A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30 degrees C (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthocyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was decreased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influenced by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg.g-fresh cell(-1) was obtained on day 9 by a temperature-shift from 30 degrees C, after 3-d culture, to 15 degrees C. The highest anthocyanin yield of 318 mg.L-1 on day 12 was achieved when the temperature was shifted from 30 degrees C, after 5-d culture, to 20 degrees C. For a global optimization of both the yield and productivity, the optimum anthocyanin yield and productivity of 272 mg.L-1 and 30.2 mg.L-1.d(-1) on day 9 were achieved by a two-stage culture with a temperature-shift from 30 degrees C after 3 d to 20 degrees C.

Characteristic infrared spectroscopic patterns in the protein bands of human breast cancer tissue

Infrared (IR) spectra of normal, hyperplasia, fibroadenoma and carcinoma tissues of human breast obtained from 96 patients have been determined and analyzed statistically. Several spectral differences were detected in the frequency regions of N-H stretching, amide I, II and III bands: (1) the bands in the region 3000-3600cm-1 shifted to lower frequencies for the carcinomatous tissue; (2) the A(3300)/A(3075) absorbance ratio was significantly higher for the fibroadenoma than for the other types of tissues; (3) the frequency of the a-helix amide I band decreased for the malignant tissue, while the corresponding beta -sheet amide I band frequency increased; (4) the A(1657)/A(1635) and A(1553)/A(1540) absorbance ratios were the highest for fibroadenoma and carcinoma tissues; (5) the A(1680)/A(1657) absorbance ratio decreased significantly in the order of normal > hyperplasia > fibroadenoma > carcinoma; (6) the A(1651)/A(1545) absorbance ratio increased slightly for the fibroadenoma and the carcinoma tissues; (7) the bands at 1204 and 1278 cm(-1), assigned to the vibrational modes of the collagen, did not appear in the original spectra as resolved peaks and were distinctly stronger in the deconvoluted spectra of the carcinoma tissue and (8) the A(1657)/A(1204) and A(1657)/A(1278) absorbance ratios, both yielding information on the relative content of collagen, increased in the order of normal < hyperplasia < carcinoma < fibroadenoma. The said differences imply that the information is useful for the diagnosis of breast cancer and malignant breast abnormalities, and may serve as a basis for further studies on conformational changes in tissue proteins during carcinogenesis. (C) 2001 Elsevier Science B.V. All rights reserved.

Instability of anthocyanin accumulation in Vitis vinifera L. var. Gamay Freaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinitera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73 +/- 0.15, 1.45 +/- 0.04, 0.77 +/- 0.024 and 0.27 +/- 0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% for V vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line, showed greater potential in enhanced the anthocyanin production.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.

Dealing with affective needs in e-learning: Contrasting two cases in two cultures.

This chapter presents and contrasts descriptions of two cases of online affective support provided to support students engaged in higher level learning tasks. The cases are set in different cultures, centre upon different intended learning outcomes, and follow different tutorial styles. One (Eastern) tutor acted as a “shepherd leader” in response to needs arising in the Confucian Heritage Culture as the teacher promoted critical thinking, according to the Western model. The other (Western) tutor provided Rogerian facilitation of reflective learning journals, kept by students seeking to develop personal and professional capabilities. In both styles, affective support features strongly. The cultural and pedagogical comparisons between the cases have proved useful to the writers. These distinctions together with the similarities between the two online styles emerge in the comparisons. Keywords: affective needs, asynchronous discussion, Confucian Heritage Culture, constructivism, critical thinking, facilitation, reflection, reflective learning journals, Rogerian, shepherd leadership, social-constructivist, student-centred, support.

A novel breast tissue density classification framework

Oliver, A., Freixenet, J., Marti, R., Pont, J., Perez, E., Denton, E. R. E., Zwiggelaar, R. (2008). A novel breast tissue density classification framework. IEEE Transactions on Information Technology in BioMedicine, 12 (1), 55-65

'Adipaging': Aging and obesity share biological hallmarks related to a dysfunctional adipose tissue

The increasing aging of our societies is accompanied by a pandemic of obesity and related cardiometabolic disorders. Progressive dysfunction of the white adipose tissue is increasingly recognized as an important hallmark of the aging process which in turn contributes to metabolic alterations, multi-organ damage, and a systemic pro-inflammatory state ('inflammaging'). On the other hand, obesity, the paradigm of adipose tissue dysfunction, shares numerous biological similarities with the normal aging process such as chronic inflammation and multi-system alterations. Accordingly, understanding the interplay between accelerated aging related to obesity and adipose tissue dysfunction is critical to gain insight into the aging process in general as well as into the pathophysiology of obesity and other related conditions. Here we postulate the concept of 'adipaging' to illustrate the common links between aging and obesity and the fact that, to a great extent, obese adults are prematurely aged individuals.

Cancer Risk Assessment Related to Anatomical Tissue Types

H. Strange, W. He, E. Denton and R. Zwiggelaar, 'Cancer Risk Assessment Related to Anatomical Tissue Types', Proceedings of the Twelfth Annual Conference on Medical Image Understanding and Analysis, 2008, p.138. Sponsorship: EPSRC

INVESTIGATION OF BUBBLE DYNAMICS AND HEATING DURING FOCUSED ULTRASOUND INSONATION IN TISSUE-MIMICKING MATERIALS

The deposition of ultrasonic energy in tissue can cause tissue damage due to local heating. For pressures above a critical threshold, cavitation will occur in tissue and bubbles will be created. These oscillating bubbles can induce a much larger thermal energy deposition in the local region. Traditionally, clinicians and researchers have not exploited this bubble-enhanced heating since cavitation behavior is erratic and very difficult to control. The present work is an attempt to control and utilize this bubble-enhanced heating. First, by applying appropriate bubble dynamic models, limits on the asymptotic bubble size distribution are obtained for different driving pressures at 1 MHz. The size distributions are bounded by two thresholds: the bubble shape instability threshold and the rectified diffusion threshold. The growth rate of bubbles in this region is also given, and the resulting time evolution of the heating in a given insonation scenario is modeled. In addition, some experimental results have been obtained to investigate the bubble-enhanced heating in an agar and graphite based tissue- mimicking material. Heating as a function of dissolved gas concentrations in the tissue phantom is investigated. Bubble-based contrast agents are introduced to investigate the effect on the bubble-enhanced heating, and to control the initial bubble size distribution. The mechanisms of cavitation-related bubble heating are investigated, and a heating model is established using our understanding of the bubble dynamics. By fitting appropriate bubble densities in the ultrasound field, the peak temperature changes are simulated. The results for required bubble density are given. Finally, a simple bubbly liquid model is presented to estimate the shielding effects which may be important even for low void fraction during high intensity focused ultrasound (HIFU) treatment.

THE ROLE OF ACOUSTIC CAVITATION IN ENHANCED ULTRASOUND-INDUCED HEATING IN A TISSUE-MIMICKING PHANTOM

A complete understanding of high-intensity focused ultrasound-induced temperature changes in tissue requires insight into all potential mechanisms for heat deposition. Applications of therapeutic ultrasound often utilize acoustic pressures capable of producing cavitation activity. Recognizing the ability of bubbles to transfer acoustic energy into heat generation, a study of the role bubbles play in tissue hyperthermia becomes necessary. These bubbles are typically less than 50μm. This dissertation examines the contribution of bubbles and their motion to an enhanced heating effect observed in a tissue-mimicking phantom. A series of experiments established a relationship between bubble activity and an enhanced temperature rise in the phantom by simultaneously measuring both the temperature change and acoustic emissions from bubbles. It was found that a strong correlation exists between the onset of the enhanced heating effect and observable cavitation activity. In addition, the likelihood of observing the enhanced heating effect was largely unaffected by the insonation duration for all but the shortest of insonation times, 0.1 seconds. Numerical simulations were used investigate the relative importance of two candidate mechanisms for heat deposition from bubbles as a means to quantify the number of bubbles required to produce the enhanced temperature rise. The energy deposition from viscous dissipation and the absorption of radiated sound from bubbles were considered as a function of the bubble size and the viscosity of the surrounding medium. Although both mechanisms were capable of producing the level of energy required for the enhanced heating effect, it was found that inertial cavitation, associated with high acoustic radiation and low viscous dissipation, coincided with the the nature of the cavitation best detected by the experimental system. The number of bubbles required to account for the enhanced heating effect was determined through the numerical study to be on the order of 150 or less.

HEATING IN VASCULAR TISSUE AND FLOW-THROUGH TISSUE PHANTOMS INDUCED BY FOCUSED ULTRASOUND

High intensity focused ultrasound (HIFU) can be used to control bleeding, both from individual blood vessels as well as from gross damage to the capillary bed. This process, called acoustic hemostasis, is being studied in the hope that such a method would ultimately provide a lifesaving treatment during the so-called "golden hour", a brief grace period after a severe trauma in which prompt therapy can save the life of an injured person. Thermal effects play a major role in occlusion of small vessels and also appear to contribute to the sealing of punctures in major blood vessels. However, aggressive ultrasound-induced tissue heating can also impact healthy tissue and can lead to deleterious mechanical bioeffects. Moreover, the presence of vascularity can limit one’s ability to elevate the temperature of blood vessel walls owing to convective heat transport. In an effort to better understand the heating process in tissues with vascular structure we have developed a numerical simulation that couples models for ultrasound propagation, acoustic streaming, ultrasound heating and blood cooling in Newtonian viscous media. The 3-D simulation allows for the study of complicated biological structures and insonation geometries. We have also undertaken a series of in vitro experiments, in non-uniform flow-through tissue phantoms, designed to provide a ground truth verification of the model predictions. The calculated and measured results were compared over a range of values for insonation pressure, insonation time, and flow rate; we show good agreement between predictions and measurements. We then conducted a series of simulations that address two limiting problems of interest: hemostasis in small and large vessels. We employed realistic human tissue properties and considered more complex geometries. Results show that the heating pattern in and around a blood vessel is different for different vessel sizes, flow rates and for varying beam orientations relative to the flow axis. Complete occlusion and wall- puncture sealing are both possible depending on the exposure conditions. These results concur with prior clinical observations and may prove useful for planning of a more effective procedure in HIFU treatments.