyces cerevisiae TC6 isolated from Thai water kefir, and Lactobacillus brevis TBRC 3003 isolated from picked cabbage. Methods: Crude microbial extracts were obtained from whole cultures (cells and broths) using ethyl acetate as extracting solvent, and the dried extracts were redissolved in ethanol before use. Cytotoxic, antiproliferative and antimigratory effects of the two microbial extracts on MCF-7, HepG2, and HeLa were tested using 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetra zolium bromide (MTT), clonogenic formation and wound healing assays. Results: Lb. brevis TBRC 3003 showed the highest cytotoxicity toward HepG2 cells (IC50 of 669.72 µg/mL), while S. cerevisiae TC6 showed the highest cytotoxicity against MCF-7 (IC50 of 691.49 µg/mL) and HeLa (IC50 of 379.16 µg/mL) based on MTT assay. Anti-colony formation test showed that S. cerevisiae TC6 was most the most effective in inhibiting colony formation of HepG2 (IC50 of 311.12 µg/mL) and HeLa (IC50 of 494.64 µg/mL), while Lb. brevis TBRC 3003 was the most potent in inhibiting colony formation of MCF-7 (IC50 of 267.88 µg/mL). Conclusion: Both microbes can potentially be implemented in functional foods as bio-therapeutics with chemopreventive properties against breast, liver and cervical cancers

Hyperendemic dengue transmission and identification of a locally evolved DENV-3 lineage, Papua New Guinea 2007-2010

Background Dengue is endemic in the Western Pacific and Oceania and the region reports more than 200,000 cases annually. Outbreaks of dengue and severe dengue occur regularly and movement of virus throughout the region has been reported. Disease surveillance systems, however, in many areas are not fully established and dengue incidence is underreported. Dengue epidemiology is likely least understood in Papua New Guinea (PNG), where the prototype DENV-2 strain New Guinea C was first isolated by Sabin in 1944 but where routine surveillance is not undertaken and little incidence and prevalence data is available. Methodology/Principal findings Serum samples from individuals with recent acute febrile illness or with non-febrile conditions collected between 2007–2010 were tested for anti-DENV neutralizing antibody. Responses were predominantly multitypic and seroprevalence increased with age, a pattern indicative of endemic dengue. DENV-1, DENV-2 and DENV-3 genomes were detected by RT-PCR within a nine-month period and in several instances, two serotypes were identified in individuals sampled within a period of 10 days. Phylogenetic analysis of whole genome sequences identified a DENV-3 Genotype 1 lineage which had evolved on the northern coast of PNG which was likely exported to the western Pacific five years later, in addition to a DENV-2 Cosmopolitan Genotype lineage which had previously circulated in the region. Conclusions/Significance We show that dengue is hyperendemic in PNG and identify an endemic, locally evolved lineage of DENV-3 that was associated with an outbreak of severe dengue in Pacific countries in subsequent years, although severe disease was not identified in PNG. Additional studies need to be undertaken to understand dengue epidemiology and burden of disease in PNG

Crystallisation of wild-type and variant forms of a recombinant plant enzyme beta-D-glucan glucohydrolase from barley (Hordeum vulgare L.) and preliminary X-ray analysis

Wild-type and variant crystals of a recombinant enzyme β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 x 250 x 375 µm at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 x 40 x 60 µm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achieved full size within 5-14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P4₃2₁2 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57-1.95 Å and the cell dimensions were between a = b = 99.2-100.8 Å and c = 183.2-183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4-3.5 ų.Da⁻¹ and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.Sukanya Luang, James R. Ketudat Cairns, Victor A. Streltsov and Maria Hrmov

Bioavailability of Glucoraphanin and Sulforaphane From High-Glucoraphanin Broccoli

Scope: Broccoli accumulates 4-methylsulphinylbutyl glucosinolate (glucoraphanin) which is hydrolyzed to the isothiocyanate sulforaphane. Through the introgression of novel alleles of the Myb28 transcription factor from Brassica villosa, broccoli genotypes have been developed that have enhanced levels of glucoraphanin. This study sought to quantify the exposure of human tissues to glucoraphanin and sulforaphane following consumption of broccoli with contrasting Myb28 genotypes. Methods and results: Ten participants were recruited into a three-phase, double-blinded, randomized crossover trial (NCT02300324), with each phase comprising consumption of 300 ml of a soup made from broccoli of one of three Myb28 genotypes (Myb28B/B, Myb28B/V, Myb28V/V). Plant myrosinases were intentionally denatured during soup manufacture. Three-fold and five-fold higher levels of sulforaphane occurred in the circulation following consumption of Myb28V/B and Myb28V/V broccoli soups, respectively. The percentage of sulforaphane excreted in 24 h relative to the amount of glucoraphanin consumed varied amongst volunteers from 2% to 15%, but did not depend on the broccoli genotype. Conclusion: This is the first study to report the bioavailability of glucoraphanin and sulforaphane from soups made with novel broccoli varieties. The presence of one or two Myb28V alleles results in enhanced delivery of sulforaphane to the systemic circulation

MODIS-derived green Noctiluca blooms in the upper Gulf of Thailand: Algorithm development and seasonal variation mapping

In recent decades, red tides of non-toxic harmful algal blooms have frequently occurred in monsoon-influenced tropical areas, particularly the green form of Noctiluca scintillans (hereafter green Noctiluca). However, our understanding of the mechanism of red tide formation is hindered by spatial and temporal constraints of field data. In this study, we used moderate resolution imaging spectroradiometer (MODIS) ocean color data along with a locally developed algal-bloom classification algorithm to investigate the seasonal variability of dominant red tides across the upper Gulf of Thailand (uGoT). During our July 2018 observation, a super green Noctiluca bloom with extraordinarily high chl-a (>1,469 mg m-3) displayed a distinct spectral reflectance characteristic among red tides in blue-to-green and red-to-near infrared wavelengths. According to the distinctive in situ hyperspectral characteristics of uGoT algal blooms, we developed a classification algorithm for MODIS normalized at 488, 531, and 667 nm, which successfully discriminated green Noctiluca in three levels of blooms, namely, super (100%), strong (>80%), and weak (>40%), from other algal blooms (i.e., dinoflagellates, diatoms, cyanobacteria, and mixed red tide species) as well as non-bloom oceanic and coastal waters using MODIS data, as confirmed by uGoT red tide reports. Monthly MODIS-based discrimination composites from 2003 to 2021 revealed seasonal variability in the surface distribution and bloom frequency of green Noctiluca and other red tides according to the Asian monsoon seasons: the southwest monsoon (May–September) and the northeast monsoon (October–January of the following year). Green Noctiluca blooms occurred farther from the shore and estuaries than other red tides (dinoflagellates and cyanobacteria), and were much more frequent than other red tides between the Tha Chin and Chao Phraya River mouths during the non-monsoon period (February to April). The frequency and distribution of green Noctiluca blooms, as well as other algal blooms, varied with the monsoon season. By comparing MODIS-derived algal blooms to monsoon-induced factors (i.e., sea surface winds, precipitation, and river discharge), we present an unprecedented overview of the spatial and temporal dynamics of red tides throughout the uGoT under Asian monsoon conditions. This research contributes to our understanding of the impact of climate change on phytoplankton dynamics