Silencing of long non­coding RNA NEAT1 inhibits hepatocellular carcinoma progression by downregulating SMO by sponging microRNA­503.

Hepatocellular carcinoma (HCC) poses an increasing threat to humans, due to its poor prognosis. Nuclear­enriched abundant transcript 1 (NEAT1), a type of long non­coding (lnc)RNA, has been found to function in a variety of cancer types. However, the role of NEAT1 in HCC is poorly understood. Reverse transcription­quantitative PCR was used to detect the expression levels of NEAT1, microRNA (miR)­503 and Smoothened (SMO) mRNA in HCC tissues and cells. MTT and flow cytometry assays were used to investigate cell viability and apoptosis, respectively, while Transwell assays were performed to investigate cell invasion and migration. StarBase and TargetScan were utilized to predict the target sequence between miR­503 and NEAT1 or SMO, the results of which were verified using a dual­luciferase reporter assay. The protein expression level of SMO was measured using western blot. The RNA expression level of NEAT1 and SMO was significantly elevated in HCC tissues and cells compared with that in the corresponding healthy tissues and cells, which was contrary to miR­503 expression level. NEAT1 silencing was found to restrict the viability, migration and invasion of the cells, while simultaneously induced apoptosis in the HCC cell line. Further studies found that miR­503 expression was negatively correlated with NEAT1 or SMO. It was also confirmed that NEAT1 directly interacted with miR­503 and miR­503 could bind to the 3'­untranslated region of SMO. Furthermore, overexpression of NEAT1 or SMO could reverse the effects of miR­503­mediated inhibition on cell viability, invasion, migration and promotion of apoptosis in the HCC cell lines. These results demonstrated that downregulation of NEAT1 impeded the viability, migration, invasion and induced apoptosis through the NEAT1/miR­503/SMO axis in the HCC cell line.

Phosphorus supply via a fed-batch strategy improves lipid heterotrophic production of Chlorella regularis.

Intracellular phosphorus (P) accumulation can improve microalgal growth and lipid synthesis. However, large excess of P causes cell poisoning. This study utilized a P-fed-batch strategy to investigate its potential to improve the utilization of the excessive P, while avoiding toxic side effects. This strategy contributed to a more complete utilization of the intracellularly stored P, which enhanced the microalgae biomass by 10-15% by upregulating the brassinosteroid growth hormone gene at a P-fed-batch frequency of 2-8. Furthermore, the lipid content increased by 4-16% via upregulation of lipid synthesis-related genes. As a result, the P-fed-batch strategy significantly increased the lipid production by 13-19%. The content of saturated fatty acid increased by ~ 100%, implying improved combustibility and oxidative stability. This is the first study of this P-fed-batch strategy and provides a new concept for the complete utilization of excessive P.

lncRNA NEAT1 mediates sepsis progression by regulating Irak2 via sponging miR-370-3p.

Sepsis is a life-threatening condition and often associated with multiple organ failure. Nuclear-enriched abundant transcript 1 (NEAT1), a member of the long non-coding RNAs (lncRNAs), was reported to be involved in the regulation of sepsis progression. However, its precise regulatory mechanism needs to be further explored. In this study, the cell-counting kit-8 assay was used to check cell viability. The quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expression levels of NEAT1, miR-370-3p and Interleukin 1 receptor associated kinase 2 (Irak2). Flow cytometry assay and ELISA were used to check cell apoptosis and the concentrations of inflammatory cytokines, respectively. The starBase was used to predict binding sites between miR-370-3p and NEAT1 or Irak2 and the dual-luciferase reporter assay was performed to verify the interaction. The protein level of Irak2 in samples was measured by western blot. The high concentration of lipopolysaccharide (LPS) led to the high death ratio of RAW 264.7 and HL-1 cells. NEAT1 and Irak2 were upregulated in sepsis tissues and LPS-induced RAW 264.7 and HL-1 cells, opposite to the expression of miR-370-3p. In addition, knockdown of NEAT1 promoted viability, suppressed apoptosis and reduced the expression of inflammatory cytokines in LPS-induced RAW 264.7 and HL-1 cells. Moreover, we found that miR-370-3p interacted with NEAT1 and targeted the 3'UTR of Irak2. Further research indicated that downregulation of miR-370-3p or upregulation of Irak2 rescued NEAT1 silencing-mediated inhibitory effect on sepsis progression. Knockdown of NEAT1 hampered sepsis progression by downregulating Irak2 via interacting with miR-370-3p in LPS-induced RAW 264.7 and HL-1 cells.

Synergistic effect and mechanisms of compound bioflocculant and AlCl3 salts on enhancing Chlorella regularis harvesting.

The high energy input required for harvesting microalgae means that commercial production of microalgal biodiesel is economically unfeasible. In this study, we investigated the flocculation efficiency and synergistic mechanisms of novel coupled flocculants, AlCl3 and compound bioflocculants (CBF), to overcome this difficulty. AlCl3 flocculation was found to be very sensitive to pH, and flocculation efficiency increased from 55 to 95 % when pH increased from 4 to 10. CBF was environmental friendly, less reliant on pH, but had a relatively low flocculation of 75 % in optimum conditions. The harvesting efficiency of Chlorella regularis can achieve a satisfactory level of 96.77 % even in neutral conditions, with a CBF dosage of 0.26 g/L, AlCl3 dosage of 0.18 g/L, and coagulant aid (CaCl2) dosage of 0.12 g/L. Interestingly, compared with the use of single flocculant, the dosage of CBF, AlCl3, and coagulant aid (CaCl2) were reduced by about 52, 49, and 66 %, respectively. Besides, the aluminum (Al) ion content of the supernatant decreased significantly to a residue of only 0.03 mg/L, therefore meeting the downstream process needs easily. Patching and bridging played key roles in coupled flocculant flocculation, in which AlCl3 mainly carried out the electrical neutralization. This work provides new insight into an efficient, economical, and environmentally friendly protocol for microalgae harvesting.

Nickel biosorption by discharged biomass from wastewater treatment bioreactor: size plays a key role.

Biomass size significantly affects the characteristics of the extracellular polymeric substances, which in turn influences the biosorption mechanisms. In this work, nickel biosorption mechanisms and the capacity of excess aerobic granules (AGs) of >850 µm, 500-850 µm, 212-500 µm, and bioflocs <212 µm, discharged during wastewater treatment operation, were investigated by elemental composition and spectroscopic analysis. Ni(2+) biosorption capacity decreased apparently from 1.28-1.53 to 0.93 mEq/g with the increase of biomass size from 0-850 to >850 µm due to their different biosorption mechanisms. Chemical binding between nickel and protein was dominant for biomass less than 850 µm, whereas nickel adsorption was mainly physical when biogranule size was greater than 850 µm, because the nickel had no opportunity to interact with protein located in the core of the biogranule. Only low levels of ion exchange (<15.22 %) and precipitation (negligible) were observed for all kinds of biomass. Thus, biomass greater and less than 850 µm presented quite different biosorption mechanisms.

Granulation, control of bacterial contamination, and enhanced lipid accumulation by driving nutrient starvation in coupled wastewater treatment and Chlorella regularis cultivation.

Bacterial contamination and biomass harvesting are still challenges associated with coupling of microalgae and wastewater treatment technology. This study investigated aggregation, bacterial growth, lipid production, and pollutant removal during bacteria contaminated Chlorella regularis cultivation under nutrient starvation stress, by supposing the C/N/P ratios of the medium to 14/1.4/1 (MB2.5) and 44/1.4/1 (MB4.0), respectively. Granules of 500-650 µm were formed in the bacteria contaminated inoculum; however, purified C. regularis were generally suspended freely in the medium, indicating that bacterial presence was a prerequisite for granulation. Extracellular polymeric substance (EPS) analysis showed that polysaccharides were dominant in granules, while protein mainly distributed in the outer layer. Denaturing gradient gel electrophoresis (DGGE) results revealed Sphingobacteriales bacterium and Sphingobacterium sp. are vital organisms involved in the flocculation of microalgae, and nitrifiers (Stenotrophomonas maltophilia) could co-exist in the granular. Both EPS and DGGE results further supported that bacteria played key roles in granulation. C. regularis was always dominant and determined the total biomass concentration during co-cultivation, but bacterial growth was limited owing to nutrient deficiency. Starvation strategy also contributed to enhancement of lipid accumulation, as lipid content in MB4.0 with a greater C/N/P led to the greatest increase in the starvation period, and the maximum lipid productivity reached 0.057 g/(L·day). Chemical oxygen demand and nitrogen removal in MB4.0 reached 92 and 96%, respectively, after 3 days of cultivation. Thus, cultivation of microalgae in high C/N/P wastewater enabled simultaneous realization of biomass granulation, bacterial overgrowth limitation, enhanced lipid accumulation, and wastewater purification.