Objective: To evaluate the effectiveness of droplet digital PCR (ddPCR) assay for detection of neuraminidase (NA) H275Y mutations in influenza A H1N1 pdm09 virus. Methods: The primers and dual probes were designed based on the sequence of the H1N1 pdm09 NA gene fragment which contained 275 amino acid sites, and the annealing temperature of ddPCR assay was optimized to establish a method for detection of H275 drug-sensitive genes and H275Y drug-resistant genes in H1N1 pdm09 virus. The sensitivity of ddPCR assay and fluorescent quantitative PCR (qPCR) assay was compared using the detection limit, and the specificity of ddPCR and qPCR assays was compared for detection of 14 respiratory virus samples. In addition, 64 clinical samples and 5 influenza isolates were tested to calculate the abundance of H275Y mutations, and the mutation abundance of 5 influenza isolates was compared with next-generation sequencing results. Results: The optimal annealing temperature was 62.2 ℃. The detection limits of ddPCR assay were 5.28 (95%CI: 4.28-7.45) copies/reaction for H1N1 pdm09 H275 drug-sensitive plasmids and 6.51 (95%CI: 5.25-9.37) copies/reaction for H1N1 pdm09 H275Y drug-resistant plasmids, and the detection limits of qPCR assay were 5.70 (95%CI: 4.83-7.45) copies/reaction for H1N1 pdm09 H275Y drug-sensitive plasmids and 7.06 (95%CI: 5.92-9.40) copies/reaction for H1N1 pdm09 H275Y drug-resistant plasmids. Both ddPCR and qPCR assays detected H275 and H275Y drug-resistant plasmids in H1N1 pdm09 viral samples but did not detect H275 and H275Y drug-resistant plasmids in other 11 respiratory virus samples, and these two assays showed consistent results. Of the 64 clinical samples, ddPCR assay detected H275Y mutation in three pharyngeal swab specimens from a severe pneumonia patients infected with H1N1 pdm09 virus, and the greatest mutation abundance was detected in samples collected on day 4 post-treatment with oseltamivir phosphate (53.37%). ddPCR assay detected 0.63, 88.93%% and 1.27% H275Y mutation abundance in samples collected on days 2, 4 and 5 post-treatment with oseltamivir phosphate, and next-generation sequencing detected 89.46% H275Y mutation abundance in samples collected on day 4 post-treatment with oseltamivir phosphate; however, no H275Y mutation was detected in samples collected on days 2 or 5 post-treatment with oseltamivir phosphate. Conclusions ddPCR presents a higher sensitivity and specificity than qPCR assay for detection of H275Y mutations in H1N1 pdm09 virus, and presents a higher sensitivity than next-generation sequencing for detection of low-frequency mutations, which is effective for quantitative detection of H275Y mutations in the NA fragment of the H1N1 pdm09 virus.

OBJECTIVETo demonstrate the effects and mechanisms of Huangkui capsule (HKC) on renal fibrosis in rats with diabetic nephropathy (DN).

METHODRats were randomly divided into 5 groups, the sham-operated group (Sham group, n = 5), the vehicle-given group (Vehicle group, n = 7), the low dose of HKC-treated group (L-HKC group, n = 7), the high dose of HKC-treated group (H-HKC group, n = 7) and the lipoic acid (LA)-treated group (LA group, n = 7). DN models were induced by intraperitoneal injection of streptozotocin (STZ,35 mg x kg(-1)) twice and unilateral nephrectomy. After models were successfully established, the rats in HKC and LA groups were daily administrated with HKC suspensions (0.75, 2 g x kg(-1)) or LA suspensions (60 mg x kg(-1)) respectively, and at the same time, the rats in Vehicle group were daily administrated with distilled water (2 mL) for 8 weeks. All rats were sacrificed at the end of week 8 to collect blood and renal tissues. UAlb, renal function, renal fibrotic morphologic characteristics, as well as oxidative stress (OS)-related markers, the protein expressions of the key signaling molecules in p38 mitogen-activated protein kinase (p38MAPK) signaling pathway, fibrogenic cytokines and inflammatory factors were examined respectively.

RESULTHKC, similar to LA, improved the general state of health, body weight, UAlb, BUN, UA and Alb in DN model rats. Of note, renal fibrosis was ameliorated in HKC groups,especially in H-HKC group which was better than that in LA group. In addition, HKC not only improved the main indexes of OS in the kidney like LA, but also down-regulated the protein expressions of phosphorylated-p38MAPK (p-p38MAPK), transforming growth factor (TGF)-β1 and tumor necrosis factor(TNF)-α in the kidney, whereas, LA only decreased the protein expression of TNF-α in the kidney in DN model rats.

CONCLUSIONHKC, similar to LA, has the actions of anti-OS in vivo. Moreover, HKC could attenuate renal fibrosis by suppressing the activation of p38MAPK signaling pathway and the protein expressions of fibrogenic cytokines and inflammatory factors in the kidney in DN model rats, which is different from LA.

Abelmoschus ; chemistry ; Animals ; Capsules ; Diabetic Nephropathies ; drug therapy ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Kidney ; drug effects ; pathology ; MAP Kinase Signaling System ; drug effects ; Male ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

In the development of diabetic nephropathy (DN), reactive oxygen specie (ROS) over much in vivo leads to oxidative stress(OS)-related renal injuries, which are characterized by the structural and functional changes in glomerular and renal tubular cells in morphology. The regulative approaches of OS involve the several signaling pathways, in which, both p38 mitogen-activated protein kinase (MAPK) signaling pathway and adenosine monophosphate-activated protein kinase (AMPK) signaling pathway play the important roles as the target of anti-oxidants. The interventional actions of Chinese herbal compound prescriptions and the extracts of single Chinese herbal medicine (CHM) on OS in the kidney in DN include regulating the balance between ROS and antioxidants, reducing the production of AGEs, inhibiting the expression of growth factors and intervening the activity of signaling pathways.
Animals ; Diabetic Nephropathies ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Kidney ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Signal Transduction ; drug effects ; Treatment Outcome

BACKGROUND: Magnetic nanoparticles have attracted tremendous attention for their diverse biomedical applications involving tumor hyperthermia, drug controlled release and magnetic resonance angiography. The interaction between biomaterials and cells is the major part in the in vitro study, and the evaluation of cellular uptake of magnetic nanoparticles is indispensable for tumor hyperthermia and drug delivery. OBJECTIVE: To review the progress of evaluation methodology of cellular uptake of magnetic nanoparticles. METHODS: The articles about magnetic nanoparticles and cellular uptake were retrieved from PubMed, SCI and Embase databases published during January 2010 to June 2017 by the first author using computer. The Mesh terms were "magnetic nanoparticles", and the key words were "cellular uptake", "internalization" or "endocytosis". The logical operator between them was "AND". RESULTS AND CONCLUSION: Qualitative quantitative assessment methods for cellular uptake of magnetic nanoparticles include Prussian blue staining, fluorescence microscopy, confocal microscopy,transmission electron microscopy. Quantitative assessment methods for cellular uptake of magnetic nanoparticles include inductively coupled plasma, magnetization measurements, colorimetry of Prussian blue staining. The development direction of the evaluation methodology is from qualitative to quantitative. As each method has its own merits and demerits, we should choose the right evaluation method based on the function and application of magnetic nanoparticles and then supply the right experimental basis for the biomedical application of magnetite nanoparticles.

BACKGROUNDCataracts is considered be formed because of an abnormal glucose metabolic pathway or oxidative stress. We explored the damaging role of ONOO- and antagonism of cholecystokinin octapeptide-8 (CCK-8) in diabetic cataractal rat lenses.

METHODSA diabetic cataractal animal model was established by peritoneal injection of streptozotocine (STZ). Thirty-six normal SD rats were taken as control group; seventy-two were given STZ (45 mg/kg) and then divided into STZ group and CCK-8 group (peritoneal injection CCK-8). STZ induced diabetic rats were treated with CCK-8 for 60 days. Lenses were examined with slit lamp at 20, 40 and 60 days. Immunofluorescent staining and Western blot analysis were used for determining nitrotyrosine (NT, a marker for ONOO-). PT-PCR and gene array analysis were used for determining the expression of inducible nitric oxide synthetase mRNA (iNOS mRNA) in lens epithelium (LEC).

RESULTSSTZ group rats developed lens opacity by 20 days that reached a high level by 60 days after STZ injection. CCK-8 group rats delayed the cataract formation. CCK-8 group rats delayed the cataract formation. There was no distinct expression of NT and iNOS mRNA in control group. In STZ group, there were distinct expression of NT and upregulation of iNOS mRNA; however, CCK-8 group showed weak expression of NT and downregulation of iNOS mRNA.

CONCLUSIONSNT, which may be a new form of oxidative stress, was expressed in diabetic rat LEC although CCK-8 could reverse NT damage in LEC. The results suggested that CCK-8 might be a useful therapeutic agent against diabetic cataract. The antagonizing mechanism of CCK-8 may be related to direct antagonism of ONOO- as well as its inhibition of the expression of iNOS mRNA for production of NO and therefore decrease in the formation of ONOO-.

Animals ; Blotting, Western ; Cataract ; etiology ; prevention & control ; Diabetes Mellitus, Experimental ; complications ; Fluorescent Antibody Technique ; Male ; Nitric Oxide Synthase Type II ; genetics ; Oxidation-Reduction ; Peroxynitrous Acid ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sincalide ; pharmacology ; Streptozocin ; Tyrosine ; analogs & derivatives ; genetics

The present study was designed to observe if puerarin decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO(-)). One hundred and eight rats were randomly divided into control group (n=36), streptozotocin (STZ) group (n=36) and STZ + puerarin group (n=36). The rats in the control group intraperitoneally (i.p.) received 0.5 ml of saline. The rats in STZ group and STZ + puerarin group received intraperitoneal injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin (140 mg/kg per day, i.p.). On days 20, 40 and 60 of the experiment, morphologic changes of lenses were observed with slit lamp. Then the animals were sacrificed for further analysis. The amount and percentage of apoptotic LECs were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO(-)) was examined by immunohistochemistry. Apoptosis-related genes (iNOS, etc.) were analyzed by gene array. The results showed that in the control group, all the lenses were clear. In STZ group, gradually severe opacity of the lens was observed on days 20, 40 and 60. But in STZ + puerarin group, mild opacity of the lens was observed on day 20 and more severe on day 40, but markedly decreased on day 60. In the control group, mild apoptosis of LECs was observed. In STZ group, time-dependent increase in apoptosis of LECs was observed. In STZ + puerarin group, mild apoptosis of LECs was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of NT in the lens in the control group, but an increased expression of NT in STZ group. In STZ + puerarin group, mild expression of NT was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of iNOS in the lens in the control group, but continuous up-regulation of iNOS expression in STZ group. In STZ + puerarin group, mild expression of iNOS was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. Except the changes of iNOS related to NO production, the other apoptosis-related genes, including BCL-2 and SOD were down-regulated, while NF-kappaB and TNFR1-FADD-caspase signal transduction way were up-regulated in STZ group. The results were opposite in STZ + puerarin group and the control group. These findings show that NT is expressed in diabetic rat lens, which proves that LEC apoptosis in diabetic lens is partly induced by ONOO(-) which may be a new oxidative damage way to form cataract. Puerarin partly decreases LEC apoptosis induced by ONOO(-) and is a potential medicine for therapy of diabetic cataract. The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO(-) in diabetic rats.
Animals ; Apoptosis ; Cataract ; chemically induced ; Diabetes Mellitus, Experimental ; Epithelial Cells ; drug effects ; Isoflavones ; pharmacology ; Lens, Crystalline ; cytology ; Nitric Oxide Synthase Type II ; metabolism ; Peroxynitrous Acid ; Rats ; Tyrosine ; analogs & derivatives ; metabolism

OBJECTIVETo develop a rapid and effective method for genomic DNA extraction with magnetic bead-based semi-automatic system.

METHODSDNA was extracted from whole blood samples semi-automatically with nucleic acid automatic extraction system.The concentration and purity of samples was determined by UV-spectrophotometer. Orthogonal design was used to analyze the main effect of lysis time, blood volume, magnetic bead quantity and ethanol concentration on the DNA yield; also the 2-way interaction of these factors.

RESULTSLysis time, blood volume, magnetic bead quantity and ethanol concentration were associated with DNA yield (P<0.05), but no interaction existed. DNA yield was higher under the condition with 15 min of lysis time, 100 μl of blood volume, 80 μl of magnetic beads and 80 % of ethanol. A significant association was found between the magnetic bead quantity and DNA purity OD260/OD280 (P=0.008). Interaction of blood volume and lysis time also existed (P=0.013). DNA purity was better when the extracting condition was 40 μl of magnetic beads, 15 min of lysis time and 100 μl of blood volume. Magnetic beads and ethanol concentration were associated with DNA purity OD260/OD230 (P=0.017 and P<0.05), the result was better when magnetic beads was 40 μl and ethanol concentration was 80 %.

CONCLUSIONThe results indicate that the optimized conditions with 40 μl magnetic beads will generate higher quality of genomic DNA from the whole blood samples.

Analysis of Variance ; DNA ; blood ; isolation & purification ; Humans ; Immunomagnetic Separation ; methods

This study analyzed the current situation and problems of two-way referral between wound healing department of general hospitals and community health service centers through stipulated interview with physicians in general hospitals and community health service centers, and patients visiting these organizations from March 2011 to April. It was found that the current two-way referral process for wound repair were facing a series of problems, including hospitals-transfer difficulty, incomplete two-way referral policy and undefined practice protocol, information-sharing obstacle between general hospitals and community health service centers. The critical countermeasures for overcoming these obstacles in two-way referral of wound ailments shall include construction of further linkage mechanism among all levels of hospitals, establishment of the drug-obtaining mechanism, establishment of an explicit two-way referral process of wound repair, and establishment of a database of diagnosis and treatment information of patients that can be accessed by doctors of different levels of hospitals, etc.
Community Health Planning ; Community-Institutional Relations ; Hospitals, Special ; Humans ; Referral and Consultation ; Wound Healing

OBJECTIVEThe present study was designed to evaluate the clinical manifestations, surgical findings, pathologic types and treatment of cardiac tumor and to analyze the echocardiographic characteristics of the cases.

METHODSRecords of 19 patients with cardiac tumors confirmed by operations and pathology at Fuwai Cardiovascular Hospital in Beijing, China between Jan, 1983 and Dec, 2003 were reviewed. Clinical and echocardiographic data of all patients were analyzed.

RESULTSThe median age of patients was 7 +/- 5 years, ranging from 5 months to 14 years. There were 8 male and 11 female cases. The surgical findings revealed that 57.9% (11 cases) of cardiac tumors were found in left heart, 36.8% (7 cases) in right heart, 5.3% (1 case) in two ventricles. The pathological study revealed that 17 cases (89.5%) were diagnosed as primary cardiac benign tumors including myxoma in 10 cases (52.6%), rhabdomyoma in 4 cases (21.1%), fibroma in 2 cases (10.5%) and lipoma in 1 case (5.3%). Two cases were diagnosed (10.5%) as cardiac malignant tumors including a primary cardiac rhabdomyosarcoma and a metastatic epithelioid sarcoma. By using echocardiography 11 cases were diagnosed as myxomas and rhabdomyoma (11/19, 57.9%), 8 cases were diagnosed as space occupying lesions of the heart or myxoma (8/19, 42.1%).

CONCLUSIONSMyxomas are the most common heart tumors seen in infancy and childhood, followed in frequency by rhabdomyomas, fibromas and lipomas. Surgery is recommended for patients with refractory and severe clinical symptoms.

Adolescent ; Child ; Child, Preschool ; Echocardiography ; Female ; Fibroma ; diagnostic imaging ; Heart Neoplasms ; diagnostic imaging ; Humans ; Infant ; Lipoma ; diagnostic imaging ; Male ; Myxoma ; diagnostic imaging ; Rhabdomyoma ; diagnostic imaging

Objective: To identify the avian influenza virus subtype from the avian and environmental samples using the Ion Torrent new-generation semiconductor sequencing technology and to establish a high-throughput sequencing method to identify unclassified influenza A virus. Methods: Virus RNA was extracted from the nine avian swab and environmental samples and real-time RT-PCR was carried out to detect universal fluA, H5N1, H7N9 and H9N2. The whole genome of influenza A virus was amplified by PathAmpFluA kit. Sequencing library was prepared using Next Fast DNA Fragmentation & Library Prep Set for Ion Torrent kit and high-throughput sequencing was done by Ion Torrent Personal Genome Machine(PGM). Data from the PGM was processed and quality evaluated using Ion TorrentSuite v3.0 software. Sequence assembly and influenza database blast were carried out by FluAtyping v4.0 and PathogenAnalyzer bioinformatics software to identify the influenza A virus subtype of these nine samples. Results: The results of real-time RT-PCR for universal fluA of these nine samples were positive but the results for H5N1, H7N9 and H9N2 were negative. Seven subtypes of influenza A virus were identified by high-throughput sequencing and bioinformatics analysis: six samples were H2N3, H5N6, H5N8, H7N1, H7N7, H11N3 subtype respectively and three samples were H6N6 subtype. Conclusions Avian influenza virus has many subtypes in the environment of Zhejiang province. Ion Torrent semiconductor sequencing technology is suitable for fast identification of unclassified influenza virus for avian influenza environment monitoring.