A rapid and accurate determination method of lipids in microalgae plays a significant role in an efficient breeding process for high-lipid production of microalgae. Using low field nuclear magnetic resonance (LF-NMR), we developed a direct quantitative method for cellular lipids in Chlorella protothecoides cells. The LF-NMR signal had a linear relationship with the lipid content in the microalgae cells for both dry cell samples and algal broth samples (R2 > 0.99). These results indicated that we could use this method for accurate determination of microalgal lipids. Although LF-NMR is a rapid and easy lipid determination method in comparison to conventional methods, low efficiency would limit its application in high throughput screening. Therefore, we developed a novel combined high throughput screening method for high-lipid content mutants of C. protothecoides. Namely, we initially applied Nile red staining method for semi-quantification of lipid in the pre-screening process, and following with LF-NMR method for accurate lipid determination in re-screening process. Finally, we adopted this novel screening method in the breeding process of high-lipid content heterotrophic cells of C. protothecoides. From 3 098 mutated strains 108 high-lipid content strains were selected through pre-screening process, and then 9 mutants with high-lipid production were obtained in the re-screening process. In a consequence, with heterotrophical cultivation of 168 h, the lipid concentration could reach 5 g/L, and the highest lipid content exceeded 20% (W/W), which was almost two-fold to that of the wild strain. All these results demonstrated that the novel breeding process was reliable and feasible for improving the screening efficiency.
Chlorophyta ; chemistry ; Heterotrophic Processes ; High-Throughput Screening Assays ; Lipids ; analysis ; Magnetic Resonance Spectroscopy ; Microalgae ; chemistry ; Staining and Labeling

A 27-year-old woman who suffered from a 2-year history of systemic lupus erythematosus (SLE) presented with a 6-month history of nodules and ulcer on the right lower extremity. Direct microscopic examination of the pus showed branched and septate hyphae and spores in a chain-like arrangement.Histopathological examination revealed yellowish brown hyphae and spores. Dark green velvety colony grew on Sabouraud dextrose agar (SDA). Slide culture showed branched, septate hyphae and spine-like annellated conidiophores. The isolate was identified as Exophiala spinifera by DNA sequence analysis. The strain was unable to liquefy gelatin, could grow at 25 ℃ to 39 ℃, and was sensitive to itraconazole, amphotericin B and terbinafine. Animal test revealed that the infection induced by Exophiala spinifera in immunocompromised mice was more severe than that in normal controls. Based on the clinical features, histopathological, fungal culture and DNA sequencing results, the patient was diagnosed with systemic lupus erythematosus accompanied by subcutaneous phaeohyphomycosis caused by Exophiala spinifera.

AIM To investigate the protective effect of polydatin on ischemia-reperfusion (I-R) injury in cardiac muscle and the possible mechanism. METHODS Langendorff technique was used to make I-R injury in rats. Male Sprague-Dawley rats were randomly divided into control, model, polydatin(25, 50 and 75 μmol·L-1), glibenclamide(Gli, 10 μmol·L-1)+polydatin(50 μmol·L-1), 5-hydroxydecanoate(5-HD, 100 μmol·L-1)+polydatin(50 μmol·L-1), and atractyloside (Atr, 20 μmol·L-1)+polydatin(50 μmol·L-1) groups. The hearts in control group were perfused with K-H solution for 110 min. Model group hearts were subjected to 30 min no-flow global ischemia followed by 60 min of reperfusion. The hearts in 3 polydatin groups were perfused with K-H solution containing different concentrations of polydatin for 10 min before I-R. The hearts in Gli+polydatin and 5-HD+polydatin groups were perfused with K-H solution containing Gli or 5-HD for 5 min firstly, then perfused with K-H solution containing both polydatin and Gli or 5-HD for 10 min before I-R. The hearts in Atr+polydatin group were perfused with K-H solution containing polydatin for 10 min before I-R and perfused with K-H solution containing Atr for 15 min after I-R. The cardiac function, including left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), the maximal rates of rise and decline of left ventricular pressure (±dp/dtmax), and coronary flow (CF), were recorded before, after 30 min no-flow global ischemia and, during 60 min reperfusion. Myocardial infarct size was assessed using 2, 3, 5-triphenyltetrazolium chloride method and myocardial ultrastructure was observed via transmission electron microscope after 60 min reperfusion. RESULTS There were no significant differences in cardiac functional parameters between control and model groups in pre-ischemia condition. Compared with model group, polydatin promoted a better recovery of cardiac function after I-R in a concentration-dependent manner. After 60 min of reperfusion, the values of LVDP, ±dp/dtmax and CF in polydatin groups were much higher, but LVEDP was lower than those in model group. Polydatin (50 μmol·L-1) also significantly reduced myocardial infarct size and relieved the I-R injury of myocardial ultrastructure. The protective effects of polydatin (50 μmol·L-1) on LVDP, LVEDP, ±dp/dtmax and CF, as well as the inhibitory effect on infarct size after I-R were abolished by Gli, 5-HD and Atr. CONCLUSION Polydatin has protective effect against I-R injury in rat hearts, which may be related with the opening of ATP-sensitive potassium channel located in both cell membrane and mitochondrial membrane, as well as inhibition of mitochondrial permeability transition pore opening.

Objective To investigate the effect of Intedeukin-18 (IL-18) on Th1/Th2 balance and its antitumor mechanism in C57BL/6 mice Lewis lung cancer model. Methods 24 C57BL/6 mice were randomly divided into three equal groups: group A(IL-18 injec-tion group, n = 8), group B (Lewis lung cancer model, n = 8) and group C (normal control group, n = 8). The Lewis lung cancer cells were cultured and implanted subcutaneously into the group A and group B. IL-18 and NS were given to group A and B respectively by intrap-eritoneal injection on the 7th day (once every day, 7 times altogether), but group C was not given any treatment. Enzyme-linked immunosor-bent assay (ELISA) was used to detect the Th1/Th2 cytokines. Health status in all the animals was evaluated; the volume and weight ofsubcutaneous tumors were measured. Results The concentration of IFN-γ in group A and C were significantly higher than those in group B (P <0.05), and the concentration of IL-4 in group A and C were significantly lower than those in group B (P<0.05), but there was no significant difference between group A and C (P>0.05). The tumor growth inhibitory rate was 75%. Conclusion IL-18 can effectively induced IFN-γ and inhibit IL-4 production, regulate Th1/Th2 balance in the C57BL/6 mice Lewis lung cancer model, and elicit the antitu-mor immunity of the host, which could obviously inhibit the growth of tumor cells and decelerate the proliferation of tumor cells.

AIM: To investigate the effect of chronic intermittent hypobaric hypoxia (CIHH) on the activity of adrenergic receptor(AR) in rabbit sino-atrial node (SAN) by intracellular recording.METHODS: New Zealand rabbits were randomly divided into three groups: control group (Con), 14 d CIHH treatment group (CIHH14) and 28 d CIHH treatment group (CIHH28). The CIHH rabbits were exposed to a simulated 5 000 m (oxygen 11.1%) hypobaric hypoxia in hypoxic chamber for 14 d or 28 d (6 h/d), respectively. SAN preparation was used and the transmembrane action potential was recorded by micropipettes. Isoproterenol hydrochloride (ISO, agonist of β-AR) and phenylephrine (PE, agonist of α_1-AR) at different concentrations (0.01, 0.1 and 1 μmol/L) were applied cumulatively to investigate the electrophysiological effect of the drugs on the rabbit SAN among Con, CIHH14 and CIHH28 groups, respectively. RESULTS: (1) CIHH didnt change the parameters of action potential of SAN recorded under basic condition. (2) ISO changed some parameters of AP significantly in a dose-dependent manner, including increases in the amplitude of AP (APA), maximal rate of depolarization (V_(max)), the velocity of diastolic (phase 4) depolarization (VDD), and rate of pacemaker firing (RPF). (3) The response of AP to ISO in CIHH rabbits was decreased significantly compared to that in Con animals. Under 1 μmol/L of ISO, the increases in VDD, RPF, APA and V_(max) in CIHH animals were smaller than those in Con animals (P<0.05). (4) No effect of PE (0.01, 0.1 and 1 μmol/L) on the parameters of action potential was observed. CONCLUSION: CIHH decreases the reactivity of β-AR, but has no effect on α_1-AR in SAN of rabbit.

Objective To understand the characteristics of problem behaviors among junior middle school students in northern Guizhou rural areas to provide reference for formulating the intervention measures .Methods The Chinese middle school students mental health scale and the self-made externalizing behavior problem questionnaire were adopted to perform the questionnaire sur-vey on left-behind kids of 6 junior middle schools .Results In the implicit problem behavior ,the scores of girls in hostility ,interper-sonal ,depression factor ,anxiety and total score were higher than those of boys ,and the each factor score was increased with grade ;in the explicit behavioral problems ,the occurrence rates of smoking ,drinking and gambling in boys were higher ,the detection rates of smoking and gambling were higher than those in students ,the occurrence rates of suicidal idea and leaving from home idea in students were 13 .1% and 22 .2% respectively ,in which the occurance rates of girls were higher than those in boys .The detection rate of sex related behaviors in boys was higher than that in girls .Conclusion The problem behaviors appear the gender and grade differences among left-behind kids in northern Guizhou rural areas .The differential mental health education should be carried out according to different target behaviors .

Objective Single voxel proton spectroscopy technique (1H-MRS)was performed in the healthy newborn brain tissue to investigate the metabolic ratio in the right basal ganglia and the right frontal lobe changes with gestational age, and to improve the understanding of the neonatal brain development. Methods In this study, 69 newborns were selected, including twenty-five premature and forty-four term neonates. By using the Gesell developmental scale to evaluate brain development , they all had normal neurologic outcomes at 12-months corrected age. A point-resolved spectroscopy sequence was used to study the metabolic data obtained from each region of interest. Results N-acetylaspartate: creatine and N-acetylaspartate:choline were positively correlated with gestational age (the right basal ganglia r = 0.27, 0.63, the right frontal lobe r = 0.44, 0.64 respectively, P < 0.05) and choline:creatine was negative (the right basal ganglia r = -0.51,the right frontal lobe r = -0.41, P < 0.05), with a linear regression relationship among variables. In the same gestational age group , the N-acetylaspartate: choline of the right basal ganglia was higher than that of the right frontal lobe , and the metabolic ratios among the anatomic locations were significantly different (P < 0.05). Conclusion Neonatal brain metabolite levels are associated with gestational age , and MRS can monitor newborn brain maturation quantitatively.

Objective:To investigate the possible role of NKT cells in unexplained recurrent spontaneous abortion by measuring the NKT cell numbers,maturity and cytokine secretion of the placenta of mice with unexplained recurrent spontaneous abortion.Methods:Normal pregnancy model in hybrid by feeding CBA / J and BABL/C in a cage,and the model of unexplained recurrent spontaneous abortion was established by feeding CBA / J and DBA2/J in a cage.The number of NKT and CD3~+T cells was determined by flow-cytometry;Th1/Th2-relative cytokines were assayed by ELISA and T-bet expression was determinded by fluorescence quantitative PCR.Results:There was not significant change of CD3~+ T cells when compared between normal pregnancy and unexplained recurrent spontaneous abortion group (P＞0.05).In the course of normal pregnancy,the IFN-γ secreted by placenta lymphocytes decreased gradually,accompanied by the decline of NKT cell number and the proportion of mature cells;whereas in the course of unexplained recurrent spontaneous abortion,it was on the opposite.There was significant difference of T-bet mRNA expression between the two groups.T-bet mRNA expression was related to the proportion of mature NKT cells or placenta IFN-γ secretion by lymphocytes.Conclusion:Unexplained recurrent spontaneous abortion may be related to NKT cells disorders,NKT cells are of low-mature proportion and inadequate secretion of IFN-γ during early pregnancy,whereas are shown high-mature proportion and excessive secretion of IFN-γ during latter pregnancy;the anomaly of T-bet mRNA expression may be one of the factors leading to NKT cells disorder.

ObjectiveTostudythemolecularmechanismoflanthanumonblocking lipopolysaccharide(LPS)-mediated activation of nuclear factor-kappaB (NF-κB)signaling in macrophages.MethodsThe RAW264.7 macrophages were cultured routinely and divided into 4 groups randomly:LaCl3 +LPS group,LPS group,LaCl3 group and control group.The nuclear translocation of p65 protein was detected by immunocytochemistry.Total,cytoplasmic and nuclear proteins were extracted respectively,and then the binding activity of p65 with the target gene was measured by ELISA.Western blot assays were also performed to detect the expression levels of the proteins,including nuclear p65,IκBα and IKK kinase,the phosphorylation status of IκBα and IKK kinase.ResultsLanthanum can block LPS-induced activation of p65 protein through various ways,such as inhibiting its nuclear translocation,reducing its expression in the nuclei and decreasing its binding activity with the target genes.Lanthanum inhibited the degradation of LPS-induced IκBα,but the phosphorylation of LPS-induced IKKβ can not be blocked by lanthanum,nor did the phosphorylation ability of p-IKKβ on IκBα.Conclusion Lanthanum inhibited the degradation of IκBα,nuclei translocation of p65 protein and its binding activity with the target genes,thereby inhibited LPS-induced NFκB activation,which might be one of the inhibition mechanisms of lanthanum on nuclear activation.

Objective: To study the changes of monocyte cytokines in peripheral blood of n-hexane neuropathy patients induced by P₀ protein, and to explore the role of autoimmunity in n-hexane neuropathy patients. Methods: In May 2018, 5 patients with peripheral neuropathy diagnosed as n-hexane poisoning were selected as case group in Shenzhen Prevention and Treatment Center for Occupational Disease in 2017. 6 workers exposure to n-hexane and 6 workers without n-hexane exposure were selected as contact group and control group. Peripheral blood mononuclear cells(PBMC) were isolated from venous blood. Results: The number of spots produced by INF-γ and IL-10 increased after stimulation with P₀ protein in case group, and the positive rate was significantly higher than control group and the contact group. Conclusion Autoimmunity induced by P₀ protein may be involved in the occurrence of myelin sheath damage in n-hexane neuropathy patients.