How tolerances, competition and dispersal shape benthic invertebrate colonisation in restored urban streams.

Biotic communities often respond poorly to river restoration activities and the drivers of community recovery after restoration are not fully understood. According to the Asymmetric Response Concept (ARC), dispersal capacity, species tolerances to stressors, and biotic interactions are three key drivers influencing community recovery of restored streams. However, the ARC remains to be tested. Here we used a dataset on benthic invertebrate communities of eleven restored stream sections in a former open sewer system that were sampled yearly over a period of eleven years. We applied four indices that reflect tolerance against chloride and organic pollution, the community's dispersal capacity and strength of competition to the benthic invertebrate taxa lists of each year and site. Subsequently, we used generalised linear mixed models to analyse the change of these indices over time since restoration. Dispersal capacity was high directly after restoration but continuously decreased over time. The initial communities thus consisted of good dispersers and were later joined by more slowly dispersing taxa. The tolerance to organic pollution also decreased over time, reflecting continuous improvement of water quality and an associated increase of sensitive species. On the contrary, chloride tolerances did not change, which could indicate a stable chloride level throughout the sampling period. Lastly, competition within the communities, reflected by interspecific trait niche overlap, increased with time since restoration. We show that recovery follows a specific pattern that is comparable between sites. Benthic communities change from tolerant, fast dispersing generalists to more sensitive, slowly dispersing specialists exposed to stronger competition. Our results lay support to the ARC (increasing role of competition, decreasing role of dispersal) but also underline that certain tolerances may still shape communities a decade after restoration. Disentangling the drivers of macroinvertebrate colonisation can help managers to better understand recovery trajectories and to define more realistic restoration targets.

Rifampicin resistant Mycobacterium tuberculosis in Vietnam, 2020-2022.

Objective: We conducted a descriptive analysis of multi-drug resistant tuberculosis (MDR-TB) in Vietnam's two largest cities, Hanoi and Ho Chi Minh city. Methods: All patients with rifampicin resistant tuberculosis were recruited from Hanoi and surrounding provinces between 2020 and 2022. Additional patients were recruited from Ho Chi Minh city over the same time period. Demographic data were recorded from all patients, and samples collected, cultured, whole genome sequenced and analysed for drug resistance mutations. Genomic susceptibility predictions were made on the basis of the World Health Organization's catalogue of mutations in Mycobacterium tuberculosis associated with drug resistance, version 2. Comparisons were made against phenotypic drug susceptibility test results where these were available. Multivariable logistic regression was used to assess risk factors for previous episodes of tuberculosis. Results: 233/265 sequenced isolates were of sufficient quality for analysis, 146 (63 %) from Ho Chi Minh City and 87 (37 %) from Hanoi. 198 (85 %) were lineage 2, 20 (9 %) were lineage 4, and 15 (6 %) were lineage 1. 17/211 (8 %) for whom HIV status was known were infected, and 109/214 (51 %) patients had had a previous episode of tuberculosis. The main risk factor for a previous episode was HIV infection (odds ratio 5.1 (95 % confidence interval 1.3-20.0); p = 0.021). Sensitivity for predicting first-line drug resistance from whole genome sequencing data was over 90 %, with the exception of pyrazinamide (85 %). For moxifloxacin and amikacin it was 50 % or less. Among rifampicin-resistant isolates, prevalence of resistance to each non-first-line drug was < 20 %. Conclusions: Drug resistance among most MDR-TB strains in Vietnam's two largest cities is confined largely to first-line drugs. Living with HIV is the main risk factor among patients with MDR-TB for having had a previous episode of tuberculosis.

The Asymmetric Response Concept explains ecological consequences of multiple stressor exposure and release.

Our capacity to predict trajectories of ecosystem degradation and recovery is limited, especially when impairments are caused by multiple stressors. Recovery may be fast or slow and either complete or partial, sometimes result in novel ecosystem states or even fail completely. Here, we introduce the Asymmetric Response Concept (ARC) that provides a basis for exploring and predicting the pace and magnitude of ecological responses to, and release from, multiple stressors. The ARC holds that three key mechanisms govern population, community and ecosystem trajectories. Stress tolerance is the main mechanism determining responses to increasing stressor intensity, whereas dispersal and biotic interactions predominantly govern responses to the release from stressors. The shifting importance of these mechanisms creates asymmetries between the ecological trajectories that follow increasing and decreasing stressor intensities. This recognition helps to understand multiple stressor impacts and to predict which measures will restore communities that are resistant to restoration.

Importance of different imperviousness measures for predicting runoff and nutrient emissions from non-urban and urban land-uses at large spatial coverage.

Growing population and urbanization challenge water resources sustainability and require stringent solutions in terms of emission measurements and pollution controls. Advancements in observation techniques have improved the availability of impervious surface data that cover both urban and non-urban areas to assess the impacts of urbanization. However, most models used in macroscale studies continue to derive surface imperviousness based on land-use classes and population data, and the contributions of non-urban impervious surfaces to runoff and nutrient emissions remain largely ignored. Effects of different impervious surface data on the predicted runoff and nutrient emissions is investigated in this study for macroscale urban and non-urban areas in tandem by means of an extended urban module MONERIS - PCRaster to enable scenarios with high-resolution imperviousness data. The results showed that approximately 70% of the total runoff and nutrient emissions nationwide originated from low-to-medium populated impervious surfaces rather than from major urban catchments. Using high-resolution imperviousness data at various aggregation levels resulted in lower biased outputs of predicted runoff and nutrient emissions when compared to results using the estimated impervious data from land-use and population information. The impervious surface shares between urban and non-urban lands revealed the opposite trends of urbanization developments in the less populated areas versus an increasing contribution of emissions from non-urban areas rather than urban centers in densely populated municipalities. Overall, the non-urban impervious surface areas contributed 5-20% of the "hidden" runoff volumes and nutrient emissions from all impervious areas. The results of this study highlight the need of model adaptations regarding the increased availability of high-resolution imperviousness data and the trend of urbanization development beyond urban areas for more accurate quantification of potential flood risks and emission hotspots of macroscale urbanized areas for sustainable water resources management.

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation.

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO2, formate, and H2 that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially Nε-acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, Nε-acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia.

Harmonized assessment of nutrient pollution from urban systems including losses from sewer exfiltration: a case study in Germany.

A growing literature indicates that untreated wastewater from leaky sewers stands among major sources of pollution to water resources of urban systems. Despite that, the quantification and allocation of sewer exfiltration are often restricted to major pipe areas where inspection data are available. In large-scale urban models, the emission from sewer exfiltration is either neglected (particularly from private sewers) or represented by simplified fixed values, and as such its contribution to the overall urban emission remains questionable. This study proposes an extended model framework which incorporates sewer exfiltration pathway in the catchment model for a better justified pollution control and management of urban systems at a nationwide scale. Nutrient emission from urban areas is quantified by means of the Modelling of Nutrient Emissions in River Systems (MONERIS) model. Exfiltration is estimated for public and private sewers of different age groups in Germany using the verified methods at local to city scales, upscaling techniques, and expert knowledge. Results of this study suggest that the average exfiltration rate is likely to be less than 0.01 L/s per km, corresponding to approximately 1 mm/m/year of wastewater discharge to groundwater. Considering the source and age factors, the highest rate of exfiltration is defined in regions with significant proportions of public sewers older than 40 years. In regions where public sewers are mostly built after 1981, the leakage from private sewers can be up two times higher than such from public sewers. Overall, sewer exfiltration accounts for 9.8% and 17.2% of nitrate and phosphate loads from urban systems emitted to the environment, which increases to 11.2% and 19.5% in the case of no remediation scenario of projected defective sewer increases due to ageing effects. Our results provide a first harmonized quantification of potential leakage losses in urban wastewater systems at the nationwide scale and reveal the importance of rehabilitation planning of ageing sewer pipes in public and private sewer systems. The proposed model framework, which incorporates important factors for urban sewer managers, will allow further targeting the important data need for validating the approach at the regional and local scales in order to support better strategies for the long-term nutrient pollution control of large urban wastewater systems.

Leveraging Immonium Ions for Targeting Acyl-Lysine Modifications in Proteomic Datasets.

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl-lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion. By implementing an analysis that accounted for features within each MS2 spectrum, the method clearly identifies peptides with short chain acyl-lysine modifications from complex lysates. Immonium ions can also be used to validate novel acyl modifications; in this study, the first examples of 3-hydroxylpimelyl-lysine modifications are reported and they are validated using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets.

N-terminal autoprocessing and acetylation of multifunctional-autoprocessing repeats-in-toxins (MARTX) Makes Caterpillars Floppy-like effector is stimulated by adenosine diphosphate (ADP)-Ribosylation Factor 1 in advance of Golgi fragmentation.

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.

"I can make more from selling medicine when breaking the rules" - understanding the antibiotic supply network in a rural community in Viet Nam.

BACKGROUND: As in many other low and middle income countries (LIMCs), Vietnam has experienced a major growth in the pharmaceutical industry, with large numbers of pharmacies and drug stores, and increasing drug expenditure per capita over the past decade. Despite regulatory frameworks that have been introduced to control the dispensing and use of prescription-only drugs, including antibiotics, compliance has been reported to be strikingly low particularly in rural parts of Vietnam. This qualitative study aimed to understand antibiotic access and use practices in the community from both supplier and consumer perspectives in order to support the identification and development of future interventions. METHODS: This qualitative study was part of a project on community antibiotic access and use (ABACUS) in six LMICs. The focus was Ba Vi district of Hanoi capital city, where we conducted 16 indepth interviews (IDIs) with drug suppliers, and 16 IDIs and 6 focus group discussions (FGDs) with community members. Drug suppliers were sampled based on mapping of all informal and formal antibiotic purchase or dispensing points in the study area. Community members were identified through local networks and relationships with the field collaborators. All IDIs and FGDs were audio-taped, transcribed and analysed using content analysis. RESULTS: We identified a large number of antibiotic suppliers in the locality with widespread infringements of regulatory requirements. Established reciprocal relationships between suppliers and consumers in drug transactions were noted, as was the consumers' trust in the knowledge and services provided by the suppliers. In addition, antibiotic use has become a habitual choice in most illness conditions, driven by both suppliers and consumers. CONCLUSIONS: This study presents an analysis of the practices of antibiotic access and use in a rural Vietnamese setting. It highlights the interactions between antibiotic suppliers and consumers in the community and identifies possible targets for interventions.

Comparison of the alternative models SOURCE and SWAT for predicting catchment streamflow, sediment and nutrient loads under the effect of land use changes.

Quantifying the water quantity and quality variations resulting from human induced activities is important for policy makers in view of increasing water scarcity and water pollution. Simple models can be robust tools in estimating the runoff from catchments, but do they also sufficiently reflect complex physio-chemical processes required for spatially-explicit simulation of soil-water interactions, and the resulting pollutant responses in catchments? Do these models respond sensitive to the impacts of different land use change representations? These questions are considered by applying the semi-distributed process-based catchment models SWAT and SOURCE to the Sixth Creek catchment in South Australia. Both models used similar data whereas inputs for SOURCE were generated from land-use based Functional Units (FUs), while FUs for SWAT were based on land use, soil and slope combinations. After satisfying calibration of both models for the outlet station of the catchment, the simulated flow by SOURCE produced high goodness of fit metrics, while nutrient loads simulated by SWAT were more realistic. Both models benefitted from using locally available Potential Evapotranspiration data for calibrating the hydrology. Scenarios of intensified land uses by two models showed more credible results for sediment and nutrient loads with the static approach when simulating the linear rather than the non-linear land use changes. The study has shown that informing decisions on the hydrology at catchment scale is well suited to less-complex models, whereas decisions on impact of land use change on water quality in catchments are better suited by models with process descriptions for soil-water interactions.

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes.

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

The A/ENTH Domain-Containing Protein AtECA4 Is an Adaptor Protein Involved in Cargo Recycling from the trans-Golgi Network/Early Endosome to the Plasma Membrane.

Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane.

Modelling the impacts of altered management practices, land use and climate changes on the water quality of the Millbrook catchment-reservoir system in South Australia.

Sustainable management of drinking water reservoirs requires taking into account the potential effects of their catchments' development. This study is an attempt to estimate the daily patterns of nutrients transport in the catchment - reservoir systems through the application of the ensemble of complementary models SWAT-SALMO. SWAT quantifies flow, nitrate and phosphate loadings originating in catchments before entering downstream reservoirs meanwhile SALMO determines phosphate, nitrate, and chlorophyll-a concentrations within the reservoirs. The study applies to the semi-arid Millbrook catchment-reservoir system that supplies drinking water to north-eastern suburbs of Adelaide, South Australia. The catchment hosts viti- and horticultural land uses. The warm-monomictic, mesotrophic reservoir is artificially aerated in summer. After validating the simulation results for both Millbrook catchment and reservoir, a comprehensive scenario analysis has been conducted to reveal cascading effects of altered management practices, land uses and climate conditions on water quality in the reservoir. Results suggest that the effect on reservoir condition in summer would be severe, most likely resulting in chlorophyll-a concentrations of greater than 40 µg/l if the artificial destratification was not applied from early summer. A 50% curbing of water diversion from an external pipeline to the catchment will slightly limit chlorophyll-a concentrations by 1.22% as an effect of reduced inflow phosphate loads. The simulation of prospective land use scenarios converting 50% of present pasture in the Millbrook catchment into residential and orchards areas indicates an increase of summer chlorophyll-a concentrations by 9.5-107.9%, respectively in the reservoir. Global warming scenarios based on the high emission simulated by SWAT-SALMO did result in earlier growth of chlorophyll-a but overall the effects on water quality in the Millbrook reservoir was not significant. However scenarios combining global warming and land use changes resulted in significant eutrophication effects in the reservoir, especially in the unmanaged condition with stratification in summer. This study has demonstrated that complementary model ensembles like SWAT-SALMO allow to comprehend more realistically cascading effects of distinct catchment processes on internal reservoir's processes, and facilitate integrated management scenarios.

Terahertz Time-Domain Spectroscopy of Thermoresponsive Polymers in Aqueous Solution.

The behavior of highly concentrated aqueous solutions of two thermoresponsive polymers poly(N-isopropylacrylamide) (PNIPAm) and poly(N-vinylcaprolactam) (PVCL) have been investigated by terahertz time-domain spectroscopy (THz-TDS). Measurements have been performed for concentrations up to 20 wt %, over a frequency range from 0.3 to 1.5 THz and for temperatures from 20 to 45 °C including the zone for lower critical solution temperature (LCST). THz-TDS enables the study of the behavior of water present in the solution (i.e., free or bound to the polymer). From these measurements, in addition to phase transition temperature, thermodynamic data such as variation of enthalpy and entropy can be inferred. Thanks to these data, further insights upon the mechanism involved during the dehydration phenomenon were obtained. These results were compared to the ones issued from dynamic light scattering, spectroscopy, or microscopy techniques to underline the interest to use THz-TDS as a powerful tool to characterize the behavior of thermoresponsive polymers in highly concentrated solutions.

Naïve CD8(+) T cell derived tumor-specific cytotoxic effectors as a potential remedy for overcoming TGF-ß immunosuppression in the tumor microenvironment.

Despite of the potential implications for cancer immunotherapy, conventional approaches using in vitro expanded CD8(+) T cells have suboptimal outcomes, mostly due to loss of functionality from cellular exhaustion. We therefore investigated the phenotypic and functional differences among in vitro activated CD8(+) T cells of three different sources, namely naïve (NTeff), memory (MTeff) and tumor-infiltrating lymphocytes (TILeff) from human and mice, to better understand mechanisms behind potent effector functions and potential for overcoming current limitations. In line with the greater proliferation activity and longer telomere lengths of NTeff populations, cells of naïve origin exhibited significantly less amounts of T cell exhaustion markers than those of MTeff and TILeff, and moreover, acquired distinct expression patterns of memory-promoting transcription factors, T-bet and Eomes, induced in a rapid and sustainable manner. NTeff cells appeared to have lower expression of Foxp1 and were refractory to apoptosis upon TGF-ß conditioning, implying better survival potential and resistance to tumor-induced immune suppression. Of CD8(+) T cell pools activated to tumor-specific CTLs, naïve cell generated effectors possessed the most potent cytotoxic activity, validating implications for use in rational design of adoptive immunotherapy.

Hyperbranched polymer mediated size-controlled synthesis of gadolinium phosphate nanoparticles: colloidal properties and particle size-dependence on MRI relaxivity.

Hyperbranched polymers based on the poly(amidoamine), HyPAM, were used to synthesize gadolinium phosphate nanowires under mild conditions. Control of the average particle size was obtained by adjusting polymer concentration. Proton relaxivity measurements reveal an optimum particle size, reaching relaxivity values as high as 55 ± 9 mM(-1) s(-1) for r1 and 67 ± 11 mM(-1) s(-1) for r2. The colloidal stability of these hybrid systems were optimized through the use of functionalized core-shell polymers containing PEG segments and C18-PEG segments, structures which also offer the possibility of imparting additional function into the polymer-particle hybrids.

Thermoresponsive Properties of PNIPAM-Based Hydrogels: Effect of Molecular Architecture and Embedded Gold Nanoparticles.

Thermoresponsive hydrogels were successfully prepared from poly(N-isopropylacrylamide)-based polymers with different architectures (linear, branched, or hyperbranched). The macromolecular architectures strongly influence the internal structure of the hydrogels, therefore modulating their thermoresponsive and rheological properties. These hydrogels were used for the in situ synthesis of gold nanoparticles. Significant changes in hydrogel microstructures and in average pore size due to the presence of gold nanoparticles were observed. Additionally, their presence significantly increases both the mechanical strength and the toughness of the hydrogel networks.

Mesomorphic ionic hyperbranched polymers: effect of structural parameters on liquid-crystalline properties and on the formation of gold nanohybrids.

Branched thermotropic liquid crystals were successfully obtained from ionic interactions between hyperbranched polyamidoamine and sodium dodecylsulfate. These complexes present columnar rectangular and lamellar thermotropic mesophases as demonstrated by polarized optical microscopy, differential scanning calorimetry, and small-angle X-ray scattering. The relationships between the structural characteristics of the polymers (size of the hyperbranched core, hyperbranched or dendritic nature of the core, and substitution ratio) and the mesomorphic properties were studied. In situ formation of gold nanoparticles was then performed. The templating effect of the liquid crystal mesophase resulted in the formation of isotropic nanoparticles, the size of which was dictated by the local organization of the mesophase and by the molar mass of the hyperbranched complex.

Inhibition of cell proliferation by an anti-EGFR aptamer.

Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K(d) = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.

Technical and biological issues relevant to cell typing with aptamers.

A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome.