Observations of linear aggregation behavior in rotifers (Brachionus calyciflorus).

Linear aggregation is present in some animals, such as the coordinated movement of ants and the migration of caterpillars and spinylobsters, but none has been reported on rotifers. The rotifers were collected and clone cultured in the laboratory at 25 ± 1°C, under natural light (light intensity ~130 lx, L:D = 14:10). The culture medium(pH = 7.3) was formulated as described by Suga et al., and rotifers were fed on the micro algae Scenedesmus obliquus grown in HB-4 medium to the exponential growth stage. When density was high (150 individuals ml-1), the behavior of rotifers was observed using a stereo microscope (Motic ES-18TZLED). In this paper, linear aggregation in Brachionus calyciflorus was found for the first time, and experiments were carried out to verify the correlation between linear aggregation and culture density of B. calyciflorus. With the increase of density, the number of aggregations increase, the number of individuals in the aggregation increased, and the maintenance time of the aggregation was also increased. Therefore, we speculate that the formation of aggregates is related to density and may be a behavioral signal of density increase, which may transmit information between density increase and formation of dormant eggs.

MicroRNA-9 overexpression suppresses vulnerable atherosclerotic plaque and enhances vascular remodeling through negative regulation of the p38MAPK pathway via OLR1 in acute coronary syndrome.

Acute coronary syndrome (ACS) is characterized by atherosclerotic plaque rupture with a high incidence of recurrent ischemic events. Several microRNAs are found to be aberrantly expressed in atherosclerotic plaques. This study aims to investigate the effects of microRNA-9 (miR-9) on vulnerable atherosclerotic plaque and vascular remodeling in ACS and underlying mechanisms. Microarray-based gene expression profiling was used to identify differentially expressed genes related to ACS and regulatory miRNAs. Oxidized low-density lipoprotein (lectin-like) receptor 1 (OLR1) was identified to be aberrantly activated in ACS and regulated by miR-9. OLR1 was verified as a target gene of miR-9 by bioinformatics prediction and dual luciferase reporter gene assay. The atherosclerotic models were induced in ApoE-/- mice, in which the agomir or antagomir of miR-9, or small interfering RNA (siRNA) against OLR1 were separately introduced. Serum lipid levels and expression of vascular remodeling and inflammatory response-related factors were determined, respectively. On the basis of the obtained results, in the atherosclerosis mice treated with the agomir of miR-9 and siRNA against OLR1, the p38-mitogen-activated protein kinase (p38MAPK) pathway was inhibited; levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol, tumor necrosis factor-α, interleukin-6, and vascular endothelial growth factor were reduced, but the high-density lipoprotein cholesterol level was increased, along with decreased vulnerable atherosclerotic plaque area and enhanced vascular remodeling. Taken together, these findings suggested an inhibitory role miR-9 acts in the formation of vulnerable atherosclerotic plaques in ACS mice, along with a promoted vascular remodeling, via a negative feedback regulation of OLR1-mediated p38MAPK pathway.

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells.

OBJECTIVES: Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood. MATERIALS AND METHODS: Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-ß-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models. RESULTS: We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo. CONCLUSIONS: Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.

Multilocus sequence analyses reveal extensive diversity and multiple origins of fluconazole resistance in Candida tropicalis from tropical China.

Candida tropicalis is among the most prevalent human pathogenic yeast species, second only to C. albicans in certain geographic regions such as East Asia and Brazil. However, compared to C. albicans, relatively little is known about the patterns of genetic variation in C. tropicalis. This study analyzed the genetic diversity and relationships among isolates of C. tropicalis from the southern Chinese island of Hainan. A total of 116 isolates were obtained from seven geographic regions located across the Island. For each isolate, a total of 2677 bp from six gene loci were sequenced and 79 (2.96%) polymorphic nucleotide sites were found in our sample. Comparisons with strains reported from other parts of the world identified significant novel diversities in Hainan, including an average of six novel sequences (with a range 1 to 14) per locus and 80 novel diploid sequence types. Most of the genetic variation was found within individual strains and there was abundant evidence for gene flow among the seven geographic locations within Hainan. Interestingly, our analyses identified no significant correlation between the diploid sequence types at the six loci and fluconazole susceptibility, consistent with multiple origins of fluconazole resistance in the Hainan population of C. tropicalis.

Neuroprotective activity of two active chemical constituents from Tinospora hainanensis.

OBJECTIVE: To determine the chemical structure of the new compound and investigate the protective effects of Tinosporaic acid A and B towards in-vitro neuro. METHODS: The structures of two new compounds were established by analyzing its 1D and 2D NMR spectra as well as HRESIMS. Their neuroprotective effects with respect to the antioxidant properties were evaluated by radical scavenging tests and hydrogen peroxide-injured oxidative stress model in PC12 cell lines. Cell morphology of treated PC12 cells was observed by phase contrast microscopy. In-vitro MTT assay, lactate dehydrogenase activity assay and oxidative stress markers (intracellular ROS production, MDA level, and caspase-3 activity) were used to evaluate the protective effects against hydrogen peroxide induced cytotoxicity in PC12 cells. RESULTS: The two new compounds, named Tinosporaic acid A and B, were isolated and identified from the stem bark of Tinospora hainanensis. Cell viability studies identified a representative concentration for each extract that was subsequently used to measure oxidative stress markers. Both extracts were able to reverse the oxidative damage caused by hydrogen peroxide, thus promoting PC12 cells survival. The concentration of Tinosporaic acid A and B were 86.34 µg/mL and 22.06 µg/mL respectively, which is neuroprotective for EC50. The results indicated that both of them significantly attenuated hydrogen peroxide-induced neurotoxicity. CONCLUSION: The two new compounds isolated from ethanol extracts of Tinospora hainanensis are the promising natural ones with neuroprotective activity and needed for further research.

[Immunological analysis of piperaquine-resistant murine model of Plasmodium berghei ANKA strain].

OBJECTIVE: To analyze the immunological characteristics of murine model of piperaquine sensitive (PQS) line and resistant (PQR) line of Plasmodium berghei (Pb) ANKA strain. METHODS: 64 Kunming mice were divided into three groups, 16 in each of groups A and C, 32 in group B (16 of 32 were used for observing survival days). Each mouse in groups A and B was infected with 1 x 10(7) erythrocytic stage parasites of PbPQS and PbPQR, respectively. Mice in group C were injected with the same volume of normal saline. On days 4, 8, 12 and 16 after inoculation, 4 mice from each group were sacrificed. Blood samples were collected for thin blood smear examination, and parasitemia rate calculated. Spleens were removed and spleen lymphocytes suspension prepared. Spleen lymphocytes were stimulated with ConA, and cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Nitrogen oxide (NO) and IFN-gamma level of spleen cell culture supernatants were detected by the Griess reagent and ELISA methods, respectively. Another 10 mice were each inoculated with 1 x 10(7) parasites of PbPQR line, and the mice were then challenged with lethal PQS line when the parasites turned into blue stained cells. The parasitemia and survival days were recorded. RESULTS: The average survival time of group A was (9.0 +/- 3.0) d, the parasitemia rate was over 50% at 6-12 days post- infection with severe anemia. On 16th day post-infection, no death was recorded in group B with a parasitemia rate of (2666 +/- 254) %. After ConA stimulation, the proliferation of spleen lymphocytes in groups A (0.65 +/- 0.08) and B (0.86 +/- 0.20) at 12 days after infection was significantly higher than that of group C (0.18 +/- 0.03) (P < 00.01). NO level in spleen cell culture supernatant increased with prolonged infection time. On 12th day post-infection, NO level of groups A [(48.80 +/- 3.49) micromol/L] and B [(54.80 +/- 2.17) micromol/L] was higher than that of group C [(7.80 +/- 0.71) micromol/L] (P < 0.01). IFN-gamma concentration in spleen lymphocytes culture supernatant increased with prolonged infection time. The highest IFN-gamma level of group A was (752.20 +/- 39.49) pg/ml on 12th day post-infection, while in group B it was (855.80 +/- 33.65) pg/ml on 8th day after infection, then decreased on 12th day [(620.20 +/- 27.11) pg/ml]. IFN-gamma level showed a significant difference between groups A and B (P < 0.01). In 10 days after challenge, the parasitemia rate in PQR group was up to (2.44 +/- 2.07)%, and gradually disappeared. No parasite was detected on 40th day after challenge and no mice died. CONCLUSION: The proliferation of spleen cells, NO and IFN-gamma levels of spleen lymphocytes culture supernatant in PbANKA strain PQR line are significantly higher than that of PQS line. PbPQR line can induce certain protective immunoreaction.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).