IMD-mediated innate immune priming increases Drosophila survival and reduces pathogen transmission.

Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can 'prime' the immune response, resulting in improved survival upon reinfection. Here, we investigated the mechanistic basis and epidemiological consequences of innate immune priming in the fruit fly Drosophila melanogaster when infected with the gram-negative bacterial pathogen Providencia rettgeri. We find that priming in response to P. rettgeri infection is a long-lasting and sexually dimorphic response. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding and spread. The enhanced survival of individuals previously exposed to a non-lethal bacterial inoculum coincided with a transient decrease in bacterial loads, and we provide strong evidence that the effect of priming requires the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericin B is the main effector of bacterial clearance, it is not sufficient for immune priming, which requires regulation of IMD by peptidoglycan recognition proteins. This work underscores the plasticity and complexity of invertebrate responses to infection, providing novel experimental evidence for the effects of innate immune priming on population-level epidemiological outcomes.

Mitochondrial perturbation in immune cells enhances cell-mediated innate immunity in Drosophila.

BACKGROUND: Mitochondria participate in various cellular processes including energy metabolism, apoptosis, autophagy, production of reactive oxygen species, stress responses, inflammation and immunity. However, the role of mitochondrial metabolism in immune cells and tissues shaping the innate immune responses are not yet fully understood. We investigated the effects of tissue-specific mitochondrial perturbation on the immune responses at the organismal level. Genes for oxidative phosphorylation (OXPHOS) complexes cI-cV were knocked down in the fruit fly Drosophila melanogaster, targeting the two main immune tissues, the fat body and the immune cells (hemocytes). RESULTS: While OXPHOS perturbation in the fat body was detrimental, hemocyte-specific perturbation led to an enhanced immunocompetence. This was accompanied by the formation of melanized hemocyte aggregates (melanotic nodules), a sign of activation of cell-mediated innate immunity. Furthermore, the hemocyte-specific OXPHOS perturbation induced immune activation of hemocytes, resulting in an infection-like hemocyte profile and an enhanced immune response against parasitoid wasp infection. In addition, OXPHOS perturbation in hemocytes resulted in mitochondrial membrane depolarization and upregulation of genes associated with the mitochondrial unfolded protein response. CONCLUSIONS: Overall, we show that while the effects of mitochondrial perturbation on immune responses are highly tissue-specific, mild mitochondrial dysfunction can be beneficial in immune-challenged individuals and contributes to variation in infection outcomes among individuals.

The Drosophila Toll Pathway in Innate Immunity: from the Core Pathway toward Effector Functions.

The fruit fly Drosophila melanogaster Toll signaling pathway has an evolutionarily conserved role in controlling immune responses. Whereas the microbial recognition mechanisms and the core signaling pathway leading to activation of the humoral immune response via the NF-κB transcription factors have been well established for many years, the mechanistic understanding of the effector functions at the molecular level is currently rapidly evolving. In this review, we discuss the current developments in elucidating the role of the Drosophila Toll signaling pathway in immunity. We discuss the emerging role of Toll in viral infections and sex-specific differences in immunity. Mainly, we focus on Toll pathway regulation, the effector molecules, and cellular immunity.

Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. METHODS: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. RESULTS: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P = .003) or control rats (SUVmean, 0.2 ± 0.03; P < .0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. CONCLUSION: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions.

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion.

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase.

The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7 kDa-11.5 kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the ß' subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always ß', many interactions are taxon-specific. ω-less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the ß' subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω-less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω-less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.

Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells.

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.

Nafamostat is a Potent Human Diamine Oxidase Inhibitor Possibly Augmenting Hypersensitivity Reactions during Nafamostat Administration.

Nafamostat is an approved short-acting serine protease inhibitor. However, its administration is also associated with anaphylactic reactions. One mechanism to augment hypersensitivity reactions could be inhibition of diamine oxidase (DAO). The chemical structure of nafamostat is related to the potent DAO inhibitors pentamidine and diminazene. Therefore, we tested whether nafamostat is a human DAO inhibitor. Using different activity assays, nafamostat reversibly inhibited recombinant human DAO with an IC50 of 300-400 nM using 200 µM substrate concentrations. The Ki of nafamostat for the inhibition of putrescine and histamine deamination is 27 nM and 138 nM, respectively For both substrates, nafamostat is a mixed mode inhibitor with P values of <0.01 compared with other inhibition types. Using 80-90% EDTA plasma, the IC50 of nafamostat inhibition was approximately 360 nM using 20 µM cadaverine. In 90% EDTA plasma, the IC50 concentrations were 2-3 µM using 0.9 µM and 0.18 µM histamine as substrate. In silico modeling showed a high overlap compared with published diminazene crystallography data, with a preferred orientation of the guanidine group toward topaquinone. In conclusion, nafamostat is a potent human DAO inhibitor and might increase severity of anaphylactic reaction by interfering with DAO-mediated extracellular histamine degradation. SIGNIFICANCE STATEMENT: Treatment with the short-acting anticoagulant nafamostat during hemodialysis, leukocytapheresis, extracorporeal membrane oxygenator procedures, and disseminated intravascular coagulation is associated with severe anaphylaxis in humans. Histamine is a central mediator in anaphylaxis. Potent inhibition of the only extracellularly histamine-degrading enzyme diamine oxidase could augment anaphylaxis reactions during nafamostat treatment.

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent.

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

Variation in mitochondrial DNA affects locomotor activity and sleep in Drosophila melanogaster.

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved among many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. Our results demonstrate that Drosophila strains from different locations differ in sleep and activity, and that females are generally more active than males. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we were able to quantify the among-line variance attributable to mitochondrial DNA, and we establish that mtDNA variation affects both activity and sleep, in a sex-specific manner. Altogether our study highlights the important role that mitochondrial genome variation plays on organismal physiology and behaviour.

Osa-Containing Brahma Complex Regulates Innate Immunity and the Expression of Metabolic Genes in Drosophila.

Negative regulation of innate immunity is essential to avoid autoinflammation. In Drosophila melanogaster, NF-κB signaling-mediated immune responses are negatively regulated at multiple levels. Using a Drosophila RNA interference in vitro screen, we identified a set of genes inhibiting immune activation. Four of these genes encode members of the chromatin remodeling Osa-containing Brahma (BAP) complex. Silencing additional two genes of the BAP complex was shown to have the same phenotype, confirming its role in immune regulation in vitro. In vivo, the knockdown of osa and brahma was shown to enhance the expression of the Toll pathway-mediated antimicrobial peptides when the flies were challenged with Gram-positive bacteria Micrococcus luteus In this setting, osa knockdown had a particularly strong effect on immune effectors that are predominantly activated by the Imd pathway. Accordingly, Drosophila NF-κB Relish expression was increased by osa silencing. These transcriptional changes were associated with enhanced survival from M. luteus + E. faecalis infection. Besides regulating the expression of immune effector genes, osa RNA interference decreased the expression of a large group of genes involved in metabolism, particularly proteolysis. Of note, the expression of the recently characterized, immune-inducible gene Induced by Infection (IBIN) was diminished in osa knockdown flies. Although IBIN has been shown to modulate metabolism upon infection, the expression of selected Osa-regulated metabolism genes was not rescued by overexpressing IBIN. We conclude that the BAP complex regulates expression of genes involved in metabolism at least partially independent or downstream of IBIN Moreover, Osa affects the NF-κB-mediated immune response by regulating Drosophila NF-κB factor Relish expression.

Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Correction: Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster.

[This corrects the article DOI: 10.1371/journal.ppat.1007504.].

Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster.

Non-coding RNAs have important roles in regulating physiology, including immunity. Here, we performed transcriptome profiling of immune-responsive genes in Drosophila melanogaster during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. We show that upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-κB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism.

Alertness Training Increases Visual Processing Speed in Healthy Older Adults.

In this study, we investigated whether alertness training in healthy older adults increases visual processing speed (VPS) and whether functional connectivity in the cingulo-opercular network predicts training gain. Using the theory of visual attention, we derived quantitative estimates of VPS before and after training. In Study 1, 75 healthy older adults participated in alertness training, active-control training, or no training (n = 25 each). A significant Group × Session interaction indicated an increase in VPS in the alertness-training group but not in the control group, despite VPS not differing significantly between groups before training. In Study 2, 29 healthy older adults underwent resting-state functional MRI and then participated in alertness training. Pretraining functional connectivity in the cingulo-opercular network correlated with the individual training-induced change in VPS. In conclusion, results indicate that alertness training improves visual processing in older adults and that functional connectivity in the cingulo-opercular network provides a neural marker for predicting individual training gain.

Digitization of neuropsychological diagnostics: a pilot study to compare three paper-based and digitized cognitive assessments.

BACKGROUND AND OBJECTIVE: The number of people suffering from dementia is increasing worldwide and so is the need for reliable and economical diagnostic instruments. Therefore, the aim of this study was to compare the processing times of the neuropsychological tests Trail Making Tests A and B (TMT-A/B) and Color-Word Interference Test (CWIT), which were performed in both digital and paper versions. METHODS: The pilot study was conducted among 50 healthy participants (age 65-83 years) using a randomized crossover design. The correlations and differences in the individual processing times of the two test versions were statistically analyzed. Further research questions concerned the influence of the individual usage of technology and the technology commitment of participants as well as the influence of the assessed usability on participants' performance. RESULTS: Between the two versions (paper-based vs. digital) statistically significant correlations were found in all tests, e.g., TMT-A r(48) = 0.63, p < 0.01; TMT-B rs(48) = 0.77, p < 0.001). The mean value comparison showed statistically significant differences, e.g., interference table (CWIT) t(49) = 11.24, p < 0.01). Correlations with medium effect were found between the differences in processing times and the individual usage of computer (e.g., rs(48) = - 0.31) and smartphone (rs(48) = - 0.29) and between the processing times of the TMT-B and the usability (rs(48) = 0.29). CONCLUSIONS: The high correlations between the test procedures appear promising. However, the differences found in the processing times of the two test versions require validation and standardization of digitized test procedures before they can be used in practice.

Genetic and functional implications of an exonic TRIM55 variant in heart failure.

BACKGROUND: To tackle the missing heritability of sporadic heart failure, we screened for novel heart failure-associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo. METHODS AND RESULTS: Heart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/- cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wildtype genotype or overexpression of WT TRIM55, respectively. CONCLUSIONS: A previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.

Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family.

Borrelia burgdorferisensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato Because B. burgdorferisensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

Crystal structure of dimeric Synechococcus spermidine synthase with bound polyamine substrate and product.

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Human Copper-Containing Amine Oxidases in Drug Design and Development.

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.