cloning-large-scale-production-high-capacity-adenoviral-vectors-based

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

University Witten/Herdecke

In this work, we prepared an in situ gel-forming composite drug delivery system (DDS) to treat colorectal peritoneal carcinomatosis. The composite DDS was based on curcumin loaded polymeric micelles (Cur-M) and thermosensitive hydrogel. Cur-M had a particle size of 27.1 ± 1.3 nm with polydisperse index of 0.149 ± 0.017, and the drug loading and encapsulation efficiency of Cur-M were 14.82 ± 0.07 and 98.83 ± 0.45%, respectively. The prepared Cur-M in thermosensitive hydrogel system (Cur-H) was a free-flowing sol at ambient temperature, and converted into non-flowing gel at body temperature, serving as a drug depot. In vitro drug release behavior suggested that Cur-H and Cur-M could release Cur in an extent period, and Cur-H showed a slower cumulative release rate. In addition, compared with free Cur, Cur-M showed higher cytotoxicity and apoptotic induction efficiency. Furthermore, colorectal peritoneal carcinomatosis mouse model was used to evaluate the anti-tumor activity of Cur-H, and the results suggested that Cur-H could inhibit tumor growth and metastasis, and prolonged survival of tumor-bearing mice. Immunohistochemical and immunofluorescent staining of tumor tissues in each group were conducted. The results demonstrated that tumors in Cur-H group showed lower proliferation activity, more apoptotic cells, and fewer microvessels. Besides, pharmacokinetic studies of Cur-H and Cur-M by intraperitoneal administration were performed. Compared with Cur-M, Cur-H showed a higher AUC and longer t½. Thus, the above results suggested that Cur-H may have potential applications in colorectal peritoneal carcinomatosis.

Improving Anti-Tumor Activity of Curcumin by Polymeric Micelles in Thermosensitive Hydrogel System in Colorectal Peritoneal Carcinomatosis Model.

The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana.

Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

Tissue adhesion is a common complication after surgery. In this work, a dexamethasone loaded polymeric micelles in thermosensitive hydrogel composite (Dex hydrogel) was prepared, which combined the anti-adhesion barrier with controlled release of anti-adhesion drug. Dexamethasone (Dex) was encapsulated in polymeric micelles (Dex micelles), and then the Dex micelles were loaded into biodegradable and thermosensitive hydrogel. The obtained Dex hydrogel showed a temperature-dependent sol-gel-sol phase transition behavior. The Dex hydrogel could form a non-flowing gel in situ upon subcutaneous injection and gradually degrade in about 20 days. In addition, Dex hydrogel was assigned for anti-adhesion studies in a more rigorous recurrent adhesion animal model. Compared with normal saline (NS) and Dex micelles group, tissue adhesions in hydrogel and Dex hydrogel group were significantly alleviated. In Dex hydrogel group, the media adhesion score is 0, which was dramatically lower than that in blank hydrogel group (2.50, P < 0.001). In histopathological examination and scanning electron microscopy (SEM) analysis, an integral neo-mesothelial cell layer with microvilli on their surface was observed, which revealed that the injured parietal and visceral peritoneum were fully recovered without the concerns of adhesion formation. Our results suggested that Dex hydrogel may serve as a potential anti-adhesion candidate.

Thermosensitive hydrogel containing dexamethasone micelles for preventing postsurgical adhesion in a repeated-injury model.

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants.

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures.

Commonly used human adenovirus type 5 (HAdV-5) based vectors are restricted by their tropism and pre-existing immunity. Here we characterized novel HAdV-5 vectors pseudotyped with hypervariable regions (HVR) and the surface domains (SD) of other human adenovirus types. Hexon-modified HAdV-5 vectors (HV-HVR5, HV-HVR12, HV-SD12, HV-SD4) could be reconstituted and amplified in human embryonic kidney cells. After infection of various cell lines, we measured transgene expression levels by performing luciferase reporter assays or coagulation factor IX ELISA. Dose-dependent studies revealed that luciferase expression levels were comparable for HV-HVR5, HV-SD12, HV-SD4, whereas HV-HVR12 expression levels were significantly lower. Next vector genome copy numbers (VCN) from genomic DNA and nuclear extracts were determined by quantitative PCR. Surprisingly, determination of cell-associated VCN revealed up to 3-fold increased VCN for HV-HVR12 compared to HV-SD12 and HV-HVR5. Increased nuclear fraction associated HV-HVR12 DNA molecules and decreased transgene expression levels were independent of the cell line used and we observed the same effect for a hexon-modified high-capacity adenoviral vector encoding human coagulation factor IX. Notably we found that nuclear import of viral DNA was increased for HV-HVR12 compared to HV-HVR5. In conclusion, studying hexon-modified adenoviruses in vitro demonstrated that HVRs but also flanking hexon regions influence uptake and transgene expression of adenoviral vectors.

Adenovirus hexon modifications influence in vitro properties of pseudotyped human adenovirus type 5 vectors.

Centromeres are defined by the presence of CENH3, a variant of histone H3. Centromeres in most plant species contain exclusively highly repetitive DNA sequences, which has hindered research on structure and function of centromeric chromatin. Several maize centromeres have been nearly completely sequenced, providing a sequence-based platform for genomic and epigenomic research of plant centromeres. Here we report a high resolution map of CENH3 nucleosomes in the maize genome. Although CENH3 nucleosomes are spaced ∼190 bp on average, CENH3 nucleosomes that occupied CentC, a 156-bp centromeric satellite repeat, showed clear positioning aligning with CentC monomers. Maize centromeres contain alternating CENH3-enriched and CENH3-depleted subdomains, which account for 87% and 13% of the centromeres, respectively. A number of annotated genes were identified in the centromeres, including 11 active genes that were located exclusively in CENH3-depleted subdomains. The euchromatic histone modification marks, including H3K4me3, H3K36me3 and H3K9ac, detected in maize centromeres were associated mainly with the active genes. Interestingly, maize centromeres also have lower levels of the heterochromatin histone modification mark H3K27me2 relative to pericentromeric regions. We conclude that neither H3K27me2 nor the three euchromatic histone modifications are likely to serve as functionally important epigenetic marks of centromere identity in maize.

Gene Expression and Chromatin Modifications Associated with Maize Centromeres.

To investigate health status of general population in environment cadmium polluted area in Hezhang County, Guizhou Province. Methods The subjects were selected by random sampling from the local resident. The urine samples were collected and detected for cadmium, N-acetyl-β-D-glucosaminidase (NAG) and β2-microglobulin (β2-MG). ICP-MS (inductively coupled plasma-mass spectrometry) was used to determine Cd concentrations. β2-MG was determined using radioimmunoassay. NAG and creatinine was measured by spectrophotometry.

[Survey on the status of health effects and relationship between indices of health effects of population in environment cadmium polluted area in Hezhang County, Guizhou Province].

To evaluate the effect of physiological dose 17-β-estrodiol (E2) replacement therapy on vascular dementia caused by cerebral chronic hypoperfusion.

[Estrogen decreases vascular damage induced by chronic hypoperfusion through upregulating VEGF expression].

A new nanocrystalline inorganic-organic hybrid semiconductor [(BV)2(Ag5Br9)]n () with an intriguing windmill-like 1-D [Ag5Br9]n(n-) polymeric chain was assembled. Great efforts were devoted to investigate its semiconducting properties and applications, such as photoluminescence, emission decay lifetime, waste water purification, electrochemistry behavior and dielectric properties.

A new nanocrystalline inorganic-organic hybrid exhibiting semiconducting properties and applications.

To explore a more accurate method for calculating the cumulative cadmium intake from rice.

[Comparison of two methods in calculating the cumulative cadmium intake (CCI) from rice in the cadmium polluted area].

We investigated the molecular epidemiologic characteristics and virulence of hypermucoviscosity-positive K. pneumoniae in mainland of China. 24.62% (16/65) hypermucoviscosity-positive strains were detected in total 65 clinical isolates. 68.75% (11/16) of these were genotype K2 and carried rmpA and iucA genes. MLST revealed five sequence types (STs): ST65 (7), ST23 (4), ST86 (3), ST412 (1), ST375 (1), while the other four isolates were defined as other STs. The order of lethal dose 50 (LD50) values of ST types was ST23 (2.19×10(3)CFU/mouse) < ST86 (1.70×10(4) CFU/mouse) < ST65 (5.05×10(7) CFU/mouse) < other STs (1.90×10(8) CFU/mouse). In conclusion, The K2 with ST65 carrying rmpA and iucA was the most predominant among in the hypermucoviscosity-positive Klebsiella pneumoniae strains from community-acquired infection cases in Harbin North China. The sequence types are better able to predict the risk of Klebsiella pneumonia infectious disease.

Multilocus sequence types and virulence determinants of hypermucoviscosity-positive Klebsiella pneumoniae isolated from community-acquired infection cases in Harbin, North China.

Article	Year
Improving Anti-Tumor Activity of Curcumin by Polymeric Micelles in Thermosensitive Hydrogel System in Colorectal Peritoneal Carcinomatosis Model. Journal of biomedical nanotechnology\| PubMed ID: 26307840	2015
Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana. Fungal genetics and biology : FG & B\| PubMed ID: 26319315	2015
Thermosensitive hydrogel containing dexamethasone micelles for preventing postsurgical adhesion in a repeated-injury model. Scientific reports\| PubMed ID: 26324090	2015
Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures. The Plant cell\| PubMed ID: 26373455	2015
Adenovirus hexon modifications influence in vitro properties of pseudotyped human adenovirus type 5 vectors. The Journal of general virology\| PubMed ID: 26519158	2015
Gene Expression and Chromatin Modifications Associated with Maize Centromeres. G3 (Bethesda, Md.)\| PubMed ID: 26564952	2015
[Survey on the status of health effects and relationship between indices of health effects of population in environment cadmium polluted area in Hezhang County, Guizhou Province]. Wei sheng yan jiu = Journal of hygiene research\| PubMed ID: 26591775	2015
[Estrogen decreases vascular damage induced by chronic hypoperfusion through upregulating VEGF expression]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University\| PubMed ID: 26607074	2015
A new nanocrystalline inorganic-organic hybrid exhibiting semiconducting properties and applications. Dalton transactions (Cambridge, England : 2003)\| PubMed ID: 26733362	2016
[Comparison of two methods in calculating the cumulative cadmium intake (CCI) from rice in the cadmium polluted area]. Wei sheng yan jiu = Journal of hygiene research\| PubMed ID: 26738387	2015
Multilocus sequence types and virulence determinants of hypermucoviscosity-positive Klebsiella pneumoniae isolated from community-acquired infection cases in Harbin, North China. Japanese journal of infectious diseases\| PubMed ID: 26743146	2016

Wenli Zhang

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