Environmental Microbiology is an important basic course of Environmental Engineering.Its characteristic is content broad,quick development and strong practicality,thus this curriculum's teaching has certain degree of difficulty.Some suggestions and concrete measures about teaching reform,which included curriculum's course content,teaching method,experiment teaching and assessment methods were proposed in this paper.

Objective To investigate the possible mechanism of endonuclease G (Endo G)-mediated non-Caspase-dependent apoptotic pathway in brain neuronal apoptosis in chronic fluorosis rats.Methods Sixty Sprague Dawley (SD) rats (half male and half female) were randomly divided into two groups:control group fed with tap water with fluoride content ＜ 0.5 mg/L and fluorine group in which sodium fluoride was added into drinking water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content ＜ 0.5 mg/kg.The experiment period was 10 months.At the end of the experiment,all the animals were sacrificed,and brain tissue was taken.Flow cytometry was used to examine apoptosis rate,immune-histochemistry was employed to detect the distribution of Endo G in brain tissue;Western blotting was used to test the protein expression of Endo G.Results Compared to the apoptosis rate of control group [(1.3 ± 0.6)％,(1.9 ± 0.3)％],the apoptosis rate in hippocampus and cortex of rats with chronic fluorosis [(2.6 ± 0.6)％,(3.1-± 0.7)％] was significantly increased (t =3.1,3.4,all P ＜ 0.05).The Endo G positive neurons and their degree of staining in CA1,CA2,CA3 and CA4 of hippocampus,frontal cortex as well as the upper layer of parietal cortex [(11.1 ± 2.2),(10.2 ± 1.9),(9.8 ± 3.1),(9.9 ± 1.6),(10.6 ± 2.9),(8.2 ± 2.4),(11.1 ± 2.8) scores] in rats with chronic fluorosis were significantly higher than those in the control group [(5.8 ± 1.8),(6.7 ± 2.6),(5.2 ± 2.4),(7.2 ± 2.1),(7.7 ± 2.6),(6.1 ± 1.9),(8.1 ± 2.6) scores,t =2.9,2.5,2.4,2.3,2.2,2.5,2.3,P ＜ 0.01 or ＜ 0.05].The protein level of Endo G in the mitochondria of rat brains with chronic fluorosis [(86.4 ± 7.2)％,(83.9 ± 6.8)％] was significantly lower than that of control group [(100.0 ± 6.1)％,(100.0 ± 5.5)％,t =2.6,2.3,all P ＜ 0.05].Meanwhile,the protein level of Endo G in the nucleus of neurons from chronic fluorosis rats [(117.5 ± 6.4)％,(115.2 ± 6.2)％] was significantly higher than that of the control [(100.0 ± 5.2)％,(100.0 ± 5.5)％,t =2.5,2.2,all P ＜ 0.05].Conclusion The high expression of Endo G and nuclear transfer are related to the neuron apoptosis in chronic fluorosis rat,which may be one of the mechanisms of brain injury of the disease.

Objective To determine the Golgi dispersal in radiation damaged cells and the protective effect of vanillin derivatives.Methods Immunofluorescence, cell cycle analysis of flow-cytometry,Western blot,and clone formation were used.Results Immunofluorescence observation showed that the Golgi dispersal caused by 2 Gy 60 Coγ-ray was significantly increased in a dose-dependent manner in the range of 4-10 Gy as was demonstrated by the fact that the Golgi area was significantly increased. When the irradiated cells were treated with the radioprotective agent VND3207, a vanillin derivative,the Golgi dispersal induced by radiation was significantly reduced.The radiation-induced Golgi dispersal was also displayed in a pattern of time-course after irradiation in the HeLa cells, and persisted at least to 36 h post-irradiation. Cell cycle test results indicated that the Golgi dispersal was not associated with the G2/M arrest triggered by radiation-induced DNA damage response.VND3207 could promote cell survival by plate colony formation assay.Conclusion The Golgi dispersal can be caused byγ-ray irradiation in a dose-and time-dependent manner, and VND3207 can provide a good protection against radiation injury associated with inhibited Golgi dispersal.

Objective To observe the pronunciation characteristics of vowels' tone in prelingual hearing-impaired children accepted cochlear implant (CI). Methods 8 prelingual hearing-impaired children accepted CI aged 3~4 years (group CI) and 8 normal-hearing children (group NH) with the same age were asked to pronounce the vowels of /a/, /o/, /e/, /i/, /u/, and /ü/ in four tones with the direction of speech training teacher. The sounds were recorded and analyzed with Praat software extract Formants (F1, F2) and duration of the sounds. Results The error of vowels in four tones was more in group CI than in group NH, but difficult level was almost the same between groups. There was significant difference in the F1 of /e/, F2 of /i/ and /u/ in tone 2, F2 of /e/ and duration of /o/, /e/, and /ü/ in tone 3 between groups (P<0.05). Conclusion Pronouncing the four tones of vowels is more difficult for the hearing-impaired children. For vowels articulation training,it is important to focus on the mouth and breath training.

Objective To investigate the effects of TLR7 on imiquimod induced apoptosis of THP-1 derived macrophages.Methods Three cell lines ( THP-1 derived macrophages, MDCK cell line and HUVEC cell line) with different capabilities of expressing TLR7 were selected.The survival rates of cells af-ter the treatment with different concentrations of imiquimod were detected by MTT assay.The levels of IL-6 in the supernatants of TLR7 inhibitor chloroquine or TLR7-siRNA treated cells were detected by enzyme-linked immunosorbent assay.The apoptosis of cells was detected by flow cytometry after inhibiting the ex-pression of TLR7.Results Imiquimod induced the apoptosis of THP-1 derived macrophages, MDCK cell lines and HUVEC cell lines.The levels of IL-6 were significantly decreased as the expression of TLR7 was inhibited by treating THP-1 derived macrophages with chloroquine or TLR7-siRNA.Treating THP-1 derived macrophages with chloroquine or TLR7-siRNA did not affect the cell apoptosis induced by imiquimod.Con-clusion Imiquimod could induce the apoptosis of THP-1 derived macrophages through TLR7 independent pathway.

In most mammalian central nervous system diseases, axons are damaged.Due to the limited ability of damaged neurons to promote axonal regeneration, the formation of glial scar and the release of inhibitory nutrients, it is difficult to regenerate axons of damaged neurons. The purpose of this study was to investigate the effect of cerebroprotein hydrolysate for injection (II) (CBL) on neuritogenesis and its underlying mechanism. Immunofluorescence staining was used to detect the axon length of mouse neuroma cells (Neuro-2a) and mouse primary cortical neuronal cells. Western blotting was used to detect the expression of phosphorylated TrkB protein in Neuro-2a cells and mouse primary cortical neuronal cells. The results showed that CBL could increase the axon length of Neuro-2a cells or mouse primary cortical neuronal cells, and that the phosphorylation level of TrkB in neuronal cells was significantly increased when 5 μg/mL CBL was applied to neuronal cells for 1 h. In conclusion, CBL can promote neuritogenesis, and increase the expression of phosphorylated TrkB, which may be related to the activation of TrkB signaling pathway.

Objective To evaluate the value of transvaginal ultrasound in diagnosing intrauterine adhesions.Methods Transvaginal ultrasound was performed in 136 patients with suspicious intrauterine adhesions and compared with hysteroscopy correspondingly .The ultrasonographic features of intrauterine adhesions on transvaginal ultrasound were summarized .Results One hundred and twenty one cases (89.0%, 121/136 ) of intrauterine adhesions were verified by hysteroscopy .The hysteroscopic findings included:(1) Forty seven cases(38.9%,47/121) were minimal intrauterine adhesions , 46 cases(38.0%, 46/121) were moderate intrauterine adhesions , and 28 cases (23.1%,28/121) were severe intrauterine adhesions.(2) Sixty one cases(50.4%,61/121) were central intrauterine adhesions , 24 cases(19.8%, 24/121) were marginal intrauterine adhesions , and 36 cases (29.8%, 36/121) were mixed type of intrauterine adhesions.The transvaginal ultrasound findings included:(1)Nineteen cases(40.4%,19/47) were minimal intrauterine adhesions ,33 cases(71.7%,33/46)were moderate intrauterine adhesions ,and 23 cases(82.1%,23/28) were severe intrauterine adhesions .(2) Thirty nine cases (63.9%,39/61) were central intrauterine adhesions ,9 cases(37.5%,9/24) were marginal intrauterine adhesions ,and 27 cases (75.0%, 27/36 ) were mixed type of intrauterine adhesions .By transvaginal ultrasound, seventy-five (62.0%,75/121) cases of intrauterine adhesions were correctly diagnosed , whereas 46 cases (38.0%, 46/121) were missed.And 3 cases ( 3.8%, 3/78 ) were misdiagnosed as intrauterine adhesions on transvaginal ultrasound,including one endometrial polyp ,one thin endometrium and one septate uterus .The sensitivity, specificity and accuracy of transvaginal ultrasound in diagnosing intrauterine adhesions were 62.0%(75/121), 80.0%(12/15) and 64.0%(87/136) respectively.There were significant statistical differences in diagnosing different degrees of intrauterine adhesions ( χ2 =15.956,P=0.000) and different parts of intrauterine adhesions( χ2 =8.792,P=0.012) by transvaginal ultrasound.Conclusions Transvaginal ultrasound is an effective, easy to perform and noninvasive technique in screening and diagnosing intrauterine adhesions.Transvaginal ultrasound is an effective way in diagnosing intrauterine adhesions showing a noninvasive and simpler way than hysteroscopy .Transvaginal ultrasound is of great value in screening and diagnosing intrauterine adhesions .

Objective To explore the role of apoptosis-inducing factors (AIF) mediated non-apoptosis classic pathway involved in neuron apoptosis of rats with chronic fluorosis.Methods Sixty Sprague Dawley (SD) rats (body weight 100-120 g) were divided into two groups (30 rats in each group,half male and half female) by random number table according to body weight.Control group was fed with tap water with fluoride content ＜ 0.5 mg/L and fluorine group was fed with water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content ＜ 0.5 mg/kg.After 10 months,all the animals were sacrificed though heart perfusion using phosphate buffer,and brain tissue was taken.Immunohistochemical method was employed to detect the distribution of AIF in brain tissue.Western blotting was used to test the protein expression of AIF,cl-caspase-3 and cl-caspase-9.Flow cytometry was used to examine apoptosis rate.Results The AIF positive distribution and degree of staining in the neurons of hippocampal CA1,CA2 and CA3,as well as the parietal cortex (7.50 ± 2.17,9.00 ± 1.63,8.00 ±0.82,10.24 ± 1.80) in rats with chronic fluorosis were significantly higher than those of the control group (5.18 ±1.66,6.27 ± 1.42,6.36 ± 1.96,6.96 ± 2.62,t =2.76,4.09,2.45,5.77,all P ＜ 0.05).The AIF protein expression of neuronal mitochondria in the cerebral tissue of rats with chronic fluorosis [(89.46 ± 8.47)％] was significantly lower than that of the control group [(100.00 ± 7.12)％,t =3.16,P ＜ 0.01],while the AIF protein expression of the neuronal nucleus [(112.80 ± 7.10)％] was significantly higher than that of the control group [(100.00 ± 8.20)％,t =3.75,P ＜ 0.01];cl-caspase-3 and cl-caspase-9 protein expression in the neurons of hippocampus and cortex from the rats with chronic fluorosis [(132.14 ± 18.66)％,(107.31 ± 2.58)％,(121.33 ± 14.86)％,(112.97 ± 7.97)％]were significantly higher than those of the control group [(100.00 ± 11.99)％,(100.00 ± 3.74)％,(100.00 ± 16.87)％,(100.00 ± 8.04)％,t =3.55,3.94,2.32,2.81,P ＜ 0.01 or ＜ 0.05].As compared with those of the control group [(1.28 ± 0.59)％,(1.88 ± 0.25)％],the apoptosis rates in hippocampus and cortex of the rats with chronic fluorosis [(2.55 ± 0.58)％,(3.05 ± 0.65)％] were significantly increased (t =3.08,3.40,all P ＜ 0.05).Conclusion Both of AIF-mediated caspase-independent apoptosis and the classic caspase-dependent apoptosis pathways have participated in neuron apoptosis in rat induced by chronic fluorosis,which may be one of the mechanisms of brain damage of the disease.

Objective Rheumatoid arthritis (RA) is a typical type Ⅲ hypersensitivity with a large number of immune complexes (IC) and complement deposits in the synovial tissue , but its specific pathogenesis is not yet clear.This article was to explore the expression of the antigenic profile of serum ICs in RA.Methods ICs were isolated from the serum of 55 patients with RA (41 cases of anti-CCP antibody [+] and 14 cases of anti-CCP antibody [-]), 41 with systemic lupus erythematosus (SLE), and another 41 healthy controls by polyethylene glycol (PEG) precipitation, separated by immunoprecipitation, digested with trypsin in gel, and then subjected to mass spectrometry for identification.The levels of total proteins were compared among different groups using Vennny 2.1.0.The protein expression was considered to be up-regulated when the total protein level of the RA group was >2 times and down-regulated when it was <0.5 times that of the control.Further functional analysis was performed on the differential proteins in RA using the STRING software.Results Totally, 277 proteins were identified in the serum ICs of the RA patients, including 162 in the anti-CCP (+) and 248 in the anti-CCP (-) RA group.Compared with the SLE and healthy control groups, only 129 proteins were found in the RA patients, including 38 in the anti-CCP (+), 109 in the anti-CCP (-) RA group, and 18 in both the two groups.Among the proteins identified in the RA patients and healthy controls, 2 and 11 were up-regulated while 17 and 21 down-regulated in the anti-CCP (+) and anti-CCP (-) RA group, respectively.Conclusion More differentially expressed proteins were identified in the anti-CCP (-) than in the anti-CCP (+) RA patients.The identification of differentially expressed proteins provides a new idea and direction for the investigation of the pathogenesis and new biomarkers of RA.

Objective To investigate the clinical significance of myasthenia gravis (MG) associated autoantibodies.Methods Titin,ryanodine receptor (RyR)and acetylcholine receptor (AChR) antibodies were examined in the sera of 74 myasthenia gravies patients by ELISA.Results AChR,Titin, RyR antibodies were detected in 77.0% (57/74),39.2% (29/74) and 32.4% (24/74) of the MG patients,respectively.For thymoma MG,AChR,Titin and RyR antibodies were detected in 76.2% (16/21),71.4% (15/21) and 52.4% (11/21) respectively.For late onset MG,Titin and RyR antibodies were detected in 77.3% (17/22) and 50.0% (11/22) respectively.With respect to the modified Osserman classification,the positve rate for Titin and RyR antibodies is much higher in more severe patients (X~2= 16.094,P=0.001;X~2=11.226,P=0.011).Titin antibodies was significantly related with RyR antibodies (r=0.380,P=0.001).Conclusions Titin and RyR antibodies show high sensitivity for thymoma MG,and the combination of serological and radiological testing can increase both sensitivity and specificity in diagnosis of thymoma MG.The levels of the two antibodies may serve as important prognosis markers in MG.The induction of the immune response against Titin and RyR and the possible pathogenic effects of the two antibodies will be further studied.