Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification / 生物医学与环境科学（英文）

Objective@#To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .@*Methods@#We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).@*Results@#CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.@*Conclusion@#The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Inactivation of Poliovirus by Ozone and the Impact of Ozone on the Viral Genome / 生物医学与环境科学（英文）

OBJECTIVE@#To investigate the mechanisms underlying ozone-induced inactivation of poliovirus type 1 (PV1).@*METHODS@#We used cell culture, long-overlapping RT-PCR, and spot hybridization assays to verify and accurately locate the sites of action of ozone that cause PV1 inactivation. We also employed recombinant viral genome RNA infection models to confirm our observations.@*RESULTS@#Our results indicated that ozone inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ozone specifically damaged the 80-124 nucleotide (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 lacking this region was non-infectious.@*CONCLUSION@#In this study, we not only elucidated the mechanisms by which ozone induces PV1 inactivation but also determined that the 80-124 nt region in the 5'-NCR is targeted by ozone to achieve this inactivation.

Effect of cadmium on mitochondrial function of hematopoietic stem cells in mouse bone marrow / 军事医学

Objective To explore the effect of cadmium chloride on mitochondrial function of hematopoietic stem cells in mouse bone marrow .Methods After being quarantined for one week , male Kunming mice weighted 20 ±2 g were randomly divided into three groups: control group , low dose cadmium-exposure group and high dose cadmium-exposure group.Mice in low dose and high dose cadmium-exposure groups were exposed to cadmium chloride solution at a dose of 7.5, 15 mg/kg body mass while those in control group were given an equal volume of distilled water through gavage administration every Monday , Wednesday and Friday for six consecutive weeks before cells in mouse bone marrow were collected at the 8th week.Mitochondrial membrane potential and ROS levels of mouse hematopoietic stem cells were detected using a flow cytometry .Results Compared with control group , the gain of body weight was significantly suppressed in cadmium-exposure group (P<0.01).Compared with control group, mitochondrial ROS levels of hematopoietic stem cells significantly increased in cadmium-exposure group and was dose-related(P<0.05,P<0.01). Besides, mitochondrial membrane potential of hematopoietic stem cells decreased in cadmium -exposure group compared with control group and was dose-related(P<0.05,P<0.01).Conclusion Cadmium exposure can lead to dose-related mitochondrial dysfunction of hematopoietic stem cells via oxidative damage in Kunming mice .

Tumor Targeting of Fluorescent Magnetic IR780-Fe3 O4 Nanoparticles for Detection of Circulating Tumor Cells / 分析化学

The tumor targeted fluorescent magnetic IR780-Fe3 O4 nanoparticles were prepared for separation and detection of circulating tumor cells ( CTCs ) . These IR780-Fe3 O4 nanoparticles were characterized by electron microscopy, fluorescence spectrometer, and superconducting quantum interferometer. The targeting effect of IR780-Fe3 O4 nanoparticles was analyzed on the tumor and normal cells by confocal microscope and flow cytometry, and the confocal microscope was used to target the location of IR780-Fe3 O4 nanoparticles in MCF-7 cells. The standard curve was drawn and evaluated accorded to the IR780-Fe3 O4 nanoparticles fluorescence intensity of tumor cells after incubation. The results showed that IR780-Fe3 O4 nanoparticles could target a variety of CTCs. Furthermore, cellular localization experiment proved that IR780-Fe3 O4 nanoparticles could target the mitochondria of tumor cells. With the method of coupling magnetic Fe3 O4 nanoparticles, IR780 could well distinguish the tumor and normal cells, which could be used for separating and detecting the CTCs in simulated blood.

Tumor Targeting of Fluorescent Magnetic IR780-Fe3 O4 Nanoparticles for Detection of Circulating Tumor Cells / 分析化学

The tumor targeted fluorescent magnetic IR780-Fe3 O4 nanoparticles were prepared for separation and detection of circulating tumor cells ( CTCs ) . These IR780-Fe3 O4 nanoparticles were characterized by electron microscopy, fluorescence spectrometer, and superconducting quantum interferometer. The targeting effect of IR780-Fe3 O4 nanoparticles was analyzed on the tumor and normal cells by confocal microscope and flow cytometry, and the confocal microscope was used to target the location of IR780-Fe3 O4 nanoparticles in MCF-7 cells. The standard curve was drawn and evaluated accorded to the IR780-Fe3 O4 nanoparticles fluorescence intensity of tumor cells after incubation. The results showed that IR780-Fe3 O4 nanoparticles could target a variety of CTCs. Furthermore, cellular localization experiment proved that IR780-Fe3 O4 nanoparticles could target the mitochondria of tumor cells. With the method of coupling magnetic Fe3 O4 nanoparticles, IR780 could well distinguish the tumor and normal cells, which could be used for separating and detecting the CTCs in simulated blood.

Changes of the mitochondrial DNA copy number and the antioxidant system in the PBMC of hepatocellular carcinoma / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the changes of the copy numbers of mtDNA in peripheral blood mono-nucle- ar cell(PBMC) and the disordered of antioxidant capacity of hepatocellular carcinoma (HCC) patients.</p><p><b>METHODS</b>The Ficoll Hypaque method was used to isolate the PBMC from blood specimens. The ND1 gene of the mitochondrial was amplified by real-time PCR; meantime β-actin was served as a quantitative standard marker; the difference of mtDNA copy number in PBMC was compared between HCC and healthy control group. The level of reactive oxygen species (ROS) in PBMC was determined by flow cytometry. The change of total antioxidant capacity (T- AOC) of plasma was detected by the biochemistry examination.</p><p><b>RESULTS</b>The copy numbers of ND1 gene in PBMC of HCC was 73% that of the healthy control group,which suggested a decrease of the copy numbers of mtDNA in HCC. The levels of ROS of PBMC in HCC was (417. 82 ± 110.62) and (301.82 ± 75.54) in control group, which showed that the levels of ROS of PBMC in HCC were significant higher than that in control group (P < 0.01).Plasma T-AOC in HCC was (1.30 ± 0.85), and (3.20 ± 1.62) in control. The T-AOC of plasma of HCC was significantly lower than in control group (P < 0.01).</p><p><b>CONCLUSION</b>There was a certain relationship between the decrease of the copy numbers of mtDNA and the disordered antioxidant capacity in hepatocellular carcinoma, which may be associated with the development of hepatocellular carcinoma.</p>

Antiviral effects of aqueous extract from Spatholobus suberectus Dunn. against coxsackievirus B3 in mice / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the antiviral effects of the aqueous extract of Spatholobus suberectus Dunn. (A.E.), a Chinese medicinal herb, against coxsackievirus B3 (CVB3).</p><p><b>METHODS</b>The antiviral effects of A.E. against CVB3 in vitro (primarily cultured myocardial cells) and in vivo (BALB/c mice) were determined. Serum pharmacological method was also adopted by in vitro experiments. The effects of A.E. inhibiting the CVB3 mRNA expression were compared by RT-PCR in mice in vivo.</p><p><b>RESULTS</b>A.E. exhibited obvious antiviral: effects in vivo, and serum samples obtained from the rats with oral administration of A.E. (10 μg/mL, 5 μg/mL), reduced the virus titers in the infected myocardial cells (3.00±0.70, 3.55±0.52, P<0.01). Meanwhile, the viral myocarditis induced by CVB3 was inhibited significantly by A.E., and the 15-day mortality was reduced to 40% and 45% (P<0.01) in mice treated with A.E. at doses of 50 mg/kg and 100 mg/kg, respectively, while the 30-day mortality was decreased to 45% and 50%, respectively (P<0.01). Moreover, the mRNA expression of Coxsackie virus B3 was significantly inhibited by A.E.</p><p><b>CONCLUSION</b>Aqueous extract of Spatholobus suberectus Dunn. (A.E.) has inhibitory effect on CVB3 both in vitro and in vivo.</p>

Expression of coxsackievirus and adenovirus receptor in non-small cell lung cancer and its significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and proten expression of coxsackievirus and adenovirus receptor (CAR) in the corresponding normal lung tissue, para-neoplastic tissue and lung cancer tissue, and the correlation of CAR expression with the carcinogenesis as well as the expression difference in various clinicopathologic parameters.</p><p><b>METHODS</b>The expression of CAR mRNA and protein in the samples from 32 lung cancer patients was determined by RT-PCR and Western blot, respectively.</p><p><b>RESULTS</b>The expression level of CAR mRNA and protein in normal lung tissue, paraneoplastic tissue and cancer tissue were 1.000 +/- 0.012, 1.048 +/- 0.035, 1.282 +/- 0.072, and 0.902 +/- 0.038, 0.944 +/- 0.042, 1.08 +/- 0.052, respectively, with a statistical significance among the groups (P = 0.022, P = 0.007, P = 0.009, P = 0.027). There was a statistically significant positive correlation between expression of CAR mRNA and that of CAR protein (r = 0.448, P = 0.026). The expression levels of CAR were significantly different among different pathological types (P = 0.012), with a high level of CAR in all 7 bronchiolo-alveolar carcinoma (BAC, P = 0.029). However, there was no statistical significance in other clinicopathologic parameters (P > 0.05), including gender, age, smoking or not, tumor size, with or without lymph node metastasis and TNM stage.</p><p><b>CONCLUSION</b>The expression of CAR mRNA and protein in cancer tissue samples are significantly higher than that in the normal and paraneoplastic samples, indicating that CAR might play a crucial role in the carcinogenesis. It may become a new potential prognostic marker for lung cancer patients.</p>

Impacts of passive smoking on learning and memory ability of mouse offsprings and intervention by antioxidants / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To determine the impact of passive smoking and the protective effect of antioxidants such as vitamin E and quercetin on learning and memory ability of mouse offsprings.</p><p><b>METHODS</b>A passive smoking model of pregnant mice was established. Learning and memory ability was evaluated by the water maze test and long term potentiation (LTP). Nitric oxide (NO), content, nitric oxide synthase (NOS), acetylcholinesteras (Ache) activity in brain, vitamin E concentration, and reactive oxygen species (ROS) in serum were determined. The latency period (the time during which the mice swim from the starting position to the ending position) and errors (the number of mice entering the blind end) in control and antioxidant intervention groups were compared with those in the smoke exposure group after 6 days.</p><p><b>RESULTS</b>The latency period as well as errors in the air, control diet, tobacco smoke (TS), and vitamin E diet groups were decreased significantly as compared with the TS and control diet groups (P<0.05). LTP was restrained in the TS and control diet groups. LTP in all the antioxidant diet groups was significantly increased compared with the TS and control diet groups. In addition, NOS and acetylcholinesteras (Ache) activitiy was significantly higher in the TS and control diet groups than in the air and control diet group. NO content was not significantly different among the different groups, and significantly lower in the TS and vitamin E diet groups than in the TS group, control diet group, quercetin diet group, and mixture diet group (P<0.05). Vitamin E concentration and ROS activity in serum were correlated with the outcome of water maze and LTP.</p><p><b>CONCLUSION</b>Passive smoking reduces LTP formation by disturbing the hippocampus function of mice, by decreasing NOS and Ache activity and increasing NO content. Antioxidants (especially vitamin E) partially improve the learning and memory ability of offsprings whose mothers are exposed to tobacco smoke during pregnancy.</p>

Microarray Application in Environmental Microbial Community Research / 微生物学通报

Microarray technology, used in microorganisms detection with its advantages of rapid detection, high sensitivity, high-throughput and low cost, has been applied in environmental microbial community research widely in past few years. It focuses on investigation of structure, diversity, function, dynamics of microbial populations within complex environmental samples. Furthermore, it also reveals their responses and adaptation to environmental perturbations such as climate change, toxic contaminants. According probe design patterns, several types of microarrays, such as phylogenetic oligonucleotide arrays (POAs), functional gene arrays (FGAs), metagenomic array(MGA) and community genome arrays (CGAs) have been constructed for environmental studies. This review discusses applications of microarrays to environmental microbial populations research along with its potential for screening of specific microorganisms, gene or expression functional gene representing different environmental microbial populations.

Directed molecular evolution of nitrite oxido-reductase by DNA-shuffling / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment.</p><p><b>METHODS</b>The norB gene coding the ndtrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria.</p><p><b>RESULTS</b>After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L x d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.</p><p><b>CONCLUSION</b>DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.</p>

Detection of RNA of SARS coronavirus in hospital sewage / 中华预防医学杂志

<p><b>OBJECTIVE</b>In order to explore the existence of SARS coronavirus (Co-V) and/or its RNA in sewage of hospitals administered SARS patients.</p><p><b>METHODS</b>A novel electropositive filter was used to concentrate the SARS-CoV from the sewage of two hospitals administered SARS patients in Beijing, including twelve 2,500 ml sewage samples from the hospitals before disinfection, and ten 25,000 ml samples after disinfection; as well as cell culture, RT-PCR and sequencing of gene to detect and identify the viruses from sewage.</p><p><b>RESULTS</b>There was no live SARS-CoV detected in the sewage in this study. The nucleic acid of SARS-CoV had been found in the 12 sewage samples before disinfection from both hospitals by semi-nested PCR. After disinfection, SARS-CoV RNA could only be detected from the samples from the 309th Hospital, and the others were negative.</p><p><b>CONCLUSION</b>It provides evidence that there is no live SARS-Cov in the sewage from hospitals with SARS patients though SARS-CoV RNA can be detected.</p>

A study on detecting and identifying enteric pathogens with PCR / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a rapid and definite diagnostic test of bacterial enteritis caused by pathogenic enterobacteria, the most frequent etiologic agent of infectious enteritis in the world.</p><p><b>METHODS</b>A set of conventional PCR assays were applied to detect and identify salmonella, shigella, and E. coli O157:H7 directly from pure culture and fecal samples. The general primers of pathogenic enterobacteria were located on the uidA gene, which were found not only in E. coli nuclear acid, but also in shigella and salmonella genes. Shigella primer was from ipaH gene whose coded invasive plasmid relative antigen existed both in plasmid and in genome. The primers of salmonella were designed from the 16SrRNA sequence. The primer of E. coli O157:H7 was taken from eaeA gene. Five random primers were selected for RAPD. The detection system included common PCR, semi-nested PCR and RAPD.</p><p><b>RESULTS</b>This method was more sensitive, specific and efficient and its processing was rapid and simple. For example, the method could be used to specifically detect and identify salmonella, shigella, and E. coli O157:H7, and its sensitivity ranged from 3 to 50 CFU, and its detection time was 4 hours.</p><p><b>CONCLUSION</b>This PCR method, therefore, can serve as a routine and practical protocol for detecting and identifying pathogenic microorganisms from clinical samples.</p>

Pseudonomas sp.W2 Metabolic Pathway of Bisphenol A / 微生物学通报

With GC-MS、LC-UV and gene analysis,we studied Pseudonomas sp.W2 metabolic pathway of bisphenol A(Bpa).It was discovered that 4'-(trimethylsiloxy)-Acetophenone、p-Hydroxy benzaldehyde and p-Hydroxy benzoic acid are medium metabolites and that the bacteria has pcaG.

Study on Escherichia coli Growth Stimulated by Yeast Extract and Echlonia kurome Okum Water Extract / 微生物学通报

The means of OD value measurement and plate counting were used to choose the bacterial growth stimulants. Among the 120 substances sorted to 13 kinds (the trace elements, REE, carbohydrate, amino acids and amino acids derivatives, vitamins, nucleosides, plant hormones, animal hormones, plant and animal extracts, Echlonia Kurome Okum water extract and yeast extract), Echlonia Kurome Okum water extract and yeast extract were found to stimulate the growth of Escherichia coli significantly.