The Blood-Brain Barrier Is Unaffected in the Ndufs4-/- Mouse Model of Leigh Syndrome.

Mitochondrial dysfunction plays a major role in physiological aging and in many pathological conditions. Yet, no study has explored the consequence of primary mitochondrial deficiency on the blood-brain barrier (BBB) structure and function. Addressing this question has major implications for pharmacological and genetic strategies aimed at ameliorating the neurological symptoms that are often predominant in patients suffering from these conditions. In this study, we examined the permeability of the BBB in the Ndufs4-/- mouse model of Leigh syndrome (LS). Our results indicated that the structural and functional integrity of the BBB was preserved in this severe model of mitochondrial disease. Our findings suggests that pharmacological or gene therapy strategies targeting the central nervous system in this mouse model and possibly other models of mitochondrial dysfunction require the use of specific tools to bypass the BBB. In addition, they raise the need for testing the integrity of the BBB in complementary in vivo models.

A Marine λ-Oligocarrageenan Inhibits Migratory and Invasive Ability of MDA-MB-231 Human Breast Cancer Cells through Actions on Heparanase Metabolism and MMP-14/MMP-2 Axis.

Sugar-based molecules such as heparins or natural heparan sulfate polysaccharides have been developed and widely studied for controlling heparanase (HPSE) enzymatic activity, a key player in extracellular matrix remodelling during cancer pathogenesis. However, non-enzymatic functions of HPSE have also been described in tumour mechanisms. Given their versatile properties, we hypothesized that sugar-based inhibitors may interfere with enzymatic but also non-enzymatic HPSE activities. In this work, we assessed the effects of an original marine λ-carrageenan derived oligosaccharide (λ-CO) we previously described, along with those of its native counterpart and heparins, on cell viability, proliferation, migration, and invasion of MDA-MB-231 breast cancer cells but also of sh-MDA-MB-231 cells, in which the expression of HPSE was selectively downregulated. We observed no cytotoxic and no anti-proliferative effects of our compounds but surprisingly λ-CO was the most efficient to reduce cell migration and invasion compared with heparins, and in a HPSE-dependent manner. We provided evidence that λ-CO tightly controlled a HPSE/MMP-14/MMP-2 axis, leading to reduced MMP-2 activity. Altogether, this study highlights λ-CO as a potent HPSE "modulator" capable of reducing not only the enzymatic activity of HPSE but also the functions controlled by the HPSE levels.

Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction.

Multicopper oxidases (MCOs) catalyze the oxidation of a variety of substrates while reducing oxygen into water through four copper atoms. As an additional feature, some MCOs display an enhanced activity in solution in the presence of Cu2+. This is the case of the hyperthermophilic laccase HB27 from Thermus thermophilus, the physiologic role of which is unknown. As a particular feature, this enzyme presents a methionine rich domain proposed to be involved in copper interaction. In this work, laccase from T. thermophilus was produced in E. coli, and the effect of Cu2+ on its electroactivity at carbon nanotube modified electrodes was investigated. Direct O2 electroreduction is strongly dictated by carbon nanotube surface chemistry in accordance with the enzyme dipole moment. In the presence of Cu2+, an additional low potential cathodic wave occurs, which was never described earlier. Analysis of this wave as a function of Cu2+ availability allows us to attribute this wave to a cuprous oxidase activity displayed by the laccase and induced by copper binding close to the Cu T1 center. A mutant lacking the methionine-rich hairpin domain characteristic of this laccase conserves its copper activity suggesting a different site of copper binding. This study provides new insight into the copper effect in methionine rich MCOs and highlights the utility of the electrochemical method to investigate cuprous oxidase activity and to understand the physiological role of these MCOs.

Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids.

Extracellular pH variations are seen as the principal endogenous signal that triggers activation of Acid-Sensing Ion Channels (ASICs), which are basically considered as proton sensors, and are involved in various processes associated with tissue acidification. Here, we show that human painful inflammatory exudates, displaying non-acidic pH, induce a slow constitutive activation of human ASIC3 channels. This effect is largely driven by lipids, and we identify lysophosphatidylcholine (LPC) and arachidonic acid (AA) as endogenous activators of ASIC3 in the absence of any extracellular acidification. The combination of LPC and AA evokes robust depolarizing current in DRG neurons at physiological pH 7.4, increases nociceptive C-fiber firing, and induces pain behavior in rats, effects that are all prevented by ASIC3 blockers. Lipid-induced pain is also significantly reduced in ASIC3 knockout mice. These findings open new perspectives on the roles of ASIC3 in the absence of tissue pH variation, as well as on the contribution of those channels to lipid-mediated signaling.

Bacterial Respiratory Chain Diversity Reveals a Cytochrome c Oxidase Reducing O2 at Low Overpotentials.

Cytochrome c oxidases (CcOs) are the terminal enzymes in energy-converting chains of microorganisms, where they reduce oxygen into water. Their affinity for O2 makes them attractive biocatalysts for technological devices in which O2 concentration is limited, but the high overpotentials they display on electrodes severely limit their applicative use. Here, the CcO of the acidophilic bacterium Acidithiobacillus ferrooxidans is studied on various carbon materials by direct protein electrochemistry and mediated one with redox mediators either diffusing or co-immobilized at the electrode surface. The entrapment of the CcO in a network of hydrophobic carbon nanofibers permits a direct electrochemical communication between the enzyme and the electrode. We demonstrate that the CcO displays a µM affinity for O2 and reduces O2 at exceptionally high electrode potentials in the range of +700 to +540 mV vs NHE over a pH range of 4-6. The kinetics of interactions between the enzyme and its physiological partners are fully quantified. Based on these results, an electron transfer pathway allowing O2 reduction in the acidic metabolic chain is proposed.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases.

Maintaining the equilibrium between saturated and unsaturated fatty acids within membrane phospholipids (PLs) is crucial to sustain the optimal membrane biophysical properties, compatible with selective organelle-based processes. Lipointoxication is a pathological condition under which saturated PLs tend to accumulate within the cell at the expense of unsaturated species, with major impacts on organelle function. Here, we show that human bronchial epithelial cells extracted from lungs of patients with Obstructive Pulmonary Diseases (OPDs), i. e. Cystic Fibrosis (CF) individuals and Smokers, display a characteristic lipointoxication signature, with excessive amounts of saturated PLs. Reconstitution of this signature in cellulo and in silico revealed that such an imbalance results in altered membrane properties and in a dramatic disorganization of the intracellular network of bronchial epithelial cells, in a process which can account for several OPD traits. Such features include Endoplasmic Reticulum-stress, constitutive IL8 secretion, bronchoconstriction and, ultimately, epithelial cell death by apoptosis. We also demonstrate that a recently-identified lipid-like molecule, which has been shown to behave as a "membrane-reshaper", counters all the lipointoxication hallmarks tested. Altogether, these insights highlight the modulation of membrane properties as a potential new strategy to heal and prevent highly detrimental symptoms associated with OPDs.

Diversity of CO2-concentrating mechanisms and responses to CO2 concentration in marine and freshwater diatoms.

The presence of CO2-concentrating mechanisms (CCMs) is believed to be one of the characteristics that allows diatoms to thrive in many environments and to be major contributors to global productivity. Here, the type of CCM and the responses to variable CO2 concentration were studied in marine and freshwater diatoms. At 400 ppm, there was a large diversity in physiological and biochemical mechanisms among the species. While Phaeodactylum tricornutum mainly used HCO3-, Thalassiosira pseudonana mainly used CO2. Carbonic anhydrase was an important component of the CCM in all species and C4 metabolism was absent, even with T. weissflogii. For all species, at 20 000 ppm, the affinity for dissolved inorganic carbon was lower than at 400 ppm CO2 and the reliance on CO2 was higher. Despite the difference in availability of inorganic carbon in marine and fresh waters, there were only small differences in CCMs between species from the two environments, and Navicula pelliculosa behaved similarly when grown in the two environments. The results suggest that species-specific differences are great, and more important than environmental differences in determining the nature and effectiveness of the CCM in diatoms.

Transcriptomics and Lipidomics of the Environmental Strain Rhodococcus ruber Point out Consumption Pathways and Potential Metabolic Bottlenecks for Polyethylene Degradation.

Polyethylene (PE), one of the most prominent synthetic polymers used worldwide, is very poorly biodegradable in the natural environment. Consequently, PE represents by itself more than half of all plastic wastes. PE biodegradation is achieved through the combination of abiotic and biotic processes. Several microorganisms have been shown to grow on the surface of PE materials, among which are the species of the Rhodococcus genus, suggesting a potent ability of these microorganisms to use, at least partly, PE as a potent carbon source. However, most of them, if not all, fail to induce a clear-cut degradation of PE samples, showing that bottlenecks to reach optimal biodegradation clearly exist. To identify the pathways involved in PE consumption, we used in the present study a combination of RNA-sequencing and lipidomic strategies. We show that short-term exposure to various forms of PE, displaying different molecular weight distributions and oxidation levels, lead to an increase in the expression of 158 genes in a Rhodococcus representative, R. ruber. Interestingly, one of the most up-regulated pathways is related to alkane degradation and ß-oxidation of fatty acids. This approach also allowed us to identify metabolic limiting steps, which could be fruitfully targeted for optimized PE consumption by R. ruber.

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states.

BACKGROUND: Like many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors. METHODS: Patch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation. RESULTS: The present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current. CONCLUSIONS: Our findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route. GENERAL SIGNIFICANCE: This work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.

The nature of the CO2 -concentrating mechanisms in a marine diatom, Thalassiosira pseudonana.

Diatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide-concentrating mechanisms (CCMs) involving transporters and carbonic anhydrases (CAs) are well known, but the contribution of a biochemical CCM involving C4 metabolism is contentious. The CCM(s) present in the marine-centric diatom, Thalassiosira pseudonana, were studied in cells exposed to high or low concentrations of CO2 , using a range of approaches. At low CO2 , cells possessed a CCM based on active uptake of CO2 (70% contribution) and bicarbonate, while at high CO2 , cells were restricted to CO2 . CA was highly and rapidly activated on transfer to low CO2 and played a key role because inhibition of external CA produced uptake kinetics similar to cells grown at high CO2 . The activities of phosphoenolpyruvate (PEP) carboxylase (PEPC) and the PEP-regenerating enzyme, pyruvate phosphate dikinase (PPDK), were lower in cells grown at low than at high CO2 . The ratios of PEPC and PPDK to ribulose bisphosphate carboxylase were substantially lower than 1, even at low CO2 . Our data suggest that the kinetic properties of this species results from a biophysical CCM and not from C4 type metabolism.

Searching for combinations of small-molecule correctors to restore f508del-cystic fibrosis transmembrane conductance regulator function and processing.

The mutated protein F508del-cystic fibrosis transmembrane conductance regulator (CFTR) failed to traffic properly as a result of its retention in the endoplasmic reticulum and functions as a chloride (Cl(-)) channel with abnormal gating and endocytosis. Small chemicals (called correctors) individually restore F508del-CFTR trafficking and Cl(-) transport function, but recent findings indicate that synergistic pharmacology should be considered to address CFTR defects more clearly. We studied the function and maturation of F508del-CFTR expressed in HeLa cells using a combination of five correctors [miglustat, IsoLAB (1,4-dideoxy-2-hydroxymethyl-1,4-imino-l-threitol), Corr4a (N-[2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']bithiazolyl-2'-yl]-benzamide), VX-809 [3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid], and suberoylamilide hydroxamic acid (SAHA)]. Using the whole-cell patch-clamp technique, the current density recorded in response to CFTR activators (forskolin + genistein) was significantly increased in the presence of the following combinations: VX-809 + IsoLAB; VX-809 + miglustat + SAHA; VX-809 + miglustat + IsoLAB; VX-809 + IsoLAB + SAHA; VX-809 + miglustat + IsoLAB + SAHA. These combinations restored the activity of F508del-CFTR but with a differential effect on the appearance of mature c-band of F508del-CFTR proteins. Focusing on the VX-809 + IsoLAB cocktail, we recorded a level of correction higher at 37°C versus room temperature, but without amelioration of the thermal instability of CFTR. The level of functional rescue with VX-809 + IsoLAB after 4 hours of incubation was maximal and similar to that obtained in optimal conditions of use for each compound (i.e., 24 hours for VX-809 + 4 hours for IsoLAB). Finally, we compared the stimulation of F508del-CFTR by forskolin or forskolin + VX-770 [N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide] with cells corrected by VX-809 + IsoLAB. Our results open new perspectives for the development of a synergistic polypharmacology to rescue F508del-CFTR and show the importance of temperature on the effect of correctors and on the level of correction, suggesting that optimized combination of correctors could lead to a better rescue of F508del-CFTR function.

[Update for cord blood unit selection in hematopoietic stem cell transplantation (workshop SFGM-TC)]. / Mise à jour des recommandations pour le choix des unités de sang placentaire en greffe de cellules souches hématopoïétiques (atelier SFGM-TC).

Changing practices and the limited use of cord blood units as a source of cells for allogeneic hematopoietic stem cell transplants (HSC) led us to reconsider the recommendations established in 2011 and 2012, and to propose an update incorporating recent bibliographic data. If HLA compatibility was until now established at low resolution for HLA-A and B loci, and at high resolution for HLA-DRB1, the recent papers are converging towards an increase in the level of resolution, making way for a compatibility now defined in high resolution for all the considered loci, and the inclusion of the HLA-C locus, in order to establish a level of HLA compatibility on 8 alleles (HLA-A, B, C and DRB1). The CD34+ dose is a determining factor in hematopoietic reconstitution but it is not correlated with the total nucleated cells content. This is why we recommend taking these two data into account when choosing a cord blood unit. The recommendations established by our group are presented as a flow chart taking into account the characteristics of the underlying pathology (malignant or non-malignant), the cell dose and the HLA compatibility criteria, as well as criteria linked to the banks in which units are stored.

Rescue of functional CFTR channels in cystic fibrosis: a dramatic multivalent effect using iminosugar cluster-based correctors.

Cystic fibrosis is caused by a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. N-butyl 1-deoxynojirimycin (N-Bu DNJ), a clinical candidate for the treatment of cystic fibrosis, is able to act as a CFTR corrector by overcoming the processing defect of the mutant protein. To explore the potential of multivalency on CFTR correction activity, a library of twelve DNJ click clusters with valencies ranging from 3 to 14 were synthesized. Significantly, the trivalent analogues were found to be up to 225-fold more potent than N-Bu DNJ and up to 1000-fold more potent than the corresponding monovalent models. These results provide the first description of a multivalent effect for correcting protein folding defects in cells and should have application for the treatment of a number of protein folding disorders. Preliminary mechanistic studies indicated that CFTR correction activity enhancement was not due to a multivalent effect in ER-glucosidase inhibition or to a different mode of action of the multivalent iminosugars.

Paper functionalization for detection of Plasmodium falciparum DNA using square waves voltammetry.

Malaria elimination is a major goal to be reached in the next decade. Significant progress were made in the past, and the prevalence decreased in many areas while the positive trend stalled in the last years. The exact number of asymptomatic carriers of Plasmodium parasites is unknown since this population is not detected by conventional diagnosis methods and participate in the maintenance of transmission. Molecular methods to detect low parasitemia are not available at point-of-care in low-income countries of malaria endemic areas. Adaptation of molecular methods such as loop-mediated isothermal amplification of DNA may provide effective tools but it required simplification of DNA detection. Square waves voltammetry, easily imbedded in small device such as cell phone, was largely described for DNA detection but support for reaction was an issue to address. Here we used an efficient functionalization method of paper-based material to facilitate the interactions between isothermal amplification product and methylene blue for easy-to-use DNA detection. The proof-of-concept of qualitative detection of very low parasitemia from malaria infected patients using newly chemically treated paper for square waves voltammetry was obtained with a sensitivity and specificity of 100% and a limit-of-detection of 0.1 parasite. µL-1 corresponding to a parasitemia of 0.000002%.

Serum Lipidomic Screen Identifies Key Metabolites, Pathways, and Disease Classifiers in Crohn's Disease.

BACKGROUND: There is an unmet medical need for biomarkers that capture host and environmental contributions in inflammatory bowel diseases (IBDs). This study aimed at testing the potential of circulating lipids as disease classifiers given their major roles in inflammation. METHODS: We applied a previously validated comprehensive high-resolution liquid chromatography-mass spectrometry-based untargeted lipidomic workflow covering 25 lipid subclasses to serum samples from 100 Crohn's disease (CD) patients and 100 matched control subjects. Findings were replicated and expanded in another 200 CD patients and 200 control subjects. Key metabolites were tested for associations with disease behavior and location, and classification models were built and validated. Their association with disease activity was tested using an independent cohort of 42 CD patients. RESULTS: We identified >70 metabolites with strong association (P < 1 × 10-4, q < 5 × 10-4) to CD. Highly performing classification models (area under the curve > 0.84-0.97) could be built with as few as 5 to 9 different metabolites, representing 6 major correlated lipid clusters. These classifiers included a phosphatidylethanolamine ether (O-16:0/20:4), a sphingomyelin (d18:1/21:0) and a cholesterol ester (14:1), a very long-chain dicarboxylic acid [28:1(OH)] and sitosterol sulfate. These classifiers and correlated lipids indicate a dysregulated metabolism in host cells, notably in peroxisomes, as well as dysbiosis, oxidative stress, compromised inflammation resolution, or intestinal membrane integrity. A subset of these were associated with disease behavior or location. CONCLUSIONS: Untargeted lipidomic analyses uncovered perturbations in the circulating human CD lipidome, likely resulting from multiple pathogenic mechanisms. Models using as few as 5 biomarkers had strong disease classifier characteristics, supporting their potential use in diagnosis or prognosis.

This study reports a comprehensive untargeted lipidomic analysis of 600 serum samples from patients with Crohn's disease and matched control subjects, identified and replicated ~70 metabolites associated with Crohn's disease, and developed highly performing classification models (area under the curve > 0.84-0.97) with as few as 5 metabolites.

Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus.

Thermus thermophilus laccase belongs to the sub-class of multicopper oxidases that is activated by the extra binding of copper to a methionine-rich domain allowing an electron pathway from the substrate to the conventional first electron acceptor, the T1 Cu. In this work, two key amino acid residues in the 1st and 2nd coordination spheres of T1 Cu are mutated in view of tuning their redox potential and investigating their influence on copper-related activity. Evolution of the kinetic parameters after copper addition highlights that both mutations play a key role influencing the enzymatic activity in distinct unexpected ways. These results clearly indicate that the methionine rich domain is not the only actor in the cuprous oxidase activity of CueO-like enzymes.

Design and development of a robotic predator as a stimulus in conditioned place aversion for the study of the effect of ethanol and citalopram in zebrafish.

Zebrafish are becoming a species of choice in psychopharmacology, laying a promising path to refined pharmacological manipulations and high-throughput behavioral phenotyping. The field of robotics has the potential to accelerate progress along this path, by offering unprecedented means for the design and development of accurate and reliable experimental stimuli. In this work, we demonstrate, for the first time, the integration of robotic predators in place conditioning experiments. We hypothesized zebrafish to be capable of forming a spatial association under a simulated predation risk. We repeatedly exposed experimental subjects to a robotic heron impacting the water surface and then evaluated their spatial avoidance within the experimental tank in a subsequent predator-free test session. To pharmacologically validate the paradigm, we tested zebrafish in drug-free conditions (control groups) or in response to three different concentrations of citalopram (30, 50, and 100 mg/L) and ethanol (0.25, 0.50, and 1.00%). Experimental data indicate that, when tested in the absence of the conditioning stimulus, zebrafish displayed a marked preference for the bottom of the test tank, that is, the farthest location from the simulated attacks by the robotic heron. This conditioned geotaxis was reduced by the administration of citalopram in a linear dose-response curve and ethanol at the low concentration. Ultimately, our data demonstrate that robotic stimuli may represent valid conditioning tools and, thereby, aid the field of zebrafish psychopharmacology.

A comprehensive study of phospholipid fatty acid rearrangements in metabolic syndrome: correlations with organ dysfunction.

The balance within phospholipids (PLs) between saturated fatty acids and monounsaturated or polyunsaturated fatty acids is known to regulate the biophysical properties of cellular membranes. As a consequence, in many cell types, perturbing this balance alters crucial cellular processes, such as vesicular budding and the trafficking/function of membrane-anchored proteins. The worldwide spread of the Western diet, which is highly enriched in saturated fats, has been clearly correlated with the emergence of a complex syndrome known as metabolic syndrome (MetS). MetS is defined as a cluster of risk factors for cardiovascular diseases, type 2 diabetes and hepatic steatosis; however, no clear correlations have been established between diet-induced fatty acid redistribution within cellular PLs and the severity/chronology of the symptoms associated with MetS or the function of the targeted organs. To address this issue, in this study we analyzed PL remodeling in rats exposed to a high-fat/high-fructose diet (HFHF) over a 15-week period. PL remodeling was analyzed in several organs, including known MetS targets. We show that fatty acids from the diet can redistribute within PLs in a very selective manner, with phosphatidylcholine being the preferred sink for this redistribution. Moreover, in the HFHF rat model, most organs are protected from this redistribution, at least during the early onset of MetS, at the expense of the liver and skeletal muscles. Interestingly, such a redistribution correlates with clear-cut alterations in the function of these organs.This article has an associated First Person interview with the first author of the paper.

Comparison between two- and three-dimensional scoring of zebrafish response to psychoactive drugs: identifying when three-dimensional analysis is needed.

Zebrafish (Danio rerio) have recently emerged as a valuable laboratory species in the field of behavioral pharmacology, where they afford rapid and precise high-throughput drug screening. Although the behavioral repertoire of this species manifests along three-dimensional (3D), most of the efforts in behavioral pharmacology rely on two-dimensional (2D) projections acquired from a single overhead or front camera. We recently showed that, compared to a 3D scoring approach, 2D analyses could lead to inaccurate claims regarding individual and social behavior of drug-free experimental subjects. Here, we examined whether this conclusion extended to the field of behavioral pharmacology by phenotyping adult zebrafish, acutely exposed to citalopram (30, 50, and 100 mg/L) or ethanol (0.25%, 0.50%, and 1.00%), in the novel tank diving test over a 6-min experimental session. We observed that both compounds modulated the time course of general locomotion and anxiety-related profiles, the latter being represented by specific behaviors (erratic movements and freezing) and avoidance of anxiety-eliciting areas of the test tank (top half and distance from the side walls). We observed that 2D projections of 3D trajectories (ground truth data) may introduce a source of unwanted variation in zebrafish behavioral phenotyping. Predictably, both 2D views underestimate absolute levels of general locomotion. Additionally, while data obtained from a camera positioned on top of the experimental tank are similar to those obtained from a 3D reconstruction, 2D front view data yield false negative findings.