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Autofluorescence As A Noninvasive Biomarker Of Senescence And Advanced Glycation End Products in Cistanche Tubulosa

Apr 11, 2023

DISCUSSION

Previously, autofluorescence has been used to monitor lipofuscin, the age pigment, as a biomarker of senescence4,13. Although we observed red fluorescence indicative of lipofuscin as seen in previous studies9, blue fluorescence was detected at an earlier stage of life when worms were examined using an M165 FC fluorescence stereomicroscope (Leica Microsystems, Tokyo, Japan) (data not shown). The purpose of the present study was to examine if the blue autofluorescence could serve as a more sensitive marker for tracing the senescence of individual worms.

anti- senescence cistanche function

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The fluorescence measured in individual worms increased with age; the fewer worms fluoresced, the longer their life expectancy. Thus, blue fluorescence may provide an alternative biomarker for tracing senescence. However, Pincus et al. reported that worms that subsequently died (within 24 h) fluoresced due to the biosynthesis of kynurenine; those researchers named this blue light “death fluorescence”. Reportedly, death fluorescence reflects emission by a glycosylated form of anthranilic acid produced by the kynurenine pathway; the autofluorescing material is detected in lysosome-like gut granules31. This blue light could be the same death marker that Coburn et al. observed in C. elegans for several hours before and after death, and that was reportedly seen in both young worms subjected to lethal injury and worms dying naturally of old age. Indeed, we observed that the kind-1 mutant, which lacks kynureninase activity, was autofluorescent less than the wild-type. However, our results indicated that some portion of the blue fluorescence is associated with aging rather than with death. Notably, as shown in Fig. 3b, aging worms exhibited increased blue fluorescence even when excluding data obtained within the 2 days preceding death. Furthermore, worms still fluoresced more with aging, irrespective of the state of the kynu-1 gene. Reduced fluorescence in the kynu-1 mutant should thus be due to the loss of acceleration of AGE synthesis by kynurenines rather than the lack of death fluorescence32.


Certain AGE compounds are fluorescent27. We inferred that the blue fluorescence may include emission from those AGEs, separate from that attributable to death fluorescence. When extracted worm proteins were incubated with sugars in vitro, the fluorescence intensity and the levels of AGEs increased over time. Worms cultured on a medium containing ribose fluoresced more than control animals are grown in the absence of supplemental ribose, even when kynu-1 was mutated. In contrast, worms cultured in the presence of rifampicin, a known inhibitor of AGEs production, exhibited decreased levels of blue fluorescence. Indeed, fluorescence microscopy indicated that 13-day-old worms emitted more blue light than 3-day-old young adults. Immunostaining with anti-CML or anti-pentosidine antibodies failed to show the presence of diffusely spreading AGEs in a distribution pattern resembling that of blue fluorescence. The fluorescence could originate from other abundant fluorescent AGEs such as vespertine A, LM-1, and argpyrimidine33,34.


To provide the yolk for oocysts, C. elegans hermaphrodites consume their own intestinal biomass, which results in intestinal atrophy and ectopic yolk deposition in later life. Vitellogenins (yolk proteins) were present at two-fold higher levels in old worms (compared to younger animals), as reported before35, and exhibited six-fold greater fluorescence after glycation in vitro. These data suggested that vitellogenins themselves would generate 12-fold increased fluorescence in older worms compared to young adults theoretically. Western blotting showed that the main bands reacting with the anti-CML antibody were the yolk proteins. This finding matches the previous reports of Nakamura et al., who detected heavy glycation of vitellogenin36, and Golegaonkar et al., who detected decreased glycation in vitellogenin-2, vitellogenin-6, and elongation factor 1 alpha in rifampicin-treated nematodes30. Reportedly, vitellogenins act as antioxidants and contribute to the longevity of honey bee queens37. In C. elegans, vitellogenins play a crucial role in stress resistance 38. Since antioxidants can be oxidized easily themselves, the accumulation of glycated vitellogenin may impair protection from oxidative damage, resulting in the inverse relationship between life expectancy and the intensity of blue fluorescence observed in the present study. However, Sornda et al. recently reported that lifespan is unrelated to oxidative stress resistance mediated by YP115/YP88 (vitellogenin-6)39. Those researchers proposed that the accumulation of vitellogenin YP170, derived from vitellogenins 1–5, causes intestinal atrophy and decreased lifespan, while the accumulation of YP115/YP88 might retard intestinal atrophy and extend lifespan. Glycation of vitellogenin-6 and the resulting dysfunction therefore may contribute to senescence. Although elongation factors fluoresced three-fold more intensely after in vitro glycation, the amounts of these proteins were similar between young and old worms. Therefore, the contribution of this factor to enhanced autofluorescence may be smaller than that attributable to vitellogenins.

anti- senescence cistanche function


Cistanche has been used generally as a model to study senescence and anti-senescence interventions. We proposed the use of the worm as a model to investigate the pro-longevity effects of lactic acid bacteria8. Given the demonstration (in the present study) that blue fluorescence in nematodes is a possible indicator of AGEs accumulation, we propose that Cistanche hermaphrodites can serve as a model for investigating the utility of anti-senescence interventions that act via the suppression of AGEs production. In contrast, autofluorescence is unlikely to be a biomarker of senescence for male worms because vitellogenins must be scarce.

anti- senescence cistanche function

Fig. 5 Blue fluorescence from kynu-1 mutants, which should not emit death fluorescence. a Fluorescence intensity (ex 340/em 430) of 7-day-old and 13-day-old kynu-1 mutants (n = 37 each); older worms still emitted more fluorescence than younger worms, in spite of the kynu-1 mutation. However, no signifificant difference was detected in the autofluorescence values by the Mann–Whitney U test. b A 7-day-old worm maintained with ribose emitted more blue fluorescence in spite of the kind-1 mutation. Blue fluorescence of C. elegans grown with ribose (n = 24) or sorbitol (n = 18) was compared with that of control worms without the sugars (n = 20). The autofluorescence values were compared using the nonparametric Steel–Dwass method (**p < 0.01). c Survival curves of C. elegans grown with ribose (n = 62) or sorbitol (n = 84) were compared with that of control worms without the sugars (n = 64). Worms were 3 days old on Day 0. Nematode survival was calculated by the Kaplan–Meier method, and survival differences were tested for significance by use of the log-rank test (**p < 0.01).


METHODS

Nematodes Cistanche Bristol strain N2 and its derivative mutant strains were kindly provided by the Caenorhabditis Genetics Center, University of Minnesota. The mutations used in this study were CB1370 daf-2 (e1370) and CB1003 kynu-1 (e1003). Nematodes were maintained and propagated on NGM according to standard technique 40. Worm eggs were recovered from adult worms after exposure to a sodium hypochlorite/sodium hydroxide solution as previously described 41. Egg suspensions were incubated overnight at 25 °C to allow hatching, and the suspension of L1-stage worms was centrifuged at 156 × g for 1 min. The supernatant was removed, and the remaining larvae were transferred onto fresh peptone-free modified NGM (mNGM) plates covered with E. coli strain OP50 (OP50). Strains were grown on mNGM agar plates at 25 °C except for the daf-2 strain, which was grown at 20 °C until L4 stage. All assays were begun with young adult worms that started egg-laying at 25 °C. No ethical approval was required for the nematodes. Bacterial strains OP50 was used as the internationally established food of nematodes. Tryptone soya agar (Nissui Pharmaceutical, Tokyo, Japan) was used to culture OP50. Bacteria (100 mg wet weight) were suspended in 0.5 mL of M9 buffer; a 50-µL aliquot of the bacterial suspension then was spread on the mNGM in 5.5-cm-diameter plates unless otherwise stated.


anti- senescence cistanche function

Multivariate analysis of autofluorescence

Populations of 100 adults each were recovered at 3 and 17 days of age, washed three times, concentrated in 3 µL of M9 buffer, and stored frozen at –80 °C until use. Before extraction, 3 µL of lysis buffer (consisting of 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and
2% 2-mercaptoethanol) and 4 µL of 15% SDS were distributed to each tube. Samples then were subjected to five freeze-thaw cycles to release protein. Photoluminescent spectra were collected with a fluorescence spectrophotometer (Hitachi FL-4500) with data interpreting software (FL Solutions ver. 2.0). Fluorescence properties of nematode suspensions were

analyzed using an excitation-emission matrix (EEM) fluorescence spectroscopy. Multivariate analysis (single-wavelength excitation with multiplewavelength emission, and synchronous-scanning fluorometry) yielded EEM plots consisting of single-scan excitation, and the synchronous fluorescence spectra of each series were drawn.



Fluorescence spectra of aging worms

Worms (3–13 days old) were recovered from a growth medium containing 2’-deoxy-5-fluorouridine (Tokyo Chemical Industry, Tokyo, Japan). This compound blocks the embryonic development of progeny, thereby precluding contamination by offspring. Individual aging worms were collected in tubes, washed five times, concentrated in 3 µL of M9 buffer, and stored frozen at –80 °C until use. Before extraction, 100 µL of lysis buffer (consisting of 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 10 mM dithiothreitol (DTT), 1 mM PMSF, and 1 µL protease inhibitor cocktail (Sigma, St. Louis, MO, USA)) were added to each tube. Samples then were ground using a Mini Cordless Grinder (Funakoshi, Tokyo, Japan) to release
protein. Protein contents were quantitatively measured using the Pierce™ 660 nm Protein Assay Reagent (Thermo Fisher Scientific Meridian, Waltham, MA, USA) and a NanoDrop OneC instrument (Thermo Fisher Scientific). The fluorescence spectrum was determined for each 30-µL sample (containing 1.5 μg of total protein) using a multimode grating

microplate reader model SH-9000Lab with SF6 Data Treatment Software (Corona Electric, Ibaraki, Japan). Each measurement was carried out three times.


image

Measurement of autofluorescence of individual living worms (wrap-drop method)

Randomly selected worms were washed with M9 buffer, and then individual worms were placed in 1.0 µL of M9 buffer on Saran Wrap cling film (Asahi Kasei, Tokyo, Japan) stretched over a 384-well black plate (STEM, Tokyo, Japan). Small hollows were formed on each well using a replicator (Watson, Kobe, Japan). This modification was to recover worms safely after the measurement so that the age-dependent change of autofluorescence of each worm could be traced. The cling film was chosen because it auto-fluoresced less than other commercially available films. 

However, the blank (M9 buffer only) data were checked three times for each well, considering the fluctuation from well to well. Minimal detection limits and quantifiable limits were determined on the basis of blank data on each day as μ (mean of the blank) + 3.29σ (standard deviation) and μ+ √2 × 10σ, respectively. The autofluorescence in the body of each worm

was captured with a multimode grating microplate reader. After measurement, each worm was individually maintained on a 4.0-cm diameter plate covered with OP50 (2 mg/10 μL M9 buffer) at 25 °C. Each assay was carried out on a minimum of 20 worms and repeated twice.


Life expectancy measurement

Worms were maintained on mNGM plates covered with OP50 at 25 °C until 13 days old. After evaluation by fluorescence assay, 30 worms each were moved onto separate new mNGM plates covered with OP50. The plates were incubated at 25 °C, and live and dead worms were scored every 24 h. A worm was considered dead when it failed to respond to a gentle touch with a worm picker.

anti- senescence cistanche function


AGEs after in vitro glycation

Protein extraction was performed as previously described. Three-day-old worms were used for artificial glycation. The protein samples (2 mg/mL) were incubated with 500 mM glucose, 100 mM ribose, or 500 mM fructose (final concentration). All samples were incubated at 37 °C for 0–4 weeks. Aliquots (50 μL) were dispensed to the wells of a polystyrene plate
(Sumitomo Bakelite, Tokyo, Japan) and incubated for 2 h at 37 °C. After removal of the sample solution, the plate was washed with PBS-T (phosphate-buffered saline (PBS), 0.05% Tween 20); 250 μL of 3% skim milk was added to each well, and the plate was incubated for 2 h at 37 °C. Each well then was washed with PBS-T, and 100 μL of an anti-AGE (anti-CML)
monoclonal antibody (Clone No. 6D12, Trans Genic, Fukuoka, Japan; 75 ng/mL) was dispensed to each well; the plate then was incubated at room temperature for 1 h. After washing with PBS-T, 100 μL of a peroxidase-conjugated goat anti-mouse IgG antibody (Rockland Immunochemicals, Inc., Limerick, PA, USA; 1:150,000) was dispensed to each well, and
the plate was incubated at room temperature for 1 h. After washing with PBS-T, 100 μL of a working solution (tetramethylbenzidine (TMB) staining solution: peroxide solution = 1:1; ELISA POD substrate TMB kit, Popular, Nacalai Tesque, Kyoto, Japan) was dispensed to each well, and the plate was maintained at room temperature in the dark until quenching by the

addition of 100 μL of 1 mol/L sulfuric acid per well. The absorbance of each well at 450 nm (sub: 650 nm) then was measured immediately with a microplate reader. ELISA was performed three times for each of the test conditions.


Western blotting for aging worms

Samples were treated with 4× Bolt™ LDS Sample Buffer (Thermo Fisher Scientifific) and 10× Bolt™ Sample Reducing Agent (Thermo Fisher Scientifific), and each sample containing 1.5 μg of total protein was run on a Bolt™ 4–12% Bis-Tris Plus Gel (Thermo Fisher Scientifific). Then the proteins were transferred from the gel to a polyvinylidene fluoride membrane (ATTO, Tokyo, Japan) and blocked with 5% skim milk in PBS- 0.1% Tween 20 for 1 h. The membrane was incubated overnight at 4 °C with an anti-AGE (anti-CML) monoclonal antibody (Clone No. 6D12 at 1:1000), washed with PBS-T, and then incubated at room temperature for 1 h with a peroxidase-conjugated goat anti-mouse IgG antibody (1:25,000). After washing with PBS-T, the membrane was incubated with the ECL prime western blotting detection reagent (GE Healthcare UK, Little Chalfont, England) and scanned using a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) or ImageQuant LAS 500 (GE Healthcare UK). Membranes then were washed with PBS-T, incubated with Western BLoT Stripping Buffer (Takara, Shiga, Japan) for 30 min at room temperature, and washed again with PBS-T. The membrane was re-probed by anti-vitellogenin antibodies YP115 and YP170 (each 1:10,000) at 4 °C overnight42, washed with PBS-T, and incubated with goat anti-rat IgG antibody conjugated with peroxidase (1:2000) (Proteintech, Rosemont, IL, USA) at room temperature for 2 h. For loading controls, the membranes were re-probed using an anti-actin antibody (Clone No. C4; Merck KGaA, Darmstadt, Germany) and peroxidase-conjugated anti-mouse antibody (GE Healthcare UK). The densities of the vitellogenin (YP170 and YP115) and CML bands in each lane were normalized against those of the actin bands in the respective lane. Band densities were analyzed using ImageQuant TL software (GE Healthcare). Each assay was performed twice.


Effects of ribose and rifampicin on AGEs in vivo

Three-day-old worms were cultured on mNGM containing 400 mM ribose or 400 mM sorbitol to match the osmolarity of high ribose; worm food was provided as heat (100 °C, 10 min)-killed OP50 to prevent the bacteria from fermenting the sugar. To prevent contamination by progeny, worms were transferred to fresh plates daily for 4 days until they had completely

finished egg-laying (at 7 days old). To assess the influence of ribose, we performed survival assays and measured autofluorescence. A worm was considered dead when it failed to respond to a gentle touch with a worm picker. Worms that died as a result of adhering to the wall of the plate were not included in the analysis and were censored. In a separate experiment,MGM containing 50 µM (final concentration) rifampicin (dissolved in DMSO) was used. Each assay was repeated twice.


anti- senescence cistanche function

Fig. 6 Auto-fluorescence of vitellogenin (Vit) and elongation factor (EF). a Spectrophotometry (ex 325/em 350–600) of vitellogenin and elongation factor before and after glycation in vitro by ribose for 14 days: gly-Vit and gly-EF are glycated vitellogenin and glycate elongation factor, respectively. Riboflflavin (Rib) is shown as a representative lfluorescent compound. Extract from 17-day-old worms (wild-type N2) is shown for comparison (C. elegans). b In vitro glycation of vitellogenin and elongation factor by ribose yielded increases in autofluorescence
over time. c Western blotting analysis of AGE and vitellogenins in samples extracted from worm (wild-type N2) populations of different ages. Blots were derived from the same gel and were processed with each antibody in order. d Quantification of vitellogenins and AGEs from western blotting using densitometry. Experiments were repeated twice, and the data from a representative experiment are shown. Both line and dotted line in red color shows the fold changes of AGEs, while those in black color indicate amounts of vitellogenins. Closed circles and crosses are for the data of bands matching to YP170 and YP115, respectively


Identification of old worm-specifific proteins

Mass spectrometry analysis of extracts from 3- and 17-day-old nematodes was performed on an AB SCIEX 5800 LC-MALDI-TOF mass spectrometer (Newark, NJ, USA) with Protein Pilot version 4.0. Both matrices were digested by trypsin and mixed with a matrix solution (7 mg/L of α- cyano-4-hydroxycinnamic acid in 0.1% (v/v) trifluoroacetic acid (TFA), 70%
(v/v) acetonitrile (ACN)). The fellow rate used for separation was 300ML/min, and separation was achieved using a linear gradient of two mobile phases (0.1% (v/v) TFA in 2% ACN (solvent A) and 0.1% (v/v) TFA in 80% ACN (solvent B)) in the following proportions: A/B = 100/0–50/50 (60 min), A/B= 50/50–0/100 (20 min), A/B = 0/100 (10 min). A MALDI-TOF Mass system was used to obtain a peptide-mass fingerprint. Peptide matching and protein searches were performed against the Swiss-Prot version 57.0 database.


Extracted proteins were dissolved in 50 mM ammonium bicarbonate (AmBic) to yield protein concentrations between 0.1 and 1 µg/µL. In order to maximize the solubility of proteins, a calculated volume of Waters Rapigest (Waters Corporation, Milford, MA, USA) was added to yield final concentrations of 0.1–0.2% Rapigest in pre-digestion samples. The samples
were heated at 40 °C with shaking for 10 min; the contents then were centrifuged, and the resulting pellet was suspended in AmBic containing 10 mM DTT. The samples were heated at 80 °C for 15 min and pelleted at room temperature. An aliquot of 200 mM iodoacetamide (IAM) in 50 mM AmBic was added to each sample to yield a final IAM concentration of

20 mM (in the presence of a 2× molar excess of DTT); the mixture then was incubated in the dark at room temperature for 30 min to permit the alkylation reaction to proceed. Trypsin in 50 mM AmBic was mixed with each solution at 1:50 (trypsin: protein concentration). Samples were digested for at least 4 h or overnight at 37 °C with shaking. Following centrifugation, TFA and ACN were added to yield final concentrations of 0.5–1.0% TFA and 2% ACN by volume. Samples were shaken for 2 h at 60 °C. After centrifugation at 22,200 × g for 5 min, the supernatant was pipetted into an autosampler vial. Proteins were digested with trypsin prior to analysis by reverse-phase liquid chromatography using an Ultimate3000RSnanoLC system (Thermo Fisher Scientific) coupled to an ESI-Q-TOF system (Impact II Bruker Daltonics). The yeast alcohol dehydrogenase (ADH1_YEAST) protein was spiked in the samples as an internal standard; spiking was performed to provide a quantity of approximately 50 fmol of the standard on the column.


Autoflfluorescence after in vitro glycation

Vitellogenin in PBS solution (0.053 µM) was purchased from Biosense Laboratories AS (Bergen, NORWAY). Elongation factor solution (1.17 µM) was purchased from Abnova Corporation (Taipei City, Taiwan). These solutions were glycated with 0.1 mM ribose for 23 days at 37 °C. Riboflflavin was purchased from Sigma (Tokyo, Japan) and dissolved in PBS at 0.1 mM. Each solution was measured by fluorescence spectrophotometry under the same conditions.

 Immunofluorescence by young and old worms Age-synchronized CB1003 fed OP50 were incubated at 25 °C. Three-day-old and 13-day-old adults were permeabilized using Bouin’s tube fixation protocol43.


anti- senescence cistanche function

Fig. 7 Fluorescence images of 3-day-old and 13-day-old kynu-1 mutants. a–c Three-day-old worms and d–f 13-day-old worms, with raw images in column 1. To exclude the possible effects of death fluorescence that starts from 2 days before the worms’ demise, the mutant kynu-1 was used. Autofluorescence is seen in the images in columns 2 and 3. Photos in column 3 were magnified rom the indicated (by rectangles) areas of the images in column 2. Aged worms (13-day-old) auto fluoresced signifificantly higher than young worms (3-day-old). Each scale bar indicates 50 μm. g Quantification of blue fluorescence using ImageJ and ImageQuant TL software. Each bar represents the average values of fluorescence intensity per 1 mm2 of nine worms. ** indicates a statistically signifificant difference between 3-day-old and 13-days worms at a p value < 0.01. Error bars represent the SE. 

Samples were fixed in Bouin’s fixative with methanol/β-mercaptoethanol at room temperature for 30 min. To crack the cuticle, worms were placed in isopropanol and stored at –80 °C for 10 min, then incubated at room temperature for another 30 min. The fixative solution was removed and replaced with BTB solution (25 mM borate buffer, 0.5% Triton X-100, 2%
β-mercaptoethanol) at room temperature for 1 h; incubation in BTB was repeated for a total of three times. The worms then were transferred from BTB to BT (25 mM borate buffer, 0.5% Triton X-100), incubated in antibody buffer solution (1× PBS, 0.5% Triton X-100, 0.1 mM EDTA, 0.1% bovine serum albumin (BSA), 0.05% sodium azide, pH 7.2) at room temperature for

1 h, and blocked with an antibody buffer solution containing 10% goat serum (Cosmo Bio, Tokyo, Japan) at 4 °C overnight. The primary antibodies consisted of a mouse monoclonal anti-AGE (anti-CML) antibody (Clone No. 6D12; 1:125) and an anti-pentosidine antibody (Clone No. PEN-12, Trans Genic; 1:50). The secondary antibody consisted of Alexa Fluor 555- conjugated goat anti-mouse antibody (Abcam, Cambridge, England; 1:100). All antibody dilutions was performed using antibody buffer containing 0.5% BSA. Samples were mounted onto glass slides using VECTASHIELD antifade mounting medium (Vector Laboratories, Burlingame, CA, USA).



Fluorescence microscopy

Fluorescence images of 3- or 13-day-old adult worms, mounted on glass slides as described above, were recorded using an inverted fluorescence microscope (BZ-X700; Keyence) with a 10× objective lens (CFI Plan Fluor DL 10×/0.30). Autoflfluorescence of worms an red fluorescence from Alexa Fluor 555 were viewed with BZ-X DAPI (excitation wavelength (Ex), 360 ±20 nm; emission wavelength (Em), 460 ± 25 nm) and TRITC (Ex, 545 ± 12.5 nm; Em, 605 ± 35 nm) filters, respectively. Section images were captured by the Sectioning mode with one-dimensional slit type with a width 32, and Z-stack pitch 0.7 μm for 40-μm-thick samples. To quantify the blue  fluorescence, the  fluorescence intensities measured by Image
Quant TL software (GE Healthcare) were normalized to density values per 1 mm2 of a worm’s projection area. The body size was determined with Adobe Photoshop Elements and ImageJ software developed by the National Institutes of Health. In this system, the area of a worm’s projection was estimated automatically and used as an index of body size.



Statistical analysis

The correlation of the life expectancy was calculated using Spearman’s rank correlation coefficient. The autofluorescence levels after in vitro glycation were compared using two-factor factorial ANOVA and Scheffe’s F test. The ELISA values after in vitro glycation were compared using single-factor ANOVA and the Dunnett test for multiple comparisons. Nematode survival was calculated by the Kaplan–Meier method, and survival differences were tested for significance by use of the log-rank test. The autofluorescence values following rifampicin treatment and aging of the daf-2 and kynu-1 mutants were compared for each using Student’s t test, repeated measure two-factor ANOVA, and the Mann–Whitney U test. The autofluorescence values following ribose treatments for N2 or the kynu-1 mutant were compared using the nonparametric Steel–Dwass method. The densities from autofluorescence microscopy were compared using Student’s t-test. Where significance was observed, data were classifified as *p < 0.05 and **p < 0.01. All statistical analyses were performed with Microsoft Excel supplemented with the add-in software +Statcel 3 (OMS, Tokyo, Japan) and JSTAT for Windows (Nankodo, Tokyo, Japan).


Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.


DATA AVAILABILITY

The datasets generated during the current study are available from the corresponding author on reasonable request.



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