Content uploaded by Alessandro Di Cerbo
Author content
All content in this area was uploaded by Alessandro Di Cerbo on Apr 17, 2018
Content may be subject to copyright.
Send Orders for Reprints to reprints@benthamscience.ae
The Open Public Health Journal, 2018, 11, 3-00 3
1874-9445/18 2018 Bentham Open
The Open Public Health Journal
Content list available at: www.benthamopen.com/TOPHJ/
DOI: 10.2174/187494450181101????
SHORT COMMUNICATION
Oxytetracycline-Protein Complex: The Dark Side of Pet Food
Alessandro Di Cerbo1,2,*, Antonio Scarano2, Federica Pezzuto3, Gianandrea Guidetti4, Sergio
Canello5, Diego Pinetti6, Filippo Genovese6 and Lorenzo Corsi1
1Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
2Department of Medical, Oral and Biotechnological Sciences, Dental School, University G. d`Annunzio of Chieti-
Pescara, Chieti, Italy
3Department of Clinical Science, University of Modena and Reggio Emilia, Modena, Italy
4SANYpet S.p.a., Research and Development Department, Bagnoli di Sopra (Padua), Italy
5Forza10 USA Corp., Research and Development Department, Orlando (FL), USA
6CIGS, University of Modena and Reggio Emilia, Modena, Italy
Received: January 09, 2017 Revised: March 27, 2018 Accepted: April 08, 2018
Abstract:
Background:
Worldwide antibiotic abuse represents a huge burden, which can have a deep impact on pet and human health through nutrition and
medicalization representing another way of antibiotic resistance transmission.
Objective:
We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and
Oxytetracycline (OTC), a widely and legally used antibiotic in zootechny, which might determine a toxic effect on K562 cells.
Method:
Cell viability and HPLC-ESI/QqToF assays were used to assess potential toxicity of bone extract derived from OTC-treated chickens
according to standard withdrawal times and from antibiotic free-treated chickens at 24, 48 and 72h of incubation.
Results:
Cell culture medium with ground bone from chickens reared in the presence of OTC resulted significantly cytotoxic at every
incubation time regardless of the bone concentration while BIO-CCM resulted significantly cytotoxic only after 72h of incubation.
HPLC-ESI/QqToF assay ruled out the possible presence of OTC main derivatives possibly released by bone within culture medium
until 1 μg/mL.
Conclusion:
The presence of a protein complex with OTC is able to exert a cytotoxic effect once released in the medium after 24-48h of
incubation.
Keywords: Antibiotic abuse, Oxytetracycline, Protein complex, Cytotoxic effect, Pro-inflammatory effect, K562 cells.
1. INTRODUCTION
Some studies reveal that the widespread antibiotic use in agriculture and aquaculture might contribute to the
* Address correspondence this author at the Department of Life Sciences, University of Modena and Reggio Emilia, Alessandro Di Cerbo, Modena,
Italy; Tel: 00390592055367; E-mail: Alessandro811@hotmail.it
4 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
development of resistance to antibiotics commonly used in human medicine [1]. This issue becomes important
particularly in the zootechnical due to its accumulation in animal feed and food with potential chronic consequences
deriving from its ingestion to the species fed with these foods [2].
Nowadays, antimicrobials use represents a serious concern particularly in two correlated fields i.e. medical and
agricultural [2 - 5]. In poultry, for instance, antibiotics are used to promote growth and to treat, control, and prevent
overcrowding diseases [2, 6, 7]. A routinely exposure to antibiotics induce a selection for resistant bacteria that can
persist on meat and in animal waste with a vertical transmission through maternal generations of breeding stocks [7].
Such bacteria can get in contact with humans in food-animal production facilities, in meat processing plants but also
consuming contaminated meat [6, 8 - 10]. Despite there is not a common consensus on the use potential antibiotic
resistance elicited by antibiotic used in animal food many countries have made substantial efforts to reduce the overall
use of antibiotics in food-producing animals, in the attempt to decrease the antibiotic resistance in animals, the
environment, and in human beings.
Moreover, Mueller et al. hypothesized that food allergens e.g. beef, fish and chicken could drag antibiotics and
hormones thus representing the cause for the onset of dermatological symptoms in cats [11]. Among pharmacologically
active substances, tetracycline (in particular oxytetracycline, OTC) and their metabolites present in meats and meat-
based foods for humans and pets were considered and studied [9, 10, 12].
We firstly hypothesized and observed the role of OTC as an underlying cause of some chronic inflammatory
pathology in vivo and in vitro as described by Di Cerbo et al. [8, 9, 13 - 17]. Due to its low cost and high efficacy [18],
OTC is widely employed in the intensive farming of poultry [2], livestock [19] and aquaculture [20]. However, OTC
has a high affinity for calcium, mainly present within bones, and a very low and long clearance in treated animals [21].
Further, pet food production, which mainly relies on poultry by-products [22], also avails itself of an important
percentage of bone meal (20-30%) [8] with a consequent dragging of OTC residues that are frequently found within
commercially available diets [10].
Despite the setting of maximum residue limits in foods by Food and Drug Administration [23] and World Health
Organization [24] OTC residues may still persist since bone is not considered as an edible tissue, thus making pet food
potentially dangerous [25].
In this paper, based on our previous results [6, 21], we aimed our research to determine possible interactions
between biological substrates, such as proteins, and OTC, which might lead to an increase or decreased toxic effect on
K562 cells mediated by the antibiotic. For this purpose we evaluated the potential toxicity of bone extract derived from
OTC-treated chickens according to standard withdrawal times and from antibiotic free-treated chickens.
2. MATERIALS AND METHODS
2.1. Cell Culture
K562 myelogenous leukemia cell line, were purchased from American Type Culture Collection (ATCC) (LGC
Standards srl, Milan, Italy). K562 cells were grown in RPMI supplemented with 10% Fetal Bovine Serum (FBS) 100
g/mL streptomycin, 100 U/mL penicillin, 2 mM glutamine (Euroclone Spa, Milan, Italy). The cells were cultured in a
humidified incubator at 37 °C with 95% and 5% CO2.
2.2. Conditioned Culture Medium Preparation
Conditioned Culture Medium (CCM) was obtained by incubating 10 mL of a RPMI 1640 cell culture medium with
124, 90, 6, 2.48 mg and 124 μg of sterilized ground bone from chickens reared in the presence (OTC-CCM) or absence
(BIO-CCM) of treatments with OTC and constantly shaken for 24-48-72 h at 37° C. After incubation, both CCMs were
filtered through 0.45 and 0.20 μm syringe filters (Sartorius Stedim Biotech, Goettingen, Germany) to remove any
residual ground bone particles and microbial contamination.
2.3. Determination of Cell Viability
Cell viability was assessed after 24-48-72 h of continuous exposure with the aforementioned concentrations. A Cell
Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) was used to measure the cytotoxicity on
K562 cells. Briefly, the K562 cells were plated on 96-well plates (Euroclone, Milan, Italy) at concentration of 5000 or
10000 cells/cm2. After exposure to desired concentrations of the different compounds, 10µl of CCK-solution was added
Protein Complex: The Dark Side The Open Public Health Journal, 2018, Volume 11 5
to each well and incubated for a period of 2 h at 37°C. Finally, absorption was measured at 450 nm using a multiplate
reader Multiscan FC (Thermo Scientific, USA). Dimethyl Sulfoxide (DMSO) 3% was used as toxic reference drug. Cell
viability was expressed as a percentage of that of the untreated cells (Control). For each concentration of tested
compounds, mean values of the mean absorbance rates from four wells were calculated.
2.4. HPLC-ESI/MS Analysis
CCM samples were analyzed using a HPLC-ESI/MS analyses were performed on a 1200 Series HPLC coupled to
either a 6520A Quadrupole/Time-of-Flight high mass spectrometer (QqToF) or a 6410B Triple Quadrupole mass
spectrometer (QqQ), (both from Agilent Technologies, California, USA) via an electrospray ion source (ESI). On the
HPLC-ESI/QqToF system, the chromatographic separation was carried out with 4 mL injection volume on an Agilent
Zorbax SB-C18 30 x 2.1 mm ID 3.5 mm ps column. Elution was performed at T=25 °C, with a flow rate of 0.3 mL/min.
were used as a mobile phase. A linear gradient elution starting at 0.5 min going from 2% (B) up to 40% (B) in 11
minutes then up to 95% (B) in 6 minutes was performed using water with 0.5% formic acid (A) and Acetonitrile (B) as
mobile phase components. The mass spectrometer was operated in positive-ion mode (ESI+) using “Auto MS/MS”
acquisition with MS and MS/MS scan ranges being 50<m/z<1000 Th and selection of the top 3 most abundant mono-
charged ions from each MS scan for their subsequent MS/MS spectra acquisition (active exclusion was enabled after 1
spectrum and released after 0.15 min). Targeted MS/MS analyses on the HPLC-ESI/QqQ included chromatographic
separation on an Agilent Zorbax SB-C18 30 x 2.1 mm ID 3.5 mm ps column at 0.3 ml/min with the following gradient
(mobile phases: A: water + 0.1 FA%, B: acetonitrile + 0.1 FA%): 0’ 98:2 A:B, kept for 0.5’ then B% was raised to 40%
in 11’, then B% was raised to 95% in 3’, hold for 6’ then columns was reconditioned at the starting conditions, for a
total runtime of 32 minutes.
ESI source was operated in positive mode at 3.5 kV, the Gas Heater was set to 300 °C, the gas flow was 8 l/min and
the nebulizer pressure was set to 25 psi.
Three transitions for OTC were chosen for Selected Reaction Monitoring, as shown in the Table (1).
Table 1. Selected Reaction Monitoring for the three transitions of OTC.
Precursor (m/z) Product (m/z) Fragmentor (V) CE (V) Dwell Time (ms)
461.5 426.4 106 17 400
461.5 286.1 106 25 100
461.5 201.2 106 29 100
A matrix-matched calibration curve was prepared, ranging from 1 ppb to 1 ppm OTC concentration on a diluted and
filtered CCM; samples were filtered, diluted at the same dilution factor of the calibration curve and analyzed. For
standard-addition method, the sample was spiked with OTC ranging from 1 ppb to 1 ppm.
2.5. Statistical Analysis
All data are presented as the mean ± SD of at least three different experiments done in quadruplicate. One-way
ANOVA analysis of variance with Dunnett’s post-test, were performed to compare differences between the groups, as
indicated in the figures (Graph-Pad 6 Software Inc., San Diego, CA, USA). P values < 0.05 were considered significant.
3. RESULT
Here we found that OTC-CCM resulted significantly cytotoxic at every incubation time regardless of the bone
concentration while BIO-CCM resulted significantly cytotoxic only after 72h of incubation Fig. (1).
Qualitative analysis was performed using the HPLC-ESI/QqToF assay in order to test for the presence of OTC (at a
concentration ≥ 1mg/mL) and its main derivatives N-Demethyloxytetracycline, N-Didemethyloxytetracycline, and
Apooxytetracycline. To this purpose, their presence was tested using their molecular ion signals eventually revealed on
their respective extracted ion chromatogram (EIC) in the ESI(+) MS experiments. Due to the QqToF high resolution
and mass accuracy (< 5 ppm) each EIC was obtained using a narrow extraction window (10 ppm) centered on the
molecular ion theorical m/z value. In order to account for the eventual suppression of OTC signals at the 1 mg/mL
concentration level, due to matrix effect in extracted samples or for its degradation/combination in the extracted sample
environment, extracted samples were as well spiked with OTC at 1 μg/mL and analyzed right away (t0) and after 24 and
48 hours (t 24, t 48). Neither OTC nor its derivatives were revealed in non-spiked OTC-CCM and BIO-CCM extracted
6 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
samples Figs. (2, 3) while OTC molecular ion ([M+H]+ at m/z= 461.155) was clearly revealed in t 0, t 24 and t 48
spiked samples with roughly the same signal intensity (within the instrument variability). This suggested the OTC
concentration being substantially below the tested 1 μg/mL level, while reasonably excluding strong
degradation/combination reactions of OTC in the extracted matrix environment at least within the tested 48 hours
period.
Fig. (1). Graphical representation of the effects of conditioned medium with bone with (OTC-CCM) and without (BIO-CCM)
oxyteracycline on K562 cell line at different incubation times (24, 48 and 72h) and at different bone concentrations (124, 90, 6, 2.48
mg and 124 μg). *p < 0.05, ***p < 0.001 vs Control (Ctrl).
Targeted MS/MS analyses were performed on the HPLC-ESI/QqQ to quantitate OTC in bone samples. Preliminary
tests showed a heavy matrix effect, thus we optimized the dilution factor and injection volumes for best results, ending
up with a 20-fold dilution and 20 μl of diluted sample injected.
Protein Complex: The Dark Side The Open Public Health Journal, 2018, Volume 11 7
Fig. (2). From top, ESI(+) MS EIC of m/z 461.155 ion shown in the retention window of OTC in OSSO BIO non-spiked, t0, t24 and
t48 samples.
Fig. (3). From the top, ESI(+) MS EIC of m/z 461.155 ion shown in the retention window of bone with OTC non-spiked at 0, 24 and
48h.
8 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
Matrix-matched calibration curve showed a good fit (r2 ≥ 0.999); unfortunately, the sample signal was below the
quantification limit. In order to increase the sensitivity because of the low concentration of OTC in analyzed samples,
standard-addition method was applied. In this case, by extrapolating the intercept of the curve on the y-axis we were
able to determine OTC concentration. By correcting the value for the dilution factor, the final determined concentration
was 200 ng/ml. As a proof of concept, we ran the same standard addition analysis on the organic bone extract; the curve
in this case showed an intercept with the y-axis significantly closer to the origin, indicating that the concentration of
OTC (if any) was significantly lower in this extract compared to the bone extract Fig. (4).
Fig. (4). Triple Quadrupole mass spectrometer (QqQ-ESI) molecular iron determination of OTC-protein complex.
4. DISCUSSION
Previously published data showed a significant cytotoxic effect of OTC powder at 24, 48 and 72h at a concentration
of 150 μg/mL (according to the amount of OTC expected to be provided to chicken during a standard treatment and
disclosed on the antibiotic package) and at 48 and 72h at 75 and 35 μg/mL [8]. Moreover, the cytotoxic effect was
clearly visible at OTC concentrations far below those previously investigated [10].
We recently claimed the unavoidable occurrence of oxytetracycline as a contaminant of meat, bone meal and
poultry by-products, which are widely present within commercially available pet diets and human foods (mainly
würstels and sausages) [8, 10]. In addition, we reported a discrepancy among OTC kibble concentration, OTC serum
concentration of dogs with dermatitis and otitis, diarrhea and general anxiety and in vitro OTC, 20% liquid or powder,
last cytotoxic concentration [10]. In fact, OTC kibble concentration was 19 μg/mL, serum oxytetracycline concentration
was 0.22 μg/mL while in vitro last cytotoxic concentration was 35 μg/mL for the OTC powder and 87.5 μg/mL for the
20% liquid OTC.
Despite our in vitro data are in agreement with those previously published [6, 10], it is worth noting that HPLC-
ESI/QqToF clearly evidenced the lack of OTC and its main derivatives within conditioned medium until 1 μg/mL.
However the results obtained with Triple Quadrupole mass spectrometer (QqQ) showed the presence of an OTC-protein
complex
This latter result helped us to rule out the toxic role of OTC itself and allowed us to speculate the presence of a
protein complex inside bone with OTC able to exert a cytotoxic and pro-inflammatory effect once released in the
medium after 24-48h of incubation. Moreover, it is reasonable to hypothesize that most of symptoms observed in the
dogs of our previous paper might be due to an overall inflammatory condition elicited by the daily intake of kibbles
made of chicken-derived bone with OTC. Although more researches are needed in order to figure out the chemical
composition of the complex and the molecular mechanism involved in the cell toxicity, our results revealed a new
possible explanation linked to the side effects of chicken-derived bone with OTC.
Protein Complex: The Dark Side The Open Public Health Journal, 2018, Volume 11 9
CONCLUSION
Our research represents a further insight into the overall landscape of antimicrobials abuse pointing out the possible
consequences on pets and humans well being with particular regard to the actual antibiotic resistance spread.
Unfortunately, WHO and FDA have set high OTC minimal residual limits within organs of farmed animals and do not
consider bone as a possible veihicle of contamination since considered as not edible [8, 26]. In this view our findings
rise new questions concerning the interactions between antibiotics, i.e. OTC, and organic substrates. Indeed our results
pointed out the formation of a complex which induce cell toxicity even more then antibiotic alone. The further question
to be answerd is wether this complex is able to induce antibiotic resitence too.
Since WHO ruled out an agenda on the study and the prevention of antimcorobial resistence (AMR) in the different
fields where the antibiotic were used, further studies with more detailed evaluations are needed to support our
preliminary observations, and to confirm a possible new scenario in the AMR.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENTS
Decleared none.
REFERENCES
[1] Tang KL, Caffrey NP, Nóbrega DB, et al. Restricting the use of antibiotics in food-producing animals and its associations with antibiotic
resistance in food-producing animals and human beings: a systematic review and meta-analysis. Lancet Planet Health 2017; 1(8): e316-27.
[http://dx.doi.org/10.1016/S2542-5196(17)30141-9] [PMID: 29387833]
[2] Palmieri B, Di Cerbo A, Laurino C. Antibiotic treatments in zootechnology and effects induced on the food chain of domestic species and,
comparatively, the human specie. Nutr Hosp 2014; 29(6): 1427-33.
[PMID: 24972484]
[3] Devaraj NK. Antibiotic Resistance: A Real Menace. Oman Med J 2017; 32(6): 531.
[http://dx.doi.org/10.5001/omj.2017.102] [PMID: 29218134]
[4] Shea KM. Antibiotic resistance: what is the impact of agricultural uses of antibiotics on children’s health? Pediatrics 2003; 112(1 Pt 2): 253-8.
[PMID: 12837918]
[5] Witte W. Medical consequences of antibiotic use in agriculture. Science 1998; 279(5353): 996-7.
[http://dx.doi.org/10.1126/science.279.5353.996] [PMID: 9490487]
[6] Di Cerbo A, Palatucci AT, Rubino V, et al. Toxicological Implications and Inflammatory Response in Human Lymphocytes Challenged with
Oxytetracycline. J Biochem Mol Toxicol 2016; 30(4): 170-7.
[http://dx.doi.org/10.1002/jbt.21775] [PMID: 26537863]
[7] Koch BJ, Hungate BA, Price LB. Food-animal production and the spread of antibiotic resistance: the role of ecology. Front Ecol Environ
2017; 15(6): 309-18.
[http://dx.doi.org/10.1002/fee.1505]
[8] Mazzeranghi F, Zanotti C, Di Cerbo A, et al. Clinical efficacy of nutraceutical diet for cats with clinical signs of cutaneus adverse food
reaction (CAFR). Pol J Vet Sci 2017; 20(2): 269-76.
[http://dx.doi.org/10.1515/pjvs-2017-0032] [PMID: 28865210]
[9] Di Cerbo A, Canello S, Guidetti G, Laurino C, Palmieri B. Unusual antibiotic presence in gym trained subjects with food intolerance; a case
report. Nutr Hosp 2014; 30(2): 395-8.
[PMID: 25208795]
[10] Di Cerbo A, Sechi S, Canello S, et al. Preliminary Study of Antibiotic Residues-related Adverse Food Reactions in Dogs fed on commercially
10 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
available Pet Food. Vet Ital 2018.
[11] Mueller RS, Olivry T, Prélaud P. Critically appraised topic on adverse food reactions of companion animals (2): common food allergen
sources in dogs and cats. BMC Vet Res 2016; 12: 9.
[http://dx.doi.org/10.1186/s12917-016-0633-8] [PMID: 26753610]
[12] Chi Z, Liu R, You H, Ma S, Cui H, Zhang Q. Probing the in vitro cytotoxicity of the veterinary drug oxytetracycline. PLoS One 2014; 9(7):
e102334.
[http://dx.doi.org/10.1371/journal.pone.0102334] [PMID: 25019386]
[13] Di Cerbo A, Centenaro S, Beribè F, et al. Clinical evaluation of an antiinflammatory and antioxidant diet effect in 30 dogs affected by chronic
otitis externa: preliminary results. Vet Res Commun 2016; 40(1): 29-38.
[http://dx.doi.org/10.1007/s11259-015-9651-4] [PMID: 26743397]
[14] Di Cerbo A, Pezzuto F, Canello S, Guidetti G, Palmieri B. Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in
Dogs. J Vis Exp 2015; (101): e52717.
[PMID: 26167807]
[15] Di Cerbo A, Palmieri B, Chiavolelli F, Guidetti G, Canello S. Functional Foods in Pets and Humans. Int J Appl Res Vet Med 2014; 12(3):
192-9.
[16] Di Cerbo A, Sechi S, Canello S, Guidetti G, Fiore F, Cocco R. Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory
Effects of a Nutraceutical Diet. J Vis Exp 2017(119).
[17] Di Cerbo A, Canello S, Guidetti G, et al. Adverse food reactions in dogs due to antibiotic residues in pet food: a preliminary study. Vet Ital In
press
[http://dx.doi.org/10.12834/VetIt.1357.7466.2] [PMID: 29633230]
[18] Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance.
Microbiol Mol Biol Rev 2001; 65(2): 232-60. [second page, table of contents.].
[http://dx.doi.org/10.1128/MMBR.65.2.232-260.2001] [PMID: 11381101]
[19] Kimera ZI, Mdegela RH, Mhaiki CJ, et al. Determination of oxytetracycline residues in cattle meat marketed in the Kilosa district, Tanzania.
Onderstepoort J Vet Res 2015; 82(1): 911.
[http://dx.doi.org/10.4102/ojvr.v82i1.911] [PMID: 26842366]
[20] Chuah LO, Effarizah ME, Goni AM, Rusul G. Antibiotic Application and Emergence of Multiple Antibiotic Resistance (MAR) in Global
Catfish Aquaculture. Curr Environ Health Rep 2016; 3(2): 118-27.
[http://dx.doi.org/10.1007/s40572-016-0091-2] [PMID: 27038482]
[21] Odore R, De Marco M, Gasco L, et al. Cytotoxic effects of oxytetracycline residues in the bones of broiler chickens following therapeutic oral
administration of a water formulation. Poult Sci 2015; 94(8): 1979-85.
[http://dx.doi.org/10.3382/ps/pev141] [PMID: 26015592]
[22] Maine IR, Atterbury R, Chang KC. Investigation into the animal species contents of popular wet pet foods. Acta Vet Scand 2015; 57: 7.
[http://dx.doi.org/10.1186/s13028-015-0097-z] [PMID: 25886611]
[23] Headquarters FaAOF. Maximum Residue Limits for Veterinary Drugs in Foods. . 2012. In: Codex Alimentarius Commission 35th Session
[Internet]. ftp://ftp.fao.org/codex/weblinks/MRL2_e_2012.pdf.;[1-40].
[24] Agency UEP. Food and Drugs 2014.http://www.ecfr.gov/
[25] Agency UEP. Food and Drugs. PART 556 -Tolerances for residues of new animal drugs in food. Subpart B-Specific Tolerances for Residues
of New Animal Drugs. 2014. In: Electronic code of federal regulations (eCFR) [Internet]. http://www.ecfr.gov/.
[26] Graham F, Paradis L, Bégin P, Paradis J, Babin Y, Des Roches A. Risk of allergic reaction and sensitization to antibiotics in foods. Ann
Allergy Asthma Immunol 2014; 113(3): 329-30.
[http://dx.doi.org/10.1016/j.anai.2014.06.029] [PMID: 25168224]
© 2018 Di Cerbo et al.
This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a
copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
