Geoffrey S. Baird

A monomeric red fluorescent protein

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

DJ-1 and α-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease

Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinsons disease. Both DJ-1 and alpha-synuclein, two proteins critically involved in Parkinsons disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and alpha-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and alpha-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and alpha-synuclein from 117 patients with Parkinsons disease, 132 healthy individuals and 50 patients with Alzheimers disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and alpha-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and alpha-synuclein levels were decreased in Parkinsons patients versus controls or Alzheimers patients when blood contamination was controlled for. In the population aged > or = 65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and alpha-synuclein, respectively, the sensitivity and specificity for patients with Parkinsons disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for alpha-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or alpha-synuclein and the severity of Parkinsons disease. Taken together, this represents the largest scale study for DJ-1 or alpha-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and alpha-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinsons disease, if variables such as blood contamination and age are taken into consideration.

Recent advances in technology for measuring and manipulating cell signals

Signal transduction research has made some glowing progress in the past 12 months. Recent advances in fluorescent proteins, small molecule fluorophores and imaging technology are generating new ways to investigate signal transduction.

Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall

Alveolar rhabdomyosarcoma may be extremely difficult to distinguish from other primitive round cell neoplasms without ancillary immunohistochemistry and/or genetic study. Particularly in adults and in the head and neck locations, the differential diagnosis of alveolar rhabdomyosarcoma includes small cell carcinoma and neuroepithelial tumors, such as esthesioneuroblastoma. We have recently seen cases of genetically confirmed alveolar rhabdomyosarcoma, which were misdiagnosed owing to expression of cytokeratins and neuroendocrine markers. We studied a large group of well-characterized alveolar rhabdomyosarcomas for expression of such markers. Forty-four alveolar rhabdomyosarcomas (18 genetically confirmed) were retrieved from our archives and immunostained for wide-spectrum cytokeratin (OSCAR), low molecular weight cytokeratin (Cam5.2), synaptophysin, chromogranin A, and CD56 using commercially available antibodies. Cases were scored as ‘negative’, ‘rare’ (<5% positive cells), ‘1+’ (5–25%), ‘2+’ (26–50%) and ‘3+’ (>51%). The tumors occurred in 23 males and 21 females at a mean age of 18 years (range, <1–64 years), and involved many sites. Fifty percent of cases (22 of 44) expressed wide-spectrum cytokeratin, and scored almost equally as rare, 1+, and 2+, but rarely 3+. Cam5.2 was positive in 52% (14 of 27). Forty-three percent of cases (16 of 37) expressed at least one of the specific neuroendocrine markers, 32% (12 of 37) expressed synaptophysin, 22% (eight of 36) expressed chromogranin A, and 11% expressed both. Expression of synaptophysin and chromogranin A was typically confined to rare cells but could be more widespread. Thirty-two percent of cases (12 of 37) expressed the wide-spectrum cytokeratin and at least one of the neuroendocrine markers, and 8% (three of 36) expressed cytokeratin and both neuroendocrine markers. CD56 expression was nearly ubiquitous. Aberrant expression of epithelial and neuroendocrine markers is relatively common in alveolar rhabdomyosarcoma, occurring in 30–40% of cases. These findings have significant implications for the diagnosis of alveolar rhabdomyosarcoma, particularly in adults and in the head and neck locations. Although expression of cytokeratin and/or synaptophysin alone does not necessarily indicate epithelial or neuroendocrine differentiation, coexpression of cytokeratin and neuroendocrine markers, and in particular the presence of chromogranin expression, suggest true epithelial and/or neuroendocrine differentiation in a subset of alveolar rhabdomyosarcomas. CD56 is not a specific neuroendocrine marker, and should not be used in the absence of synaptophysin/chromogranin. These findings emphasize the need to employ a panel of markers, to include desmin, myogenin/MyoD1, and genetic study in the diagnosis of primitive round cell neoplasms in all age groups and in all locations.

Where are all the aptamers

Oligonucleotide aptamers, first reported 20 years ago by Tuerk and Gold1 and Ellington and Szostak,2 are short, single strands of DNA or RNA that assume 3-dimensional configurations that facilitate specific binding interactions with other chemical species. In Tuerk and Gold’s1 seminal article, the nucleic acid target of an RNA-binding protein (specifically, the 8-base RNA sequence favored by T4 DNA polymerase) was selectively amplified from a pool containing all possible random 8-base RNA sequences. It is interesting that the technique found not only T4 DNA polymerase’s native RNA target sequence in the pool but also another sequence that bound the protein with similar affinity, indicating that their technique was a generalizable method for finding nucleic acids that bound to nonnucleotide targets. This work and Ellington and Szostak’s2 demonstration that RNA ligands could be found for small molecule dyes were the first of countless studies showing that Systematic Evolution of Ligands by EXponential enrichment, or SELEX, can be applied to nearly any molecular target to generate high-affinity, specific oligonucleotide ligands. In SELEX, one begins with a chemically synthesized pool of randomized oligonucleotides flanked on either end by defined primer-binding sequences, operating on the hypothesis that a high-affinity binding species is contained somewhere in that pool. This hypothesis may seem farfetched, especially when considering that there are only 4 natural nucleotide bases in DNA, in contrast with the 20 natural amino acids possible in an antibody’s variable region. However, even small random DNA libraries can be incredibly diverse—a theoretical 25-mer random DNA oligonucleotide pool still contains 425, or more than a million billion, different species—implying that relatively short sequences of randomized DNA can be sufficiently diverse to contain a wide variety of potential binding ligands. After preparing the SELEX pool, one proceeds to …

Protein signature of lung cancer tissues.

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment.

Age-Dependent Changes in the Cerebrospinal Fluid Proteome by Slow Off-Rate Modified Aptamer Array

An important precondition for the successful development of diagnostic assays of cerebrospinal fluid (CSF) biomarkers of age-related neurodegenerative diseases is an understanding of the dynamic nature of the CSF proteome during the normal aging process. In this study, a novel proteomic technology was used to quantify hundreds of proteins simultaneously in the CSF from 90 cognitively normal adults 21 to 85 years of age. SomaLogics highly multiplexed proteomic platform can measure more than 800 proteins simultaneously from small volumes of biological fluids using novel slow off-rate modified aptamer (SOMAmer) protein affinity reagents with sensitivity, specificity, and dynamic ranges that meet or exceed those of enzyme-linked immunosorbent assays. In the first application of this technology to CSF, we detected 248 proteins that possessed signals greater than twofold over background. Several novel correlations between detected protein concentrations and age were discovered that indicate that both inflammation and response to injury in the central nervous system may increase with age. Applying this powerful proteomic approach to CSF provides potential new insight into the aging of the human central nervous system that may have utility in discovering new disease-related changes in the CSF proteome.

Isopropanol Protein Precipitation for the Analysis of Plasma Free Metanephrines by Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND High-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS)1 analysis of plasma free metanephrines is the most diagnostically sensitive and specific screening test for the diagnosis of pheochromocytoma. We sought to develop an in-house method for this expensive test METHODS We used off-line isopropanol protein precipitation of plasma to remove interfering substances before LC-MS/MS analysis. We compared the extraction efficiency and limits of quantification of protein precipitation to those of previously reported solid-phase techniques. RESULTS The new method had limits of quantification of 0.09 nmol/L and 0.17 nmol/L for metanephrine and normetanephrine, respectively. Method comparison with a previously described solid-phase extraction method revealed Deming regression slopes of 0.904 and 0.994, intercepts of 0.007 and 0.023, and SEs of the residuals (S(y/x)) of 0.071 and 0.284 for metanephrine and normetanephrine, respectively. Extraction efficiency of isopropanol protein precipitation was 66% for metanephrine and 35% for normetanephrine, results that were superior to the efficiencies of 4% and 1% for our adapted solid-phase extraction method. No ion suppression was observed at the retention times for metanephrine and normetanephrine. CONCLUSIONS Isopropanol protein precipitation is a novel and effective off-line sample preparation method for metanephrines that offers a less expensive alternative to on-line solid-phase extraction for low-volume testing and requires a sample volume of only 200 microL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.

Rapid Histochemistry Using Slow Off-rate Modified Aptamers With Anionic Competition

Immunohistochemistry is used in both research and clinical settings to identify proteins in tissue samples. Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. Aptamers can circumvent these limitations, but their application has been hindered by nonspecific binding to cellular components, particularly in the nucleus. Here we describe unique slow off-rate modified aptamers that facilitate rapid and selective binding to target proteins in tissue. Specifically, we have developed a fluorescent aptamer that binds to the human epidermal growth factor receptor 2 (HER2) in breast carcinomas quickly and specifically, and we have shown that the slow off-rate of the aptamer from the HER2 protein contributes to its selectivity. These findings open the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.

The laboratory test utilization management toolbox

Efficiently managing laboratory test utilization requires both ensuring adequate utilization of needed tests in some patients and discouraging superfluous tests in other patients. After the difficult clinical decision is made to define the patients that do and do not need a test, a wealth of interventions are available to the clinician and laboratorian to help guide appropriate utilization. These interventions are collectively referred to here as the utilization management toolbox. Experience has shown that some tools in the toolbox are weak and other are strong, and that tools are most effective when many are used simultaneously. While the outcomes of utilization management studies are not always as concrete as may be desired, what data is available in the literature indicate that strong utilization management interventions are safe and effective measures to improve patient health and reduce waste in an era of increasing financial pressure.