Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues.

A better method for determination of shikimate in plant tissues is needed to monitor exposure of plants to the herbicide glyphosate [N-(phosphonomethyl)glycine] and to screen the plant kingdom for high levels of this valuable phytochemical precursor to the pharmaceutical oseltamivir. A simple, rapid, and efficient method using microwave-assisted extraction (MWAE) with water as the extraction solvent was developed for the determination of shikimic acid in plant tissues. High performance liquid chromatography was used for the separation of shikimic acid, and chromatographic data were acquired using photodiode array detection. This MWAE technique was successful in recovering shikimic acid from a series of fortified plant tissues at more than 90% efficiency with an interference-free chromatogram. This allowed the use of lower amounts of reagents and organic solvents, reducing the use of toxic and/or hazardous chemicals, as compared to currently used methodologies. The method was used to determine the level of endogenous shikimic acid in several species of Brachiaria and sugarcane (Saccharum officinarum) and on B. decumbens and soybean (Glycine max) after treatment with glyphosate. The method was sensitive, rapid and reliable in all cases.

Expressions and clinic significance of miRNA-143, miRNA- 34A, miRNA-944, miRNA-101 and miRNA-218 in cervical cancer tissues

Purpose: To search for novel biomarkers for early diagnosis of cervical cancer, as well as novel therapeutic target for cervical cancer. Methods: A total of 96 cervical tissue specimens were collected from patients in the Second Affiliated Hospital of Zhengzhou University, out of which 10 were normal control. The remaining specimens (86) were cervical cancer specimens and were divided into 4 groups (A - D) based on tumor-biomarker levels of CA125 and SCC. Quantitative real-time polymerase chain reaction technology (qRT-PCR) was used to detect the expressions of miRNA-143, miRNA-34A, miRNA-944, miRNA-101 and miRNA-218 in the cervical cancer tissues. Results: The levels of CA125 (U/mL) and SCC (ug/L) expressed in normal control group and groups A - D were 11.75 and 0.73 (n = 10), 382 and 2.72 (n = 25), 912.9 and 3.93 (n = 21), 1675 and 5.87 (n = 29), and 2120 and 6.66 (n = 11), respectively. Furthermore, qRT-PCR results showed that the expressions of miRNA-944 and miRNA-218 in cervical cancer tissues were markedly up-regulated compared to normal control tissues (p < 0.01). In contrast, the expression level of miRNA-143, miRNA-34A, and miRNA-101 were significantly decreased (p < 0.01). Conclusion: The biomarkers, miRNA-143, miRNA-34A, miRNA-944, miRNA-101 and miRNA-218, can be considered novel for early diagnosis of cervical cancer.

Movement effects on the flow physics and nutrient delivery in engineered valvular tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-:τ: explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-:τ: environments.^

Micronutrient contents in healthy and Crinipellis perniciosa infected tissues of Theobroma grandiflorum.

Micronutrient contents in leaf and branch tissues of cupuassu plants (Theobroma grand iflorum) infected by Crinipellis perniciosa, the causal agent of witches' broom disease, were determined to support further studies on the effect of nutritional status in the disease progress. Lower contents of boron and manganese and higher content of copper were found in infected leaf tissues. There was not any statistical difference in the copper content of branch tissues, even though higher copper content was found in healthy branches than in healthy leaf tissues. No changes in contents of iron and zinc in healthy of infected tissues were detected.

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues.

2008

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

Radiobiological model comparison of 3D conformal radiotherapy and IMRT plans for the treatment of prostate cancer

The main aim of radiotherapy is to deliver a dose of radiation that is high enough to destroy the tumour cells while at the same time minimising the damage to normal healthy tissues. Clinically, this has been achieved by assigning a prescription dose to the tumour volume and a set of dose constraints on critical structures. Once an optimal treatment plan has been achieved the dosimetry is assessed using the physical parameters of dose and volume. There has been an interest in using radiobiological parameters to evaluate and predict the outcome of a treatment plan in terms of both a tumour control probability (TCP) and a normal tissue complication probability (NTCP). In this study, simple radiobiological models that are available in a commercial treatment planning system were used to compare three dimensional conformal radiotherapy treatments (3D-CRT) and intensity modulated radiotherapy (IMRT) treatments of the prostate. Initially both 3D-CRT and IMRT were planned for 2 Gy/fraction to a total dose of 60 Gy to the prostate. The sensitivity of the TCP and the NTCP to both conventional dose escalation and hypo-fractionation was investigated. The biological responses were calculated using the Källman S-model. The complication free tumour control probability (P+) is generated from the combined NTCP and TCP response values. It has been suggested that the alpha/beta ratio for prostate carcinoma cells may be lower than for most other tumour cell types. The effect of this on the modelled biological response for the different fractionation schedules was also investigated.

Percutaneous insertion of Dual Growth Rods using a new mobile bearing implant

Growth rods are commonly used for the treatment of scoliosis in the immature spine. Many variations have been proposed but breakage of implants is a common problem. Growth rod insertion commonly involves large exposures at initial insertion followed by multiple smaller procedures for lengthening. We present our early experiences using a percutaneous technique of insertion of a new titanium mobile bearing implant (Medtronic Inc). The implant allows some rotatory motion in the middle of the construct thus reducing construct stresses and thus possibly reducing rod breakage risk. Based on this small initial series with 12 months follow-up, percutaneous insertion of growth rods using the new implant is a safe and reliable technique although the infection rate in our sample was of note. This may be related to the titanium wear and inflammation seen in the soft tissues at time of operation and visualised on histology. No implants have required removal due to infection, and all infections were treated with debridement at next lengthening and suppressive antibiotics. Propionibacterium is one of the commonest infections seen with spinal implants and sometimes does not respond to simple antibiotic suppression. The technique allows preservation of the soft tissues until definitive fusion is needed and may lead to a decrease in hospital stay. The implant is low profile and seems to offer advantages over other systems on the market. Further follow up is needed to look at longer term outcomes with this new implant type.

The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

Anisotropy of spin relaxation of water protons in cartilage and tendon

Transverse spin relaxation rates of water protons in articular cartilage and tendon depend on the orientation of the tissue relative to the applied static magnetic field. This complicates the interpretation of magnetic resonance images of these tissues. At the same time, relaxation data can provide information about their organisation and microstructure. We present a theoretical analysis of the anisotropy of spin relaxation of water protons observed in fully hydrated cartilage. We demonstrate that the anisotropy of transverse relaxation is due almost entirely to intramolecular dipolar coupling modulated by a specific mode of slow molecular motion: the diffusion of water molecules in the hydration shell of a collagen fibre around the fibre, such that the molecular director remains perpendicular to the fibre. The theoretical anisotropy arising from this mechanism follows the “magic-angle” dependence observed in magnetic-resonance measurements of cartilage and tendon and is in good agreement with the available experimental results. We discuss the implications of the theoretical findings for MRI of ordered collagenous tissues.

Development of a biaxial compression device for biological samples : preliminary experimental results for a closed cell foam

Biological tissues are subjected to complex loading states in vivo and in order to define constitutive equations that effectively simulate their mechanical behaviour under these loads, it is necessary to obtain data on the tissue's response to multiaxial loading. Single axis and shear testing of biological tissues is often carried out, but biaxial testing is less common. We sought to design and commission a biaxial compression testing device, capable of obtaining repeatable data for biological samples. The apparatus comprised a sealed stainless steel pressure vessel specifically designed such that a state of hydrostatic compression could be created on the test specimen while simultaneously unloading the sample along one axis with an equilibrating tensile pressure. Thus a state of equibiaxial compression was created perpendicular to the long axis of a rectangular sample. For the purpose of calibration and commissioning of the vessel, rectangular samples of closed cell ethylene vinyl acetate (EVA) foam were tested. Each sample was subjected to repeated loading, and nine separate biaxial experiments were carried out to a maximum pressure of 204 kPa (30 psi), with a relaxation time of two hours between them. Calibration testing demonstrated the force applied to the samples had a maximum error of 0.026 N (0.423% of maximum applied force). Under repeated loading, the foam sample demonstrated lower stiffness during the first load cycle. Following this cycle, an increased stiffness, repeatable response was observed with successive loading. While the experimental protocol was developed for EVA foam, preliminary results on this material suggest that this device may be capable of providing test data for biological tissue samples. The load response of the foam was characteristic of closed cell foams, with consolidation during the early loading cycles, then a repeatable load-displacement response upon repeated loading. The repeatability of the test results demonstrated the ability of the test device to provide reproducible test data and the low experimental error in the force demonstrated the reliability of the test data.

Reduction of Secondary Degeneration after Spinal Cord Injury by Acute Delivery of Proinflammatory Growth Factors

INTRODUCTION Inflammation is a protective attempt to facilitate the removal of damaged tissue and to initiate the healing response in other tissues. However, after spinal cord injury (SCI), this response is prolonged leading to secondary degeneration and glial scarring. Here, we investigate the potential of sustained delivery of pro-inflammatory factors vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) to increase early inflammatory events and promote inflammatory resolution. Method Animal ethics approval was obtained from the Queensland University of Technology. Adult Wistar-Kyoto rats (12-16 weeks old) were subjected to laminectomies and T10 hemisections. Animals were then randomised to treatment (implantation of osmotic pump (Alzet) loaded with 5ug VEGF & 5 ug PDGF) or control groups (lesion control or lesion plus pump delivering PBS). Rats were sacrificed at one month and the spinal cords were harvested and examined by immunohistology, using anti-neurofilament-200(NF200) and anti- ionized calcium binding adapter molecule 1 (Iba1). One way ANOVA was used for statistic analysis. Results At 1 month, active pump-treated cords showed a high level of axonal filament throughout the defects as compared to the control groups. The mean lesion size, as measured by NF200, was 0.47mm2 for the lesion control, 0.39mm2 for the vehicle control and 0.078mm2 for the active pump group. Significant differences were detected between the active pump group and the two control groups (AP vs LC p= 0.017 AG vs VC p= 0.004). Iba-1 staining also showed significant differences in the post-injury inflammatory response. Discussion We have shown that axons and activated microglia are co-located in the lesion of the treated cord. We hypothesise the delivery of VEGF/PDGF increases the local vessel permeability to inflammatory cells and activates these along with the resident microglia to threshold population, which ultimately resolved the prolonged inflammation. Here, we have shown that maintaining the inflammatory signals for at least 7 days improved the morphology of the injured cord. Conclusion This study has shown that boosting inflammation, by delivery VEGF/PDGF, in the early phase of SCI helps to reduce secondary degeneration and may promote inflammation resolution. This treatment may provide a platform for other neuro-regenrative therapies.

Cyclic strain disrupts endothelial network formation on Matrigel

Most forms of tissue healing depend critically on revascularisation. In soft tissues and in vitro, mechanical stimuli have been shown to promote vessel-forming activity. However, in bone defects, increased interfragmentary motion impairs vascular regeneration. Because these effects seem contradictory, we aimed to determine whether a range of mechanical stimuli exists in which angiogenesis is favoured. A series of cyclic strain magnitudes were applied to a Matrigel-based “tube formation” assay and the total lengths of networks formed by human microvascular endothelial cells measured at 24 h. Network lengths were reduced at all strain levels, compared to unstretched controls. However, the levels of pro-angiogenic matrix metalloproteases-2 and -9 in the corresponding conditioned media were unchanged by strain, and vascular endothelial growth factor was uniformly elevated in stretched conditions. By repeating the assay with the addition of conditioned media from mesenchymal stem cells cultivated in similar conditions, paracrine stimuli were shown to increase network lengths, but not to alter the negative effect of cyclic stretching. Together, these results demonstrate that directly applied periodic strains can inhibit endothelial organisation in vitro, and suggest that this may be due to physical disruption rather than biochemical modulation. Most importantly, the results indicate that the straining of endothelial cells and their assembly into vascular-like structures must be studied simultaneously to adequately characterise the mechanical influence on vessel formation.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.