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Improving the drought resistance of rice is of great significance for expanding the planting area and improving the stable yield of rice. In our previous work, we found that ROLLED AND ERECT LEAF1 (REL1) protein promoted enhanced tolerance to drought stress by eliminating reactive oxygen species (ROS) levels and triggering the abscisic acid (ABA) response. However, the mechanism through which REL1 regulates drought tolerance by removing ROS is unclear. In this study, we identified REL1 interacting protein 5 (RIP5) and found that it directly combines with REL1 in the chloroplast. We found that RIP5 was strongly expressed in ZH11 under drought-stress conditions, and that the rip5-ko mutants significantly improved the tolerance of rice plants to drought, whereas overexpression of RIP5 resulted in greater susceptibility to drought. Further investigation suggested that RIP5 negatively regulated drought tolerance in rice by decreasing the content of ascorbic acid (AsA), thereby reducing ROS clearance. RNA sequencing showed that the knockout of RIP5 caused differential gene expression that is chiefly associated with ascorbate and aldarate metabolism. Furthermore, multiple experimental results suggest that REL1 is involved in regulating drought tolerance by inhibiting RIP5. Collectively, our findings reveal the importance of the inhibition of RIP5 by REL1 in affecting the rice's response to drought stress. This work not only explains the drought tolerance mechanism of rice, but will also help to improve the drought tolerance of rice. [ABSTRACT FROM AUTHOR]

Abstract Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters.

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. B acillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ ZosA) as a P1B4-type ATPase in the PerR regulon that serves as an Fe( II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfe T mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn( II). Conversely, a pfe T mutant is more resistant to Mn( II) overload. In vitro, the PfeT ATPase is activated by both Fe( II) and Co( II), although only Fe( II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4-type ATPases previously implicated in bacterial pathogenesis. [ABSTRACT FROM AUTHOR]

In this paper, we investigate the multiple attribute decision making problems with dual hesitant fuzzy information, in which the information about attribute weights is incompletely known, and the attribute values take the form of dual hesitant fuzzy values. We first introduce some approaches to obtaining the weight information of attributes, and then establish an optimization model based on the positive ideal point of attribute values, by which the attribute weights can be determined. For the special situations where the information about attribute weights is completely unknown, we establish another optimization model. By solving this model, we get a simple and exact formula, which can be used to determine the attribute weights. We compute the correlation coefficient between each alternative and positive ideal alternative, and then rank the alternatives by means of the correlation coefficient between each alternative and positive ideal alternative. Finally, we shall present a numerical example to show the pattern recognition problems about the classification of metal materials with dual hesitant fuzzy information in order to illustrate the method proposed in this paper. [ABSTRACT FROM AUTHOR]

In this work, the medium containing ionic products of dicalcium silicates (Ca2SiO4) for culturing MG63 cells was prepared by immersing a titanium alloy plate with the plasma sprayed Ca2SiO4 coatings in DMEM solution. The effect of the ionic products on cellular differentiation, collagen production, and local growth factors (prostaglandin E2 [PGE2] and transforming growth factor-β [TGF-β1]) of osteoblast-like MG63 cells were investigated. The normal DMEM was also used to culture MG63 cells as the control group. Differentiation of cell was evaluated by detecting alkaline phosphatase (ALP) activity and osteocalcin (OC) synthesis as well as their gene expression. Collagen production was analyzed by Sircol assay. The levels of PGE2 and TGF-β1 in culture medium were measured using enzyme-linked immunosorbent assay (ELISA). The gene expressions of TGF-β receptors (TGF-β RI and TGF-β RII) were also measured by real-time PCR technology. MG63 cells cultured in DMEM containing ionic products of Ca2SiO4 coating showed enhanced differentiation and increased collagen production. The results obtained from ELISA showed that the levels of PGE2 and TGF-β1 in experimental group were higher than that in control. The gene expression of TGF-β receptors was upregulated, indicating that more TGF-β1 bonded to their receptors which produce more effects on the osteoblastic activity, leading to enhanced differentiation and synthetic activity of osteoblast. It is concluded that ionic products of Ca2SiO4 coating may enhance cellular differentiation and collagen production by influencing TGF-β1 pathway. [ABSTRACT FROM AUTHOR]

An astaxanthin-overproducing (∼1000 μg g) strain of Phaffia rhodozyma, termed MK19, was established through 1-methyl-3-nitro-1-nitrosoguanidine and Co60 mutagenesis from wild-type JCM9042 (merely 35-67 μg g). The total fatty acid content of MK19 was much lower than that of the wild type. Possible causes of the astaxanthin increase were studied at the gene expression level. The expression of the carotenogenic genes crtE, crtI, pbs, and ast, which are responsible for astaxanthin biosynthesis from geranylgeranyl pyrophosphate, was highly induced at the mRNA level, leading to excessive astaxanthin accumulation. In contrast, transcription levels of the genes ( hmgs, hmgr, idi, mvk, mpd, fps), responsible for the initial steps in the terpenoid pathway, were essentially the same in wild type and MK19. Although fatty acid and total ergosterol content were reduced by 40-70 mg g and 760.3 μg g, respectively, in MK19 as compared with the wild type, but the transcription levels of rate-limiting genes in fatty acid and ergosterol pathways such as acc and sqs were similar. Because fatty acids and ergosterol are two branch pathways of astaxanthin biosynthesis in P. rhodozyma, our findings indicate that enhancement of astaxanthin in MK19 results from decreased fatty acid and ergosterol biosynthesis, leading to precursor accumulation, and transfer to the astaxanthin pathway. Strengthening of the mevalonate pathway is suggested as a promising metabolic engineering approach for further astaxanthin enhancement in MK19. [ABSTRACT FROM AUTHOR]

Phosphate (P) availability often limits biological nitrogen fixation (BNF) by diazotrophic bacteria. In soil, only 0.1% of the total P is available for plant uptake. P solubilizing bacteria can convert insoluble P to plant-available soluble P (ionic P and low molecular-weight organic P). However, limited information is available about the effects of synergistic application of diazotrophic bacteria and P solubilizing bacteria on the nitrogenase activity of rhizosphere and nifH expression of endosphere. In this study, we investigated the effects of co-inoculation with a diazotrophic bacterium ( Paenibacillus beijingensis BJ-18) and a P-solubilizing bacterium ( Paenibacillus sp. B1) on wheat growth, plant and soil total N, plant total P, soil available P, soil nitrogenase activity and the relative expression of nifH in plant tissues. Co-inoculation significantly increased plant biomass (length, fresh and dry weight) and plant N content (root: 27%, shoot: 30%) and P content (root: 63%, shoot: 30%). Co-inoculation also significantly increased soil total N (12%), available P (9%) and nitrogenase activity (69%) compared to P. beijingensis BJ-18 inoculation alone. Quantitative real-time PCR analysis showed co-inoculation doubled expression of nifH genes in shoots and roots. Soil nitrogenase activity and nifH expression within plant tissues correlated with P content of soil and plant tissues, which suggests solubilization of P by Paenibacillus sp. B1 increased N fixation in soils and the endosphere. In conclusion, P solubilizing bacteria generally improved soil available P and plant P uptake, and considerably stimulated BNF in the rhizosphere and endosphere of wheat seedlings., Competing Interests: The authors declare that they have no competing interests., (© 2020 Li et al.)

Paenibacillus is a large genus of Gram-positive, facultative anaerobic, endospore-forming bacteria. The genus Paenibacillus currently comprises more than 150 named species, approximately 20 of which have nitrogen-fixation ability. The N 2 -fixing Paenibacillus strains have potential uses as a bacterial fertilizer in agriculture. In this study, 179 bacterial strains were isolated by using nitrogen-free medium after heating at 85 °C for 10 min from 69 soil samples collected from different plant rhizospheres in different areas. Of the 179 bacterial strains, 25 Paenibacillus strains had nifH gene encoding Fe protein of nitrogenase and showed nitrogenase activities. Of the 25 N 2 -fixing Paenibacillus strains, 22 strains produced indole-3-acetic acid (IAA). 21 strains out of the 25 N 2 -fixing Paenibacillus strains inhibited at least one of the 6 plant pathogens Rhizoctonia cerealis , Fusarium graminearum , Gibberella zeae , Fusarium solani , Colletotrichum gossypii and Alternaria longipes . 18 strains inhibited 5 plant pathogens and Paenibacillus sp. SZ-13b could inhibit the growth of all of the 6 plant pathogens. According to the nitrogenase activities, antibacterial capacities and IAA production, we chose eight strains to inoculate wheat, cucumber and tomato. Our results showed that the 5 strains Paenibacillus sp. JS-4, Paenibacillus sp. SZ-10, Paenibacillus sp. SZ-14, Paenibacillus sp. BJ-4 and Paenibacillus sp. SZ-15 significantly promoted plant growth and enhanced the dry weight of plants. Hence, the five strains have the greater potential to be used as good candidates for biofertilizer to facilitate sustainable development of agriculture., Competing Interests: The authors declare there are no competing interests., (©2019 Liu et al.)

Magnetotactic bacteria synthesize intracellular membrane-enveloped magnetite bodies known as magnetosomes which have been applied in biotechnology and medicine. A series of proteins involved in ferric ion transport and redox required for magnetite formation have been identified but the knowledge of magnetosome biomineralization remains very limited. Here, we identify a novel OxyR homolog (named OxyR-Like), the disruption of which resulted in low ferromagnetism and disfigured nano-sized iron oxide crystals. High resolution-transmission electron microscopy showed that these nanoparticles are mainly composed of magnetite accompanied with ferric oxide including α-Fe 2 O 3 and 𝜀-Fe 2 O 3 . Electrophoretic mobility shift assay and DNase I footprinting showed that OxyR-Like binds the conserved 5'-GATA-N{9}-TATC-3' region within the promoter of pyruvate dehydrogenase ( pdh ) complex operon. Quantitative real-time reverse transcriptase PCR indicated that not only the expression of pdh operon but also genes related to magnetosomes biosynthesis and tricarboxylic acid cycle decreased dramatically, suggesting a link between carbon metabolism and magnetosome formation. Taken together, our results show that OxyR-Like plays a key role in magnetosomes formation.

Magnetotactic bacteria are capable of forming nanosized, membrane-enclosed magnetosomes under iron-rich and oxygen-limited conditions. The complete genomic sequence of Magnetospirillum gryphiswaldense strain MSR-1 has been analyzed and found to contain five fur homologue genes whose protein products are predicted to be involved in iron homeostasis and the response to oxidative stress. Of these, only the MGMSRv2&#95;3149 gene (irrB) was significantly downregulated under high-iron and low-oxygen conditions, during the transition of cell growth from the logarithmic to the stationary phase. The encoded protein, IrrB, containing the conserved HHH motif, was identified as an iron response regulator (Irr) protein belonging to the Fur superfamily. To investigate the function of IrrB, we constructed an irrB deletion mutant (ΔirrB). The levels of cell growth and magnetosome formation were lower in the ΔirrB strain than in the wild type (WT) under both high-iron and low-iron conditions. The ΔirrB strain also showed lower levels of iron uptake and H2O2 tolerance than the WT. Quantitative real-time reverse transcription-PCR analysis indicated that the irrB mutation reduced the expression of numerous genes involved in iron transport, iron storage, heme biosynthesis, and Fe-S cluster assembly. Transcription studies of the other fur homologue genes in the ΔirrB strain indicated complementary functions of the Fur proteins in MSR-1. IrrB appears to be directly responsible for iron metabolism and homeostasis and to be indirectly involved in magnetosome formation. We propose two IrrB-regulated networks (under high- and low-iron conditions) in MSR-1 cells that control the balance of iron and oxygen metabolism and account for the coexistence of five Fur homologues., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel., Results: Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%., Conclusions: Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

Background: Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF., Results: A novel lipase Est&#95;p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est&#95;p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est&#95;p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 μmol∙min⁻¹∙mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est&#95;p6 has safety for commercial LMF flavor production and food manufacturing processes., Conclusions: The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est&#95;p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.

The bacterial strain Magnetospirillum gryphiswaldense MSR-1 does not produce siderophores, but it absorbs a large amount of ferric iron and synthesizes magnetosomes. We demonstrated previously the presence of six types of ferric reductase isozymes (termed FeR1 through FeR6) in MSR-1. Of these isozymes, FeR5 was the most abundant and FeR6 showed the highest ferric reductase activity. In the present study, we cloned the fer5 and fer6 genes from MSR-1 and expressed them separately in Escherichia coli. FeR5 and FeR6 were shown to be bifunctional enzymes through analysis of amino acid sequence homologies, structural predictions (using data from GenBank), and detection of enzyme activities. FeR5 is a thioredoxin reductase and FeR6 is a flavin reductase, in addition to being ferric reductases. To elucidate the functions of the enzymes, we constructed two single-gene-deletion mutant strains (Δfer5 and Δfer6 mutants) and a double-gene-deletion mutant strain (Δfer5 Δfer6 [Δfer5+6] mutant) along with its complemented strains (C5 and C6). An evaluation of phenotypic and physiological properties did not reveal significant differences between the wild-type and single-gene-deletion strains, whereas the double-gene-deletion strain showed reduced iron absorption and no magnetosome synthesis. Complementation of the double-gene-deletion strain using either fer5 or fer6 resulted in the partial recovery of magnetosome synthesis. Quantitative real-time PCR analysis of fer5 and fer6 transcriptional levels in the wild-type and complemented strains demonstrated consistent transcription of the two genes and confirmed that FeR5 and FeR6 are bifunctional enzymes that play complementary roles during the process of magnetosome synthesis in MSR-1.

Objective: To explore the surgical technique and the effectiveness of reconstructing the hip center in total hip arthroplasty (THA) for developmental dysplasia of hip (DDH)., Methods: Between January 2004 and January 2010, 29 patients (32 hips) with DDH underwent THA. There were 6 males (6 hips) and 23 females (26 hips), aged 45-67 years (mean, 50.6 years). The locations were left side in 22 hips and right side in 10 hips. According to Crowe classification, 12 hips were rated as Crowe I and 20 hips as Crowe II. The patients had limb length discrepancy of 1.9-4.4 cm. The Harris score was 50.7 +/- 8.6., Results: All incisions healed by first intension. Posterior hip dislocation occurred in 1 case (1 hip) after operation. All patients were followed up 2 years-4 years and 6 months (mean, 2.3 years). The lower limbs were lengthened by 0.5-5.8 cm (mean, 2.5 cm). The Harris score was 87.7 +/- 5.9 at 1 year after operation, showing significant difference when compared with preoperative score (t = 21.77, P = 0.00). X-ray films showed loosening of acetabular component in 1 case (1 hip) and displacement of acetabular component with too large abduction angle in 1 case (1 hip); no loosening and subsidence of acetabular or femoral components occurred in the other patients during follow-up. The horizontal location of hip center, the vertical distance between teardrop and the hip center, the vertical acetabular abduction angle, and the femoral offset were better than those before operation (P < 0.05)., Conclusion: For DDH patients, reconstructing the hip center in THA can lengthen the limb, improve the joint function, and reduce the failure rate of joint replacement.

Magnetotactic bacteria (MTB) synthesize unique organelles, the magnetosomes, which are intracellular nanometer-sized, membrane-enveloped magnetite. The biomineralization of magnetosomes involves the uptake of large amounts of iron. However, the iron metabolism of MTB is not well understood. The genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense strain MSR-1 contains two ferrous iron transport genes, feoB1 and feoB2. The FeoB1 protein was reported to be responsible mainly for the transport of ferrous iron and to play an accessory role in magnetosome formation. To determine the role of feoB2, we constructed an feoB2 deletion mutant (MSR-1 ΔfeoB2) and an feoB1 feoB2 double deletion mutant (MSR-1 NfeoB). The single feoB2 mutation did not affect magnetite crystal biomineralization. MSR-1 NfeoB had a significantly lower average magnetosome number per cell (∼65%) than MSR-1 ΔfeoB1, indicating that FeoB2 plays a role in magnetosome formation when the feoB1 gene is deleted. Our findings showed that FeoB1 has a greater ferrous iron transport ability than FeoB2 and revealed the differential roles of FeoB1 and FeoB2 in MSR-1 iron metabolism. Interestingly, compared to the wild type, the feoB mutants showed increased sensitivity to oxidative stress and lower activities of the enzymes superoxide dismutase and catalase, indicating that the FeoB proteins help protect bacterial cells from oxidative stress.

Five of 8 fungal lipases screened were found to effectively hydrolyze astaxanthin esters from Haematococcus pluvialis algal cell extracts. Among these, an alkaline lipase from Penicillium cyclopium, expressed in Pichia pastoris, had the highest enzymolysis efficiency. Tween80 was shown to be an effective emulsifier in this lipase hydrolysis system for the 1st time. A series of experiments were performed to find optimal conditions for hydrolysis (pH, temperature, reaction time, lipase dosage). In the optimal reaction system, Tween80 and H. pluvialis extracts (mass ratio 1:1) were emulsified and added to the above lipase at a dosage of 4.6 U/μg (relative to total carotenoids), in phosphate buffer (0.1 M, pH 7.0), and incubated at 28 °C for 7 h, with agitation at 180 rpm. The free astaxanthin recovery ratio under these conditions was 63.2%., (© 2011 Institute of Food Technologists®)