Sol Cristians

Douwe Dekker
email: d.dekker@umcutrecht.nl

Emiel Jacob

Henri Bartels

Mohamed Amiche
email: mohamed.amiche@u-pec.fr

Dermaseptins, Multifunctional Antimicrobial Peptides: A Review of Their Pharmacology, Effectivity, Mechanism of Action, and Possible Future Directions

Keywords: dermaseptin, Phyllomedusa bicolor, peptide, amphibian, infectious, antimicrobial, tumor

Dermaseptins are a group of α-helical shaped polycationic peptides isolated from the Hylid frogs, with antimicrobial effects against bacteria, parasites, protozoa, viruses in vitro. Besides, anti-tumor effects have been demonstrated. However, few animal experiments and no clinical trials have been conducted thus far. This review summarizes the current knowledge on the pharmacology, ethno pharmacology, effectivity against infectious pathogens and tumors cells and the mechanism of action of the Dermaseptins. Future research should focus on further clarification of the mechanisms of action, the effectivity of Dermaseptins against several cancer cell lines and their applicability in humans.

INTRODUCTION

Dermaseptins (DRSs) are a family of peptides that are part of the skin secretions of several Hylid frogs, particularly from the Agalychnis and Phyllomedusa family [START_REF] Nicolas | The dermaseptins[END_REF][START_REF] Amiche | A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae[END_REF]. In 1991, the first DRS was identified and characterized as a peptide rich in basic amino acids with a high propensity to adopt an α-helical structure in a hydrophobic medium [START_REF] Mor | Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin[END_REF]. The DRS-S1 was purified by reverse phase high-pressure liquid chromatography (RP-HPLC) from a skin extract of the Phyllomedusa sauvagii and sequenced by Edman's method. By now, there are more than a hundred DRS-like peptides classified in the large family of DRSs that share a strong identity in the cDNA sequences encoding their biosynthetic precursors [START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF].

DRSs are often classified as antimicrobial peptides (AMPs), since they show effectivity in vitro against some gram positive and gram negative bacteria, parasites, yeasts, protozoa, viruses and display immune modulatory effects (Mor et al., 1994a;Mor et al., 1994b;[START_REF] Strahilevitz | Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes[END_REF][START_REF] Charpentier | Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics[END_REF][START_REF] Brand | Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells[END_REF][START_REF] Navon-Venezia | Antibacterial properties of dermaseptin S4 derivatives with in vivo activity[END_REF][START_REF] Brand | Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)[END_REF][START_REF] Conceicao | Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis[END_REF][START_REF] Conlon | Peptides with differential cytolytic activity from skin secretions of the lemur leaf frog Hylomantis lemur (Hylidae: Phyllomedusinae)[END_REF][START_REF] Leite | Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Secondary structure, antimicrobial activity, and mammalian cell toxicity[END_REF][START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helix-hinge-helix structure and membrane curvature strain[END_REF][START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF][START_REF] Jiang | Specificity determinants" improve therapeutic indices of two antimicrobial peptides piscidin 1 and dermaseptin s4 against the gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa[END_REF][START_REF] Zairi | In vitro activities of dermaseptins K4S4 and K4K20S4 against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa planktonic growth and biofilm formation[END_REF][START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF]. Beside these antimicrobial properties, DRSs show activity against several human cancer types [START_REF] Charpentier | Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics[END_REF][START_REF] Conlon | Peptides with differential cytolytic activity from skin secretions of the lemur leaf frog Hylomantis lemur (Hylidae: Phyllomedusinae)[END_REF][START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF][START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helix-hinge-helix structure and membrane curvature strain[END_REF][START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF][START_REF] Shi | Two novel dermaseptin-like antimicrobial peptides with anticancer activities from the skin secretion of Pachymedusa dacnicolor[END_REF][START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF][START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF][START_REF] Zhu | Discovery of two skinderived dermaseptins and design of a TAT-fusion analogue with broad-spectrum antimicrobial activity and low cytotoxicity on healthy cells[END_REF] and can therefore be classified as an anticancer peptide as well.

In view of the current increasing bacterial resistance to conventional antibiotics [START_REF] Laxminarayan | Antibiotic resistance-the need for global solutions[END_REF] the demand for novel antibacterial pharmaceuticals is high. Likewise, there is a need for novel anti-tumor treatments as cancer is rapidly becoming the leading cause of death in the Western world and conventional therapeutics are both cytotoxic and prone to therapy resistance due to microevolution of the tumor tissue [START_REF] Corrie | Cytotoxic chemotherapy: clinical aspects[END_REF][START_REF] Colak | Cancer stem cells-important players in tumor therapy resistance[END_REF].

The aim of this review is to summarize the current knowledge on DRSs, to elaborate on the ethnopharmacology, the potential therapeutic values with respect to their anti-microbial and antitumor potency, and to suggest future directions for research. We thereby restricted ourselves to DRSs (sensu stricto) according to the nomenclature proposed by [START_REF] Amiche | A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae[END_REF].

ETHNOPHARMACOLOGY IN PEPTIDE DISCOvERY

Many plants and animal products have yet found their way from traditional use to Western medicine leading to the discovery of for example morphine, codeine, quinine, aspirin, curare, pilocarpine and ACE-inhibitors [START_REF] Bisset | One man's poison, another man's medicine?[END_REF][START_REF] Alves | The faunal drugstore: animal-based remedies used in traditional medicines in Latin America[END_REF][START_REF] Dias | A historical overview of natural products in drug discovery[END_REF]. Likewise, DRS-B's from the Phyllomedusa bicolor secretions are yet known to be applied for human use in traditional medicine. This Phyllomedusa bicolor is an Amazonian amphibian found in the forests of Brazil, the Guianas, Venezuela, Colombia, Peru, and Bolivia. The secretions of this frog are referred to by 'Kambo' , 'Kampu, or 'Sapo' , and are used by natives as a medicine and part of a cleansing ritual. The use of Kambo by South American Indians was first described by Constantin Tastevin in 1925 in the Kachinaua, Kurina, and the Kanamari tribes [START_REF] Lima | A expansão urbana do kampo (Phyllomedusa bicolor): notas etnográficas1[END_REF]. Later, the traditional use of kambo was also documented in the Katukina tribe, the Mayoruna tribe, and the Matse tribe [START_REF] Lima | A expansão urbana do kampo (Phyllomedusa bicolor): notas etnográficas1[END_REF][START_REF] Gorman | Sapo in my soul[END_REF]. Other DRS secreting frogs have not been documented to be useful in rituals.

The Kambo-ritual is characterized by an immediate and short-lived physical response followed by a longer lasting mental and minor physical effects. The Indians 'harvest' the frog by collecting the secretion from a live frog and transfer its secretions to a bamboo stick. The medicine may then be used to cure or prevent illness, to expel 'panema' (bad spirit), or even to induce an abortion [START_REF] Gorman | Sapo in my soul[END_REF]. When unlucky in hunting Kambo reportedly increases stamina, and sharpens senses during long hunts. Application involves burning dots on the skin, usually on arms and/or legs, and sticking a small dose (10 mg) 'dot' on the open wound. The symptoms are severe and immediate; violent nausea, vomiting, diarrhea, edema of the face and headaches. Symptoms last until the secretion is removed from the wound usually after 15-20 minutes.

Besides its traditional use in the Amazon, Kambo has found its way into the Western alternative healing scene as well. While the reports on the beneficial effects of this ritual are numerous and range from relieving symptoms of pain syndromes, autoimmune diseases, skin disease, and cancer to substance abuse and depression [START_REF] Hesselink | Kambô: A Shamanic Medicine-Personal Testimonies[END_REF], so are the accounts of adverse effects of participating in a rite, with or without experienced guidance. These include a transient syndrome of inappropriate antidiuretic hormone secretion [START_REF] Leban | The syndrome of inappropriate antidiuretic hormone secretion after giant leaf frog (Phyllomedusa bicolor) venom exposure[END_REF], presumed drug induced liver injury in a chronic alcoholic [START_REF] Pogorzelska | Toxic hepatitis caused by the excretions of the Phyllomedusa bicolor frog -a case report[END_REF], sudden death upon chronic kambo use in which autopsy revealed underlying heart disease possibly related to reduced myocardial perfusion [START_REF] Aquila | The Biological Effects of Kambo: Is there a relationship between its administration and sudden death?[END_REF] and delayed kambo related symptoms in a 24-year-old woman 22 hours after the ritual [START_REF] Li | Prolonged toxicity from Kambo cleansing ritual[END_REF].

The short-lasting effects of kambo are related to a diversity of biologically active peptides besides DRS including Phyllokinin (a bradykinin), Phyllomedusa (a tachykinin), Sauvagine (vasodilator), Caerulin (a CCK like peptide) and Deltorphin/ Demorphin (opioid receptor agonists). Studies on these peptides have contributed greatly to our knowledge concerning the μ/δ-opioid and serotonin receptor [START_REF] Erspamer | Pharmacological studies of 'sapo' from the frog Phyllomedusa bicolor skin: a drug used by the Peruvian Matses Indians in shamanic hunting practices[END_REF], but are beyond the scope of this review. Many of the immediate effects of Kambo can be explained by peptides in the frogsecretion. Vasoactive properties of some of these peptides for example might contribute to the rapid absorption of the secretion into the fresh burn. Amongst these peptides, DRS is the primary candidate for the anti-microbial effects reported upon the use of Kambo.

STRUCTURAL AND PHARMACOLOGICAL ANALYSIS OF THE DRS FAMILY

Even though each DRS has a unique amino acid sequence and selectivity patterns towards microorganisms and tumors, there are also a lot of pharmacological similarities. Figure 1 shows the amino acid sequence alignment of 57 DRSs sequences extracted from the antimicrobial peptide databases (http://aps.unmc.edu/AP/main.php). There is amino-acid sequence similarity within DRS from the same frog (DRS-B1-B6; 33-62%) [START_REF] Charpentier | Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics[END_REF], but also between DRS from different species (DRS-B1 and DRS-S1; 81%, DRS-B2, and DRS-D 84.8%) (Mor et al., 1994b;[START_REF] Auvynet | Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9[END_REF]. They are mostly rather short peptides (21-34 residues) with a highly-preserved tryptophan residue on the 3rd position from the N-terminus [START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF] except for DRS-S10, DRS-S13, DRS-C3, and DRS-A4. DRSs can be fitted into an amphipathic α-helix with their hydrophobic residues on one face and the polar cationic residues in cluster on the opposite face. They usually do this is an anionic environment, or under the influence of certain phospholipids [START_REF] Hoskin | Studies on anticancer activities of antimicrobial peptides[END_REF]. These charged clusters tend to differ among DRS, for example; DRS-B1 has a narrow polar face of a mean radial angle of 115°, DRS-B2 has a polar face covering almost half of the helix; 175° and DRS-S1 a polar face of 145° (Mor et al., 1994a). Furthermore, they show great variation in net charge and density of charge. DRS-S9 seems to be an exception as it has a highly hydrophobic core flanked by cationic residues [START_REF] Lequin | Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini[END_REF][START_REF] Caillon | Biophysical investigation of the membrane-disrupting mechanism of the antimicrobial and amyloid-like peptide dermaseptin S9[END_REF]. Thus far, several DRS have been in vitro tested for their activity against various microorganisms [START_REF] Lorin | The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro[END_REF][START_REF] Savoia | In vitro activity of dermaseptin S1 derivatives against genital pathogens[END_REF][START_REF] De Moraes | Evaluation of the in vitro activity of dermaseptin 01, a cationic antimicrobial peptide, against Schistosoma mansoni[END_REF]van Zoggel et al., vitro activity against gram positive and negative bacteria with various specificities [START_REF] Strahilevitz | Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes[END_REF]. Derivatives of DRS-S4, DRS-CA1, DRS-DU1 and DRS-PH show in vitro activity against Staphylococcus aureus (including the methicillin resistant strain), Pseudomonas aeruginosa and E. coli, even when they are formed in biofilm [START_REF] Navon-Venezia | Antibacterial properties of dermaseptin S4 derivatives with in vivo activity[END_REF][START_REF] Zairi | In vitro activities of dermaseptins K4S4 and K4K20S4 against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa planktonic growth and biofilm formation[END_REF][START_REF] Liu | Discovery of phylloseptins that defense against gram-positive bacteria and inhibit the proliferation of the non-small cell lung cancer cell line, from the skin secretions of Phyllomedusa frogs[END_REF][START_REF] Zhu | Discovery of two skinderived dermaseptins and design of a TAT-fusion analogue with broad-spectrum antimicrobial activity and low cytotoxicity on healthy cells[END_REF]. Though less cytotoxic compared to conventional antibiotics [START_REF] Zairi | In vitro activities of dermaseptins K4S4 and K4K20S4 against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa planktonic growth and biofilm formation[END_REF], the DRS-S4 derivatives used demonstrated similar or even higher efficacy in vitro [START_REF] Porat | Acyl-substituted dermaseptin S4 derivatives with improved bactericidal properties, including on oral microflora[END_REF][START_REF] Rotem | Physicochemical properties that enhance discriminative antibacterial activity of short dermaseptin derivatives[END_REF][START_REF] Marynka | In vitro discriminative antipseudomonal properties resulting from acyl substitution of N-terminal sequence of dermaseptin s4 derivatives[END_REF][START_REF] Jiang | Specificity determinants" improve therapeutic indices of two antimicrobial peptides piscidin 1 and dermaseptin s4 against the gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa[END_REF]. These results were confirmed in vitro and in vivo with mice infected with P. aeruginosa [START_REF] Navon-Venezia | Antibacterial properties of dermaseptin S4 derivatives with in vivo activity[END_REF][START_REF] Marynka | In vitro discriminative antipseudomonal properties resulting from acyl substitution of N-terminal sequence of dermaseptin s4 derivatives[END_REF] and in various incubation media varying in temperature and acidity [START_REF] Rydlo | Antibacterial properties of dermaseptin S4 derivatives under extreme incubation conditions[END_REF]. Table 1 provides an overview of the activity of a few DRSs against selected pathogens frequently related to infections and associated with anti-biotic resistance. For a complete list of DRSs and pathogens with corresponding MIC and source, we refer to Supplementary Table 1s.

Antiviral Activity of Peptides From the DRS Family

In addition to the antibacterial efficacy, DRS-S1 (and derivatives) show activity against pathogens causing genital infections such as human papilloma virus (HPV) and herpes simplex virus (HSV) [START_REF] Savoia | In vitro activity of dermaseptin S1 derivatives against genital pathogens[END_REF]. Furthermore, modified DRS-S4 has shown antiviral activity against Herpes Simplex Virus 1 & 2, including the acyclovir resistant strain in vitro [START_REF] Belaid | In vitro antiviral activity of dermaseptins against herpes simplex virus type 1[END_REF][START_REF] Bergaoui | In vitro antiviral activity of dermaseptin S(4) and derivatives from amphibian skin against herpes simplex virus type 2[END_REF]. For the activity peaks in case of DRS administration prior to incubation with the virus, interference early in the viral replication cycle is hypothesized [START_REF] Belaid | In vitro antiviral activity of dermaseptins against herpes simplex virus type 1[END_REF][START_REF] Mechlia | Dermaseptins as potential antirabies compounds[END_REF]. DRS-S4 and S9 both demonstrate in vitro activity against HIV-1 virus [START_REF] Lorin | The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro[END_REF][START_REF] Wang | Identification of novel human immunodeficiency virus type 1-inhibitory peptides based on the antimicrobial peptide database[END_REF] by inhibition of HIV attachment to endometrial cells, uptake by the dendritic cells and subsequent transmission to T-cells [START_REF] Lorin | The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro[END_REF]. Again, interference of DRS-S4 in an early phase of virus replication is suggested as less reduction of HIV is observed once the T-cells have been infected. Substitution of methionine for lysine on the 4th position DRS-S4 to reduce cytotoxicity to mammalian cells, did not affect the anti-HIV activity observed [START_REF] Lorin | The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro[END_REF]. More recently, DRS-S4 has shown effectivity against Rabies virus in mice [START_REF] Mechlia | Dermaseptins as potential antirabies compounds[END_REF].

Antifungal Activity of Peptides From the DRS Family

DRSs also show activity against fungi in vitro. So far, DRS-B1-B2, DRS-S1-S5 DRS-O1, DRS-CA1 DRS-DU1 all demonstrate cytotoxicity against Candida albicans [START_REF] Strahilevitz | Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes[END_REF]Mor et al., 1994a;Mor et al., 1994b;[START_REF] Leite | Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Secondary structure, antimicrobial activity, and mammalian cell toxicity[END_REF][START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF][START_REF] Shi | Two novel dermaseptin-like antimicrobial peptides with anticancer activities from the skin secretion of Pachymedusa dacnicolor[END_REF][START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF][START_REF] Zhu | Discovery of two skinderived dermaseptins and design of a TAT-fusion analogue with broad-spectrum antimicrobial activity and low cytotoxicity on healthy cells[END_REF]. In particular, DRS-S3 shows anti-fungal activity by means of triggering apoptosis [START_REF] Morton | An amphibian-derived, cationic, alpha-helical antimicrobial peptide kills yeast by caspase-independent but AIF-dependent programmed cell death[END_REF]. In Aspergillus fumigates, cytotoxic activity was demonstrated for DRS-B1-B2 and DRS-S1, but not DRS-S5. Minimal inhibitory

DRS-B1 DRS-B2 DRS-B3 DRS-B4 DRS-S1 DRS-S2 DRS-S3 DRS-S4 DRS-S4 K4-S4(1-16) DRS-S5 DRS-PH DRS-H3 DRS-L1 DRS-O1 DRS-DI06 DRS-CA1 DRS-DU1 DRS-PD1 DRS-PD2 DRS-PS4
This table gives an overview of the effectivity of DRS activity against a selected number of pathogens. Cells are colored dark green if: more than one study agrees on high activity (MIC <10 μM); light green if: 1 study finds high activity (MIC <10 μM); orange if studies do not agree on MIC; red if one or more studies agree on low activity (MIC > 10 μM); white indicates that there was no published data available. In the Human erythrocyte column, the opposite is instinctively true; green color indicates low, and red indicates high activity [START_REF] Strahilevitz | Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes[END_REF]Mor et al., 1994a;Mor et al., 1994b;Carpentier et al., 1998;[START_REF] Brand | Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells[END_REF][START_REF] Navon-Venezia | Antibacterial properties of dermaseptin S4 derivatives with in vivo activity[END_REF][START_REF] Conceicao | Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis[END_REF][START_REF] Brand | Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)[END_REF][START_REF] Conlon | Peptides with differential cytolytic activity from skin secretions of the lemur leaf frog Hylomantis lemur (Hylidae: Phyllomedusinae)[END_REF][START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helix-hinge-helix structure and membrane curvature strain[END_REF][START_REF] Zairi | In vitro activities of dermaseptins K4S4 and K4K20S4 against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa planktonic growth and biofilm formation[END_REF][START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF][START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF][START_REF] Leite | Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Secondary structure, antimicrobial activity, and mammalian cell toxicity[END_REF][START_REF] Jiang | Specificity determinants" improve therapeutic indices of two antimicrobial peptides piscidin 1 and dermaseptin s4 against the gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa[END_REF][START_REF] De Assis | Assessing antibacterial potential of components of Phyllomedusa distincta skin and its associated dermal microbiota[END_REF][START_REF] Shi | Two novel dermaseptin-like antimicrobial peptides with anticancer activities from the skin secretion of Pachymedusa dacnicolor[END_REF][START_REF] Belmadani | Dermaseptin-S1 decreases Candida albicans growth, biofilm formation and the expression of hyphal wall protein 1 and aspartic protease genes[END_REF][START_REF] Zhu | Discovery of two skinderived dermaseptins and design of a TAT-fusion analogue with broad-spectrum antimicrobial activity and low cytotoxicity on healthy cells[END_REF][START_REF] Shams | Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity[END_REF] for more information, see Supplementary Table S1.

concentrations (MIC) ranging from 3.1 μM to 30 μM were observed. In addition, others [START_REF] Belmadani | Dermaseptin-S1 decreases Candida albicans growth, biofilm formation and the expression of hyphal wall protein 1 and aspartic protease genes[END_REF] found that DRS-S1 inhibits C. albicans in biofilm formation when using concentrations of 100 μM. These findings are summarized in Table 1.

Antiparasitic Activity of Peptides From the DRS Family

Finally, DRS-S3 and S4 derivatives appear able to target malarial parasites within a host erythrocyte without disrupting the host [START_REF] Ghosh | Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis[END_REF][START_REF] Krugliak | Antimalarial activities of dermaseptin S4 derivatives[END_REF][START_REF] Dagan | In vitro antiplasmodium effects of dermaseptin S4 derivatives[END_REF].

In addition DRS from the Phyllomedusa oreades (DRS-O1) has shown activity against Schistosoma mansoni (de Moraes et al., 2011), Trypanosoma cruzi [START_REF] Brand | Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells[END_REF][START_REF] Leite | Phylloseptins: a novel class of anti-bacterial and anti-protozoan peptides from the Phyllomedusa genus[END_REF] and Leishmania amazonesis [START_REF] Brand | Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)[END_REF].

RESEARCH IN ONCOLOGY

Parallel to the efficacy against microbials, efficacy against tumor cells has been studied as well [START_REF] Hoskin | Studies on anticancer activities of antimicrobial peptides[END_REF][START_REF] Balandin | Molecular mechanisms of antitumor effect of natural antimicrobial peptides[END_REF]. Table 2 summarizes the data published on the impact of DRS on selected cancer cell lines. DRS-B2 for example, shows a dose dependent growth inhibition of prostatic adenocarcinomas (GI50 = 0.71-2.65µM) and some pancreatic cancer cell lines. Administration of 1µM DRS-B2 was enough for 50% reduction in colony formation in both prostate adenoma and mammary carcinoma cell lines [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF]. No growth inhibition on glioblastoma and mammary carcinoma cell lines was observed [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF][START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF]. At a concentration of 15 µM, no activity against stromal prostate fibroblasts and skin fibroblasts was observed, indicating low cytotoxicity in surrounding tissue [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF][START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF]. In vivo, DRS did not arrest human PC3 tumor growth in mice, but inhibited growth with more than 50% vs. controls attributed to a 24% reduction of angiogenesis in tumors of treatment groups, quantified by CD34+ positive stained endothelial cells. No side-effects or differences in total blood count were observed [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF].

Furthermore, DRS-PH shows IC50's of 0.69 µM, 2.01 µM, and 2.36 µM, against breast cancer adenoma (MCF-7), non-small cell lung carcinoma (NSCLC) (H157), and glioblastoma (U251MG) in vitro respectively [START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF]. For DRS-PH less efficacy was observed against PC3 cell lines, compared to the activity profile of DRS-B2. DRS-PH did also show some cytotoxicity against human dermal endothelium, and mammalian red blood cells [START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF].

MECHANISM OF ACTION

The mode of action by which antimicrobial peptides kill microbes is mainly known for α-helix cationic peptides which have been extensively studied [START_REF] Amiche | Dermaseptins as Models for the Elucidation of Membrane-Acting Helical Amphipathic Antimicrobial Peptides[END_REF][START_REF] Melo | The mechanism of action of antimicrobial peptides: Lipid Vesicles vs Bacteria[END_REF][START_REF] Bahar | Antimicrobial peptides[END_REF]. Two models explaining the interaction of α-helical cationic AMPs with membranes have been proposed: the barrel-stave model [START_REF] Ehrenstein | Electrically gated ionic channels in lipid bilayers[END_REF] and the carpet or carpet model [START_REF] Pouny | Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes[END_REF], both taken over by [START_REF] Shai | Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell nonselective membrane-lytic peptides[END_REF][START_REF] Shai | Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell nonselective membrane-lytic peptides[END_REF]. The cationic antimicrobial peptides, destructured in aqueous media, adopt an α-helical structure in contact with the plasma membranes of the host cell and then interact with the negative charges of the components of the membrane surface [START_REF] Zasloff | Antimicrobial peptides in health and disease[END_REF]. After binding, the peptide will disrupt the permeability of the membrane and either cause the death of the microorganism or enter the cell compartment and interact with intracellular targets. Note that most cationic antimicrobial peptides have a direct action on the membrane of bacteria, but some such as buforine II act intracellularly [START_REF] Park | Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions[END_REF]. In most of the mechanisms described, the binding of antimicrobial peptide to the membrane is followed by permeabilization of the membrane, which alone can cause cell death, or as a step in more complex processes.

In addition, very few studies on the anti-tumour action mechanism(s) of antimicrobial peptides have been conducted. The most important results show that their oncolytic mechanisms include: (i) induction of necrosis via cell membrane lysis, (ii) This table gives an overview of the DRS that have been tested for activity against some human cancer cell lines in vitro. Cells are colored in green if: high activity (< 10 μM to reach EC50) was found, orange if: two studies did not agree on high activity, red if they agreed on low activity (> 10 μM to reach EC50), white if: no published data was available. In the Human erythrocyte column, the opposite is instinctively true; green color indicates low, and red indicates high activity [START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF][START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF]Mor et al., 1994b;[START_REF] Nicolas | The dermaseptin superfamily: a gene-based combinatorial library of antimicrobial peptides[END_REF][START_REF] Charpentier | Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics[END_REF][START_REF] Conlon | Peptides with differential cytolytic activity from skin secretions of the lemur leaf frog Hylomantis lemur (Hylidae: Phyllomedusinae)[END_REF][START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helix-hinge-helix structure and membrane curvature strain[END_REF][START_REF] Shi | Two novel dermaseptin-like antimicrobial peptides with anticancer activities from the skin secretion of Pachymedusa dacnicolor[END_REF][START_REF] Huang | Dermaseptin-PH: a novel peptide with antimicrobial and anticancer activities from the skin secretion of the south american orange-legged leaf frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF][START_REF] Zhu | Discovery of two skinderived dermaseptins and design of a TAT-fusion analogue with broad-spectrum antimicrobial activity and low cytotoxicity on healthy cells[END_REF] initiation of apoptosis via mitochondrial membrane rupture and (iii) non-membranolytic modes of action [START_REF] Ellerby | Anti-cancer activity of targeted proapoptotic peptides[END_REF][START_REF] Chen | RGD-Tachyplesin inhibits tumor growth[END_REF][START_REF] Deslouches | Antimicrobial peptides with selective antitumor mechanisms: prospect for anticancer applications[END_REF].

Below, we will elaborate on how DRSs disrupt the lipid bilayer of microbes and cancer cells, and which features are possibly responsible for DRSs affinity to microbes and cancer cells. Additionally, we will discuss how DRSs modulate host immune systems and pathogen's gene expression. Last, we summarize the evidence of DRS acting as a receptor (ant)agonist.

Disruption of the Lipid Bilayer

One common feature DRSs often demonstrate is their disruption of the lipid bilayer of a target cell. Early evidence for this is the depolarization of bacterial membranes in vitro [START_REF] Fleury | Combined resistivity and capillary pressure measurements using micropore membrane technique[END_REF]. To do this, DRSs likely form tetramers in their quaternary structure that form toroidal pores. Using the planar lipid bilayer technique, it was shown that the DRS peptides accumulate in a carpet like manner on the outside of a lipid bilayer until a threshold concentration is reached, causing them to form pores in which the peptides are intercalated with the phospholipid headgroups of the membrane [START_REF] Duclohier | Bilayer lipid composition modulates the activity of dermaseptins, polycationic antimicrobial peptides[END_REF]. The selectivity of these pores appears to be determined by the phospholipid headgroups of the plasma membrane. The carpet of DRSs on the outside of the lipid bilayer as well as tetramer channel with intercalated phospholipid headgroups causes the membrane to lose its integrity [START_REF] Yeaman | Mechanisms of antimicrobial peptide action and resistance[END_REF]. The reason why DRS favor binding to some pathogens and tumors is still under investigation and several hypotheses exist, discussed here are the possible influence of; membrane charge, membrane sulfatation and membrane fluidity.

First DRSs are generally rather cationic peptides and thus more prone to bind negatively charged membranes. Bacteria and cancer cell membranes typically have a net negative charge, the former due to negatively charged phospholipids on inner (gram negative) and single (gram positive) membranes (Shai, 2002), the latter due to expression of phosphatidylserine and negatively charged mucin proteins [START_REF] Utsugi | Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes[END_REF][START_REF] Hoskin | Studies on anticancer activities of antimicrobial peptides[END_REF]. Interestingly, erythrocytes have net negatively charged membranes as well, but are generally less affected by DRS [START_REF] Caillon | Biophysical investigation of the membrane-disrupting mechanism of the antimicrobial and amyloid-like peptide dermaseptin S9[END_REF].

Second the sulfatation of glycosaminoglycans (GAG)'s on the membrane surface seems to be of high importance for DRS-B2's effectivity against prostate cancer cell lines (PC3) [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF]. Low concentrations of Chondroitin Sulfate C (CS-C), which is a sulfated GAG, contribute to the α-helical shape of DRS-B2, which is its biological active form. Interestingly, sulfated GAG's seem to be essential to the effectivity of cell penetrating peptides (CPPs) as well [START_REF] Yang | Interaction of amphiphilic alpha-helical cell-penetrating peptides with heparan sulfate[END_REF]. Furthermore, when [START_REF] Zhu | Discovery of two skinderived dermaseptins and design of a TAT-fusion analogue with broad-spectrum antimicrobial activity and low cytotoxicity on healthy cells[END_REF] investigated the activity of DRS-DP1 and 2, they found that by introducing a TAT (GRKKRRQRRR) peptide at the N-terminal, affinity to the cell membrane and interaction with GAG's increased.

Third, cancer cells often have increased membrane fluidity and irregularities of cell surface which contributes to membrane destabilization and could affect receptor binding and other communications between cancer cell and environment [START_REF] Hoskin | Studies on anticancer activities of antimicrobial peptides[END_REF]. This may account for DRSs being more effective against one cancer type (PC3) than another [START_REF] Leuschner | Membrane disrupting lytic peptides for cancer treatments[END_REF]van Zoggel et al., 2012a). It has been observed that membrane environment with a strong positive curvature strain influences the DRS-B2 into a state that facilitates insertion into the membrane [START_REF] Galanth | Mechanism of antibacterial action of dermaseptin B2: interplay between helix-hinge-helix structure and membrane curvature strain[END_REF].

Modulation of the Host Immune System

Besides the membrane disrupting activity, DRS likely modulates the host defense system as well. By introducing DRS-S1, neutrophils of rat and humans stimulate their microbicidal activities such as their production of reactive oxygen species [START_REF] Ammar | Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes[END_REF]. Moreover, DRS-S9 is chemotactic for human leukocytes [START_REF] Auvynet | Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9[END_REF]. On the other hand, DRS-S4 has been observed to bind Lipopolysaccharides (LPS), which would rather suppress activation of macrophages and decrease the production of inflammatory cytokines [START_REF] Navon-Venezia | Antibacterial properties of dermaseptin S4 derivatives with in vivo activity[END_REF]. Furthermore, DRS also shows angiostatic activities, which may influence tumor growth [START_REF] Van Zoggel | Antitumor and angiostatic peptides from frog skin secretions[END_REF][START_REF] Van Zoggel | Antitumor and angiostatic activities of the antimicrobial peptide dermaseptin B2[END_REF].

Modulation of the Pathogen's Gene Expression

We previously mentioned that DRSs can permeate membranes of different types of cells and cell nuclei, however it is also possible that DRS changes gene expression in a pathogen cell as well. DRS-S1 Modulates the expression of C. albicans genes, such as the Hyphal wall protein 1 (HWP1) gene [START_REF] Belmadani | Dermaseptin-S1 decreases Candida albicans growth, biofilm formation and the expression of hyphal wall protein 1 and aspartic protease genes[END_REF]. In vitro, its expression was downregulated which likely accounts for the modified cell morphology that was found. Furthermore, changes in aspartic protease genes were found.

Interaction with Cell Membrane Receptors

Lastly, it seems unlikely that there is a partner protein on the surface or cytoplasm of the tumor cells that can account for the effects of DRS. This is illustrated by the example in which DRS-B2's activity on PC3 cell lines is almost identical when composed of only amino acids in D configuration, [START_REF] Santos | Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans[END_REF]. Since receptors are usually stereo-selective, it is unlikely DRS-B2 works through a receptor. However, there is evidence that some DRS can inhibit adenosine triphosphate (ATP) production through receptor binding [START_REF] Laughlin | Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides[END_REF].

FUTURE PERSPECTIvES

As DRSs act against a wide variety of pathogens and tumor cell lines, it is tempting to speculate on their therapeutic potential. Nevertheless, current knowledge on DRS is fragmented with respect to mechanism of action and diverse with respect to pathogens and cancer cell lines studied. Consecutively, we will discuss remaining questions on the mechanism of action, potential clinical applications and safety.

Future Research on Available DRSs

For research on DRSs to move forward on the already investigated DRSs described in this paper, it is essential to understand the mechanism of action of a specific DRS against a pathogen to finetune this DRS into a molecule that can target a specific clinical problem. These potential clinical therapies include treatment of infections caused by specific micro-organisms, the treatment of infections caused by multiple micro-organisms such as skin infections, and as a component of cancer treatment. Furthermore, we discuss the current use of DRS in biotechnology.

It seems likely that the mode of action of DRSs relies, similarly to many AMPs, in part on membrane disruption of target cells. Important factors in this include the cationic nature of the peptide, and its ability to retain an α-helical shape. The presence of certain phospholipids, GAGs, and proteins contribute to this. However, the means to identify more of the cell membrane factors, including the shape and fluidity of the cell membrane are not readily available, and would require innovative tools and techniques at the interface of chemistry biology and biophysics.

DRSs can be tested for specific clinical problems in vivo. For example, some DRSs act against specific, anti-biotic resistant pathogens. From Table 1 we can infer that DRS-B4, DRS-O1, DRS-DI06, DRS-CA1, DRS-DU1, DRS-PD2 and DRS-PS4 are all promising candidates for specifically targeting P. aeruginosa, a pan-resistant bacterium notorious for causing severe infections in hospital settings. Another example is DRS-O1 which has thus far been the only DRS to show activity against the S. mansoni, a neglected tropical disease in need of treatment possibilities.

On the other hand, there are some DRSs that can act against a range of pathogens. This makes some DRSs ideal for clinical applications in which several pathogens need to be targeted. Skin infections, for example in diabetic foot ulcers or catheter infections are caused by organisms such as gram-positive bacteria as well as some fungi [START_REF] Schittek | The role of antimicrobial peptides in human skin and in skin infectious diseases[END_REF]. From Table 1 we can conclude that DRS-S3, DRS-S5, DRS-CA1, DRS-DU1, DRS-PS4, DRS-PD2 and DRS-O1 all show activity against the S. aureus (some including the methicillin resistant variant), as well as the C. albicans fungus, without damaging erythrocytes. Gomes and colleagues (2015) have already positively assessed DRS's incorporation in cotton gauzes and show potential in fast-release medical applications. Therefore they are potential starting points for therapeutic applications in skin infections. Some even speculate that there is potential for DRS as a contraceptive, as DRS-S4 is spermicidal [START_REF] Zairi | Spermicidal activity of dermaseptins[END_REF]. DRS-S4 shows activity against HIV, and several other genital pathogens [START_REF] Lorin | The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro[END_REF][START_REF] Savoia | In vitro activity of dermaseptin S1 derivatives against genital pathogens[END_REF]. However, it appears the recent literature prefers antimicrobials that are within (or close to) the human genome for the use as contraceptives [START_REF] Tanphaichitr | Potential use of antimicrobial peptides as vaginal spermicides/ microbicides[END_REF]. Moreover, the clinical relevance of such a contraceptive as well as the high degree of effectivity that would be demanded are questionable.

There is the potential of DRS as an aiding therapeutic in cancer treatment by working in tandem with conventional chemotherapies. This idea finds its roots in a hypothesis regarding the evolutionary origins of DRSs in frogs. The idea is that DRSs function as an accessory protein that lyses cells and penetrates tissue to allow effectiveness of other neuromodulators and enzymes in frog secretions [START_REF] Konig | The diversity and evolution of anuran skin peptides[END_REF]. This contrasts with the view that DRSs are part of an innate immune system. Indeed, there is striking similarity in the amino acid sequences of preproDRS and precursors of demorphin and deltorphin [START_REF] Amiche | Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins[END_REF] (which are powerful opioid agonists), suggesting these peptides work together to achieve a common goal. This idea might extend to clinical practice as well. Potentially DRS could be part of a drug delivery system and play similar role to other cell penetrating peptides in cancer research [START_REF] Bolhassani | Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer[END_REF][START_REF] Bolhassani | Improvement of different vaccine delivery systems for cancer therapy[END_REF][START_REF] Yang | Interaction of amphiphilic alpha-helical cell-penetrating peptides with heparan sulfate[END_REF]. DRS would penetrate and disintegrate cell membranes of specific cancer cells and (attached) conventional cytostatic agents may then -more effectively -affect the tumor tissue. Some support for this idea comes from a study using Cecropin A and conventional chemotherapy on leukemic patients, Cecropin A increased the effect of the conventional therapy [START_REF] Hui | The combined effects of antibacterial peptide cecropin A and anti-cancer agents on leukemia cells[END_REF]. Possibly the same idea can be used when targeting microbial pathogens [START_REF] Balaban | A chimeric peptide composed of a dermaseptin derivative and an RNA III-inhibiting peptide prevents graft-associated infections by antibioticresistant staphylococci[END_REF]. Potentially, red blood cells or chitosan nanoparticles could function as a potential carrier for DRS [START_REF] Feder | Affinity driven molecular transfer from erythrocyte membrane to target cells[END_REF][START_REF] Medeiros | Chitosan nanoparticles for dermaseptin peptide delivery toward tumor cells in vitro[END_REF].

Meanwhile the field of applied biotechnology has moved forward on these developments and researchers have incorporated DRS in the genetic material of potato and citrus plants. The plants can express these peptides and protect the crops from disease [START_REF] Rivero | Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens[END_REF][START_REF] Furman | Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease[END_REF]. More recently, DRS-B1 was modified (N-terminally modified and recombined respectively) to protect poplar plants and tobacco plants from infections by inserting the DRS protein in the genome of the host [START_REF] Yevtushenko | Enhancing disease resistance in poplar through modification of its natural defense pathway[END_REF][START_REF] Shams | Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity[END_REF]. On the one hand pesticides can now be avoided on these crops, on the other hand studies already report resistance of bacteria to anti-microbial peptides by producing positively charged molecules on the membrane and pumping AMP's out of the cells [START_REF] Joo | Bacterial strategies of resistance to antimicrobial peptides[END_REF][START_REF] Andersson | Mechanisms and consequences of bacterial resistance to antimicrobial peptides[END_REF]. Genetically altering plants on a large scale could potentially endanger DRS's use as an antibiotic in humans.

Lastly, the safety of DRS in humans remains to be investigated. Even though several DRS have been administered to mice and rats, to our knowledge, no phase-1 clinical trials have been conducted on the safety of any DRS in humans thus far. Encouragingly, many of the documented short-term adverse effects of the Kambo ritual such as nausea and tachycardia can be ascribed to other molecules in the cocktail [START_REF] Erspamer | Pharmacological studies of 'sapo' from the frog Phyllomedusa bicolor skin: a drug used by the Peruvian Matses Indians in shamanic hunting practices[END_REF], nevertheless the safety of the DRS remain to be shown. Researchers in many fields could benefit from the knowledge of a safe DRS peptide.

Future Research on Novel DRSs

The investigation of other Anuran species is likely to yield anti-microbial or DRS like peptides that can contribute to illuminating the mechanism of action of these peptides and provide starting points for therapeutical treatments. According to some estimations, skin compounds have been detected and isolated from 400 anuran species, which means that more than 90% of all documented frog species still await screening [START_REF] Konig | The diversity and evolution of anuran skin peptides[END_REF]. The isolated peptides from different species thus far are very often unique and sometimes useful which makes it very likely that new, useful and novel biomolecules await discovery.

To ensure these new molecules contribute to scientific literature in a less fragmented way, it is important to identify the questions that remain on the mechanism of action of DRS. Whenever a new DRS or a modification is tested, ideally these mechanisms should be evaluated: 1) disruption of plasma/mitochondrial membranes; 2) necrosis; 3) apoptosis; 4) mechanisms of mediated immunity; 5) membrane receptor involvement; 6) inhibition of DNA synthesis; 7) anti-angiogenic effects [START_REF] Gaspar | From antimicrobial to anticancer peptides. a review[END_REF]. Additionally, their activity against human erythrocytes and epithelial cells should be evaluated to assess clinical relevance and help understand how DRS recognize target cells.

DRS are a complex family of bioactive peptides. Accumulating evidence suggests their efficacy in a wide variety of medical applications. Despite the still puzzling mechanisms of action, DRSs are extremely suitable for specific medical problems.

FIGURE 1 |

 1 FIGURE 1 | Amino acid sequence alignment of 57 DRS peptides (http://aps.unmc.edu/AP/main.php) using CLUSTAL O (1.2.4) multiple sequence alignment (https://www.ebi.ac.uk/Tools/msa/clustalo/). The letters represent an amino acid according to conventional nomenclature. The dashes are introduced to optimize amino-acids alignments. Blue colors indicate a well-preserved amino acid (>55% of identity), green colors indicate higher rates of preservation within this DRS group (>80% of identity).

TABLE 1 |

 1 Effectivity of Dermaseptins in vitro against various pathogens.

	E. coli	E. faecalis	P. aeruginosa	S. aureus	C. albicans	A. fumigatus	Human
							erythrocytes

TABLE 2 |

 2 Overview of the activity DRSs against various cancer cell types.

	HEPG2	MCF-7	U251MG	H157	MDA-MB-435S	PC-3	Human
							erythrocytes
	DRS-B2
	DRS-B3
	DRS-B4
	DRS-PH
	DRS-L1
	DRS-CA1
	DRS-DU1
	DRS-PD1
	DRS-PD2
	DRS-PS4

Frontiers in Pharmacology | www.frontiersin.org November 2019 | Volume 10 | Article 1421

AUTHOR CONTRIBUTIONS

EB is first author and writer of this manuscript. DD supervised the initial draft of this manuscript, helped identify the aims and research question and came up with the initial structure. MA critically reviewed the drafts several times, elaborated on the mechanisms of action, revised structure and added figures.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2019.01421/ full#supplementary-material

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Jeff Avery
email: jeffery.avery@uwaterloo.ca

Daniel Vogel
email: dvogel@uwaterloo.ca

Edward Lank
email: lank@uwaterloo.ca

Damien Masson
email: dmasson@uwaterloo.ca

Hanae Rateau
email: hrateau@uwaterloo.ca

Holding Patterns: Detecting Handedness With A Moving Smartphone At Pickup Détection de la manualité via les capteurs d'orientation du smartphone lors de la prise en main

Figure 1: Smartphone holding postures: our method detects which hand is holding the phone prior to first interaction.

hand they are using to hold the device, and use this information to optimize the interaction. We introduce a method to use built-in orientation sensors to detect which hand is holding a smartphone prior to first interaction. Based on logs of people picking up and unlocking a smartphone in a controlled study, we show that a dynamic-time warping approach trained with user-specific examples achieves 83.6% accuracy for determining which hand is holding the phone, prior to touching the screen.

CCS CONCEPTS

• Human-centered computing → HCI theory, concepts and models; Pointing; Visualization techniques. KEYWORDS interaction technique, models, handedness, smartphone, unlock RÉSUMÉ En fonction de la tâche et du contexte, les utilisateurs de smartphone ont pour habitude de changer de main pour tenir leur appareil. Idéalement, nous souhaiterions connaître la main utilisée afin d'optimiser l'interaction. A cet effet, nous introduisons une méthode utilisant les capteurs d'orientation intégrés afin de déterminer la main tenant le smartphone avant toute interaction. Nous montrons, par l'analyse des données de participants prenant et déverrouillant leurs smartphones durant une expérience contrôlée, qu'une approche utilisant l'algorithme Dynamic-Time Warping permet d'obtenir une précision de 83.6% afin de détecter la main utilisée.

MOTS CLÉS techniques d'interaction, modèles

INTRODUCTION

Smartphones are often held one-handed with the thumb used for touch input [START_REF] Hoober | How Do Users Really Hold Mobile Devices[END_REF]. The shift towards larger phones makes this style of interaction challenging: reaching far corners of a large screen with a thumb while holding a phone in the same hand is uncomfortable and awkward. Researchers have recognized this issue and introduced methods to make single-handed use on large devices easier. For example, Kim et al. [START_REF] Kim | Interaction Techniques for Unreachable Objects on the Touchscreen[END_REF] introduce Edge and Large touch for interacting with large screens, and Apple's Reachability feature "shifts" the interface down to make the furthest corners of the screen accessible. However, these techniques require explicit activation, which can be inefficient since people switch hands based on what they are doing and what they are holding [START_REF] Hoober | How Do Users Really Hold Mobile Devices[END_REF][START_REF] Kim | Interaction Techniques for Unreachable Objects on the Touchscreen[END_REF], requiring continual readjustment of their hand posture. A more proactive solution would dynamically recognize how the phone is held and adapt the interface accordingly. Many attempts have been made to optimize interfaces based on detecting handedness, but most require special sensors, or utilize touch data collected during the initial interaction. We feel that the idea mechanism would detect handedness before the user had attempted to interact with the screen.

TouchMe, for example, place additional capacitive sensors along the sides of the case to determine hand position [START_REF] De Luca | Touch Me Once and I Know It's You!: Implicit Authentication Based on Touch Screen Patterns[END_REF]. In Pre-Touch [START_REF] Hinckley | Pre-Touch Sensing for Mobile Interaction[END_REF], a self-capacitance touchscreen is used to leverage aspect of the touch happening before an actual contact. Similar to our work, their prototype can detect the hand holding the phone prior to the first interaction. They also show how this information can be used to augment interfaces in the context of one-handed interaction. Our approach differs in that it uses information already available in modern smartphones and does not need specific hardware. ContextType detects which hand is holding the phone only after a firm tap on the side of case [START_REF] Goel | ContextType: Using Hand Posture Information to Improve Mobile Touch Screen Text Entry[END_REF]. GripSense [START_REF] Goel | GripSense: Using Built-in Sensors to Detect Hand Posture and Pressure on Commodity Mobile Phones[END_REF] combines touch characteristics like capacitive pressure with internal motion sensor thresholds to distinguish between holding a phone with the left hand, the right hand, or both hands with 84.3% accuracy. Park et al. [START_REF] Park | A Study on Grasp Recognition Independent of Users' Situations Using Built-in Sensors of Smartphones[END_REF] extend GripSense with accelerometer data to improve accuracy to 87.7%. However, GripSense requires 4 to 5 taps or swipes to achieve these levels of accuracy, which takes time, and is only valid for that specific interaction.

Lochtefeld et al. use DTW on swipe data captured during device unlock to achieve an impressive 98.5% accuracy in detecting handedness, but are restricted to working with data captured as part of the unlock swipe gestures [START_REF] Löchtefeld | Detecting Users Handedness for Ergonomic Adaptation of Mobile User Interfaces[END_REF]. There is a recent move towards using biometric unlock mechanisms to unlock devices (e.g. gaze to unlock on recent Apple iPhones, or fingerprint identification). If we wish to support these mechanisms, we need to consider approaches that do not rely on touch-screen gestures to identify handedness.

We contribute a method to detect which hand is holding a smartphone by analyzing the built-in orientation sensor stream as the phone is lifted and unlocked, before the user touches the screen. Our insight is that the movement of the phone as the user removes it from their pocket can be recast as an implicit motion gesture [START_REF] Ruiz | DoubleFlip: A Motion Gesture Delimiter for Mobile Interaction[END_REF]. We use dynamictime warping (DTW) trained on these "pre-unlock" motions performed by the same person. With this setup, we can detect whether a phone is held in the left or right hand with 83.6% accuracy, prior to the user touching the screen. Our method is as accurate as GripSense's holding hand detection, and we show that two training examples may be sufficient to achieve this level of accuracy, so user-specific configuration in a real deployment is practical. While our method cannot detect a change of hand after unlock, it could be combined with GripSense that predicts the holding hand after a few taps or swipes, effectively detecting when the phone is held in a different hand.

SMARTPHONE HOLDING PATTERNS

Before describing our data gathering experiment and detection method, we review Hoober's field survey of smartphone holding postures [START_REF] Hoober | How Do Users Really Hold Mobile Devices[END_REF] since it defines hand posture terminology and characterizes real world occurrence.

Hoober classified 1,333 observations of people using smartphones in public into 5 holding postures. We change IHM'19, December 10-13, 2019, Grenoble, France Hoober's class names to emphasize which hand is holding the phone and what style of hold is used (Figure 1): held in the left hand with left thumb input ("left-single"), held in the right hand with right thumb input ("right-single"), cradled in the left hand with right finger input ("left-cradled"), cradled in the right hand with left finger input ("right-cradled"), and two-handed with two thumb input ("two-handed").

Hoober found that most interactions were single-handed (49%) or cradled (36%), with people using the two-handed posture predominantly for typing (15%). Given a strong mapping of two-hands to typing [START_REF] Mastin | Handedness Statistics[END_REF] and its relatively low occurrence, we focus only on single-handed holding postures. For single-handed postures, 67% were right-single and 33% were left-single. This does not correlate with the estimated proportion of left-handed individuals in a population (between 3% to 12% [START_REF] Mastin | Handedness Statistics[END_REF]), suggesting some level of non-dominant hand interaction. Hoober, and Ng et al. [START_REF] Ng | The Effects of Encumbrance and Mobility on Touch-Based Gesture Interactions for Mobile Phones[END_REF], both observed people holding their smartphone with different hands based on the context (e.g. when opening a door, they may wish to use their dominant hand).

DATA COLLECTION

Each time a person grasps their smartphone and unlocks it, there is a predictable series of events: the power switch is pressed, the phone is lifted up to face the user, and a touch screen unlock widget is moved. Our hypothesis is that the motion during this "pre-unlock" sequence varies depending on which hand is used to hold the phone, and systematic differences can be detected using a classifier. The goal of this experiment is to collect example motion data from the accelerometer, gravity, and orientation sensors in the period between power-on and unlock. To our knowledge, no previous work has explored this unlocking motion. Truong et al. [START_REF] Khai | Slide to X: Unlocking the Potential of Smartphone Unlocking[END_REF] augment the unlock gesture itself and Hintze et al. [START_REF] Hintze | Diversity in Locked and Unlocked Mobile Device Usage[END_REF] examine unlocked and locked usage focusing on touch screen interactions before and after unlocking.

Participants 16 people were paid $2 to participate (8 females, 1 left-handed, mean age 23.8 years, SD 6.1). All had prior experience with smartphones and using swipe to unlock.

Apparatus

The experiment was conducted using a Nexus 5 phone running Android 5.02. A custom Android application was written to simulate the appearance, position, and behaviour of standard unlock widgets, but with greater instrumentation. All sensor data streams were logged at the highest available sample rate (100 Hz). From an initial pilot with 6 participants, we observed similar motion data regardless whether a person is sitting or standing, whether the phone starts in a pocket or laying on table. This observation would need further investigation, but it suggests that our results should generalize to all contexts even if the collected data is limited to one of these conditions. For this reason, the smartphone was placed face on a table down beside participants, prior to starting each task.

Task

The experimental task required participants to pick up the smartphone from the table and unlock it using a specific hand posture. Before each trial, the phone was locked and placed on a table beside them. Three seconds later, a chime rang prompting the participant to pick up the phone, turn on the power, and swipe an unlock slider. This completed the trial, and the process repeated (Figure 2). The unlock slider was displayed in one of ten combinations of position and direction representing the most common configurations of current devices (Figure 3). The unlock slider target was a 15 mm diameter circle with a 15 mm long slider track, similar to standard unlock widgets. Based on the initial target position, the slider was either centered in the display, or positioned 8 mm from the bot-tom, top, or side to approximate positions of unlock widgets on commercial devices.

Design

The independent variables are the HAND used to hold the phone (LEFT or RIGHT) and the type of HOLD used (SIN-GLE or CRADLE) which combine into a hand holding POS-TURE factor with four levels: LEFT-SINGLE, LEFT-CRADLE, RIGHT-SINGLE, RIGHT-CRADLE (Figure 1). The dependent variables were X, Y, and Z values for three sensor data streams: accelerometer (ACC), gravity (GRAV) and orientation (ORIENT). These values were logged from power-on until the unlock widget was fully engaged. Each participant completed all 10 positions and directions of the unlock widget with each POSTURE in one contiguous block. The order of POSTURE was a 4 x 4 Latin-square. The data-gathering portion of the study was approximately 15 minutes per participant.

Results

In total, 640 power-on to unlock sequences were logged (40 per participant, 10 per POSTURE per participant). The logs revealed that sensors sometimes needed to "warm up" after power-on (values were all 0 otherwise), so we use the first stable X, Y, Z values updated after power-on (usually within 100 ms of power-on). The mean time of logged sequences was 1403 ms (SD 736).

Distribution of Sensor Readings at Power-on and Unlock. We examined the distribution of each component for each sensor reading at power-on (the start of the movement) and unlock (the end of the movement). If a sensor reading was sufficient to classify postures, we would expect to see a visual separation of the values by hand posture when the sensor readings were plotted (Figure 4). Although we found no clear separation between LEFT and RIGHT HANDS, or SIN-GLE and CRADLED HOLDS, GRAV-Y showed some minimal separation by HAND. Previous work has essentially used thresholds on values like these to detect hands, so we explore this possibility in the next section. Sequence of Sensor Readings from Power-on to Unlock. Visual inspection of the ORIENT X, Y, and Z sensor values during a typical unlock sequence suggests a subtle, but distinguishable pattern (see a comparison of single trials example in Figure 5). The Y and Z values converge for LEFT-SINGLE, but diverge for RIGHT-SINGLE. The X and Z values are offset but similar for RIGHT-CRADLE, but notice-able diverge at the beginning of the trial for LEFT-CRADLE. These kinds of observations suggest pre-unlock motion sequences differ, according to which hand is holding the device. These are the types of patterns we leverage for classification.

CLASSIFICATION MODELS

We test two methods to detect which hand is holding a phone using the motion data from our experiment; simple thresholds, based on measuring sensor values at key points, and dynamic-time warping, based on the full sequence of sensor values between power on and device unlock.

Simple Thresholds

Our first attempt at classification looked at the distribution of sensor values at power-on and unlock, to determine if any single set of sensors could be used to classify hand POS-TURE at a given point-in-time. We tested simple thresholds for each sensor value (ACC-X, ACC-Y, . . . ). Thresholds were determined as a "midpoint" between hand POSTURE distributions. Specifically, the mean of the third-quartile of the lowest factor, and the first quartile of the highest factor. This threshold was used to classify the POSTURE using two-way cross validation, and the analysis was repeated using each sensor's X, Y, and Z. This failed to produce useful results: the best POSTURE detection was below chance. Collapsing SINGLE and CRADLED postures together to detect which IHM'19, December 10-13, 2019, Grenoble, France HAND is holding the phone (i.e. LEFT or RIGHT hand regardless of HOLD) improved accuracy, but the results were poor, ranging from near chance (51.8% for ORIENT X) to 62.5% (for ACC X). For this reason, simple thresholds are deemed insufficient for HAND or POSTURE classification.

Dynamic Time Warping

In a typical pre-unlock sequence, we observed possible characteristic patterns depending on the POSTURE used (Figure 5). To classify sensor values over time, we use dynamic-time warping (DTW), a nearest-neighbor pattern-matching algorithm commonly used in speech [START_REF] Sakoe | Dynamic programming algorithm optimization for spoken word recognition[END_REF] and gesture recognition [START_REF] Bemdt | Using Dynamic Time Warping to Find Patterns in Time Series[END_REF][START_REF] De Luca | Touch Me Once and I Know It's You!: Implicit Authentication Based on Touch Screen Patterns[END_REF]. Using DTW, we compare a sequence of sensor values against labelled model data representing a known hand posture; the closest matching path returned from DTW is used to classify the hand posture.

We implemented DTW using Euclidean distances for each measurement in our calculations. One potential issue using DTW is that motion sequences vary in length; unlocking a phone can take a variable amount of time, so paths contain a variable number of points. To make paths easier for DTW to match, we first normalize them by interpolating the data over a 3 second window sampled at 100Hz. Normalizing to 3 seconds maintains a reasonable representation of all sequences (since the mean sequence time plus two standard deviations is 2.9 seconds.)

To determine what combination of sensor readings to use, we ran DTW with each sensor and each combination of X, Y, and Z values (i.e. testing ACC X, ACC Y, . . . , ACC XYZ, ORIENT X, ORIENT Y, . . . , ORIENT XYZ, GRAV X, GRAV Y, . . . ). The best sensor combination, ORIENT X,Y,Z is used in subsequent results. Generic Classification. A generic classifier would require no per-participant configuration in a real system. To test if a generic classifier is possible, we split our data evenly into across training and test sets regardless of participant. For each task in the training set, we generated sequences according to the combination of sensor readings being tests (e.g. ACC-X, ORIENT-XYZ, . . . ). This provided us with a set of paths for each task that we could use as a model. Using DTW, we found the closest matching gesture and the POSTURE associated with this match was used for classification.

We tested using two-way cross validation, and generated confusion matrices for a four-class model (treating each POSTURE separately), as well as a binomial classifier (col-lapsing HOLD, so classes were LEFT and RIGHT hand only). The highest recognition rates were found using ORIENT Z (see four-class results in Table 1). Accuracy for a specific hand posture is low, with LEFT-CRADLE having the highest recognition rate at 46.0%. If we group single and cradled holds together, recognition rates improve to 55.3% for LEFT hand, and 79.4% for RIGHT hand detection, for a mean recognition rate of 67.4%. This is an improvement over using simple thresholds, but falls short of techniques like GripSense [START_REF] Goel | GripSense: Using Built-in Sensors to Detect Hand Posture and Pressure on Commodity Mobile Phones[END_REF].

Our informal observations during the data gathering suggest individuals may have distinct pre-unlock motions, which reduces the feasibility of a generic model and indicates that a machine learning approach trained on the user would be User-Specific Classification. We split data for each participant evenly into training and test sets, and used two-way cross validation to assess each participant's individual performance. We repeated this for every participant, and calculated the mean for each cell in our confusion matrix (see four-class results in Table 2).

Hand POSTURE detection is no greater than 63.0% (for LEFT-SINGLE hand detection) and would require further investigation. However, focusing on HAND detection alone greatly increases accuracy. By combining SINGLE and CRADLED classes, accuracy improves to 74.5% for LEFT hand and 92.8% for RIGHT hand, an overall accuracy of 83.6%. This is comparable to holding hand detection accuracy rates reported by GripSense [START_REF] Goel | GripSense: Using Built-in Sensors to Detect Hand Posture and Pressure on Commodity Mobile Phones[END_REF].

A per-user model requires training data for each user. We examined how varying the size of the training set would impact our results. We re-ran our tests using DTW with leave-n-out cross-validation, and training set sizes of n = 2 to 8 samples. A training set size of 5 samples was optimal, with other sizes varying in accuracy between 76% (n=2) and 77% (n=8). This suggests as few as two training samples could achieve reasonable accuracy.

DISCUSSION

These are encouraging accuracy rates, but what are the implications for actual deployment?

The first consideration is what does it really mean to detect which hand is holding the phone when we do not know if the thumb of the same hand is used (single) or if the finger of the other hand is used (cradle). We argue that an interface compensating for single-handed use does not mean the interface becomes unusable while the phone is cradled and the other hand's finger is used. In fact, people often switch between single and cradle while continuing to hold the phone with the same hand [START_REF] Hoober | How Do Users Really Hold Mobile Devices[END_REF]. The main implication is that interface adjustment techniques should be designed with this in mind. For example, if the system knows which hand is holding the phone, a menu of commands can be positioned on the correct side to be near that hand's thumb, but the rest of the interface may be designed to be used with both single and cradle holds.

Our attempt at creating a generic classifier was unsuccessful mainly because individuals have distinct pre-unlock motions. Therefore, a deployed system would require some kind of training and configuration stage where the user demonstrates a set of unlock motions with different postures to train the classifier. This would only need to be done once. Alternately, a system could collect per-unlock motion data and then ask the user to verify what their posture was. This would gradually build up a set of unlock motion paths. Since two examples of each posture provides 76% accuracy, this process would not be too arduous.

An issue with any detection is addressing classification errors. Even 84% accuracy means an incorrect classification about 1 in 7 times. If the goal is to adjust the interface for a particular hand, an error means it would be in a suboptimal state. One approach is to use the DTW distance metric to determine the detection confidence, to help decide when an interface adjustment should be made. A second approach is to turn this implicit pre-unlock gesture into an explicit one. Users could move their phone with a particular flourish to simultaneously distinguish between hands and trigger inter-face adjustment. This is related to error correcting motion gestures like double-flip [START_REF] Ruiz | DoubleFlip: A Motion Gesture Delimiter for Mobile Interaction[END_REF]. In future work, we plan to investigate this and similar refinements.

Finally, we believe this type of pre-unlock detection is highly complementary to post-unlock detection techniques like GripSense. Our method cannot detect changes of hand after unlock, but on a basic level, our method could be used to condition a prior probability for the GripSense method to increase combined detection accuracy. This would benefit holding hand detection, but also other kinds of detection supported by GripSense since an a priori estimate of what hand is holding the phone can be exploited as a constraint.

CONCLUSION

We contribute a method to detect which hand is holding a smartphone by analyzing the built-in orientation sensor stream as the phone is lifted and unlocked, prior to first interaction, without the use of external sensors. Our insight is that we can treat the recognition problem as an implicit motion gesture [START_REF] Ruiz | DoubleFlip: A Motion Gesture Delimiter for Mobile Interaction[END_REF], and use dynamic-time warping (DTW) to classify hand based on lift-and-unlock motions per-formed by the same person. With this setup, we can detect whether a phone is held in the left or right hand with 83.6% accuracy. Our method can be used standalone and achieve effective results with 2 to 5 training samples collected during during typical device usage, making user configuration and training in a real deployment feasible.

Figure 2 :

 2 Figure 2: Experimental task. From a resting position (a), the participant picked up the phone and pressed the power button (b), then used the unlock widget to unlock the phone (c).

Figure 3 :

 3 Figure 3: Unlock slider positions. Sliders were presented in one of ten common unlock widget positions.

Figure 4 :

 4 Figure 4: Key sensor component readings at power-on and unlocking for each POSTURE.

Figure 5 :

 5 Figure 5: Orientation sensor values during a typical unlock sequence: each graph represents a single trial.

Holding Paerns :

 Paerns Detecting Handedness With A Moving Smartphone… IHM'19, December 10-13, 2019, Grenoble, France 7:5

Table 1 :

 1 General Model: Observed vs. Predicted Accuracy for Left, Right {L,R}, Single, Cradled {S,C} Postures

		LS	LC	RS	RC
	Left-Single (LS)	31.1% 19.8% 24.5% 24.5%
	Left-Cradle (LC)	14.0% 46.0% 20.0% 20.0%
	Right-Single (RS)	9.5% 11.1% 39.7% 39.7%
	Right-Cradle (RC) 9.5% 11.1% 39.7% 39.7%

Table 2 :

 2 User-Specific Model: Observed vs. Predicted Accuracy for Left, Right {L,R}, Single, Cradled {S,C} Postures

		LS	LC	RS	RC
	Left-Single (LS)	63.0% 15.2% 10.9% 10.9%
	Left-Cradle (LC)	10.4% 60.4% 14.6% 14.6%
	Right-Single (RS)	1.3%	5.9% 46.4% 46.4%
	Right-Cradle (RC) 1.3%	5.9% 46.4% 46.4%
	more appropriate. To test this possibility, we explore the use
	of user-specific models.

ACKNOWLEDGMENTS

Funding for this research was provided by the Natural Science and Engineering Research Council of Canada and the National Research Council of Canada. Experiments described in this paper were reviewed by the Office of Research Ethics at the University of Waterloo. We thank participants for volunteering for our study.


Haniel Barbosa
email: haniel-barbosa@uiowa.edu

Jasmin Christian Blanchette
email: j.c.blanchette@vu.nl

Mathias Fleury
email: mathias.fleury@mpi-inf.mpg.de

Pascal Fontaine
email: pascal.fontaine@loria.fr

Hans-Jörg Schurr
email: hans-jorg.schurr@loria.fr

Better SMT Proofs for Easier Reconstruction

Proof assistants are used in verification, formal mathematics, and other areas to provide trustworthy, machine-checkable formal proofs of theorems. Proof automation reduces the burden of proof on users, thereby allowing them to focus on the core of their arguments. A successful approach to automation is to invoke an external automatic theorem prover, such as a satisfiability-modulo-theories (SMT) solver [START_REF] Barrett | Satisfiability modulo theories[END_REF], reconstructing any generated proofs using the proof assistant's inference kernel. The success rate of reconstruction, and hence the usefulness of this approach, depends on the quality of the generated proofs.

We report on the experience gained by working on reconstruction of proofs generated by an SMT solver while also improving the solver's output. By doing so, we were able to understand some practical constraints of reconstruction systems and find areas that require attention in the documentation of the proof output. We also discovered bugs in the proof generation code.

Proof generation from SMT solvers has attracted attention in the past [START_REF] Barrett | Proofs in satisfiability modulo theories[END_REF]. The SMT solvers CVC4 [START_REF] Barrett | CVC4[END_REF] and Z3 [START_REF] De | Z3: An efficient SMT solver[END_REF] produce proofs, but CVC4's output format does not record quantifier reasoning, whereas Z3 does not always produce fine-grained steps, notably for skolemization. The SMT solver we work with, veriT [START_REF] Bouton | veriT: An open, trustable and efficient SMT-solver[END_REF], was recently extended with a more fine-grained proofproducing module [START_REF] Barbosa | Scalable fine-grained proofs for formula processing[END_REF] that records skolemization and other preprocessing steps in a detailed fashion. Proofs produced by veriT [START_REF] Besson | A flexible proof format for SMT: A proposal[END_REF] take the form of a list of steps with optional annotations for term sharing. The syntax is based on SMT-LIB [START_REF] Barrett | The Satisfiability Modulo Theories Library (SMT-LIB)[END_REF].

Many proof assistants reconstruct proofs generated by automatic theorem provers. Examples include the SMTCoq plugin [START_REF] Ekici | SMTCoq: A plug-in for integrating SMT solvers into Coq[END_REF], which reconstructs proofs from CVC4 and veriT inside Coq, and Isabelle's smt tactic [START_REF] Böhme | Proving Theorems of Higher-Order Logic with SMT Solvers[END_REF], which reconstructs Z3 proofs. We extended this tactic to support veriT proofs as well. The smt tactic first translates the current higher-order proof goal to a first-order SMT problem. Then the external SMT solver is invoked. If the solver reports "unsatisfiable," the tactic will attempt to replay the generated proof in Isabelle.

Our experience emphasizes the importance of complete documentation. When veriT is called with the option --proof-format-and-exit, it generates a list of proof rules and a description of their semantics. Furthermore, earlier publications [START_REF] Barbosa | Scalable fine-grained proofs for formula processing[END_REF][START_REF] Besson | A flexible proof format for SMT: A proposal[END_REF][START_REF] Déharbe | Quantifier inference rules for SMT proofs[END_REF] provide background documentation on the proof calculus. Nevertheless, this documentation was lacking, especially concerning implicit steps performed by veriT. To replay the proof, the implicit steps must also be performed on Isabelle's side. Such implicit transformations appear in two places. First, veriT ignores the orientation of equalities in the input. The simple solution was to print the input assertions after this normalization to allow Isabelle to link atoms with their normalized form. Second, veriT performs some simplifications immediately before printing a proof step. This includes eliminating repeated literals and double negation from clauses. Now that this behavior is precisely documented, Isabelle can reconstruct these implicit steps most of the time at the cost of some automatic search. We plan to make this implicit normalization optional in future versions of veriT.

The size of the generated proofs is a practical constraint we initially overlooked. During skolemization, veriT introduces Hilbert choice terms in place of skolemized variables. Thus, ∃x. p(x) is skolemized to p(εx. p(x)). While this is elegant in theory, the choice term can be prohibitively large, especially when it is repeated in the output, leading to reconstruction failures. A solution is to use term sharing in the generated proofs: veriT adds a name annotation to every term and subsequently uses the name instead of the term. Sharing can have a dramatic impact on size: a 62 MB proof was compressed to 192 KB.

We encountered some difficulties with the replacement of constants by choice terms. Instead of choice terms, for efficiency reasons veriT uses fresh constants during solving. These constants must be replaced by the corresponding choice terms in the proof output. When choice terms were nested, the proof output did not fully replace constants inside choice terms. Since the choice functions are often quite long, such errors are hard to detect by a human reader, but instantly prohibit reconstruction.

We also observed a phenomenon we call proof rot. During the development of an automatic prover, we might inadvertently introduce small discrepancies with respect to the documented behavior. For example, the instantiation rule used by veriT is slightly stronger than published [START_REF] Déharbe | Quantifier inference rules for SMT proofs[END_REF] and documented. The documented form is (∀x. ϕ) → ϕ[t/x], but in practice it sometimes takes the form (∀x.

ϕ 1 ∧ • • • ∧ ϕ n ) → ϕ i [t/x]
. Such changes accumulate and complicate reconstruction. During the implementation of the reconstruction procedure, each change had to be either documented or corrected. Now that it is in place, proof reconstruction serves as a safeguard to prevent such changes from being accidentally reintroduced.

Prospect Proofs are meant to be replayed. Implementing the reconstruction during the development of the proof-producing routines ensures that proofs can be replayed in practice. Given the flexibility of the SMT language, the proofs generated by SMT solvers need to account for a wide variety of theories and language features, which results in complex proofs with many possibilities for errors. These errors can be found by reconstructing proofs.

The quality of veriT's proofs remains unsatisfactory. Simple input problems often produce long, unwieldy proofs; yet, many proof steps are too coarse. A rule for linear arithmetic produces a certificate of the unsatisfiability of a set of inequalities using Farkas's lemma without providing explicit coefficients. This means that reconstruction must rely on a decision procedure to refind the proof, which sometimes fails or is slow. Furthermore, term sharing is required to keep proofs to a reasonable size, but also results in unreadable proofs. A good balance has yet to be found.

A call to an automated prover from inside a proof assistant can fail. Often this is because the prover could not find a proof, but sometimes the proof cannot be reconstructed. This should never happen.

Acknowledgments The work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 713999, Matryoshka). Previous experiments were carried out using the Grid'5000 testbed (https://www.grid5000.fr/), supported by a scientific interest group hosted by Inria and including CNRS, RENATER, and several universities as well as other organizations.