Preserved striatal innervation maintains motor function despite severe loss of nigral dopaminergic neurons.

Degeneration of dopaminergic neurons in the substantia nigra and their striatal axon terminals causes cardinal motor symptoms of Parkinson's disease. In idiopathic cases, high levels of mitochondrial DNA alterations leading to mitochondrial dysfunction are a central feature of these vulnerable neurons. Here we present a mouse model expressing the K320E-variant of the mitochondrial helicase Twinkle in dopaminergic neurons, leading to accelerated mitochondrial DNA mutations. These K320E-TwinkleDaN mice showed normal motor function at 20 months of age, although ∼70% of nigral dopaminergic neurons had perished. Remaining neurons still preserved ∼75% of axon terminals in the dorsal striatum and enabled normal dopamine release. Transcriptome analysis and viral tracing confirmed compensatory axonal sprouting of the surviving neurons. We conclude that a small population of substantia nigra dopaminergic neurons is able to adapt to the accumulation of mitochondrial DNA mutations and maintain motor control.

Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing.

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.

Zinc Ammonio-dodecaborates: Synthesis, Lewis Acid Strength, and Reactivity.

Two series of zinc salts, [EtZn][A] and Zn[A]2, with weakly coordinating anions [A]- as counterions have been prepared, and their activities as catalysts for hydrosilylation reactions of 1-hexene, benzophenone, and acetophenone have been investigated. The counterions and per- and partially chlorinated 1-ammonio-closo-dodecaborate anions [Me3NB12Cl11]- [1]-, [Pr3NB12H5Cl6]- [2]-, [Bu3NB12H4Cl7]- [3]-, and [Hex3NB12H5Cl6]- [4]- were chosen as potential and more readily available alternatives to carborate anions such as [CHB11Cl11]- and [HexCB11Cl11]-. The basicity of anion [4]- was determined as being close to that of the triflimide anion [N(SO2CF3)2]-, and the fluoride ion affinities (FIAs) of compounds [EtZn][2] and Zn[2]2 are lower than those of the Lewis acids B(C6F5)3 and Zn[HexCB11Cl11]2. The higher anion basicity and the resulting lower Lewis acidity of the zinc centers result in low activity in 1-hexene hydrosilylation catalysis and only moderate activity in the hydrosilylation catalysis of benzophenone and acetophenone.

A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac.

Increasing contamination of environmental waters with pharmaceuticals represents an emerging threat for the drinking water quality and safety. In this regard, fast and reliable analytical methods are required to allow quick countermeasures in case of contamination. Here, we report the development of a magnetic bead-based immunoassay (MBBA) for the fast and cost-effective determination of the analgesic diclofenac (DCF) in water samples, based on diclofenac-coupled magnetic beads and a robust monoclonal anti-DCF antibody. A novel synthetic strategy for preparation of the beads resulted in an assay that enabled for the determination of diclofenac with a significantly lower limit of detection (400 ng/L) than the respective enzyme-linked immunosorbent assay (ELISA). With shorter incubation times and only one manual washing step required, the assay demands for remarkably shorter time to result (< 45 min) and less equipment than ELISA. Evaluation of assay precision and accuracy with a series of spiked water samples yielded results with low to moderate intra- and inter-assay variations and in good agreement with LC-MS/MS reference analysis. The assay principle can be transferred to other, e.g., microfluidic, formats, as well as applied to other analytes and may replace ELISA as the standard immunochemical method.

Squash bees host high diversity and prevalence of parasites in the northeastern United States.

The squash bee Eucera (Peponapis) pruinosa is emerging as a model species to study how stressors impact solitary wild bees in North America. Here, we describe the prevalence of trypanosomes, microsporidians and mollicute bacteria in E. pruinosa and two other species, Bombus impatiens and Apis mellifera, that together comprise over 97% of the pollinator visitors of Cucurbita agroecosystems in Pennsylvania (United States). Our results indicate that all three parasite groups are commonly detected in these bee species, but E. pruinosa often exhibit higher prevalences. We further describe novel trypanosome parasites detected in E. pruinosa, however it is unknown how these parasites impact these bees. We suggest future work investigates parasite replication and infection outcomes.

Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss of Nigral Dopaminergic Neurons.

Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of dopaminergic neurons (DaNs) in the substantia nigra (SNc), whereas DaNs in the neighboring ventral tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage calcium channels to generate action potentials, which lead to oxidant stress by yet unknown pathways. To determine the molecular mechanisms linking calcium load with selective cell death in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the mitochondrial membrane potential in mice of both sexes with genetically induced, severe mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox-sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all DaNs, exclusively SNc neurons showed an oxidized redox-system, i.e., a low reduced/oxidized glutathione (GSH-GSSG) ratio. This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of reactive oxygen species rather unlikely. Surprisingly, a high mitochondrial inner membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into the cell and into mitochondria, respectively, rescued the disturbed redox ratio and induced further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs to mitochondrial defects.SIGNIFICANCE STATEMENT Parkinson's disease is characterized by the preferential degeneration of dopaminergic neurons (DaNs) of the substantia nigra pars compacta (SNc), resulting in the characteristic hypokinesia in patients. Ubiquitous pathological triggers cannot be responsible for the selective neuron loss. Here we show that mitochondrial impairment together with elevated calcium burden destabilize the mitochondrial antioxidant defense only in SNc DaNs, and thus promote the increased vulnerability of this neuron population.

MRPS25 mutations impair mitochondrial translation and cause encephalomyopathy.

Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient's fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.

Tailored Mobility in a Zeolite Imidazolate Framework (ZIF) Antibody Conjugate*.

Zeolitic imidazolate framework (ZIF) hybrid fluorescent nanoparticles and ZIF antibody conjugates have been synthesized, characterized, and employed in lateral-flow immunoassay (LFIA). The bright fluorescence of the conjugates and the possibility to tailor their mobility gives a huge potential for diagnostic assays. An enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP) as label, proved the integrity, stability, and dispersibility of the antibody conjugates, LC-MS/MS provided evidence that a covalent link was established between these metal-organic frameworks and lysine residues in IgG antibodies.

Operative temperature analysis of the honey bee Apis mellifera.

A key challenge for linking experiments of organisms performed in a laboratory environment to their performance in more complex environments is to determine thermal differences between a laboratory and the energetically complex terrestrial ecosystem. Studies performed in the laboratory do not account for many factors that contribute to the realized temperature of an organism in its natural environment. This can lead to modelling approaches that use experimentally derived data to erroneously link the air temperature in a laboratory to air temperatures in energetically heterogenous ecosystems. Traditional solutions to this classic problem assume that animals in an isotropic, isothermal chamber behave either as pure heterothermic ectotherms (body temperature=chamber temperature) or homeothermic endotherms (body temperature is entirely independent of chamber temperature). This approach may not be appropriate for endothermic insects which exist as an intermediate between strongly thermoregulating endotherms and purely thermoconforming species. Here, we use a heat budget modelling approach for the honey bee Apis mellifera to demonstrate that the unique physiology of endothermic insects may challenge many assumptions of traditional biophysical modelling approaches. We then demonstrate under modelled field-realistic scenarios that an experiment performed in a laboratory has the potential to both overestimate and underestimate the temperature of foraging bees when only air temperature is considered.

Fluorescence polarization immunoassay for the determination of diclofenac in wastewater.

Pharmacologically active compounds are often detected in wastewater and surface waters. The nonsteroidal anti-inflammatory drug diclofenac (DCF) was included in the European watch list of substances that requires its environmental monitoring in the member states. DCF may harmfully influence the ecosystem already at concentrations ≤ 1 µg L-1. The fast and easy quantification of DCF is becoming a subject of global importance. Fluorescence polarization immunoassay (FPIA) is a homogeneous mix-and-read method which does not require the immobilization of reagents. FPIA can be performed in one phase within 20-30 min, making it possible to analyse wastewater without any complicated pre-treatment. In this study, new tracer molecules with different structures, linking fluorophores to derivatives of the analyte, were synthesized, three homologous tracers based on DCF, two including a C6 spacer, and one heterologous tracer derived from 5-hydroxy-DCF. The tracer molecules were thoroughly assessed for performance. Regarding sensitivity of the FPIA, the lowest limit of detection reached was 2.0 µg L-1 with a working range up to 870 µg L-1. The method was validated for real wastewater samples against LC-MS/MS as reference method with good agreement of both methods. Graphical abstract.

Honey bee viruses are highly prevalent but at low intensities in wild pollinators of cucurbit agroecosystems.

Managed and wild bee populations are in decline around the globe due to several biotic and abiotic stressors. Pathogenic viruses associated with the Western honey bee (Apis mellifera) have been identified as key contributors to losses of managed honey bee colonies, and are known to be transmitted to wild bee populations through shared floral resources. However, little is known about the prevalence and intensity of these viruses in wild bee populations, or how bee visitation to flowers impacts viral transmission in agroecosystems. This study surveyed honey bee, bumble bee (Bombus impatiens) and wild squash bee (Eucera (Peponapis) pruinosa) populations in Cucurbita agroecosystems across Pennsylvania (USA) for the prevalence and intensity of five honey bee viruses: acute bee paralysis virus (ABPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and slow bee paralysis virus (SBPV). We investigated the potential role of bee visitation rate to flowers on DWV intensity among species in the pollinator community, with the expectation that increased bee visitation to flowers would increase the opportunity for transmission events between host species. We found that honey bee viruses are highly prevalent but in lower titers in wild E. pruinosa and B. impatiens than in A. mellifera populations throughout Pennsylvania (USA). DWV was detected in 88% of B. impatiens, 48% of E. pruinosa, and 95% of A. mellifera. IAPV was detected in 5% of B. impatiens and 4% of E. pruinosa, compared to 9% in A. mellifera. KBV was detected in 1% of B. impatiens and 5% of E. pruinosa, compared to 32% in A. mellifera. Our results indicate that DWV titers are not correlated with bee visitation in Cucurbita fields. The potential fitness impacts of these low viral titers detected in E. pruinosa remain to be investigated.

Towards Naked Zinc(II) in the Condensed Phase: A Highly Lewis Acidic ZnII Dication Stabilized by Weakly Coordinating Carborate Anions.

The employment of the hexyl-substituted anion [HexCB11 Cl11 ]- allowed the synthesis of a ZnII species, Zn[HexCB11 Cl11 ]2 , 3, in which the Zn2+ cation is only weakly coordinated to two carborate counterions and that is soluble in low polarity organic solvents such as bromobenzene. DOSY NMR studies show the facile displacement of at least one of the counterions, and this near nakedness of the cation results in high catalytic activity in the hydrosilylation of 1-hexene and 1-methyl-1cyclohexene. Fluoride ion affinity (FIA) calculations reveal a solution Lewis acidity of 3 (FIA=262.1 kJ mol-1 ) that is higher than that of the landmark Lewis acid B(C6 F5 )3 (FIA=220.5 kJ mol-1 ). This high Lewis acidity leads to a high activity in catalytic CO2 and Ph2 CO reduction by Et3 SiH and hydrogenation of 1,1-diphenylethylene using 1,4-cyclohexadiene as the hydrogen source. Compound 3 was characterized by multinuclear NMR spectroscopy, mass spectrometry, single crystal X-ray diffraction, and DFT studies.

Mitochondrial DNA mutations induce mitochondrial biogenesis and increase the tumorigenic potential of Hodgkin and Reed-Sternberg cells.

Functioning mitochondria are crucial for cancer metabolism, but aerobic glycolysis is still considered to be an important pathway for energy production in many tumor cells. Here we show that two well established, classic Hodgkin lymphoma (cHL) cell lines harbor deleterious variants within mitochondrial DNA (mtDNA) and thus exhibit reduced steady-state levels of respiratory chain complexes. However, instead of resulting in the expected bioenergetic defect, these mtDNA variants evoke a retrograde signaling response that induces mitochondrial biogenesis and ultimately results in increased mitochondrial mass as well as function and enhances proliferation in vitro as well as tumor growth in mice in vivo. When complex I assembly was impaired by knockdown of one of its subunits, this led to further increased mitochondrial mass and function and, consequently, further accelerated tumor growth in vivo. In contrast, inhibition of mitochondrial respiration in vivo by the mitochondrial complex I inhibitor metformin efficiently slowed down growth. We conclude that, as a new mechanism, mildly deleterious mtDNA variants in cHL cancer cells cause an increase of mitochondrial mass and enhanced function as a compensatory effect using a retrograde signaling pathway, which provides an obvious advantage for tumor growth.

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels.

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.

Wash-Free Multiplexed Mix-and-Read Suspension Array Fluorescence Immunoassay for Anthropogenic Markers in Wastewater.

Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers. Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer. For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.

Parasitic gut infection in Libellula pulchella causes functional and molecular resemblance of dragonfly flight muscle to skeletal muscle of obese vertebrates.

We previously demonstrated the existence of a naturally occurring metabolic disease phenotype in Libellula pulchella dragonflies that shows high similarity to vertebrate obesity and type II diabetes, and is caused by a protozoan gut parasite. To further mechanistic understanding of how this metabolic disease phenotype affects fitness of male L. pulchella in vivo, we examined infection effects on in situ muscle performance and molecular traits relevant to dragonfly flight performance in nature. Importantly, these traits were previously shown to be affected in obese vertebrates. Similarly to obesity effects in rat skeletal muscle, dragonfly gut infection caused a disruption of relationships between body mass, flight muscle power output and alternative pre-mRNA splicing of troponin T, which affects muscle calcium sensitivity and performance in insects and vertebrates. In addition, when simulated in situ to contract at cycle frequencies ranging from 20 to 45 Hz, flight muscles of infected individuals displayed a left shift in power-cycle frequency curves, indicating a significant reduction in their optimal cycle frequency. Interestingly, these power-cycle curves were similar to those produced by flight muscles of non-infected teneral (i.e. physiologically immature) adult L. pulchella males. Overall, our results indicate that the effects of metabolic disease on skeletal muscle physiology in natural insect systems are similar to those observed in vertebrates maintained in laboratory settings. More generally, they indicate that study of natural, host-parasite interactions can contribute important insight into how environmental factors other than diet and exercise may contribute to the development of metabolic disease phenotypes.

Convenient Access to Gallium(I) Cations through Hydrogen Elimination from Cationic Gallium(III) Hydrides.

Although gallium hydrides XnGaH3-n (X = monoanionic substituent) are usually stable compounds, cationic arene-solvated species [H2Ga(arene)2]+ spontaneously eliminate dihydrogen at room temperature to afford the arene-solvated gallium(I) compounds [Ga(PhF)2][CHB11Cl11] (1) and [Ga(Ph3CH)][B(C6F5)4] (3). A key requirement appears to be the presence of a weakly coordinating anion. Use of the more basic triflimide anion, [NTf2]-, reverses the stability, i.e., the gallium(III) hydride H2GaNTf2 (4) is more stable than the gallium(I) compound GaNTf2 (5). The experimental results are supported by DFT calculations. Compounds 1 and 3 can be used as catalysts for the oligomerization of 2,4,4-trimethyl-1-pentene and the hydrosilylation of benzophenone and 1-hexene.

Improving the Course of Depressive Symptoms After Inpatient Psychotherapy Using Adjunct Web-Based Self-Help: Follow-Up Results of a Randomized Controlled Trial.

BACKGROUND: We recently showed in a randomized controlled trial that Web-based self-help as an adjunct improved the effectiveness of multimodal inpatient psychotherapy for depression. OBJECTIVE: The aims of this study were (1) to determine whether a Web-based self-help adjunctive to multimodal inpatient psychotherapeutic treatment could also improve the course of depressive symptoms and (2) to identify predictors of residual depressive symptoms at follow-up. METHODS: Overall, 229 patients were randomized either to the Web-based self-help intervention group (Deprexis) or an active control group (Web-based information about depression and depressive symptoms) in addition to multimodal inpatient psychotherapy. Participants in both groups were able to access their respective Web-based programs for 12 weeks, which meant that they typically had access after discharge from the inpatient unit (mean hospitalization duration: 40 days, T1). Follow-up was performed 6 months after study intake (T3). RESULTS: At follow-up, participants of the Web-based self-help group had considerably lower symptom load regarding depressive symptoms (d=0.58) and anxiety (d=0.46) as well as a better quality of life (d=0.43) and self-esteem (d=0.31) than participants of the control group. Nearly 3 times as many participants of the intervention group compared with the control group achieved remission in accordance with less deterioration. The number needed to treat based on the Beck Depression Inventory-II (BDI-II) improved over time (T1: 7.84, T2: 7.09, and T3: 5.12). Significant outcome predictors were BDI at discharge and treatment group. CONCLUSIONS: Web-based self-help as an add-on to multimodal inpatient psychotherapy improved the short-term course of depressive symptoms beyond termination. Residual symptoms at discharge from inpatient treatment and utilization of the Web-based self-help were the major predictors of depressive symptoms at follow-up. Challenges and barriers (eg, costs, therapists' concerns, or technical barriers) of adding Web-based interventions to inpatient treatment have to be addressed. TRIAL REGISTRATION: ClinicalTrials.gov NCT02196896; https://clinicaltrials.gov/ct2/show/NCT02196896.

[Tonsil surgery in Brandenburg-trends between 2013 and 2017]. / Operationen an den Gaumenmandeln im Land Brandenburg ­ Entwicklungen von 2013 bis 2017.

BACKGROUND: The frequency of tonsil surgery in Brandenburg (BB) during the period from 2013 to 2017 is analyzed. Causes and possible consequences of the trends are discussed. MATERIALS AND METHODS: An OPS code-based survey is carried out in the 10 ENT hospitals in BB and using the data from the federal states (Federal Statistical Office). In addition, the respective heads of ENT departments are interviewed concerning the indication for tonsillectomy (TE). The average number of operations in 2013/2014 is compared to that in 2017 (BB) and 2016 (federal states). RESULTS: In 9/10 participating hospitals in BB, 10,302 operations were performed, thereof 58.4% as TE, 19.0% as tonsillotomy (TT), 13.7% as abscess tonsillectomy (abscess TE), and 8.9% other. In BB in 2017 in comparison to 2013/2014, because of the decrease in TE by 45.6%, a total of 21.1% less operations were performed, while TT and abscess TE increased by 32% and 18%, respectively. The average age at surgery was 28.1, 38.1, and 5.9 years for TE, abscess TE, and TT, respectively. The entire trend for Germany until 2016 on the basis of 395,674 cases reflects a decrease in TE (30.7%) and abscess TE (14%), while TT was performed 27.5% more often, resulting in a total decrease of 21.3%. The interviewed persons follow the "justifiable operation indications" of the German guideline from 2015 and adapted their previous practice. CONCLUSION: Our survey and results indicate that the massive decrease of TE in BB is associated with the "justifiable operation indications" of the 2015 German guidelines on inflammatory diseases of the tonsils. TE is mainly performed in adults. Therefore, research concerning conservative versus surgical treatment for recurrent tonsillitis is urgently needed for this age profile.

Novel Electrochemical Paper-Based Immunocapture Assay for the Quantitative Determination of Ethinylestradiol in Water Samples.

We report a novel and innovative electrochemical paper-based immunocapture assay (EPIA) to address the need for ultrasensitive detection of emerging pollutants without regulatory status and whose effects on environment and human health are not completely yet understood. In particular, we present the application of this system toward highly sensitive detection of the emerging pollutant ethinyl estradiol (EE2). The EPIA approach is based on the use of paper microzones modified with silica nanoparticles (SNs) and anti-EE2 specific antibodies for capture and preconcentration of EE2 from river water samples. After the preconcentration procedure, the paper microzones are placed onto a screen-printed carbon electrode modified with electrochemically reduced graphene (RG). The bound EE2 is subsequently desorbed adding a diluted solution of sulfuric acid on the paper microzones. Finally, recovered EE2 is electrochemically detected by OSWV. The proposed novel methodology showed an appropriate LOD and linear range for the quantification of EE2 for water samples with different origins. The nonsophisticated equipment required, the adequate recovery values obtained (from 97% to 104%, with a RSD less than 4.9%), and the appropriate LOD and linear range value (0.1 ng L-1 and 0.5-120 ng L-1, respectively) achieved by our immunocapture sensor present significant analytical figures of merit, particularly when the routine quantification of EE2 is considered. In addition, our system was based on electrochemical paper-based technology, which allows obtainment of portable, easy-to-use, inexpensive, and disposable devices. The EPIA can also serve as a general-purpose immunoassay platform applicable to quantitation of other drugs and emerging pollutants in environmental samples.