The role of HDAC6 in enhancing macrophage autophagy via the autophagolysosomal pathway to alleviate legionella pneumophila-induced pneumonia.

Legionella pneumophila (L. pneumophila) is a prevalent pathogenic bacterium responsible for significant global health concerns. Nonetheless, the precise pathogenic mechanisms of L. pneumophila have still remained elusive. Autophagy, a direct cellular response to L. pneumophila infection and other pathogens, involves the recognition and degradation of these invaders in lysosomes. Histone deacetylase 6 (HDAC6), a distinctive member of the histone deacetylase family, plays a multifaceted role in autophagy regulation. This study aimed to investigate the role of HDAC6 in macrophage autophagy via the autophagolysosomal pathway, leading to alleviate L. pneumophila-induced pneumonia. The results revealed a substantial upregulation of HDAC6 expression level in murine lung tissues infected by L. pneumophila. Notably, mice lacking HDAC6 exhibited a protective response against L. pneumophila-induced pulmonary tissue inflammation, which was characterized by the reduced bacterial load and diminished release of pro-inflammatory cytokines. Transcriptomic analysis has shed light on the regulatory role of HDAC6 in L. pneumophila infection in mice, particularly through the autophagy pathway of macrophages. Validation using L. pneumophila-induced macrophages from mice with HDAC6 gene knockout demonstrated a decrease in cellular bacterial load, activation of the autophagolysosomal pathway, and enhancement of cellular autophagic flux. In summary, the findings indicated that HDAC6 knockout could lead to the upregulation of p-ULK1 expression level, promoting the autophagy-lysosomal pathway, increasing autophagic flux, and ultimately strengthening the bactericidal capacity of macrophages. This contributes to the alleviation of L. pneumophila-induced pneumonia.

The enrichment characterisation of Nitrospira under high DO conditions.

Nitrite-oxidizing bacteria (NOB) are crucial to nitrification and nitrogen elimination in wastewater treatment. Mass reports exist on the links between NOB and other microorganisms, for instance, ammonia-oxidizing bacteria (AOB). However, a few studies exist on the enrichment characterisation of NOB under high dissolved oxygen (DO) conditions. In this study, NOB was designed to be enriched individually under high DO conditions in a continuous aeration sequencing batch reactor (SBR), and the kinetic characterisation of NOB was evaluated. The analysis revealed that the average NO2--N removal rate was steady above 98%, with DO and NO2--N being 3-5 mg L-1 and 50-450 mg L-1, respectively. The NO2--N removal efficiency of the system was significantly enhanced and better than in other studies. The high-throughput sequencing suggested that Parcubacteria_ genera_incertae_sedis was the first dominant genus (21.99%), which often appeared in the NOB biological community with Nitrospira. However, the dominant genus NOB was Nitrospira rather than Nitrobacter (8.49%). This result suggested that Nitrospira was capable of higher NO2--N removal. But lower relative abundance indicated that excessive NO2--N had an adverse effect on the enrichment and activity of Nitrospira. In addition, the nitrite half-saturation constant (KNO2) and the oxygen half-saturation constant (KO) were 1.71 ± 0.19 mg L-1 and 0.95 ± 0.10 mg L-1, respectively. These results showed that the enriched Nitrospira bacteria had different characteristics at the strain level, which can be used as a theoretical basis for wastewater treatment plant design and optimisation.

Characterization of melanoidins in thermal hydrolysis sludge and effects on dewatering performance.

Thermal hydrolysis pretreatment (THP) of sludge can form the refractory brown melanoidins due to the occurrence of the Maillard reaction, which adversely involves the subsequent sludge anaerobic digestion (AD) process. However, details of the generation pattern of melanoidins and how they affect the sludge dewaterability remain largely unknown. This work aims to determine an approach to characterize and quantify the melanoidins created by THP of sludge. On this basis, the effect of melanoidins on sludge dewatering performance was revealed by adding synthetic melanoidins to the mixed sludge. Experimental results showed that three-dimensional fluorescence-region integration (3DEEM-FRI) could effectively distinguish melanoidins from other organic substances and achieve semi-quantitative characterization in sludge. The melanoidins significantly deteriorated the sludge dewaterability, and the lowest solids content of the filter cake (TS) was only 17.78% at the addition of 480 mg (g TS)-1, which was a drop of about 20% compared to the control group. The mechanism investigations indicated that the internal structure of sludge becoming particularly complicated and the opportunities for molecules to collide with each other enlarged because of the contribution of melanoidins, resulting in the increment of the sludge apparent viscosity and consistency coefficient (k), a decline of the flow behavior index (n) and a weakening of flowability. Melanoidins could capture massive water molecules and carry negative charges with the decrease of sludge particle size and zeta potential value, which enhanced the electrostatic repulsion between sludge particles and abated the flocculation ability, thus further aggravating the sludge dewatering performance.

Using a combination of different conditioners to promote dewatering of digested sludge: Rheological characteristics.

The dewatering capacity of sludge is a key factor in sludge disposal and reuse. In this study, the effects of conditioning with three conditioners (polyacrylamide (PAM), poly aluminum sulfate (PAS), and sludge biochar (SAC)) and their combined conditioning effect at different doses on the dewatering performance of digested sludge were systematically investigated. The mechanism of change in dewatering performance was analyzed based on rheological principles. A Box‒Behnken multifactor experiment based on the response surface method (RSM) was also used to establish a quadratic multiple prediction model for the solids content of filter cake to obtain the optimal ratio of coupled treatment. The results showed for individual conditioner use, PAM with a dose of 3‰ had the best effect on sludge dewatering, and the dewatering effect of the combined conditioner sludge treatment was better than that of the sludge treated with individual conditioners, with the solids content of the filter cake exceeding 35%. The Herschel-Bulkley model was used to fit the rheological data, and the results showed that the yield stress decreased with increasing PAM dose and gradually increased with increasing PAS and SAC doses. The thixotropy of sludge after SAC conditioning was evident compared to that after PAM and PAS conditioning. The yield stress of sludge decreased and flowability deteriorated after combined conditioning. There was a linear relationship between the dewatering performance of conditioned sludge and thixotropy and yield stress, which indicated the feasibility of using rheological indices to evaluate changes in sludge dewatering performance.

Rheological properties and dewaterability of anaerobic co-digestion with sewage sludge and food waste: effect of thermal hydrolysis pretreatment and mixing ratios.

Anaerobic co-digestion (co-AD) of sewage sludge (SS) and food waste (FW) converts municipal organic waste into renewable energy, which plays an important role in achieving carbon emissions reduction. The existing anaerobic digestion (AD) treatment projects often have problems such as low organic conversion and unstable performance. SS and FW were used as raw materials to explore the effects of thermal hydrolysis pretreatment (THP) and mixing ratios on the dewaterability and rheological properties of the digestate. The results showed that co-digestion of FW and SS in a ratio of 1:1 obtained the highest biogas production (255.14 mL/g VS), which was 1.53 times and 14.5 times higher than that of mono-digestion of FW and thermal hydrolysis pretreatment sewage sludge (THSS), respectively. However, the dewaterability of this ratio deteriorated sharply after co-digestion, with a decrease of 54.92%. The groups containing a higher proportion of THSS had improved dewaterability after AD. The apparent viscosity and shear stress were reduced by co-digestion compared with mono-digestion of THSS and FW, indicating a higher flow property of the co-digestion matrix. After the Herschel-Bulkley model fitting, there were linear correlations between rheological indices and soluble chemical oxygen demand (SCOD), and digestate dewaterability.

Anaerobic co-digestion of sewage sludge pretreated by thermal hydrolysis and food waste: gas production, dewatering performance, and community structure.

Anaerobic co-digestion can effectively break the limitations of mono-digestion. However, there are still some problems such as long residence time, unsatisfactory methane yield, and unstable performance for co-digestion of sewage sludge (SS) and food waste (FW). Therefore, the SS in the reactor treating co-digestion of SS and FW is considered to be pretreated by thermal hydrolysis. In this work, the anaerobic co-digestion of SS of thermal hydrolysis pretreatment (THP) and FW significantly improved the stability, methane production of the digestive reactor, and dewaterability of the digested sludge. The R6 obtained the most cumulative methane production (315.76 mL/g VS). In addition, compared to R3, the cumulative methane production and maximum methane production rate of R5 increased by 9.93% and 14.56%, respectively. The dewaterability of R4, R5, and R6 was improved, while the dewatering performance of the R3 decreased to a greater extent. The results of the kinetic model fitting were consistent with the experimental results. Among them, the hydrolysis constants (Kh) of anaerobic co-digestion of THP-SS and FW were 0.121, 0.130, and 0.114 d-1, respectively, which were higher than those of other groups. And the estimated lag time (λ) of co-digestion was also lower than that of mono-digestion groups. Microbial community analysis indicated that the bacterial diversity and richness of anaerobic co-digested groups of THP-SS and FW were enhanced, while the methanogens with acetoclastic pathway became the main methanogenic microorganisms. This work provides essential information on anaerobic co-digestion containing different THP-SS contents.

Effects of Iron Powder Addition and Thermal Hydrolysis on Methane Production and the Archaeal Community during the Anaerobic Digestion of Sludge.

The conventional anaerobic digestion of sludge has the disadvantages of long digestion time and low methane production. Pretreatment is often used to mitigate these problems. In this study, three pretreatment methods, namely, the addition of iron powder, high-temperature thermal hydrolysis, and a combination of these methods, were compared for application with conventional continuous anaerobic digestion reactors. The results showed that pretreatment improved methane yield by 18.2-22.9%, compared to the control reactor (conventional anaerobic digestion). Moreover, it was recognized that the archaeal community in the sludge underwent significant changes after pretreatment. Specifically, the addition of iron powder reduced the diversity in the archaeal community, but increased the abundance of hydrogenotrophic methanogens without changing the community composition. Thermal hydrolysis at high temperatures had the reverse effect, as it increased the diversity of the archaeal community but inhibited the growth of acetoclastic methanogens. In the case of the combined pretreatment, the thermal hydrolysis had a dominant influence on the archaeal community. By comparing the changes in functional gene content, it was found that the functional abundance of the archaeal community in the transport and metabolism of carbohydrates, lipids, and amino acids was higher after pretreatment than in the control group.

[Mechanism of histone deacetylase inhibitor tubastatin A promoting autophagy of Legionella pneumophila infected RAW264.7 cells].

Objective To investigate the role of HDAC6 in the interference of Legionella pneumophila on the autophagy of macrophages and its mechanism. Methods RAW264.7 macrophages were treated with 10 µmol/L, 5 µmol/L, and 2.5 µmol/L tubastatin A (TubA). CCK-8 assay was used to detect the proliferative activity of RAW264.7 macrophages, and the half maximal inhibitory concentration (IC50) of TubA was determined. A model of RAW264.7 macrophages infected with Legionella pneumophila was established and divided into TubA free groups (further divided into cell control group, inactivated bacteria group, and live bacteria group) and TubA treatment groups (10 µmol/L, 5 µmol/L, 2.5 µmol/L, each further divided into cell control group, inactivated bacteria group, and live bacteria group). The cells were collected at 6, 12, 24, and 48 h after Legionella pneumophila infection. The bacterial proliferation assay was conducted to detect the proliferation of Legionella pneumophila in RAW264.7 macrophages; RAW264.7 macrophages were transfected with pmCherry-C1-EGFP-LC3B plasmid to detect autophagic flux changes in each group; real-time quantitative PCR and Western blot were used respectively to detect the mRNA and protein expression levels of histone deacetylase 6 (HDAC6), sequestosome 1(SQSTM1/P62), microtubule associated protein 1 light chain 3 (LC3), α-tubulin, valosin containing protein (p97/VCP), heat shock protein 90 (HSP90), HSP70, heat shock transcription factor 1 (HSF1), and filamentous actin (F-actin). Results IC50 of TubA was 50 µmol/L. Compared with those in the RAW264.7 normal control group, the proliferation of Legionella pneumophila in mouse macrophages was significantly reduced after the addition of TubA. In the groups without the HDAC6 inhibitor, the live bacteria group had a stronger inhibiting effect on autophagic flux than the inactivated bacteria group compared with the normal control group. In the TubA groups with the HDAC6 inhibitor, the green fluorescence bright spots decreased and the autophagic flux increased in the live bacteria group. After the RAW264.7 macrophages were treated with inactivated and live Legionella pneumophila for 6, 12, 24, and 48 h, the mRNA and protein expression levels of HDAC6, α-tubulin, p97-VCP, and P62 decreased in the TubA group interfered with the Legionella pneumophila compared with the RAW264.7 normal control group. Conclusion The interference of Legionella pneumophila on the autophagy of macrophages is associated with the signal pathways of HDAC6/P62/LC3B and HDAC6/p97/HSF1.

Effect of high-temperature thermal hydrolysis on rheological properties and dewaterability of sludge.

The disposal processes like dewatering and anaerobic digestion (AD) are commonly utilized to reduce the volume of sludge and recover energy. Thermal hydrolysis process is widely used as a pretreatment for sludge AD, which can change rheological properties and dehydration of sludge irreversibly. The experiment was designed to evaluate the effect of high-temperature thermal hydrolysis (120, 130, 145, 160 and 170°C) for 60 min on sludge rheological properties, as well as its dewaterability. Rheological tests were performed at (20 ± 0.1)°C with a HAAKE Viscotester 550 Rotary Viscometer. Both raw and thermal hydrolysis sludge has a considerable reduction on apparent viscosity and yield stress, but a somewhat increase in thixotropy. With the increase of temperature, the sludge flow behaviour index n increases linearly, while the consistency coefficient k follows the law of linear decline, showing that thermal hydrolysis can weaken the non-Newtonian fluid properties and then improve sludge fluidity. The dewaterability increases linearly with the temperature. Besides, the dewaterability of high-temperature thermal hydrolysis processes sludge was always significantly better than raw sludge as its mud cake has a much larger solid content. Notably, the increase in flow performance index n and the decrease of thixotropy kinetic coefficient K caused by thermal hydrolysis are all linear with the enhancement of dewaterability, which demonstrates that rheological indicators can be a new tool to evaluate the dewaterability of sludge.

Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus.

The present study aimed to examine the effects of FcγRIIB on systemic lupus erythematosus (SLE) and to investigate the underlying mechanisms. For this purpose, lentiviral vector carrying the membrane­bound type FcγRIIB gene (mFcγRIIB lentivirus) and soluble FcγRIIB (sFcγRIIB) protein were used to treat B cells from patients with SLE. The B cells were treated with calf thymus DNA (ctDNA) and anti­calf thymus DNA­immune complexes (anti­ctDNA­IC). mFcγRIIB lentivirus and sFcγRIIB protein were also injected into MRL/lpr SLE mice. The results revealed that anti­ctDNA­IC treatment significantly downregulated the IgG antibody secretion of B cells treated with mFcγRIIB lentivirus. mFcγRIIB and sFcγRIIB decreased the phosphorylation level of Bruton's tyrosine kinase (BTK) in B cells, and increased the phosphorylation level of Lyn proto­oncogene (Lyn), docking protein 1 (DOK1) and inositol polyphosphate­5­phosphatase D (SHIP). mFcγRIIB promoted the apoptosis of B cells. Following the treatment of MRL/lpr SLE mice with mFcγRIIB lentivirus, the levels of urinary protein, serum anti­nuclear and anti­dsDNA antibodies were decreased, while the levels of mFcγRIIB in B cells were increased. mFcγRIIB ameliorated the pathologies of the kidneys, liver and lymph node tissues of the MRL/lpr SLE mice. Following treatment of the MRL/lpr SLE mice with sFcγRIIB, the levels of urinary protein, serum anti­dsDNA antibody and BTK and SHIP phosphorylation levels in B cells were decreased, while the serum sFcγRIIB and sFcγRIIB­IgG levels were increased. On the whole, the findings of the present study demonstrate that recombinant FcγRIIB inhibits the secretion of IgG antibody by B cells from patients with SLE, ameliorates the symptoms of SLE in mice, and alters the phosphorylation levels of downstream proteins of the FcγRIIB signaling pathway in B cells. These results suggest that FcγRIIB may play preventive and therapeutic roles in SLE by inhibiting B cell activation via the FcγRIIB signaling pathway, which provides a novel theory and strategy for the prevention and treatment of SLE.

[Legionella pneumophila activates Nrf2-Keap1 pathway to inhibit autophagy of mouse RAW264.7 macrophages].

Objective To investigate the effect of Legionella pneumophila (LP) on the autophagy flux of RAW264.7 macrophages and explore the molecular mechanism of the expression changes of autophagy-related factors. Methods Live LP and inactivated LP (MOI=10, 50, 100) were separately used to affect RAW264.7 for 1, 2 and 3 hours so as to screen the optimum condition of LP infection. The optimal condition for LP infection was MOI=50 and the infection time was 2 hours. After affected by rapamycin (RAPA) for 12 hours, RAW264.7 cells were then treated by live and inactivated LP for another 2 hours. Normal control group, RAPA group, live LP group, inactivated LP group, RAPA-treated live LP group, RAPA-treated inactivated LP group were designed. The pmCherry-C1-EGFP-LC3B double fluorescent labeling protein method was used to monitor the changes of autophagy flux. The relevant factor CLN3, histone deacetylase 6 (HDAC6), regulator of G protein signaling 19 (RGS19), tumor necrosis factor (TNF), cathepsin B (CTSB), GABA type A receptor associated protein like 2 (GABARAPL2), P62, microtubule-related protein 1 light chain 3 (LC3) were screened by gene array analysis. In order to validate the results of gene array, real-time quantitative PCR (RT-qPCR) was used to detect the mRNA levels of nuclear factor erythroid derived 2 like 2 (Nrf2), beclin1 and kelch like ECH associated protein 1 (Keap1); Western blot analysis was performed to measure the protein levels of Nrf2, beclin1 and Keap1. Results Both the live LP group and the inactivated LP group inhibited the autophagy flux compared with the normal control group and the RAPA group. Gene array analysis showed that in the live LP and inactivated LP groups, LC3 expression was down-regulated and P62 expression was up-regulated. The results of RT-qPCR and Western blot analysis were consistent with the gene array. The mRNA and protein levels of Keap1, beclin1, and Nrf2 significantly decreased, while the mRNA and protein levels of Nrf2 significantly increased. Conclusion LP can inhibit the autophagy of macrophage via activating Nrf2-Keap1 signaling pathway.

[Operation Characteristics of the Biofilm CANON Reactor During the Temperature Reduction Process].

The focus of this paper, was low temperature, high ammonia nitrogen wastewater. The operation characteristics of the biofilm CANON process during the temperature reduction process were determined, by continuously adjusting different operating conditions. The aim was to explore the methods needed for the CANON process to obtain stable shortcut nitrification and a good nitrogen removal effect, when the influent NH4+-N concentration is high and the temperature low. The results showed that, ① compared with the biofilm CANON reactor temperature changing from medium to low temperature directly (30℃±1℃→19℃), it was more conducive to adapt the nitrogen-removing bacteria to the low-temperature environment, while the temperature was gradually lowered. Moreover, the extent of each reduction should be minimized. Besides, the operating conditions should be adjusted to ensure the nitrogen removal effect. ② The temperature was gradually reduced to about 19℃ after 25 d, and then decreased to about 15℃ after another 18 d. The NH4+-N and TN removal rates could be respectively stable at 90% and 70% over a long period of time. The TN removal rate and removal load could still reach 72.52% and 0.78 kg·(m3·d)-1, respectively, even when the temperature dropped to 12℃. ③ When adapting biological CANON sludge during the temperature reduction process, shortcut nitrification should be given priority. A stable shortcut nitrification effect should be obtained by maintaining a certain concentration of residual NH4+-N, and by strictly controlling the DO concentration to restrain NOB activity.

A rapid and sensitive UHPLC-MS/MS method for the determination of celosin A in rat plasma with application to pharmacokinetic study.

Celosin A (CA), a natural compound isolated from Celosia argentea L., has been shown significant hepatoprotective effect on AHNP-induced liver injury. This study described a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for determination of CA in rat plasma. Methanol-mediated precipitation was used for sample pretreatment. Chromatographic separation was achieved on a T3 column with gradient elution using water and acetonitrile as mobile phase. Determination was obtained using an electrospray ionization source in negative selected reaction monitoring mode at the transitions of m/z 793.3 → m/z 661.2 and m/z 955.6 → m/z 793.2 for CA and IS, respectively. The assay was linear over the concentration range 0.25-2500 ng/mL (r > 0.995) with a lowest limit of quantification (LLOQ) of 0.25 ng/mL. The intra- and inter-day precisions (RSD) were 1.65-9.84 and 2.46-13.49%, respectively, while accuracy (RR) ranged from 96.21 to 99.45%, respectively. The recovery ranged from 95.09 to 102.22% and the matrix effect from 98.29 to 100.13%. The analyte was stable under the tested storage conditions. The method has been successfully applied to a preclinical pharmacokinetic study in rats after a single intravenous (2 mg/kg) or oral (50 mg/kg) administration. The oral bioavailability of CA was ~1.94%; in addition, there was no difference between male and female rats. This is the first time of the use of an UHPLC-MS/MS method for determination of CA concentration in rat plasma and for evaluation of its pharmacokinetic behavior.

[MOMP of Legionella inhibits phagocytosis of RAW264.7 macrophages and enhances their chemotaxis by activating NOD2/RIP2 signaling pathway].

Objective To investigate the effect of the main outer membrane protein (MOMP) of Legionella on the phagocytosis and chemotaxis of RAW264.7 macrophages and explore its mechanism. Methods MOMP and RAW264.7 macrophages were cultured in vitro. The toxicity of MOMP to RAW264.7 macrophages was detected by CCK-8 assay and 50% inhibitory concentration (IC50) was determined. The RAW264.7 macrophages were treated by MOMP (1.14, 0.57, 0.28) µg/mL and the control group was established. The cells and cultivate supernatants were collected 24, 48 and 72 hours after the RAW264.7 macrophages were treated by MOMP. The phagocytic function of macrophages was detected by the neutral red phagocytosis experiment; the chemotaxis function of macrophage was examined by TranswellTM assay, and the levels of monocyte chemoattractant protein 1 (MCP-1) and interleukin 10 (IL-10) in cell culture supernatant monocytes were detected by ELISA. Real-time quantitative PCR was used to check the mRNA level of macrophage nucleotide-binding oligomerization domain 1 (NOD1), NOD2 and receptor-interacting protein 2 (RIP2). The protein levels of NOD1, NOD2 and RIP2 were detected by Western blot analysis. Results The IC50 of MOMP on RAW264.7 macrophages was 5.69 µg/mL. Compared with the control cells, MOMP treatment caused a decrease of RAW264.7 macrophage phagocytosis in a dose- and time-dependent manner. With the increase of MOMP dosage, the chemotaxis of macrophages and the secretory levels of MCP-1 and IL-10 in the cell culture supernatant increased, and peaked in 36 hours. The mRNA and protein expression levels of NOD2, RIP2 also increased, mRNA levels of NOD2 and RIP2 peaked in 12 hours, and protein levels peaked in 24 hours. Conclusion MOMP can inhibit the phagocytosis of RAW264.7 macrophages and enhance its chemotaxis function, which is related to the activation of NOD2/RIP2 signaling pathway.

[Impact of C/N Ratio on Nitrogen Removal Performance and N2O Release of Granular Sludge CANON Reactor].

To Explore a suitable C/N ratio for efficient nitrogen removal and simultaneously achieving N2O release reduction, ammonia-rich wastewater with sodium acetate as an organic carbon source in a granular sludge completely autotrophic nitrogen removal over nitrite (CANON) reactor under different C/N water conditions were studied to determine the reactor's nitrogen removal performance and N2O release. The results showed that the total nitrogen (TN) removal rate and the removal load tended to increase gradually with the increase of C/N, ranging from 0 to 2.0. When C/N=0, the TN removal rate was 56.50 mg·L-1 in 7 h; the highest TN removal efficiency was 49%. When C/N=2.0, the highest TN removal rate was 71.42 mg·L-1 in 7 h; the highest TN removal efficiency was 59.52%, and the contribution of CANON to nitrogen removal gradually decreased, whereas the denitrification contribution gradually increased. When △NO3--N/△TN=0.086, the contribution of CANON nitrogen removal was only 51.48% and that of denitrification was 48.52%. The N2O release volume and release ratio decreased with increasing C/N. When C/N=0, the N2O release volume and rate were the highest, namely 3.60 mg and 2.13%, respectively. The lowest N2O release volume and rate were 1.61 mg and 0.75%, respectively, when C/N=2.0.

[Optimization of the Mainstream Anaerobic Ammonia Oxidation Process and Its Changes of the Microbial Community].

The completely autotrophic ammonium removal over nitrite(CANON)biofilm reactor acclimated by high-strength ammonia wastewater was used to treat low-strength ammonia wastewater. The treatment can be divided into three stages:① the nitrogen removal efficiency of anaerobic ammonia oxidation was low during the continuous aeration stage with inorganic wastewater as raw water (0-59 d) and with an aeration amount of 30 mL·min-1 and ammonia concentration of 80 mg·L-1 (until day 56), the TN removal load was only 0.13 kg·(m3·d)-1; ② during the continuous aeration stage with domestic wastewater as raw water (60-110 d), the addition of organic carbon improved the TN removal load to 0.22 kg·(m3·d)-1 on day 79; the removal rate of NH4+-N then reached 100% when the aeration volume improved to 100 mL·min-1 on day 103; however, the TN removal efficiency and TN removal load decreased to 42.36% and 0.14 kg·(m3·d)-1, respectively. ③ To increase both the NH4+-N and TN removal efficiency during the intermittent aeration stage with domestic wastewater as raw water (110-160 d), the aeration amount was increased to 50 mL·min-1, while aeration was continued for 30 min and was stopped for the next 30 min; on day 131, the NH4+-N removal efficiency increased to 86.34%, the TN removal efficiency and removal load reached 85.87% and 0.3 kg·(m3·d)-1 respectively; on day 141, the aeration was increased to 100 mL·min-1 and the removal efficiency of NH4+-N reached 100%, while the removal efficiency and removal load of TN were 64.28% and 0.22 kg·(m3·d)-1, respectively, indicating that the intermittent aeration strategy effectively improves the nitrogen removal performance of the CANON reactor. To analyze the variation of the microbial community during different stages, the samples of three stages (0, 56, and 152 d) were analyzed using high-throughput sequencing technology. The results show that:① Candidatus Brocadia is less affected than Candidatus Kuenenia during the low-strength ammonia stages with inorganic and domestic wastewater as raw water; ② Nitrosominas and Nitrospira were the dominant bacteria of AOB(ammonia oxidizing bacteria) and NOB (nitrite oxidizing bacteria), respectively. Domestic wastewater had a greater impact on Nitrosomonas than on Nitrospira; ③ Denitrifying bacteria were present during the whole stage; Pseudomonas and Paracoccus were the most adaptable, even though their relative abundances during each stage were below 0.5%.

[Recombinant Legionella pneumophila flagella protein A (rflaA) induces the secretion of IL-6 and IL-1ß in RAW264.7 cells in vitro].

Objective To investigate the effect of recombinant Legionella pneumophila flagella protein A (rflaA) on the secretion of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) by RAW264.7 macrophage and the possible mechanism. Methods RAW264.7 cells were treated with 0.000, 0.125, 0.250, 0.500, 1.000, 2.000, 4.000 and 8.000 µg/mL rflaA to determine the EC50 of rflaA using CCK-8 assay. Secretion of IL-6 and IL-1ß were measured by ELISA at 24, 36 and 48 hours after treatment of the cells with 0.04, 0.08 and 0.16 µg/mL rflaA. At 6, 12, 24, 36 and 48 hours after treatment of the cells with 0.04, 0.08 and 0.16 µg/mL rflaA, the expressions of IL-6, IL-1ß, NOD-like receptor protein 3 (NLRP3) and caspase-1 mRNAs were detected by quantitative real-time PCR, and the expressions of NLRP3 and caspase-1 proteins were tested by Western blotting. Results RflaA enhanced the expressions of IL-6 and IL-1ß, and the higher concentration of rflaA was more potential. The expressions of IL-6 and IL-1ß reached peak when the cells were treated with 0.16 µg/mL rflaA for 36 hours. Treatment of RAW264.7 cells with rflaA promoted the expressions of IL-6 and IL-1ß, NLRP3 and caspase-1 mRNA, and 0.16 µg/mL rflaA was the most potential at 12 hours after treatment. Expressions of NLRP3 and caspase-1 protein increased after treatment with rflaA, and 0.16 µg/mL rflaA induced the highest expression of both proteins at 24 hours after treatment. Conclusion RflaA could enhance the secretion of IL-6 and IL-1ß by promoting the expressions of NLRP3 and caspase-1 in RAW264.7 cells.

[Chemotherapy increases FcγR⠡B content in peripheral blood of the patients with lung squamous cell carcinoma].

Objective: To detect the FcγRⅡB content of monocytes and B cells,the concentration of soluble FcγRⅡB( s FcγRⅡB) and its antibodies in the peripheral blood of patients with lung squamous cell carcinoma( LSCC) before and after chemotherapy. Methods: The expression and localization of FcγR Ⅱ B on monocytes and B cells were detected by immunofluorescence technique; the expression of FcγR Ⅱ B mRNA was examined by quantitative real-time PCR; Western blotting was performed to detect the expression of FcγRⅡB protein; the serum concentrations of s FcγRⅡB,s FcγRⅡB-Ig G complex,anti-FcγRⅡB autoantibody were determined by ELISA. Results: The expression of FcγRⅡB on monocytes and B cells,the total s FcγRⅡB and anti-FcγRⅡB antibody content in the sera of LSCC patients were lower than those in healthy volunteers. Chemotherapy enhanced the expression of FcγRⅡB both on the detected cell surface and in the peripheral blood of patients with LSCC,and increased the serum concentration of anti-FcγR Ⅱ B autoantibodies as well. In addition,the concentration of s FcγR Ⅱ B-Ig G in the peripheral blood was higher in LSCC patients than that in healthy volunteers,and it decreased after chemotherapy. Conclusion: The level of FcγRⅡB decreases in LSCC patients,but it rises after chemotherapy.

[Preparation of human soluble FcepsilonR1α and detection of serum FcepsilonR1α antibodies in patients with allergic rhinitis].

OBJECTIVE: To induce the expression of human soluble Fc epsilon receptor I alpha (sFcepsilonR1α) in a prokaryotic expression vector, purify the recombinant human sFcepsilonR1α protein, detect its binding affinity for human serum IgE antibodies and detect the levels of sFcepsilonR1α, sFcepsilonR1α-IgE and FcepsilonR1α antibodies. METHODS: The FcepsilonR1α extracellular region gene was amplified using nested polymerase chain reaction (PCR) and was expressed in a prokaryotic expression vector pET-sFcepsilonR1α using recombinant DNA technology under optimal conditions. The human sFcepsilonR1α protein was purified using iminodiacetic acid (IDA) His binding resin and identified using Western blotting. The affinity between the recombinant human sFcepsilonR1α and serum IgE antibodies and the levels of total sFcepsilonR1α, sFcepsilonR1α-IgE and FcepsilonR1α antibodies were measured using ELISA. RESULTS: The amplified gene corresponding to the extracellular region FcepsilonR1α was approximately 600 bp. PCR, double enzyme digestion and sequencing confirmed the correct sequence of the expression vector pET-sFcepsilonR1α. After human sFcepsilonR1α protein was induced in the expression vector pET-FcepsilonR1α and purified, Western blotting showed that its relative molecular mass (Mr) was approximately 42,000. ELISA revealed that the human sFcepsilonR1α bound with a high affinity to serum IgE, and the lower levels of total sFcepsilonR1α and sFcepsilonR1α-IgE and higher levels of serum anti-FcepsilonR1α antibodies in the patients with allergic rhinitis than in the normal subjects. CONCLUSION: We successfully synthesized human sFcepsilonR1α which had a strong binding affinity for human serum IgE. The higher levels of serum anti-FcepsilonR1α antibodies in the patients with allergic rhinitis than the normal subjects.

[Up-regulated expression of FcγRIIB inhibits phagocytosis and chemotaxis of macrophages].

OBJECTIVE: To construct an inducible lentivirus vector of FcγRIIB and observe its effect on rat macrophages. METHODS: FcγRIIB gene fragment was obtained from rat liver mRNA as the template by reverse transcription PCR, and then cloned into tetracycline response element (TRE) plasmid to establish recombinant plasmid TRE-FcγRIIB. Lentiviral packaging plasmid was transfected into HEK293T cells together with the recombinant plasmid TRE-FcγRIIB and Tet plasmid, respectively. Then the titers of the above lentiviruses were measured. Rat macrophages were co-infected by FcγRIIB-lentivirus and Tet-lentivirus, thereafter induced by doxycycline (DOX) of gradient concentrations. The mRNA and protein levels of FcγRIIB were measured by immunofluorescence, real-time quantitative PCR (qRT-PCR) and Western blotting. After induced by DOX, macrophages were detected in phagocytic and chemotactic function. RESULTS: The recombinant plasmid we constructed was confirmed correct by PCR, enzyme digestion and sequencing. The titers of FcγRIIB-lentivirus and Tet-lentivirus reached 10(6) TU/mL. FcγRIIB was expressed under DOX induction in macrophages which were co-infected by the packaged lentiviruses. In addition, FcγRIIB level was positively correlated with the concentration of DOX, but was negatively correlated with phagocytosis and chemotaxis of macrophages. CONCLUSION: The up-regulation of FcγRIIB expression in macrophages might suppress cell phagocytosis and chemotaxis.