[Establishment and application of HPLC-MS/MS method for detecting m6A level in RNA].

Objective: Explore the establishment of a fast, stable and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detecting the level of m6A modification in RNA and its application. Methods: The degree of m6A in RNA can be expressed as the ratio of m6A and adenosine (A) in concentration, which can be determined by ESI source positive ion multiple reaction monitoring (MRM) mode. The established method was verified by analyzing three quality control samples (m6A: 4, 40, 400 nmol/L; A: 40, 400, 4 000 nmol/L) with three different concentrations of low, medium, and high. The method was used to detect the degree of m6A in RNA from mouse spleen T cells treated in different ways. The t test was used to compare the differences between the two groups of data. Results: The established method had a good Linearity (R2>0.99) in a range of 1-500 nmol/L for m6A and 10-5 000 nmol/L for A. The limit of detection (LOD) was 1 nmol/L for m6A and 10 nmol/L for A. The recoveries were between 98.9% and 116.5%. The intra-day (n=5) RSDs and the inter-day (n=15, 5 days) RSDs were 2.4%-9.5% and 4.4%-9.6%, respectively. And this method was used to detect the degree of m6A in the RNA from mouse spleen T cells cultured in different conditions. The results showed that the m6A modification level in the RNA of primary CD8+T cell was 0.271 5±0.017 9, and the m6A modification level in the RNA of primary CD8+T cell with IL-27 was 0.251 7±0.015 0, indicating that primary CD8+T cells have a higher level of RNA methylation. Conclusion: This research has established a fast, simplemethylation degree in RNA with HPLC-MS/MS. This method is easy to be popularized and is suitable for the detection of large quantity of samples, and of great significance in analyzing the relationship between methylation and diseases.

LncRNA MIAT stimulates oxidative stress in the hypoxic pulmonary hypertension model by sponging miR-29a-5p and inhibiting Nrf2 pathway.

OBJECTIVE: To elucidate the potential biological functions of long non-coding RNA (lncRNA) MIAT in the development of hypoxic pulmonary hypertension (HPH) and the underlying mechanism. MATERIALS AND METHODS: Twenty Sprague Dawley (SD) rats were randomly assigned into normoxia group (n=10) and hypoxia group (n=10), respectively. In vivo HPH model in rats was established by hypoxic induction. Expression levels of MIAT and miR-29a-5p in rats were detected. Meanwhile, hemodynamic indicators in rats were examined. In vitro HPH model was conducted in hypoxia-induced HPAECs. The interaction between MIAT and miR-29a-5p was assessed by Dual-Luciferase reporter assay. Moreover, their regulatory effects on viability, migratory ability, oxidative stress, and the Nrf2 pathway in hypoxia-induced HPAECs were examined. RESULTS: MIAT was upregulated in both in vivo and in vitro HPH models, while miR-29a-5p was downregulated. Knockdown of MIAT suppressed viability, migratory ability, and oxidative stress in hypoxia-induced HPAECs. MiR-29a-5p was the target gene binding MIAT, and silence of miR-29a-5p partially relieved the inhibitory effects of MIAT on the above regulations in HPAECs. CONCLUSIONS: MIAT promotes proliferative and migratory abilities, as well as oxidative stress in hypoxia-induced HPAECs by targeting miR-29a-5p, thus aggravating the development of HPH.

Effects of lamivudine on cell proliferation of liver cancer and expressions of HBsAg, HBeAg, and MMP-9 in patients.

OBJECTIVE: To explore the effects of lamivudine on cell proliferation of liver cancer and expressions of HBsAg, HBeAg, and MMP-9 in hepatoma cells. MATERIALS AND METHODS: In the intervention group, HepG2.2.15 cells were cultured with lamivudine at 100, 200, and 300 µmol/L for 24 hours, 48 hours, and 72 hours. In the control group, HepG2.2.15 cells were cultured without lamivudine. MTT assay was used to assess the proliferative activity of cells after the intervention by lamivudine for 24 hours, 48 hours, and 72 hours. ELISA was used to measure the expression levels of HBsAg, HBeAg, and MMP-9 after the intervention by lamivudine for 48 hours and 72 hours. RESULTS: There was no significant difference between the intervention group and the control group in the proliferation activity of cells (p>0.05). After 48 hours and 72 hours of intervention by lamivudine, the expressions of MMP-9, HBsAg, and HBeAg in the control group were statistically lower than those in the intervention groups with lamivudine at 100 µmol/L, 200 µmol/L, and 300 µmol/L (p<0.05). The expressions of MMP-9, HBsAg, and HBeAg in HepG2.2.15 gradually decreased with the increase of intervention concentration and intervention time of lamivudine (p<0.05). CONCLUSIONS: Lamivudine cannot directly inhibit the proliferation of liver cancer cells, but it can reduce the expressions of MMP-9, HBsAg, and HBeAg in hepatoma cells, inhibit the replication of HBV disease in hepatoma cells, and suppress tumor growth.

LncRNA PVT1 knockdown affects proliferation and apoptosis of uveal melanoma cells by inhibiting EZH2.

OBJECTIVE: To detect the expression of long non-coding ribonucleic acid (lncRNA) plasmacytoma variant translocation gene 1 (PVT1) in uveal melanoma (UM) tissues, and to investigate its influence on the proliferation and apoptosis of UM cells as well as its mechanism. PATIENTS AND METHODS: 40 cases of UM tissues and 40 cases of adjacent tissues surgically resected in our hospital from October 2015 to April 2018 were collected. The expression level of lncRNA PVT1 in these tissues was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Stable knockdown of lncRNA PVT1 was constructed in human UM cell line OCM-1 using small interfering RNA (siRNA). The impact of lncRNA PVT1 on UM cell proliferation was detected by Cell Counting kit-8 (CCK-8) and colony formation assay. Flow cytometry was applied to measure the apoptotic level of UM cells in the blank control group and lncRNA PVT1 knockdown group. Meanwhile, the expression level of enhancer of zeste homologue 2 (EZH2) was determined by Western blotting. RESULTS: The expression level of lncRNA PVT1 in UM tissues was remarkably higher than that in the adjacent tissues (p<0.05). UM cell proliferation was notably repressed after lncRNA PVT1 knockdown by siRNA. Flow cytometry results indicated that the number of apoptotic UM cells in lncRNA PVT1 knockdown group significantly increased compared with that in the blank control group (p<0.05). The protein expression of EZH2 was suppressed after lncRNA PVT1 knockdown (p<0.05). CONCLUSIONS: LncRNA PVT1 knockdown in UM cells can repress the proliferation of UM cells and promote their apoptosis by regulating EZH2 expression.

[Relations between high risk sexual behavior and HIV infection among men who have sex with men in ways of meeting male partners].

Objective: To understand the relations between high risk sexual behavior and HIV infection among MSM in ways of finding male partners in Ningbo. Methods: A cross-sectional study was conducted in Ningbo between April and November in 2018. Data related to socio-demographics, ways of finding male partners, adoption of gay apps and sexual behaviors were collected by snowball method. Blood samples were drawn for HIV antibody testing. Classified data was evaluated by chi-square test. Related factors on HIV infection were analyzed by multivariate logistic regression. Results: A total of 735 participants were included in this study. Ways of finding male partners would through gay apps (60.8%, 447/735), QQ/Wechat (32.3%, 237/735) and gay-places (6.9%, 51/735). Related information on high risk sexual behavior and HIV infection among gay apps users were found as: 16.8%(75) had sexual behavior once per week in the past 6 months, 41.8% (187/447) had multiple sexual partners, 12.1% (54/447) had unprotected anal intercourse in the last time, 52.3% (234/447) having had unprotected anal intercourse in the past 6 months. The overall HIV prevalence rate was 12.1%(54/447). Among the HIV cases who got infected within the two years, 68.6%(24/35) of them had used gay apps for less than two years. Results from the, multivariate logistic regression analysis showed that gay apps users were more susceptible to infected HIV than those who used the QQ/Wechat (OR=3.03, 95%CI: 1.30-7.07). Conclusions: Gay apps was popularly known among the Ningbo MSM, and was associated with the high risk sexual behaviors and HIV infection. HIV control and prevention programs should be strengthened in the MSM population who used the gay apps. Related surveillance and intervention programs for MSM, who use the gay apps, need to be further reinforced.

[Clinical analysis of orbital ectopic meningiomas].

Objective: To investigate the clinical features, imaging features, diagnoses and therapeutic regimens of orbital ectopic meningiomas. Methods: A retrospective study. All the clinical data of 10 patients (10 eyes) with orbital ectopic meningiomas were analyzed retrospectively from August 1999 to October 2016. They included imaging data such as color Doppler ultrasound, CT and MRI, and information of diagnosis, pathology, therapeutic regimen and prognosis. Results: The age of orbital ectopic meningiomas was 7 to 68 years old. There were 4 males and 6 females. The clinical manifestations were mainly with swollen eyelid (8 cases), exophthalmos (7 cases), visual impairment (2 cases) and accidentally found a mass in the orbit with physical examination(2 cases). The tumor was located in (5 cases) or outside (5 cases) the muscular funnel of the orbit. The imaging features of CT included the irregular shape, unclear border, and extraocular muscular adhesions (10 cases). T1 weighted image of MRI showed low and medium signals and T2 weighted image showed medium and high signals(8 cases). There was no definite diagnosis before surgery. Ten patients were treated with surgery, and it was confirmed intraoperatively that the tumors were not adjacent to the optic nerve and orbital periosteum. The pathological diagnoses were mostly epithelial meningiomas (9 cases). There were no significant changes in visual acuity, but temporary eye movement disorders occurred postoperatively (10 cases). The patients were followed for 6 months to 6 years. Two cases underwent recurrent at 3 months and 2 years after operation, and received treatment of γ-knife radiation therapy. The tumor reduced after γ-knife radiotherapy, and had no change in 2 years and 6 years, respectively. Conclusions: As a kind of rare orbital tumors, it is difficult to diagnose orbital ectopic meningiomas accurately. The clinical features and imaging findings can help to detect the disease, but lacking the characteristics of optic nerve sheath meningiomas and periosteal meningiomas. Surgical resection could achieve a good prognosis without visual impairment. If the tumor recurs after pathological diagnosis, γ-knife conformal radiation therapy may still be effective for tumor recurrence. (Chin J Ophthalmol, 2018, 54: 665-670).

[Clinical analysis of patients with orbital nonspecific inflammatory response diseases whose CT or MR images show enlargement of the infraorbital nerves].

Objective: To discuss the clinical features, imaging features, pathological patterns, treatment principles and prognosis of the orbital nonspecific inflammatory response diseases patients whose CT or MR images show enlargement of the infraorbital nerves. Methods: A retrospective case series study. Seven orbital disease patients who were treated at Tianjin Medical University Eye Hospital between March 2013 and May 2017 were included. All patients, imaging pictures showed enlargement of the infraorbital nerve. The medical histories, clinical featuers, imaging features, pathologies, serological examinations, therapeutic processes and prognosis were collected and analyzed. Results: The 7 patients included 4 males and 3 females aged from 55 to 68 years (the average age was 60). Bilateral involvement was present in 5 of 7 patients. The main clinical manifestations include proptosis, increasing of orbital pressure, impairment of visual functions, reduction in ocular motility, facial sensation, periocular ache, involvement of lymph node and salivary gland, etc. CT results showed enlargement of infraorbital nerve, accompanied with or without the destruction of bone. The inflammatory response may involve with extraocular muscles and lacrimal glands, or were shown as lesions with irregular shape and blurred borders. The MR images generally showed equal T(1) and equal T(2) signal, accompanied with evident enlargement of the infraorbital nerve. Of all the 7 patients, 6 underwent operation, and the pathology confirmed that 2 of the 6 were involved with inflammatory pseudotumors and the rest 4 were involved with IgG4-related ophthalmic disease (IgG4-ROD). The level of IgG4 in the serum were detected for 4 patients, the results of 3 were high and the other was normal. Of all the 7 patients,1 patient underwent conservative treatment, but disease recurred for several times, and the serum IgG4 level for the patient was higher than normal;1 inflammatory pseudotumor patient was cured completely only by surgery, and has a favourable prognosis; 1 IgG4-ROD patient accepted glucocorticoid, surgery, radiotherapy and chemotherapy, but disease recurred several times; The rest of the patients accepted glucocorticoid and operation, 2 were sensitive to glucocorticoid, 2 were not sensitive, and 1 involved with recurrence. Conclusions: If orbital nonspecific inflammation response was accompanied with enlargement of infraorbital nerve, it is suggested that patient is more likely to be involved with IgG4-ROD;Pathology and serological tests can be used for the diagnosis of IgG4-ROD, however treatment effect appears to be poor for most patients, and patients prone to relapse. (Chin J Ophthalmol, 2018, 54: 515-519).

[Clinical analysis of orbital and periorbital abscess in 17 patients].

Objective: To discuss the etiology, clinical features and treatment principles of the orbital and periorbital abscess. Methods: A retrospective case series of 17 cases with orbital and periorbital abscess between July 2010 and November 2015 were conducted. All patients(,) clinical data including medical history, etiology, abscess location, paranasal sinus involvement, eye involvement, microbiological test results, imaging features, treatment and prognosis were summarized and analyzed. Results: In all 17 patients, there were 10 males and 7 females with age from 3.0 to 71.0 years (the average age was 33.9 years).Eight patients(,) bacterial cultures of the pus and secretion were positive in all 17 patients. Orbital and periorbital abscess patients could manifest decreased vision, redness and swelling of eyelid, conjunctival congestion and edema, ocular motility disorders, displacement of eyeball, increased orbital pressure, abscess rupture etc. CT showed us the soft tissue mass, accompanied with sinusitis or paranasal sinus mass. MR performed with the long T(1) and T(2) signals. The signals of the abscess cavity were not uniform. For the etiology,11 cases were secondary to sinusitis, including 1 case of diabetes; 2 cases with orbital fractures.One case was secondary to orbital fracture repairment surgery. One case was secondary to the remnant of sequestrum and foreign bodys in the wound after repairment surgery. One case was injured by the hard object. One case was secondary to paranasal sinuses large B-cell lymphoma. One case had diabetic history and the blood sugar was controlled unstablly. For the treatment, 7 cases were treated by the drainage surgery which was performed via the sinus with endoscopic and abscess resection performed via the skin.Two cases were treatment by the abscess resection only.One case was treated by the drainage surgery performed via the sinus with endoscopic only. Six cases were treated by the drainage surgery performed via the skin. One case was only administered intravenous antibiotic. Sixteen cases acquired well prognosis without serious complications except 1 case which occurred central retinal artery and vein occlusion. Conclusions: The orbital and periorbital abscess is mainly a complication of paranasal sinus infection, or secondary to trauma, surgery, tumor, etc; Orbital and periorbital abscess always manifest inflammatory neoplastic clinical features, the key of the diagnosis is to make sure the etiology; Incision and drainage of the abscess is the main treatment method when necessary. We can do the surgery with other departments to avoid the occurrence of serious complications.(Chin J Ophthalmol, 2017, 53: 588-593).

[Re-evaluate the value of orbital decompression for thyroid associated ophthalmopathy].

Thyroid-associated ophthalmopathy (TAO) is a common autoimmune syndrome affecting the thyroid and orbit. It can result in the fibrosis of extraoular muscles and hyperplasia of adipose tissue. The mechanism of TAO is not clear and there was few effective treatment. Recently, orbital decompression was performed on more and more patients. The surgery can improve the appearance of the patient and relieve the symptoms caused by the high orbital pressure. Therefore, it is necessary to re-evaluate the clinical value of the orbital decompression and surgical indications. The principles and techniques of orbital decompression with relative factors were discussed in this review. (Chin J Ophthalmol, 2017, 53: 401-403).

[The value of DCE-MRI in assessing the course of thyroid associated ophthalmopathy].

Objective: To explore the feasibility of the semi-quantitative rectus extraocular muscle (EOM) parameters of dynamic contrast-enhanced magetic resonance imaging (DCE-MRI) in assessing the clinical course of thyroid associated ophthalmopathy (TAO). Methods: It was a retrospective case series study. A total of 136 cases of TAO were recruited from March 2011 to October 2012 in the Tianjin the first center hospital including 63 males and 72 females, aged 24.0-65.0 years, with an average age of (40.5±10.9) years. Forty healthy volunteers were recruited as control group (CG). According to clinical activity score (CAS), all TAO patients were divided into 2 groups, activity group (AG) and inactivity group (IAG). MRI and DCE-MRI orbit scan were performed in each subject. Drew time-intensity curves (TIC) by Siemens 3.0 MR (syngo) post-processing workstation. The semi-quantitative parameters of DCE-MRI were calculated. The semi-quantitative paramters based on TIC include early enhancement coefficient (EEC), maximum enhancement coefficient (Emax) and wash-out coefficient (WC(5min)). Kruskal-Wallis H rank test was used for comparing signal intensity among 3 groups, and Nemenyi test for pairwise comparison between groups. The DCE-MRI parameters (EEC, Emax, WC(5 min)) among groups were compared by one-way ANOVA, and Bonferroni t test is for pairwise comparison between groups. The diagnostic value of mean EEC, mean Emax, WC(5min) for assessment of the clinical course in TAO was analyzed by ROC curve. Results: There were significant difference in signal intensity (SI) of rectus EOM on T(2)WI among CG, AG and IAG, which is significantly different in 88 cases of AG including 45 cases of high intensity 51.1%, 23 cases of moderate intensity 26.1%, and 20 cases of low intensity 22.7%,compared with CG and IAG. EEC (P<0.05), Emax and WC5min values of rectus EOM of TAO group were significantly lower than those of CG(P<0.05), which values of rectus EOM of TAO active group of EEC are 0.63±0.06、0.61±0.05、0.56±0.09、0.57±0.09, and values of rectus EOM of TAO inactive group of EEC are 0.49±0.05、0.50±0.08、0.57±0.10、0.55±0.09. The values of rectus EOM of TAO active group of Emax are 1.35±0.09、1.28±0.09、1.21±0.17、1.25±0.10, and the values of rectus EOM of TAO inactive group of Emax are 1.04±0.06、1.05±0.10、1.20±0.19、1.16±0.11. The values of rectus EOM of TAO active group of WC(5 min) are 0.13±0.03、0.13±0.03、0.13±0.06、0.13±0.03 and the values of rectus EOM of TAO inactive group of WC5min are 0.08±0.02、0.79±0.03、0.11±0.06、0.09± 0.03. EEC (χ(2)=9.20, P<0.05), Emax and WC(5min) values of rectus EOM of TAO group were significantly lower than those of CG (P<0.05). EEC, Emax and WC(5min) values of medial rectus and inferior rectus EOM of IAG were significantly lower than those of AG(P<0.05). WC(5min) values of superior rectus EOM of IAG were significantly lower than those of AG (P<0.05). There were no differences in EEC and Emax values of lateral rectus and superior rectus EOM between IAG and AG (P>0.05). There were no differences in WC(5min) values of lateral rectus EOM between IAG and AG (P>0.05). The area under the curve (AUC) were 0.771, 0.879, 0.898 for mean EEC, mean Emax, and mean WC(5min), respectively. Conclusion: The semi-quantitative paramters of DCE-MRI can show the clinical activity of TAO patients and can be considered as the quantitative index of TAO activity staging. (Chin J Ophthalmol, 2017, 53: 430-435).

[Bone marrow-derived fibrocytes and thyroid-associated opthalmopathy].

Thyroid-associated opthalmopathy(TAO) is a common autoimmune syndrome affecting the thyroid and orbit, which can result in the fibrosis of extraoular muscles and hyperplasia of adipose tissue. Advanced TAO patients could even lose vision caused by cornea ulcer and congestion of optic nerve from expansion of the extraocular muscles and orbit fat. Currently, there are no therapies shown to prevent it, because its cellular and molecular mechanisms are not clear. Some studies have recently implicated bone marrow-derived fibroblast-like, called fibrocytes are involved in the pathogenesis of TAO. We reviewed and summarized the research advances of TAO and also the relationship between the fibrocytes and pathogenesis of TAO in the paper. (Chin J Ophthalmol, 2017, 53: 470-473).

SOST silencing promotes proliferation and invasion and reduces apoptosis of retinoblastoma cells by activating Wnt/ß-catenin signaling pathway.

This study aimed to investigate the effects of SOST and the Wnt/ß-catenin signaling pathway on the proliferation, migration, invasion, and apoptosis of human retinoblastoma cells. Fifty-five retinoblastoma and 21 normal retinal tissue samples were collected as the case group and control group, respectively. HXO-RB44 and SO-RB50 cells were selected and assigned into blank, negative control (NC), siRNA 1, siRNA 2, siRNA 3, IWR-1-endo 1, IWR-1-endo 2 and IWR-1-endo 3 groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of SOST, Wnt-1, and ß-catenin in the collected tissue samples. MTT assay, flow cytometry, transwell assay and the starch test were employed to determine the cell proliferation, cell cycle, apoptosis, invasion and migration after transfection. The qRT-PCR and western blotting were also used to detect the mRNA and protein expressions of SOST, Wnt-1, ß-catenin, C-myc, Cyclin D1, MMP-2 and MMP-9. The tumor formation in nude mice was conducted to evaluate the effects of SOST on the growth of a transplanted tumor. Compared with normal retinal tissues, the retinoblastoma tissues exhibited a downregulation of SOST but an upregulation of Wnt-1 and ß-catenin. The proliferation, invasion and migration of HXO-RB44 and SO-RB50 cells in the SOST-siRNA group were significantly higher than the cells in the blank and NC groups. The expressions of Wnt-1, ß-catenin, C-myc, Cyclin D1, MMP-2 and MMP-9 in the three SOST-siRNA groups were elevated, but the SOST decreased when compared with the blank and NC groups. SOST silencing promoted the growth of transplanted tumors in nude mice. These findings indicate that SOST silencing promotes the proliferation, invasion and migration, and decreases the apoptosis of human retinoblastoma cells by activating the Wnt/ß-catenin signaling pathway.

Hybrid biofilm-membrane bioreactor (Bf-MBR) for minimization of bulk liquid-phase organic substances and its positive effect on membrane permeability.

Four biofilm membrane bioreactors (Bf-MBRs) with various fixed carrier volumes (C:M) were operated in parallel to investigate the effect of attached-growth mode biomass involvement to the change of liquid-phase organics characteristics and membrane permeability, by comparing with conventional MBR. The experiments displayed that C:M and co-existence of biofilm with suspended solids in Bf-MBRs resulted in slight difference in pollutants removal effectiveness, and in rather distinct biomass properties and bacterial activities. The membrane permeability and specific resistance of bulk suspension of Bf-MBRs related closely with the liquid-phase organic substance, including soluble microbial products (SMP) and biopolymer cluster (BPC). Compared with conventional MBR, Bf-MBR with proper C:M had a low total biomass content and food-chain, where biofilm formation and its dominance affected liquid-phase organics, especially through reducing their content and minimizing strongly and weakly hydrophobic components with small molecular weight, and thus to mitigate membrane fouling significantly.

Immune protective mechanism of rMOMP protein ophthalmic vaccine regarding intraocular hypertension and retinal optic nerve injury in rats.

The aim of this study was to explore the immune protective mechanism of rMOMP protein vaccine in intraocular hypertension and retinal optic nerve injury in rats. The rMOMP protein ophthalmic vaccine was prepared and quality-controlled. Sixty normal adult SD rats were randomly divided into two groups to establish a chronic ocular hypertension model and an optic nerve injury model. The model rats were vaccinated with rMOMP-CS ophthalmic vaccine. Fluorogold retrograde tracing was used to observe retinal ganglion cells, and an immunofluorescence method to determine the expression of retinal GAP43, CD3, BDNF, and GDNF. rMOMP protein ophthalmic vaccine met the requirements for medicinal use. The number of retinal ganglion cells (RGCs) of the rMOMP-CS group in the chronic ocular hypertension model was significantly higher than that of the CS group (P < 0.05). The count of RGCs of the rMOMP-CS group in the optic nerve clamping injury model was significantly higher than that of the CS group (P < 0.01). Thus, rMOMP protein ophthalmic vaccine can induce an increase in the expression of retinal neurotrophic factors, thereby exerting a protective effect on damaged retinal optic nerve.

VEGF attenuated increase of outward delayed-rectifier potassium currents in hippocampal neurons induced by focal ischemia via PI3-K pathway.

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway.

Investigation of the shape change of bio-flocs and its influence on mass transport using particle image velocimetry.

In this laboratory study, an advanced flow visualization technique - particle image velocimetry (PIV) - was employed to investigate the change of shape of activated sludge flocs in water and its influence on the material transport characteristics of the flocs. The continuous shape change of the bio-flocs that occurred within a very short period of time could be captured by the PIV system. The results demonstrate that the fluid turbulence caused the shift of parts of a floc from one side to the other in less than 200 ms. During the continuous shape change, the liquid within the floc was forced out of the floc, which was then refilled with the liquid from the surrounding flow. For the bio-flocs saturated with a tracer dye, it was shown that the dye could be released from the flocs at a faster rate when the flocs were swayed around in water. The experimental results indicate that frequent shape change of bio-flocs facilitates the exchange of fluid and materials between the floc interior and the surrounding water. This mass transfer mechanism can be more important than molecular diffusion and internal permeation to the function and behavior of particle aggregates, including bio-flocs, in natural waters and treatment systems.

Stabilization of source-separated urine by biological nitrification process: treatment performance and nitrite accumulation.

A laboratory study on nitrification of high-strength source-separated urine was conducted by means of sequencing batch reactors (SBR) and membrane bioreactors (MBR). The highest influent ammonia concentration for SBR and MBR reached more than 2,400 and 1,000 mg N/L, while the maximum pH was about 9 and 8.9, respectively. The ammonia oxidizing efficiency in both SBRs and MBRs was around 50%, which was restrained mainly by the deficiency of alkalinity in bulks. Meanwhile, the nitrite accumulation did also dominate in these two systems, and the major factor to inhibit the nitrite oxidization was thought to be the high free ammonia and free nitrous acid content in bulks. Hence, an ammonia nitrite solution was achieved with concentration ratio of 1:1; after that ammonia oxidation was restrained owing to the deficiency of alkalinity in urine. The temperature and influent ammonia content have no great influence on the nitrification process in both kinds of bioreactors. The nitrification can be progressed under a solids retention time (SRT) longer than 30 d; however, termination of ammonia oxidization was observed as the SRT fell below 20 d. The nitrifier biomass showed an excellent settleability, such that the suspended solids (SS) in effluent was of a low average, about 60 mg/L. This study on the stabilization of human urine will be useful to understand the process of urine separation from source.

Evaluation of the importance of various operating and sludge property parameters to the fouling of membrane bioreactors.

A single-fibre microfiltration system was employed to investigate the importance of various operating and sludge property parameters to the membrane fouling during sludge filtration. The sludge was obtained from a submerged membrane bioreactor (SMBR). A series of comparative and correlative filtration and fouling tests were conducted on the influence of the operating variables, sludge properties and the liquid-phase organic substances on the membrane fouling development. The test results were analysed statistically with Pearson's correlation coefficients and the stepwise multivariable linear regression. According to the statistical evaluation, the membrane fouling rate has a positive correlation with the biopolymer cluster (BPC) concentration, sludge concentration (mixed liquor suspended solids, MLSS), filtration flux and viscosity, a negative correlation with the cross-flow velocity, and a weak correlation with the extracellular polymeric substances and soluble microbial products. BPC appear to be the most important factor to membrane fouling development during the sludge filtration, followed by the filtration flux and MLSS concentration. The cross-flow rate also is important to the fouling control. It is argued that, during membrane filtration of SMBR sludge, BPC interact with sludge flocs at the membrane surface to facilitate the deposition of the sludge cake layer, leading to serious membrane fouling.

A membrane bioreactor for an innovative biological nitrogen removal process.

A hybrid system has been developed for biological nitrogen removal through nitrification-denitrification. The system includes an aerobic tank and an anoxic tank with an intermediate sludge settler connected to a membrane bioreactor (MBR) with a submerged 0.4 microm hollow-fiber membrane module. The laboratory system has a total working volume of 6.5 L treating a glucose-based synthetic wastewater. The experimental results demonstrate that the new process is highly effective for simultaneous organic and nitrogen removal. During the stationary operation, a sludge SS (suspended solids) concentration of 6 g/L or higher can be maintained in the reactors. The system has a COD (chemical oxygen demand) loading rate of up to 2,100 mg/L-d and a total nitrogen loading rate of up to 170 mg N/L-d. More than 95% COD can be degraded, and the total nitrogen removal efficiency can be 90% or higher as the nitrogen is reduced from 100 to around 7.5 mg/L. A high quality effluent is produced with a SS of less than 1 mg/L. With the MBR, organic degradation, nitrogen removal and sludge-liquid separation can be well achieved within a short HRT of about 10 hr.

Excision repair cross-complementing 1 expression protects against ischemic injury following middle cerebral artery occlusion in the rat brain.

To study the effects of excision repair cross-complementing 1 (ERCC1) on the pathophysiological process of brain ischemia, we examined the changes in ERCC1 expression, as well as the functional significance of ERCC1 in the rat brain following middle cerebral artery occlusion (MCAO). The results were as follows: (1) ERCC1 immunopositive cells were widely distributed in various brain regions. ERCC1 expression was localized to the nuclei of neurons and astrocytes. (2) ERCC1 expression, as determined by western blot, increased at 3 days, remaining until 14 days, in the ipsilateral cortex and striatum following MCAO. Immunohistochemical analysis demonstrated that ischemia induced increased ERCC1 expression within the periinfarct core, with increasingly less expression toward the core. (3) Knockdown of ERCC1 expression by intraventricular injection of antisense plasmids increased DNA damage and infarct volume in the ischemic brain. (4) ERCC1 overproduction, by injection of expression plasmids, significantly reduced infarct volume and the accumulation of DNA-damaged neurons. Taken together, these results indicate that both endogenous ERCC1 and exogenous ERCC1 have an important neuroprotective function in the brain. In addition, administration of ERCC1 to the brain could prove to be a successful strategy for neuronal protection against ischemic injury.