Next-generation sequencing combined with serological tests based pathogen analysis for a neurocysticercosis patient with a 20-year history:a case report.

BACKGROUND: Neurocysticercosis (NCC) is the most common helminthic infection of the central nervous system (CNS) caused by the larval stage of Taenia solium. Accurate and early diagnosis of NCC remains challenging due to its heterogeneous clinical manifestations, neuroimaging deficits, variable sensitivity, and specificity of serological tests. Next-generation sequencing (NGS)-based pathogen analysis in patient's cerebrospinal fluid (CSF) with NCC infection has recently been reported indicating its diagnostic efficacy. In this case study, we report the diagnosis of a NCC patient with a symptomatic history of over 20 years using NGS analysis and further confirmation of the pathology by immunological tests. CASE PRESENTATION: This study reports the clinical imaging and immunological features of a patient with a recurrent headache for more than 20 years, which worsened gradually with the symptom of fever for more than 7 years and paroxysmal amaurosis for more than 1 year. By utilizing NGS technique, the pathogen was detected in patient's CSF, and the presence of Taenia solium-DNA was confirmed by a positive immunological reaction to cysticercus IgG antibody in CSF and serum samples. The symptoms of the patient were alleviated, and the CSF condition was improved substantially after the anti-helminthic treatment. CONCLUSIONS: This study suggests that combining CSF NGS with cysticercus IgG testing may be a highly promising approach for diagnosing the challenging cases of NCC. Further studies are needed to evaluate the parasitic DNA load in patients' CSF for the diagnosis of disease severity, stage, and monitoring of therapeutic responses.

Apoptotic Effect of 1800 MHz Electromagnetic Radiation on NIH/3T3 Cells.

To investigate the effect of 1800 MHz electromagnetic radiation (EMR) on apoptosis, we exposed NIH/3T3 cells at 1800 MHz with a specific absorption rate (SAR) of 2 W/kg intermittently for 12, 24, 36, and 48 h. After exposure, Cell Counting Kit-8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis; the expression of p53, a molecule with the key role in apoptosis, was measured by real-time qPCR, western blot, and immunofluorescence; and images of the structure of the mitochondria, directly reflecting apoptosis, were captured by electron microscopy. The results showed that the viability of cells in the 12, 36, and 48 h exposure groups significantly decreased compared with the sham groups; after 48 h of exposure, the percentage of late apoptotic cells in the exposure group was significantly higher. Real-time qPCR results showed that p53 mRNA in the 48 h exposure group was 1.4-fold of that in the sham group; significant differences of p53 protein fluorescence expression were observed between the exposure groups and the sham groups after 24 h and 48 h. The mitochondrial swelling and vesicular morphology were found in the electron microscopy images after 48 h exposure. These findings demonstrated 1800 MHz, SAR 2 W/kg EMR for 48 h may cause apoptosis in NIH/3T3 cells and that this apoptosis might be attributed to mitochondrial damage and upregulation of p53 expression.

Recombinant DENV 2 NS5: An effective antigen for diagnosis of DENV infection.

Dengue fever is a mosquito-borne viral disease with dramatically increasing morbidity rate worldwide in decades. Since there is no specific treatment to date, early diagnosis is important for providing proper timely medical care to minimize mortality, and for the prompt initiation of public health control measures. NS5 is a potential biomarker for dengue virus infection due to its highly conserved and immunogenic properties. In this study, the DENV 2 NS5 full-length and the DENV 2 NS5 C-terminus RNA-dependent RNA polymerase domain fragment (NS5-C70) expression plasmids were constructed, and the 104 kDa full-length NS5 and the 70 kDa NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products were found to react with the sera of patients infected with dengue virus when analyzed by an enzyme-linked immunosorbent assay (ELISA), which resulted in significantly higher absorption values than those of control sera. The recombinant DENV 2 NS5 exhibited strong reactivity to each of the four types of sera, whereas the NS5-C70 showed strong reactivity only to DENV 2 and 4. In comparison, the positive agreement value of recombinant NS5-based assay with either MyBioSource or Panbio assay was higher than that of the two commercially available IgG indirect ELISA kits. These results suggest that the recombinant DENV 2 NS5 be an effective antigen for detection of dengue virus infection. The recombinant NS5-C70 may also be used as an auxiliary antigen for diagnostic purposes.

Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene.

The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747µM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents.

Tanshinol protects hippocampus and attenuates vascular dementia development.

Tanshinol (3-(3',4'-dihydroxyphenyl)-(2R)-lactic acid, TSL) is widely used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. Here, we assessed whether TSL protected hippocampus and attenuated vascular dementia (VD) development in rats. The behavioral analysis showed that TSL could decrease the distance and latency time, and increase the swim speed in water maze in rats subjected to VD. TSL remarkably increased acetylcholine level and decreased acetylcholinesterase activity in rats subjected to VD. Likewise, TSL remarkably decreased malondialdehyde and increased superoxide dismutase levels in rats subjected to VD. Furthermore, treatment with TSL reduced the level of dead neurons in dentate gyrus. In addition, TSL upregulated growth-associated protein 43 (GAP43) and vascular endothelial growth factor (VEGF) expression and downregulated phosphorylated Akt (p-AKt) and phosphorylated glycogen synthase kinase (p-GSK3ß) expression in hippocampus in rats subjected to VD. These results suggest that TSL may be a potential compound in VD model.

[Inhibition of HIV-1 in vitro by combination of vpr and tat specific short hairpin RNA via lentiviral vectors].

Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.

[Investigation on inhibition of HIV III B virus with extractions of Juglans regia].

OBJECTIVE: To study the effect of anti-HIV III B virus with extractions from Juglans regia, so as to searching for the new and efficacious leading compound of AIDS therapy. METHOD: Phytochemical and chromatographical techniques were used to isolate compounds from J. regia; MT4 cells and HIV III B virus were used to study the effect of anti-HIV activity in vitro. BIACORE 3000 molecule coupled equipment were used for the target research also. RESULT: Two extractions (B&E) were isolated from J. regia which possess the effect of anti-HIV activity. Targets study found that extraction B could affected on HIV-1 gp-41 fusing protein and extraction E could affected on HIV-1 integrase respectively. CONCLUSION: J. regia is a traditional Chinese medicine with active anti-HIV activity and worth to be studied further.

[Subcellular localization of APOBEC3G by confocal laser scanning microscope (CLSM)].

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) cDNA was amplified from total RNA prepared from nonpermissive H9 cells by RT-PCR. APOBEC3G cDNA is 1155nt long, encoding 384 amino acids. The APOBEC3G gene was then cloned into the eukaryotic expression vector pEGFP-C3. The generated pEGFP-3G construct was then transfected into CD4+ HeLa cell to determine the expression and the subcellular localization of GFP-APOBEC3G fusion protein. Under CLSM the localization of the expressed GFP-APOBEC3G in the cytoplasm of CD4+ HeLa cells was observed.

[Comparative evaluation of Hebei HIV-1 p24 kit for the detection of human immunodeficiency virus].

OBJECTIVE: To probe into the feasibility of screening anti-HIV compounds by using HIV-1 p24 detection kit made by Hebei Medical University. METHODS: The sensitivity, reproducibility and efficacy of the Hebei p24 kit were evaluated compared with the commercially available Vironostika HIV-1 Antigen Microelisa System (Biomerieux). RESULTS: Hebei p24 kit had high sensitivity and good reproducibility. In vitro screening demonstrated that there was no statistically significant difference (P greater than 0.05) between these two kits in assessing anti-HIV compounds. CONCLUSION: Hebei p24 kit could be used as an easily affordable alternative method for detection of HIV-1 in screening anti-HIV compounds.

[Recent progress on anti-HIV research of traditional Chinese medicine and components].

This paper summarized the recent 6 years' progress of anti-HIV compounds and traditional Chinese medicines by searching international network and reviewing the domestic and foreign literature. Traditional Chinese medicinal appeared to be a rich source of potentially useful materials for the treatment of human immunodeficiency virus infection. Some of them are much more potent in anti-HIV activity. And some components extracted from the herbs are even more tonic than the crude herb medicines. It has been proved that some active components such as alkaloids, proteins, flavonoids, quercetin, terpene, lignanoid are able to work on anti-HIV. People should pay more attention to the study of traditional Chinese medicine and the leading compounds on anti-HIV/AIDS in the clinic and in the laboratory. So searching for high efficacy and low toxicity anti-HIV drug from traditional Chinese medicine is an important and prospective research direction in the future.

[Immortalization of human embryonic cervical epithelial cells induced by E6, E7 genes of human papillomavirus 16].

OBJECTIVE: To establish an immortalized cell line derived from the embryonic cervical epithelium by infection with the recombinant adeno-associated virus (rAAV) containing human papillomavirus (HPV)16 E6, E7, and to study the biological features of cervical cancer cell line. METHODS: Human embryonic cervical tissues were cultured in keratinocyte free serum (K-FS) medium and infected with rAAV containing HPV16 E6, E7. Morphological features and growth rate were examined by light, electronic and fluorescence microscopies. The fragments of E6, E7 were detected by polymerase chain reaction (PCR) and laser confocal microscopy. The biological characteristics of human cervical epithelium were observed by soft agar culture, scid mice inoculation and chromosome analysis. Cell proliferative dynamics was plotted by flow cytometry. RESULTS: After a long-term culture, the phenotype kept the characteristics of primary epithelial cells. They showed monolayer, anchorage-dependent and attachment-inhibited growth without forming colonies in soft agar culture. They were non-oncogenic when inoculated into scid mice. The tonofilament expression in the cervical cancer cells was inspected by electronic microscopy, demonstrating that the cells were squamous epithelium in origin. The cell line contained HPV16 E6, E7 genes by PCR and laser confocal detection. Chromosome analysis disclosed that the karyotype was diploid or polyploid. The 11th chromosome was assumed to be the integration site by rAAV containing HPV16 E6, E7. CONCLUSIONS: Establishment of the immortalized cervical epithelial cell line by infection with rAAV containing HPV16 E6, E7, supports that HPV16 E6, E7 may be the primary etiology of cervical cancer. It will facilitate further research on the etiology and pathogenesis of cervical cancer.

[Construction and analysis of activity of an HIV-1/bovine immunodeficiency virus chimeric clone cDNA].

OBJECTIVE: Chimeric human/bovine immunodeficiency virus (HBIV) cDNA was constructed by replacing HIV tat and LTR with bovine immunodeficiency virus (BIV) tat and LTR to study the activity of BIV tat and LTR in the chimerae. METHODS: The target fragments of BIV tat, LTR and HIV gag, pol, env were respectively amplified by using PCR and sequentially inserted into pBluescript SK(+) vector. The chimeric clone was transfected into human MT4 cells. The transcript and gene expression of the HBIV chimeric virus were detected by using RT-PCR and a reverse transcriptase assay, respectively. RESULTS: BIV tat mRNA and HIV gag mRNA were detected. The reverse transcriptase activity of the chimeric virus was analyzed in the fluctuation curve. CONCLUSIONS: In chimeric HBIV cDNA transfected MT?4 cells, BIV tat and HIV gag were transcripted. The reverse transcriptase of the chimeric virus had biological activity. These data suggest that in MT4 cells, BIV LTR had promoter activity and BIV tat had the function of transactivation in the chimeric virus. The study of the chimeric virus with infectivity is in progress.