[Clinical study of constructing nomogram model based on multi-dimensional clinical indicators to predict prognosis of knee osteoarthritis].

OBJECTIVE: To analyze the factors affecting the prognosis of patients with knee osteoarthritis, and to construct a nomogram prediction model in conjunction with multi-dimensional clinical indicators. METHODS: The clinical data of 234 patients with knee osteoarthritis who were treated in our hospital from January 2015 to June 2021 were retrospectively analyzed, including 126 males and 108 females；age more than 60 years old for 135 cases, age less than 60 years old for 99 cases. Lysholm knee function score was used to evaluate the prognosis of the patients, and the patients were divided into good prognosis group for 155 patients and poor prognosis group for 79 patients according to the prognosis. The clinical data of the subjects in the experimental cohort were analyzed by single factor and multiple factors. The patients were divided into experimental cohort and verification cohort, the results of the multiple factor analysis were visualized to obtain a nomogram prediction model, the receiver operating characteristic curve（ROC）, calibration curve and decision curve were used to evaluate the model's discrimination, accuracy and clinical benefit rate. RESULTS: The results of multivariate analysis showed that smoking, pre-treatment K-L grades of Ⅲ to Ⅳ, and high levels of interleukin 6 （IL-6） and matrix metallo proteinase-3 （MMP-3） were risk factors for the prognosis of patients with knee osteoarthritis. ROC test results showed that the area under the curve of the nomogram model in the experimental cohort and validation cohort was 0.806[95%CI（0.742, 0.866）] and 0.786[（95%CI（0.678, 0.893）], respectively. The results of the calibration curve showed that the Brier values of the experimental cohort and verification cohort were 0.151 points and 0.134 points, respectively. When the threshold probability value in the decision curve was set to 31%, the clinical benefit rates of the experimental cohort and validation cohort were 51% and 56%, respectively. CONCLUSION: The prognostic model of patients with knee osteoarthritis constructed based on multi-dimensional clinical data has both theoretical and practical significance, and can provide a reference for taking targeted measures to improve the prognosis of patients.

Transcriptomics analysis and fed-batch regulation of high astaxanthin-producing Phaffia rhodozyma/Xanthophyllomyces dendrorhous obtained through adaptive laboratory evolution.

Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma. ONE-SENTENCE SUMMARY: A high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.

Aroma enhancement of instant green tea infusion using ß-glucosidase and ß-xylosidase.

ß-Glucosidase and ß-xylosidase were investigated for their ability to improve the aroma of instant green tea. The aroma and corresponding contributors were analyzed by sensory evaluation, gas chromatography-mass spectrometry, and odor activity value. Their specific contributions to aroma attributes were further examined by aroma reconstruction and omission experiments. The ß-glucosidase treatment significantly enhanced floral and grassy notes, on account of the increases of geraniol, nonanal, and cis-3-hexen-1-ol, and weakened the caramel note, attributable to the increases of nonanal, cis-3-hexen-1-ol, geraniol, methyl salicylate, and decanal. The co-treatment with ß-glucosidase and ß-xylosidase further enhanced the grassy note, with further increase in nonanal and cis-3-hexen-1-ol, and further weakened the caramel note, with additional increase in nonanal, cis-3-hexen-1-ol, methyl salicylate, and decanal. The synergistic action of ß-glucosidase and ß-xylosidase provides new clues to the production of instant green tea infusions with high aroma quality.

Preparation of isoquercitrin by biotransformation of rutin using α-L-rhamnosidase from Aspergillus niger JMU-TS528 and HSCCC purification.

Isoquercitrin is a flavonoid with important applications in the pharmaceutical and nutraceutical industries. However, a low yield and high production cost hinders the industrial preparation of isoquercitrin. In the present study, isoquercitrin was prepared by biotransformation of rutin using α-L-rhamnosidase from Aspergillus niger JMU-TS528 combined with high-speed countercurrent chromatography (HSCCC) purification. As a result, the optimum transformation pH, temperature, and time were pH 4.0, 60 °C, and 60 min, respectively. The Km and Vmax were 0.36 mM and 0.460 mmol/min, respectively. For isoquercitrin production, the optimal rutin concentration and transformation time were approximately 1000 mg/L and 60 min. The rutin transformation rate reached 96.44%. The isoquercitrin was purified to a purity of 97.83% using one-step purification with HSCCC. The isoquercitrin was identified using UPLC-Q-TOF-MS. The comprehensive results indicated that the biotransformation procedure using the α-L-rhamnosidase from A. niger JMU-TS528 combined with HSCCC was a simple and effective process to prepare isoquercitrin, which might facilitate the production of isoquercitrin for industrial use.

Suppressive Interaction Approach for Masking Stale Note of Instant Ripened Pu-Erh Tea Products.

The unpleasant stale note is a negative factor hindering the consumption of instant ripened Pu-erh tea products. This study focused on investigating volatile chemicals in instant ripened Pu-erh tea that could mask the stale note via sensory evaluation, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-olfactometry (GC-O) analyses. GC-MS and GC-O analyses showed that linalool, linalool oxides, trans-ß-ionone, benzeneacetaldehyde, and methoxybenzenes were the major aroma contributors to the simultaneous distillation and extraction (SDE) extract of instant ripened Pu-erh tea. Sensory evaluation showed that the SDE extract had a strong stale note, which was due to methoxybenzenes. By investigating suppressive interaction among flavour components, the stale note from methoxybenzenes was shown to have reciprocal masking interactions with sweet, floral, and green notes. Moreover, the validation experiment showed that the addition of 40 µg/mL of trans-ß-ionone in the instant ripened Pu-erh tea completely masked the stale note and improved the overall aromatic acceptance. These results elucidate the volatile chemicals that could mask the stale note of instant ripened Pu-erh tea products, which might help to develop high quality products made from instant ripened Pu-erh tea.

Enhancement of the thermostability of Aspergillus niger α-l-rhamnosidase based on PoPMuSiC algorithm.

α-l-Rhamnosidase is a biotechnologically important enzyme in food industry and in the preparation of drugs and drug precursors. To expand the functionality of our previously cloned α-l-rhamnosidase from Aspergillus niger JMU-TS528, 14 mutants were constructed based on the changes of the folding free energy (ΔΔG), predicted by the PoPMuSiC algorithm. Among them, six single-site mutants displayed higher thermal stability than wild type (WT). The combinational mutant K573V-E631F displayed even higher thermostability than six single-site mutants. The spectra analyses displayed that the WT and K573V-E631F had almost similar secondary and tertiary structure profiles. The simulated protein structure-based interaction analysis and molecular dynamics calculation were further implemented to assess the conformational preferences of the K573V-E631F. The improved thermostability of mutant K573V-E631F may be attributed to the introduction of new cation-π and hydrophobic interactions, and the overall improvement of the enzyme conformation. PRACTICAL APPLICATIONS: The stability of enzymes, particularly with regards to thermal stability remains a critical issue in industrial biotechnology and industrial processing generally tends to higher ambient temperature to inhibit microbial growth. Most of the α-l-rhamnosidases are usually active at temperature from 30 to 60°C, which are apt to denature at temperatures over 60°C. To expand the functionality of our previously cloned α-l-rhamnosidase from Aspergillus niger JMU-TS528, we used protein engineering methods to increase the thermal stability of the α-l-rhamnosidase. Practically, conducting reactions at high temperatures enhances the solubility of substrates and products, increases the reaction rate thus reducing the reaction time, and inhibits the growth of contaminating microorganisms. Thus, the improvement on the thermostability of α-l-rhamnosidase on the one hand can increase enzyme efficacy; on the other hand, the high ambient temperature would enhance the solubility of natural substrates of α-l-rhamnosidase, such as naringin, rutin, and hesperidin, which are poorly dissolved in water at room temperature. Protein thermal resistance is an important issue beyond its obvious industrial importance. The current study also helps in the structure-function relationship study of α-l-rhamnosidase.

Enhanced bioreduction of iron and arsenic in sediment by biochar amendment influencing microbial community composition and dissolved organic matter content and composition.

Biochar derived from the pyrolysis at 500 °C with fresh biogas slurry and residue, was conducted to investigate its potential role in mediating the speciation and mobilization of As(V) and Fe(III) from arsenic-contaminated tailing mine sediment, with consideration of the changes in microbial populations and dissolved organic matter (DOM). The reduction of As(V) (10-13%) and Fe(III) (12-17%) were partly in response to biochar abiotically causing desorption and reduction effect, but were predominantly (87-90% and 83-88% for As(V) and Fe(III)) attributed to biochar stimulating biological reduction. The level of As(III) released from sediment upon biochar amendment (656.35±89.25 µg L(-1)) was significantly higher than the level released without biochar amendment (98.06±19.38 µg L(-1)) after 49 days incubation. Although a low level of Fe(II) (0.81±0.07 mg L(-1)) was determined in the solution when amending with biochar, most of released Fe(II) (166.25±40.25 mg L(-1)) was formed as biochar-Fe(II)minerals composite. More importantly, biochar stimulated the DOM bioavailability in association with bacterial activities mediating As(V) and Fe(III) reduction. High-throughput sequencing results indicated biochar application shifted the soil microbial community and increased the relative abundance of As(V)-/Fe(III)-reducing bacteria, mostly Geobacter, Anaeromyxobacter, Desulfosporosinus and Pedobacter. The discovery of biochar-bacteria-DOM consortium may broaden new understanding into speciation and mobilization of metals, which arouses attention to exploit feasible bioremediation for metal-contaminated sediment.

Electrochemical reclamation of silver from silver-plating wastewater using static cylinder electrodes and a pulsed electric field.

Silver was reclaimed from silver-plating wastewater by using a pulsed electric field (PEF) combined with static cylinder electrodes (SCE). The conditions that produced the maximal silver recovery rate (RR(Ag)) (99%) were as follows: average retention time of 10 min, interelectrode gap of 50mm, solution pH of 9.0, temperature of 45 degrees C, initial Ag(I) concentration of 1000 mg L(-1), PEF pulse frequency of 1200 Hz, current density of 5.0 A m(-2) and a pulse duty cycle of 60%. Compared with the conventional direct current (DC) technology, the PEF process exhibited improvements in the silver recovery rate (RR(Ag)), total energy consumption (TEC) and physical properties of the silver deposits, especially for low Ag(I) concentrations, for example, from 500 to 1000 mg L(-1). For an initial Ag(I) concentration of 500 mg L(-1), the PEF process produced an RR(Ag) of up to 99%, and the TEC was 4.56 kWh (kg Ag)(-1). In comparison, the RR(Ag) and TEC were 90% and 5.66 kWh (kg Ag)(-1), respectively, in the DC process. The results of SEM observation and XRD analysis indicated that the silver deposits formed by the PEF process were smaller, denser, and of a higher purity than those produced by the DC process. Therefore, the presented method was effective for reclaiming silver from silver-plating wastewater.

[Expression of the soluble human Fas ligand in Dictyostelium discoideum].

An expression system is described for high-yield production of recombinant soluble human FasL (shFasL) in Dictyostelium discoideum cells. DNA encoding amino acids 141 - 281 of hFasL was PCR amplified from cDNA derived from activated human neutrophils. The resulting product was fused with a DNA fragment encoding hCG-beta signal peptide and cloned in the expression vector pMB12neo. Dictyostelium strain AX3 was transfected with this plasmid, yielding a recombinant strain called AX3-pCESFL95-H3. In order to improve the shFasL expression level, pMB12neo was optimized by replacing its transcriptional terminator/ polyadenylation segment of the 2H3 gene with an actin8 terminator/polyadenylation segment, yielding derived expression vector pMB74. The recombinant Dictyostelium strain called AX3-pLu8 was generated with this new plasmid. When the recombinant cells were cultivated in a complex HL-5C medium, a cell density of (1.5 - 2) x 10(7)/mL was reached, and the shFasL level expressed by strains AX3-pCESFL95-H3 and AX3-pLu8 was 23.5 microg/L and 206 microg/L, respectively. By using a newly developed synthetic medium called SIH as culture medium, higher cell density of (4 - 5) x 10(7)/mL was achieved. Correspondently, 111 microg/L and 420 microg/L shFasL were secreted by recombinant strains AX3-pCESFL95-H3 and AX3-pLu8, respectively.

[Uptake of nickel from industrial wastewater by genetically engineered Escherichia coli JM109].

Heavy metal wastewater poses a serious threat to the environment. In comparison to the existing methods of chemical precipitation, ion exchange and carbon adsorption, biosorption is an attractive alternative for the recovery of heavy metals from industrial effluents. However, nickel ion, different from other heavy metal ions, is a more recalcitrant pollutant and has low affinity to many metal tolerant microorganisms. In this study, Escherichia coli JM109 was genetically engineered to simultaneously express a Ni2+ transport system (the product of nixA gene) andoverexpress metallothionein (MT). NixA protein has a high affinity for Ni2+, and metallothioneins (MTs) are capable of binding a variety of heavy metals including Ni2+ . The Ni2+ bioaccumulation performance of the genetically engineered E. coli JM109 was evaluated. Time-course test showed that the bioaccumulation rate was rapid, and 95% of the accumulation was achieved within the first 10 minutes. The maximum Ni2+ bioaccumulation by genetically engineered E. coli cells was dramatically increased from 1.54 mg/g to 10.11mg/g, a more than five-fold increase than that of the original E. coli strain. The isotherm was of Langmuir type. Within the tested pH range (pH 4-10), the engineered cells displayed more resistance to pH variation, retaining up to 80% of the Ni2+ binding capacity at pH 4, while the original E. coli host cells lost 80% of Ni2+ binding capacity at pH 4. The presence of Na+ and Ca2+ affected Ni2+ bioaccumulation, but the effects were not serious, as 71% and 66% of the Ni2+ binding capacities were retained respectively at the concentrations of 1000 mg/L Na+ and 1000 mg/L Ca2+ . However, Mg2+ exerted a severe adverse effect on Ni2+ bioaccumulation, 83% of Ni2+ accumulating capacity was lost when Mg2+ concentration reached 200 mg/L. The effects of different kinds of heavy metals on Ni2+ accumulating were different. The genetically engineered E. coli cell lost less than 45% of its Ni2+ bioaccumulation activity in the presence of 50 mg/L lead or cadmium, 66% in the presence of 25mg/L mercury and 84% in the presence of 40 mg/L copper. The presence of glucose did not improve Ni2+ uptake. Our study suggests that the genetically engineered E. coli JM109 has potential application for effective and efficient recovery of nickel from aqueous solutions.

Biosorption of lead by Phanerochaete chrysosporium in the form of pellets.

The growth of Phanerochaete chrysosporium(ATCC 24725) in pellets was influenced by culture time, medium pH, C/N, surfactant concentration, spore number in inoculum, and shaking rate. The removal of Pb2+ from aqueous solution by this kind of mycelial pellets was studied. The results indicated that many factors affected biosorption. These factors included pH, Pb2+ concentration, co-ion, adsorption time, and chemical pretreatments of biomass. Under optimum biosorption conditions(pH 4.5, 27 degrees C, 16 h), the highest lead uptake of 108 mg/g, was observed with mycelial pellets of 1.5-1.7 mm in diameter which were treated with 0.1 mol/L NaOH solution before adsorption. Pretreatment of biomass with NaOH further increased its biosorption capacity.