Fourier transform infrared spectroscopy, a powerful tool to monitor biopharmaceuticals production

A Escherichia coli é o microorganismo mais usado como hospedeiro para a produção de produtos recombinantes, tais como plasmídeos usados para terapia génica e vacinação de ADN. Desta forma, torna-se importante compreender as relações metabólicas complexas e a bioprodução de plasmídeo, que ocorre em ambientes de cultura dinâmicos, a fim de controlar e optimizar o desempenho do sistema de expressão recombinante. O objectivo principal deste trabalho consiste em avaliar a potencialidade da espectroscopia FT-IR para monitorizar e caracterizar a produção do plasmídeo pVAX-LacZ em culturas recombinantes de E. coli, nomeadamente para extrair informação relacionada com as variáveis críticas (biomassa, plasmídeo, fontes de carbono e acetato) e informação metabólica da célula hospedeira E. coli. Para tal, culturas de E. coli com diferentes concentrações de glucose e glicerol e diferentes estratégias de cultivo (batch e fed-batch) foram monitorizadas por espectroscopia de infravermelho perto (NIR) e de infravermelho médio (MIR). Tanto a espectroscopia NIR com a MIR permitiram extrair informação sobre as variáveis críticas do bioprocesso, através da construção de modelos de regressão por mínimos quadrados parciais, que resultaram em elevados coeficientes de regressão e baixos erros de previsão. A abordagem NIR apresenta a vantagem de aquisição em tempo real das variáveis do bioprocesso, já a abordagem MIR permite a leitura simultânea de centenas de amostras de várias culturas ao mesmo tempo através do uso multi-microplacas, sendo muito vantajosa nos casos de micro-bioreactores usados para optimização. Para além disso, como os espectros MIR apresentam mais informação do que os espectros NIR, uma vez que representam os modos de vibração fundamentais das biomoléculas, enquanto que os espectros NIR representam sobreposições e combinações de vibrações, os dados espectrais MIR também permitiram a aquisição de informação bioquímica ao longo das culturas de E. coli a partir da análise das componentes principais (PCA) bem como do estudo das características bioquímicas, tais como as reservas de glicogénio e os níveis de transcrição aparente. Portanto, a espectroscopia FT-IR apresenta assim características relevantes para a compreensão e monitorização do processo de produção de culturas recombinantes, sendo, de acordo com Quality-by-Design e Process Analytical Technology, muito importante para fins de controlo e optimização.Escherichia coli is the most used microorganism as host for the production of recombinant products, such as plasmids used for gene therapy and DNA vaccination. Therefore, it is important to understand the complex metabolic relationships and the plasmid bioproduction process occurring in dynamic culture environments, in order to control and optimize the performance of the recombinant expression system. The main goal of this work is to evaluate the potential of Fourier Transform Infrared (FT-IR) spectroscopy to monitor and characterize recombinant E. coli cultures producing the plasmid model pVAX-LacZ, namely to extract information concerning the critical variables (biomass, plasmid, carbon sources and the by-product acetate) and metabolic information regarding the host E. coli. To achieve that cultures of E. coli conducted with different mixture of glucose and glycerol and different cultivation strategies (batch and fed-batch) were monitored in-situ by a fiber optic probe in near- infrared (NIR) and of the cell pellets in at-line in high-throughput mode by mid-infrared (MIR) spectroscopy. Both NIR and MIR spectroscopy setup enabled to extract information regarding the critical variables of the bioprocess by the implementation of partial least square regression models that result in high regression coefficients and low prediction errors. The NIR setup presents the advantage of acquiring in real time the knowledge of the bioprocess variables, where the at-line measurements with the MIR setup presents more advantageous in cases of micro-bioreactors used in optimization protocols, enabling the simultaneously information acquisition of hundreds samples by using multi-microplates. Furthermore, as the MIR spectra presents more information than the NIR spectra, since it represents the fundamental vibration modes of biomolecules while the NIR spectra represents overtones and combinations of vibrations, the MIR data also enabled to acquire biochemical information along the E. coli cultures as pointed out in an principal component analysis and by the estimation of biochemical features as glycogen reserves and apparent transcriptional levels. Therefore, FT-IR spectroscopy presents relevant features towards the understanding and monitoring of the production process of recombinant cultures for control and optimization purposes, in according to the Quality-by-Design and the Process Analytical Technology

Role of stem cells in cancer therapy and cancer stem cells: a review

For over 30 years, stem cells have been used in the replenishment of blood and immune systems damaged by the cancer cells or during treatment of cancer by chemotherapy or radiotherapy. Apart from their use in the immuno-reconstitution, the stem cells have been reported to contribute in the tissue regeneration and as delivery vehicles in the cancer treatments. The recent concept of 'cancer stem cells' has directed scientific communities towards a different wide new area of research field and possible potential future treatment modalities for the cancer. Aim of this review is primarily focus on the recent developments in the use of the stem cells in the cancer treatments, then to discuss the cancer stem cells, now considered as backbone in the development of the cancer; and their role in carcinogenesis and their implications in the development of possible new cancer treatment options in future

Lowering the apoptotic threshold in colorectal cancer cells by targeting mitochondria

Background: Colorectal cancer is the third most-common cancer and the second most-common cause of cancer related death in UK. Although chemotherapy plays significant role in the treatment of colorectal cancer, morbidity and mortality due to drug resistance and cancer metastasis are yet to be eliminated. Recently, doxycycline has been reported to have cytotoxic and anti-proliferating properties in various cancer cells. In this study, whether doxycycline was apoptosis threshold lowering agent in colorectal cancer cells by targeting mitochondria was answered.Results: This study showed dose-dependent cytotoxic effects of cisplatin, oxaliplatin and doxycycline in HT29 colorectal cancer cells. Doxycycline showed inhibition of cytochrome-c-oxidase activity in these cells over a time period. The pre-treatment of doxycycline reported statistically significant increased cytotoxicity of cisplatin and oxaliplatin compared to cisplatin and oxaliplatin alone. The caspase studies revealed significantly less expression and activity of caspase 3 in HT29 cells pre-treated with doxycycline compared to the cells treated with cisplatin and oxaliplatin alone.Conclusions: It was concluded that doxycycline lowered the apoptotic threshold in HT 29 cells by targeting mitochondria. This also raised possible caspase-independent mechanisms of apoptosis in HT29 cells when pre-treated with doxycycline however this needs further research work

Superconductivity in Co-doped LaFeAsO

Here we report the synthesis and basic characterization of LaFe1-xCoxAsO for several values of x. The parent phase LaFeAsO orders antiferromagnetically (TN ~ 145 K). Replacing Fe with Co is expected to both electron dope the system and introduce disorder in the FeAs layer. For x = 0.05 antiferromagnetic order is destroyed and superconductivity is observed at Tconset = 11.2 K. For x = 0.11 superconductivity is observed at Tc(onset) = 14.3 K, and for x = 0.15 Tc = 6.0 K. Superconductivity is not observed for x = 0.2 and 0.5, but for x = 1, the material appears to be ferromagnetic (Tc ~ 56 K) as judged by magnetization measurements. We conclude that Co is an effective dopant to induce superconductivity. Somewhat surprisingly, the system appears to tolerate considerable disorder in the FeAs planes.Comment: 19 pages, 9 figure

Cyclooxygenase/lipoxygenase shunting lowers the anti-cancer effect of cyclooxygenase-2 inhibition in colorectal cancer cells

BACKGROUND: Arachidonic acid metabolite, generated by cyclooxygenase (COX), is implicated in the colorectal cancer (CRC) pathogenesis. Inhibiting COX may therefore have anti-carcinogenic effects. Results from use of non-steroidal anti-inflammatory drugs inhibiting only COX have been conflicting. It has been postulated that this might result from the shunting of arachidonic acid metabolism to the 5-lipoxygenase (5-LOX) pathway. Cancer cell viability is promoted by 5-LOX through several mechanisms that are similar to those of cyclooxygenase-2 (COX-2). Expression of 5-LOX is upregulated in colorectal adenoma and cancer. The aim of this study was to investigate the shunting of arachidonic acid metabolism to the 5-LOX pathway by cyclooxygenase inhibition and to determine if this process antagonizes the anti-cancer effect in colorectal cancer cells. METHODS: Three colorectal cancer cell lines (HCA7, HT-29 & LoVo) expressing 5-LOX and different levels of COX-2 expression were used. The effects of aspirin (a non-selective COX inhibitor) and rofecoxib (COX-2 selective) on prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) secretion were quantified by ELISA. Proliferation and viability were studied by quantifying double-stranded DNA (dsDNA) content and metabolic activity. Apoptosis was determined by annexin V and propidium iodide staining using confocal microscopy, and caspase-3/7 activity by fluorescent substrate assay. RESULTS: COX inhibitors suppressed PGE(2) production but enhanced LTB(4) secretion in COX-2 expressing cell lines (P <0.001). The level of COX-2 expression in colorectal cancer cells did not significantly influence the anti-proliferative and pro-apoptotic effects of COX inhibitors due to the shunting mechanism. CONCLUSIONS: This study provides evidence of shunting between COX and 5-LOX pathways in the presence of unilateral inhibition, and may explain the conflicting anti-carcinogenic effects reported with use of COX inhibitors

Inducing apoptosis of human colon cancer cells by an IGF-I D domain analogue peptide

Background: The resistance of tumour cells to apoptosis is a major contributor to the limited effectiveness of chemotherapies. Insulin-like growth factor I (IGF-I) has potential to protect cancer cells from variety of apoptotic challenges. This study was carried out to investigate the effect of a novel IGF-I receptor antagonist on apoptosis in colon cancer cells.Results: We have designed and synthesised a novel antagonist of IGF-I receptor. The effect of this antagonist on human colon cancer cell proliferation was examined by a non-radioactive assay; the apoptosis was revealed by determining the activities of cellular caspases3/7, 8 and 9. The apoptosis pathways were investigated by examining the levels of pro-apoptosis proteins with Western blotting. Following 40 hours treatment with the novel antagonist peptide, colon cancer cell Caspase 3/7 activities increased 2-7 times; Caspase 8 activities increased 2-5 times and Caspase 9 increased 1.2-1.6 times. The proliferation of cancer cell was inhibited by 14-15%. The data showed that the antagonist induced colon cancer cell apoptosis and inhibited cancer cell proliferation. The different changes of Caspase 3/7, 8 and 9 activities suggested that the extrinsic pathways may play a major role in the antagonist peptide-induced apoptosis.Conclusion: This is the first report on this novel antagonist to induce human colon cancer cell apoptosis and inhibit cancer cell proliferation. These results suggest that IGF-I receptor antagonists may have the potential to be developed as a novel therapy for colon cancers in the future

Thermoelectric Figure of Merit of Strongly Correlated Superlattice Semiconductors

We solved the Anderson Lattice Hamiltonian to get the energy bands of a strongly correlated semiconductor by using slave boson mean field theory. The transport properties were calculated in the relaxation-time approximation,and the thermoelectric figure of merit was obtained for the strongly correlated semiconductor and its superlattice structures. We found that at room temperature $ZT$ can reach nearly 2 for the quantum wire lattice structure.We believe that it is possible to find high values of thermoelectric figure of merit from strongly correlated semiconductor superlattice systems.Comment: 4 pages, 3 figure

Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes

Modelo de negocio para el desarrollo de un aplicativo de ayuda emocional: SanaMente

El presente trabajo comprende el desarrollo de un plan de negocio para la aplicación denominada SanaMente, la cual busca establecer una conexión entre psicólogos y pacientes. En ese sentido, este proyecto busca ofrecer beneficios para ambas partes, en el caso de los psicólogos brindaremos de oportunidades crecimiento profesional y la posibilidad de ampliar su cartera de clientes, mientras que por el lado de los usuarios (pacientes) se les brindará un servicio de apoyo emocional personalizado en un ambiente confiable y seguro. Durante el proceso de validación recurrimos a entrevistas con psicólogos clínicos y personas que hayan presentado problemas de estrés y ansiedad, con la finalidad de verificar si la solución corresponde a un problema real y se realizaron experimentos para ambos segmentos. Posteriormente se realizaron los concierges, cuyo fin fue conocer el precio más adecuado para cada uno de los servicios de SanaMente y determinar la cantidad de ventas, según el canal. Finalmente, para evaluar la viabilidad y rentabilidad del proyecto se realizó un análisis financiero, el cual incluye el pronóstico de ventas, análisis del COK y WACC, inversiones, costo de operaciones, plan de operaciones y el presupuesto de recursos económicos.This research work includes the development of the business plan for the application called Mental Health, which has the purpose of connecting psychologists and patients. In that sense, this project aims to offer benefits for both sides, in the case of psychologists, we will provide opportunities for professional growth and the possibility of expanding their client portfolio, while on the side of the users (patients), they will be offered a personalized emotional support service in a safe and secure environment. During the validation process we resorted to interviews with clinical psychologists and people who have presented stress and anxiety problems, in order to verify if the solution corresponds to a real problem and we also did experiments for both segments. Moreover, the concierges were carried out, and their purpose was to know the most appropriate price for each of Metal Health’s services Finally, in order to evaluate the viability and profitability of the project, we did a financial analysis which includes the sales forecast, COK and WACC analysis, investments, cost of operations, plan of operations and the budget of economic resourcesTrabajo de investigació