Ci a ion: Lo ca, C.;
Fe nández-Rhodes, M.; Sánchez
Milán, J.A.; Mule , M.; Elo za, F.;
Ramos-Miguel, A.; Callado, L.F.;
Meana, J.J.; Mu , M.; Ba alla, I.; e al.
Nex -Gene a ion P o eomics o B ain
Ex acellula Vesicles in Schizoph enia
P o ide New Clues on he Al e ed
Molecula Connec ome. Biomedicines
2024,12, 129. h ps://doi.o g/
10.3390/biomedicines12010129
Academic Edi o s: Meenakshi
Ahluwalia, Kuma Vaibha ,
Pankaj Gau and Pankaj Ahluwalia
Recei ed: 20 Decembe 2023
Re ised: 30 Decembe 2023
Accep ed: 5 Janua y 2024
Published: 8 Janua y 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
biomedicines
A icle
Nex -Gene a ion P o eomics o B ain Ex acellula Vesicles
in Schizoph enia P o ide New Clues on he Al e ed
Molecula Connec ome
C is ina Lo ca 1,2,† , Ma ía Fe nández-Rhodes 1,2,†, Jose An onio Sánchez Milán1,2, Ma ía Mule 1,2,
Félix Elo za 3, Al edo Ramos-Miguel 4,5,6 , Luis F. Callado 4,5,6 , J. Ja ie Meana 4,5,6 , Ma ia Mu 7,
Iolanda Ba alla 7, Elisabe Vilella 6,8,* , Aida Se a 2,*,‡ and Xa ie Galla -Palau 1,9,*,‡
1Biomedical Resea ch Ins i u e o Lleida D . Pi a éFounda ion (IRBLLEIDA), Neu oscience A ea,
+Pec P o eomics Resea ch G oup (+PPRG), Uni e si y Hospi al A nau de Vilano a (HUAV), 80 A . Ro i a
Rou e, 25198 Lleida, Spain; c is inalo [email p o ec ed] (C.L.); [email p o ec ed] (M.F.-R.);
[email p o ec ed] (J.A.S.M.); [email p o ec ed] (M.M.)
2Depa men o Medical Basic Sciences, Biomedical Resea ch Ins i u e o Lleida D . Pi a é
Founda ion (IRBLLEIDA), +Pec P o eomics Resea ch G oup (+PPRG), Uni e si y o Lleida (UdL),
25198 Lleida, Spain
3P o eomics Pla o m, CIC bioGUNE, Basque Resea ch and Technology Alliance (BRTA), CIBERehd, Science
and Technology Pa k o Bizkaia, 48160 De io, Spain; [email p o ec ed]
4Depa men o Pha macology, Uni e si y o he Basque Coun y UPV/EHU, 48940 Leioa, Spain;
[email p o ec ed] (A.R.-M.); [email p o ec ed] (L.F.C.); ja ie [email p o ec ed] (J.J.M.)
5Bioc uces Bizkaia Heal h Resea ch Ins i u e, 48903 Ba akaldo, Spain
6Cen o de In es igación Biomédica en Red en Salud Men al CIBERSAM, Ins i u o de Salud Ca los III,
43206 Reus, Spain
7Psychia y Depa men , Hospi al Uni e si a i San a Ma ia, Medicine Depa men , Uni e si a de
Lleida (UdL), 25198 Lleida, Spain; [email p o ec ed] (M.M.); [email p o ec ed] (I.B.)
8Hospi al Uni e si a i Ins i u Pe e Ma a, Ins i u In es igacióSani à ia Pe e Vi gili (IISPV)-CERCA,
Uni e si a Ro i a i Vi gili, 43206 Reus, Spain
9Depa men o Psychology, Uni e si y o Lleida (UdL), 25001 Lleida, Spain
*Co espondence: [email p o ec ed] (E.V.); [email p o ec ed] (A.S.); [email p o ec ed] (X.G.-P.)
†These au ho s con ibu ed equally o his wo k.
‡These au ho s con ibu ed equally o his wo k.
Abs ac : Ex acellula esicles (EVs) a e iny memb anous s uc u es ha media e in e cellula com-
munica ion. The ole(s) o hese esicles ha e been widely in es iga ed in he con ex o neu ological
diseases; howe e , hei po en ial implica ions in he neu opa hology subjacen o human psychia ic
diso de s emain mos ly unknown. He e, by using nex -gene a ion disco e y-d i en p o eomics, we
in es iga e he po en ial ole(s) o b ain EVs (bEVs) in schizoph enia (SZ) by analyzing hese esicles
om he h ee pos -mo em ana omical b ain egions: he p e on al co ex (PFC), hippocampus
(HC), and cauda e (CAU). The esul s ob ained indica e ha bEVs om SZ-a ec ed b ains con ain
egion-speci ic p o eins ha a e associa ed wi h abno mal GABAe gic and glu ama e gic ansmis-
sion. Simila ly, hese esicles om he analyzed egions we e implica ed in synap ic decay, abno mal
b ain immuni y, neu on s uc u al imbalances, and impai ed cell homeos asis. Ou indings also
p o ide e idence, o he i s ime, ha ne wo ks o molecula exchange (in ol ing he PFC, HC,
and CAU) a e po en ially ac i e and media ed by EVs in non-diseased b ains. Addi ionally, hese
bEV-media ed ne wo ks seem o ha e become pa ially e e sed and la gely dis up ed in he b ains
o subjec s a ec ed by SZ. Taken as a whole, hese esul s open he doo o he unco e ing o new
biological ma ke s and he apeu ic a ge s, based on he composi ions o bEVs, o he bene i o
pa ien s a ec ed by SZ and ela ed psycho ic diso de s.
Keywo ds: ex acellula esicles; molecula exchange; neu oin lamma ion; sys ems biology;
immunoglobulins; b ain an ibodies; psychia y; psycho ic spec um
Biomedicines 2024,12, 129. h ps://doi.o g/10.3390/biomedicines12010129 h ps://www.mdpi.com/jou nal/biomedicines
Biomedicines 2024,12, 129 2 o 20
1. In oduc ion
Schizoph enia (SZ) is a se e e men al diso de ha in ol es acu e psycho icism and
is es ima ed o a ec 21 million people wo ldwide [
1
]. Indi iduals diagnosed wi h SZ
commonly show impai men o he supe io abili ies o hinking, speech, emo ional eg-
ula ion, and social cogni ion [
2
]. The p e alence o he diso de shows a global ising
endency and in ol es a high disabili y a e o he a ec ed indi iduals [
1
]. Thus, he
pe sonal and social bu den o SZ is de as a ing in mul iple ace s, and nea ly one-hal
o he subjec s p esen e ac o y o ms o he disease [
3
], which ha dly espond o any
a ailable pha macological ea men s.
Mul iple epidemiological culp i s seem o be implica ed in he o igin o he diso de ,
including gene ic, biologic, and en i onmen al ac o s [
3
]. Ea ly childhood auma, he
accumula ion o auma ic e en s, and d ug abuse a e conside ed highly ele an ac o s in
he e osion o he human capaci y o esilience and in igge ing i s -episode psychosis
(FEP), a s age o illness o en p eceding SZ [
4
]. Indeed, se e al la ge-scale gene ic s udies
ha e al eady been pe o med o deciphe he highly he i able a e o he diso de [
5
,
6
].
Mul iple gene ic a ian s, including common a ian s and a e mu a ions, ha e been
associa ed wi h SZ. Common gene ic a ian s, including hose wi h weak o no s a is ically
signi ican associa ion, can explain mo e han 20% o he liabili y o he diso de [
5
]. Ra e
mu a ions in genes such as SETD1A, CUL1, XPO7, GRIA3, GRIN2A, and RB1CC1 ha e
also been iden i ied o subs an ially inc ease he isk o SZ, as p e iously e iewed [
7
], and
la ge copy numbe a ian s (CNVs) wi h la ge e ec sizes ha e also been implica ed in he
diso de [
8
]. Las ly, new indings abou gene ic loci linked o SZ suscep ibili y ha e shed
ligh on he pa hophysiology o he condi ion [9].
Simila ly, neu ochemical a emp s o cha ac e ize he molecula basis o he diso de
ha e also been conduc ed, and he al e a ion o he dopamine gic b ain ne wo ks ha e been
encoun e ed [
10
,
11
]. SZ seems o also be associa ed wi h s a es o inc eased in lamma ion
and oxida i e s ess, as well as wi h al e a ions o he inna e immuni y [
12
]. A a wide
scope, pa ien s wi h SZ p esen neu oana omical changes, including a p og essi e loss o
b ain g ay ma e , which appea s o be consis en wi h inc eased abe an p uning and a
consequen dec eased numbe o co ical glu ama e gic synapses [
13
]. Simila ly, pa ien s
wi h SZ display whi e ma e mic os uc u al abno mali ies ac oss mul iple b ain egions,
wi h a signi ican educ ion in ac ional aniso opy and widesp ead connec i i y de ici s,
indica ing ha he diso de may esul om an impai ed b ain connec ome [
14
]. Al hough
connec ome al e a ions in ol ing key neu oana omical s uc u es, such as he p e on al
co ex (PFC), hippocampus (HC), and cauda e (CAU), ha e been la gely iden i ied in he
b ains o subjec s wi h SZ [
15
], by using
in i o
imaging app oaches, any easible molecula
subs a e o hese connec ome al e a ions emains elusi e.
Ex acellula esicles (EVs) a e iny esicles ha ac as in e cellula ca ie s and
ac i ely pa icipa e in molecula exchange and communica ion wi hin he cen al ne -
ous sys em [
16
]. We, and o he colleagues, ha e la gely s udied hese esicles using
sys ems biology and ha e desc ibed hei implica ions in mul iple neu ophysiological unc-
ions associa ed wi h aging-degene a i e demen ias and i s he apeu ic po en ial [
17
–
21
].
P o eome-wide analyses ha e shown ha EV biogenesis is al e ed du ing p eclinical AD,
wi h he p esence o speci ic p o eins such as MHC class- ype ma ke s, p ion p o ein (P P),
and amyloid p o ein (APP) [
17
]. Changes in he le els o AD- ela ed p o eins in ol ed in
esicle endocy osis and he sec e o y pa hways ha e been obse ed in bo h p eclinical and
symp oma ic AD cases [
17
,
18
]. Fu he mo e, EVs om AD pa ien s ha e been ound o
con ain al e ed le els o cy okines, pS396 au, A
β
1-42, and mic oRNA, which may e lec
disease se e i y [
22
,
23
]. EVs ha e also been p oposed as excellen pla o ms o nano ec o s
in neu ologic diseases gi en hei capaci y o c oss he blood–b ain ba ie [
19
,
24
,
25
]. I
ollows, hence, ha hese esicles could play c ucial unc ions in he al e ed b ain connec-
ome ha is ex ensi ely ound in SZ, while hei ole(s) in ha diso de a e s ill mos ly
unknown [26].
Biomedicines 2024,12, 129 3 o 20
In o de o shed ligh on he po en ial unc ions o EVs in he b ain connec ome o SZ,
we pe o m he e an in-dep h cha ac e iza ion o he p o eome composi ions o pos -mo em
b ain EVs (bEVs) om h ee di e en neu oana omical egions ha a e b oadly implica ed
in illness- ela ed al e a ions in b ain connec i i y. The ob ained da a e lec he po en ial
oles o hese esicles wi hin he complexi y o he psychia ic diso de and poin owa ds
he pa icipa ion o bEVs in ac i e b ain ne wo ks o molecula exchange ha become
speci ically al e ed in SZ.
2. Ma e ials and Me hods
2.1. Chemicals and Reagen s
All chemicals and sol en s we e pu chased om Sigma-Ald ich (S . Louis, MO, USA)
unless o he wise speci ied. Wa e and ace oni ile (ACN) o liquid ch oma og aphy (HPLC)
g ade we e pu chased om The mo Fishe Scien i ic (The mo Fishe Chemical, Wal ham,
MA, USA). Sequencing-g ade modi ied ypsin was pu chased om P omega (Madison,
WI, USA).
2.2. Human B ain Samples
All pos -mo em b ain specimens we e collec ed du ing au opsies pe o med be ween
2010 and 2018 a he Basque Ins i u e o Legal Medicine (Bilbao, Spain). Only samples wi h
pos -mo em delay < 24 h we e included. G ey ma e specimens om he do sola e al
PFC (DLPFC, app oxima ing B odmann a ea (BA) 9), hippocampus (HC), and cauda e
nucleus we e ca e ully dissec ed, a oiding he whi e ma e , and immedia ely s o ed a
−80 ◦C un il assay. Re ospec i e sea ch in o subjec s’ medical eco ds was conduc ed o
an emo em diagnoses o SZ mee ing DSM-IV o ICD-10 c i e ia. Clinical diagnoses o
SZ we e all pe o med by a boa d-ce i ied psychia is o he Basque Heal hca e Sys em
(Osakide za). Cases wi h addi ional psychia ic o neu ologic diagnoses, including a his o y
o subs ance abuse, we e excluded. Samples om 15 SZ cases mee ing he abo e c i e ia
we e assayed in he p esen s udy. Each SZ case was pai ed o a ma ched (con ol, C) subjec ,
ma ching case’s sex, age, and pos -mo em delay, and wi h no e idence o psychia ic o
neu ological condi ions, acco ding o a ailable an emo em medical eco ds. Demog aphic
cha ac e is ics o all C and SZ subjec s a e summa ized in Table 1.
Table 1. De ails o he human pos -mo em b ains analyzed in his s udy.
a
PMD e e s o pos -mo em
delay exp essed in hou s.
Subjec Gende Age a Dea h (y.o.) PMD a(h) Clinical G oup
1 M 58 6 SZ
2 M 57 3 C
3 M 51 18 SZ
4 M 50 2 C
5 M 58 24 SZ
6 M 58 20 C
7 M 58 16 SZ
8 M 56 15 C
9 M 60 17 SZ
10 M 60 14 C
11 F 46 12 SZ
12 F 48 21 C
13 M 43 6 SZ
14 M 44 21 C
15 F 50 14 SZ
16 F 49 18 C
17 M 63 20 SZ
18 M 63 20 C
19 M 48 13 SZ
20 M 47 20 C
Biomedicines 2024,12, 129 4 o 20
Table 1. Con .
Subjec Gende Age a Dea h (y.o.) PMD a(h) Clinical G oup
21 M 60 7 SZ
22 M 60 19 C
23 M 52 24 SZ
24 M 52 23 C
25 M 52 11 SZ
26 M 51 16 C
27 F 52 10 SZ
28 F 51 10 C
29 M 41 17 SZ
30 M 41 15 C
2.3. P epa a ion o B ain Tissues P io o EVs Ob en ion
B ain issues om each espec i e and p e iously de ailed neu oana omical egion
and subjec we e dissec ed, and any emaining meninges and la ge blood essels we e
ca e ully inspec ed and emo ed. Dissec ed issues we e subsequen ly washed h ee imes
wi h 1
×
PBS o 30 min. B ain issues om each neu oana omical egion and subjec we e
andomly pooled o o m h ee independen biological g oup eplica es (n= 5; ~150 mg) pe
condi ion and neu oana omical egion, as we p e iously indica ed [
18
,
27
]. Homogeniza ion
o dissec ed b ain issues was pe o med as p e iously desc ibed by ou g oup [
16
] by
using a Bulle Blende issue homogenize (Nex Ad ance, Rayme own, NY, USA). B ie ly,
each sample was suspended in sa e-lock ubes in a de e gen - ee homogeniza ion bu e
consis ing o 100 mM ammonium ace a e (AA) a pH 6.5 (500
µ
L), supplemen ed wi h
p o ease inhibi o , and mixed wi h homogeniza ion beads (150 mg, 0.9–2.00 mm magne ic
pa icles) ha we e p e iously washed h ee imes wi h 1
×
PBS o 30 min. Homoge-
niza ion was hen conduc ed in ou cycles o 5 min each. A he end o each cycle, he
homogena e was cen i uged a 15,000
×
g o 10 min, and he supe na an s we e collec ed.
A e wa ds, 300
µ
L o homogeniza ion bu e was added, and he p ocess was epea ed.
The in ensi y o he i s wo cycles was medium, and maximum in ensi y was used in he
las wo cycles. All o he p ocedu es we e pe o med a 4 ◦C.
2.4. En ichmen o B ain EVs by PROSPR
bEVs we e en iched om he de e gen - ee b ain homogena es by using PROSPR
as we p e iously de ailed [
16
]. B ie ly, EV-con aining homogena es we e mixed wi h a
ou old olume o chilled ace one (
−
20
◦
C), o exed and cen i uged a 5000
×
g o
<1 min. Supe na an s con aining he hyd ophobic EV ac ion we e hen concen a ed
o nea -d yness using a acuum concen a o (Eppendo AG, Hambu g, Ge many) and
s o ed a −80 ◦C un il u he use.
2.5. Cha ac e iza ion o B ain EVs using Nanopa icle T acking Analysis
bEV ac ions ob ained om subjec s wi h SZ and C subjec s we e subjec ed o in-
dep h cha ac e iza ion using a nanopa icle acking analysis (NTA) as we p e iously
desc ibed [
18
]. B ie ly, bEV p epa a ions we e isualized and analyzed using a Nanosigh
NS300 wi h a sCMOS came a (Mal e n Panaly ical, Mal e n, UK). Analysis pa ame e s
we e se as ollows: 60 s acquisi ion ime, came a le el 4, slide shu e o 50, slide gain
o 100, FPS 32.5, sy inge pump speed o 100, o al olume pe sample o 1 mL, iscosi y
o 0.906–0.910 cP, and empe a u e o ~24
◦
C. NTA was pe o med wi hou es ablishing
any es ic ed a eas o he image ields by allowing o imaging and analysis o he ull
sample con en .
2.6. Ul as uc u al Cha ac e iza ion o B ain EVs
Rep esen a i e bEVs ac ions om SZ and C subjec s we e moun ed on Cu-Fo m a -
ca bon g ids and kep o 20 min a oom empe a u e (RT). G ids we e hen washed wi h
Biomedicines 2024,12, 129 5 o 20
HPLC wa e , and bEV p epa a ions we e ixed by using 1% glu a aldehyde in PBS o
5 min. The bEVs we e subsequen ly s ained wi h u anyl oxala e o 5 min, embedded
in me hyl-cellulose-u anyl-oxala e, and subsequen ly d ied o pe manen p ese a ion.
Elec on mic og aphs we e hen imaged by using a Jeol Jem 1010 elec on mic oscope a
80 kV
. The ob ained ul as uc u al mic og aphs we e hen scale-calib a ed, bi-le eled, and
u he analyzed by using he open so wa e ImageJ (Na ional Ins i u es o Heal h (NIH),
Be hesda, MD, USA).
2.7. P ocessing o B ain EVs o Nex -Gene a ion Label-F ee P o eomics
bEV samples we e suspended in a lysis bu e composed o 16 M u ea in 100 mM
ammonium bica bona e (ABB), subsequen ly incuba ed o 20 min a RT, and inally dilu ed
1:1 wi h HPLC wa e , as we p e iously indica ed [
17
]. The bEV p o eomes we e hen
subjec ed o yp ic diges ion, as p e iously desc ibed [
19
]. B ie ly, bEV p o eins we e
educed using 10 mM di hio h ei ol a 30
◦
C o 3 h, ollowed by alkyla ion using 20 mM
iodoace amide (IAA) o 1 h a oom empe a u e in da kness. Nex , u ea was dilu ed o
<1 M wi h 25 mM ABB, and he p o eins we e diges ed wi h ypsin o e nigh a 30
◦
C,
wi h a 1:20 p o ein- o-enzyme a io (w/w). Acidi ica ion wi h 0.5% inal concen a ion o
o mic acid (FA) was used o quench he eac ion. Wa e s Sep-Pak 50 mg C18 ca idges
(Wa e s, Mil o d, MA, USA) we e hen used o o desal he pep ides, and pep ide eco e y
was achie ed by using a 70% ACN concen a ed bu e . Finally, elu ed pep ides we e
concen a ed by using a acuum concen a o .
2.8. Fou -Dimensional P o eomics o B ain EVs
Desal ed bEVs and diges ed p o eome samples we e esuspended in 0.1% FA p io o
LC-MS/MS analysis by using an EVOSEP liquid ch oma og aphic ins umen (EVOSEP,
Odense, Denma k) a 300 nL
·
min
−1
. Samples we e un by pe o ming an 88 min g adien
(15 samples pe day). The EVOSEP liquid ch oma og aphe was coupled online o he
s a e-o - he-a imsTOF P o mass spec ome e (B uke Dal onics, Bille ica, MA, USA), and
he samples we e analyzed by using ou -dimensional (4D) pa allel accumula ion–se ial
agmen a ion (PASEF) da a acquisi ion, as p e iously de ailed [19,28].
2.9. Bioin o ma ics and Da a Analysis
Bioin o ma ic analysis o he ob ained bEV 4D p o eomics aw da a was ca ied ou ,
as p e iously indica ed [
29
], by using he specialized p o eomics sui e so wa e PEAKS
S udio X (Bioin o ma ics Solu ions INC., Wa e loo, Canada). P ecu so ion ole ance
was se o 10 ppm, and agmen ion ole ance was se o 0.05 Da. T ypsin was se as
p o eoly ic enzyme o da abase sea ching, and ca bamidome hyla ion o Cys esidues was
se as ixed modi ica ion. The human Unip o da abase (downloaded on 3 Feb ua y 2023,
con aining 140,065 p o ein sequences) was used o he iden i ica ion o p o eins. Decoy
usion,
FDR < 1%
, was es ablished o p o ein iden i ica ion in all samples, and ypsin
wi h clea age on a leas one end was se as a p o eoly ic enzyme. The bEV p o eome da a
we e expo ed o Mic oso Excel CSV iles, and in-house-gene a ed mac os we e c ea ed
and used o u he analysis.
Label- ee ela i e quan i ica ion o bEV p o eins be ween condi ions and b ain egions
was pe o med based on spec al coun , as p e iously epo ed [
30
–
33
], and he equali y
o a iances was assessed using Le ene’s es . The e e ed da a we e analyzed using
pa ame ic wo-way ANOVA and by pe o ming mul iple compa isons and Fishe ’s Leas
Signi ican Di e ence es s, wi h s a is ical signi icance se a p< 0.05 (95% con idence
in e al). A e wa ds, addi ional Bon e oni co ec ion o mul iple compa isons was ap-
plied
(p< 0.05)
. P o eins p esen in all h ee egions analyzed we e subjec ed o co ela ion
analysis, and only Pea son’s co ela ion coe icien s ≥ ±0.8 we e conside ed as indica o s
o s ong in e ac ion be ween he analyzed a iables.
Addi ionally, Bioconduc o “o g.Hs.eg.db” package ( e sion 3.16.0) and “clus e P o-
ile ” package ( e sion 4.6.2) we e ins alled in R so wa e ( e sion 4.2.3) o Gene On ology
Biomedicines 2024,12, 129 6 o 20
unc ional analysis and KEGG (Kyo o Encyclopedia o Genes and Genomes) pa hway
en ichmen analysis.
2.10. Da a A ailabili y
All p o eomics da a gene a ed o his s udy ha e been made publicly a ailable
h ough he specialized eposi o y, PRIDE, wi h he ollowing iden i ie : PXD042732. The
ee a ailabili y and e-usage o human b ain gene a ed sys ems biology da a, such as hese
in his s udy, ha e been encou aged by he scien i ic communi y o openly con ibu e o he
p og ess o he neu oscien i ic unde s anding o he human b ain and i s diseases [34].
3. Resul s
3.1. Mo phome ic Cha ac e is ics o bEVs in Schizoph enia
To ini ially de ine he speci ic p ope ies o bEVs, we pe o med an in-dep h mo -
phome ic cha ac e iza ion o hese esicles in he b ains o he con ols (C) and o he
subjec s wi h SZ. The bEVs showed a mean concen a ion o 1.14
×
10
12
pa icles/mL and
an a e age diame e size o 255.73 nm (Figu e 1A). Rega ding he diame e size, he e
we e no disce nible a ia ions be ween he g oups unde analysis; ne e heless, he mean
concen a ion o bEVs showed a signi ican di e ence, wi h he C g oup’s bEVs exhibi ing
a g ea e concen a ion o pa icles (Figu e 1A). Subsequen ly, we pe o med an ul a-
s uc u al analysis o bEVs using ansmission elec on mic oscopy (TEM) o de ine he
p edominan mo phology and pu i y o he analyzed bEV p epa a ions. As shown in he
ep esen a i e mic og aphs o Figu e 1B, he EVs ob ained showed p edominan sphe ical
mo phology wi hou any appa en di e ences obse ed be ween he C and subjec s wi h
SZ. O no e, he ul as uc u al s udy also con i med he absence o any app eciable pa icle
con amina ion in he ob ained bEV p epa a ions (Figu e 1B).
Biomedicines 2024, 12, x FOR PEER REVIEW 7 o 21
Figu e 1. Mo phological and ul as uc u al cha ac e iza ion o b ain ex acellula esicles (bEVs)
ob ained om pos -mo em b ain issues. (A) A e age size dis ibu ion p o iles o bEVs ob ained
using nanopa icle acking analysis (NTA) o bEVs om he p e on al co ex (PFC) egion o con-
ols (C) and subjec s wi h schizoph enia (SZ). Cap u es e e o he a e age dis ibu ion ob ained
om en independen size/concen a ion dis ibu ion uns. S anda d de ia ion o he mean is
shaded in blue. (B) Rep esen a i e mic og aphs o bEVs, ob ained by ansmission elec on mic os-
copy (TEM) om he PFC, hippocampus (HC), and cauda e (CAU) egions o C subjec s (uppe
mic og aphs) and o subjec s wi h SZ (lowe mic og aphs). Scale ba in Figu e 1B ep esen s 100
nm. ** indica es signi ican s a is ical diffe ences (p < 0.0001).
3.2. Molecula Composi ions o bEVs in Schizoph enia
We hen pe o med a ou -dimensional unbiased disco e y-d i en cha ac e iza ion
o he ob ained bEV p o eomes o de ine he molecula composi ion(s) o hese esicles in
he h ee analyzed b ain egions (PFC, HC, and CAU) om he C g oup and subjec s wi h
Figu e 1. Con .
Biomedicines 2024,12, 129 7 o 20
Biomedicines 2024, 12, x FOR PEER REVIEW 7 o 21
Figu e 1. Mo phological and ul as uc u al cha ac e iza ion o b ain ex acellula esicles (bEVs)
ob ained om pos -mo em b ain issues. (A) A e age size dis ibu ion p o iles o bEVs ob ained
using nanopa icle acking analysis (NTA) o bEVs om he p e on al co ex (PFC) egion o con-
ols (C) and subjec s wi h schizoph enia (SZ). Cap u es e e o he a e age dis ibu ion ob ained
om en independen size/concen a ion dis ibu ion uns. S anda d de ia ion o he mean is
shaded in blue. (B) Rep esen a i e mic og aphs o bEVs, ob ained by ansmission elec on mic os-
copy (TEM) om he PFC, hippocampus (HC), and cauda e (CAU) egions o C subjec s (uppe
mic og aphs) and o subjec s wi h SZ (lowe mic og aphs). Scale ba in Figu e 1B ep esen s 100
nm. ** indica es signi ican s a is ical diffe ences (p < 0.0001).
3.2. Molecula Composi ions o bEVs in Schizoph enia
We hen pe o med a ou -dimensional unbiased disco e y-d i en cha ac e iza ion
o he ob ained bEV p o eomes o de ine he molecula composi ion(s) o hese esicles in
he h ee analyzed b ain egions (PFC, HC, and CAU) om he C g oup and subjec s wi h
Figu e 1. Mo phological and ul as uc u al cha ac e iza ion o b ain ex acellula esicles (bEVs)
ob ained om pos -mo em b ain issues. (A) A e age size dis ibu ion p o iles o bEVs ob ained
using nanopa icle acking analysis (NTA) o bEVs om he p e on al co ex (PFC) egion o con ols
(C) and subjec s wi h schizoph enia (SZ). Cap u es e e o he a e age dis ibu ion ob ained om
en independen size/concen a ion dis ibu ion uns. S anda d de ia ion o he mean is shaded in
blue. (B) Rep esen a i e mic og aphs o bEVs, ob ained by ansmission elec on mic oscopy (TEM)
om he PFC, hippocampus (HC), and cauda e (CAU) egions o C subjec s (uppe mic og aphs)
and o subjec s wi h SZ (lowe mic og aphs). Scale ba in Figu e 1B ep esen s 100 nm. ** indica es
signi ican s a is ical di e ences (p< 0.0001).
3.2. Molecula Composi ions o bEVs in Schizoph enia
We hen pe o med a ou -dimensional unbiased disco e y-d i en cha ac e iza ion
o he ob ained bEV p o eomes o de ine he molecula composi ion(s) o hese esicles
in he h ee analyzed b ain egions (PFC, HC, and CAU) om he C g oup and subjec s
wi h SZ (Supplemen a y Da ase s S1 and S2). The ob ained bEV p o eomes da a we e
subsequen ly ma ched o he da a cu a ed in he specialized EV da abase eposi o ies,
Exoca a and Vesiclepedia, as we p e iously indica ed [
16
,
17
], o de ine he quali y o he
bEV p epa a ions and iden i y any po en ial di e ences ega ding he po ion o exosomes
and mic o esicles p esen . Consis en iden i ica ion be ween 70 and 90 pe cen o he
op 100 exosomal and mic o esicle ma ke s, espec i ely cu a ed in hese da abases, was
achie ed h oughou he analyzed g oups and b ain egions (Figu e 2A,B). Addi ionally,
no signi ican di e ences we e obse ed ega ding he p esence o EV ma ke s be ween
he analyzed g oups and b ain egions (Figu e 2A,B).
Biomedicines 2024,12, 129 8 o 20
Biomedicines 2024, 12, x FOR PEER REVIEW 9 o 21
Figu e 2. Molecula cha ac e iza ion pe o med by ou -dimensional liquid ch oma og aphy p o e-
omics o b ain ex acellula esicles (bEVs) o he h ee analyzed b ain egions, p e on al co ex
(PFC), hippocampus (HC), and cauda e (CAU). (A,B) P esence o mic o esicle ma ke s in he p o-
eomes o bEVs ob ained om subjec s wi h schizoph enia (SZ) and con ol (C) subjec s. Pa allel
analysis o he ob ained p o eome da a was pe o med in (A) wi h he op 100 p o ein ma ke s cu-
a ed in he specialized mic o esicle da a eposi o y, Vesiclepedia, and in (B), i was pe o med wi h
he specialized exosomal da a eposi o y, Exoca a. (C) Diag am able indica ing he numbe o com-
mon and unique p o eins p esen in he bEV p o eomes o he analyzed b ain egions, PFC, HC, and
CAU, om C subjec s and subjec s wi h SZ. Salmon ones ep esen common p o eins o he PFC
egion. Pu ple ones ep esen common p o eins o he HC egion, and blue ones ep esen com-
mon p o eins o he CAU egion. ALL indica es he numbe o p o eins commonly p esen in he
h ee e alua ed egions, and ANY indica es he o al numbe o p o eins conside ing all iden i ied
p o eins be ween he h ee e alua ed egions. (D) Mean a e age cumula i e p o eome le els in bEVs
Figu e 2. Molecula cha ac e iza ion pe o med by ou -dimensional liquid ch oma og aphy p o-
eomics o b ain ex acellula esicles (bEVs) o he h ee analyzed b ain egions, p e on al co ex
(PFC), hippocampus (HC), and cauda e (CAU). (A,B) P esence o mic o esicle ma ke s in he p o-
eomes o bEVs ob ained om subjec s wi h schizoph enia (SZ) and con ol (C) subjec s. Pa allel
analysis o he ob ained p o eome da a was pe o med in (A) wi h he op 100 p o ein ma ke s
cu a ed in he specialized mic o esicle da a eposi o y, Vesiclepedia, and in (B), i was pe o med
wi h he specialized exosomal da a eposi o y, Exoca a. (C) Diag am able indica ing he numbe o
common and unique p o eins p esen in he bEV p o eomes o he analyzed b ain egions, PFC, HC,
and CAU, om C subjec s and subjec s wi h SZ. Salmon ones ep esen common p o eins o he PFC
egion. Pu ple ones ep esen common p o eins o he HC egion, and blue ones ep esen common
p o eins o he CAU egion. ALL indica es he numbe o p o eins commonly p esen in he h ee
e alua ed egions, and ANY indica es he o al numbe o p o eins conside ing all iden i ied p o eins
be ween he h ee e alua ed egions. (D) Mean a e age cumula i e p o eome le els in bEVs o he
h ee analyzed b ain egions, PFC, HC, and CAU, om C subjec s and subjec s wi h SZ. *** indica es
s a is ical signi icance a p< 0.001. E o ba s ep esen s anda d de ia ion o he mean.
Biomedicines 2024,12, 129 9 o 20
The p esence o common and unique speci ic p o eins ac oss g oups and b ain egions
was also analyzed in he p o iled bEV p epa a ions (Figu e 2C). No di e ences ega ding
he numbe o p o eins p esen in bEV p o eomes h oughou he analyzed g oups we e
de ec ed, wi h a o al o 1258 p o eins being iden i ied in he bEV p o eomes o he C
subjec s and 1252 p o eins being iden i ied in he bEV p o eomes o subjec s wi h SZ
(Figu e 2C; Supplemen a y Da ase s S1 and S2). Howe e , whe eas 672 p o eins we e
iden i ied as common o all b ain egions in he bEV p o eomes o he C subjec s, only
537 we e encoun e ed in he bEV p o eomes o he subjec s wi h SZ (Figu e 2C). O no e,
201 p o eins o hese ha a e common o all b ain egions we e unique o he bEVs o
he C subjec s, whils 66 p o eins we e unique o he bEVs om he subjec s wi h SZ
(Figu e 2C). Simila ly, 57 p o eins we e uniquely iden i ied in he PFC-bEVs, 206 p o eins
we e uniquely iden i ied in he HC-bEVs, and 86 p o eins we e uniquely iden i ied in he
CAU-bEVs o subjec s wi h SZ (Figu e 2C). Comple e lis s o hese common and unique
p o eins iden i ied in bEVs a e, espec i ely, included in Supplemen a y Da ase S3 and
Supplemen a y Tables S1–S5.
The o al p o eome bEVs le els we e also analyzed in he h ee b ain egions o he
C g oup and subjec s wi h SZ. A signi ican up egula ion a ec ing he o al p o eome o
bEVs in he PFC o subjec s wi h SZ was obse ed (Figu e 2D), whils he bEV p o eomes
om he es o b ain egions sc u inized did no show signi ican al e a ions.
3.3. Schizoph enia-Linked Al e a ion o bEV P o eomes
We hen analyzed whe he some o he bEV p o eins in hese subjec s al e hei
le els h oughou he h ee analyzed b ain egions. The p o eins ha we e iden i ied
wi h signi ican ly al e ed egula ion in hese analyses a e de ailed in Figu e 3A. These
p o eins, he ea e e e ed o as SZ-al e ed bEV p o eins, we e subsequen ly subjec ed
o unc ional ca ego iza ion conside ing hei ole(s) in speci ic molecula unc ions and
biological p ocesses, as shown in Figu e 3B,C. We ound ha la ge po ions o hese SZ-
al e ed bEV p o eins con ibu e o cell g ow h and homeos a ic main enance as biological
p ocesses and o he main enance o cell s uc u e as molecula unc ion (Figu e 3B). A
la ge subse o hese p o eins was also associa ed wi h he mal unc ion o cell me abolism,
b ain immuni y, and calcium homeos asis (Figu e 3C).
Speci ically, as shown in Figu e 3A, he signi ican down egula ion o s uc u al p o-
eins in bEVs om subjec s wi h SZ, including ac in (ACTA1), ubulins (TUBA1A, TUBA1B,
TUBA1C, TUBB4A, TUBB6, and TUBB8B), cell adhesion (NCAM1), and mic o ubule-linked
p o eins (MAP1B, MAP4, MAP6, and MAPT), was obse ed. O no e, abno mal down egu-
la ion in he bEVS o subjec s wi h SZ was also a ec ing essen ial s uc u al and esicula
p o eins associa ed wi h ac i e synap ic densi ies and spines, including bassoon (BSN),
synap opodin (SYNPO), synapsins (SYN1 and SYN2), and SNAP 25 (Figu e 3A). Con-
e sely, he up egula ion o immunoglobulins (IGHA1, IGHA2, IHKC, IGHG1, IGLC2,
IGLC3, IGHG4, and IGHG2) was clea ly iden i ied in he diso de ed bEV p o eomes o
subjec s wi h SZ (Figu e 3A). Wo hy o no e, hese iden i ied bEV p o ein al e a ions
om subjec s wi h SZ we e p imo dially obse ed in he PFC bEV p o eomes and we e
no mi o ed in he o he b ain egions ha we e analyzed (Figu e 3A). Finally, i is wo -
hy o men ion ha signi ican down egula ions o he myelin p o ein (MBP) and he
as oglial ma ke (GFAP) we e also obse ed in he bEV p o eomes o he subjec s wi h SZ
(Figu e 3A).
A unc ional ca ego iza ion o he signi ican ly al e ed bEV p o eomes o he subjec s
wi h SZ was also pe o med, as shown in Figu e 3D. The up egula ion o a subse o bEV
p o eins in ol ed in immuni y, an igen binding, and cellula anspo was p edominan in
he PFC egion o subjec s wi h SZ (Figu e 3D). Fu he mo e, he up egula ion o a subse
o bEV p o eins in ol ed in cell mo ili y, cell main enance, and cell s uc u e was also
p edominan in he HC egion o subjec s wi h SZ (Figu e 3D). Simila ly, he up egula ion
o a high po ion o bEV p o eins in ol ed in he main enance o cell s uc u e, cell g ow h
and main enance, cellula calcium homeos asis, cellula me abolism, and cellula anspo
Biomedicines 2024,12, 129 16 o 20
also con ibu e o explain he dicho omy ha has been ound in he b ains o subjec s wi h
SZ ega ding he hype exci abili y o he HC egion [
50
] and he hypoexci abili y o he PFC
egion [51], which is a ac ha we also conside o be ex emely wo h u he explo ing.
Ou da a also indica e an exace ba ed up egula ion o an ibody immunoglobulins in
he bEVs o he PFC egion in SZ b ains. These molecules a e ypically p oduced by B cells
and kep away om he cen al ne ous sys em (CNS) in no mal neu ologic condi ions [
52
].
Mo eo e , i s p esence wi hin he CNS has been associa ed wi h he appa i ion o psycho ic
symp oma ology seconda y o au oimmune diseases [
53
,
54
]. Al hough he p esence o
au o-an ibodies in he b ains o subjec s a ec ed by SZ has been p e iously epo ed, he
mechanisms in ol ed in speci ic blood–b ain ba ie pe meabili y o hese molecules
emains unknown [
55
]. The ac ha bEVs become a sou ce ha explains he a i al
mechanism and inc ease in immunoglobulins in he PFC o b ains a ec ed by SZ, based
on he indings he e, is highly compelling, and a e u he alida ion, i holds p omise o
con ibu e o iden i ying new he apeu ic a ge s o he diso de .
Al hough bEVs seem o undoub edly implica e in b ain in e egional molecula ex-
change, as ecen ly indica ed [
35
], clea e idence o ha ou come in humans is s ill lacking.
O no e, he indings epo ed in his s udy s ongly suppo his ele an ac , o he i s
ime, bo h in heal hy and diseased b ains. This s udy has shown ha he e is in e ac ion
a ec ing he le els o ce ain p o eins ha a e commonly iden i ied in bEVs in he h ee
analyzed b ain egions. This inding was in e p e ed as he unco e ing o a po en ial ac i e
ne wo k o molecula exchange media ed by bEVs in he analyzed b ains. Fu he mo e,
ou da a show ha bEVs in he CAU egion show ac i e exchange po en iali y wi h HC
and PFC in he cogni i ely no mal b ain, which is a signi ican disco e y ha needs mo e
e i ica ion because i may ha e signi ican e ec s on how he b ain communica es be-
ween i s cells and domains unde no mal ci cums ances. S ikingly, we also obse ed ha
he po en ial ac i e ne wo ks o molecula exchange be ween he h ee analyzed egions
become al e ed in he analyzed b ains om subjec s wi h SZ, hus indica ing ha hese
ac i e ne wo ks media ed by bEVs become dis up ed in he diso de . Speci ically, he
myelin p o ein, MBP; he collapsin, DPYSL2; and he Tau p o ein, MAPT, we e he ele an
p o eins clea ly iden i ied o be in ol ed in ha dis up ed b ain ne wo k media ed by EVs.
All o hese e e ed p o eins we e hus showing e e sed s ong co ela ions wi h he bEVs
o subjec s wi h SZ compa ed o hose o C subjec s.
5. Conclusions
Collec i ely, he indings epo ed in his s udy iden i y c ucial p o eins linked o he
bEVs in SZ and ein o ce he ini ial hypo hesis abou he implica ion o hese esicles in
he neu opa hology o he diso de . Addi ionally, ou esea ch iden i ies speci ic p o ein
ma ke s in b ain EVs wi h he abili y o ci cula e in biological luids. These ma ke s may
p o e o be use ul in s a i ying clinical pa ien s and co ela ing he symp oma ology o
hei diso de s wi h he p edominance o neu opa hology in speci ic b ain egions. The
ob ained esul s also con ibu e o he e idence sugges ing ha bEVs o ches a e molecula
exchange in he human b ain, in oducing he no el y ha al e a ions o his p ocess occu
in psychia ic diseases, pa icula ly SZ. As such, hese esicles migh also cons i u e he
b ain’s bluep in o he connec ome changes p e iously disco e ed by neu oimaging
s udies. Las ly, hey o e a esh and ex emely aluable sou ce o po en ial he apeu ic
a ge s o he illness, which, al oge he , me i s u he esea ch.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps:
//www.mdpi.com/a icle/10.3390/biomedicines12010129/s1, Supplemen a y Da ase S1: Lis o
iden i ied p o eins in bEVs o C b ains in each b ain egion. Supplemen a y Da ase S2: Lis o
iden i ied p o eins in bEVs o SZ samples in each b ain egion. Supplemen a y Da ase S3: Lis o
iden i ied p o eins in bEVs o con ol and SZ samples common o all b ain egions; Supplemen a y
Da ase S4: Da a o he connec ome analysis shown in Figu e 4. On he igh , a e age and s anda d
de ia ion (SD) o e e y signi ica i e p o ein o bEVs consis en ly iden i ied in he h ee analyzed
ana omical b ain egions. On he le , co ela ion ma ix. Table S1: P o eins exclusi ely iden i ied in
Biomedicines 2024,12, 129 17 o 20
b ain ex acellula esicles (bEVs) o subjec s a ec ed by schizoph enia (SZ) in all analyzed b ain
egions. Table S2: P o eins exclusi ely iden i ied in b ain ex acellula esicles (bEVs) o con ol (C)
subjec s in all analyzed b ain egions. Table S3: P o eins exclusi ely iden i ied in bEVs o he PFC
egion in SZ. Table S4: P o eins exclusi ely iden i ied in bEVs o he HC in SZ. Table S5: P o eins
exclusi ely iden i ied in bEVs o he CAU in SZ.
Au ho Con ibu ions: Concep ualiza ion: C.L., M.F.-R., A.R.-M., E.V., A.S. and X.G-P.; Me hodology:
C.L., M.F.-R., J.A.S.M., M.M. (Ma ía Mule ), F.E., M.M. (Ma ia Mu ) and I.B.; W i ing: C.L., M.F.-R.,
A.S. and X.G.-P.; B ain Samples: A.R.-M., L.F.C. and J.J.M.; Re iew and Edi ing: E.V., A.S. and X.G.-P.;
Funding Acquisi ion: A.S. and X.G.-P.; Supe ision: A.S. and X.G.-P. All au ho s ha e ead and
ag eed o he published e sion o he manusc ip .
Funding: Suppo o his wo k was p o ided by he Na ional Ins i u e o Heal h/Ins i u o de Salud
Ca los III-ISCIII, Spain (PI22/00443 o X.G.-P.) (g an co- unded by he Eu opean Union); he Minis y
o Science and Inno a ion-MCIN, Spain and he Na ional Resea ch Council/Agencia Es a al de
In es igación-AEI, Spain (PID2020-114885RB-C21 o A.S.) unded by MCIN/AEI/10.13039/501100011033.
This esea ch was also co- inanced by he Spanish Minis y o Science and Inno a ion wi h unds
om he Eu opean Union Nex Gene a ionEU; om he Reco e y, T ans o ma ion and Resilience
Plan (PRTR-C17.I1); and om he Au onomous Communi y o Ca alonia wi hin he amewo k o
he Bio echnology Plan Applied o Heal h ((EVBRAINTARGET-Y7340-ACPPCCOL007 o X.G.-P.,
A.S., M.Mu , and A.R.-M.) coo dina ed by he Ins i u e o Bioenginee ing o Ca alonia (IBEC));
he Dipu acióde Lleida, Spain (PIRS22/03 o X.G.-P. & I.B. and PIRS23/02 o A.S.); he Ca alan
Resea ch Council-AGAUR (AGAUR 21SGR010065 o E.V.; 2023 LLAV 00056 o X.G.-P.; and 2022
DI 100 o X.G.-P.); and he Basque Go e nmen (IT211/19 and IT1512/22 o J.J.M and L.F.C.). X.G.-
P. acknowledges a Miguel Se e p og am enu e ack con ac (CP21/00096) om he ISCIII,
awa ded on he 2021 call unde he Heal h S a egy Ac ion, co- unded by he Eu opean Union (FSE+).
A.S. acknowledges a Ramón y Cajal p og am enu e ack con ac (RYC2021-030946-I) unded
by MCIN/AEI/10.13039/501100011033 and by he “Eu opean Union Nex Gene a ionEU/PRTR”;
A.R.-M. acknowledges a Ramón y Cajal p og am enu e ack con ac (RYC-2016-19282) unded
by MCIN/AEI/10.13039/501100011033. M.F.-R.’s pos doc o al con ac is unded by PRTR-C17.I1
and EVBRAINTARGET-Y7340-ACPPCCOL007. C.L.’s PhD is unded by he Eu opean Social Fund
o he ec ui men o p edoc o al esea che s (PEJD-2019-PRE/BIO-16475); M.M.’s PhD is unded
by he MCIN-AEI (PR2021-097934); and J.A.S.M.’s PhD is unded by AGAUR (2023 FI-1 00054),
and J.A.S.M.’s con ibu ions we e also suppo ed by Dipu acióde Lleida ‘Aju s al Talen en In-
es igacióBiomèdica”. IRBLLEIDA, J.A.S.M., X.G.-P., and A.S. a e co- unded by he CERCA P o-
g am/Gene ali a de Ca alunya. J.J.M., A.R.-M., and X.G.-P. a e membe s o he ExoPsyCog Conso -
ium, unded by IKUR-Neu obiosciences—Basque Go e nmen .
Ins i u ional Re iew Boa d S a emen : All o he expe imen al p ocedu es we e app o ed by he
e hical commi ee o he Uni e si y Hospi al A nau de Vilano a—IRBLLEIDA, Spain (Re # XI2022)
and we e pe o med in s ic acco dance wi h ins i u ional guidelines, he Decla a ion o Helsinki,
and he Spanish O ganic Law 3/5 Decembe 2018 o P o ec ion o Pe sonal Da a (LOPD). Subjec
iden i ica ion codes we e kep anonymized in all cases.
In o med Consen S a emen : In o med consen o he dona ion o he included b ain issues was
ob ained om all subjec s and/o hei ep esen a i es a he Basque Ins i u e o Legal Medicine.
Da a A ailabili y S a emen : All p o eomics da a gene a ed o his s udy ha e been made publicly
a ailable h ough he specialized eposi o y, PRIDE, wi h he ollowing iden i ie : PXD042732.
Acknowledgmen s: The au ho s ex end hei app ecia ion o Aina Iuliana Onoiu o he aluable
assis ance in he da a analysis du ing he culmina ion o he unde g adua e inal yea p ojec wi hin
ou g oup a IRBLLEIDA/UdL. Addi ionally, ou g a i ude goes o he s a membe s o he Basque
Ins i u e o Legal Medicine o hei coope a i e spi i and gene ous suppo . We also hank ou pee s
o he en iching and aluable eedback p o ided a he ollowing in e na ional scien i ic con e ences:
ISMND2022 in A hens, G eece; ISFMS2022 in Flo ence, I aly; Ex acellula Vesicles: F iends and Foes
II 2023 a he Weizmann Ins i u e in Is ael; WCN 2023 in Mon eal, Canada; and ASCB-EMBO Cell
Bio 2023 in Bos on, MA, USA. Finally, we exp ess g a i ude o he al uis ic dono s and ela i es
whose gene osi y has made his s udy possible.
Con lic s o In e es : The au ho s decla e ha hey ha e no compe ing in e es s wi h ega d o he
da a and conclusions epo ed. The in e p e a ions p o ided in his wo k a e based on he ob ained
Biomedicines 2024,12, 129 18 o 20
scien i ic da a and a e hose o he au ho s and no necessa ily hose o he public bodies ha unded
he s udy.
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Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
