Identification of a high-efficiency baculovirus DNA replication origin that functions in insect and mammalian cells.

UNLABELLED: The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCE: Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.

Promotion of wound healing using adipose-derived stem cells in radiation ulcer of a rat model.

BACKGROUND: Wound healing is a complex biologic process that involves the integration of inflammation, mitosis, angiogenesis, synthesis, and remodeling of the extracellular matrix. However, some wounds fail to heal properly and become chronic. Although some simulated chronic wound models have been established, an efficient approach to treat chronic wounds in animal models has not been determined. The aim of this study was to develop a modified rat model simulating the chronic wounds caused by clinical radiation ulcers and examine the treatment of chronic wounds with adipose-derived stem cells. RESULTS: Sprague-Dawley rats were irradiated with an electron beam, and wounds were created. The rats received treatment with adipose-derived stem cells (ASCs), and a wound-healing assay was performed. The wound sizes after ASC treatment for 3 weeks were significantly smaller compared with the control condition (p < 0.01). Histological observations of the wound edge and immunoblot analysis of the re-epithelialization region both indicated that the treatment with ASCs was associated with the development of new blood vessels. Cell-tracking experiments showed that ASCs were colocalized with endothelial cell markers in ulcerated tissues. CONCLUSIONS: We established a modified rat model of radiation-induced wounds and demonstrated that ASCs accelerate wound-healing.

Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect.

INTRODUCTION: The promotion of wound healing using dermal substitutes has become increasingly widespread, but the outcomes of substitute-assisted healing remain functionally deficient. Adipose-derived stem cells (ASCs) have been investigated widely in regenerative medicine and tissue engineering, and they have the potential to enhance wound healing. In this study, we focused on investigating the effects and mechanism of ASCs combined with an acellular dermal matrix (ADM) to treat full-thickness cutaneous wounds in a murine model. METHODS: The ADM was prepared from the dorsal skin of nude mice by decellularization by treatment with trypsin followed by Triton X-100. The human ASCs were isolated and cultured from abdominal lipoaspirate. We created a rounded, 8-mm, full-thickness cutaneous wound in nude mice and divided the mice into the following 4 groups: silicon sheet cover only, silicon sheet with spreading ASCs, ADM only, and ASCs seeded on ADM. The granulation thickness was evaluated by histology after 7 days. Further comparisons between the ADM only and ASC-seeded ADM groups were undertaken by assessing the reepithelialization ratio and blood vessel density at postoperative days 9 and 14. Statistical analyses were conducted using Student 2-tailed t test. Immunofluorescent histology and ASC labeling were also performed to identify possible mechanisms. RESULTS: The ADM was successfully prepared, and the cytometry analysis and differentiation assay provided the characterization of the human ASCs. A marked improvement in granulation thickness was detected in the ADM-ASC group in comparison with other 3 groups. A significantly increased rate of reepithelialization in the ADM-ASC group (80 ± 6%) compared to the ADM only group (60 ± 7%) was noted on postoperative day 9. The blood vessel density was evidently increased in the ADM-ASC group (7.79 ± 0.40 vessels per field) compared to the ADM only group (5.66 ± 0.23 vessels) on day 14. Cell tracking experiments demonstrated that labeled ASCs were colocalized with staining for VEGF or endothelial cell maker vWF after the transplantation of ADM-ASCs on postoperative day 14. CONCLUSIONS: Adipose-derived stem cells seeded on an ADM can enhance wound healing, promote angiogenesis, and contribute to newly formed vasculature, and VEGF-expressing ASCs can be detected after transplantation. This model could be used to improve the other clinical applications of ASCs and to decipher the detailed mechanism by which ASCs interact with wound tissue.

[Construction of a double functional recombinant strain of Pichia pastoris co-expressing phytase and mannanase and the enzymatic analyses].

Co-expression of phytase and mannanase in Pichia pastoris is a useful way to reduce the production cost in feedstuff industry. Based on the published DNA sequences of phytase gene and mannanase gene, primers were designed and genes phyA and man were cloned by PCR from Aspergillus terreus and the plasmid pHBM1201, respectively. Then the two fragments were treated and inserted into the same expression vector pHBM907C, which contains both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pHBM907C-phyA and the plasmid pHBM907C-man. The phyA expression cassette was combined to the expression vector pHBM907C-man which contains the expression cassette of mannanase, so pHBM907C-phyA-man was obtained. The recombinant expression plasmid pHBM907C-phyA-man was digested by Sal I and introduced into the chromosomes of Pichia pastoris GS115 by using the LiCl/PEG method. Following transformation, several parameters that demonstrated the expression of phytase and mannanase were measured. Firstly, the two different Petri dishes that contain enzymatic substrates such as calcium phytate and mannose were screened simultaneously, thus 100 clones were found to be positive on both of these plates. Secondly, 6 clones among them were chosen for induced expression at shakeflasks showing the probability of high expression. After that, some relative enzymatic properties were measured. At 72 hours' induction in the condition of shake cultivation, the enzyme activity of phytase in supernatant was 120.6 U/mL, while the enzyme activity of mannanase in supernatant was 39.7 U/mL. The expression product phytase was active under pH 2.0-6.5, and the activity was up to the highest under pH 5.5. And the other expression product mannanase was active under pH 5.5-10.5, and the activity was up to the highest under pH 7.5. The optimal temperatures for the two enzyme were both around 52 degrees C: the optimal temperature for phytase activity was 50 degrees C, and that for mannanase was 55 degrees C. At last, stability test of the engineered yeast was taken, and the engineered yeast still showed an excellent stability even after 10 generation growth in the absence of selective pressure. The stable double functional engineered yeast simultaneously expressing extracellular phytase and mannanase is obtained. It will satisfy the demand for industrialized production in some degree.

Importin alpha1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1.

Zac1, a novel seven-zinc-finger transcription factor, preferentially binds GC-rich DNA elements and has intrinsic transactivation activity. To date, the NLS (nuclear localization signal) of Zac1 has not been empirically determined. We generated a series of EGFP (enhanced green fluorescence protein)-tagged deletion mutants of Zac1 and examined their subcellular localization, from which we defined two NLSs within the DNA-binding (or zinc-finger) domain. Fusion proteins consisting of the two EGFP-tagged zinc-finger clusters (zinc finger motifs 1-3 and 4-7) were located exclusively in the nucleus, demonstrating that each of the zinc-finger clusters is sufficient for nuclear localization. Physical interactions between these two zinc-finger clusters and importin alpha1 were demonstrated using an in vitro glutathione S-transferase pull-down assay. Finally, our results indicate that the association of Zac1 with importin alpha1 is also involved in regulating the transactivation activity of Zac1 on the p21WAF1/CIP1 gene and protein expression.

[Surgical treatment of anterior circulation aneurysm via pterion keyhole approach].

OBJECTIVE: To investigate the feasibility of pterion keyhole approach with minimal invasion for treatment of the anterior circulation aneurysm. METHOD: Aneurysm clipping through the pterion keyhole approach was performed in patients with anterior circulation aneurysms, including 9 with posterior communication artery aneurysms, 3 with middle cerebral artery aneurysms and 6 with anterior communication artery aneurysms, who were in stages I to III according to Hunt-Hess scale. RESULT: All the aneurysms were clipped successfully. One patient with a left posterior communication artery aneurysm developed transient sensory aphasia and motor aphasia after surgery due to intraoperative aneurysm rupture. No facial paralysis occurred due to damage of the facial nerve. CONCLUSION: The pterion keyhole approach is a very useful surgical approach for treatment of anterior circulation aneurysms on the basis of cautious determination of indications and careful operation planning.

[Experimental study of a biological dural graft in rabbits].

OBJECTIVE: To evaluate the safety and efficacy of a dural graft prepared using porcine membrane in duraplasty. METHODS: Eighteen New Zealand rabbits were randomly divided into groups A (n=4), B (n=4), C (n=5), and D (n=5) sacrificed 3, 14, 30 and 90 d after duraplasty, respectively. Each animal underwent bilateral parietal craniectomy behind the coronal suture and beside the midline to expose the dura, which was cut on the right side and substituted with the dural graft. The exposed dura on the left was kept intact as control. The rabbits were observed for WBC counts before the operation and before sacrifice by transcardiac formalin perfusion, respectively. The meninges and brain tissues were histologically examined after sacrifice. RESULTS: The WBC count varied little after the operation (P>0.05). Microscopic examination demonstrated tissue repair on both the implantation side and control side, without graft adhesion to the cortical surface. In group A, a large number of leukocytes were seen gathering on the lateral dura, suggesting acute tissue repair. In group B, endothelial cells covering the inner surface of the graft could be seen. Fibroblasts and fibrocytes were seen in the grafts between collagen fibers in group C, and in group D, fibroblasts and fibrocytes increased between the collagen fibers and the suture healed. CONCLUSION: The dura graft is safe and applicable for dural defect repair.

[Origin and clinical significance of the vertebral arterial fenestration: report of a case].

Fenestration of the vertebral artery, different from duplication, is a rare vascular variation, which can be usually classified into extracranial, intracranial and extracranial-intracranial types. We report a case of extracranial fenestration detected by cerebral angiography, and analyze the embryologic pathogenesis and clinical significance of this variation.