Direct Dynamic Protein-Affinity Selection Mass-Spectrometry

A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERα-LBD). In-solution incubation is performed of the analyte and the His-tagged ERα-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein–ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%

Demographic survey of pediatric patients presenting to a chiropractic teaching clinic

<p>Abstract</p> <p>Background</p> <p>Considering the increasing use of alternative therapies for children, it is appropriate to determine the demographic profile of pediatric patients entering a chiropractic clinic.</p> <p>Methods</p> <p>Collection of demographic data including age, gender, condition at presentation, previous clinicians consulted and medical referral rates of pediatric patients presenting to a chiropractic teaching clinic between 2006 and 2010.</p> <p>Results</p> <p>Over-all, 20.5% of patients were aged between two days and 15 years and classified as pediatric patients. The most common presenting complaint was musculoskeletal (35%). Excess crying (30%) was the most common complaint in the largest presenting age group which was under 12 weeks of age (62.3%). All children had previously presented for medical care for the same condition. Most (83%) of the infant patients under 12 weeks of age were referred for care by a medical practitioner.</p> <p>Conclusion</p> <p>Parents commonly presented their child for care at this chiropractic clinic with a recommendation from a medical practitioner. The most common complaints were musculoskeletal and excessive crying conditions and the most prevalent age group was under 12 weeks of age.</p

Recent developments in protein–ligand affinity mass spectrometry

This review provides an overview of direct and indirect technologies to screen protein–ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

Lohr V, Haedicke O, Genzel Y, et al. The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis. BMC Biotechnology. 2014;14(1): 72.Background: In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results: To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions: A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes

Influence of antioxidants in virgine olive oil on the formation of heterocyclic amines in fried beefburgers.

An association between the intake of heterocyclic amines (HAs) and the development of cancer has been observed in some epidemiological studies, while in other studies no such correlation has been found. HAs are mutagenic/carcinogenic compounds formed at low levels via the Maillard reaction and a free radical mechanism during cooking of animal tissue. The addition of pure antioxidants or foods containing antioxidants has previously been shown to decrease the amount of HAs formed during cooking. In this study, beefburgers were fried in six different oils: refined olive oil, virgin olive oil, virgin olive oil depleted of phenols, rapeseed oil, virgin olive oil with rosemary extract and refined olive oil with rosemary extract. The content of antioxidative compounds in the virgin olive oil and the rosemary extract was determined. The beefburgers were analysed with regards to 12 different HAs by solid phase extraction and HPLC analysis. MeIQx, 4,8-DiMeIQx, PhIP, Harman and Norharman were detected in all beefburgers fried in the different oils, but the relative amounts varied. Frying in virgin olive oil reduced the formation of HAs compared with refined olive oil. This effect is probably due to the content of phenols in the virgin olive oil. The HA-reducing effect of virgin olive oil decreased during storage, but the addition of rosemary extract may prevent this decrease

Model for Quality Evaluation and Improvement of Higher Distance Education based on Information Technology

The article describes a proposed model for assessing the quality of higher distance education based on the technologies of the Information and Communication. This model can be adopted by higher education institutions in order to ensure proper use of Information Technologies and Communication in teaching and learning processes, and strategic processes that support the distance education. This research will use models and success stories from other countries as a basis for generating a preliminary model. Furthermore, this research contains initial reports and results as to justify the contribution of it

Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads

A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 mu L of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2005/04473-4