Catch and measure–mass spectrometry‐based immunoassays in biomarker research

Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage

Introduction

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.

Methods

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.

Results

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.

Conclusion

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.     

Approach for an equation of state for adsorbed protein surfaces.

A surface equation of state for polymer interfaces is obtained by equating the chemical potentials of the solvent in the bulk and surface phases. This equation of state contains the fraction of the surface covered with polymer and the surface activity coefficient as parameters. These parameters may be obtained by measuring the thermodynamic modulus of elasticity. Moreover, if the amount of protein in the interface is known the degree of folding in the surface may be evaluated. The present theory was applied to adsorbed beta-lactoglobulin in surfaces. The results are in agreement with qualitative equations and results from other sources.     

Type IV Pilus Biogenesis,Twitching Motility,and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF,PilT1, and PilT2

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whether T. thermophilus has any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that in T. thermophilus, T4P and natural transformation are linked but distinct systems.     

Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus: ROLE IN COMPLEX STABILITY,PILIATION, ADHESION,TWITCHING MOTILITY,AND NATURAL TRANSFORMATION*

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

Strengthening Community-Based Vital Events Reporting for Real-Time Monitoring of Under-Five Mortality: Lessons Learned from the Balaka and Salima Districts in Malawi

Background

Malawi ratified a compulsory birth and death registration system in 2009. Until it captures complete coverage of vital events, Malawi relies on other data sources to calculate mortality estimates. We tested a community-based method to estimate annual under-five mortality rates (U5MR) through the Real-Time Monitoring of Under-Five Mortality (RMM) project in Malawi. We implemented RMM in two phases, and conducted an independent evaluation of phase one after 21 months of implementation. We present results of the phase two validation that covers the full project time span, and compare the results to those of the phase one validation.

Methods and Findings

We assessed the completeness of the counts of births and deaths and the accuracy of disaggregated U5MR from the community-based method against a retrospective full pregnancy history for rolling twelve-month periods after the independent evaluation. We used full pregnancy histories collected through household interviews carried out between November 2013 and January 2014 as the validation data source. Health Surveillance Agents (HSAs) across the 160 catchment areas submitted routine reports on pregnancies, births, and deaths consistently. However, for the 15-month implementation period post-evaluation, average completeness of birth event reporting was 76%, whereas average completeness of death event reporting was 67% relative to that expected from a comparable pregnancy history. HSAs underestimated the U5MR by an average of 21% relative to that estimated from a comparable pregnancy history.

Conclusions

On a medium scale, the community-based RMM method in Malawi produced substantial underestimates of annualized U5MR relative to those obtained from a full pregnancy history, despite the additional incentives and quality-control activities. We were not able to achieve an optimum level of incentive and support to make the system work while ensuring sustainability. Lessons learned from the implementation of RMM can inform programs supporting community-based interventions through HSAs in Malawi.