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The high cell density culture of baker's yeast FX-2 was investigated in a 50 L(A) automatic bioreactor. Herein, it was found firstly that the Crabtree effect clearly existed in batch fermentation with higher glucose content, then the critical initial glucose content range (≤2.00 g L-1 ) was reasonably ascertained to effectively avoid Crabtree effect. In the next fed-batch fermentations with different strategies, the second strategy (maintain ethanol concentration lower than 0.10% and pH around 4.80) was confirmed to be more beneficial to yeast growth than the first strategy (keep reducing sugar not more than 2.00 g L-1 and control steady Carbon/Nitrogen ratio 3.05:1.00). After that, one optimal control strategy (maintain pH around 4.80 and keep respiratory quotient in the range of 0.90-1.00) was constructed to further enhance cell yield. Under an optimal control strategy, four schemes with the aim of achieving pH-stat were compared, and yeast extract instead of other alkaline materials was selected as a better regulator. As a result, 148.37 g L-1 dry cell weight, 38.25 × 108 mL-1 living cells, and 8.24 g L-1 h-1 productivity were harvested, which respectively elevated 23.74%, 135.38%, and 24.47% compared to that obtained under the traditional scheme (regulate pH with ammonia); meanwhile, the maximum oxygen uptake rate and carbon dioxide excretion rate were both more than 250.00 mmol L-1 min-1 .

1′,4′-trans-diol-ABA is a key precursor of the biosynthesis of abscisic acid (ABA) biosynthesis in fungi. We successfully obtained the pure compound from a mutant of Botrytis cinerea and explored its function and possible mechanism on plants by spraying 2 mg/L 1′,4′-trans-diol-ABA on tobacco leaves. Our results showed that this compound enhanced the drought tolerance of tobacco seedlings. A comparative transcriptome analysis showed that a large number of genes responded to the compound, exhibiting 1523 genes that were differentially expressed at 12 h, which increased to 1993 at 24 h and 3074 at 48 h, respectively. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were primarily enriched in pathways related to hormones and resistance. The DEGs of transcription factors were generally up-regulated and included the bHLH, bZIP, ERF, MYB, NAC, WRKY and HSF families. Moreover, the levels of expression of PYL/PYR, PP2C, SnRK2, and ABF at the ABA signaling pathway responded positively to exogenous 1′,4′-trans-diol-ABA. Among them, seven ABF transcripts that were detected were significantly up-regulated. In addition, the genes involved in salicylic acid, ethylene and jasmonic acid pathways, reactive oxygen species scavenging system, and other resistance related genes were primarily induced by 1′,4′-trans-diol-ABA. These findings indicated that treatment with 1′,4′-trans-diol-ABA could improve tolerance to plant abiotic stress and potential biotic resistance by regulating gene expression, similar to the effects of exogenous ABA.

Five previously undescribed lanostane-type triterpenoids, including two triterpenoids with a rearranged side chain (applanoic acids E and F), one C21 nortriterpenoid (16,17-dehydroapplanone E), as well as two highly oxygenated lanostane triterpenoids (methyl applaniate B and applanoic acid G), were isolated from the fruiting bodies of Ganoderma applanatum (Pers.) Pat. Their structures were elucidated on the basis of spectroscopic analysis, X-ray crystallography and ECD data. Applanoic acid E, 16,17-dehydroapplanone E, and methyl applaniate B showed inhibitory effects on the release of NO by LPS-induced BV-2 cells.

The Pd-based nanomaterials with advanced structures have shown remarkable activity for direct alcohol fuel cells (DAFCs). However, the dissolution of Pd and alloyed base metals, as well as the instability of Ostwald ripening and the formation of the poisoning species in fuel cell operation environments could cause catalyst degradation, which still remains an issue. Herein we report a graphene-confined thermal conversion strategy to fabricate high stability PdFe-embedded porous carbon nanofibers (PdFe@G-CNFs) for ethanol electrooxidation. Benefiting from the unique porous carbon-encapsulated structure and highly alloyed small-sized PdFe component, the resultant samples exhibit excellent electrocatalytic activity and stability for ethanol electrooxidation in alkaline media. Especially, the Pd2Fe1@G-CNFs exhibits enhanced carbon monoxide tolerance and higher peak current density (1896 mA mg−1) than Pd@G-CNFs (860 mA mg−1) and Pd2Fe1@CNFs (1276 mA mg−1). This study provides a new insight into designing high-efficiency and stable Pd-based electrocatalysts for DAFCs.

Nervilia fordii (Hance) Schltr. has been widely used as a medicinal and edible herb in Southwest China and Southeast Asia. In this study, NFP-1, a new water-soluble polysaccharidewith a purity of 97.8%, was purified from water extract of Nervilia fordii by DEAE-cellulose and Sephadex G-100 chromatography. NFP-1 has a relative molecular weight of 950 kDa determined by high performance gel-permeation chromatography (HPGPC). Its monosaccharide compositions were analyzed by high performance liquid chromatography (HPLC) after pre-column derivatizing its hydrolysate with 1-phenyl-3-methyl-5-pyrazolone (PMP). NFP-1 mainly consists of galactose, arabinose, rhamnose, and galacturonic acid. Based on FT-IR, methylation and GC–MS analysis, and NMR, the structure unit of NFP-1 was established as →4)-α-Rhap-(2→ 4)-α-GalpA-(1→2)-α-Rhap-(1→2)-α-Rhap-(4→1)-β-Galp-T containing two branch chains of →2,4)-α-Rhap-(1→5)-α-Araf-(1→3)-α-Araf-(1→, and →2,4)-α-Rhap-(1→4)-β-Galp-(1→. The immunomodulatory assays revealed the dual-functionalities of NFP-1. NFP-1 could significantly induce the secretion of nitric oxide (NO), and promote the secretions of TNF-α, IL-6, and IL-1β in RAW264.7 macrophages. NFP-1 could also significantly inhibit the production of NO, depress the secretions of TNF-α, IL-6 and IL-1β in RAW264.7 macrophages activated by lipopolysaccharide (LPS), and promote the production of IL-10 meanwhile. Our study suggested that Nervilia fordii could be an ideal medicinal or functional food due to its dual immunomodulatory activities., Graphical abstract Unlabelled Image

Fatty acid synthase (FAS) is highly expressed in human adipocytes and cancer cells and is considered as a dual therapeutic target for obesity and cancer treatment. Garcinone E is a natural xanthone and exists in the pericarp of Garcinia mangostana. In previous studies, xanthones were reported to be highly active inhibitors of FAS. In the present study, the detailed inhibitory mechanism of garcinone E on FAS was investigated. We found that garcinone E inhibited the activity of FAS in a concentration-dependent manner with a half-inhibitory concentration value of 3.3 μM. The inhibition kinetic results showed that the inhibition of FAS by garcinone E was competitive with respect to acetyl-CoA, mixed competitive and noncompetitive with respect to malonyl-CoA, and noncompetitive to NADPH. In addition, garcinone E showed irreversible inhibition on FAS, which was different from all other xanthones. Since FAS is believed to be a therapeutic target for obesity and cancer treatment, these findings suggest the clinical potential of garcinone E in the prevention and treatment of both obesity and cancer.

In this work, the influence of annealed ITO thin film as a transparent electrode layer in pulse laser deposition (PLD) Cu2ZnSnS4 (CZTS) thin-film solar cell was investigated. The proposed annealed ITO thin film exhibits a high degree of crystallinity and large and evenly distributed grains. Thus, low resistivity and wide and high transmitted band in the UV/VIS light range with ideal optical band gap of 3.8 eV can be obtained. Devices built with these annealed ITO thin films on our PLD CZTS solar cell exhibit a significant improvement in power conversion efficiency from 0.2% to 1.92%.

Bacillus subtilis ZK0, which was isolated from cotton, produces a type of lipopeptide antibiotic iturin A that inhibits the growth of pathogenic fungi on agricultural crops. However, the low level of iturin A production by B. subtilis ZK0 does not support its large-scale application. In this study, B. subtilis ZK0 was subjected to genetic manipulation to improve iturin A production. By the independent or simultaneous overexpression of two regulatory genes (comA and sigA), iturin A production by B. subtilis ZK0 was significantly increased. When both genes were simultaneously overexpressed under optimal conditions, iturin A production increased up to 215 mg l−1 (an approximate 43-fold increase compared with B. subtilis ZK0). Moreover, overexpression of both genes was unexpectedly found to inhibit biofilm formation by B. subtilis ZK0, indicating that the phenomenon of ‘stuck fermentation’ would be avoided during B. subtilis ZK0 fermentation. In conclusion, a genetic manipulation method that improves iturin A production and inhibits biofilm formation in B. subtilis ZK0 is reported for the first time and this method has the potential to be widely applied in B. subtilis ZK0 commercial fermentation. Significance and Impact of the Study This study provides new perspectives on improving iturin A production by Bacillus subtilis. Our newly engineered strains could be applied to commercial fermentation by enhancing yields of iturin A and reducing the rate of ‘stuck fermentation’. Increased production would facilitate more widespread application of this powerful antibiotic.

High dose intake of fluoride results in fluorosis and our previous experiments at single-molecule level using magnetic tweezers have proved that fluoride anions destabilize DNA hairpin and its equilibrium critical force decreases with increasing concentration of fluoride anions. One possible mechanism is that a fluoride anion competes with a DNA base and forms a hydrogen bond with its complementary base. The Isothermal Titration Calorimetry experiments suggest that the fluoride anion does not form a hydrogen bond with a DNA base. With increasing concentration of fluoride anions, decreasing Zeta potentials for different oligonucleotides enhance the electrostatic repulsion between complementary oligonucleotides, which accounts for the decreasing association constant and destabilization of DNA hairpin. Molecule dynamics simulation was further performed to determine the ion distribution around the oligonucleotide, radial distribution functions (RDFs) indicate that the fluoride anion distributes far from the oligonucleotide in comparison with the chloride anion, which is the possible mechanism that decreases Zeta potentials with the increasing concentration of fluoride anions.

International audience; Antimony selenide (Sb2Se3) is a potential absorber material for environment-friendly and cost-efficient photovoltaics and has achieved considerable progress in recent years. However, the severe open-circuit voltage (Voc) deficit ascribed to the interface and/or bulk defect states has become the main obstacle for further efficiency improvement. In this work, Sb2Se3 absorber layer was prepared by an effective combination reaction involving sputtered and selenized Sb precursor thin films. The self-assembled growth of Sb2Se3 thin films with large crystal grains, benign preferential orientation, and accurate chemical composition were successfully fulfilled under an appropriate thickness of Sb precursor and an optimized selenization scenario. Substrate structured Sb2Se3 thin-film solar cells, a champion device with a power-conversion efficiency of 6.84%, were fabricated. This device is comparable to state-of-the-art ones and represents the highest efficiency of sputtered Sb2Se3 solar cells. Importantly, the high Voc of 504 mV is closely related to the reduced deep level defect density for the Sb2Se3 absorber layer, the passivated interfacial defects for Sb2Se3/CdS heterojunction interface, and the additional heterojunction heat treatment-induced Cd and S inter-diffusion. This significantly improved Voc demonstrates remarkable potential to broaden its scope of applications for Sb2Se3 solar cells. © 2020 Elsevier Ltd

Biomass-derived carbon composites (e.g., metal oxide/biocarbon) have been used as promising electrode materials for energy storage devices owing to their natural abundance and simple preparation process. However, low loading content/inhomogeneous distribution of metal oxides and inefficient cracking of biocarbon (BC) are intractable obstacles that impede the efficient utilization of biomass. In this work, hierarchical porous MnO/BC composites were prepared by a facile molten-salt-assisted strategy based on the superior salt-water absorption ability of agaric. The addition of NaCl induces a liquid reaction medium by formation of a molten salt mixture at high temperature to effectively realize the activation and cracking of the bulk carbon, and it also acts as a recyclable sacrificial template to form mesopores and macropores in the as-prepared hierarchical porous MnO/BC composites. The highly porous and uniform BC framework effectively enhances ion diffusion and electron-transfer ability, serves as a protective layer to prevent fracturing and agglomeration of MnO, and thus enables superior rate performance and cycling stability of the MnO/BC composite for both supercapacitor electrodes (94 % capacity retention at 20 mA cm-2 after 5000 cycles) and lithium-ion battery anodes (783 mA h g-1 after 1000 cycles). Notably, considering the simple and low-cost preparation process, this work opens a promising avenue for the large-scale production of advanced metal oxide/BC hybrid electrode materials for electrochemical energy storage.

Abscisic acid (ABA) is one of the five classical phytohormones involved in increasing the tolerance of plants for various kinds of stresses caused by abiotic or biotic factors, and it also plays important roles in regulating the activation of innate immune cells and glucose homeostasis in mammals. For these reasons, as a "stress hormone," ABA has recently received attention as a candidate drug for agriculture and biomedical applications, prompting significant development of ABA synthesis. Some plant-pathogenic fungi can synthesize natural ABA. The fungus Botrytis cinerea has been used for biotechnological production of ABA. Identification of the transcription factors (TFs) involved in regulation of ABA biosynthesis in B. cinerea would provide new clues to understand how ABA is synthesized and regulated. In this study, we defined a novel Cys2His2 TF, BcabaR1, that regulates the transcriptional levels of ABA synthase genes (bcaba1, bcaba2, bcaba3, and bcaba4) in an ABA-overproducing mutant, B. cinerea TBC-A. Electrophoretic mobility shift assays revealed that recombinant BcabaR1 can bind specifically to both a 14-nucleotide sequence motif and a 39-nucleotide sequence motif in the promoter region of bcaba1 to -4 genes in vitro A decreased transcriptional level of the bcabaR1 gene in B. cinerea led to significantly decreased ABA production and downregulated transcription of bcaba1 to -4 When bcabaR1 was overexpressed in B. cinerea, ABA production was significantly increased, with upregulated transcription of bcaba1 to -4 Thus, in this study, we found that BcabaR1 acts as a positive regulator of ABA biosynthesis in B. cinereaIMPORTANCE Abscisic acid (ABA) could make a potentially important contribution to theoretical research and applications in agriculture and medicine. Botrytis cinerea is a plant-pathogenic fungus that was found to produce ABA. There has been a view that ABA is related to the interaction between pathogenic fungi and plants. Identification of regulatory genes involved in ABA biosynthesis may facilitate an understanding of the underlying molecular mechanisms of ABA biosynthesis and the pathogenesis of B. cinerea Here, we present a positive regulator, BcabaR1, of ABA biosynthesis in B. cinerea that can affect the transcriptional level of the ABA biosynthesis gene cluster, bcaba1 to -4, by directly binding to the conserved sequence elements in the promoter of the bcaba1 to -4 genes. This TF was found to be specifically involved in regulation of ABA biosynthesis. This work provides new clues for finding other ABA biosynthesis genes and improving ABA yield in B. cinerea.

Background Neonatal hypoxic-ischemic brain damage, characterized by tissue loss and neurologic dysfunction, is a leading cause of mortality and a devastating disease of the central nervous system. We have previously shown that vitexin has been attributed various medicinal properties and has been demonstrated to have neuroprotective roles in neonatal brain injury models. In the present study, we continued to reinforce and validate the basic understanding of vitexin (45 mg/kg) as a potential treatment for epilepsy and explored its possible underlying mechanisms. Methods P7 Sprague-Dawley (SD) rats that underwent right common carotid artery ligation and rat brain microvascular endothelial cells (RBMECs) were used for the assessment of Na+-K+-Cl− co-transporter1 (NKCC1) expression, BBB permeability, cytokine expression, and neutrophil infiltration by western blot, q-PCR, flow cytometry (FCM), and immunofluorescence respectively. Furthermore, brain electrical activity in freely moving rats was recorded by electroencephalography (EEG). Results Our data showed that NKCC1 expression was attenuated in vitexin-treated rats compared to the expression in the HI group in vivo. Oxygen glucose deprivation/reoxygenation (OGD) was performed on RBMECs to explore the role of NKCC1 and F-actin in cytoskeleton formation with confocal microscopy, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide, and FCM. Concomitantly, treatment with vitexin effectively alleviated OGD-induced NKCC1 expression, which downregulated F-actin expression in RBMECs. In addition, vitexin significantly ameliorated BBB leakage and rescued the expression of tight junction-related protein ZO-1. Furthermore, inflammatory cytokine and neutrophil infiltration were concurrently and progressively downregulated with decreasing BBB permeability in rats. Vitexin also significantly suppressed brain electrical activity in neonatal rats. Conclusions Taken together, these results confirmed that vitexin effectively alleviates epilepsy susceptibility through inhibition of inflammation along with improved BBB integrity. Our study provides a strong rationale for the further development of vitexin as a promising therapeutic candidate treatment for epilepsy in the immature brain.

A two-step cathodic electrochemiluminescence (ECL) of meso-tetra(4-carboxyphenyl) porphyrin (TCPP) in aqueous solution has been reported for the first time. ECL generates upon sweeping from -0.2 V to -1.5 V using potassium peroxydisulfate (K2S2O8) as a reductive–oxidative coreactant. Two cathodic ECL peaks are found simultaneously. One appears at -0.8 V which is corresponding to the reduction of TCPP, another is at -1.1 V that is attributed to the reduction of K2S2O8. ECL efficiency (φecl) is 0.22 for TCPP using Ru(bpy)32+/S2O82− as the standard (φecl = 1). Under the optimum conditions, ECL intensity has a linear relationship with the concentration of TCPP in the range of 2.0 × 10−7 to 1.0 × 10−5 mol L−1 with the detection limit of 1.7 × 10−8 mol L−1. The practical application to determination pheophorbide which is obtained from fresh spinach leaves has been accomplished. The results demonstrate the facility and effectivity of our method for quantitative analysis of porphyrin. At the same time of building this method, the possible ECL mechanism is discussed in detail.

The first nucleosomes in the downstream of transcription starting sites are called +1 nucleosomes, which are expected to be readily unwrapped for DNA transcription. To investigate DNA accessibility in +1 nucleosomes, MNase-seq experiments were carried out with 20 reconstituted +1 nucleosomes of budding yeast. Although MNase has been known for its sequence preference in DNA digestions, we confirmed that this sequence preference is overwhelmed by DNA accessibility by identifying the sequence-driven and accessibility-driven cleavages. Specifically, we find that sequences favoured by MNase at the end regions such as TA dinucleotide are prohibited from cleavage at the internal sites in the early stage of digestion. Nevertheless, sequences less favoured by MNase at the end regions such as AA/TT dinucleotide are predominantly cleaved at the internal sites in the early stage of digestion. Since AA/TT is known as a rigid dinucleotide step resistant to DNA bending, these internal cleavages reflect the local site exposures induced by DNA mechanics. As the DNA entry site of +1 nucleosomes in yeast is found AA/TT-rich, this sequence element may play a role in gene activation by reducing DNA–histone affinities along the direction of DNA transcription.

A highly efficient copper(I) iodide/N,N,N′,N′-tetramethylethylenediamine (CuI/TMEDA) catalytic system for the synthesis of ynones has been developed. A variety of terminal alkynes, including 4-iodophenylacetylene, were smoothly coupled with acid chlorides in the presence 2 mol% CuI and 5 mol% TMEDA to afford the corresponding ynones in good to excellent yields in a short time. It is noteworthy that the reaction is conducted under solvent-free conditions at room temperature.

To introduce DNA into Streptomyces noursei xinao-4, which produces xinaomycins, we explored an intergeneric conjugal transfer system. High efficiency of conjugation (8 × 10−3 exconjugants per recipient) was obtained when spores of S. noursei xinao-4 were heat-shocked at 50 °C for 10 min, mixed with Escherichia coli ET12567 (pUZ8002/pSET152) in the ratio of 1:100, plated on 2CMY medium containing 40 mmol/L MgCl2, and incubated at 30 °C for 22 h. With this protocol, the plasmids pKC1139 and pSET152 were successfully transferred from E. coli ET12567 (pUZ8002) with different frequencies. Among all parameters, the ratio of donor to recipient cell number had the strongest effect on the transformation efficiency. In order to validate the above intergeneric conjugal transfer system, a glycosyltransferase gene was cloned and efficiently knocked out in S. noursei xinao-4 using pSG5-based plasmid pKC1139.

Computer modeling of very large biomolecular systems, such as long DNA polyelectrolytes or protein-DNA complex-like chromatin cannot reach all-atom resolution in a foreseeable future and this necessitates the development of coarse-grained (CG) approximations. DNA is both highly charged and mechanically rigid semi-flexible polymer and adequate DNA modeling requires a correct description of both its structural stiffness and salt-dependent electrostatic forces. Here, we present a novel CG model of DNA that approximates the DNA polymer as a chain of 5-bead units. Each unit represents two DNA base pairs with one central bead for bases and pentose moieties and four others for phosphate groups. Charges, intra- and inter-molecular force field potentials for the CG DNA model were calculated using the inverse Monte Carlo method from all atom molecular dynamic (MD) simulations of 22 bp DNA oligonucleotides. The CG model was tested by performing dielectric continuum Langevin MD simulations of a 200 bp double helix DNA in solutions of monovalent salt with explicit ions. Excellent agreement with experimental data was obtained for the dependence of the DNA persistent length on salt concentration in the range 0.1–100 mM. The new CG DNA model is suitable for modeling various biomolecular systems with adequate description of electrostatic and mechanical properties. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Prototype foamy virus encodes a transactivator called Bel1 that enhances viral gene transcription and is essential for PFV replication. Nuclear localization of Bel1 has been reported to rely on two proximal basic motifs R(199)H(200) and R(221)R(222)R(223) that likely function together as a bipartite nuclear localization signal. In this study, we report that mutating R(221)R(222)R(223), but not R(199)H(200), relocates Bel1 from the nucleus to the cytoplasm, suggesting an essential role for R(221)R(222)R(223) in the nuclear localization of Bel1. Although not affecting the nuclear localization of Bel1, mutating R(199)H(200) disables Bel1 from transactivating PFV promoters. Results of EMSA reveal that the R(199)H(200) residues are vital for the binding of Bel1 to viral promoter DNA. Moreover, mutating R(199)H(200) in Bel1 impairs PFV replication to a much greater extent than mutating R(221)R(222)R(223). Collectively, our findings suggest that R(199)H(200) directly participate in Bel1 binding to viral promoter DNA and are indispensible for Bel1 transactivation activity.

The formation of authigenic carbonates at cold seep is closely related to underlying methane seepage and anaerobic oxidation of methane (AOM), and these processes can be reflected in the geochemical characteristics of pore water in the sediments. In terms of petrological, mineralogical and geochemical analyses of research materials, including authigenic carbonates and pore water samples from the middle Okinawa Trough, East China Sea,these results show that authigenic carbonate minerals are mainly composed of high-magnesium calcite, low-magnesium calcite, dolomite, and iron dolomite and exhibit extremely negative carbon isotopic compositions (δ13C = −52.30‰ to −31.10‰ V-PDB) and positive oxygen isotopic compositions (δ18O = 3.36‰ to 7.27‰ V-PDB). Geochemical analyses of pore water suggest linear sulfate gradients and steep increase of methane and total alkalinity concentrations with depth, which indicate sulfate-induced AOM activities. Based on the sulfate concentration reduction, carbon isotope characteristics of dissolved inorganic carbon (DIC) and increased methane concentration, the sulfate-methane interface (SMI) is considered to be shallow. These data, combined with the variations in ion concentrations in pore water, suggest that the leakage of biogenic methane and intense AOM resulted in the formation of abundant authigenic carbonates, which inherited light carbon (12C)-enriched isotopic signature of methane in the shallow sediments. Combined geochemical characteristics of pore water and authigenic carbonates, intense methane seep activities and associated AOM can be inferred both in present-day and ancient geological records on the western slope of the middle Okinawa Trough.

Efficient and stable quenching of electrochemiluminescence of Ru(bpy)3(2+)/TPA by malachite green(MG) and crystal violet(CV) at the glass carbon (GC) electrode is reported. A novel quenching mechanism has been proposed. Resonance energy transfer from the excited-state luminophore Ru(bpy)3(2+*) to MG/CV and dynamic quenching are suggested as the mechanism for quenching ECL. The quenching mechanism is discussed in detail based on UV-visible absorption spectra, cyclic voltammograms, ECL curves and fluorescence methods. MG shows more efficient quenching than CV. Moreover, the quenched ECL intensity versus the concentration of MG and CV are linear over the concentration ranges of 8 × 10(-10)-8 × 10(-7)M and 3.46 × 10(-9)-5.5 × 10(-7)M, respectively. The corresponding limit of detection (LOD) was 1.0 × 10(-10)M for MG and 1.1 × 10(-10)M for CV (S/N=3).

Abscisic acid (ABA) is a classical phytohormone which plays an important role in plant stress resistance. Moreover, ABA is also found to regulate the activation of innate immune cells and glucose homeostasis in mammals. Therefore, this ‘stress hormone’ is of great importance to theoretical research and agricultural and medical applications. Botrytis cinerea is a well-known phytopathogenic ascomycete that synthesizes ABA via a pathway substantially different from higher plants. Identification of the functional genes involved in ABA biosynthesis in B. cinerea would be of special interest. We developed an ABA-overproducing mutant strain, B. cinerea TBC-A, previously and obtained a 41.5-Mb genome sequence of B. cinerea TBC-A. In this study, the transcriptomes of B. cinerea TBC-A and its ancestral strain TBC-6 were sequenced under identical fermentation conditions. A stringent comparative transcriptome analysis was performed to identify differentially expressed genes participating in the metabolic pathways related to ABA biosynthesis in B. cinerea. This study provides the first global view of the transcriptional changes underlying the very different ABA productivity of the B. cinerea strains and will expand our knowledge of the molecular basis for ABA biosynthesis in B. cinerea.

The aldehyde dehydrogenase (ALDH) superfamily of NAD(P) + -dependent enzymes, in general, oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes to their corresponding carboxylic acids and play an essential role in detoxification of reactive oxygen species (ROS) accumulated under the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Oryza sativa , a homologue of ALDH gene (OsALDH22) was isolated and characterized. OsALDH22 is conserved in eukaryotes, shares high homology with the orthologs from aldehyde dehydrogenase subfamily ALDH22. The OsALDH22 encodes a protein of 597 amino acids that in plants exhibit high identity with the orthologs from Zea mays, Sorghum bicolor, Hordeum vulgare and Arabidopsis thaliana , respectively, and the conserved amino acid characteristics for ALDHs are present, including the possible NAD + binding site (F-V-G-S-P-G-V-G), the catalytic site (V-T-L-E-L-G-G-K) and the Cys active site. Semi-quantitative PCR and real-time PCR analysis indicates that OsALDH22 is expressed differentially in different tissues. Various elevated levels of OsALDH22 expression have been detected when the seedlings exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Transgenic rice plants overexpressing OsALDH22 show elevated stresses tolerance. On the contrary, down-regulation of OsALDH22 in the RNA interference (RNAi) repression transgenic lines manifests declined stresses tolerance.

Various abilities to synthesize and accumulate glycine betaine (GB) are crucial for angiosperms to develop salt and drought tolerances. In higher plants, GB is synthesized by a two-step oxidation of choline via an intermediate form of betaine aldehyde, and catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). In this study, numerous truncated and/or recombinant transcripts of two BADH homologs resulting from an unusual posttranscriptional processing were detected in rice (Oryza sativa) and other cereal crops, including maize (Zea mays), wheat (Triticum aestivum), and barley (Hordeum vulgare). The observed events took place at the 5′ exonic region, and led to the insertion of exogenous gene sequences and a variety of deletions that resulted in the removal of translation initiation codon, loss of functional domain, and frame-shifts with premature termination by introducing stop codon. By contrast, the BADH transcripts from dicotyledonous species, such as spinach (Spinacia oleracea), Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum), had correctly processed mRNA. This suggests the differentiation of posttranscriptional processing in BADH genes potentially contributes to the variation of GB-synthesizing capacities among various plant species. In addition, comprehensive sequence analyses demonstrated that extensive sequence similarities (named as short, direct repeats) are of paired presence surrounding the junctions of both the deletion and/or insertion sites in the unusual BADH transcripts. The site selection for the deletion/insertion was altered in response to the stress conditions. This indicates that the sequence elements of short, direct repeats are probably required for the recognition of the deletion/insertion sites.

In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 107 transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production.

Ligand-stabilized human telomeric G-quadruplex DNA is believed to be an anticancer agent, as it can impede the continuous elongation of telomeres by telomerase in cancer cells. In this study, five well-established human telomeric G-quadruplex DNA models were probed on their binding behaviors with thioflavin T (ThT) via both conventional molecular dynamics (MD) and well-tempered metadynamics (WT-MetaD) simulations. Novel dynamics and characteristic binding patterns were disclosed by the MD simulations. It was observed that the K(+) promoted parallel and hybridized human telomeric G-quadruplex conformations pose higher binding affinities to ThT than the Na(+) and K(+) promoted basket conformations. It is the end, sandwich, and base stacking driven by π-π interactions that are identified as the major binding mechanisms. As the most energy favorable binding mode, the sandwich stacking observed in (3 + 1) hybridized form 1 G-quadruplex conformation is triggered by reversible conformational change of the G-quadruplex. To further examine the free energy landscapes, WT-MetaD simulations were utilized on G-quadruplex-ThT systems. It is found that all of the major binding modes predicted by the MD simulations are confirmed by the WT-MetaD simulations. The results in this work not only accord with existing experimental findings, but also reinforce our understanding on the dynamics of G-quadruplexes and aid future drug developments for G-quadruplex stabilization ligands.

Glycine betaine (GB) is a compatible quaternary amine that enables plants to tolerate abiotic stresses, including salt, drought and cold. In plants, GB is synthesized through two-step of successive oxidations from choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Rice is considered as a typical non-GB accumulating species, although the entire genome sequencing revealed rice contains orthologs of both CMO and BADH. Several studies unraveled that rice has a functional BADH gene, but whether rice CMO gene (OsCMO) is functional or a pseudogene remains to be elucidated. In the present study, we report the functional characterization of rice CMO gene. The OsCMO gene was isolated from rice cv. Nipponbare (Oryza sativa L. ssp. japonica) using RT-PCR. Northern blot demonstrated the transcription of OsCMO is enhanced by salt stress. Transgenic tobacco plants overexpressing OsCMO results in increased GB content and elevated tolerance to salt stress. Immunoblotting analysis demonstrates that a functional OsCMO protein with correct size was present in transgenic tobacco but rarely accumulated in wild-type rice plants. Surprisingly, a large amount of truncated proteins derived from OsCMO was induced in the rice seedlings in response to salt stresses. This suggests that it is the lack of a functional OsCMO protein that presumably results in non-GB accumulation in the tested rice plant. Key message Expression and transgenic studies demonstrate OsCMO is transcriptionally induced in response to salt stress and functions in increasing glycinebetaine accumulation and enhancing tolerance to salt stress. Immunoblotting analysis suggests that no accumulation of glycinebetaine in the Japonica rice plant presumably results from lack of a functional OsCMO protein.

The master two-dimensional computer database of rat liver epithelial (RLE) cellular proteins (Wirth et al., Electrophoresis 1991, 12, 931-954) has been expanded to include detailed information concerning 1100 nucleoplasmic (cytosolic) and 850 particulate associated [35S]methionine labeled as well as 215 nucleoplasmic and 269 particulate associated [32P]orthophosphate labeled RLE nuclear polypeptides, respectively. The RLE nuclear protein database developed using the Elsie 5 gel analysis system contains both qualitative and quantitative annotations including polypeptide identification number, protein name (if known), molecular weight and pI information, quantitation and polypeptide spot shape, subcellular location, as well as specific information regarding transformation (chemical and spontaneous) and growth-related characteristics. Microsequencing of polypeptides directly from two-dimensional (2-D) blotted membranes has recently been established in our laboratory and provides a highly efficient and rapid means of polypeptide identification in the absence of specific antibodies. At present the RLE protein database is still in the developmental stage and is continually being updated as additional information is obtained. Nonetheless, it is anticipated that knowledge obtained concerning the identification and characterization of specific transformation and/or growth regulatory proteins in the RLE in vitro cell system will not only have direct application to other rodent and human 2-D protein databases currently under development but will also complement them.

The methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [32P]orthophosphate and [35S]methionine, performing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2-D PAGE electrophoretograms using two films placed back-to-back. The first film, which is positioned in direct contact with the dried silver-stained gel, visualized both exposure to 35S and 32P while the second film recorded exposure to only 32P due to the differential energy levels of the two isotopes. The juxta-positioning of the silver-stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver-stained and methionine-labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type beta 1 (TGF-beta 1)-mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI 4.95/M(r) 97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF-beta 1. The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involving protein phosphorylation.

It is generally accepted that cancer development is a multistage process involving a variety of cell types (Farber and Cameron 1980). Over the last 20 years considerable effort has been made to define the genetic events involved in the etiology of cancer development. Indeed, molecular genetics has been highly successful in the demonstration that development of certain forms of human cancers, in particular, colorectal, lung, and breast, may arise following a series of four to six distinct genetic alterations in specific target tissues (Fearon and Vogelstein 1990). These mutations may involve either the activation of one or more cellular protooncogene(s) and/or inactivation of one or more tumor suppressor gene(s) and are thought to initiate a cascade of biochemical events that culminate in the dysregulation of normal control mechanisms governing cellular growth and differentiation (Weinberg 1989; Hunter 1991). Regardless of the nature of the oncogenic agent, the observed phenotypic effects on the host cells are remarkably similar. These include characteristic alterations in cellular morphology, adhesiveness and motility, and cell-to-cell communication, as well as dysregulation of cellular growth and differentiation, including loss of contact growth inhibition in vitro, acquisition of anchorage-independent growth in soft agar, and tumorigenicity in susceptible animals.

Background Aromatic rice is popular worldwide because of its characteristic fragrance. Genetic studies and physical fine mapping reveal that a candidate gene (fgr/OsBADH2) homologous to betaine aldehyde dehydrogenase is responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of OsBADH2 is lacking. To elucidate the physiological roles of OsBADH2, sequencing approach and RNA interference (RNAi) technique were employed to analyze allelic variation and functions of OsBADH2 gene in aroma production. Semi-quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as gas chromatography-mass spectrometry (GC-MS) were conducted to determine the expression levels of OsBADH2 and the fragrant compound in wild type and transgenic OsBADH2-RNAi repression lines, respectively. Results The results showed that multiple mutations identical to fgr allele occur in the 13 fragrant rice accessions across China; OsBADH2 is expressed constitutively, with less expression abundance in mature roots; the disrupted OsBADH2 by RNA interference leads to significantly increased 2-acetyl-1-pyrroline production. Conclusion We have found that the altered expression levels of OsBADH2 gene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin.