Gene and noncoding RNA regulation underlying photoreceptor protection: microarray study of dietary antioxidant saffron and photobiomodulation in rat retina.

Abstract

To identify the genes and noncoding RNAs (ncRNAs) involved in the neuroprotective actions of a dietary antioxidant (saffron) and of photobiomodulation (PBM).
We used a previously published assay of photoreceptor damage, in which albino Sprague Dawley rats raised in dim cyclic illumination (12 h 5 lux, 12 h darkness) were challenged by 24 h exposure to bright (1,000 lux) light. Experimental groups were protected against light damage by pretreatment with dietary saffron (1 mg/kg/day for 21 days) or PBM (9 J/cm(2) at the eye, daily for 5 days). RNA from one eye of four animals in each of the six experimental groups (control, light damage [LD], saffron, PBM, saffronLD, and PBMLD) was hybridized to Affymetrix rat genome ST arrays. Quantitative real-time PCR analysis of 14 selected genes was used to validate the microarray results.
LD caused the regulation of 175 entities (genes and ncRNAs) beyond criterion levels (p<0.05 in comparison with controls, fold-change >2). PBM pretreatment reduced the expression of 126 of these 175 LD-regulated entities below criterion; saffron pretreatment reduced the expression of 53 entities (50 in common with PBM). In addition, PBM pretreatment regulated the expression of 67 entities not regulated by LD, while saffron pretreatment regulated 122 entities not regulated by LD (48 in common with PBM). PBM and saffron, given without LD, regulated genes and ncRNAs beyond criterion levels, but in lesser numbers than during their protective action. A high proportion of the entities regulated by LD (>90%) were known genes. By contrast, ncRNAs were prominent among the entities regulated by PBM and saffron in their neuroprotective roles (73% and 62%, respectively).
Given alone, saffron and (more prominently) PBM both regulated significant numbers of genes and ncRNAs. Given before retinal exposure to damaging light, thus while exerting their neuroprotective action, they regulated much larger numbers of entities, among which ncRNAs were prominent. Further, the downregulation of known genes and of ncRNAs was prominent in the protective actions of both neuroprotectants. These comparisons provide an overview of gene expression induced by two neuroprotectants and provide a basis for the more focused study of their mechanisms.

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Gene and noncoding RNA regulation underlying photoreceptor
protection: microarray study of dietary antioxidant saffron and
photobiomodulation in rat retina
Riccardo Natoli,1,2 Yuan Zhu,2,5 Krisztina Valter,1,2 Silvia Bisti,2,3,5 Janis Eells,2,4 Jonathan Stone2,5
1Division of Biomedical Sciences & Biochemistry, Research School of Biology, Australian National University; Sydney, Australia;
2ARC Centre of Excellence in Vision Science, Sydney, Australia; 3Department of Science and Biomedical Technology, University
of L’Aquila, Coppito II, Via Vetoio, L’Aquila, Italy; 4Department of Biomedical Sciences University of Wisconsin Milwaukee,
Milwaukee, WI; 5Bosch Institute, Discipline of Physiology and Save Sight Institute, University of Sydney, Sydney, Australia
Purpose: To identify the genes and noncoding RNAs (ncRNAs) involved in the neuroprotective actions of a dietary
antioxidant (saffron) and of photobiomodulation (PBM).
Methods: We used a previously published assay of photoreceptor damage, in which albino Sprague Dawley rats raised
in dim cyclic illumination (12 h 5 lux, 12 h darkness) were challenged by 24 h exposure to bright (1,000 lux) light.
Experimental groups were protected against light damage by pretreatment with dietary saffron (1 mg/kg/day for 21 days)
or PBM (9 J/cm2 at the eye, daily for 5 days). RNA from one eye of four animals in each of the six experimental groups
(control, light damage [LD], saffron, PBM, saffronLD, and PBMLD) was hybridized to Affymetrix rat genome ST arrays.
Quantitative real-time PCR analysis of 14 selected genes was used to validate the microarray results.
Results: LD caused the regulation of 175 entities (genes and ncRNAs) beyond criterion levels (p<0.05 in comparison
with controls, fold-change >2). PBM pretreatment reduced the expression of 126 of these 175 LD-regulated entities below
criterion; saffron pretreatment reduced the expression of 53 entities (50 in common with PBM). In addition, PBM
pretreatment regulated the expression of 67 entities not regulated by LD, while saffron pretreatment regulated 122 entities
not regulated by LD (48 in common with PBM). PBM and saffron, given without LD, regulated genes and ncRNAs beyond
criterion levels, but in lesser numbers than during their protective action. A high proportion of the entities regulated by
LD (>90%) were known genes. By contrast, ncRNAs were prominent among the entities regulated by PBM and saffron
in their neuroprotective roles (73% and 62%, respectively).
Conclusions: Given alone, saffron and (more prominently) PBM both regulated significant numbers of genes and
ncRNAs. Given before retinal exposure to damaging light, thus while exerting their neuroprotective action, they regulated
much larger numbers of entities, among which ncRNAs were prominent. Further, the downregulation of known genes
and of ncRNAs was prominent in the protective actions of both neuroprotectants. These comparisons provide an overview
of gene expression induced by two neuroprotectants and provide a basis for the more focused study of their mechanisms.
The photoreceptors (rods and cones) of mammalian retina
are the most specialized, metabolically active and fragile of
the nerve cells of the retina [1–3]. Photoreceptors are also the
most vulnerable of retinal cells to genetic stress, induced by
mutations in genes whose expression is specific to
photoreceptors, and in ubiquitously expressed genes [4,5].
The breakdown of photoreceptor stability is a major element
of age-related retinal disease, and therefore of age-related
blindness [6].
The stress-induced death of photoreceptors is
accompanied by damage to the survivors [7–9]. Both death
and damage appear to be caused by oxidative stress, i.e., by
the damaging effects of partially reduced forms of oxygen,
Correspondence to: Riccardo Natoli, Research School of Biology,
Robertson Building (Bldg 46), Sullivans Creek Road, The Australian
National University, Canberra ACT 0200, Australia; Phone: 61 2
6125 8559; FAX: 61
riccardo.natoli@anu.edu.au
2 6125 8680, email:
often called reactive oxygen species. Absorption of light (the
normal function of photoreceptor outer segments) increases
oxidation of their lipids, creating morphological and
functional damage as light exposure is increased [10–12]. The
idea that light-induced damage is caused by oxidative stress
is supported by evidence that levels of endogenous
antioxidants increase following light damage [13–15], and
that exogenous antioxidants are protective [15–21], for cones
[22,23] as well as rods.
We have explored the neuroprotective potential of the
ancient spice saffron, which shows a strong protective effect
against light-induced damage of photoreceptors [24]. The
stigmata of Crocus sativus contain powerful antioxidants
(crocin, crocetin) in biologically high concentrations [25];
their multiple C=C bonds give the stigmata their color,
fragrance, taste, and antioxidant
concentration in saffron may be an evolutionarily special case,
as the plant is a sterile triploid bred by vegetative propagation
for its fragrance, taste, color, and medicinal properties. In a
potential. Their
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Received 23 February 2010 | Accepted 31 August 2010 | Published 3 September 2010
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recent double blind clinical trial [26], saffron (2 μg/day over
12 weeks) induced a partial but consistent recovery of the
electroretinogram elicited from the macula, and of visual
acuity. We have also pioneered the use of photobiomodulation
(PBM) as a retinal neuroprotectant. Red to infrared (600–
1,000 nm) light at low intensities promotes wound healing in
skin and oral mucosa [27], and protects photoreceptors from
toxin- [28], genetic- [29], and light-induced [30] damage.
Furthermore, it reduces laser-induced retinal scarring. PBM
delivered transcranially reduces cerebral pathology in animal
models of brain damage [31–33] and in human ischemic
stroke [34]. PBM acts partly by repairing mitochondrial
function and upregulating oxidative phosphorylation [35].
Again, no harmful side effects have been reported at the doses
used in this in vivo work (daily doses of 5 J/cm2 or less). To
develop the understanding of these neuroprotective effects,
we have used microarray techniques to identify the genes
regulated by saffron and PBM in their protective actions.
METHODS
Experimental organization: The protective potential of
dietary saffron, and of PBM, was tested using a light damage
assay. Animals were treated in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision
Research, and with protocols approved by the ANU Animal
Ethics Committee. Young adult Sprague Dawley rats aged
P80–120 were reared in 5 lux cyclic light, and prepared in six
groups. Each group comprised two males and two females.
Control—These animals were raised in 5 lux cyclic light,
as above. They were routinely fed a vegetable (potato or rice)
matrix, developed as a biodegradable packaging material, and
we used the same matrix as vehicle for feeding them with
saffron.
Saffron-exposed only—Animals were fed saffron at
1 mg/kg/day for 3 weeks. Saffron (stigmata of Crocus
sativus, from the Abbruzzo region in Italy) was soaked in
water (at 2 mg of spice/ml H2O) and 12 h was allowed for the
major antioxidants, which are water-soluble [25], to dissolve
fully. The solute was then fed to the rats by injecting a small
volume into a piece of the vegetable matrix, which the animal
readily ingested. The volume for each daily feed was
calculated to provide the solutes from 1 mg of saffron/kg
bodyweight. Tissue was collected 24 h after the last feed.
Photobiomodulation-exposed only—Animals were
exposed to 670 nm red light from a WARP 75 source (60mW/
cm2, Quantum Devices Inc., Barneveld, WI). Animals were
handled gently over several days until they were adapted to
handling. Each was then gently restrained with a towel and
held under a Plexiglas platform with the head ~2.5 cm below
the platform. The WARP75 device was placed on top of the
platform and turned on for 3 min. This arrangement provided
a fluence of 9 J/cm2 at the eye. The animals did not hide from
or appear agitated by the red light. Animals were treated in
this way once daily for 5 days at 9:00 AM. Tissue was
collected 24 h after the last treatment.
Light-damaged only—The
individually in Plexiglas cages, with food kept on the floor of
the cages and water offered from transparent containers, to
ensure uniform exposure. After overnight dark adaptation,
animals were exposed to bright (1,000 lux) light for 24 h, from
a white fluorescent source. Exposure began and ended at 9:00
AM
animals were kept
TABLE 1. TAQMAN PROBES USED FOR QPCR
Name
angiotensinogen (serpin peptidase inhibitor, clade A, member 8)
Beta actin
carnitine O-octanoyltransferase
chemokine (C-C motif) ligand 2
endothelin 2
fatty acid binding protein 5, epidermal
fibroblast growth factor 2
glyceraldehyde-3-phosphate dehydrogenase
glial fibrillary acidic protein
glutathione peroxidase 3
heme oxygenase (decycling) 1
optineurin
signal transducer and activator of transcription 3
suppressor of cytokine signaling 3
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily d, member 1
Gene symbol
Agt
Actb (Control)
Crot
Ccl2
Edn2
Fabp5
Fgf2
Gapdh (Control)
Gfap
Gpx3
Hmox1
Optn
Stat3
Socs3
Smarcd1
TaqMan assay ID
Rn00593114_m1
Rn00667869_m1
Rn00583174_m1
Rn01456716_g1
Rn00561135_m1
Rn00821817_g1
Rn00570809_m1
Rn99999916_s1
Rn00566603_m1
Rn00673916_g1
Rn01536933_m1
Rn00595346_m1
Rn00562562_m1
Rn00585674_s1
Rn01533317_m1
????????Listing of all TaqMan probes used in this project including the reference genes Gapdh and Beta Actin.
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Saffron light damaged—Animals in this group were fed
saffron for 3 weeks, as above. At 9:00 AM on the last day of
feeding, they were exposed to damaging light for 24 h, as
above. Tissue was collected at the end of this 24 h period.
Photobiomodulation light damaged—Animals in this
group were exposed to PBM, as above, for 5 days. Beginning
at 9:00 AM on the last day of treatment, they were exposed to
damaging light for 24 h, as above. Tissue was collected at the
end of this 24 h period.
Tissue collection: At the points in the protocol specified
above, animals were euthanized with Lethabarb (60 mg/kg
intraperitoneally). The retina from one eye of each animal was
dissected free immediately, and placed in an individual tube
containing RNAlater (Ambion Biosystems, Austin, TX), and
stored at 4 °C overnight. The following day, tubes were
transferred to –80 °C. The fellow eye was fixed by immersion
in 4% (W/V) paraformaldehyde for examination of
morphology and immunohistochemistry.
Fellow eyes were marked on the superior aspect with
indelible pen for future orientation, enucleated and
immersion-fixed in 4% (W/V) paraformaldehyde for 3 h,
washed in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4 at pH of 7.4) thrice, then
cryoprotected by immersion in 15% (W/V) sucrose overnight.
Eyes were sectioned at 12 μm on a cryostat in the superior-
inferior axis.
RNA extraction and analysis: RNA was extracted and purified
using previously published methods [36]. To determine the
Figure 1. Photoreceptor rescue by
saffron and photobiomodulation.
Images show inner and outer nuclear
layer of the retina, and the extent of
damage caused by light damage to a
control animal (A), to a animal
subjected to light damage (LD; B) and
to an animal
photobiomodulation and then subjected
to LD (C). The red label, applied with
the TdT-mediated dUTP nick end
labeling (TUNEL) technique, marks
cells whose DNA is undergoing the
fragmentation characteristic
apoptotic death. TUNEL-positive cells
are confined to the ONL, i.e., they are
the somas of photoreceptors. The
number of TUNEL-positive cells is
reduced by PBM pretreatment. D: Mean
numbers of TUNEL-positive cells per
mm of outer nuclear layer, for control,
LD, SafLD, and PBMLD groups. The
reductions in cell death caused by
pretreatment with saffron and PBM
were statistically significant.
pre-treated with
of
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quantity and purity of the sample, RNA was analyzed on an
ND-1000 spectrophotometer (Nanodrop Technologies,
Wilmington, DE) and a 2100-Bioanalyzer (Agilent
Technologies, Santa Clara, CA). RNA samples were used
only if the A260/A280 ratio was above 1.8 and the RNA integrity
number was greater than 8.5.
Microarray analysis: To study the changes in gene expression
induced in the six experimental groups, we used 18
Affymetrix (Santa Clara, CA) Rat Genome ST arrays. These
microarrays contain over
oligonucleotide features representing 27,342 genes. Labeling,
hybridization, washing, and scanning of the microarray were
performed at the Australian Cancer Research Foundation
(ACRF) Biomolecular Resource Facility at the John Curtin
School of Medical Research, Australian National University,
following the manufacturers’ specifications. The arrays were
scanned on the Affymetrix GeneChip 3000 7G high resolution
scanner and analyzed using the GeneSpring GX v10 software
(Agilent Technologies) and Partek Genomic Suite 6.4
Software (Partek Inc., St. Louis, MO). The hierarchical
clustering was performed using GeneSpring on the full entity
list (genes plus noncoding RNA [ncRNA]) for each of the six
groups. Normalization was performed using the Robust
Multichip Average (RMA) algorithm and only gene
expression levels with statistical significance (p<0.05) were
recorded as being “present” above background levels. Genes
with expression levels below this statistical threshold were
considered “absent.” For the box and whisker plot, we first
ran a multivariate ANOVA (ANOVA) analysis on the six
groups to identify genes whose expression was significantly
varied (p<0.05, fold-change >2). This yielded a list of 187
entities, from which the box and whisker plot was generated.
The Partek Genomic Suite was used to identify genes and
ncRNAs whose expression differed between experimental
groups, typically between one experimental group and one
control group. Data in the form of a computerized version of
the .DAT file (CEL) files were imported and gene expression
values were derived using the RMA algorithm on the “core”
metaprobe list, which represents RefSeq genes and full-length
GenBank mRNAs. For each comparison between treatment
and control group, two-sample Student t tests were used to
calculate the probability P that the expression of a gene had
not changed. Genes and ncRNAs whose expression was
significantly changed by treatment were selected using the
criteria that p<0.05 and the fold-change in expression >2. The
microarray data discussed in this publication have been
uploaded to the National Center for Biotechnology
Information (NCBI’s) Gene Expression Omnibus [37] and are
accessible through gene expression omnibus (GEO) Series
accession number GSE22818.
Quantitative polymerase chain reaction: RNA for
quantitative polymerase chain reaction (qPCR) was handled
in the same way as RNA extracted for the GeneChip®
700,000 twenty-five-mer
experiments. Three biologic groups were used, with one
animal in each treatment group. Superscript III and the
accompanying standard protocol (Invitrogen, Carlsbad, CA)
were used to convert 1 µg of retinal RNA to cDNA (cDNA).
TaqMan® (Applied Biosystems, Foster City, CA) Gene
Expression Mastermix (Cat# 4369514) and probes (Table 1)
were used to assess the validity of gene expression changes
identified in the microarray experiment using a StepOne Plus
qPCR machine and StepOne software v2.1 (Applied
Biosystems). Assays were performed in duplicate (to account
for individual sample variability) and biologic triplicate (to
account for biologic variability), with fold changes
determined using comparative cycle threshold (Ct; delta-delta
ct). Both glyceraldehyde 3-phosphate dehydrogenase
(Gapdh) and β-actin (Actb) were used as reference genes in
all qPCR experiments.
Figure 1 shows the protection of light-stressed
photoreceptors in rat retina achieved in the current work,
confirming previous reports for saffron [24] and PBM [30].
Light stress caused the death of photoreceptors, shown as
TUNEL-labeling of cells in the ONL (Figure 1B).
Pretreatment with saffron or PBM reduced the number of
TUNEL-positive cells in the ONL (Figure 1C, for PBM), as
well as reducing the light-induced thinning of the ONL (data
not shown). When quantitative data were pooled (Figure 1D),
significant differences were apparent between the LD group
on the one hand, and the saffron-treated and PBM-treated
groups on the other (control versus LD, p<0.002 on two-tailed
t test; LD versus saffron LD, p<0.0025; LD versus PBMLD,
p<0.002).
TdT-mediated dUTP nick end labeling and quantification:
Cell death was assessed by the TdT-mediated dUTP nick end
labeling (TUNEL) technique to identify the fragmentation of
DNA characteristic of apoptotic cells, following a previously
published protocol [38] but using a fluorophore, Alexa 594,
to visualize the enzymatic reaction. TUNEL-labeled sections
were scanned from superior to inferior edge in 1 mm steps and
the number of TUNEL-positive profiles in each 1 mm of the
outer nuclear layer (ONL) was recorded. The frequency of
TUNEL-positive profiles per mm of ONL was averaged from
at least two sections per animal, and three or four animals were
analyzed for each condition. The Student t test was used to
compare the effects of different treatment conditions.
To demonstrate cell survival, the DNA-specific dye
bisbenzimide (Calbiochem, La Jolla, CA) was used. Sections
were incubated in the dye, diluted 1:10,000 in 1× PBS for 2
min at room temperature.
RESULTS
Global analyses of gene expression: Four approaches were
used to gain an overview of entity (gene and ncRNA)
expression changes in the present data.
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Figure
diagram. This diagram shows the degree
of similarity/difference between the 18
samples used in this study. Each column
represents a sample; there were three
control samples, three samples from
retinas (each retina from a different
animal) treated only with saffron, three
from retinas/animals treated only with
photobiomodulation (PBM), three from
retinas/animals treated only with light
damage (LD), three from retinas/
animals treated with PBM and LD, and
three from retinas/animals treated with
saffronLD. The columns are arranged so
that the most similar ones are next to
each other. The branching lines at the
top indicate in more detail the columns/
samples that are most similar/different.
A: With two exceptions, the three
samples from each experimental group
resembled each other more than samples
in other experimental groups. The
exceptions were PBMLD1, which
resembled the PBM samples more
closely than the other two PBMLD
groups; saffronLD1, which resembled
the PBMLD samples more closely than
the other saffron LD groups. Of the three
treatments used (PBM, saffron, LD), LD
induced the most variable response by
all assessments used. B: When
expression values in the three samples
of each of the six experimental groups
were averaged, a distinct pattern of
similarities emerged. The three saffron-
only samples were closer to control than
the PBM-only, suggesting that saffron
by itself regulates fewer genes/entities
than PBM. The LD-treated groups
clustered together, with the two treated
groups (PBMLD and SaffronLD)
resembling each other more closely than
the LD group. That is, treatment by
PBM and Saffron before LD had
broadly similar effects on the LD-
induced regulation of genes/entities.
2. Hierarchical clustering
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Hierarchical clustering analysis—The hierarchical
clustering of individual replicates (Figure 2A) indicates that
the patterns of gene expression in the three samples of each
group were highly reproducible. Of the 18 samples (3 samples
in each of 6 groups), 16 clustered most closely with samples
from the same group. One exception was PBMLD1, which
clustered with the PBM samples; the other was saffronLD1
(SafLD1), which clustered with two of the PBMLD samples.
Because the saffron and PBM samples clustered closely
within their respective groups, the two exceptions suggest
some variability in the impact of LD on gene expression.
The pattern of clustering obtained when the group
replicas were averaged is shown in Figure 2B. The three
samples exposed to LD cluster together, separate from the
three groups not exposed, indicating that LD has a strong
impact on retinal gene expression. In the three non-LD groups,
the saffron-treated sample clustered closer to control retina,
suggesting that PBM alone has a stronger effect on retinal
gene expression than saffron alone. Within the three LD-
exposed groups, the retinas also exposed to photoreceptor-
protective treatment (PBMLD, SafLD1) show gene
expression closer to each other than to the LD group,
suggesting that PBM and saffron modify the gene expression
induced by LD in broadly similar ways.
Distributions of gene expression in the six averaged
samples—the box and whisker plot—An overview of gene
expression in our six experimental groups is gained from the
“box and whisker” plot in Figure 3. There were 187 genes
included in these analyses; these were selected by a multi-
ANOVA analysis of the six experimental groups (p<0.05, fold
change [FC]>2).
For each sample, the plot shows the median expression
value of these genes as the horizontal line across the box. The
upper and lower ends of the box mark the first and third
quartile values, so that the box “contains” half of the sample
value; the extensions show 1.5xIQR, where IQR is the
interquartile range for the sample. Expression values outside
the extensions are considered outlying values, and are shown
in red.
LD caused the median expression value to rise from the
control value, with the expression of many entities (genes or
ncRNAs) lying in outlier regions (12 above, 16 below).
Saffron has relatively little effect on the distribution of gene
expression levels, but PBM narrows the distribution and
creates outliers. These two protective treatments thus seem to
have distinctive effects. Finally, the effect of PBM and saffron
given before LD was to reduce the LD-induced increase of the
median and to reduce the number of outliers (to none in
PBMLD, one in saffron LD).
Venn diagram analysis: entities associated with
neuroprotection A third overview of entity regulation
associated with the neuroprotective actions of PBM and
saffron is given by a Venn diagram analysis (Figure 4);
numbers are shown separately for known genes and ncRNAs.
The diagram is applied to three sets of regulated entities—
those regulated by LD (compared to control); those regulated
Figure 3. “Box and whisker” plots of the
distributions of entity expression in the
six experimental groups (replicates
averaged). There were 187 genes
included in these analyses; these were
selected by a multi-ANOVA analysis of
the six experimental groups (p<0.05, FC
> 2). For each sample, the plot shows the
median expression value of these genes
as the horizontal line across the box. The
upper and lower ends of the box mark
the first and third quartile values, so that
the box “contains” half of the sample
value; the extensions show 1.5xIQR,
where IQR is the interquartile range for
the sample. The red lines indicate
“outliers,” genes or ncRNAs whose
expression level was greater or less than
1.5xIQR from the median.
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by LD when preceded by PBM (compared to control): and
those regulated by LD when preceded by saffron feeding
(compared to control). LD regulated 175 entities. Of these, 50
(44 known genes, 6 ncRNAs) were not regulated beyond
criterion when LD was preceded by conditioning with PBM
(PBMLD) or with saffron (SafLD). That is, the expression of
these 50 entities (listed in Table 2) was suppressed by both
PBM and saffron conditioning. Their suppression may be
important in the protective actions of PBM and saffron.
When saffron was given to the animal before light
damage (SafLD), the expression of a large number of entities
(48 in common with PBM and 74 unique to saffron) were
regulated, and were not regulated by LD; i.e., their regulation
can be attributed to saffron and may be important in its
protective effect. Similarly, when the retina was conditioned
by PBM before exposure to LD, the expressions of 67 entities
(48 in common with saffron and 19 unique to PBM) was
regulated, which were not regulated by LD. Their regulation
can be attributed to PBM and may be important in the
protective effect of PBM. The entities regulated by saffron
and PBM given before LD, and not by LD, are listed in Table
3.
By separating known genes from ncRNAs, the Venn
diagram analysis draws attention to the prominence of
ncRNAs among the entities regulated by both saffron and
PBM when they are exerting their protective actions. For
example, LD regulated 175 entities, of which only 13 (7.5%)
were ncRNAs. Saffron preceding LD regulated 244 entities,
of which 83 (34%) were ncRNAs; while PBM preceding LD
regulated 116 entities, of which 51 (44%) were ncRNAs.
Among the 48 entities regulated by PBM and saffron, but not
by LD, and which are therefore potentially neuroprotective
entities, 39 (81%) were ncRNAs.
Expression changes: identified genes and noncoding
RNA Given the prominence of ncRNAs among the entities
regulated by saffron and PBM when conditioning LD, we
surveyed the relative numbers of genes and ncRNAs in the
seven comparisons shown in Figure 5A. As already noted, LD
regulated a large number of known genes, but few ncRNAs.
Conversely, ncRNAs outnumber known genes in the action
Figure 4. Venn Diagram showing
similarity and differences between
genes. The diagram is applied to three
sets of regulated entities: those regulated
by light damage (LD; compared to
control); those regulated by LD when
preceded by photobiomodulation
(PBM; compared to control); and those
regulated by LD when preceded by
saffron feeding (compared to control).
These sets were selected by two-sample
Student t test analysis (p<0.05) and fold-
change (FC>2).
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TABLE 2. GENES AND NCRNA SIGNIFICANTLY REGULATED BY TREATMENT WITH PHOTOBIOMODULATION AND SAFFRON.
Probeset ID
Gene assignment
Gene symbol
RefSeq
p-value
FC (LD/C)
10901166
angiopoietin-like 4
Angptl4
NM_199115
0.046613
2.20489
10738477
ADP-ribosylation factor 4-like
Arf4l
NM_001107052
0.027718
−2.12332
10865442
complement component 1, s subcomponent
C1s
NM_138900
0.027086
2.0311
10847761
Cd44 molecule
Cd44
NM_012924
0.017188
2.40207
10771649
chemokine (C-X-C motif) ligand 11
Cxcl11
NM_182952
0.041843
2.71709
10827231
cysteine-rich, angiogenic inducer, 61
Cyr61
NM_031327
0.024324
2.07091
10890654
estrogen receptor 2 (ER beta)
Esr2
NM_012754
0.009894
2.32768
10714890
Fas (TNF receptor superfamily, member 6)
Fas
NM_139194
0.01695
2.9447
10886031
FBJ osteosarcoma oncogene
Fos
NM_022197
0.008085
2.43039
10797527
growth arrest and DNA-damage-inducible, gamma
Gadd45 g
NM_001077640
0.01519
2.2081
10784120
gap junction protein, beta 6
Gjb6
NM_053388
0.033887
−2.2151
10849841
interleukin 1 beta
Il1b
NM_031512
0.046205
2.18761
10862908
interleukin 23 receptor
Il23r
XM_001067609
0.03848
2.92018
10733553
interferon regulatory factor 1
Irf1
NM_012591
0.005382
2.70651
10867306
hypothetical protein LOC683514
LOC683514
NM_001127569
0.028303
2.1278
10934056
moesin
Msn
NM_030863
0.031765
2.04966
10896814
myelocytomatosis oncogene
Myc
NM_012603
0.007411
2.7792
10920860
myeloid differentiation primary response gene 88
Myd88
NM_198130
0.014129
2.34728
10926588
nuclear factor of kappa light polypeptide gene enhancer i
Nfkbie
NM_199111
0.014731
2.17613
10750848
nuclear factor of kappa light polypeptide gene enhance
Nfkbiz
NM_001107095
0.00217
2.32035
10823365
purinergic receptor P2Y, G-protein coupled 12
P2ry12
NM_022800
0.017115
−2.38721
10792421
plasminogen activator, tissue
Plat
NM_013151
0.004342
2.38492
10911484
protogenin homolog (Gallus gallus)
Prtg
NM_001037651
0.027196
2.04521
10842475
protein tyrosine phosphatase, non-receptor type 1
Ptpn1
NM_012637
0.002949
2.18916
10821581
similar to hypothetical protein MGC42105
RGD1308116
ENSRNOT00000021964
0.013255
−2.2474
10710930
similar to hypothetical protein DKFZp434I2117
RGD1308215
NM_001106296
0.016796
−2.2475
10803006
similar to hypothetical protein B230399E16
RGD1559694
ENSRNOT00000020858
0.034301
−2.49615
10882514
RGD1560224
RGD1560224
ENSRNOT00000009292
0.048646
−2.31446
10855681
similar to hypothetical protein
RGD1562590
ENSRNOT00000015469
0.006687
2.09732
10800434
ring finger protein 125
Rnf125
NM_001108424
0.037824
2.3249
10893918
strawberry notch homolog 2 (Drosophila)
Sbno2
NM_001108068
0.007136
2.23202
10765195
selectin, platelet
Selp
NM_013114
0.03163
2.27171
10704505
solute carrier family 1 (neutral amino acid transporter),
Slc1a5
NM_175758
0.028834
2.70661
10736795
schlafen 2
Slfn2
NM_001107031
0.048056
2.03465
10717935
superoxide dismutase 2, mitochondrial
Sod2
NM_017051
0.039527
2.05701
10781273
stanniocalcin 1
Stc1
NM_031123
0.048406
2.26891
10869149
T-cell acute lymphocytic leukemia 2
Tal2
NM_001109462
0.012626
2.32825
10783880
transglutaminase 1, K polypeptide
Tgm1
NM_031659
0.012214
2.7564
10887306
tumor necrosis factor, alpha-induced protein 2
Tnfaip2
NM_001137633
0.038454
2.32289
10858967
tumor necrosis factor receptor superfamily, member 1a
Tnfrsf1a
NM_013091
0.013832
2.36185
10829313
transient receptor potential cation channel, subfamily
Trpm2
NM_001011559
0.009704
−2.04814
10802422
tubulin, beta 6
Tubb6
NM_001025675
0.015119
2.4974
10802995
zinc finger protein 516
Znf516
ENSRNOT00000021768
0.000278
2.01399
10813949
zinc finger protein 622
Znf622
ENSRNOT00000014423
0.011767
2.11
10821585
—

—
0.001925
−2.09732
10802710
—

—
0.005024
2.05529
10857403
—

—
0.007403
2.00653
10813885
—

—
0.00781
2.73034
10859195
—

—
0.025583
2.49356
10752799
—

—
0.026753
3.52422
Genes and ncRNAs (44 known genes, 6 ncRNAs) whose expression was significantly regulated by light damage (LD), and whose regulation was reduced below
criterion when the retina was conditioned by photobiomodulation (PBM) and by saffron. These reductions in regulation may be important for the protective effects
of PBM and saffron.
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TABLE 3. GENES AND NCRNA REGULATED BY PHOTOBIOMODULATION AND SAFFRON DURING LIGHT DAMAGE BUT NOT REGULATED BY LIGHT DAMAGE ALONE.
Probeset ID
Gene_assignment
Gene symbol
RefSeq
p-value
FC (PBMLD/C)
FC (SafLD/C)
10786710
biotinidase
Btd
NM_001012047
0.002593
−2.58996
−2.00118
10727084
cysteinyl-tRNA synthetase
Cars
NM_001106319
0.000472
2.04587
2.27983
10754902
discs, large homolog 1 (Drosophila)
Dlg1
NM_012788
0.007578
2.02011
2.20452
10814281
fatty acid binding protein 12
Fabp12
NM_001134614
0.03715
−2.09977
−2.09843
10797597
isoleucyl-tRNA synthetase
Iars
NM_001100572
0.01099
2.21568
2.14011
10796326
optineurin
Optn
NM_145081
0.001857
−2.04049
−2.16666
10810322
similar to calmegin
RGD1310572
BC097408
0.011866
2.57427
2.03946
10753017
similar to Putative protein C21orf45
RGD1310778
BC167102
0.000554
−2.95657
−2.95334
10758134
ubiquitin C
Ubc
NM_017314
0.006248
−2.2729
−2.14387
10765728
—

—
0.000316
−2.05098
−3.55913
10722459
—

—
0.000643
−4.83475
−5.7297
10722449
—

—
0.000759
−4.77591
−4.75198
10722435
—

—
0.000872
−4.53877
−3.87361
10838282
—

—
0.001154
−3.51509
−4.57167
10703224
—

—
0.001264
−3.60718
−4.66873
10722429
—

—
0.00139
−3.95495
−4.50258
10722465
—

—
0.00144
−4.49582
−5.45683
10722473
—

—
0.00149
−2.89842
−2.9913
10862359
—

—
0.00159
−4.16679
−6.62332
10894268
—

—
0.001622
−3.30485
−2.65319
10932269
—

—
0.001633
−2.51937
−3.05085
10722423
—

—
0.001744
−3.54235
−2.91368
10722461
—

—
0.002014
−3.20546
−3.46585
10722437
—

—
0.002127
−3.81721
−4.06358
10722481
—

—
0.002143
−4.18134
−4.50928
10855946
—

—
0.002276
−4.8482
−5.97919
10722433
—

—
0.002438
−2.97454
−3.52979
10722471
—

—
0.002612
−4.01264
−4.18106
10722453
—

—
0.002883
−2.32871
−2.48323
10721700
—

—
0.002935
−2.66889
−2.35149
10839872
—

—
0.003128
−3.58942
−4.13962
10722431
—

—
0.003286
−2.28992
−2.57576
10722443
—

—
0.003413
−3.36447
−3.16986
10722451
—

—
0.00356
−3.75565
−3.75383
10932228
—

—
0.003868
−2.39044
−2.32665
10722425
—

—
0.004662
−2.86663
−3.06065
10722427
—

—
0.004665
−4.23993
−4.9848
10722479
—

—
0.004887
−2.12953
−2.2299
10870039
—

—
0.005491
−2.02844
−2.04155
10722467
—

—
0.006418
−2.28354
−2.11141
10867318
—

—
0.007967
−3.77635
−2.33207
10722409
—

—
0.012757
−2.28142
−2.24389
10820008
—

—
0.014354
−2.331
−3.03034
10722383
—

—
0.017588
−2.07462
−2.12019
10722379
—

—
0.017621
−2.03053
−2.08741
10707643
—

—
0.020362
−3.27802
−3.23128
10722417
—

—
0.024253
−2.16311
−2.33892
10722387
—

—
0.028341
−2.62369
−2.64182
Genes and ncRNAs regulated by photobiomodulation (PBM) and saffron conditioning, but not by light damage (LD) alone (9 known genes, 39 ncRNAs). Their
regulation by PBM and saffron conditioning suggests that they are important in the protective effects of both PBM and saffron
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of PBM on the control retina (PBM versus control); in the
action of PBM when exerting its protective action against LD
(PBMLD versus LD); and in the protective action of saffron
(saffronLD versus LD). It seems likely that the regulation of
ncRNAs accounts for a significant part of the protective effect.
This suggestion is supported by the difference
comparison in Figure 5B. Measuring only changes in the
numbers of genes and ncRNAs whose expression was
significantly regulated by saffron or PBM before LD, the
protective actions of saffron and PBM are both associated with
increases in the number of ncRNAs regulated, and decreases
in the numbers of identified genes whose expression was
regulated.
As a final step, we considered the directions of entity
expression changes in these several conditions (Figure 5C,
Figure 4D). The most striking outcome of this separation is
that the protective effects of PBM and saffron are associated
with a decrease in the number of known genes upregulated,
and an increase in the number of ncRNAs downregulated.
Validation by real-time PCR: Thirteen genes were chosen for
RT–PCR validation of the microarray outcomes; those chosen
were strongly regulated and/or retina-relevant. Five genes
(Crot, Optn, Edn2, Smarcad1, Gpx3) were significantly
regulated by saffron in the LD assay. Crot and smarcad1 are
involved in fatty acid metabolism, Edn2 in retinal signaling
in response to injury, and Gpx3 in antioxidative activity.
Optn acts as an mgluR1 receptor on retinal bipolar cells.
Fabp5 is also saffron-regulated, and related to fatty acid
metabolism. Fgf and GFAP are proteins upregulated by stress;
Stat3 and Socs3 are related to transduction pathways, ccl2 to
inflammatory responses, and Agt and heme oxygenase 1
(Hmox1) to cardiovascular control.
Figure 6 shows a comparison for each of the 13 genes
between its regulation as assessed by the microarray
procedure and its regulation as assessed by RT–PCR. The
correlation between the two techniques appears particularly
close for ccl2, Socs3, Stat2, Cro, Edn2, Hmox1, Fabp5, and
smarcad. Common trends, with quantitative differences at
some sample points, are evident for Optn, GFAP, Agt, Fgf2,
and Gpx3. Overall, the correlation between the two techniques
seems strong.
Entities associated with the protective actions of saffron and
photobiolmodulation listed:
Light damage–induced regulation inhibited by
photobiolmodulation or saffron—The genes and ncRNAs
whose regulation by LD was inhibited by PBM or saffron are
listed in Table 2; as noted above, this inhibition affected
principally (88%) known genes (44 known genes, 6 ncRNAs).
All 50 entities were upregulated by LD; they are therefore
candidates for genes and regulatory elements whose
upregulation is damaging to photoreceptors.
Regulation by photobiolmodulation and saffron, but
not LD—Table 3 lists genes and ncRNAs that were not
regulated by LD but were regulated by PBM and saffron when
conditioning (protecting) photoreceptors challenged by LD.
Figure 7 shows that the effects of PBM and saffron on their
regulation were highly correlated. The entity regulation
shown in Table 3 contrasts in two ways with the pattern of
regulation in Table 2: Most of the entities whose regulation
was changed by saffron and PBM conditioning were ncRNAs
Figure 5. Analysis of entities regulated
(known genes versus ncRNAs) and
direction of regulation. A: Numbers of
genes and ncRNAs regulated in seven
comparisons among the experimental
groups. SaffLD is the group given
saffron before light damage (LD). B:
Effects of
photobiomodulation (PBM) on the
numbers of LD-induced expression
changes of known genes and ncRNAs.
C: Direction of regulation of known
genes by PBM and saffron when given
as pretreatments to LD. D: Direction of
regulation of ncRNAs by PBM and
saffron when given as pretreatments to
LD.
saffron and
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(81%), and all the ncRNAs and half the known genes were
downregulated.
Regulation by PBM or saffron, but not light damage
—Further candidates for genes and ncRNAs protective to
Figure 6. Comparisons, for thirteen
selected genes, of expression changes in
the six experimental groups, assessed by
qPCR and microarray analysis.
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photoreceptors can be found in 74 entities (37 known genes,
37 ncRNAs) regulated by saffron (but not by PBM) when
conditioning/protecting photoreceptors (Table 4), and in the
19 entities (9 known genes, 10 ncRNAs) regulated by PBM
(but not by saffron) when conditioning/protecting
photoreceptors (Table 5).
Regulation by LD, SaffronLD, and PBMLD—Genes
found to be regulated by SaffronLD and LD (Table 6),
PBMLD and LD (Table 7), and SaffronLD, PBMLD, and LD
(Table 8) are shown in the corresponding tables. These genes
are not discussed as the changes in expression levels are likely
due to LD and not saffron or PBM.
DISCUSSION
The present results provide an overview of gene and ncRNA
regulation associated with the neuroprotective actions of PBM
and saffron. The analyses used were chosen partly to provide
validation of the method, for example the hierarchical
clustering analysis in Figure 2 and the microarray-PCR
comparison in Figure 6. In addition, they allow a compare-
and-contrast discussion of the possible actions of saffron and
PBM.
The box-and-whisker presentation in Figure 3 suggests
that PBM and saffron acting on the retina in the absence of a
light challenge have distinct effects. Saffron has relatively
little effect on the expression of genes by the retina, but when
given as pretreatment to LD, saffron reduced the large changes
in gene expression induced by LD. PBM by itself had a much
more significant effect on retinal gene expression than saffron,
narrowing the distribution of entity expression changes and
generating many “outliers.” PBM given as pretreatment to LD
reduced the gene expression caused by LD toward control
levels.
The Venn diagram analysis allowed a logical separation
of lists of genes and ncRNAs whose regulation appears to
contribute to neuroprotection; it also draws attention to the
prominence of ncRNAs (rather than known genes) among the
entities regulated during the protective action of PBM and
saffron.
Figure 7. Evidence of similarity in the
actions of
photobiomodulation. This graph shows
the correlation for 48 entities (9 known
genes and 39 ncRNAs) between the
change in gene expression associated
with photobiomodulation (PBM) and
saffron pre-treatments.
saffron and
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TABLE 4. GENES NCRNA REGULATED BY SAFFRON PRE-CONDITIONING BUT NOT PHOTOBIOMODULATION OR LIGHT DAMAGE.
Probeset ID
Gene_assignment
Gene symbol
RefSeq
p-value
FC (SafLD/C)
10808041
alanyl-tRNA synthetase
Aars
NM_001100517
0.009419
2.12845
10920371
coiled-coil domain containing 72
Ccdc72
NM_001126048
0.000362
−2.15161
10753771
Cd47 molecule
Cd47
NM_019195
0.004538
2.18358
10840895
cytochrome c oxidase subunit IV isoform 2
Cox4i2
NM_053472
0.006367
−2.04762
10860548
carnitine O-octanoyltransferase
Crot
NM_031987
0.000412
2.42699
10871623
endothelin 2
Edn2
NM_012549
0.003864
2.39144
10791631
ectonucleotide pyrophosphatase/phosphodiesterase 6
Enpp6
NM_001107311
0.048871
−3.40078
10740135
fascin homolog 2, actin-bundling protein, retinal (Stro
Fscn2
NM_001107072
0.002804
2.03501
10938219
glycerol kinase
Gk
NM_024381
0.000123
2.16424
10732439
guanine nucleotide binding protein (G protein), gamma 1
Gng13
NM_001135918
0.006565
−2.23127
10733680
glutathione peroxidase 3
Gpx3
NM_022525
0.00182
−2.17933
10715200
helicase, lymphoid specific
Hells
NM_001106371
0.000882
2.2618
10863430
hexokinase 2
Hk2
NM_012735
0.029119
−2.24022
10733056
interferon gamma inducible protein 47
Ifi47
NM_172019
0.001063
2.31444
10714903
interferon-induced protein with tetratricopeptide repea
Ifit3
NM_001007694
0.004673
3.02663
10753784
intraflagellar transport 57 homolog (Chlamydomonas)
Ift57
NM_001107093
0.000101
2.67228
10815873
interleukin 12a
Il12a
NM_053390
0.003212
−2.04998
10804187
leucyl-tRNA synthetase
Lars
NM_001009637
0.000471
2.05987
10932310
mediator complex subunit 14
Med14
XM_228713
0.02603
2.23949
10923270
oligonucleotide/oligosaccharide-binding fold containin
Obfc2a
NM_001014216
8.85E-05
2.0681
10855114
olfactory receptor 820
Olr820
NM_001000974
0.025967
2.01366
10830003
pterin-4 alpha-carbinolamine dehydratase/dimerization c
Pcbd1
NM_001007601
0.009857
−2.05998
10708281
phosphodiesterase 8A
Pde8a
NM_198767
0.005571
−2.00121
10889475
peroxidasin homolog (Drosophila)
Pxdn
ENSRNOT00000060139
0.00349
−2.6691
10803138
RNA binding motif, single stranded interacting protein
Rbms2
NM_001025403
0.002005
−2.15568
10716415
similar to enolase (46.6 kDa) (2J223)
RGD1308333
NM_001134505
0.015495
2.04964
10820002
similar to Ac1147
RGD1563254
XM_577969
0.029198
−2.23707
10771190
similar to ATP-binding cassette, sub-family G (WHI
RGD1564709
NM_001107205
0.044266
2.04858
10797566
sphingosine-1-phosphate receptor 3
S1pr3
ENSRNOT00000019473
0.00326
2.25911
10750282
solute carrier family 5 (inositol transporters), member 3
Slc5a3
NM_053715
0.002179
2.1573
10842440
solute carrier family 9 (sodium/hydrogen exchanger), m
Slc9a8
NM_001025281
0.000656
2.12805
10899174
SWI/SNF related, matrix associated, actin dependent r
Smarcd1
NM_001108752
0.002766
−2.04334
10831606
transporter 1, ATP-binding cassette, sub-family B (MDR/TAP)
Tap1
NM_032055
0.011581
2.37179
10902375
TBC1 domain family, member 15
Tbc1d15
ENSRNOT00000005207
0.000945
2.0265
10858370
ubiquitin specific peptidase 18
Usp18
NM_001014058
0.019532
2.19315
10907681
zinc finger protein 385A
Zfp385a
NM_001135088
0.000479
−2.14878
10846652
zinc finger protein 385B
Zfp385b
NM_001107736
0.001897
−2.11791
10840061
—

—
0.047419
2.15179
10924441
—

—
0.044499
2.20978
10891487
—

—
0.042933
2.01488
10886190
—

—
0.040809
−2.64706
10898158
—

—
0.040237
−2.11912
10930226
—

—
0.039217
−2.1554
10915105
—

—
0.033403
−2.30406
10886854
—

—
0.032145
2.23693
10731193
—

—
0.031476
2.4581
10843907
—

—
0.030096
2.01264
10875117
—

—
0.027972
2.25392
10801781
—

—
0.026332
2.81764
10825167
—

—
0.022483
−2.8835
10722375
—

—
0.019218
−2.05422
10803001
—

—
0.018227
2.34996
10819500
—

—
0.017801
2.16908
10766880
—

—
0.015099
2.10145
10938891
—

—
0.013364
2.13311
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TABLE 4. CONTINUED.
Probeset ID
Gene_assignment
Gene symbol
RefSeq
p-value
FC (SafLD/C)
10776604
—

—
0.012016
−2.84767
10757702
—

—
0.009955
−2.06188
10867008
—

—
0.009246
−2.29587
10891878
—

—
0.008961
−2.10309
10830454
—

—
0.008641
−2.44031
10827830
—

—
0.006903
2.30678
10897004
—

—
0.006062
2.01807
10742429
—

—
0.005794
2.98095
10834602
—

—
0.005352
−2.59778
10926624
—

—
0.005005
2.14084
10781982
—

—
0.004882
2.31978
10896630
—

—
0.003343
−2.13243
10930622
—

—
0.002504
2.29803
10756086
—

—
0.002238
2.23035
10923938
—

—
0.00188
2.22066
10938893
—

—
0.001545
2.49702
10899788
—

—
0.000365
−2.13694
10766722
—

—
8.76E-05
2.60553
10752219
—

—
1.94E-05
−2.05925
Genes and ncRNAs regulated saffron conditioning, but not by photobiomodulation (PBM) and not by light damage (LD) alone (37 known genes, 37 ncRNAs).
Their regulation by saffron conditioning suggests that they are important in the protective action of saffron, and not of PBM.
Molecular Vision 2010; 16:1801-1822 <http://www.molvis.org/molvis/v16/a196> © 2010 Molecular Vision
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TABLE 5. GENES AND NCRNA REGULATED BY PHOTOBIOMODULATION PRE-CONDITIONING BUT NOT SAFFRON OR LIGHT DAMAGE ALONE.
Probeset ID
Gene_assignment
Gene symbol
RefSeq
p-value
FC (NIRLD/C)
10916920
ATP synthase, H+ transporting, mitochondrial F0 complex, s
Atp5l
NM_212516
0.03394
−2.14869
10867731
calbindin 1
Calb1
NM_031984
0.03827
−2.09147
10770342
epoxide hydrolase 1, microsomal
Ephx1
NM_001034090
0.019435
−2.16344
10892381
nudix (nucleoside diphosphate linked moiety X)-type mo
Nudt14
NM_001106760
0.018954
−2.0385
10847156
olfactory receptor 673
Olr673
NM_001000351
0.015388
2.0757
10801260
protocadherin gamma subfamily B, 6
Pcdhgb6
ENSRNOT00000060466
6.03E-05
2.18939
10891322
polyglutamine-containing protein
Pqcp
NM_001012470
0.018634
2.00327
10796307
similar to calcium/calmodulin-dependent protein ki
RGD1560691
NM_001107365
0.019657
2.35325
10817552
thioredoxin interacting protein
Txnip
NM_001008767
0.018274
−2.32009
10886988
—

—
0.000109
25.1775
10718602
—

—
0.000392
2.10568
10721694
—

—
0.011105
−2.00257
10919224
—

—
0.011219
−2.57253
10758033
—

—
0.021858
−2.84458
10840318
—

—
0.028594
2.23714
10878967
—

—
0.031786
−2.35082
10713602
—

—
0.036424
−2.41324
10886894
—

—
0.04513
−2.03777
10797671
—

—
0.049361
−2.07756
Genes and ncRNAs regulated by photobiomodulation (PBM) conditioning, but not saffron and not by light damage (LD) alone (9 known genes, 10 ncRNAs). Their
regulation by PBM conditioning suggests that they are important in the protective effects of PBM, but not in the protective action of saffron. Entities regulated by
PBM, when exerting its protective action (9 known genes, 10 nc RNAs)
Molecular Vision 2010; 16:1801-1822 <http://www.molvis.org/molvis/v16/a196> © 2010 Molecular Vision
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