Aptamer-based multiplexed proteomic technology for biomarker discovery

Background

The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.

Methodology/Principal Findings

We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.

Conclusions/Significance

We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.     

Regulation of ion transport by pH and [HCO3-] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 +/- 22.5 microequiv.kg(-1).h(-1)) in controls to base secretion (84.7 +/- 14.4 microequiv.kg(-1).h(-1)). The HCO3(-)-induced Vte increase was inhibited by basolateral acetazolamide (200 micromol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3(-) did not take place in Cl(-)-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 +/- 18.4 microequiv kg(-1) h(-1). This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl(-)-free saline. Therefore, the cellular response to low pH is different from the HCO3(-)-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3(-)-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3(-)-dependent cotransporters mediate the response to low pH.     

Ligand binding kinetic studies on the hybrid hemoglobin alpha (human):beta (carp): a hemoglobin with mixed conformations and sequential conformational changes

Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) alpha (human):beta (carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human alpha-chain is R state and the carp beta-chain is T state. We have no evidence at this pH that the carp beta-chain ever assumes the R conformation. At pH 6, the human alpha-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp beta-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human alpha-chains are in the R state, the two beta-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the beta-chains first assume T-state properties, followed by the alpha-chains. The rate of the R-T conformational change for the carp beta-chains is at least 300 times greater than that for the human alpha-chains. At pH 9, the R----T conformational transition rate is at least 200 times slower than that for human hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F

The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (K(a)) and average binding sites (n) for VPg were (8.51 +/- 0.21) x 10(6) M(-1) and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative DeltaH degrees (-29.32 +/- 0.13 kJmol(-1)) and positive DeltaS degrees (36.88 +/- 0.25 Jmol(-1)K(-1)). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m(7)G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 +/- 1.27 and 75.37 +/- 2.95 kJmol(-1) respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.     

Lipid phenotyping of lung epithelial lining fluid in healthy human volunteers

Background

Lung epithelial lining fluid (ELF)—sampled through sputum induction—is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood.

Objectives

To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort.

Methods

Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes.

Results

The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender.

Conclusions

We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

     

Longitudinal associations of the endocrine environment on fat partitioning in postmenopausal women

Among postmenopausal women, declining estrogen may facilitate fat partitioning from the periphery to the intra-abdominal space. Furthermore, it has been suggested that excess androgens contribute to a central fat distribution pattern in women. The objective of this longitudinal study was to identify independent associations of the hormone milieu with fat distribution in postmenopausal women. Fifty-three healthy postmenopausal women, either using or not using hormone replacement therapy (HRT) were evaluated at baseline and 2 years. The main outcomes were intra-abdominal adipose tissue (IAAT), subcutaneous abdominal adipose tissue, and total thigh fat analyzed by computed tomography scanning and leg fat and total body fat mass measured by dual-energy X-ray absorptiometry. Serum estradiol, estrone, estrone sulfate, total testosterone, free testosterone, androstenedione, dehydroepiandrosterone sulfate), sex hormone-binding globulin (SHBG), and cortisol were assessed. On average, in all women combined, IAAT increased by 10% (10.5 cm(2)) over 2 years (P < 0.05). Among HRT users, estradiol was inversely associated with, and estrone was positively associated with, 2-year gain in IAAT. Among HRT nonusers, free testosterone was inversely associated with, and SHBG was positively associated with, 2-year gain in IAAT. These results suggest that in postmenopausal women using HRT, greater circulating estradiol may play an integral role in limiting lipid deposition to the intra-abdominal cavity, a depot associated with metabolically detrimental attributes. However, a high proportion of weak estrogens may promote fat partitioning to the intra-abdominal cavity over time. Furthermore, among postmenopausal women not using HRT, greater circulating free testosterone may limit IAAT accrual.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum

The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which are membrane intrinsic proteins showing high homology to the light harvesting complexes (LHC) of higher plants. In the present study, we used a mild solubilization of P. tricornutum thylakoid membranes in combination with sucrose density gradient centrifugation or gelfiltration and obtained an oligomeric FCP complex (FCPo). The spectroscopic characteristics and pigment stoichiometries of the FCPo complex were comparable to FCP complexes that were isolated after solubilization with higher detergent per chlorophyll ratios. The excitation energy transfer between the FCP-bound pigments was more efficient in the oligomeric FCPo complexes, indicating that these complexes may represent the native form of the diatom antenna system in the thylakoid membrane. Determination of the molecular masses of the two different FCP fractions by gelfiltration revealed that the FCP complexes consisted of trimers, whereas the FCPo complexes were either composed of six monomers or two tightly associated trimers. In contrast to vascular plants, stable functional monomers could not be isolated in P. tricornutum. Both types of FCP complexes showed two protein bands in SDS-gels with apparent molecular masses of 18 and 19 kDa, respectively. Sequence analysis by MS/MS revealed that the 19 kDa protein corresponded to the fcpC and fcpD genes, whereas the 18 kDa band contained the protein of the fcpE gene. The presence of an oligomeric antenna in diatoms is in line with the oligomeric organization of antenna complexes in different photoautotrophic groups.     

The Bloom's syndrome protein (BLM) interacts with MLH1 but is not required for DNA mismatch repair

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.