Collision-Induced Unfolding Studies of Proteins and Protein Complexes using Drift Tube Ion Mobility-Mass Spectrometer.

Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) is emerging as an important biophysical technique owing to its high sensitivity, rapid analysis time, and ability to interrogate sample complexity or heterogeneity and the ability to probe protein structure dynamics. Here, a commercial IMS-MS platform has been modified for static native ESI emitters and an extended mass-to-charge range (20 kDa m/z) and its performance capabilities and limits were explored for a range of protein and protein complexes. The results show new potential for this instrument platform for studies of large protein and protein complexes and provides a roadmap for extending the performance metrics for studies of even larger, more complex systems, namely, membrane protein complexes and their interactions with ligands.

Enhanced Diffusion and Oligomeric Enzyme Dissociation.

The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but experimental support has involved oligomeric enzymes, often studied under conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds kM, the Michaelis constant. Urease, a hexamer of subunits, has been considered to be the gold standard demonstrating enhanced diffusion. Here we show that urease and certain other oligomeric enzymes dissociate above kM into their subunits that diffuse more rapidly, thus providing a simple physical mechanism that contributes to enhanced diffusion in this regime of concentrations. Mindful that this conclusion may be controversial, our findings are supported by four independent analytical techniques: static light scattering, dynamic light scattering (DLS), size-exclusion chromatography (SEC), and fluorescence correlation spectroscopy (FCS). Data for urease are emphasized and the conclusion is validated for hexokinase, acetylcholinesterase, and aldolase. For hexokinase and aldolase no enhanced diffusion is observed except under conditions when these oligomeric enzymes dissociate. At substrate concentration regimes below kM at which acetylcholinesterase and urease do not dissociate, our finding showing up to 10% enhancement of the diffusion coefficient is consistent with various theoretical scenarios in the literature.

Modular organization of cerebellar climbing fiber inputs during goal-directed behavior.

The cerebellum has a parasagittal modular architecture characterized by precisely organized climbing fiber (CF) projections that are congruent with alternating aldolase C/zebrin II expression. However, the behavioral relevance of CF inputs into individual modules remains poorly understood. Here, we used two-photon calcium imaging in the cerebellar hemisphere Crus II in mice performing an auditory go/no-go task to investigate the functional differences in CF inputs to modules. CF signals in medial modules show anticipatory decreases, early increases, secondary increases, and reward-related increases or decreases, which represent quick motor initiation, go cues, fast motor behavior, and positive reward outcomes. CF signals in lateral modules show early increases and reward-related decreases, which represent no-go and/or go cues and positive reward outcomes. The boundaries of CF functions broadly correspond to those of aldolase C patterning. These results indicate that spatially segregated CF inputs in different modules play distinct roles in the execution of goal-directed behavior.

Plasmodium falciparum Parasitemia and Band Sensitivity of the SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test in Madagascar.

Current malaria rapid diagnostic tests (RDTs) contain antibodies against Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2), Plasmodium lactate dehydrogenase (pLDH), and aldolase in various combinations. Low or high parasite densities/target antigen concentrations may influence the accuracy and sensitivity of PfHRP2-detecting RDTs. We analyzed the SD Bioline Malaria Ag P.f/Pan RDT performance in relation to P. falciparum parasitemia in Madagascar, where clinical Plasmodium vivax malaria exists alongside P. falciparum. Nine hundred sixty-three samples from patients seeking care for suspected malaria infection were analyzed by RDT, microscopy, and Plasmodium species-specific, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Plasmodium infection positivity by these diagnostics was 47.9%, 46.9%, and 58%, respectively. Plasmodium falciparum-only infections were predominant (microscopy, 45.7%; LDR-FMA, 52.3%). In all, 16.3% of P. falciparum, 70% of P. vivax, and all of Plasmodium malariae, Plasmodium ovale, and mixed-species infections were submicroscopic. In 423 P. falciparum mono-infections, confirmed by microscopy and LDR-FMA, the parasitemia in those who were positive for both the PfHRP2 and pan-pLDH test bands was significantly higher than that in those who were positive only for the PfHRP2 band (P < 0.0001). Plasmodium falciparum parasitemia in those that were detected as P. falciparum-only infections by microscopy but P. falciparum mixed infections by LDR-FMA also showed similar outcome by the RDT band positivity. In addition, we used varying parasitemia (3-0.0001%) of the laboratory-maintained 3D7 strain to validate this observation. A positive pLDH band in high P. falciparum-parasitemic individuals may complicate diagnosis and treatment, particularly when the microscopy is inconclusive for P. vivax, and the two infections require different treatments.

Proteomics and microstructure profiling of goat milk protein after homogenization.

This study investigated the protein changes in goat milk during the homogenization process using label-free quantification. We quantified 310 and 315 proteins in the control group (CG) and homogenized group (HG), respectively, and 16 proteins were significantly different between the 2 groups. For HG, the goat milk protein particle sizes were smaller and more evenly distributed and exhibited an increase in the regular arrangement of the secondary structures. Proteomics analysis verified that xanthine dehydrogenase and asparaginase-like 1 expression in CG were higher than in HG, whereas the opposite was observed for fructose-bisphosphate aldolase, κ-casein, and ß-casein. Significant changes were found in the homogenization-treated goat milk proteome that were related to goat milk glycolysis/gluconeogenesis metabolism. This work provides updated information on the current proteome characteristics of homogenized goat milk, which may be important for applying the protein component of goat milk to human nutrition and health.

Aldolase A overexpression is associated with poor prognosis and promotes tumor progression by the epithelial-mesenchymal transition in colon cancer.

There is increasing evidence that glycolysis is involved in cancer progression. Aldolase is a glycolytic enzyme that catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Disruption of the aldolase genes also plays a role in the progression of multiple types of cancer. However, the underlying mechanism of the action of aldolases in colon cancer progression remains elusive. In this study, aldolase A expression was investigated and found to be upregulated along with human colon cancer progression and metastasis at both the mRNA and protein levels in human colon cancer tissues. In addition, silencing aldolase A suppressed colon cancer cell proliferation and invasion and inhibited the EMT phenotype. Aldolase A protein expression in colon cancer was related to tumor location, tumor clinical stage and survival. Kaplan-Meier analysis showed that high aldolase A protein expression was associated with an unfavorable outcome. Moreover, aldolase A affected the development of colon cancer not only by affecting the glucose metabolism but also by interacting with the HIF-1 and other EMT-related signaling pathways; silencing aldolase A resulted in the reduced activity of these signaling pathways. These results indicate that aldolase A has additional non-glycolytic functions in transcriptional EMT regulation and may therefore have potential as a therapeutic target or a biomarker for identifying patients at risk for poorer survival.

Electrochemical Analysis of Enzyme Based on the Self-Assembly of Lipid Bilayer on an Electrode Surface Mediated by Hydrazone Chemistry.

In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of a lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility and it can be applied for real sample analysis. What is more, because the lipid bilayer is the universal basis for cell-membrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.

Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase.

BACKGROUND: Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay's high specificity. METHODOLOGY/PRINCIPAL FINDINGS: The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. CONCLUSIONS/SIGNIFICANCE: The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays.

Parasitological correlates of Plasmodium ovale curtisi and Plasmodium ovale wallikeri infection.

BACKGROUND: Malaria, due to Plasmodium ovale, can be challenging to diagnose due to clinically mild disease and low parasite burden. Two genetically distinct sub-species of P. ovale exist: Plasmodium ovale curtisi (classic) and Plasmodium ovale wallikeri (variant). It is presently unknown if the sub-species causing infection affects performance of malaria diagnostic tests. The aim of this work was to understand how the genetically distinct sub-species, P. o. curtisi and P. o. wallikeri, affect malaria diagnostic tests. METHODS: Plasmodium ovale-positive whole blood specimens were sub-speciated by PCR and sequencing of 18S rRNA and dhfr-ts. Parasitaemia, morphology, pan-aldolase positivity, 18S copy number, and dhfr-ts sequences were compared between sub-species. RESULTS: From 2006 to 2015, 49 P. ovale isolates were identified, of which 22 were P. o. curtisi and 27 P. o. wallikeri; 80% were identified in the last five years, and 88% were acquired in West Africa. Sub-species did not differ by parasitaemia, 18S copy number, or pan-aldolase positivity. Lack of Schüffner's stippling was over-represented among P. o. wallikeri isolates (p = 0.02). Several nucleotide polymorphisms between the sub-species were observed, but they do not occur at sites believed to relate to antifolate binding. CONCLUSIONS: Plasmodium ovale is increasing among travellers to West Africa, although sub-species do not differ significantly by parasitologic features such as parasitaemia. Absence of Schüffner's stippling may be a feature specific to P. o. wallikeri and is a novel finding.

Electrochemical biosensor based on enzyme substrate as a linker: Application for aldolase activity with pectin-thionine complex as recognization element and signal amplification probe.

A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate, thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe. The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p-phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability.

An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase.

BACKGROUND: Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. METHODOLOGY/PRINCIPAL FINDINGS: An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. CONCLUSIONS/SIGNIFICANCE: The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases.

Use of the rapid BinaxNOW malaria test in a 24-hour laboratory associated with accurate detection and decreased malaria testing turnaround times in a pediatric setting where malaria is not endemic.

The impact of implementing the BinaxNow malaria test was evaluated. From 288 tests, 34 malaria cases were detected. Laboratory turnaround time decreased from 9.8 to 1.7 h for report of any Plasmodium spp., 10.2 to 1.6 h for P. falciparum, and 8.6 to 1.1 h for any result.

High content screening of cortical neurons identifies novel regulators of axon growth.

Neurons in the central nervous system lose their intrinsic capacity for axon regeneration as they mature, and it is widely hypothesized that changes in gene expression are responsible. Testing this hypothesis and identifying the relevant genes has been challenging because hundreds to thousands of genes are developmentally regulated in CNS neurons, but only a small subset are likely relevant to axon growth. Here we used automated high content analysis (HCA) methods to functionally test 743 plasmids encoding developmentally regulated genes in neurite outgrowth assays using postnatal cortical neurons. We identified both growth inhibitors (Ephexin, Aldolase A, Solute Carrier 2A3, and Chimerin), and growth enhancers (Doublecortin, Doublecortin-like, Kruppel-like Factor 6, and CaM-Kinase II gamma), some of which regulate established growth mechanisms like microtubule dynamics and small GTPase signaling. Interestingly, with only one exception the growth-suppressing genes were developmentally upregulated, and the growth-enhancing genes downregulated. These data provide important support for the hypothesis that developmental changes in gene expression control neurite outgrowth, and identify potential new gene targets to promote neurite outgrowth.

Melanoma-associated retinopathy: a paraneoplastic autoimmune complication.

OBJECTIVES: To study 11 patients with melanoma-associated retinopathy (MAR) to clarify the reliability of various methods of diagnostic testing, to determine the underlying antigenic retinal proteins, and to study the clinical histories and types of associated melanomas. METHODS: Clinical data were obtained from patients with melanoma who developed marked visual problems. Testing included electroretinography, kinetic visual fields, comparative studies of Western blots, and indirect immunohistologic examination to detect antiretinal antibodies, as well as proteomic studies to identify underlying antigenic retinal proteins. RESULTS: Patients with MAR typically have rapid onset of photopsias, scotomata, and loss of central or paracentral vision. Ophthalmoscopy seldom shows significant changes early, but electroretinograms are abnormal. Results of Western blots and immunohistologic examination can show antiretinal antibodies but not always. Most patients (9 of 11) had a strong family history of autoimmune disorders. Any type of melanoma (cutaneous, choroidal, ciliary body, or choroidal nevi) may be associated with this paraneoplastic autoimmune reactivity. MAR may precede or follow the diagnosis of melanoma. Patients with MAR have the same antigenic retinal proteins that have been associated with cancer-associated retinopathy. In addition, 2 new antigenic retinal proteins, aldolase A and aldolase C, were found. CONCLUSIONS: There was a high prevalence of positive family histories of autoimmune disease in patients with MAR. To confirm the disorder, multiple clinical and serum diagnostic techniques (Western blot or indirect immunohistologic examination) are needed. Two newly observed antigenic retinal proteins, aldolase A and aldolase C, are associated with MAR.

Development of a stable positive control to be used for quality assurance of rapid diagnostic tests for malaria.

The objective of this study is to develop and evaluate a simple, cheap, and stable positive control for the quality control and quality assurance (QA) of rapid diagnostic tests (RDT) for the diagnosis of malaria. Plasmodium falciparum in vitro culture of known parasite concentrations was dried on a protein saver card, that is, dried blood spots (DBSs). The cards were stored at temperatures ranging from 27 to 60 degrees C from 1 day up to 6 months. Antigens were subsequently eluted from the card giving final concentrations ranging from 30 000 parasites to 300 parasites/microL and tested for stability against RDT based on the antigens parasite lactate dehydrogenase (pLDH), aldolase, and histidine-rich protein 2 (HRP-2). HRP-2 antigens were stable throughout the whole study and yielded positive results irrespective of parasite concentration, storage duration, or temperature, although band intensity differences could be observed when high parasites were compared with low parasite densities. Aldolase was able to generate positive signals for up to 4 weeks irrespective of the storage conditions. Thereafter, intensities decreased proportionally to increasing temperature and storage duration. Thirty thousand parasites per liter could give a signal up to 16 weeks when stored at a temperature of maximum 45 degrees C. However, densities of 300 parasites/microL were not able to generate a signal during the study. pLDH, the least stable of the 3 antigens, was not able to generate a signal after 1 week of storage. The DBS method yields a very stable positive control for quality control and QA of RDTs based on HRP-2. RDTs based on aldolase may also benefit from this method although to a lesser extent because that particular antigen is less stable in the DBS system.

Isolamento, diferenciação e aspectos bioquímicos de células-tronco de líquido amniótico / Isolation, differentiation and biochemical aspects of amniotic fluid stem cell

As células-tronco mesenquimais (MSCs) são células com grande potencial de diferenciação e estão sendo recentemente introduzidas na clínica para tratamento de várias doenças. Possuem várias vantagens incluindo sua estabilidade fenotípica in vitro. OBJETIVO: isolamento das MSCs de líquido amniótico, sua expansão e a demonstração da sua capacidade de se diferenciar em células miogênicas e adipogênicas, sem alterar a estabilidade cromossomal em meio de cultura. MÉTODOS: a fim de avaliar a mudança funcional destas células, foram avaliados parâmetros bioquímicos nas células adipogênicas já diferenciadas e antes da diferenciação através da dosagem de triglicérides. A diferenciação em células musculares foi avaliada comparando os níveis de creatinofosfoquinase - CK, desidrogenase lática - LDH e aldolase produzidas por estas células antes e após diferenciação. RESULTADOS: os níveis de triglicérides foram significativamente maiores nas células diferenciadas, mostrando ainda a formação de grânulos intracitoplasmáticos. Todos os outros valores obtidos foram significativamente maiores nas células miogênicas diferenciadas quando comparadas às não diferenciadas. CONCLUSÃO: os resultados sugerem que estes protocolos podem ser usados para avaliar diferenciação de células-tronco em células adipogênicas e miogênicas, e que o líquido amniótico pode ser uma fonte para obtenção destas células.

The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and miogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The miogenic nature of the differentiated cells was analyzed comparing the creatine kinase - CK, lactic dehydrogenase - LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracitoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated miogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.

Glycolitic enzymes are targets of oxidation in aged human frontal cortex and oxidative damage of these proteins is increased in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder pathologically characterized by neuronal loss and gliosis mainly in specific subcortical nuclei, but also in the cerebral cortex. In addition to neuron loss, hyperphosphorylated tau deposition is found in neurons, astrocytes and coiled bodies. Limited studies have shown that certain oxidative products are increased in the PSP brain. The present study examines oxidative damage in the frontal cortex in 7 PSP compared with 8 age-matched controls. Western blotting of the frontal cortex showed increased 4-hydroxy-2-nonenal (HNE)-immunoreactive bands between 40 and 50 kDa in PSP cases. Bi-dimensional gel electrophoresis and Western blotting, together with mass spectometry, were used to identify HNE-modified proteins. Oxidized phosphoglycerate kinase 1 (PGK-1) and fructose bisphosphate aldolase A (aldolase A) were identified in all cases and 4 of 7 PSP cases, respectively. In contrast, PGK-1 and aldolase A were oxidized in 3 of 8 controls. Immunohistochemistry revealed the localization of aldolase A in neurons and astrocytes, and PGK-1 mainly in astrocytes. These findings show that PGK-1 and aldolase A are targets of oxidation in the frontal cortex in the aged human cerebral cortex and that oxidative damage of these proteins is markedly increased in the frontal cortex in PSP.

[Isolation, differentiation and biochemical aspects of amniotic fluid stem cell]. / Isolamento, diferenciação e aspectos bioquímicos de células-tronco de líquido amniótico.

UNLABELLED: The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and myogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The myogenic nature of the differentiated cells was analyzed comparing the creatine kinase--CK, lactic dehydrogenase--LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracytoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated myogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.