As a world leader in serving science, ThermoFisher Scientific, Inc. (MA, USA) has recently announced an internal process improvement initiative within its global laboratory informatics line of business. The initiative is known as the Customer Achievement through Responsive Excellent Service (CARES) Initiative and includes all phases of product development, such as sales and service, demonstrating the division’s commitment to exceeding customer expectations across all operational levels. This initiative was recently launched at the Thermo Informatics World (TIW) North American and European Meetings that took place October 15–19, 2007.
The main highlight of the initiative is the platform based on seven core principles, which demonstrate the division’s commitment to customer satisfaction, in areas such as product performance, technical support and sales and account management. In place, is reliable and rigorous external product testing, which includes benchmarking to measure quality, and multiuser environments. In addition, an eServices portal will offer customers a wide-range of support, including online incident logging and access to the business Knowledge Base.
Thermo customers will also benefit from an advanced issue resolution system, which uses patented technology to capture a log of user actions and systems events to efficiently determine the cause of unexpected application behavior. Furthermore, 24-h support is offered to ensure that customers have access to assistance wherever the business is located.
Dave Champagne, Vice President of ThermoFisher Scientific, Inc. informatics commented on the initiative, “The CARES Initiative has been developed to enhance our delivery system across our product development, sales and service organizations. We are committed to providing nothing but excellence to all of our customers, no matter where their business may be located.”
Source: ThermoFisher Scientific, Inc. www.thermofisher.com/global/en/home.asp
A molecular insight into schizophrenia
Schizophrenia is a widespread disease, affecting approximately 1% of the population of the world. It can strike young people in their prime, most often between the ages of 16 and 25 years, and is the largest single cause of permanent disability in the young. Approximately 30% of sufferers attempt suicide and, unfortunately, 5–10% of them succeed.
The classical symptoms of schizophrenia are divided into three types: positive symptoms include unusual thoughts such as movement disorders, hallucinations and delusions. Negative symptoms include a reduced ability to plan, speak and express emotion and are often mistaken for depression, laziness or moody behavior. The third type consists of cognitive disorders, such as problems with memory and attention.
To date, the causes of schizophrenia have not been discovered and there is no cure; however, it can be treated and controlled with antipsychotic drugs that ease the positive symptoms and these may be supplemented with antidepressants.
Extensive postmortem research on schizophrenic brains have identified abnormalities within the corpus cal-losum, which is the white matter that connects the right and left cerebral hemispheres. It has also been proposed that these abnormalities may affect inter-hemisphere communication. In addition, the normal hemisphere asymmetry observed in healthy individuals is reduced in the left-front portion of the corpus callosum, also known as the left genu, of schizophrenic patients.
Researchers in Australia have been prompted by these observations to investigate whether there are any changes in the protein expression profile of the genu of corpus callosum induced by schizophrenia. Sinthuja Sivagnanasundaram and colleagues from the Schizophrenia Research Institute in Sydney and the University of Sydney (Australia) compared the protein profiles of ten schizophrenic patients with those of ten nonpsychiatric controls. The cases were matched for age, gender, brain hemisphere (left or right), pH and postmortem interval.
The researchers extracted proteins from brain tissue and separated them by 2D-gel electrophoresis. The spots were stained and their abundances determined by image analysis. Proteins that were found to be differentially expressed between controls and patients, or between the left and right corpus callosum genu, were digested with trypsin and subjected to mass spectrometry analysis.
A total of 864 protein spots were identified, with 64 spots differentially expressed in the genu of schizophrenic patients. These proteins correspond to 34 proteins due to multiple spots from single proteins caused by the presence of isoforms or post-translational modifications. Of these, 45 spots (21 proteins) were found to be downregulated and 19 spots (14 proteins and one unidentified spot) were upregulated.
Once the proteins associated with pre- and post-mortem factors were eliminated, the remaining proteins fitted into three functional pathways, implicating these processes in the neuropathology of schizophrenia. The majority of the protein spots (64%) were connected to cytoskeletal structure and function, which suggests that their disease-affected expression might produce abnormal cytoskeletal structure or glial cell function. This would be consistent with “the observed irregular fiber tract organization and coherence in imaging studies of schizophrenia brains,” say the researchers.
Proteins involved in neuroprotection were also discovered to be differentially expressed and the particular proteins involved may suggest that oxidative stress, brought on by an as of yet unidentified process, combined with a reduced anti-oxidant defense, might contribute to schizophrenia pathology.
A third class of proteins discovered to be differentially expressed were involved in energy metabolism, which indicates that abnormal energy pathways and/or impaired glycolytic pathway were affected. The researchers stated that a reduction in energy metabolism in the genu “might lead to decreased synaptic activity and plasticity.”
The researchers also observed that, in both controls and patients, the same protein classes were differentially expressed in the left genu compared with the right. They assume that these proteins could be involved in normal corpus callosum asymmetry, as well as in the reduced asymmetry observed in schizophrenic brains.
This initial set of protein candidates and implicated functional pathways should be confirmed by different techniques, such as western blotting, and on an extended number of samples. However, they provide a starting point for exploring the molecular mechanisms involved in schizophrenia and for linking particular proteins to the disease process.
Source: Sivagnanasundaram S, Crossett B, Dedova I, Cordwell S, Matsumoto I. Abnormal pathways in the genu of the corpus callosum in schizophrenia pathogenesis: a proteome study. Proteomics. Clin. App. 1(10), 1291–1305 (2007).
A possible cure for endometriosis?
Endometriosis is a common disease in women, characterized by the growth of cells outside of the uterus that are very similar to endometrial cells that line the uterus. The growths can attach themselves to different locations, such as the ovaries, fallopian tubes, pelvic cavity and intestines. Approximately 5 million women in the USA are affected, thus making it one of the most common female health problems.
One of the major problems of endometriosis are physical symptoms, such as severe abdominal, lower back and pelvic pain, heavy menstrual cramps and, in some cases, infertility.
The disease is also posing a problem in the medical world as no one knows why or how endometriosis develops. There are several theories proposed, but there is no firm pathogenesis. Consequently, there is no cure and endometriosis, at present, can only be suppressed. Current treatments include pain killers, nutritional therapy, hormone treatment and surgery; however, there is no overwhelming medical support for these.
Research has shown that the micro anatomy of the endometrium in sufferers is different to that of normal patients. Now researchers in Australia have used this as a starting point to try to identify proteins that are affected by the onset of endometriosis.
Sara ten Have and colleagues affiliated with the University of Sydney (Australia) have performed a proteomic study on the eutopic endometrium removed during routine surgery from six women diagnosed with endometriosis. They then compared the results with those from 12 women without the disease. All samples were collected during the secretory phase of the menstrual cycle.
Standard proteomic procedures were then carried out on the tissues and extracted proteins were separated by 2D-gel electrophoresis. The isolated spots were stained and further quantified by image analysis. The team discovered a total of 820 proteins expressed in both types of tissues, with 119 proteins either up- or downregulated in the diseased samples. From these, the 50 showing the greatest difference in abundance were identified by mass spectrometry and 21 were found solely in the endometriosis tissue.
The majority of the 21 proteins found solely in the endometriosis tissue were identified as structural or immune reaction proteins; this supports a previous assertion that endometriosis, occurring outside the uterus, is an endometrial disease, associated with apoptosis, oxidative stress, estrogen increase, increased angiogenesis and proteolytic activity.
A further set of differentially expressed proteins found in both types of tissues included actins, which are structural proteins. The researchers suggested that they are involved in the pathogenesis of endometriosis, representing “a more robust cell structure and motility properties of the eutopic endometrium found in endometriosis-affected patients.” The researchers presume that these proteins may contribute to the persistence of shed endometrial tissue after it has left the uterus.
The research will continue as part of a larger investigation that intends to also examine the menstrual and proliferative phases of the reproductive cycle, as well as ectopic lesions produced by endo-metriosis. Their long-term aim is to identify the biochemical pathways involved in the disease, followed by the design of specific drug treatments that will target particular steps in the pathway to stop its progression.
Source: ten Have A, Fraser I, Markham R, Lam A, Matsumoto I. Proteomic analysis of protein expression in the eutopic endometrium of women with endometriosis. Proteomics Clin. App. 1(10), 1243–1251 (2007).
Protein biomarkers for anabolic steroids in calves detected by LC-MS
Since cattle are an important food source, many countries around the world permit the use of growth-promoting hormones in order to increase the animal’s size and yield of meat. Both the USA and Canada follow this practice; however, the EU has banned the use of hormones in cattle owing to health concerns.
Enforcing this ban across the EU can be difficult, despite the ban being in place. In addition, detection can be evaded if the tests are carried out too late, as once the steroids are metabolized their anabolic functions are no longer detectable. There are also some steroids that will metabolize more quickly than others and, therefore, will disappear without trace.
There are two common steroid hormones currently used to bulk up cattle: 1,4-androstadiene-17β-ol-3-one (β-BOL), known as boldenone, and its 3,17-dione analogue boldione; their metabolisms are closely related. Since boldione is a direct precursor of β-BOL and has been identified as a precursor and metabolite of α-BOL (inactive epimer of β-BOL), α-BOL is currently used as a marker for steroid misuse.
EU authorities have declared that the presence of α-BOL conjugates in cattle urine at levels greater than 2 ng/ml should be regarded as suspicious. However, the presence of β-BOL conjugates at any concentration is also considered as evidence of misuse. To enforce these limits, liquid chromatography (LC)–mass spectrometry (MS) methods have been developed to detect both boldidone and α- and β-BOL epimers in cattle urine, as well as in feedstuffs, skin swabs and feces.
Italian researchers from the University of Genoa (Italy) noted that there is no published method for the detection and measurement of BOL conjugates in bovine plasma, and so, they started developing one. Elisabetta Cosulich and colleagues treated veal calves orally with a boldenone/boldione mixture based on a steroid cocktail seized from a farm by the Italian police. After which, both urine and blood were collected for up to 36 h after the administration of a single dose.
In order to measure the three compounds in urine and in plasma before and after deconjugation, the team developed an LC-MS/MS method with atmospheric pressure chemical ionization. Boldione was not detected at all in either fluid despite detection capabilities of 0.3 ng/ml. Both BOL epimers were detected initially in both fluids, but were undetectable in plasma 2 h after administration and in urine after 24 h.
Since these represent short intervals for animal testing, the team then decided that a proteomics approach would be more rewarding. The researchers reasoned that the administration of steroid hormones might change the production of one or more proteins in some way. So, the proteins in plasma collected before and after drug dosing, were separated by 2D-gel electrophoresis, and the protein spots were stained and their intensities compared by image analysis.
After 36 h of sampling, one protein stood out from the hundreds visualized. The protein was absent before drug administration, but was observed in the first post-drug sample at 2 h and steadily increased over time up to 36 h. The protein was identified as an N-truncated form of apolipoprotein A1 (ApoA1).
Following the identification of ApoA1, the research team then confirmed the post-dose variation via western blot analyses using an antibody against ApoA1. Western blotting confirmed that two ApoA1-related bands were observed before drug treatment, but a third band corresponding to the N-truncated form appeared after treatment at the correct molecular mass value and its intensity increased with time.
The researchers speculated on the possible role of N-terminal proteolysis of ApoA1 in the presences of anabolic steroids and suggested that the steroids may induce conformational changes in the protein, thereby exposing its N-terminus, making it vulnerable for proteolytic attack.
The researchers acknowledge that more extensive and rigorous testing must be carried out over a longer timescale before this truncated ApoA1 can be accepted as a biomarker for the misuse of boldenone/boldione in animal husbandry. Immunoblotting may provide a more simple way forward for such large-scale screening and dose-dependent tests.
If the effect is confirmed, then the appearance of ApoA1 in bovine serum will serve to signify the illegal use of these hormones when the plasma or urinary levels of the steroids themselves have returned to normal levels.
Source: Draisci R, Montesissa C, Santamaria B et al. Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression. Proteomics 7(17), 3184–3193 (2007).