Protective effect of oxytocin on vincristine-induced gastrointestinal dysmotility in mice.

Aims: Vincristine (VCR), an antineoplastic drug, induces peripheral neuropathy characterized by nerve damage, limiting its use and reducing the quality of life of patients. VCR causes myenteric neuron damage, inhibits gastrointestinal motility, and results in constipation or paralytic ileus in patients. Oxytocin (OT) is an endogenous neuropeptide produced by the enteric nerve system, which regulates gastrointestinal motility and exerts neuroprotective effects. This study aimed to investigate whether OT can improve VCR-induced gastrointestinal dysmotility and evaluate the underlying mechanism. Methods: Mice were injected either with saline or VCR (0.1 mg/kg/d, i. p.) for 14 days, and OT (0.1 mg/kg/d, i.p.) was applied 1 h before each VCR injection. Gastrointestinal transit and the contractile activity of the isolated colonic segments were assessed. The concentration of OT in plasma was measured using ELISA. Immunofluorescence staining was performed to analyze myenteric neurons and reactive oxygen species (ROS) levels. Furthermore, the indicators of oxidative stress were detected. The protein expressions of Nrf2, ERK1/2, P-ERK1/2, p38, and P-p38 in the colon were tested using Western blot. Results: VCR reduced gastrointestinal transit and the responses of isolated colonic segments to electrical field stimulation and decreased the amount of neurons. Furthermore, VCR reduced neuronal nitric oxide synthase and choline acetyltransferase immunopositive neurons in the colonic myenteric nerve plexus. VCR increased the concentration of OT in plasma. Exogenous OT pretreatment ameliorated the inhibition of gastrointestinal motility and the injury of myenteric neurons caused by VCR. OT pretreatment also prevented the decrease of superoxide dismutase activity, glutathione content, total antioxidative capacity, and Nrf2 expression, the increase of ROS levels, and the phosphorylation of ERK1/2 and p38 MAPK following VCR treatment. Conclusion: Our results suggest that OT pretreatment can protect enteric neurons from VCR-induced injury by inhibiting oxidative stress and MAPK pathways (ERK1/2, p38). This may be the underlying mechanism by which it alleviates gastrointestinal dysmotility.

Potential and benefits of biochar production: crop straw management and carbon emission mitigation in Shaanxi Province, China.

Shaanxi Province is an important agricultural province in western China. Its profit-oriented management of crop residues remains a concern in the agriculture sector. Aiming to accelerate the valorization of agricultural straw and offer potential solutions for profit-oriented use of crop residues in Shaanxi, this study estimated the quantity of resources and collectable amount of crop straw by using the grain-to-straw ratio, analyzed the carbon emission reduction potential considering biochar energy and soil uses with the help of a life cycle assessment (LCA) model, and calculated the economic benefits of biochar production using waste and abandoned straw in Weinan (a city of Shaanxi). The theoretical resources and collectible amount of crop straw in Shaanxi showed an overall growth trend from 1949 to 2021, reaching 1.47 × 107 and 1.26 × 107 t in 2021 respectively. In 2021, straw from corn, wheat, and other grains accounted for 94.32% of the total straw. Among the 11 cities in Shaanxi, Weinan had the largest straw resources of 2.82 × 106 t, Yulin had the largest per capita straw resources of 0.72 t/person, and Yangling had the highest resource density of 7.60 t/hm2. The total carbon emission reduction was 3.11 × 104 t under scenario A with crop straw used for power generation. The emission reduction ranged from 1.25 × 107 to 1.27 × 107 CO2e t under scenario B with biochar production for energy and soil use. By using waste and abandoned straw in Weinan for biochar production, carbon emissions could be reduced by up to 2.07 × 105 t CO2e. In terms of the economic benefit from straw pyrolysis, the actual income was estimated to range from 0.67 × 108 to 1.33 × 108 ¥/a with different carbon prices. This study sheds light on the economic and environmental benefits of agricultural straw valorization through pyrolysis in Shaanxi, and provided an important basis for promoting the agricultural straw utilization in view of its potential for carbon emission reduction.

Remediation of fomesafen contaminated soil by Bacillus sp. Za: Degradation pathway, community structure and bioenhanced remediation.

Fomesafen is a diphenyl ether herbicide used to control the growth of broadleaf weeds in bean fields. The persistence, phytotoxicity, and negative impact on crop rotation associated with this herbicide have led to an increasing concern about the buildup of fomesafen residues in agricultural soils. The exigent matter of treatment and remediation of soils contaminated with fomesafen has surfaced. Nevertheless, the degradation pathway of fomesafen in soil remains nebulous. In this study, Bacillus sp. Za was utilized to degrade fomesafen residues in black and yellow brown soils. Fomesafen's degradation rate by strain Za in black soil reached 74.4%, and in yellow brown soil was 69.2% within 30 days. Twelve intermediate metabolites of fomesafen were identified in different soils, with nine metabolites present in black soil and eight found in yellow brown soil. Subsequently, the degradation pathway of fomesafen within these two soils was inferred. The dynamic change process of soil bacterial community structure in the degradation of fomesafen by strain Za was analyzed. The results showed that strain Za potentially facilitate the restoration of bacterial community diversity and richness in soil samples treated with fomesafen, and there were significant differences in species composition at phylum and genus levels between these two soils. However, both soils shared a dominant phylum and genus, Actinobacteriota, Proteoobacteria, Firmicutes and Chloroflexi dominated in two soils, with a high relative abundance of Sphingomonas and Bacillus. Moreover, an intermediate metabolite acetaminophen degrading bacterium, designated as Pseudomonas sp. YXA-1, was isolated from yellow brown soil. When strain YXA-1 was employed in tandem with strain Za to remediate fomesafen contaminated soil, the degradation rate of fomesafen markedly increased. Overall, this study furnishes crucial insights into the degradation pathway of fomesafen in soil, and presents bacterial strain resources potentially beneficial for soil remediation in circumstances of fomesafen contamination.

[Temporal and Spatial Distribution, Utilization Status, and Carbon Emission Reduction Potential of Straw Resources in China].

Based on the crop yield data of China and each region from 1981 to 2020 (excluding data from Hong Kong, Macao, and Taiwan), by using the grain-straw ratio method, this study estimated the total amount of crop straw and collectable amount of crops, including corn, rice, wheat, other cereals, cotton, rapeseeds, peanuts, beans, tubers, sesame, fiber crops, sugarcane, and beetroots, and the spatial and temporal distribution characteristics of resource density and per capita resources of crop straw were analyzed. This study analyzed the current utilization mode, development, and change of crop straw in China. Finally, we used the life circle assessment (LCA) method to estimate the carbon emission reduction potential of biochar prepared from crop straw. The main findings were:from 1981 to 2020, the temporal distribution trend of theoretical crop straw resources and collectable straw resources in China generally showed a steady growth trend, and the two increased from 3.33×108 t and 3.04×108 t in 1981 to the highest values of 7.70×108 t and 6.63×108 t in 2020, with a net increase of 4.37×108 t and 3.59×108 t, respectively. The net increase in rice, wheat, and corn straw resources was 3.69×108t, accounting for between 77% and 85% of the total crop straw and always occupying the main position of straw resources in China. The proportion of wheat straw in the total amount of straw was maintained at approximately 20%, rice straw resources decreased from 44% to 28.4%, and corn straw increased from 19.9% to 34.2% from 1981 to 2020. In 2020, the total theoretical resources of crop straw in China were 7.72×108 t, and the source structures were:rice 28.4%, wheat 21.45%, corn 31.45%, other cereals 1.4%, beans 3.4%, tubers 0.82%, cotton 2.28%, peanuts 2.97%, rapeseeds 3.4%, sesame 0.12%, fiber crops 0.06%, beetroots 0.67%, and sugarcane 0.84%. As to the spatial distribution of crop straw resources in China in 2020, the locations with straw resources ≥ 60 million tons included Heilongjiang, Henan, and Shandong, of which Henan had as much as 88.56 million tons; those with between 40 million and 60 million tons included Hebei, Inner Mongolia, Jiangsu, and Anhui; those with between 20 million and 40 million tons included Liaoning, Jilin, Jiangxi, Hubei, Hunan, Sichuan, Yunnan, and Xinjiang; and the straw resources in the rest of the region were below 20 million tons. Rice straw was mostly distributed in the middle and lower reaches of the Yangtze River and the Northeast region, of which the amount of Heilongjiang rice straw was the largest, with 31.86 million tons; wheat straw was mainly distributed in North China, with Henan having the most abundant resources (48.04 million tons). Corn straw was mainly distributed in Northeast China and North China, of which Heilongjiang and Inner Mongolia corn straw resources were relatively rich, with 33.18 million tons and 29.90 million tons, respectively. Crop straw resource density and per capita resources were shared in 2020 in China. The average density of crop straw resources in China was 4.61 t·hm-2, and the average densities of crop straw resources in various agricultural areas were 5.39 t·hm-2 in Northeast China, 5.42 t·hm-2 in North China, 4.45 t·hm-2 in the Mengxin Region, 4.44 t·hm-2 in the middle and lower reaches of the Yangtze River, 3.92 t·hm-2in Tibet, 3.40 t·hm-2 in the Loess Plateau, 3.08 t·hm-2 in South China, and 2.91 t·hm-2 in Southwest China. The average per capita share of straw resources was 0.55 t. The average values of per capita straw resources in each region were:1.46 t in the Northeast area, 1.20 t in the Mengxin Region, 0.47 t in North China, 0.44 t in the middle and lower reaches of the Yangtze River, 0.40 t in the Loess Plateau, 0.37 t in the Southwest area, 0.33 t in the Qinghai-Tibet area, and 0.20 t in the South China area. The utilization of crop straw in China was diversified. Fertilizer and feed were the main utilizations, accounting for 62.1% and 15.4%, respectively. In 2020, collectable crop straw resources for the preparation of biochar totaled 2.04×108 t in China. Renewable energy replaced fossil fuels in the process of preparing biochar, which could reduce CO2e(CO2e:CO2 equivalent) emissions by 1.45×108 t. Biochar could sequester approximately 4.63×108 t of CO2e; biochar application was able to reduce chemical fertilizer application to achieve a CO2 emission reduction of 8.58×105 t; and biochar application could promote crop yield in order to reduce CO2e emissions by approximately 7.77×106 t. The inhibition of N2, respectively. In the process of biochar preparation and application, the total greenhouse gas emission was 3.32×107 t, and the net greenhouse effect emission reduction reached 5.86×108 t, i.e., it could sequester 0.88 t CO2e per ton of raw materials. The net greenhouse gas emission reduction of unused straw was 6.73×107 t in 2020. With the continuous harvest of grain crops in China, the potential of biochar preparation and carbon sequestration will increase yearly. Using crop straw to prepare biochar has great potential and will be one of the most effective ways to achieve carbon emission reduction in agriculture. It is suggested that government departments should pay attention to the preparation of biochar, support the field experiments of biochar application effects after applying soil on policy and funds, and then introduce relevant biochar standards to ensure the scientific application of biochar prepared by crop straw according to local conditions, so as to achieve the dual benefits of carbon emission reduction and soil remediation and yield increase.

A fast and efficient method for isolating and culturing enteric neural precursor cells from adult mouse colon.

BACKGROUND: The enteric neural precursor cells (ENPCs) are important for researching the pathogenesis of enteric nervous system (ENS)-related diseases, especially in adulthood. Because primary ENPCs are difficult to isolate and survive, easy to contaminate and low-yielding, a rapid and effective method to isolate and cultivate ENPCs from adult mice is necessary. NEW METHODS: The longitudinal muscle myenteric plexus (LMMP) was isolated from the adult mouse colon. The papain and collagenase Ⅱ were chosen to increase the yield of ENPCs. The growth and proliferation of ENPCs could be promoted by using polylysine precoated culture plates and reasonable cell seeding density. The ENPCs were identified by Nestin and the proliferative properties were verified by EDU. The transgenic Nestin-cre:tdTomato mice were used to observe the proliferation of ENPCs more intuitively in vitro. RESULTS: Our method to isolate the ENPCs from adult mouse colon was effective and high-yielding. The ENPCs were identified as Nestin positive. The Nestin-positive ENPCs could proliferate rapidly and had a tendency to differentiate into neurons and glial cells in vitro without any inducing factors. COMPARISON WITH EXISTING METHODS: Previous studies about the ENPCs focused on experiment in vivo or were limited to the embryonic mice, and had limitations of low yield and long experiment time. The ENPCs from adult mice were isolated quickly by our method, and had a high yield and purity. CONCLUSION: We described a rapid, efficient, step by step method for isolation and culture of ENPCs from the adult mouse colon, which was simple and obtained high yield of ENPCs.

Precise Regulation of Differential Transcriptions of Various Catabolic Genes by OdcR via a Single Nucleotide Mutation in the Promoter Ensures the Safety of Metabolic Flux.

Synergistic regulation of the expression of various genes in a catabolic pathway is crucial for the degradation, survival, and adaptation of microorganisms in polluted environments. However, how a single regulator accurately regulates and controls differential transcriptions of various catabolic genes to ensure metabolic safety remains largely unknown. Here, a LysR-type transcriptional regulator (LTTR), OdcR, encoded by the regulator gene odcR, was confirmed to be essential for 3,5-dibromo-4-hydroxybenozate (DBHB) catabolism and simultaneously activated the transcriptions of a gene with unknown function, orf419, and three genes, odcA, odcB, and odcC, involved in the DBHB catabolism in Pigmentiphaga sp. strain H8. OdcB further metabolized the highly toxic intermediate 2,6-dibromohydroquinone, which was produced from DBHB by OdcA. The upregulated transcriptional level of odcB was 7- to 9-fold higher than that of orf419, odcA, or odcC in response to DBHB. Through an electrophoretic mobility shift assay and DNase I footprinting assay, DBHB was found to be the effector and essential for OdcR binding to all four promoters of orf419, odcA, odcB, and odcC. A single nucleotide mutation in the regulatory binding site (RBS) of the promoter of odcB (TAT-N11-ATG), compared to those of odcA/orf419 (CAT-N11-ATG) and odcC (CAT-N11-ATT), was identified and shown to enable the significantly higher transcription of odcB. The precise regulation of these genes by OdcR via a single nucleotide mutation in the promoter avoided the accumulation of 2,6-dibromohydroquinone, ensuring the metabolic safety of DBHB. IMPORTANCE Prokaryotes use various mechanisms, including improvement of the activity of detoxification enzymes, to cope with toxic intermediates produced during catabolism. However, studies on how bacteria accurately regulate differential transcriptions of various catabolic genes via a single regulator to ensure metabolic safety are scarce. This study revealed a LysR-type transcriptional activator, OdcR, which strongly activated odcB transcription for the detoxification of the toxic intermediate 2,6-dibromohydroquinone and slightly activated the transcriptions of other genes (orf419, odcA, and odcC) for 3,5-dibromo-4-hydroxybenozate (DBHB) catabolism in Pigmentiphaga sp. strain H8. Interestingly, the differential transcription/expression of the four genes, which ensured the metabolic safety of DBHB in cells, was determined by a single nucleotide mutation in the regulatory binding sites of the four promoters. This study describes a new and ingenious regulatory mode of ensuring metabolic safety in bacteria, expanding our understanding of synergistic transcriptional regulation in prokaryotes.

Mutations of Asn321 and Glu322 Improve Resistance of 4-Hydroxyphenylpyruvate Dioxygenase SpHPPDm to Topramezone.

As a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, topramezone is an ideal target for herbicide-resistant genetic engineering. In this study, two mutants, K-19 (N321Y) and K-63 (Q166R/E322V), with topramezone resistance increased by 205.3 and 58.5%, respectively, were screened from the random mutation library of SpHPPDm, a topramezone-resistant HPPD mutant that we previously obtained. Sites N321 and E322 were identified as key sites for increased topramezone resistance by single-site mutation analysis. A mutant KB-145 (N321Y/E322K) was further obtained by saturation mutation at sites N321 and E322. The topramezone resistance of KB-145 increased by 955.3% compared to mutant SpHPPDm. In conclusion, this study identifies two new sites that significantly affect the topramezone resistance of SpHPPDm, which provides new insights into the molecular mechanism of herbicide resistance of HPPD, and the acquired mutants have great application potential in the construction of herbicide-resistant crops.

Genetic redundancy of 4-hydroxybenzoate 3-hydroxylase genes ensures the catabolic safety of Pigmentiphaga sp. H8 in 3-bromo-4-hydroxybenzoate-contaminated habitats.

Genetic redundancy is prevalent in organisms and plays important roles in the evolution of biodiversity and adaptation to environmental perturbation. However, selective advantages of genetic redundancy in overcoming metabolic disturbance due to structural analogues have received little attention. Here, functional divergence of the three 4-hydroxybenzoate 3-hydroxylase (PHBH) genes (phbh1~3) was found in Pigmentiphaga sp. strain H8. The genes phbh1/phbh2 were responsible for 3-bromo-4-hydroxybenzoate (3-Br-4-HB, an anthropogenic pollutant) catabolism, whereas phbh3 was primarily responsible for 4-hydroxybenzoate (4-HB, a natural intermediate of lignin) catabolism. 3-Br-4-HB inhibited 4-HB catabolism by competitively binding PHBH3 and was toxic to strain H8 cells especially at high concentrations. The existence of phbh1/phbh2 not only enabled strain H8 to utilize 3-Br-4-HB but also ensured the catabolic safety of 4-HB. Molecular docking and site-directed mutagenesis analyses revealed that Val199 and Phe384 of PHBH1/PHBH2 were required for the hydroxylation activity towards 3-Br-4-HB. Phylogenetic analysis indicated that phbh1 and phbh2 originated from a common ancestor and evolved specifically in strain H8 to adapt to 3-Br-4-HB-contaminated habitats, whereas phbh3 evolved independently. This study deepens our understanding of selective advantages of genetic redundancy in prokaryote's metabolic robustness and reveals the factors driving the divergent evolution of redundant genes in adaptation to environmental perturbation.

Improving the herbicide resistance of 4-hydroxyphenylpyruvate dioxygenase SpHPPD by directed evolution.

Topramezone, a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibitor herbicide, is an ideal target for herbicide-resistant genetic engineering. However, there is still a lack of HPPD gene that is highly resistant to topramezone. In previous studies, we obtained a topramezone-resistant HPPD (SpHPPDm) gene from Sphingobium sp. TPM-19, however, its resistance strength still could not meet the requirements for construction of herbicide-resistant crop. In this study, random mutagenesis (error-prone PCR) was employed to improve the topramezone resistance of SpHPPDm. Two mutants with improved resistance, K-28 (E322R) and K-113 (K249R, G327C), were screened from the random mutation library of SpHPPDm. The catalytic efficiency (kcat/Km) of mutants K-28 and K-113 only slightly decreased by approximately 2%. The half-maximal inhibitory concentration (IC50) of topramezone increased by 58.5% and 195.5% for mutants K-28 and K-113, respectively. Furthermore, mutant K-113 also showed significantly improved resistance to mesotrione and DKN (the active ingredient of isoxaflutole) with the IC50 increasing by 60.3% and 167.5%, respectively; while mutant K-28 only showed increased resistance to mesotrione with IC50 increasing by 77.6%, but reduced resistance to DKN with IC50 declining by 20.9%. Site-directed mutation assays revealed that G327C, but not K249R, contributed to topramezone resistance in mutant K-113. This study provides genetic resources for the genetic engineering of HPPD-inhibitor-resistant crops and a basis for further research on HPPD resistance mechanisms.

Role of Education and Mentorship in Entrepreneurial Behavior: Mediating Role of Self-Efficacy.

Farmers have been very precious for societies for ages. Their active experiments, valuable knowledge about their surroundings, environment, and crops' requirements have been a vital part of society. However, the psychological perspectives have been a hole in the loop of farming. Hence, this study has investigated the antecedents of entrepreneurial behaviors of farmers with the mediating risk of their entrepreneurial self-efficacy (ESE). The population chosen for this study was the farming community of suburbs of China, and a sample size of 300 was selected for the data collection. This is a survey study, where a structured questionnaire was adapted on a five-point Likert scale. The data were collected from the farming community to know their psychological and behavioral preferences about their profession. This study has produced interesting results that education, training, and intrinsic motivation play a vital role in farmers' ESE, affecting their entrepreneurial behaviors. This study will add to the body of knowledge and provide an eminent path for emerging entrepreneurs to find more mentorship opportunities to overcome the limitations in upcoming endeavors influencing education and training.

The effect of macrophage polarization on the expression of the oxytocin signalling system in enteric neurons.

BACKGROUND: The aim of the current study was to investigate the effect of macrophage polarization on the expression of oxytocin (OT) and the oxytocin receptor (OTR) in enteric neurons. METHODS: In this study, we used a classic colitis model and D-mannose model to observe the correlation between macrophage polarization and OT signalling system. In order to further demonstrate the effect of macrophages, we examined the expression of OT signalling system after depletion of macrophages. RESULTS: The data showed that, in vitro, following polarization of macrophages to the M1 type by LPS, the macrophage supernatant contained proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) that inhibited the expression of OT and OTR in cultured enteric neurons; following macrophage polarization to the M2 type by IL4, the macrophage supernatant contained anti-inflammatory cytokines (TGF-ß) that promoted the expression of OT and OTR in cultured enteric neurons. Furthermore, M1 macrophages decreased the expression of the OT signalling system mainly through STAT3/NF-κB pathways in cultured enteric neurons; M2 macrophages increased the expression of the OT signalling system mainly through activation of Smad2/3 and inhibition of the expression of Peg3 in cultured enteric neurons. In a colitis model, we demonstrated that macrophages were polarized to the M1 type during the inflammatory phase, with significant decreased in the expression of OT and OTR. When macrophages were polarized to the M2 type during the recovery phase, OT and OTR expression increased significantly. In addition, we found that D-mannose increased the expression of OT and OTR through polarization of macrophages to the M2 type. CONCLUSIONS: This is the first study to demonstrate that macrophage polarization differentially regulates the expression of OT and OTR in enteric neurons.

The neuroprotective effect of oxytocin on vincristine-induced neurotoxicity in mice.

Vincristine (VCR) is commonly used to treat a variety of hematological malignancies and solid tumors in pediatric and adult patients. However, peripheral neuropathy is a dose-limiting side effect that leaves some patients with functional disability and long-term pain. Oxytocin (OT) has demonstrated analgesic and anti-inflammatory properties, but there is no evidence regarding its effects on VCR-induced neurotoxicity. Therefore, we evaluated the potential protective effects of OT on VCR-induced neurotoxicity. In vitro, VCR (0.005 ∼ 0.1 µmol/l) and OT (10-8 ∼ 10-5 mol/l) were added into cultured primary dorsal root ganglion (DRG) neurons of mice. The length of neurites was counted by using immunofluorescence. In vivo, neurotoxicity was induced in mice by administration of VCR (0.1 mg/kg, intraperitoneal injection for 14 days) with or without pretreatment of OT (0.1 mg/kg or 1 mg/kg). Atosiban, an OT receptor (OTR) antagonist and OTR knockout (KO) mice were used for evaluating effects of OTR. Mechanical hyperalgesia was measured by using von Frey filaments. Histology of plantar skin, sciatic nerve and DRG was observed by using transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining. Results indicated that OT alleviated VCR-induced neurite damage in cultured primary DRG neurons in vitro. In vivo, OT ameliorated VCR-induced hyperalgesia. Histologically, OT attenuated the VCR-induced damages of nerve endings, myelin sheaths and Schwann cells in sciatic nerve and DRG. These effects were antagonized by atosiban. In addition, OTR knockout mice exhibited more severe hyperalgesia than wild-type mice. Globally, these results indicated that OT may have neuroprotective effects on vincristine-induced neurotoxicity in mice.

Pedobacter puniceum sp. nov. Isolated from Sludge.

Strain HX-22-1T was isolated from the sludge collected from the outlet of the biochemical treatment facility of an agricultural chemical factory in Nanjing city, Jiangsu province, PR China. Strain HX-22-1T is Gram-staining-negative, rod-shaped, non-spore-forming and non-flagellated. The 16S rRNA gene-based phylogenetic analysis indicates that the strain HX-22-1T belongs to the genus Pedobacter, closely related to Pedobacter glucosidilyticus KCTC 22438T (98.63% similarity). The genomic DNA G+C content of the strain was 34.4 mol%. Strain HX-22-1T was able to grow at 16-37 °C (optimum at 30 °C), at pH 6.0-8.0 (optimum at pH 7.0), and with 0-1% (w/v) NaCl (optimum at 0). Predominant fatty acid constituents were iso-C15:0 and summed feature 3 (iso-C16:1ω7c and/or C16:1ω6c). The predominant respiratory ubiquinone was MK-7. The polar lipid profile is composed of phosphatidylethanolamine (PE), aminophospholipid (APL), aminolipid (AL), and phospholipids (PL). The ANI and dDDH values obtained between the genomes of HX-22-1T and P. glucosidilyticus KCTC 22438T were 89.6 and 38.8%, respectively. On the basis of data from phenotypic, chemotaxonomic, and genotypic analysis, strain HX-22-1T represents a novel species of the genus Pedobacter, for which the name Pedobacter puniceum sp. nov. is proposed. The type strain is HX-22-1T (= KCTC 72655T = CCTCC AB 2019348T).

Crenobacter caeni sp. nov. Isolated from Sludge.

Strain HX-7-9T was isolated from the activated sludge collected from the outlet of the biochemical treatment facility of agricultural chemical plant in Nanjing, Jiangsu province, PR China. Strain HX-7-9T is Gram staining-negative, rod-shaped, non-spore-forming and flagellated. The 16S rRNA gene-based phylogenetic analysis indicate that strain HX-7-9T belongs to the genus Crenobacter, moderately related to Crenobacter luteus YIM-78141T (94.8% similarity). The genomic DNA G+C content of the strain was 67.5 mol%. Strain HX-7-9T was able to grow at 16-45 °C (optimum at 37 °C), at pH 6.0-8.0 (optimum at pH 7.0) and with 0-1% (w/v) NaCl (optimum at 0). Predominant fatty acid constituents were C16:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The respiratory ubiquinone was Q-8. The polar lipid profile is composed of diphosphatidylglycerol (DPG), phosphatidylmonomethylethanolamine (PME), phosphatidylethanolamine (PE), phospholipids (PL), glycolipid (GL) and aminophospholipid (APL). The ANI and dDDH values obtained between the genomes of HX-7-9T and C. luteus YIM-78141T were 79.8 and 19.1%, respectively. On the basis of data from phenotypic, chemotaxonomic and genotypic analysis, strain HX-7-9T represents a novel species of the genus Crenobacter, for which the name Crenobacter caeni sp. nov. is proposed. The type strain is HX-7-9T (= KCTC 72654T = CCTCC AB 2019349T).

Improved Herbicide Resistance of 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19 through Directed Evolution.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted extensive interest as a promising target for the genetic engineering of herbicide-resistant crops. However, naturally occurring HPPDs are generally very sensitive to HPPD inhibitors. In this study, random mutagenesis was performed to increase the HPPD inhibitors' resistance of Sphingobium sp. HPPD (SpHPPD). Two mutants, Q258M and Y333F, with improved resistance were obtained. Subsequently, a double-mutant (Q258M/Y333F) was generated through combined mutation. Q258M/Y333F exhibited the highest resistance to four HPPD inhibitors [topramezone, mesotrione, tembotrione, and diketonitrile (DKN)]. The enzyme fitness of Q258M/Y333F to topramezone, mesotrione, tembotrione, and DKN was increased by 4.0-, 4.1-, 4.2-, and 3.2-folds, respectively, in comparison with that of the wild-type. Molecular modeling and docking revealed that Q258M mutation leads to the decrease of enzyme-inhibitor-binding strength by breaking the hydrogen bond between the enzyme and the inhibitor, and Y333F mutation changes the conformational balance of the C-terminal helix H11, which hinders the binding of the inhibitor to the enzyme and thus would contribute to improved herbicide resistance. This study helps to further elucidate the structural basis for herbicide resistance and provides better genetic resources for the genetic engineering of herbicide-resistant crops.

High-level stable expression of gene for preparation of chlorothalonil hydrolytic dehalogenase and its application in elimination of chlorothalonil inhibition on bioconversion of lignocellulosic biomass.

To achieve the high-level stable expression of chlorothalonil hydrolytic dehalogenase (Chd), the gene chd was first integrated into the chromosome of Bacillus subtilis WB800. High generation stability was achieved by almost no gene lost after six generations but Chd activity decreased. aprE promoter alteration, translation initiation region modification and multi-copy chromosome integration were studied and these modifications could increase Chd activity by 270%, 2304% and 25%. Chlorothalonil residual exhibited inhibition on bioconversion of lignocellulosic biomass. The addition of Chd crude enzyme (60 µL per g wheat straw) could increase glucose production by 36.10% and 39.65% in synergistic hydrolysis and separate hydrolysis by laccase and cellulase with 120 mg/L residual chlorothalonil. Filter paper activity and carboxymethyl cellulase activity were enhanced by 12.84% and 23.95%, and biomass of Trichoderma reesei was increased by 76.67% under 50 µg chlorothalonil/g dry straw in solid-state fermentation. Thus, the high-level stable expressed Chd effectively eliminated chlorothalonil inhibition on enzymatic hydrolysis and solid-state fermentation. It showed promising potential for bioremediation of chlorothalonil pollution and improving conversion efficiency of lignocellulose.

Anaerobic biodegradation of acetochlor by acclimated sludge and its anaerobic catabolic pathway.

Acetochlor is a chloroacetamide herbicide that has been widely used for weed control in recent decades. The contamination from its residue in the environment has raised major serious concerns. The aerobic degradation of acetochlor has been well studied; however, little is known regarding its anaerobic degradation. In the study, anaerobic sludge with high acetochlor degradation efficiency was obtained by pressure acclimation in a continuous flow anaerobic reactor. The acetochlor degradation dynamics followed a first-order kinetic reaction equation. The acclimated sludge could degrade six chloroacetamide herbicides with the degradation efficiencies observed as alachlor > acetochlor > propisochlor > butachlor > pretilachlor > metolachlor, and the N-alkoxyalkyl structure of these herbicides significantly affected their biodegradability. Five metabolites, 2-ethyl-6-methyl-N-(ethoxymethyl)-acetanilide, N-(2-methyl-6-ethylphenyl) acetamide, N-2-ethylphenyl acetamide, N-2-ethylphenyl formamide and 2-ethyl-N-carboxyl aniline were identified, and a putative anaerobic acetochlor degradation pathway, initiated by dechlorination, was subsequently proposed. During acclimation, the community diversity of both eubacteria and archaea in the anaerobic sludge decreased, while the abundance of microbes belonging to genera Sporomusa, Sporobacterium, Dechloromonas, Azotobacter and Methanobacterium were significantly increased and dominated the acclimated sludge, and showing a positive correlation with the acetochlor degradation capacity. These findings should be valuable to elucidate the mechanisms associated with the anaerobic catabolism of acetochlor and facilitate the engineering application of anaerobic treatment for removing acetochlor from wastewater.

Effect of pesticide residues on simulated beer brewing and its inhibition elimination by pesticide-degrading enzyme.

Four different pesticide residues used in barley planting were selected to investigate their effects on simulated beer brewing. The influences were found to be different by varied agricultural chemicals. Among the four types of pesticides, at 25 µg/mL, triadimefon or carbendazim barely affected the brewing progress. However, ethametsulfuron-methyl and carbaryl (15 µg/mL and 2.5 µg/mL, respectively) exhibited slightly inhibition on saccharification and significantly negative impacts on yeast growth and alcohol fermentation. After pretreated by 50 µL carbaryl-degrading enzyme with the Kcat value of 2.12 s-1 at 30°C for 90 min, the negative influence on simulated beer brewing brought by carbaryl can be eliminated in the fermentation system containing 2.5 µg/mL carbaryl. The efficiency of ethanol fermentation was improved, and the removal rate of carbaryl in the brewing system was greatly accelerated. Taken together, this study suggested a potential method for solving the fermentation inhibition by pesticide residues.

Rhizobium terrae sp. nov., Isolated from an Oil-Contaminated Soil in China.

A Gram-stain-negative, facultative aerobic, non-spore-forming, non-motile, non-flagellated, rod-shaped bacterium, designated strain NAU-18T was isolated from an oil-contaminated soil in China. Strain NAU-18T could grow at 10-42 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, 7.0) and in the presence of 0-2.0% (w/v) NaCl (optimum, 0.5% NaCl in R2A). The predominant fatty acids were C18:1ω7c (71.2%) and Summed feature 2 (5.1%), representing 76.3% of the total fatty acids. The major respiratory quinones were Q9 and Q10. The DNA G + C content of strain NAU-18T was 61.4 mol% based on its draft genome sequence. Genome annotation of strain NAU-18T predicted the presence of 6668 genes, of which 6588 are coding proteins and 80 are RNA genes. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NAU-18T was a member of the genus Rhizobium and showed 96.93% (with 93.2% coverage) and 96.81% (with 100% coverage) identities with those of Neorhizobium alkalisoli CCBAU 01393T and Rhizobium oryzicola ZYY136T, respectively. In the phylogenetic analysis, strain NAU-18T and R. oryzicola ZYY136T are consistently placed in the same branch. Strain NAU-18T represents a novel species within the genus Rhizobium, for which the name Rhizobium terrae sp. nov. is proposed, with the type strain NAU-18T (=KCTC 62418T = CCTCC AB 2018075T).

Chitiniphilus eburneus sp. nov., a novel chitinolytic bacterium isolated from sludge.

A novel Gram-stain-negative, curved rod-shaped, motile and non-endospore-forming strain, designated HX-2-15T, was isolated from activated sludge of agricultural chemical plant in Nanjing, Jiangsu province, PR China (32° 03' N, 118° 46' E) . Growth was observed at 15-37 °C (optimum between 25 and 30 °C), at pH 6.0-8.0 (optimum at pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum at 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain showed closest affiliation to Chitiniphilus shinanonensis SAY3T, with a sequence similarity of 99.0 %. The predominant cellular fatty acids were C16:0, C17:0 cyclo and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The major quinone was ubiquinone Q-8 . The polar lipid profile was composed of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids, one unidentified lipid and one unidentified aminophosphoglycolipid . The genomic DNA G+C content of the strain was 63.6 mol%. The ANI and dDDH values obtained between the genomes of HX-2-15T and C. shinanonensis SAY3T were 85.3 and 29.3 % respectively. On the basis of data from phenotypic, chemotaxonomic and genotypic analysis, strain HX-2-15T represents a novel species of the genus Chitiniphilus, for which the name Chitiniphilus eburneus sp. nov. is proposed. The type strain is HX-2-15T (=KCTC 72286T=CCTCC AB 2019178T).