1,11 1, We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We 昀؀rst explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing 昀؀re昀؀y luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced in昀؀ammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.

Germline light-producing transgenic mice where lucfierase expression is controlled by an endogenouos mproter, a surrogate promoter or by a minimal promoter dotrweanms of tandem, synthetic, transcription factorndbing elements, are used to provide anin vivo readout of physiological and pathological pro1c,2e.sOsense of the advantages of this technology is that every cell willnctoain a copy of the luciferase transgene and theorerfe provide a whole-body transgene expression profle under the control of a specifc promoter of choice. Howduevcienr,gpro germline transgenics requires frequent backcrossinagnd therefore becomes a time-consuming and cosptrloycess, using many rodents.

We have previously developed a novel technology cwhhaillows the generation of light-producing somatic transgenic rodents, using lentiviral vectors as arpoof-of-concept sys3teamnd have validated this technology both in vitro4,5 and in vivo1,6. We have also demonstrated that signalling pathwasyin diseased organs can be mon-i tored specifcally, continually and in a non-invaseivmanner1,2. Exploiting the immune tolerance of neonatal mice

Neonatal intravenous administration of AAV8-CMV-GFP vector. We sought to determine the bio-distribution of GFP expression following naelo(Pnoatst-neonatal day 1, P1) intravenous administtriaon of AAV8-CMV-eGFP. On the day of birth, mice receivedμ4l0of vector (×1 1013 vector genomes/ml) via the supe-r fcial temporal vein. One month later, mice were havrested and GFP expression was analysed, using a DFC420 microscope.

Prior to gross dissection and further fxation, swkains removed and mice were viewed under a stereoscpoic fluorescence microscope. Strong, extensive and wsipdreead GFP expression was observed throughout the body, although the highest levels of expression ewmerost noticeable in the musculature (Fig1. ). Strong tran-s duction was observed within the heart (B), liver ( C), kidney (D), skeletal muscle (E), eye (F), brain (G), and myenteric plexus (H). At higher magnifcation, we osberved a majority of GFP positive hepatocyte ceilnlsthe liver (Supplementary Fig. 1).

In order to assess the expression profle in the CN,tShe brains from injected and non-injected contrmoilce were sectioned and immunohistochemistry was conduedctfor GFP expression. Vis revealed extensive andiwdespread GFP expression (Supplementary Fig. 2). Further examination under higher magnifcation of disceraetreas of the brain including the primary motor cortex, the somatosensory barrel feld (S1BF), piriform cortaetxe, den gyrus, cerebellum, and the gigantocellular nucleursevealed transduction of cells with both neuronnaldaglial morphology (Supplementary Fig. 2).

Further to this we investigated whether AAV vectoorr GFP transgene expression triggers an infammatory response ager neonatal intracranial injections. Mriocglial activation was examined in all brains colclteed at 35 days of development and compared to brain tissfureom Ppt−1/− (palmitoyl protein thioesterase 1) mice in which profound microglial activation oc1c2u,rasnd therefore serve as a positive control forromgiclial markers. Extensive microglial engorgement and activation was observed in the P−p/t−1mice, and no noticeable activation of microglia was observed in the non-injected and AAV8 injected brains (Supplementary Fig. 3). Production of AAV8 biosensors. An AAV8 producer plasmid was created containing aaGteway® accepter site (Invitrogen). Ve Gateway® sequence was cloned into the backbone and was plaedc upstream of a minimal promoter driving a codon-optimised luciferase tgraennse and an enhanced GFP linked by a bicistronic lniker, T2A (Supplementary Fig. 4). We have now assembled an extensive library of transcription factor bindienlgements in pENTR shuttle plasmids and these are shown in Supplementary Fig. 4. We selected the NFκB response element and an SFFV viral promoter for the insertion into the AAV gateway backbone. Vese two were chosen as they have been validated by bothin vitro and in vivo means in our lentiviral system3.

AAV8 biosensor vectors were generated using the A8A-VSFFV-Luc-T2A-eGFP and AAV8- NκFB -Luc-T2A-eGFP backbones.

Neonatal administration of AAV8 biosensors. Having observed widespread transgene expre-s sion ager a single neonatal administration of anVAA8-CMV-GFP vector, we chose to investigate theκNBF signalling expression profle by neonatal injectioonf the AAV8-NκFB-Luc-2A-GFP biosensor. We selected AAV8-SFFV-Luc-2A-GFP as a constitutively expressecdontrol and to allow comparison with previous e-xper iments using lentivirus vector3s.

At P1 of development, mice received a 3μ0l intravenous (IV) administration of AAV8 SFFVAoVr 8ANFκB biosensor (1× 1013 vg/ml). Mice underwent whole-body bioluminescencimeaging over the course of deve-lop ment to quantify luciferase expression.

Following IV injection of the AAVκ8BNbFiosensor, luciferase expression was strongest in the spine, thorax, paws, lower abdominal and the mouth (Fig2A.  ). In contrast, IV injection of the AAV8 SFFV nbsiosreresulted in whole-body luciferase expression but with strontgeexspression in the lower abdomen (Fig2A.  ). Ve luciferase expression was quantifed and showed stable transgene expression over development (F2iBg,.C ).

Additionally, to restrict the expression profle within the CNS and PNS, intracranial (IC) injections wereoals performed with AAV8 SFFV and AAV8κNBFbiosensors. P1 pups received 5μl (1 × 1013 vg/ml) of either bi-o sensor, and whole-body bioluminescence imaging wuansdertaken over the course of development. Lucifsera expression from the IC injected AAV8 NκBF biosensors was similar to that seen in the IVeicntjed mice, predominantly in the spine, thorax, paws, mouth, eyes and tail (Supplementary Fig. 6A).

A month ager IC or IV injection of AAV8 SFFV κoBr bNioFsensor, the mice were harvested, immun-o histochemistry and immunofuorescence with neuronaanld astrocytic markers were performed to detect GFP expression in the brain. Ager IC injection of AAVSF8FV, GFP expression was predominantly neuronal whiint the dentate gyrus and CA1 and CA3 regions of thephpiocampus (Supplementary Figs. 7A and 8A), with so me astrocytic cell targeting (Supplementary Fig. 8B)B. oth neuronal and astrocytic cells were transducebdy IC AAV8 NFκB and IV AAV8 SFFV (Supplementary Fig. 8A,B). Howere, vfollowing IV AAV8 SFFV, a mixture of cells were found to be positive for GFP within the hippocamrepgailon ager immunoperoxidase staining (Supplemenrtya Fig. 7B). Ager co-staining these sections with neuronal and astrocytic markers, we found no co-slaotiocanli with GFP (Supplementary Fig. 8A,B).

Further to this, on a separate cohort of intravesnlyoiunjected mice, we were able to show extensive whole-body bioluminescence at 240 days of development (Fi3g).  Validation of the AAV8 NFκB biosensor. To assess whether the AAV8 NκFB could be exploited to report on NκFB signalling in pathological states, mice receiveudltra-pure lipopolysaccharide (LPS) which only acts through toll-like receptor 4 to induce trancsalotion of NκFB from the cytosol to the nucl1e3u. sAt day 132 ager AAV administration, a single dose of LPS wdasmainistered intraperitoneally to both IV and IC einctjed groups. Bioimaging was taken at 4, 24, 48, 72 and69hours before (to correct for any perturbanceheinsitgnal caused by multiple imaging) and ager LPS administration. A single administration of LPS resulted insiagnifcant increase in luciferase expression in IV (Fig4;. p < 0.0001, n = 7) and IC AAV8 NFκB mice (Supplementary Fig. 9; p= 0.006, n = 7). Ve luciferase expression for individual micehwich received IV injections is shown in Supplementary Fig. 10.

In this study, we have shown for the frst time thalitght-producing somatic-transgenic rodents can pbreoduced by a single neonatal administration of an AAV8 biosensor. Specifcally, we have demonstrated that AAcVto8rsve cross the blood brain barrier (BBB) and mediate styesmic transgene expression ager a single neonatanltiravenous or intracranial administration.

Our results showed a systemic spread of transgenexperession by a neonatal intravenous administratioonf a self-complementary AAV8 vector. Vis data agrees hwiptrevious work conducted by Nakaeit al. which demonstrated that AAV8 vectors cross the BBB and resuilnt transduction of both neuronal and glial cellsthine brain, albeit with a low eociency14. Vese authors also presented transduction of cewllisthin the liver, skeletal muscle, smooth muscle, cardiac muscle and pancrea1s4. However, we were able to show a much more extensive spread of transgene expression within the CNS and visceral ograns such as the kidney and myenteric plexus. Ve strong and widespread GFP expression observed in our studmy aybe due to a number of factors including the uosfe a diferent promoter and route of administration.

Our analysis also demonstrated an improved safetyropfile within the CNS after a neonatal intravenous administration. It has been reported that lentiviral vector mediated expressionf oGFP elicits toxicity in Purkinje cells of the developing mouse cerebellum. Vis toitxyicwas attributed mainly to GFP overexpression ahlotugh the authors could not discount the lentiviral vectopersr se, or genotoxicity caused by vector integration einurnonal DNA as aggregating facto1r5s.In contrast, we detected no microglial activati oinnthe CNS suggesting that infammation or gliosis caused by the injection, the presence of vector, or expnreossfioboth luciferase and GFP did not occur. Vis suggests that the AAV system we describme ay be preferable to our previously described leinvitral vector system2.

We performed neonatal intracranial injections of our AAV8 biosensors to monitor whether we tcroicutld res the expression profle to the CNS and PNS. Howeveor,ur results revealed that the intracranial injectedmice showed a similar expression profle to the mice whicreceived an intravenous administration. Previouwsork has shown that ager a single adult intravenous injcetions of AAV8, transgene expression was observietdhiwn the CNS and also within the cervical, thoracic, and lumbar spinal cord sections. However, the expressionfplerowas not signifcant compared to other serotypes such as AAV1r6.h.8

In order to overcome this wide distribution profle, future experiments would involve the assessmfeandtdoitional AAV capsids. Vese would include AAV-PHP.Bh,iwch is a new variant AAV capsid that transduces the CNS 40-fold greater than AAV9 ager a single adunlttriavenous administration and targets astrocytes adnneurons17. Furthermore, AAV-PHP.eB an enhanced variant of AVA-PHP.B, eociently transduces the CNS and PNS greater than the AAV-PHP.B and predominately targsetneurons18. Adult injections would further restrict the transgene expression in the CNS. Kostouelat al. demonstrated CNS specifc luciferase expression aegr adult stereotactic injections of AAV8-GFAP-luciferaseointhte hippocampu1s9. Verefore, in order to achieve CNS restricted and cell specifc transduction profles dfierent confgurations of AAV capsids and adult intijoecns would have to be used.

We have established a unique technology which alloswthe production of somatic-transgenic light-emnigtti rodents. We have synthesised a library o>f20 response elements,>10 promoters, most of which we have v-ali dated in the context of a lentiviral backboninevitro, and several of themin vivo. To exploit the advantages of AAV, we gene synthesised an AAV backbone containing a tGewaay acceptor site, and strategically placed mult-icloning sites with unique restriction sites to permit easy cloning.

As proof-of principle we inserted the κNBFresponse element, consisting of octuplet cognbainteding elements, into the AAV backbone. A potential concerinthwintroducing such sequences into the viral backob ne is the loss of tandem repeats following propagaatniod namplifcation in bacteria20. However, sequencing of large-scale DNA preparations of this backbone confrmed that the repeats remained intact.

Here we demonstrate that administration of AAV biosensors by a single neonatal intravenous administration resulted in widespread luciferase expression, in cmoparison to our lentiviral biosensors which onalynstrduced the liver3. Ve bio-distribution of luciferase expression dreifdebetween the constitutive AAV8 SFFV biosensor and the NFκB biosensor. A previous study demonstrated NκBF biosensor activity following administration of AAV by pancreatic infusion in adult mi2c1e. Baseline expression rose rapidly in those mice but fell to modes-t lev els by 1 month. Vis may be attributable to an anti-luciferase or anti-capsid cytotoxic T-cell respolnimseienating transduced cells. In contrast, we observed stablexepression for at least fve months; it has been shonwthat neonatal gene transfer administration results in immune tolerization to the transgenic protein when using retro2v2,irus AAV23 and adenovirus24 vectors.

We also conducted additional studies to test anorthAeAV8 biosensoinr vivo. We administered an AAV8 biosensor containing a hypoxic response element (EH),Rdriving luciferase via tail vein injections t oadult mice (8 weeks). At day 77 post injection, the mice were subjected to brief hypoxia (10% oxy g25e.nA) burst of biolum- i nescence imaging was taken pre- and post-hypoxia and the results revealed no diference in luciferase expression ager hypoxia (Supplementary Fig. 11). Verefore, hhi glighting that certain AAV biosensors aren9t as sietnive as germline transgenic mice in detecting subtle changes in cellular signalling.

Here we present for the frst time the generation of somatic-transgenic mice with the use of AAV viral vectors delivered to neonatal mice. We have also shown wisdperead transgene expression ager a single intracranial or intravenous neonatal administration of the AAV biosensors. We have validated the AAV biosenshothrsewuiste of agonists. Vis technology not only complemeinstisnegxgermline transgenic rodents but also maximeiss the use and reduces the numbers of animals used in biomedical research26. We have generated a non-invasive gene marking technology, which can be applied to systemcdi isease models. With the use of Gatewa®ycloning the assessment of diferent response elements can easily be achieved.

All methods were performed in accordance with the relevant guidelines and regulations.

Generation of the AAV biosensor plasmids. Ve AAV8-Gateway®-Luc-T2A-eGFP was synthesised by Aldevron (Aldevron, North Dakota, USA). Vis AAVspmlaid consisted of a Gatewa®ysequence, placed upstream of a codon-optimised luciferase transgene, linked to an enhanced GFP by a bicistroniecr,liTn2kA. Ve response element NFκB and constitutive promoter SFFV was cloned into an pENT plas3m.Bidy using the Gateway® cloning kit (Invitrogen, Manchester, UK) and using themanufactures guidelines, the response element NκFB, HRE and constitutive promoter SFFV were individuallyseinrted into the AAV backbone to generate the foilnlogw plasmids; pAAV8-SFFV-Luc-T2A-eGFP, pAAV8-NκBF-Luc-T2A-eGFP and pAAV8-HRE-Luc-T2A-eGFP. Recombinant AAV production. Recombinant AAV was produced, purifed and titeredsiung standard procedures. Briefy, HEK293T cells were double transfected with the pAAV8-SFFV-Luc-2A-eGFP or pAAV8κ-BNF -Luc-2A-eGFP or pAAV8-HRE-Luc-T2A-eGFP plasmid, an dthe pDG8 plasmid expressing AAV2 Rep, AAV8 Cap gene and adenovirus 5 helper functions (PlasmiFdactory, Bielefeld, Germany) using polyethylenimein (PEImax, Polysciences Inc). Ager 72 hours of incubation at 37 °C, the cells were harvested by centrifugation and then lysed by freeze-thawing in lysis bufer. In parallel, the virus-containing supernatant was harvesetd and precipitated by using ammonium sulphate salt. Cell lyaste and precipitated supernatant were treated with benzonase, clarifed by centrifugation and fltered at 0.2μ2m before purifcation.

Ve recombinant AAV virus preparations were purifebdy iodixanol step gradient: the viral preparationaws overlaid with increasing concentrations of iodixla(n15o%, 25%, 40% and 60%, OptiPrep; Sigma-Aldrich,Dorset, UK). Ve tubes were centrifuged at 40,000 rpm forh3ours at 18 °C in a Sorvall Discovery 90SE ultracetrnifuge using a TH641 rotor (Vermo Scientifc, Paisley, UKV). e vector was extracted from the 40% fraction wiath 19-gauge needle. Purifed vector fractions were dialysed against PBS overnight.

Real-time PCR and alkaline gel electrophoresis were used to assess the viraelngome titers and integrity of the viral genome27,28, the capsid titers were determined by SDS PAGE electrophoresi2s9.

AAV8 containing the enhanced GFP gene driven by thceytomegalovirus promoter (AAV8-CMV-eGFP) in a self-complementary confguration was obtained frtohemUPenn Vector Core facility (Philadelphia, USAat)a titre of 1× 1013 vg/ml.

Animal procedures. Outbred CD1 mice and MF1 mice used in this study were supplied by Charles Rivers Laboratories. All procedures were performed undenriUted Kingdom Home Ooce Project License 70/8030, approved by the ethical review committee and folloedwinstitutional guidelines at University CollegeoLndon. All methods were performed in accordance with the relevant guidelines and regulations.

Neonatal Intracranial and Intravenous injections. For intracranial injections, mice (random mix of males and females) were subjected to brief hypothmeric anaesthesia on the day of birth, followed byiluatneral injections of concentrated AAV vectoμrl(i5n total; 1× 1013 vector genomes/ml) into the cerebral lateral veni-tr cles using a 33 gauge Hamilton needle (Fisher Scietnifc, Loughborough, UK), following co-ordinatveisdpedro by Kim et al.30. For intravenous injections, pups were subjectedot brief hypothermic anaesthesia followed by intravenous injections of AAV vectors into thersfucpieal temporal vein31, with a total volume of μ25l(1 × 1013 vg/ml). Ve neonates were then allowed to return to normal pteemrature before placing them back with the dam. Adult tail vein injections. Adult male CD1 mice were placed in an chamber set at 37 °C, ager which they were anaesthetised with isofurane with 21% oxygenA(bbotts Laboratories, London, UK). A total volumfe o 100 μl (1 × 1010 vg/ml) of AAV8-HRE-Luc-T2A-eGFP was administerediavtail vein to each mouse. Ve mice were then placed back into their cages.

Whole-body bioluminescence imaging. Where appropriate, mice were anaesthetised with isofurane with 21% oxygen. D-luciferin (Gold BiotechnologSiTesL,ouis, USA) was administered by intraperitoneal injection at a concentration of 15 mg/mL. Mice wiemreaged 5 minutes ager luciferin injection using caooled charged-coupled device camera, (IVIS Lumina II, Perkin Elmer, Coventry, UK) for between 1 second and 5-min utes. Photon output of the whole-body was measuurseidng Living Image Sogware (Perkin Elmer) and light output quantifed and expressed as photons per secodnper centimetre squared per steradian (photons/seocnd/ cm2/sr).

Hypoxia. Adult mice (19 week old mice) were placed inside a hypoxic chamber which was air tight. Ve mice were subjected to 10% oxygen mixed with nitrogenrf2ohours following protocols mentioned in Ketaradl.a2 and Nakada et al.25. Ve mice were then returned to their dams.

Collection of brain tissues. Mice were anaesthetised at day 35 of development uinsg Isofurane and the right cardiac atrium was incised, followed by injteicon of heparinized PBS into the leg cardiac venctlrei. Ve brains were removed and fxed in 4% paraformaldehyde (PFA) and then cryoprotected in 30% sucrose in 50 mM PBS. Brains were sectioned using a sliding microtoem(Carl Zeiss, Welwyn Garden City, UK) to generate40 μm transverse sections31. Sections were stored in 30% sucrose in TBS, ethylene glycol and 10% sodium azide. Immunoperoxidase immunohistochemistry. To visualise CD68 and GFP immunoreactivity, sectiosn were treated with 30% H2O2 in TBS for 30 minutes. Vey were blocked with 15%abrbit serum for CD68 (Vector Laboratories, Cambridge, UK) and goat serum for G(FVPector Laboratories) in Tris bufered saline andwTeen 20 (TBST) for 30 minutes. Vis was followed by thdedaition of primary antibody, rat anti-mouse CD68:1(010; Biorad, Hertfordshire, UK), mouse anti-GFP (1:10,00;0Abcam, Cambridge, UK) in 10% serum and TBST and incubated over night at 4 °C with constant gentlegagitation. Ve following day the sections were terdeawt ith the secondary rat anti-rabbit antibody for CD68 (1:1000; Vector Laboratories) and goat antti-(r1a:b1b0i00 dilution; Vector Laboratories) in 10% rat or goat seirnuTmBST for 2 hours. Ve sections were incubatedrfaofurther 2 hours with Vectastain ABC (Vector Laboraetso).r0i.05% of 3,′3-diaminobenzidine (DAB) was added and leg for a couple of minutes. Sections were tranrsefderto ice cold TBS. Individual brain sections werme ounted onto chrome gelatine-coated Superfrost-plus sli(dVesWR, Poole, UK) and leg to dry for 24 hours. Vide ssl were dehydrated in 100% ethanol and placed in Histoclear (National Diagnostics, Yorkshire, UK) for i5nmutes before adding a coverslip with DPX mounting dmiuem (VWR). DAB stained sections in Supplementary Figs. 1 and 2 were viewed using an Axioskop 2 Mot microscope (Carl Zeiss Ltd.) and representative images were captured using an Axiocam HR camera and Axiovisio4n.2 sogware (Carl Zeiss Ltd.). DAB stained sectionsin Supplementary Fig. 7 were viewed using Leica MZ16F microscope sogware.

Fluorescence immunohistochemistry. A similar protocol was followed as with the immuenroopxidase stain; however H2O2 treatment was omitted. Followed protocol mentioned in Keatrdaal.2.

Quantitative analysis of immunohistological staining. GFP and CD68 expression was quantifed by thresholding analysis as previously describe1d2,32. Briefy, 40 non-overlapping RGB images were takefnrom four consecutive sections through the somatosenbsoarryel feld (S1BF), caudate putamen (Cpu), the Cornu Ammonis region 1 of the hippocampus, (CA1), piriform cxo(rptieriform cort), and the 10Cb region of the- cer ebellum (10Cb). Ve images were captured using a live video camera (JVC, 3CCD, KY-F55B) mounted onto a Zeiss Axioplan microscope using the x40 objectiveensl. All camera and microscope calibration and seitntgs were kept constant throughout the image capture pioerd. Images were analysed for optimal segmentatiaonnd immunoreactive profles were determined usingImage-Pro Plus (Media Cybernetics). Foreground immun o-s taining was accurately defned according to averagginof the highest and lowest immunoreactivities wiinththe sample population for a given immunohistochemicarlkmer (per colour/flter channel selected) and measrued on a scale from 0 (100% transmitted light) to 255 (0% transmitted light) for each pixel. Vis threshold setting was constant to all subsequent images analyzed for tahnetigen used. Immunoreactive profles were discrimianted in this manner to determine the specifc immunoreactivearea (the mean grey value obtained by subtractingthe total mean grey value from non-immunoreacted value per defned feld). Macros were recorded to transfer the data to a spreadsheet for subsequent statistical analysis. Statistics. Ve data from Supplementary Figs. 2 and 3 were ploetdt graphically as the mean percentage area of immune-reactivity per feld± S.D or S.E.M. for each region. Two-way ANOVA wanitdh a Sidak9s multiple comparison was performed in Fi4ga. nd Supplementary Fig. 9.

R.K. and S.N.W. received funding from MRC grants M/PR026494/1 and MR/R015325/1, and from SPARKS grant 17UCL01. A.A.R. and S.N.W. received funding from UK MRC grant MR/N026101/1. T.R.M., A.A.R. and S.N.W. received part funding from UK NC3Rs grant NC/L00107/81. S.N.W., D.P.P., T.R.M. and S.M.K.B. received funding from ERC grant S<omabio= 260862. D.P.P. received funding from UK MRC grant MR/N019075/1. NS received a Clinical Research Training Fellowship from Wellbeing of Women. J.A.D. is supported by CONICYTsBCehcaile Doctoral Fellowship program 72160294. J.N., J.B. and M.T. received funding from MRC. J.Rc.Cei.vreed funding from NIHR GOSH BRC grant 17BX23. Vis research was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. Ve views expressed are those of the authors and not necessyatrhilose of the NHS,

Ve authors declare no competing interests.

Supplementary information is available for this paper ahtttps://doi.org/10.1038/s41598-020-59075-.3