Clinical features of twenty-three cases of adenoviral encephalitis in children / 中华传染病杂志

Objective:To investigate the clinical features of adenoviral encephalitis (AE), and to provide reference for clinical diagnosis and treatment of adenoviral encephalitis.Methods:From January 2012 to December 2020, 1 185 cerebrospinal fluid (CSF) samples of hospitalized children with suspected central nervous system infection in the Second Affiliated Hospital of Shantou University Medical College were collected for the detection of 22 common respiratory pathogens and common pathogens for encephalitis by polymerase chain reaction. Records of patients with adenovirus positive in CSF were reviewed and relevant clinical manifestations, laboratory tests and imaging examination results were collected for analysis.Results:Among 1 185 CSF samples, 242 samples were positive for viral nucleic acid, with detection rate of 20.4%, including 1.9%(23/1 185) of adenovirus. As for 23 children diagnosed with AE, 18 were male, five were female, with the age of (44.8±35.9) months, ranging from two months and 19 days to 10 years. Of 23 children, 21(91.3%) presented with fever, followed by convulsions (16 cases, 69.6%), headache (four cases, 17.4%), vomiting (11 cases, 47.8%), consciousness change (11 cases, 47.8%) and emotion disturbance (three cases, 13.0%). Among 23 children, eight cases had white blood cell counts (WBC) of (6 to <10)×10 9/L, 10 cases had WBC of (10 to 20)×10 9/L and the white blood cell classification was mainly neutrophils (21 cases, 91.3%), and C reactive protein of 20 cases (87.0%) was in the normal range. Cerebrospinal fluid examination showed that WBC were less than 15×10 6/L in 20 cases (87.0%), and WBC ≥15×10 6/L in three cases, which were up to 500×10 6/L; the protein of 19 cases was in the normal range, the glucose of 15 cases was in the normal range, and the chloride of 19 cases was in the normal range. Among 16 cases with brain magnetic resonance imaging examination, eight cases did not show abnormality, six cases with local meningeal linear enhancement, one case with small intracranial malacia, and one case with extensive intracranial lesions. For 13 cases who received electroencephalogram (EEG) test, seven cases showed normal EEG or marginal state, four cases showed extensive medium and high amplitude slow wave, one case showed spike wave or spike slow wave and one case had both of the above two changes. Among 23 children, 22 cases recovered including one case had secondary epilepsy, and the remaining one case had severe brain dysfunction and was unable to suck when discharged, with an indwelling gastric tube and accompanied by secondary epilepsy. Conclusions:The clinical manifestations and auxiliary examinations of children with AE have no obvious specificity. Most children with AE have a good prognosis, but a small number of them may have serious sequelae.

MicroRNA-206 Reduces Osteosarcoma Cell Malignancy In Vitro by Targeting the PAX3-MET Axis

PURPOSE: This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development. MATERIALS AND METHODS: Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation, metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threonine phosphorylation of Erk1/2, respectively. RESULTS: Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significant decreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or MET overexpression, but only partially attenuated by HGF treatment. CONCLUSION: miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.

Posterior debridement, interbody fusion, internal fixation for treatment of lumbar discitis / 中国骨伤

<p><b>OBJECTIVE</b>To evaluate the clinical effects of posterior debridement, interbody fusion with internal fixation in the treatment of lumbar discitis.</p><p><b>METHODS</b>The clinical data of 13 patients with lumbar discitis treated from January 2005 to June 2012 was retrospectively analyzed. There were 9 males and 4 females, aged from 31 to 68 years old with an average of 56 years old. There were 2 cases on L3, 4, 4 cases on L4, 5, and 7 cases on L5S1. Two cases complicated with diabetes, 4 cases with hypertension, and 1 case with obsolete pulmonary tuberculosis. ESR level of 13 cases was 12-89 mm/h with an average of 42 mm/h; and C reactive protein fluctuations level was level 8-114 ng/L with an average of 47 ng/L. All the patients denied history of operation or injection, and the main symptom was severe pain and limitation of motion in lumbar, with no efficacy for conservative methods. Preoperative VAS was from 5 to 10 points with an average of 7.8 points. All patients were treated with posterior debridement, interbody fusion, and internal fixation.</p><p><b>RESULTS</b>All the patients left hospital after wound healing, and the effective antibiotics were continuously used for 4 weeks intravenously and 2 weeks for orally. All patients were followed up from 7 to 24 months with an average of 18 months. VAS decreased for 0-1 point. No internal fixation breakage, and recurrence were found. Bone graft got fusion, and postoperative pathology showed phlogistic changes.</p><p><b>CONCLUSIONS</b>One-stage posterior debridement, interbody fusion with internal fixation was an effective method in treating lumbar discitis, and it lead to quicker relived pain relief and earlier mobilization.</p>

Antibody-drug conjugates and their application in the treatment of hematological malignancies / 药学学报

Monoclonal antibody-targeted therapy has been a hot spot in current clinical cancer treatment. As current antibody drugs have large molecule sizes leading to poor tissue penetration, and high dosage in clinical application leading to high cost, to overcome the problems, the development of new antibody drugs with miniaturization and high potency has become a new trend. In recent years, the conjugates of monoclonal antibodies and cytotoxins, called antibody-drug conjugates (ADCs), have entered the arsenal of anti-cancer drugs, becoming a new format of antibody drugs and attracting extensive attentions. The ADC molecule usually consists of antibody, linker and effector molecule. According to different effector molecules, ADCs can be divided into three categories as chemo-conjugates, immunotoxins and radio-conjugates. When ADC molecules are internalized into cancer cells, cytotoxins will be released by chemical, enzyme degradation or by action of lysosomal proteases, then kill targeted cells by inhibiting protein synthesis, depolymerizing microtubules or breaking double-strand DNA. Recently, two ADC drugs have been approved by the US FDA and more ADC drug candidates are in clinical phase II or III trials which show significantly clinical effects and attracting much attention and competition of pharmaceutical enterprises. In this review, antibody conjugates in the past and present will be summarized and the future development trends and challenges of this type of antibody drugs will be discussed.

Development of antibody drugs targeting against HER2 for cancer therapy / 药学学报

Human epidermal growth factor receptor 2 (HER2) belongs to the transmembrane glycoprotein receptor family. Overexpression of HER2 could directly lead to tumorigenesis and metastasis. This phenomenon could be observed in the breast cancer, ovarian cancer, gastric cancer, lung cancer and prostate cancer. Compared with the conventional chemotherapy, the targeted treatment of antibody is more specific and has lower side effects. This review describes the current status of monotherapy and combination therapies of anti-HER2 antibodies, trastuzumab and pertuzumab, with chemotherapeutic drugs. The development trends of new formats of anti-HER2 antibody drugs such as bispecific antibody, immunotoxin are also discussed.

Development of therapeutic antibodies for gastric and colorectal cancers / 浙江大学学报·医学版

With the elucidation of structures and functions, antibodies are widely applied in the diagnosis and treatment of diseases. Today, therapeutic antibodies have played ever increasing roles in the treatment of cancers. In fact, there are over 20 monoclonal antibodies which have been approved by the U.S.Food and Drug Administration (FDA) for the therapeutic use in cancers. For the gastric and colorectal cancers, there are at least 9 antibodies have been approved for cancer therapy or for clinical trials. These antibody drugs target to tumor associate antigens and can destroy the cancer cells through several mechanisms such as antibody-dependent cell cytotoxicity, complement-dependent cytotoxicity, blockage of blood nutrition and crucial signaling pathways. With the progress in gene engineering technology, the diverse structures of antibodies can be created. In addition, the antibody-conjugates with radioisotopes, toxins and cytotoxins, are also designed for targeted therapy of gastric and colorectal cancers. In this article, we review the trends in the clinical development and application of antibody drugs for future research and development of the rapidly expanding therapeutic modality in gastric and colorectal cancers.

Expression of recombinant ribosome inactivating protein MAP30 in E.coli and its biological activity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon), and to evaluate the biological activity of the recombinant protein.</p><p><b>METHODS</b>The DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR, the target DNA fragments were sequenced after T-A cloning. The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a. MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L. The recombinant MAP30 was identified by SDS-PAGE, and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.</p><p><b>RESULT</b>The nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30. The solubility of recombinant protein was analyzed by SDS-PAGE, and the MAP30 was mainly produced in soluble form. The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.</p><p><b>CONCLUSION</b>The gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.</p>

Analysis on bioactivity of HIV-1 integrase by ELISA method / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop an ELISA-based method for analyzing biologic activities of HIV-1 integrase and for high throughput screening of integrase inhibitors.</p><p><b>METHODS</b>After expression, renaturation and purification of integrase, the bioactivity of integrase and the inhibition of luffin-a were evaluated with an in vitro assay based on biotin-avidin EILSA and chemiluminescent substrates.</p><p><b>RESULT</b>(1) The specific activity of the purified integrase was 54.92 units/mg of protein. (2)IC(50) (concentration causing 50% inhibition of integrase) of luffin-a was (0.63 +/- 0.026) micromol/L.</p><p><b>CONCLUSION</b>The non-radioactive assay can be used for analysis of bioactivities and high throughput screening of inhibitors of HIV-1 integrase.</p>

Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana / 生物工程学报

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.

Progress in Research on HIV-1 Integrase and Its Inhibitors / 中国生物工程杂志

HIV-1 integrase enzyme is a 32kDa protein encoded by HIV pol gene. It is responsible for integration of viral cDNA into host chromosomal DNA, which is indispensable for HIV replication.Since there was no functional equivalent for this enzyme in human cells, inhibition of integrase will bring little side effect to human body. Thus HIV integrase has become an attractive and rational target for therapy of AIDS after reverse transcriptase and protease.The Recent research on HIV-1integrase structure,inhibitors and new therapeutic method target at HIV integrase was reviewed.

Study on lysosomes degradation of ricin A chain / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study lysosomes involvement in the degradation of ricin A chain.</p><p><b>METHODS</b>A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B. The cytotoxicity of recombinant proteins was measured by the MTT method.</p><p><b>RESULTS</b>Recombinant RTA-KFERQ was 49.87%, 54.18% and 88.68% less cytotoxic than RTA itself on the three cell lines HEPG2, Hela and A549, respectively.</p><p><b>CONCLUSION</b>Lysosomes can degrade, but not completely inactivate RTA in different cells, suggesting cells may have other degradation pathways for RTA.</p>

Purification and anti-cancer activity of ricin / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To extract and purify ricin from castor beans and to evaluate its anti-cancer activity.</p><p><b>METHODS</b>Ricin was purified from castor beans according the modified method of Nicolson and Blaustin. The lectins were extracted in 0.01 mol/L phosphate buffered saline and isolated in the 40% to 80% fraction of ammonium sulfate precipitation. The dialyzed fractionated preparation was applied with a Sepharose 4B column. The lectins were eluted with a linear lactose gradient (0.01 mol/L approximately 0.5 mol/L). Ricin was separated from the ricinus agglutinin by gel filtration on a Sephadex G-100. MTT was applied to analyze the cytotoxicity with different dosage of ricin in different cancer cell lines.</p><p><b>RESULTS</b>There was no difference between the killing effect of normal cells and that of colon cancer cells by using the high dosage of ricin (5 x 10(-8) mol/L approximately 5 x 10(-10) mol/L). However, the cytotoxicity was significant different in those cells with the low dosage of ricin (5 x 10(-11) mol/L approximately 5 x 10(-13) mol/L). Meanwhile ricin had the similar cytotoxicity to leukemia cell K562 and colon cancer cell SW480.</p><p><b>CONCLUSION</b>Ricin is able to kill tumor cells selectively at low concentration, but the selectivity does not appear at high concentrations.</p>

Identification of peptides binding to Pisum sativum agglutinin from a phage-displayed random peptide library / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To obtain peptides binding specifically to Pisum sativum agglutinin (PSA) from a phage-displayed random peptide library.</p><p><b>METHODS</b>(1) A phage-displayed random hexapeptide library was screened with PSA as target. (2) Dot blot was used to analyze the influence of the alpha-Met-D-mannoside on binding between PSA and phage-displayed peptides. (3) Three peptides (RMWSF, RYDYSY, LRLRQL) were selectively synthesized, and different concentrations were used to inhibit PSA and ConA binding to the HRP.</p><p><b>RESULTS</b>The enrichment occurred obviously after three rounds of screening. The insert sequences of amino acids, displayed on 22 phage DNAs from the third round of screening, were divided into three groups. The binding of phage-displayed peptides to PSA was specific as shown by dot blot and could be inhibited by alpha-Met-D-mannoside. LRLRQL was not dissolved in water. ARMWSF and RYDYSY inhibited binding of PSA to HRP, but failed to inhibit binding ConA to HRP.</p><p><b>CONCLUSION</b>The binding site of peptides ARMWSF and RYDYSY is different to that of alpha-Met-D-mannoside.</p>

Construction and expression of ricin A chain and green fluorescent protein fusion gene in E. coli / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the expression and purification of a fusion protein of ricin A chain (RTA) and green fluorescent protein (GFP).</p><p><b>METHODS</b>The DNA sequence encoding ricin A chain was inserted into pEGFPC1 first to make the template sequence of the fusion protein. The fusion gene was amplified from the plasmid pEGFP-RTA by PCR, and directly subcloned into T vector. The fusion gene then was cloned into expression vector pET-28a(+), and the sequence was confirmed by sequencing. Expression was induced by IPTG in E. coli BL21(DE3). The fusion protein was purified by metal chelated affinity chromatography. The cytotoxicity of fusion protein was analyzed by the MTT assay in HepG2 and Hela cells.</p><p><b>RESULTS</b>The fusion protein of ricin A chain and GFP could be produced in E. coli transformed with the expression plasmid of pET-28a(+)-GFP-RTA. The molecular weight of the recombinant protein was measured by SDS-PAGE. The fusion protein showed a green fluorescence and had a similar cytotoxicity of RTA.</p><p><b>CONCLUSION</b>A recombinant fusion protein of RTA and GFP expressed in E. coli is possessed of similar biological activity of individual GFP and RTA, which could be used in study of the intracellular trafficking and translocation of RTA.</p>

Cloning and expression of luffin-a gene from the seeds of Luffa cylindrical / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To clone luffin-a cDNA from the seeds of Luffa cylindrical, and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a (+) in E. coli.</p><p><b>METHODS</b>The cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. The target DNA fragments were sequenced after T-A cloning. The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a (+). Luffin-a was expressed in E. coli by addition of IPTG into final concentration 1.0 mmol/L. The recombinant luffin-a was identified by SDS-PAGE. The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis.</p><p><b>RESULTS</b>In comparison with the reported luffin-a, the homology of nucleotide sequence of the cloned luffin-a gene was 99.73%, while their amino acid sequences were identical. The solubility of recombinant protein was analyzed by SDS-PAGE, and the luffin-a was mainly produced in inclusion bodies. The recombinant luffin-a, renatured by dialysis of the denatured products, showed a similar cytotoxicity to ricin A chain.</p><p><b>CONCLUSION</b>The cDNA of luffin-a has been successfully cloned. The recombinant luffin-a protein expressed by E. coli is bioactive.</p>

Polymorphic analysis of DYS287 and DYS440 in She ethnic in Zhejiang province China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the polymorphisms of DYS287 and DYS440 in Zhejiang She population.</p><p><b>METHODS</b>Detection of DYS287 and DYS440 polymorphisms was performed in 100 She individuals by using polymerase chain reaction amplification and 2% agarose gel electrophoresis.</p><p><b>RESULTS</b>With 150 bp product in all She individuals, YAP(+) was absent. DYS440*3 was present in 10 She individuals (10%); DYS440*4 was present in the other 90 She individuals(90%).</p><p><b>CONCLUSION</b>The polymorphisms of DYS287 and DYS440 in Zhejiang She population were different from those in the other populations that belong to Sino-Tibetan Language Family. So both DYS440 and DYS287 are important and stable genetic markers, they can provide reliable evidence in human evolution study.</p>