Brain-transportable dipeptides across the blood-brain barrier in mice
Brain-transportable dipeptides across the blood-brain barrier in mice
Mitsuru t anaka
Fuyuko t akata
t akashi Nirasawa
t oshiro Matsui tmatsui@agr
Apart from nutrients required for the brain, there has been no report that naturally occurring peptides can cross the blood-brain barrier (BBB). the aim of this study was to identify the BBB-transportable peptides using in situ mouse perfusion experiments. Based on the structural features of Gly-Nmethylated Gly (Gly-Sar), a reported BBB-transportable compound, 18 dipeptides were synthesized, and were perfused in the mouse brain for two minutes. Among the synthesized dipeptides, Gly-Sar, Gly-pro, and t yr-pro were transported across the BBB with Ki values of 7.60 ? 1.29, 3.49 ? 0.66, and 3.53 ? 0.74 ?L/g?min, respectively, and accumulated in the mouse brain parenchyma. Additionally, using MALDI-Ms/Ms imaging analysis of t yr-pro-perfused brain, we provide evidence for t yr-pro accumulation in the hippocampus, hypothalamus, striatum, cerebral cortex, and cerebellum of mouse brain.
The physiological preference of peptide uptake has been demonstrated in human and animal studies. In subjects
with mild hypertension the intake of a dipeptide, Val-Tyr, for a month modulated blood pressu1.rTehe dipeptide,
Trp-His showed an apparent anti-atherosclerotic effect in apo E-deficient mice, while no effect was observed on
administrating a mixture of Trp and His2. We also demonstrated that the aforementioned bioactive peptides are
absorbed in their intact form across intestinal membrane into blood3,4. Peptide transport across the epithelial
layer in animal organs is mainly regulated by diverse proton-coupled oligopeptide transporters of the SLC15
family, which have a selective preference for di-/tripeptide5s. Peptide transporter 1 (PepT1) in the intestine and
PepT2 in the kidney are known to be involved in the absorption and reabsorption of di-/tripeptide6s,7. Thus far, it
has been reported that transporters for peptide incorporation are expressed in multiple organs such as the liver,
blood vessels, muscle, brain, intestine, and kidney. However, no studies report the intact absorption of dipeptides
beyond the blood-brain barrier (BBB).
It is well known that the brain selectively absorbs useful food compounds such as amino acids, glucose, i-nor
ganic compounds, and vitamins, but tightly inhibits the uptake of any other substrates, since the BBB can limit
paracellular permeation by providing a meshwork of non-fenestrated microvessel endothelial cells surrounded
by pericytes, astrocytes and neurons8. Although it has been reported that cell penetrating peptides (CPP) mostly
composed of >10 amino acids possessing cationic, amphipathic or hydrophobic properties can enter the central
nervous system (CNS) by energy-independent passive penetration or energy-dependent endocytosis
penetration pathways9, there are no studies showing the intact transport of small hydrophilic peptides (i.e.,
di-/tripeptides) beyond the BBB system. In the BBB system, the transporters of PepT2 and peptide/histidine transporter
1 (PHT1), but not PepT1, were expressed at the blood-cerebrospinal fluid (CSF)-barrier composed of the
choroid plexus epithelium7. The brain PepT2 may play a role in pumping out metabolites produced from endog-e
nous neuronal dipeptides for their clearance from the CSF7,10. An in vivo pharmacokinetic study using Pht1-null
mice11 revealed that PHT1 is expressed throughout the brain including choroid plexus, and preferably
recognizes histidines. Although Hu et a.l12 also studied Gly-N-methylated Gly (Gly-Sar) uptake into the brain through
PHT1 in adult rodents, information on brain PHT1 substrates is limited. However, recent in vivofindings on the
improvement of delayed memory score in patients with mild cognitive impairmen1t3 and on the suppression of
cognitive decline induced by neurotrophic factors in SAMP8 mice by a diet containing di-/tripeptide1s4 strongly
led us to speculate the possible intact transport of di-/tripeptides beyond the BBB.
Thus, in the present in situ mouse brain perfusion study, we demonstrate that small peptides (in this study,
dipeptides) can be transported across the BBB and accumulate in the brain. We also performed a mass
spectrometry (MS)-visualization experiment with phytic acid-aided matrix assisted laser desorption ionization (MALDI)?
MS/MS imaging15 to provide a direct evidence of the accumulation of dipeptides in the brain.
Identification of dipeptides capable of intact transport across the mouse BBB. In order to
elucidate the entry of dipeptides into the brain, Gly-Sar, a model substrate for SLC15 family of peptide transporte1r6s,
was selected to validate the present in situmouse brain perfusion experiments. The transport of Gly-Sar across
mouse BBB was determined in brain homogenates by using 2,4,6-trinitrobenzensulfonate (TNBS) aided-liquid
chromatography-time-of-flight (LC-TOF)/MS technique described previousel1y7, wherein the target peptide was
derivatized with TNBS to form a trinitrophenyl (TNP)-peptide. Since Gly-Sar ([M+ H]+: 358.0630 m/z) has the
same molecular mass as endogenous Gln, a stable isotope labele1d3C[2,15N]Gly-Sar ([M+ H]+: 361.0630 m/z),
which was used for selective MS detection of perfused Gly-Sar. As shown in Fig1.?a, a time-dependent increase
in the uptake of [13C2,15N]Gly-Sar was clearly observed during the indicated perfusion time of 0 tom2 in. Brain/
perfusate ratio of [13C2,15N]Gly-Sar and the influx rate constant ( Kivalue: 7.60? 1.29 ? L/g?min) were significantly
(P < 0.01) higher than those of fluorescein isothiocyanate conjugated (FITC)-albumin, a compound that cannot
cross the BBB18 (Fig.?1b). This observation strongly suggested that a dipeptidic compound, Gly-Sar, may transport
across the BBB in the blood-to-brain direction. No increase in the brain/perfusate ratio of FITC-albumin during
the co-perfusion with Gly-Sar (Fig.?S1) clearly indicated that Gly-Sar transport did not cause any BBB disruption
After confirming the intact transport of dipeptidic Gly-Sar across the mouse BBB, further in situmouse brain
perfusion experiments for BBB transportable peptides were performed on the basis of the dipeptide skeleton.
Gly-Pro, Ala-Gln, His-Leu, Trp-His, Val-Tyr, Met-Tyr, Ile-Tyr, and Leu-Tyr were selected, since they have been
reported to be transported in an intact form through the intestinal membrane via carrier-mediated route3s,4,19.
Additionally, by considering that the hydrophobicity may assist the transcellular diffusive transp2o0,r2t1, Trp-Leu,
Leu-Trp, Trp-Met, Trp-Ala, and Trp-Tyr with log Pvalues of 1.62, 1.02, 1.00, 0.25, and 0.21, respectively, were
also assayed. After 2.0-min perfusion of all the 13 peptides, only Gly-Pro showed a significant<(P0.001) uptake
with a brain/perfusate ratio of 10.9 ? 0.1 ?L/g-brain, compared to FITC-albumin (Fig.?2a). This indicates that
hydrophobicity is not a key factor in determining the intact BBB transport of dipeptides within the present
experimental conditions. The observation that a dipeptide, Gly-Pro, as well as Gly-Sar can be transported in
their intact forms across the mouse BBB, led us to speculate that dipeptides with an imino bond could be
possible BBB-transportable candidates. Hence, four dipeptides containing Pro at the C-terminal including Gly-Pro,
His-Pro, Ser-Pro, and Tyr-Pro, were targeted for further screening of BBB-transportable dipeptides. As shown in
Fig.?2b, it was clear that Tyr-Pro was a BBB-transportable dipeptide (brain/perfusate ratio: 10.5? 1.3 ?L/g-brain),
with efficiency comparable to that of Gly-Pro (10.9? 0.1 ?L/g-brain). No increase in brain uptake for Pro-Tyr
(Fig.?2b) strongly suggested the importance of positioning of Pro at the C-terminus of dipeptide skeleton. In situ
mouse brain perfusion experiments revealed for the first time that two dipeptides, Gly-Pro and Tyr-Pro, with
significant transport capacity (Ki value) of 3.49? 0.66 and 3.53? 0.74 ?L/g?min, respectively, are capable of crossing
the controlled BBB system (Fig3.?) (c.f., Ki of Gly-Sar: 7.60? 1.29 ?L/g?min, Fig.? 1).
BBB transport of Gly-sar and t yr-pro into brain parenchyma. To validate the intact BBB transport of
dipeptides and accumulation of perfused 1[3C2,15N]Gly-Sar and Tyr-Pro after 2.0-min perfusion, and also to rule
out the non-specific binding of the dipeptides with the capillaries in the brain, the mouse brain parenchyma was
collected by brain capillary depletion technique using dextran density centrifugati2o2.nAs shown in Fig.?4, both
[13C2,15N]Gly-Sar and Tyr-Pro were enriched in the mouse brain parenchyma, as detected by the TNBS-LC-TOF/
MS, but were undetectable in the brain microvessel fraction, suggesting that both Gly-Sar and Tyr-Pro were
transported across the BBB in intact form into the mouse brain parenchyma.
The possible BBB transport route of both dipeptides was examined by co-perfusion with His [as reported
substrate of brain PHT15 and/or l-type amino acid transporter 1 (LAT1)23] or Gly-Sar (as PHT1 substrate)12. As
shown in Fig.?5a,b, His significantly reduced the brain/perfusate ratios of [13C2,15N]Gly-Sar and Tyr-Pro,
suggesting that both dipeptides might be transported across the BBB via a common carrier, PHT1. However, no altered
uptake of Tyr-Pro by Gly-Sar and reduced uptake by l-DOPA were obtained (Fig.?5b). Moreover, the
brain/perfusate ratio of 2.0 min perfusion of Gly-Sar at 5mM (18.1 ? 0.5 ? L/g-brain) in the present experimental condition
was significantly lower than that at 20 0?M (24.5 ? 1.8 ?L/g-brain), which indicated the concentration of Gly-Sar
used in the present co-perfusion experiments was enough to be saturated for BBB transport of Gly-Sar. These
results strongly suggested that an LAT1 (or other) transport route(s) cannot be ruled out for the BBB transport of
Tyr-Pro. BBB transport route(s) of the dipeptides are now in investigation using brain capillary endothelial cells.
Location of t yr-pro in mouse brain by MALDI-Ms imaging. Based on the evidence that Tyr-Pro is
transported across the BBB into brain parenchyma (Fig.?4), MALDI-MS imaging visualization analyses were pe-r
formed to determine the regions of peptide accumulation. The brains were monitored after 2.0-min and 10-min
perfusions of Tyr-Pro, respectively. l-DOPA that can be transported across the BBB23 was selected as the positive
control to validate the visualization analysis. [3D]l-DOPA was used for perfusion to distinguish it from
endogenous l-DOPA during MS detection. As shown in Fig.?6a?c, the distribution of [D3]l-DOPA could be effectively
visualized in the mouse brain tissues upon increasing the perfusion time up to 10min. MALDI-MS/MS imaging
indicated that Tyr-Pro (target mass of fragment Pro: [M+ H]+, 279.1 > 116.0 m/z) was transported across the
BBB and accumulates in the hippocampus, hypothalamus, striatum, cerebral cortex, and cerebellum of the mouse
brain (Fig.?6d?f: sagittal slice, g and h: coronal slice).
MALDI-MS imaging technique to elucidate the distribution of BBB-transportable dipeptides in the mouse brain.
In this study, we demonstrate the localization of perfused Tyr-Pro for 2.0min and 10min, respectively, in the
hippocampus, hypothalamus, cerebral cortex, and cerebellum of the mouse brain (Fig6.d? ?f: sagittal slices, g and h:
coronal slices). Thus far, there have been no reports on the distribution of dipeptides in brain, or on the beneficial
effects of the same on the brain, except for the evidential improvement of delayed memory score in patients with
mild cognitive impairment by the intake of protein hydrolysat1e3. Although Tyr-Pro, which was present in
soybean hydrolysate at 0.47 mg/g-hydrolysate (Fig.?S2), showed in vitroangiotensin I-converting enzyme inhibition34
and antioxidant effects35, there is no study supporting its in vivo benefits. Therefore, the current observation of the
accumulation of in vivoTyr-Pro in the regions of the brain involved in memory consolidation and spatial memory
(hippocampus36), food intake control (hypothalamus), and cognition (cerebrum and cerebellu3m7) suggests that
the localization of dipeptides could have a potential physiological role in the brain. It will, therefore, be interesting
to examine the physiological effects and bioavailability of orally administered Tyr-Pro in animal models.
In conclusion, this study provides evidence for the transport of intact Gly-Pro and Tyr-Pro, along with
dipeptidic Gly-Sar, across the BBB into the mouse brain parenchyma. Moreover, we also show the selective
accumulation of Tyr-Pro in the hippocampus, hypothalamus, striatum, cerebral cortex, and cerebellum of mouse brain
by phytic acid-aided MALDI-MS imaging technique. Although further studies are needed to point out the
physiological roles of the BBB-transportable dipeptides in the brain, we establish dipeptides as molecules capable of
penetrating the BBB.
Chemicals and reagents. Gly-Sar, aprotinin, and FITC-labeled albumin were obtained from Sigma-Aldrich
(St. Louis, MO, USA). [13C2,15N]-labeled Gly-Sar and13[C5,15N]-labeled Tyr-Pro were synthesized by Scrum Co.
(Tokyo, Japan). [D3]-labeledl-DOPA was obtained from Cambridge Isotope Laboratories Inc. (Tewksbury, MA,
USA). Chymostatin was purchased from Peptide Institute Inc. (Osaka, Japan). Dipeptides (Gly-Sar, Gly-Pro,
AlaGln, His-Leu, Trp-His, Val-Tyr, Met-Tyr, Ile-Tyr, Leu-Tyr, Trp-Leu, Leu-Trp, Trp-Met, Trp-Ala, Trp-Tyr, His-Pro,
Ser-Pro, Tyr-Pro, and Pro-Tyr) were purchased from Kokusan Chemical Co. (Osaka, Japan). TNBS was
purchased from Nacalai Tesque Co. (Kyoto, Japan). 1,5-Diaminonaphthalene (1,5-DAN) was procured from Tokyo
Chemical Industry Co. (Tokyo, Japan). Distilled water, methanol (MeOH), acetonitrile (ACN), and formic acid
(FA); each reagent of LC-MS grade, were purchased from Merck Co. (Darmstadt, Germany). All other reagents
used, were of analytical grade and were used without further purification.
Animals. Seven to nine-week old male ICR mice with 30?40g body weight (Jcl:ICR, CLEA Japan, Tokyo,
Japan) were used in this study. All mice were housed for 1 week under controlled temperature at 2?11 ?C,
humidity at 55? 5%, and light scheduled for a twelve-hour period from 8:00 am to 8:0p0m. The mice were fed
the laboratory diet (CE-2, CLEA Japan) and water ad libitum. All animal experiments in this study were handled
in accordance with the Proper Conduct of Animal Experiments and Related Activities in Academic Research
Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology in
Japan. The Ethics Committee on Animal Experiments at Fukuoka University approved all experimental protocols
(permit number: 1606938 and 1702014).
In situ transcardiac mouse brain perfusion experiments. In situ mouse brain perfusion experiments
were conducted as previously described38 with slight modifications. Briefly after mice were anesthetized with
40% urethane (Sigma-Aldrich, St. Louis, MO, USA), the descending thoracic aorta was ligated, and at the start
of perfusion, left jugular was sectioned. Freshly prepared perfusion fluid [12m0M NaCl, 4mM KCl, 2.5mM
CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.8 mM anhydrous MgCl2, 5.5 mM d-glucose, and 1% BSA],
containing FITC-albumin (200? M), peptides (200 ? M) or (D3)l-DOPA (200 ? M)] was infused in the left ventricle of
the heart by inserting a 26-gauge butterfly needle at a rate of 2.0mL/min for 0?10 min (n = 3/FITC-albumin and
n = 4/peptide group). For co-perfusion of peptides with Gly-Sar (5mM), His (5 mM) or l-DOPA (5 mM), freshly
prepared perfusion fluid containing each compound was infused as mentioned above for 2.0min (n = 4 for each
group). After perfusion, whole brain was removed from the mice by decapitation and weighed. Brain samples
for quantitative analysis by LC-TOF/MS were immediately frozen in liquid nitrogen, whereas the samples for
MALDI-MS imaging analysis were immediately frozen using powdered dry ice to avoid any degradation of tissue
shape. All brain samples were stored at ?8?0C until analysis was performed.
preparation of brain parenchyma and microvessel fractions. The brain parenchyma and
microvessel fractions were prepared as described by Triguero et a.l22. Brain was obtained by decapitation after 2.0-min
dipeptide perfusion, and arachnoid membranes were peeled away. The isolated brain was minced using a glass
homogenizer in 0.5 mL physiological buffer used for preparation of the perfusion fluid, followed by further
homogenization after the addition of 1.0mL of 26.5% dextran at 4?C. The homogenate thus obtained was centrifuged at
5,400 ? g for 15 min at 4 ?C. The supernatant representing the parenchyma fraction, and the pellet representing the
microvessel fraction were carefully collected (Fig.?S3). One milliliter of the physiological buffer was then added
to the supernatant and centrifuged at 5,400? g for 15 min at 4 ?C to obtain the brain parenchyma. The absence of
parenchyma debris contamination in the brain microvessel fraction was confirmed by microscopy (Fig.?S3). For the
pellet, 1.5mL of 17.7% dextran was added, followed by centrifugation at 5,40?0 g for 15 min at 4 ?C to obtain the
microvessel fraction. Both parenchyma and microvessel fractions were frozen and stored at ?80 ?C.
Quantification of perfused dipeptides in brain by liquid chromatography-mass spectrometry.
Frozen whole brain, brain parenchyma and microvessel fractions after the perfusion of dipeptides were
lyophilized and mashed with a BioMasher II (Nippi. Inc., Tokyo, Japan). An aliquot (1m0g) of the obtained
homogenous powder was dissolved in 1.0mL of homogenized buffer (0.1% NaCl and 0.1% FA) containing
[13C5,15N]Tyr-Pro (60 pmol/mL) as the internal standard (IS) and protease inhibitors (0.5mg/mL
ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), 0.1mg/mL aprotinin, and 0.1mg/mL chymostatin). The
sample solution was sonicated using a SONIFIRE 250 (Branson Ultrasonics, Emerson Japan Co., Kanagawa, Japan)
with an output control of 3 for 10s ? 3 times at 4 ?C, following the homogenization with a Polytron homogenizer
(KINEMATICA AG, Luzern, Switzerland) with 20,000rpm for 30 s ? 3 times at 4 ?C. After the centrifugation of
the homogenate at 14,000 ? g for 15 min at 4 ?C, the supernatant obtained was subjected to ultrafiltration using
an Amicon Ultra 0.5-mL-3K centrifugal filter (Millipore, Carrigtwohill, Ireland) at 14,?00g0for 30 min at 4 ?C.
The collected filtrate was evaporated till it dried. The dry filtrate was then subjected to a TNBS derivatiza1t7ion
to obtain TNP-derivatives. 50?L of TNBS solution (150 mM, pH 10) was added to the filtrate, and incubated for
30 min at 30 ?C. After the addition of 50? L of 0.2% FA solution to stop the TNBS reaction, an aliquot (2 0? L) of
the solution was subjected to LC-TOF/MS analysis.
LC-TOF/MS analysis was performed as follows: LC separation was performed using an Agilent 1200 series
system (Agilent, Waldbronn, Germany) on a Biosuite Peptide column (2.?1 150 mm, 3 ? m, Waters, Milford, MA,
USA) at 40 ?C with a linear gradient elution of MeOH (0?100% over 20min) containing 0.1% FA at a flow rate of
0.25 mL/min. Electrospray ionization (ESI)-TOF/MS analysis was carried out using a microTOF II equipment
(Bruker Daltonics, Bremen, Germany) in positive mode. The ESI conditions were as follows: drying gas (2N),
8.0 L/min; drying temperature, 200 ?C; nebulizing gas (N2), 1.6 bar; capillary voltage, 4,500V; and mass range,
100?1,000 m/z. All data acquisition and analyses were performed by using Bruker Data Analysis 3.2 software. A
calibration solution containing 10mM sodium formate in 50% ACN was injected at the beginning of each run,
and all spectra were internally calibrated.
Typical calibration graphs obtained with the aforementioned MS conditions were used to determine the
amount of dipeptides in dry brain weight (g) as follows:1[3C2,15N]Gly-Sar,y = 2,086x + 16 (r = 0.994); Gly-Pro,
y = 1,656x + 45 (r = 0.987); Ala-Gln,y = 1,675x + 13 (r = 0.926); His-Leu, y = 10,263x ? 39 (r = 0.974); Trp-His,
y = 1,304x ? 1 (r = 0.999); Val-Tyr, y = 1,453x + 9 (r = 0.996); Met-Tyr, y = 1,332x + 9 (r = 0.994); Ile-Tyr,
y = 6,822x + 53 (r = 0.979); Leu-Tyr, y = 4,313x + 169 (r = 0.994); Trp-Leu, y = 3,258x ? 11 (r = 0.999); Leu-Trp,
y = 2381x + 46 (r = 0.959), Trp-Met, y = 1,436x ? 13 (r = 0.978); Trp-Ala,y = 4,732x + 21 (r = 0.997); Trp-Tyr,
y = 2,667x ? 18 (r = 0.990);?His-Pro, y = 1,843x ? 10 (r = 0.979); Ser-Pro, y = 2,135x + 46 (r = 0.994); Tyr-Pro,
y = 2,058x ? 3 (r = 0.999), and Pro-Tyr, y= 1,932x ? 1 (r = 0.999) [where y is the peak area ratio (observed peak
area of the target to that of IS) and x is the peptide concentration between 0?18 nmol/g-dry brain].
Brain/perfusate ratio of the perfused peptide was calculated by the peptide concentrations in wet brain (converted from
dry brain weight) as follows: brain/perfusate ratio (?L/g wet brain) = concentration of perfused peptide in wet
brain (nmol/g wet brain)/concentration of peptide in perfusate (nmol/?L). Ki value (? L/g?min) was obtained by
calculating the slope of brain/perfusate ratio against perfusion time. The kinetics experiments were performed 4
times each, at the individual time intervals of 0.5, 1.0, 1.5, and 2.0min, respectively.
MALDI-Ms imaging analysis. Distribution of the perfused Tyr-Pro and [D3]l-DOPA in the mouse brain
was analyzed using the proposed phytic acid-aided MALDI-MS imaging techniqu1e5. A frozen whole brain after
peptide perfusion experiments was sliced into 12-?m-thick sections at both sagittal and coronal faces using a
CM1850 Leica Cryomicrotome (Leica, Wetzler, Germany). Each section was thaw-mounted on an indium-tin
oxide (ITO)-coated conductive glass slide (Bruker Daltonics) and dried under the2Ngas flow. DHB was used
as MALDI matrix, for the detection of Tyr-Pro in positive MS mode. For matrix preparation, DHB (50mg/mL)
was dissolved in MeOH/water (1:1, v/v) containing 50mM phytic acid15 and 250mM (NH4)2SO4, while 1,5-DAN
(10 mg/mL) in 70% ACN was used for the detection of [D3]l-DOPA in negative MS mode. Each matrix was
sprayed with an ImagePrep automatic matrix sprayer (Bruker Daltonics) over the ITO-glass slide. MALDI-MS
imaging analysis for brain tissue-mounted ITO-glass slide was performed using an ultrafleXtreme mass
spectrometer equipped with a smartbeam II Laser (Bruker Daltonics) in reflector and LIFT modes. MS/MS data
were acquired with monoisotopic isolation of 279.1> 116.0 m/z for Tyr-Pro, while MS data were acquired with
199.1 m/z for [D3]l-DOPA. MS parameters were as follows: ion source voltage 20.0k0V; reflector voltage, 20.8kV;
lens voltage, 6.50kV; number of shots, 100 shots/spot; laser frequency, 1000Hz; laser focus, medium. MS imaging
analysis was performed with a spatial resolution of 90m? . The image data were constructed for visualization with
mass filters of ?0.01 m/z for [D3]l-DOPA in MS mode, and ?0.3 m/z for Tyr-Pro in MS/MS mode by Bruker
flexImaging software (ver. 4.1).
statistical analyses. Results are expressed as the mean? standard error of the mean (s.e.m.). Statistical
evaluation between two groups was performed by using an unpaired two-tailed Student?s t-test. A one-way
analysis of variance (ANOVA) was performed to analyze the difference among more than three groups, followed by
Dunnett?s t-test for post hoc analysis. A two-way ANOVA was performed to analyze the difference between brain/
perfusate ratios of FITC-albumin and peptide groups over time, followed by Bonferroni post hotcest. A P value
of <0.05 was considered significant. All statistical analyses were carried out using GraphPad Prism 5 software
(GraphPad Software, La Jolla, CA, USA).
The data supporting the findings reported herein are available on request, from the corresponding author.
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Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42099-9.
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