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ObjectiveThe aim of this study was to detect botulinum toxin and Clostridium botulinum （C.botulinum） in 30 batches of infant formula milk powder obtained from an enterprise and to analyze the whole genome of the strain of C. botulinum type B isolated from the samples.MethodsThe samples were subjected to C. botulinum isolation and botulinum toxin typing experiments according to the “National standard for food safety microbiological examination of C. botulinum and botulinum toxin” （GB 4789.12—2016）. The whole genomes of the isolated strains were sequenced and their genetic characteristics were analyzed.ResultsBotulinum toxin was not detected in any of the 30 batches of samples. After intraperitoneal injection of the enrichment solution into mice， four batches of milk powder samples induced symptoms of botulism， but C. botulinum was isolated from only one batch of milk powder samples. Genome sequencing revealed that the bacterium was C. botulinum group Ⅰ， type B， the toxin gene cluster was type Ha， and the toxin gene was subtype B2.ConclusionThe detection of C. botulinum in infant formula milk powder with a complex background of microorganisms should not be based on the isolation of bacteria as the gold standard but should be confirmed by a mouse toxicity test of the enrichment solution combined with a botulinum toxin antiserum protection test. Whole genome sequencing can accurately identify strains and analyze relevant genetic characteristics， providing reliable technical support for handling poisoning events.

ObjectiveTo explore the maximum allowable concentration of six chemical reagents and three enzyme lysates in a polymerase chain reaction （PCR） amplification system and the differences of IGEPAL CA-630 cleaving different species.MethodsPrepare different kinds of working solutions， including six chemical reagents （IGEPAL CA-630， CTAB， SDS， EDTA， guanidine isothiocyanate， and NaOH） and three enzymes （lysozyme， lysostaphin， and protease K） with different concentrations. Add the working solutions to the fluorescent quantitative PCR system and then analyze the differences in Ct values. Streptococcus thermophilus， Staphylococcus aureus， and Escherichia coli were lysed with IGEPAL CA-630 solutions （1% and 0.1% with TE buffer）， TE buffer and deionized water， respectively， and the differences in PCR results were analyzed.ResultsIn the PCR system， the maximum allowable final concentrations of IGEPAL CA-630， CTAB， SDS， EDTA， guanidine isothiocyanate， and NaOH were 0.800‰， 0.256‰， 0.012 8‰， 0.8 mmol/L， 0.012 8 mol/L， and 0.64 mmol/L respectively. The maximum allowable concentrations of lysozyme， lysostaphin， and protease K were 0.512， 1.28， and 0.512 μg/mL. In the lysing of S. thermophilus and S. aureus with IGEPAL CA-630， the Ct values of TE solution， 0.1% and 1% IGEPAL CA-630 decreased successively， with no significant difference between the TE solution and deionized water. There was no significant difference in Ct values of E. coli treated with deionized water， TE solution， 0.1% and 1% IGEPAL CA-630.ConclusionThis study established the maximum allowable concentration of the above nine components in the PCR amplification solution. Additionally， it was found that increasing the concentration of IGEPAL CA-630 could enhance DNA extraction from gram-positive bacteria.

In this article, we study the evolution of immersed locally convex plane curves driven by anisotropic flow with inner normal velocity V=1αψ(x)καV=\frac{1}{\alpha }\psi \left(x){\kappa }^{\alpha } for α1\alpha \gt 1, where x∈[0,2mπ]x\in \left[0,2m\pi ] is the tangential angle at the point on evolving curves. For −1≤α1\alpha \gt 1, we show only type I singularity arises in the flow, and the rescaled flow has subsequential convergence, i.e. for any time sequence, there is a time subsequence along which the rescaled curvature of evolving curves converges to a limit function; furthermore, if the anisotropic function ψ\psi and the initial curve both have some symmetric structure, the subsequential convergence could be refined to be full-time convergence.

BACKGROUND The extractable state of soil heavy metal elements is an important index to measure their bioavailability, but its content changes with soil acid-base properties and other environmental conditions. When determining which method to use for the extractable state of soil heavy metal elements, there are more than 7 kinds of extraction reagents involved in relevant standard methods or technical specifications. These include hydrochloric acid of pH=5.8, 0.1mol/L hydrochloric acid, 0.43±0.02mol/L nitric acid, 0.11mol/L acetic acid, 1mol/L ammonium nitrate solution, 0.005mol/L DTPA leaching agent, 0.01mol/L calcium chloride solution. Different scholars have different research conclusions on different extractants. There is no systematic research report on extractants suitable for different soil types. OBJECTIVES To select and determine the general extractant suitable for the extractable state of heavy metal elements in soil with different acid-base properties. METHODS Using typical farming soil samples as the research objective, 7 kinds of extracting agent were used to extract 8 heavy metals (Cd, Ni, Cu, Zn, Cr, Pb, As and Hg). Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine Cd, Cr, Cu, Pb, Zn, Ni, whereas atomic fluorescence spectrometry (AFS) was used to determine As and Hg. The extraction rates of 7 kinds of extractants were compared, and the effects of soil acid and alkali properties on the extraction rate of heavy metal elements were studied. RESULTS Dilute acid solution had a strong ability of leaching heavy metals from soil, and it was not related to the acidity and alkalinity of the soil. Although the extraction ability of 1mol/L ammonium nitrate solution for cadmium indicated that the activity of cadmium in acidic soil was much higher than that in alkaline soil, its extraction rate of lead in alkaline soil was higher than that of cadmium. The extraction rate of DTPA extractant for soil heavy metal elements was significantly higher than that of 0.01mol/L calcium chloride solution, especially for lead, copper and zinc. Moreover, the extraction rate of DTPA extractant for soil heavy metal elements did not change significantly with soil acid-base properties. 0.01mol/L calcium chloride solution had the highest extraction rate of cadmium in soil, which was closely related to the acid-base properties of soil. CONCLUSIONS 0.01mol/L calcium chloride solution is an ideal general extractant for the extractable state of soil heavy metal elements.

ObjectiveTo prepare and evaluate Enterotoxigenic Escherichia coli（ETEC） reference.MethodsThe standard strain CMCC（B）43208 was identified by biochemical reaction， matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF MS） and gene sequencing. 600 ETEC reference samples with 103 CFU/sample were prepared by freeze-drying. 20 samples were selected randomly for uniformity test， and samples were tested transportation stability by simulating 25 and 37 ℃， and storage stability by stroing samples at -20 and 4 ℃. Three laboratories were organized for collaborative calibration. 20 kinds food were chosen as the substrates to evaluate the applicability. The strain CMCC（B）43208 was identified as Escherichia coli by all of biochemical reaction， MALDI-TOF MS and 16 s RNA gene sequencing， and was identified as ETEC by lt，stp and sth gene. The uniformity result was F=1.48

In the tumor microenvironment, tumor-infiltrating immune cells (TIICs) are a key component. Different types of TIICs play distinct roles. CD8+ T cells and natural killer (NK) cells could secrete soluble factors to hinder tumor cell growth, whereas regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) release inhibitory factors to promote tumor growth and progression. In the meantime, a growing body of evidence illustrates that the balance between pro- and anti-tumor responses of TIICs is associated with the prognosis in the tumor microenvironment. Therefore, in order to boost anti-tumor response and improve the clinical outcome of tumor patients, a variety of anti-tumor strategies for targeting TIICs based on their respective functions have been developed and obtained good treatment benefits, including mainly immune checkpoint blockade (ICB), adoptive cell therapies (ACT), chimeric antigen receptor (CAR) T cells, and various monoclonal antibodies. In recent years, the tumor-specific features of immune cells are further investigated by various methods, such as using single-cell RNA sequencing (scRNA-seq), and the results indicate that these cells have diverse phenotypes in different types of tumors and emerge inconsistent therapeutic responses. Hence, we concluded the recent advances in tumor-infiltrating immune cells, including functions, prognostic values, and various immunotherapy strategies for each immune cell in different tumors.

ObjectiveCombined the traditional isolation culture, ITS sequencing and metagenomics sequencing technology to establish a new method for the analysis of fungal community composition and species diversity in Pu'er tea at retail markets.MethodsPu'er tea samples were collected at retail markets and tested for cultivable fungi in accordance with "National food safety standard Food microbiological examination: Counting of Molds and Yeasts" (GB 4789.15—2016), and the isolated strains were sequenced by ITS and analyzed in the NCBI database; the fungal genomic DNA from Pu'er tea samples was extracted by CTAB method, and performed PCR amplification and amplicon sequencing; high-throughput sequencing data was used to build OTUs clustering, and carried out species composition and diversity analysis.ResultsTwelve fungi were isolated and cultured from Pu'er tea samples, mainly Aspergillus and Lichtheimia, of which Aspergillus niger had the highest proportion (25%, 3/12). The result of species distribution based on metagenomics showed that the diversity and uniformity of fungal species in Pu'er tea samples were low, and there were relatively high number of dominant species; the highest proportion of fungi was Ascomycota (74.03%), and at the genus level, the fungi in Pu'er tea were dominated by Blastobotrys (49.43%) while Aspergillus accounted for a relatively low percentage (14.04%).ConclusionThis study comprehensively applied traditional isolation culture, molecular biology and high-throughput metagenomics sequencing technology to establish a new method for the analysis of fungal community composition and species diversity in Pu'er tea at retail markets. It provided ideas and references for further research on the microbial community structure in Pu'er tea and its impacts on quality and safety.

ObjectiveTo prepare Cronobacter spp. (Enterobacter sakazakii) proficiency test samples which meet the requirements of uniformity and stability, and use them for proficiency test.MethodsThe background strains and Cronobacter. spp were identified by biochemical and matrix-assisted laser desorption ionization time of flight mass spectrometry method. Freeze-drying was used to prepare the samples with 104CFU/bottle. According to Guidance on Evaluating the Homogeneity and Stability of Samples Used for Proficiency Testing, 20 samples were selected randomly for uniformity test. The samples were stored at -20 and 4 ℃ respectively to evaluate the storage stability, and at 25 and 37 ℃ to evaluate the transport stability. According to GB 4789.40—2016, samples were distributed to participating laboratories with operation instruction, and then the feedback result were analyzed.ResultsThe identified result of all the related strains were correct and consistent with expectations. The result of uniformity and stability tests met relevant requirements. All of the experiment result submitted by 25 laboratories within the required time were correct, and the satisfaction rate was 100%.ConclusionThe samples of Cronobacter spp. Enterobacter sakazakii in milk powder proficiency test can meet the relevant requirements, and this proficiency test provides external quality control assessment result for the participating labratories simultaneously.

Abstract Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

Objective To investigate the protective effects and primary mechanism of ginsenoside Rg1 on cardiac muscle of aging mice induced by D-galactose (D-gal). Methods C57BL/6 J mice were randomly divided into normal control, model, Rg1 intervention and negative control groups, with 10 animals in each group. D-gal (200 mg/kg) was injected intraperitoneally to the mice for 42 d to establish the model group, and the mice of the Rg1 intervention group was given 40 mg/kg Rg1 per day for 26 d intraperitoneally from the 16th day of model establishment. The mice of the normal control group were given same amount of normal saline for 42 d, and those of the negative control group were treated with normal saline after model establishment. On the 2nd day after treatment, the hearts were taken to measure the organ index. HE staining and transmission electron microscopy were performed to observe the morphology and micro-structures of the heart tissue from each group. The activities of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total superoxide dismutase (T-SOD), catalase (CAT) and contents of malondialdehyde (MDA) in the myocardial tissue homogenate were tested by enzyme-labeled colorimetry; The expression levels of Nrf2, Keap1 and HO-1 in the heart tissue were detected by Western blotting. Results Compared with the control group, the model group had significantly increased heart index (P < 0.01); larger amount of deposited collagen fibers between myocardial fibers, disordered myofibril bundles, thicker myocardial fibers, and abnormally swollen mitochondria; enhanced AST and LDH activities, declined T-SOD and CAT activities, and increased MDA content (P < 0.05); remarkably down-regulation of Nrf2 and HO-1 (P < 0.05), and no changed Keap1 level. While in the Rg1 intervention group, all the above phenomena were greatly improved after the intervention, with obviously decreased heart index (P < 0.01), not notable morphological changes with normal control mice, reversed AST, LDH, T-SOD and CAT activities and MDA contents(P < 0.05), and elevated protein levels of Nrf2 and HO-1, and decreased level of Keap1 (P < 0.01). Conclusion Rg1 effectively alleviates the damage of D-gal to the morphology and ultrastructure of mouse myocardium, and its mechanism may be related to the inhibition of oxidative stress and the up-regulation of the Nrf2 antioxidant signal pathway.

Objective To investigate the possible mechanism of kinesin family member 3A (KIF3A) taking part in the regulation of asthma airway inflammation, so as to provide a new target and direction for asthma treatment. Methods Female C57BL/6 mice were randomly divided into asthma group (n=10) and control group (n=10). Western blotting and immunohistochemistry (IHC) were performed to detect the expression of KIF3A in the 2 groups. After human bronchial epithelial 16HBE cells were intervened using phosphate buffer solution (PBS) and house dust mite (HDM), Western blotting and RT-PCR were adopted to detect the KIF3A expression. Moreover, the lentiviral vectors of KIF3A overexpression (LV-KIF3A-45894-J2, LV-KIF3A) and knock-down (LV-KIF3A-RNAi-81183-1, RNAi) were constructed and then transfected into 16HBE cells, and the blank lentiviral vectors and those containing negative control sequence served as controls, respectively. The binding of KIF3A to β-arrestin in the overexpressed cells was detected by immunoprecipitation, while the expression level of β-catenin in each group of cells was determined by Western blotting and RT-PCR. The effect of differential expression of KIF3A on the expression of chemokines CCL-17 and CCL-26 was also determined. Results In animal models, the expression of KIF3A was significantly lower in the asthma mice than the control mice (P < 0.05). In vitro study indicated that HDM intervention decreased the expression of KIF3A at protein and mRNA levels in 16HBE cells, though there was no statistical difference (P>0.05). The RT-PCR and Western blotting results showed that the expression of KIF3A were increased in the LV-KIF3A transfected cells (P < 0.05) and decreased in the RNAi treated cells (P < 0.05). Immunoprecipitation confirmed the binding of KIF3A to β-arrestin. No significant difference was found in the total amount of β-catenin protein between the LV-KIF3A transfected cells and the RNAi treated cells. Compared with the corresponding cells, the mRNA level of β-catenin was significantly lower in the RNAi treated cells (P < 0.0001), but no such difference was seen in the LV-KIF3A transfected cells. The RT-PCR results suggested that the levels of chemokines CCL-17 and CCL-26 were increased in the RNAi treated cells (P < 0.001), while decreased in the LV-KIF3A transfected cells (P < 0.05) as compared with their control cells. Conclusion KIF3A binds to β-arrestin in the Wnt/β-catenin pathway, and is involved in asthmatic airway epithelial inflammation by regulating the expression of chemokines CCL-17 and CCL-26.