OBJECTIVE To investigate the ameliorative effect and potential mechanism of imperatorin （IMP） on doxorubicin （DOX） resistance in breast cancer. METHODS The effects of maximum non-toxic concentration （100 μg/mL） of IMP combined with different concentrations of DOX （12.5， 25， 50， 75， 100 μg/mL） on the proliferation of MCF-7/DOX cells were determined by MTT method. MCF-7/DOX cells were divided into blank control group （1‰ dimethyl sulfoxide）， DOX group （50 μg/mL）， IMP+DOX group （100 μg/mL IMP+50 μg/mL DOX） and IMP group （100 μg/mL）. mRNA and protein expressions of multidrug resistance protein 1 （MDR1） and multidrug resistance-associated protein 1 in each group were measured. The relevant pathways and targets involved in the improvement of DOX resistance in breast cancer cells by IMP were screened and validated by using transcriptome sequencing technology， along with gene ontology （GO） enrichment analyses and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses. RESULTS Compared with DOX alone， the combination of IMP and DOX reduced the half inhibitory concentration of DOX on MCF-7/DOX cells from 81.965 μg/mL to 43.170 μg/mL， the reverse fold was 1.90， and the mRNA expression of MDR1 was significantly down-regulated （P＜0.05）. The results of GO enrichment analyses and KEGG pathway enrichment analyses indicated that the reversal of DOX resistance in breast cancer by IMP was mainly associated with the regulation of biological processes such as detoxification， multiple biological processes， and cell killing. The main pathway involved was the p53 signaling pathway， and the key targets mainly included constitutively photomorphogenic protein 1 （COP1）， cyclin E1 （CCNE1）， growth arrest and DNA damage-inducible protein 45A E-mail：tangxilan1983@163.com （GADD45A） and GADD45B. The results of the verification experiments showed that compared with DOX group， there was a trend of up-regulation of COP1 mRNA， and significant down- regulation of CCNE1， GADD45A， and GADD45B mRNA expression in IMP+DOX group （P＜0.05）. CONCLUSIONS The effect of IMP in ameliorating DOX resistance in breast cancer is related to its regulation of COP1， CCNE1， GADD45A and GADD45B targets in the p53 signaling pathway.

Acute liver failure (ALF) is lack of broadly approved therapeutic strategy except liver transplantation. As a glycogen metabolic intermediate, UDP-glucose (UDP-G) has been considered to accelerate liver repairment. Nevertheless, the role of UDP-G and its receptor P2Y purinoceptor 14 (P2Y₁₄R) in ALF remains unknown. The present study aims to investigate the role and underlying mechanisms of UDP-G/P2Y₁₄R axis in ALF. In this study, hepatic P2Y₁₄R is significantly increased in TAA-induced and partial hepatectomy-induced ALF, while knockout of whole-body P2Y₁₄R aggravates liver failure, manifested by inhibiting β-Catenin-mediated liver regeneration. Consistently, P2Y₁₄R deficiency exhibits impaired liver regeneration in mice suffer partial hepatectomy. Importantly, only hepatocellular specific deletion of P2Y₁₄R (P2Y₁₄R ^flox/flox Alb ^cre/+ ) mice shows a similar phenomenon, rather than stellate cell specific deletion of P2Y₁₄R (P2Y₁₄R ^flox/flox Lrat ^cre/+ ) mice. Mechanistically, P2Y₁₄R induction regulates methylation of Dact-2 through CREB/DNMT3b signals in hepatocytes, subsequently inhibiting the expression of Dact-2 which is a stabilizer of β-Catenin degradation complex, leading to the activation of β-Catenin -mediated liver regeneration. Interestingly, the administration of exogenous UDP-G can accelerate liver regeneration and liver function recovery after partial hepatectomy in hepatocellular carcinoma mice. Together, the findings propose an unrecognized role of P2Y₁₄R in ALF and provide an effective adjuvant strategy for treatment of ALF.

The plant F-box protein more axillary growth 2 (MAX2) is a key factor in the signal transduction of strigolactones (SLs) and karrinkins (KARs). As the main component of the SKP1-CUL1-FBX (SCF) complex ubiquitin ligase E3, MAX2 is responsible for specifically recognizing the target proteins, suppressor of MAX2 1/SMAX1-like proteins (SMAX1/SMXLs), which would be degraded after ubiquitination. It can thereby regulate plant morphogenesis and stress responses. There exist homologous genes of MAX2 in the important grain and oil crop soybean (Glycine max). However, its role in plant defense responses has not been investigated yet. Here, GmMAX2b, a homologous gene of MAX2, was successfully cloned from stressed soybean. Bioinformatics analysis revealed that there were two MAX2 homologous genes, GmMAX2a and GmMAX2b, with a similarity of 96.2% in soybean. Their F-box regions were highly conserved. The sequence alignment and cluster analysis of plant MAX2 homologous proteins basically reflected the evolutionary relationship of plants and also suggested that soybean MAX2 might be a multifunctional protein. Expression analysis showed that plant pathogen infection and salicylic acid treatment induced the expression of GmMAX2b in soybean, which is consistent with that of MAX2 in Arabidopsis. Ectopic expression of GmMAX2b compensated for the susceptibility of Arabidopsis max2-2 mutant to pathogen, indicating that GmMAX2b positively regulated plant disease resistance. In addition, yeast two hybrid technology was used to explore the potential target proteins of GmMAX2b. The results showed that GmMAX2b interacted with SMXL6 and weakly interacted with SMXL2. In summary, GmMAX2b is a positive regulator in plant defense responses, and its expression is induced by pathogen infection and salicylic acid treatment. GmMAX2b might exert its effect through interaction with SMXL6 and SMXL2. This study expands the theoretical exploration of soybean disease resistant F-box and provides a scientific basis for future soybean disease resistant breeding.
Glycine max/metabolism* ; Disease Resistance/genetics* ; Plant Diseases/immunology* ; Plant Proteins/genetics* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; F-Box Proteins/genetics* ; Arabidopsis/genetics* ; Phylogeny

Objective:To clone the Helicobacter pylori(Hp)hp0169 gene and conduct the crystallographic study,and to clarify its secondary and tertiary structures.Methods:The hp0169 gene and its encoded protein sequence of the Hp NCTC26695 strain were retrieved from the UniProt database.Bioinformatics method was used to analyze the physicochemical properties of the Hp recombinant protease(HpPrtC)protein;SOPMA and DNAStrar softwares were used to predict the secondary structure characteristics of HpPrtC protein;SWISS-MODEL software was used to construct the tertiary structure of the HpPrtC protein;IEDB and ABCpred softwares were used to predict the antigenic epitopes of the B lymphocytes HpPrtC protein;SYFPEITMI website was used to predict the antigenic epitopes of the T lymphocytes of HpPrtC protein;the expert pool(EP)and random forest(RF)algorithms were used to predict the crystallizability of the HpPrtC protein;the HpPrtC recombinant protein was expressed in the prokaryotic system;the HpPrtC recombinant protein was purified by Ni2+affinity chromatography and size-exclusion chromatography;the crystallization conditions for HpPrtC were screened by crystallization kit.Results:The hp0169 gene contained 1 269 base pairs and encoded the protein of 422 amino acids,the theoretical isoelectric point was 7.64 and the relative molecular weight was 47 300.HpPrtC was a hydrophilic and soluble protein.The number of amino acids of alpha helices of HpPrtC accounted for 35.78%,beta sheets 18.72%,beta turns 6.87%,and random coils 38.63%.The antigen epitope analysis results showed that HpPrtC contained five dominant linear epitopes of B lymphocytes,three conformational epitopes,and multiple potential dominant epitopes of T lymphocytes.The homology modeling results showed that HpPrtC formed a dimer,and each monomer displayed a barrel structure surrounded by β sheets,alpha helices,and random coils.HpPrtC was predicted to have moderate crystallizability without signal peptides and transmembrane helices.Small clustered needle-like crystals of HpPrtC were obtained under the conditions of 0.2 mol·L-1 magnesium chloride,0.1 mol·L-1 tris(hydroxymethyl)amino methane(Tris),3.4 mol·L-1 hexanediol,and pH=8.5.Conclusion:HpPrtC is a hydrophilic protein that forms a dimeric structure and crystallizes into small clustered needle-like crystals under suitable conditions.HpPrtC contains dominant antigenic epitopes of the T lymphocytes and B lymphocytes and can serve as an antigen for the design of Hp vaccines to establish the multivalent fusion vaccines or multi-epitope vaccines;the results provide an experimental basis for the prevention and control of Hp.

OBJECTIVE To develop a method for simultaneous content determination of menthone, menthol, 2-methoxy-4-vinylphenol, 3-butenyl phthalide, atractyline, ligustilide in Honghua Xiaoyao tablets. METHODS The quantitative analysis was carried out on a capillary of HP-5(30 m×250 μm, 0.25 μm) column combined with an oven temperature program; inlet port temperature 250 ℃; detector temperature 280 ℃, split sampling at a split ratio 10∶1. RESULTS The 6 components were completely separated and could be well separated from other components; menthone, menthol, 2-methoxy-4-vinylphenol, 3-butenyl phthalide, atractyline, ligustilide showed a good linear relationship with the chromatographic peak area within the range of 1.37-87.38, 13.67-874.55, 2.05-65.49, 3.35-107.20, 3.49-111.60, 6.95-444.60 μg·mL-1(r≥0.999 0). The average recovery was 97.63%(RSD=3.27%), 94.25%(RSD=1.80%), 97.54%(RSD=2.39%), 103.81%(RSD=1.75%), 94.30% (RSD=2.32%), 98.97%(RSD=2.36%)(n=6), respectively. And its content in 39 batches of Honghua Xiaoyao tablets were determined. CONCLUSION This method is easy to operate, accurate, reliable, and has good repeatability, can be used to supplement the insufficient quality control of Honghua Xiaoyao tablets.

Type 2 diabetes mellitus （T2DM） is a heterogeneous disease with insulin deficiency and insulin resistance （IR） as the main etiology and is often accompanied by complications. Volatile oil is a volatile oily liquid extracted from natural plants， which has many pharmacological effects such as regulating Qi， relieving pain， inhibiting bacteria， and reducing inflammation. In recent years， there have been numerous reports on the treatment of T2DM by natural plant volatile oil and its effective components， which has become one of the new directions in the treatment of T2DM. With natural plant essential oil and its active components as the starting point， this paper comprehensively analyzed and summarized the material basis， mechanism， and signaling pathways of essential oil in the treatment of T2DM and its complications in China and abroad in recent years， and focused on the inhibitory effect of essential oil and its active components， such as carvacrol， paeonol， and β-caryophylene， on IR to improve T2DM by protecting pancreatic β-cells， inhibiting α-glucosidase activity， regulating the abundance and diversity of intestinal microbiota， and regulating glucose transporter protein type4 （GLUT4）， adenylate 5′-monophosphate-activated protein kinase （AMPK）， phosphatidylinositol-3 kinase （PI3K）/protein kinase B （Akt） signaling pathways to provide some references for the volatile oil intervention in T2DM and the development of new green antidiabetic drugs.

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Alleles ; Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Circular/metabolism* ; Embryo, Nonmammalian ; Gene Editing/methods* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; Genotyping Techniques ; Green Fluorescent Proteins/metabolism* ; Integrases/metabolism* ; Luminescent Proteins/metabolism* ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/metabolism*

Objective: To explore the relationship between physiological parameters changes and severe heatstroke induced by 5-km armed cross-country training. Methods: A total of 521 male officers and soldiers from a special team who participated in the summer training of 5-km armed cross-country training from year 2016 to 2017 were enrolled. All trainees participated in 5-km armed cross-country training in high temperature and humidity environment of ambient temperature > 32 ℃and (or) relative humidity > 65%. The trainees were divided into two groups according to the incidence of severe heatstroke in the course of training. The age, enlistment time, constitution score, body mass index (BMI), external environment (ambient temperature, relative humidity, wind speed, heat index) of trainees of the two groups, and the change rates of arterial blood oxygen saturation (SaO₂), body temperature, pulse and blood pressure within 5 minutes after the 5-km armed cross-country training were compared between the two groups. The risk factors of severe heatstroke were screened by two classified Logistic regression analysis, and the predictive value of various risk factors of severe heatstroke was analyzed by the receiver operator characteristic curve (ROC). Results: In 521 trainees of 5-km armed cross-country training, 29 trainees suffered from severe heatstroke accounting for 5.57%. There was no significant difference in the age, enlistment time, constitution score, BMI, or external environment during 5-km armed cross-country training between severe heatstroke group and non-severe heatstroke group. Compared with those without severe heatstroke, the descending rates of body temperature, pulse, blood pressure and SaO₂ increased rate within 5 minutes after 5-km armed cross-country training of severe heatstroke trainees were significantly decreased [temperature descending rate: (0.67±0.30)% vs. (1.43±1.28)%, pulse descending rate: (7.53±5.21)% vs. (13.48±8.07)%, blood pressure descending rate: (9.28±6.84)% vs. (19.42±7.73)%, SaO₂ increased rate: (0.51±0.39)% vs. (1.50±1.43)%, all P < 0.01]. Two classification Logistic regression analysis showed that the temperature descending rate [odds ratio (OR) = 0.485, 95% confidence interval (95%CI) = 0.289-0.817], pulse descending rate (OR = 0.903, 95%CI = 0.845-0.965), blood pressure descending rate (OR = 0.841, 95%CI = 0.790-0.896), and SaO₂ increased rate (OR = 0.421, 95%CI = 0.250-0.711) were the risk factors for severe heatstroke during 5-km armed cross-country training (all P < 0.01). ROC curve analysis showed that temperature descending rate [area under ROC curve (AUC) = 0.659, 95%CI = 0.604-0.714], pulse descending rate (AUC = 0.730, 95%CI = 0.762-0.900), blood pressure descending rate (AUC = 0.831, 95%CI = 0.659-0.801), SaO₂ increased rate (AUC = 0.711, 95%CI = 0.655-0.767) could be used for the incidence of severe heatstroke prediction during 5-km armed cross-country training (all P < 0.01), and the predicted value was the same. Conclusions Under the same conditions, the severe heatstroke during 5-km cross-country training is closely related to the descending rates of body temperature, pulse, and blood pressure as well as SaO₂ increased rate within 5 minutes after the training, whose predictive values for severe heatstroke were the same.

Objective To investigate the effects of heat acclimatization training on the inflammatory reaction and multiple organ dysfunction syndrome (MODS) in patients with exertional heat stroke (EHS). Methods 600 officers and soldiers from a special team who participated in 5 km armed wild training from June to July 2017 were selected as the research object, while 30 healthy officers and men who did not participate in armed wild training during the same period were selected as normal controls. The officers and soldiers who participated in 5 km armed wild training (ambient temperature > 35 ℃, humidity > 65%, 2-3 times a week for 3 weeks) were divided into heat acclimatization group and non-acclimatization group, with 300 in each group. The heat acclimatization group first took part in the heat acclimatization training of wild or long distance running (the initial temperature was 30 ℃, gradually transferred to the hot time of 37 ℃), 2 hours each time, twice a day, and 5 days a week, for a total of 2 weeks. Venous blood was taken before and after heat acclimatization training, before armed wild training, and after the last training or EHS onset, and the contents of serum interleukin (IL-1β, IL-10), tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) were detected by enzyme linked immunosorbent assay (ELISA). The occurrence of EHS and MODS in EHS patients were recorded. Results There was no significant difference in serum inflammatory factors between the officers and soldiers who participated in the training and the healthy control group before heat training or cross-country training. Compared with those before heat training, IL-1β, TNF-α, IFN-γ were significantly increased in all participants of heat acclimatization training while IL-10 was significantly decreased. For those who experienced premonitory (6 cases) and mild (2 cases) heatstroke during training, they could return to normal without severe heatstroke or EHS within 10-30 minutes after being immediately put in a cool and ventilated place and given anti- heatstroke drugs and other interventions. Compared with those before wild training, the levels of inflammatory factors in the two groups of officers and soldiers also changed after wild training, but the increase or decrease of inflammatory indexes in the heat acclimatization group were significantly smaller than those in the non-acclimatization group [IL-1β (ng/L): 10.65±5.18 vs. 12.13±7.91, TNF-α (ng/L): 14.60±5.79 vs. 16.27±8.52, IFN-γ (ng/L): 13.66±5.43 vs. 15.33±8.71, IL-10 (ng/L):8.22±2.68 vs. 7.13±2.63, all P < 0.05]. During armed wild training, a total of 27 cases of EHS occurred. The incidence of EHS in the heat acclimatization group was significantly lower than that in the non-acclimatization group [2.67% (8/300) vs. 6.33% (19/300), χ2= 4.693, P = 0.030]. In patients with EHS, IL-1β, TNF-α, IFN-γ after wild training in the heat acclimatization group were also significantly lower than those in the non-acclimatization group, and IL-10 was significantly higher [IL-1β (ng/L): 34.50±3.74 vs. 39.53±4.51, TNF-α (ng/L): 43.75±2.87 vs. 46.79±2.66, IFN-γ (ng/L): 40.25±1.75 vs. 46.58±1.92, IL-10 (ng/L): 7.50±2.45 vs. 5.42±1.80, all P < 0.01], and the incidence of MODS and organ involvement of EHS patients in the heat acclimation group were significantly lower than that in the non-acclimatization group [50.00% (4/8) vs. 89.47% (17/19), χ2= 5.075, P = 0.024; 28.13% (9/32) vs. 47.79% (65/136), χ2=4.066, P=0.044]. Conclusion Heat acclimatization training before high strength training in high temperature and humidity environment can effectively reduce the degree of inflammation reaction of EHS, protect the physiological functions of EHS organs, and reduce the incidence of MODS.