Multidisciplinary approach to diagnosis and management of osteosarcoma – a review of the St Vincent's Hospital experience

Background

Osteosarcoma is the most common primary malignant bone tumour in children and young adults. Despite advances in the diagnosis and management of osteosarcoma, there have been few recent studies describing the experiences of tertiary referral centres. This paper aims to describe and discuss the clinical features, pre-operative work-up, management and outcomes of these patients at St Vincent's Hospital (Melbourne, Australia).

Methods

Retrospective study of fifty-nine consecutive patients managed for osteosarcoma at St Vincent's Hospital between 1995 and 2005.

Results

Median age at diagnosis was 21 (range, 11–84) years. Gender distribution was similar, with thirty-one male and twenty-eight female patients.Twenty-five patients had osteosarcoma in the femur, eleven each were located in the humerus and tibia, six were identified in the pelvis, and one each in the clavicle, maxilla, fibula, sacrum, ulna and radius.Pre-operative tissue diagnosis of osteosarcoma was obtained through computed tomography-guided percutaneous biopsy in over ninety percent of patients.Following initial therapy, over fifty percent of patients remained relapse-free during the follow-up period, with twelve percent and twenty-seven percent of patients documented as having local and distant disease recurrence, respectively. Of patients with recurrent disease, sixty-two percent remained disease-free following subsequent surgical intervention (most commonly, pulmonary metastatectomy).

Conclusion

Patient outcomes can be optimised through a multidisciplinary approach in a tertiary referral centre. At St Vincent's Hospital, survival and relapse rates of patients managed for osteosarcoma compare favourably with the published literature.

     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.     

Nitric Oxide Signalling In Plants

Nitric oxide (NO) is an important molecule that acts in many tissues to regulate a diverse range of physiological processes. It is becoming apparent that NO is a ubiquitous signal in plants. Since the discovery of NO emission by plants in the 1970s, this gaseous compound has emerged as a major signalling molecule involved in multiple physiological functions. Research on NO in plants has gained significant awareness in recent years and there is increasing indication on the role of this molecule as a key-signalling molecule in plants. The investigations about NO in plants have been concentrated on three main fields: The search of NO or any source of NO generation, effects of exogenous NO treatments, NO transduction pathways. However we have limited information about signal transduction procedures by which NO interaction with cells results in altered cellular activities. This article reviews recent advances in NO synthesis and its signalling functions in plants. First, different sources and biosynthesis of NO in plants, then biological processes involving NO signalling are reviewed. NO signalling relation with cGMP, protein kinases and programmed cell death are also discussed. Besides, NO signalling in plant defense response is also examined. Especially NO signalling between animal and plant systems is compared.     

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Role of Chimeric Antigen Receptor T Cell Therapy in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common primary malignant cancer of brain, which is extremely aggressive and carries a dreadful prognosis. Current treatment protocol runs around radiotherapy, surgical resection, and temozolomide with median overall survival of around 12–15 months. Due to its heterogeneity and mutational load, immunotherapy with chimeric antigen receptor (CAR) T cell therapy can be a promising treatment option for recurrent glioblastoma. Initial phase 1 studies have shown that this therapy is safe without dose-limiting side effects and it also has a better clinical outcome. Therefore, CAR T cell therapy can be a great future tool in our armamentarium to treat advanced GBM. In this article, we have explained the structure, mechanism of action, and rationale of CAR T cell therapy in GBM; we also discussed various antigenic targets and clinical outcome of initial studies of this novel therapy.     

Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K₂, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl₂ before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.     

The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds

Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO) and gamma irradiation on the sterilization of sericin scaffolds. The influence of these sterilization methods on the physical properties such as pore size, scaffold dimensions, swelling and mechanical properties, as well as the amount of sericin released from sericin/polyvinyl alcohol/glycerin scaffolds, were also investigated. Ethanol treatment was ineffective for sericin scaffold sterilization whereas gamma irradiation was the most effective technique for scaffold sterilization. Moreover, ethanol also caused significant changes in pore size resulting from shrinkage of the scaffold. Gamma-irradiated samples exhibited the highest swelling property, but they also lost the greatest amount of weight after immersion for 24 h compared with scaffolds obtained from other sterilization methods. The results of the maximum stress test and Young’s modulus showed that gamma-irradiated and ethanol-treated scaffolds are more flexible than the EtO-treated and untreated scaffolds. The amount of sericin released, which was related to its collagen promoting effect, was highest from the gamma-irradiated scaffold. The results of this study indicate that gamma irradiation should have the greatest potential for sterilizing sericin scaffolds for skin tissue engineering.