Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.
Aquaporins ; Aquaporin 1 ; Ascorbic Acid ; bcl-2-Associated X Protein ; Blood-Brain Barrier ; Brain ; Brain Edema* ; Brain Injuries ; Brain Ischemia* ; Caspase 3 ; Cell Death ; Claudin-5 ; Dehydroascorbic Acid* ; Edema ; Neurons ; Nitric Oxide Synthase Type II ; Oxidative Stress ; Post-Synaptic Density ; Stroke

O ⁶-carboxymethyl guanine(O ⁶-CMG) is a highly mutagenic alkylation product of DNA that causes gastrointestinal cancer in organisms. Existing studies used mutant Mycobacterium smegmatis porin A (MspA) nanopore assisted by Phi29 DNA polymerase to localize it. Recently, machine learning technology has been widely used in the analysis of nanopore sequencing data. But the machine learning always need a large number of data labels that have brought extra work burden to researchers, which greatly affects its practicability. Accordingly, this paper proposes a nano-Unsupervised-Deep-Learning method (nano-UDL) based on an unsupervised clustering algorithm to identify methylation events in nanopore data automatically. Specially, nano-UDL first uses the deep AutoEncoder to extract features from the nanopore dataset and then applies the MeanShift clustering algorithm to classify data. Besides, nano-UDL can extract the optimal features for clustering by joint optimizing the clustering loss and reconstruction loss. Experimental results demonstrate that nano-UDL has relatively accurate recognition accuracy on the O ⁶-CMG dataset and can accurately identify all sequence segments containing O ⁶-CMG. In order to further verify the robustness of nano-UDL, hyperparameter sensitivity verification and ablation experiments were carried out in this paper. Using machine learning to analyze nanopore data can effectively reduce the additional cost of manual data analysis, which is significant for many biological studies, including genome sequencing.
Deep Learning ; Guanine ; Nanopore Sequencing ; Nanopores ; Porins/genetics*

OBJECTIVETo investigate the expression of aquaporin (AQP)-1, 3, 8, 9 in human fetal membrane and their role in the human amniotic fluid circulation.

METHODSRT-PCR was employed for detection of the expressions of AQP-1, 3, 8, 9 mRNA in human amnion and chorion from 20 women with normal term pregnancy.

RESULTSAQP-1, 3, 8, 9 mRNA expression was detected in both human amnion and chorion, and no significant difference was found in their expression levels or between the amnion and chorion (P>0.05).

CONCLUSIONAQP-1, 3, 8, 9 can be associated with intramembranous transport and volume regulation of amniotic fluid.

Adult ; Amnion ; embryology ; metabolism ; Aquaporin 1 ; genetics ; Aquaporin 3 ; genetics ; Aquaporins ; genetics ; Chorion ; embryology ; metabolism ; Electrophoresis, Agar Gel ; Extraembryonic Membranes ; embryology ; metabolism ; Female ; Gene Expression Regulation, Developmental ; Humans ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction

Aquaporins (AQPs) are expressed in myocardium and the implication of AQPs in myocardial water balance has been suggested. We investigated the expression patterns of AQP subtypes in normal myocardium and their changes in the process of edema formation and cardiac dysfunction following myocardial infarction (MI). Immunostaining demonstrated abundant expression of AQP1, AQP4, and AQP6 in normal mouse heart; AQP1 in blood vessels and cardiac myocytes, AQP4 exclusively on the intercalated discs between cardiac myocytes and AQP6 inside the myocytes. However, neither AQP7 nor AQP9 proteins were expressed in CD1 mouse myocardium. Echocardiography revealed that cardiac function was reduced at 1 week and recovered at 4 weeks after MI, whereas myocardial water content determined by wet-to-dry weight ratio increased at 1 week and rather reduced below the normal at 4 weeks. The expression of cardiac AQPs was up-regulated in MI-induced groups compared with sham-operated control group, but their time-dependent patterns were different. The time course of AQP4 expression coincided with that of myocardial edema and cardiac dysfunction following MI. However, expression of both AQP1 and AQP6 increased persistently up to 4 weeks. Our findings suggest a different role for cardiac AQPs in the formation and reabsorption of myocardial edema after MI.
Animals ; Aquaporin 1/metabolism ; Aquaporin 4/metabolism ; Aquaporin 6/metabolism ; Aquaporins/*metabolism ; Edema/pathology ; Immunohistochemistry ; Mice ; Muscle Cells/metabolism ; Myocardial Infarction/*metabolism/pathology/ultrasonography ; Myocardium/metabolism/pathology ; Time Factors

OBJECTIVETo investigate the significance of aquaporin-1(AQP-1) and aquaporin-3(AQP-3) in the development of colorectal carcinoma.

METHODSThe expression of AQP-1 and AQP-3 was investigated using immunohistochemical staining with Streptavidin Peroxidase in tissues from colorectal adenoma (CRA, n=25), colorectal cancer (CRC, n=50), and adjacent mucosa (CRT, n=50).

RESULTSThe positive rate of AQP-1 was 64%(32/50) in CRC, significantly higher than that in CRT (38%, 19/50) and CRA(32%, 8/25)(P<0.05). The expression of AQP-1 was associated with depth of invasion and lymph node metastasis in CRC patients(P<0.05). The positive rate of AQP-3 was 56% in CRT, 44% in CRA, and 52% in CRC. There were no significant differences (P>0.05). The expression of AQP-3 was associated with age, tumor diameter, and depth of invasion (P<0.05). No significant correlation between the expression of AQP-1 and AQP-3 in CRC was shown by Spearman correlation analysis(P>0.05).

CONCLUSIONSAQP-1 expression is increased in CRC while the expression of AQP-3 is not. There is no correlation between the expression of AQP-1 and AQP-3 in CRC.

Adult ; Aged ; Aquaporin 1 ; metabolism ; Aquaporin 3 ; metabolism ; Colorectal Neoplasms ; metabolism ; Female ; Humans ; Male ; Middle Aged

OBJECTIVETo investigate the expression of aquaporin 3, 4, and 8 in the colonic mucosa of rat models with slow transit constipation (STC).

METHODSSTC rat model was established by giving the rats the compound solution of diphenoxylate. Real time polymerase chain reaction (RT-PCR) was used to measure the expression of aquaporin mRNA in colonic mucosa of STC rat models (study group,n=16) and normal rats (control group,n=16). Gray scale ratio of aquaporin to β-action (internal reference) was used for quantification.

RESULTSRT-PCR revealed that the mean gray scale ratios of aquaporin 3 in the proximal colon of the study group and control group were 0.344 and 0.602 (P<0.05), and were 0.419 and 0.509 in the distal colon (P>0.05), respectively. The mean gray scale ratios of aquaporin 4 in the proximal and the distal colon were 0.764 and 0.759 in the study group (P>0.05), and were 0.776 and 0.736 in the control group (P>0.05), respectively. However, there was no expression of aquaporin 8 in the proximal and the distal colon in either the study group or the control groups.

CONCLUSIONSExpression of aquaporin 3 in the proximal colon of STC rat models is down-regulated, which regulates water absorption. There are no significant changes in the expressions of aquaporin 4 and 8.

Animals ; Aquaporin 3 ; metabolism ; Aquaporin 4 ; metabolism ; Aquaporins ; metabolism ; Colon ; metabolism ; Constipation ; metabolism ; Disease Models, Animal ; Female ; Intestinal Mucosa ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the expressions of aquaporins (AQPs) in the mouse prostatic tissue and their significance, and to provide some evidence for a deeper insight into the physiological function and regulation of AQP expressions in normal and diseased prostatic tissues.

METHODSThe mRNA expressions of AQP0 - 4 in the mouse prostatic tissue were determined by RT-PCR, and the expressions and localizations of AQP1 and AQP3 proteins were characterized by Western blot and immunohistochemistry.

RESULTSRT-PCR exhibited the mRNA expressions of AQP1 and AQP3, but not those of AQP0, AQP2 and AQP4 in the prostate tissue, while Western blot showed the expression of the AQP1 protein with the relative molecular mass (RMM) of 28 000 and those of the glycosylated and non-glycosylated AQP3 proteins with the RMM of 35 000 and 27 000, respectively. Immunohistochemistry indicated the strong expression of AQP1 in the cyst and plasma membrane of the secretary cells and that of AQP3 in the stroma cells of the prostate.

CONCLUSIONThe AQP1 and AQP3 genes were expressed in the secretary epithelia of the mouse prostate tissue, which suggests that AQP1 and AQP3 may play an important role in the secretion of prostatic fluid.

Animals ; Aquaporin 1 ; genetics ; metabolism ; Aquaporin 3 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Prostate ; metabolism ; pathology

OBJECTIVETo construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB.

METHODSA pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting.

RESULTSRestriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli.

CONCLUSIONThe prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Molecular Sequence Data ; Neisseria gonorrhoeae ; genetics ; metabolism ; Plasmids ; Porins ; genetics ; metabolism

OBJECTIVETo construct a recombinant eukaryotic expression plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene, and evaluate the Ct MOMP-specific humoral and cellular immune response induced by pcDNA3.1-Ct MOMP168 in BALB/c mice.

METHODSRecombinant plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene was constructed. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1-Ct MOMP168 or pcDNA3.1 or PBS (n = 12, 100 microg/time per mouse), and the same immunization schedule was repeated for the third time at 2 week intervals. The titers of anti-Ct MOMP antibody and its antibody subtypes in sera, the cytotoxicity of Ct MOMP-specific cytotoxic T lymphocyte (CTL) in spleen, and the level of cytokine (IFN-gamma, IL-4, IL-10)-producing CD3(+) T cells in spleen were detected by ELISA, LDH release assays and intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS), respectively.

RESULTSThe recombinant plasmid pcDNA3.1-Ct MOMP168 was able to induce Ct-specific antibody response (A(490) = 0.973 +/- 0.136; serum titer was 1:1000) as compared with pcDNA3.1 (A(490) = 0.180 +/- 0.025) and PBS (A(490) = 0.110 +/- 0.015), and the major antibody subtype was IgG2a with statistical significance (F = 106.884, P < 0.05). When the ratio of effector cells and target cells reached to 50:1, the activity of cytotoxic T-lymphocyte in pcDNA3.1-Ct MOMP168 immunized mice (41.71% +/- 8.34%) was significantly higher (F = 22.315, P < 0.05) than that in pcDNA3.1 immunized mice (18.40% +/- 3.45%) and PBS immunized mice (14.50% +/- 2.42%). The levels of CD3(+) IFN-gamma(+) T cells in pcDNA3.1-Ct MOMP168 immunized mice (1.15% +/- 0.16%) were significantly higher (F = 99.638, P < 0.05) than that in pcDNA3.1 immunized mice (0.12% +/- 0.08%) and PBS immunized mice (0.09% +/- 0.03%), while the significant difference in the levels of IL-4(+) CD3(+) T cells and IL-10(+) CD3(+) T cells was not observed (F = 0.886 and 1.112, P > 0.05) between pcDNA3.1-Ct MOMP168 immunized mice (0.13% +/- 0.08% and 0.14% +/- 0.08%) and pcDNA3.1 (0.07% +/- 0.05% and 0.13% +/- 0.06%) or PBS immunized mice (0.08% +/- 0.04% and 0.07% +/- 0.04%).

CONCLUSIONIn BALB/c mice, the recombinant plasmid pcDNA3.1-Ct MOMP168 might induce not only the generation of Ct-specific antibody, but also the high level of Ct MOMP-specific CD3(+) IFN-gamma(+) T cells.

Animals ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Porins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a (+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD= 0.992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Neisseria gonorrhoeae ; genetics ; Plasmids ; biosynthesis ; genetics ; Porins ; biosynthesis ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; Vaccines, Synthetic