Investigation of high-risk antibiotic resistance bacteria and their associated antibiotic resistance genes in different agricultural soils with biogas slurry from China.

High-risk antibiotic-resistant bacteria (ARB) and their accompanying antibiotic resistance genes (ARGs) seriously threaten public health. As a crucial medium for ARB and ARGs spread, soils with biogas slurry have been widely investigated. However, few studies focused on high-risk multi-drug resistant bacteria (MDRB) and their associated ARGs. This study examined ARB distribution in different agricultural soils with biogas slurry across 12 districts in China. It identified high-risk MDRB in various soil backgrounds, elucidating their resistance and spread mechanism. The findings revealed that diverse cultured ARB were enriched in soils with biogas slurry, especially soil ciprofloxacin ARB, which were enriched (>2.5 times) in 68.4 % of sampling sites. Four high-risk MDRB isolated from Hebei, Zhejiang, Shanxi, and Gansu districts were identified as severe or opportunistic pathogens, which carried abundant mobile genetic elements (MGEs) and 14 known high risk ARGs, including aac(3)-IId, aac(6')-Ib3, aph(6)-Id, aac(6')-Ib3, aadA1, blaOXA-10, blaTEM-1B, dfrA12, dfrA14, cmlA1, sul1, floR, tet(M) and tet(L). The antibiotics accumulation, diverse ARGs and MGEs enrichment, and proliferation of pathogenic bacteria could be potential driving factors of their occurrence and spread. Therefore, the coexistence of the high-risk MDRB and ARGs combined with the associated MGEs in soils with biogas slurry should be further investigated to develop technology and policy for reducing their negative influences on the effectiveness of clinical antibiotics.

Co-application of concentrated biogas slurry and pyroligneous liquor mitigates ammonia emission and sustainably releases ammonium from paddy soil.

Biogas production causes vast amounts of biogas slurry (BS). Application of BS to croplands can substitute chemical fertilizers while result in higher ammonia emissions. Tremendous variation of ammonium concentration in different BSs induces imprecise substitution, while concentrated BS holds higher and more stable ammonium. Pyroligneous liquor, an acidic aqueous liquid from biochar production, can be used with concentrated BS to reduce ammonia emission. However, the effects of combining concentrated BS with pyroligneous liquor on ammonia emission and soil (nitrogen) N transformation have been poorly reported. In this study, a field experiment applying concentrated BS only, or combining with 5 %, 10 %, and 20 % pyroligneous liquor (v/v) for substituting 60 % N of single rice cultivation was conducted by contrast with chemical fertilization. The results showed that substituting chemical N fertilizers with concentrated BS increased 24.6 % ammonia emission. In comparison, applying 5 %, 10 %, and 20 % pyroligneous liquor with concentrated BS reduced 4.9 %, 20.3 %, and 24.4 % ammonia emissions, respectively. Applying concentrated BS with more pyroligneous liquor preserved higher ammonium and dissolved organic carbon in floodwater, and induced higher nitrate concentration after fertilization. Whereas soil ammonium and nitrate contents were decreased along with more pyroligneous liquor application before and after the topdressing and exhibited sustainable release until rice harvest. In comparison, the soil N mineralization and nitrification rates were occasionally elevated, while the activities of soil urease, protease, nitrate reductase, and nitrite reductase had multiple responses. Applying concentrated BS only, or combining with 5 %, 10, and 20 % pyroligneous liquor, have little effect on soil basic properties but inorganic N. In summary, applying concentrated BS with >10 % pyroligneous liquor could preserve more N with sustainable release and potentially lower N loss to the atmosphere, and we proposed that applying 13.5 % pyroligneous liquor in concentrated BS could achieve maximum soil fertility and minimum ammonia emission.

Manganese losses induced by severe soil acidification in the extensive Lei bamboo (Phyllostachys violascens) plantation stands in Eastern China.

Manganese (Mn) is a critical element in soils, essential to plant growth. Long-term and intensively managed Lei bamboo (Phyllostachys violascens) stands are usually subjected to severe soil acidification and Mn activation. However, Mn migration from topsoil to deep soil induced by severe soil acidification was poorly recognized and studied. The distribution and changes of the total and the operationally defined Mn forms in soil profiles and its potential stress and environmental effect were investigated in a chronosequence of Lei bamboo stands (0, 2, 6, 11, and 16 years of stand age). The results showed that the Mn amount was significantly decreased in topsoil and accumulated in subsoil with the long-term and intensive fertilizer application. Soil exchangeable Mn and superphosphate extractable Mn demonstrated large different variation to total Mn, whereas their sum was largely higher than and highly correlated with 8-hydroxyquinoline (HQN) extractable Mn. Soil organic carbon, pH value, exchangeable bases, and soil redox simultaneously controlled soil Mn depletion. In conclusion, long-term and intensive fertilizer application leads to soil acidification and accelerated soil Mn depletion in bamboo stand soil, promoting Mn accumulation in bamboo shoots.

Spectroscopic investigation of conformational changes in urease caused by interaction with humic acid.

Urease in soil interacts with humic acid (HA), which results in a change of the enzymatic activity and stability. However, knowledge on the conformational change of urease in the presence of HA is still lacking. Therefore, the structure of urease (net zero charge at pH 5.2) interacting with HA and the microenvironments of the tyrosine (Tyr) and tryptophane (Trp) residues were investigated at pH 6.7 and 8.0 and 0.5 and 50 mmol L-1 KCl using spectroscopic techniques. Fluorescence intensity of urease was progressively inhibited by HA with increasing mass ratio f of HA/urease. Moreover, quenching of urease fluorescence by HA was strongest at pH 6.7 (and 50 mmol L-1 KCl) where the hydrophobic attraction was counteracted by only a weak electrostatic repulsion. HA exerted only a minor effect on the positions of the maximum excitation bands for Tyr and Trp residues, indicating insignificant changes in the microenvironment of these residues in the presence of HA. At pH 6.7, the amide I and amide II bands were inhibited by HA. Curve-fitting of the amide I band of urease in complexes indicated that the percentages of α-helix, ß-sheet and ß-turn were changed. At pH 8 HA had little effect on the circular dichroism and attenuated total reflectance Fourier transform infrared spectra of urease. At this pH the interaction between urease and HA was weak due to the relatively strong electrostatic repulsion and the conformational change was insignificant. The present results increase our understanding of negatively charged protein behavior in natural environments dominated by humic substances.

Effect of humic acid on lysozyme interaction with montmorillonite and kaolinite.

Humic acid (HA) as a soil natural organic matter (NOM) can participate in the interaction between proteins and clay minerals, depending on clay type, HA and protein content, and solution conditions. The effect of HA on the interaction of lysozyme (LSZ) with kaolinite (Kao) and montmorillonite (Mont) was investigated at (initial) pHi 5 and 8. In the solutions containing both HA and LSZ, HA/LSZ complexes were formed with a net charge density depending on pH and HA/LSZ mass ratio f. LSZ adsorption on clays in the presence of HA is dominated by adsorption of HA/LSZ complexes. The HA/LSZ mass ratio (fIEP,pHi) at the isoelectric point (IEP) is pH dependent. At f fIEP,pHi the HA/LSZ complexes were negative, both conditions caused relatively high equilibrium concentrations of LSZ in solution that decreased with increasing initial LSZ concentration. The present results enhance our insight in protein soil interactions for the case that clay particles are brought in contact with aqueous solutions that contain modest amounts of both NOM and protein and stress the importance of the NOM/protein mass ratio and solution pH.

Development of a Nomogram Combining Clinical Risk Factors and Dual-Energy Spectral CT Parameters for the Preoperative Prediction of Lymph Node Metastasis in Patients With Colorectal Cancer.

OBJECTIVE: This study aimed to develop a dual-energy spectral computed tomography (DESCT) nomogram that incorporated both clinical factors and DESCT parameters for individual preoperative prediction of lymph node metastasis (LNM) in patients with colorectal cancer (CRC). MATERIAL AND METHODS: We retrospectively reviewed 167 pathologically confirmed patients with CRC who underwent enhanced DESCT preoperatively, and these patients were categorized into training (n = 117) and validation cohorts (n = 50). The monochromatic CT value, iodine concentration value (IC), and effective atomic number (Eff-Z) of the primary tumors were measured independently in the arterial phase (AP) and venous phase (VP) by two radiologists. DESCT parameters together with clinical factors were input into the prediction model for predicting LNM in patients with CRC. Logistic regression analyses were performed to screen for significant predictors of LNM, and these predictors were presented as an easy-to-use nomogram. The receiver operating characteristic curve and decision curve analysis (DCA) were used to evaluate the clinical usefulness of the nomogram. RESULTS: The logistic regression analysis showed that carcinoembryonic antigen, carbohydrate antigen 199, pericolorectal fat invasion, ICAP, ICVP, and Eff-ZVP were independent predictors in the predictive model. Based on these predictors, a quantitative nomogram was developed to predict individual LNM probability. The area under the curve (AUC) values of the nomogram were 0.876 in the training cohort and 0.852 in the validation cohort, respectively. DCA showed that our nomogram has outstanding clinical utility. CONCLUSIONS: This study presents a clinical nomogram that incorporates clinical factors and DESCT parameters and can potentially be used as a clinical tool for individual preoperative prediction of LNM in patients with CRC.

Development of a dual-energy spectral computed tomography-based nomogram for the preoperative discrimination of histological grade in colorectal adenocarcinoma patients.

BACKGROUND: The usefulness of a dual-energy spectral computed tomography (DESCT)-based nomogram in discriminating between histological grades of colorectal adenocarcinoma (CRAC) is unclear. This study aimed to develop such a nomogram and assess its ability to preoperatively discriminate between histological grades in CRAC patients. METHODS: Primary tumors monochromatic CT value, iodine concentration (IC) value, and effective atomic number (Eff-Z) in the arterial (AP) and venous phases (VP) were retrospectively compared between patients with high-grade (n=65) and low-grade (n=108) CRAC who underwent preoperative abdominal DESCT. Univariate analysis was used to compare the DESCT parameters and clinical factors between these two patient groups. Statistically significant features in the univariate analysis were included in the multivariate logistic regression model to identify the indicators for building a nomogram that could discriminate between histological grades in CRAC patients. The clinical usefulness of the nomogram and its value for predicting overall survival were statistically evaluated. RESULTS: The logistic regression analysis showed that age, clinical T stage, clinical N stage, and IC values in AP and VP were significant independent predictors for high-grade CRAC. A quantitative nomogram developed based on these predictors showed excellent performance for discriminating between the histological grades, with an area under the curve (AUC) of 0.886 and excellent agreement in the calibration curve. The Kaplan-Meier curve for overall survival showed that our nomogram identified a significant difference between the high- and low-risk groups [hazard ratio (HR), 2.188; 95% CI, 1.072-4.465; P=0.027). CONCLUSIONS: This study presents a nomogram that incorporates DESCT parameters and clinical factors and can potentially be used as a clinical tool for individual preoperative prediction of CRAC histological grade.

Liq_ccRCC: Identification of Clear Cell Renal Cell Carcinoma Based on the Integration of Clinical Liquid Indices.

Currently, preoperative diagnosis and differentiation of renal clear cell carcinoma and other subtypes remain a serious challenge for doctors. The liquid biopsy technique and artificial intelligence have inspired the pursuit of distinguishing clear cell renal cell carcinoma using clinically available test data. In this work, a method called liq_ccRCC based on the integration of clinical blood and urine indices through machine learning approaches was successfully designed to achieve this goal. Clinically available biochemical blood data and urine indices were collected from 306 patients with renal cell carcinoma. Finally, the integration of 18 top-ranked clinical liquid indices (13 blood samples and 5 urine samples) was proven to be able to distinguish renal clear cell carcinoma from other subtypes of renal carcinoma by cross-valuation with an AUC of 0.9372. The successful introduction of this identification method suggests that subtype differentiation of renal cell carcinoma can be accomplished based on clinical liquid test data, which is noninvasive and easy to perform. It has huge potential to be developed as a promising innovation strategy for preoperative subtype differentiation of renal cell carcinoma with the advantages of convenience and real-time testing. liq_ccRCC is available online for the free test of readers at http://lishuyan.lzu.edu.cn/liq_ccRCC.

Energy spectrum computed tomography improves the differentiation between benign and malignant solitary pulmonary nodules.

PURPOSE: To evaluate the diagnostic efficiency of computed tomography (CT) values at the 40~140 keV monochromatic level for the differential diagnosis of solitary pulmonary nodules (SPNs). METHODS: Energy spectrum CT data of 44 patients with SPNs were analyzed retrospectively; 24 patients with malignant SPNs served as the malignant group and 20 patients with benign SPNs served as the benign group. The basic material concentration and the enhancement degree differences in double-phase enhanced scans were compared between the two groups. The sensitivity and specificity were calculated to determine diagnosis, and were compared with the pathology results. RESULTS: The CT values at the 40~90 keV monochromatic level and the iodine concentrations of malignant group were higher than those of benign group in the arterial phase (all P < 0.05). The enhancement degree of the malignant group was higher than that for the benign group in the arterial phase (36.36 ± 33.18 HU vs 16.93 ± 24.17 HU t = 2.243, P = 0.030); however, the enhancement degrees of the two groups were similar in the venous phase (21.99 ± 15.87 HU vs 17.62 ± 24.15 HU t = 0.694, P = 0.493). The area under the curve of the enhancement degree in the arterial phase was 0.792. CONCLUSIONS: Monochromatic imaging and base material concentration of energy spectrum CT can help differentiate between benign and malignant SPNs.

Potential sources and associated risk assessment of potentially toxic elements in paddy soils of a combined urban and rural area.

The contamination of combined urban and rural area is a growing environmental issue due to rapid industrialization in China. This study investigated the eight potentially toxic elements (PTEs) arsenic (As), lead (Pb), cadmium (Cd), mercury (Hg), chromium (Cr), copper (Cu), nickel (Ni), and zinc (Zn) in paddy fields in a combined urban and rural area in Shaoxing city, Zhejiang province. Concentrations of the PTEs were determined in 60 topsoil samples (0-20 cm) collected from paddy fields in a combined urban and rural area in Shaoxing city, Zhejiang province, southeast China. The average contents of Pb, Cu, Zn, Cd, Hg, and As in the paddy soils increased by 23.7, 6.9, 17.0, 79.8, 62.5, and 47.9%, respectively, relative to their corresponding natural background concentrations. The mean contents of Cr and Ni did not exceed their background values. Multivariate statistical and geostatistical methods were used to distinguish between the PTEs (Cd, As, Pb, Zn, and Hg) contaminating the soils of the study area from those (Cr, Cu, and Ni) at background concentrations. About 17.2% of the soils were moderately polluted with PTEs as a result of urbanization. The results may be useful to policy makers in agricultural and urban management.

Rapid initiation of guided bone regeneration driven by spatiotemporal delivery of IL-8 and BMP-2 from hierarchical MBG-based scaffold.

Initiation of endogenous repair mechanisms, including key steps of stem cell recruitment and cartilage intermediate formation in endochondral ossification, is vital to regeneration of large bone defects. To biomimetically promote a rapid initiation and ensuing osteogenic stimulation, exogenous chemokine IL-8 and growth factor BMP-2 were orchestrated in a mesoporous bioactive glass (MBG)-based spatiotemporal delivery system, to achieve a rapid release of IL-8 followed by a long-term sustained release of BMP-2. The synergistic effect of IL-8 and BMP-2 on initiation stage of bone healing and underlying mechanism were thoroughly investigated in vitro and in vivo. Intriguingly, apart from its superiority in stem cell recruitment to BMP-2, IL-8 not only endowed a histological "prep-state" of endochondral ossification by up-regulating chondrogenic genes and inducing the formation of extensive cartilage tissues, facilitating rapid bone transformation by BMP-2, but also triggered a cellular "prep-state" with high expression of BMP receptors, enhancing the osteoinductivity of BMP-2. With the spatiotemporal delivery system, orchestrated signal stimuli of IL-8 and BMP-2 induced a rapid initiation including efficient stem cell recruitment and a "chondrogenic/osteogenic balance" at the first stage of endochondral ossification, and the scaffold facilitated sufficient osteoconductivity, together resulting in early extensive bone mineralization and an advanced regeneration throughout the repair of large bone defect. We believe this new idea could provide insights toward designing bone-repairing biomaterials with higher regenerative efficiency.

Biochar reduces cadmium accumulation in rice grains in a tungsten mining area-field experiment: effects of biochar type and dosage, rice variety, and pollution level.

Cadmium (Cd)-contaminated rice (Oryza sativa) in Southern China is a great threat to food security, and the paddy soil remediation is urgently needed to reduce Cd accumulation in rice. Application of biochar could effectively immobilize soil Cd and reduce Cd uptake by rice. Fields that were applied with soil treatments including control and 15 and 30 t ha-1 each hickory nut shell-derived biochar (KC) or maize straw-derived biochar (MC), and grown with two rice varieties (hybrid rice and late japonica rice) were selected for this study. The long-term effect of biochars on decreasing Cd bioavailability in paddy soils was evaluated. The results showed when MC was applied at 15 t ha-1, DTPA-Cd (soil cadmium extracted by diethylenetriamine pentaacetic acid) was reduced by 20.0 and 34.5% in Field A (slightly Cd pollution) and B (moderately Cd pollution), respectively. In Field B, soil DTPA-Cd concentrations with application of 30 t ha-1 biochars were all lower than that of 15 t ha-1 biochar, but there were no significant differences between the two types of biochars. Cd concentration in rice grains and straws of hybrid rice are two times more than those of late japonica rice. Cd bio-concentration factor both of grains and straw was significantly increased by biochar application, which in Field A was higher than that in Field B. Our results suggest that biochars reduce Cd accumulation in rice grains by immobilizing soil Cd. KC has a higher potential in lowering Cd bioavailability than MC. Hybrid rice should be prohibited to cultivate in these areas.

Improvement of extracellular lipase production by a newly isolated Yarrowia lipolytica mutant and its application in the biosynthesis of L-ascorbyl palmitate.

Yarrowia lipolytica Wt-11 producing an extracellular lipase was isolated and identified. To improve the lipase production, Y. lipolytica Wt-11 was subjected to low-energy ion implantation mutation breeding, and a best mutant, Y. lipolytica Mut-96, was obtained after screening. Under the optimal cultivation conditions, the scaled-up production of lipases were performed, and the lipase activity of Y. lipolytica Mut-96 was enhanced nearly 5.5-fold compared with that of Y. lipolytica Wt-11. After fermentation, the lipases were purified, and the characteristics of the purified lipases were studied. The optimum temperatures and pHs for lipases from Wt-11and Mut-96 were 30°C and 8.0, respectively. The purified lipases were stable between pH 7.0 and 8.5 and unstable at temperatures above 40°C. The lipase activities were enhanced by Ca2+, Ba2+, Mn2+, Fe2+ and SDS. The synthesis of L-ascorbyl palmitate via esterification with L-ascorbic acid and palmitic acid by immobilized lipases from Wt-11 and Mut-96 in organic media was investigated, and the L-ascorbyl palmitate can be respectively produced at levels of 14.8 and 27.5g/L.

MBG scaffolds containing chitosan microspheres for binary delivery of IL-8 and BMP-2 for bone regeneration.

Biomimetic delivery of chemokines and growth factors based on stem cell recruitment and endochondral ossification, as the key steps in natural regenerative process, has been an area of intense research in recent years. An inflammatory chemokine, interleukin-8 (IL-8), was recently reported with high recruitment efficiency of bone marrow stem cells, chondrogenic inductivity and immune regulatory functions. In this study, the effect of IL-8 action duration on bone morphogenetic protein-2 (BMP-2)-induced bone regeneration was studied to achieve an optimal synergism of these two proteins. Herein, a mesoporous bioactive glass (MBG)-based scaffold with BMP-2 entrapment and IL-8-loaded chitosan microspheres (CMs) was developed. The MBG scaffold with size-matched mesopores was adopted for the long-term sustained delivery of BMP-2; and CMs with different sizes, prepared using a modified ionotropic gelation method, were customized to match the optimal action time of IL-8. The results indicated that CMs of 100 µm diameter and medium crosslinking density exhibited an 85% release of IL-8 in 7 days and the MBG substrate exhibited a long-term sustained release of BMP-2. Furthermore, the binary delivery system exhibited excellent biocompatibility and synergistically enhanced osteoinductivity. In an in situ bone regeneration model of a rabbit radius large segmental defect, the system efficiently accelerated the whole regenerative process, with the highest bone formation amount from an early stage and the highest degree of regenerative completion. Since delivery systems for multiple cytokines have been in great demand due to the requirement of complicated biological processes, we believe that this new binary delivery system could be customized to design other dual delivery systems for improving bone-repairing biomaterials with higher regenerative efficiency.

Application and comparison in biosynthesis and biodegradation by Fusarium solani and Aspergillus fumigatus cutinases.

In this study, two synthesized cutinase genes from Fusarium solani and Aspergillus fumigatus were expressed in Pichia pastoris X33. The characteristics of these two cutinases were investigated and compared. The results indicated that F. solani and A. fumigatus cutinases hydrolyzed p-nitrophenyl substrates with different carbon chain lengths. A. fumigatus cutinase predominately hydrolyzed p-nitrophenyl butyrate, but F. solani cutinase preferred p-nitrophenyl decanoate. The abilities of polymer synthesis and bioplastic degradation were tested and compared between F. solani and A. fumigatus cutinases. The results showed that F. solani cutinase had degradation ability on poly(ε-caprolactone) (PCL) and synthesized polymer with a molecular weight (MW) of 2300 in organic solvent. However, A. fumigatus cutinase completely degraded PCL and synthesized molecules with a MW of 25,000, suggesting that A. fumigatus cutinase has more promising applications.

Spectral Computed Tomography Imaging of Gastric Schwannoma and Gastric Stromal Tumor.

Gastric schwannomas (GSs) and gastrointestinal stromal tumors (GSTs) are grossly similar submucosal neoplasms with different prognoses. We explored the value of spectral computed tomography (CT) to distinguish between them. METHODS: Patients diagnosed with GS or GST at Lanzhou University Second Hospital, China, between May 2013 and June 2015 were included retrospectively. The subjects underwent spectral CT examination before surgery and had histologically confirmed diagnosis of GS or GST. RESULTS: Twelve patients with GS (3 men; 9 women; mean [SD] age, 47.0 [11.5] years) and 20 with GST (7 men; 13 women; mean [SD] age, 54.7 [9.9]) showed significant differences in terms of arterial phase (AP) at 70 keV (P < 0.001), portal phase (PP) at 70 keV (P = 0.002), AP iodine concentration, PP iodine concentration, AP water concentration, AP slope of spectral curve, and PP slope of spectral curve (all P < 0.001). CONCLUSIONS: Spectral CT may be useful for noninvasive diagnosis of submucosal tumors.

RhBMP-2 loaded MBG/PEGylated poly(glycerol sebacate) composite scaffolds for rapid bone regeneration.

With the worldwide rising need of severe bone defect treatment, the development of available bone substitutes with optimal mechanical strength, sustained drug release, cell affinity and osteoinductivity remains a great challenge. In this study, an rhBMP-2 loaded polymer-coated mesoporous bioactive glass (MBG) composite scaffold was developed. The uncrosslinked poly(glycerol sebacate) (PGS) or PEGylated PGS (PEGS) coating modification had enhanced the mechanical strength of the composite scaffold, solved the brittleness problem of the MBG matrix, increased the cell affinity of the material surface, and diminished the initial burst release of rhBMP-2 from mesopores of MBG. The results indicated that the PGS coating promoted the proliferation of rat bone marrow stem cells (rBMSCs), while the PEGS coating exhibited an enhancement in the osteogenic differentiation of rBMSCs. The in vivo ectopic bone formation results provide strong evidence that the rhBMP-2-loaded MBG/PEGS composite scaffolds exhibited a rapid bone forming capacity and might yield extraordinary achievements in the field of bone tissue engineering. The design considerations can be extended to other artificial scaffolds and are expected to provide new thoughts on the development of future tissue engineering materials.