s2fieldsofstudy: Biology Chemistry Medicine

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A New Approach to Reduce Toxicities and to Improve Bioavailabilities of Platinum-Containing Anti-Cancer Nanodrugs

Platinum (Pt) drugs are the most potent and commonly used anti-cancer chemotherapeutics. Nanoformulation of Pt drugs has the potential to improve the delivery to tumors and reduce toxic side effects. A major challenge for translating nanodrugs to clinical settings is their rapid clearance by the reticuloendothelial system (RES), hence increasing toxicities on off-target organs and reducing efficacy. We are reporting that an FDA approved parenteral nutrition source, Intralipid 20%, can help this problem. A dichloro (1, 2-diaminocyclohexane) platinum (II)-loaded and hyaluronic acid polymer-coated nanoparticle (DACHPt/HANP) is used in this study. A single dose of Intralipid (2 g/kg, clinical dosage) is administrated [intravenously (i. v.), clinical route] one hour before i.v. injection of DACHPt/HANP. This treatment can significantly reduce the toxicities of DACHPt/HANP in liver, spleen, and, interestingly, kidney. Intralipid can decrease Pt accumulation in the liver, spleen, and kidney by 20.4%, 42.5%, and 31.2% at 24-hr post nanodrug administration, respectively. The bioavailability of DACHPt/HANP increases by 18.7% and 9.4% during the first 5 and 24 hr, respectively.

A major limitation for both approved and in-development nanodrugs is their rapid clearance by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system, in particular liver and spleen, which can increase their toxicity to these off-target organs and reduce their efficacy 13,15,22 . Strategies that decrease RES uptake and increase the bioavailability of nanomedicines can improve tumor targeting and decrease the side effects. Many studies have been conducted to decrease RES clearance and to increase the tumor targeting of nanomedicines by modifying nanoparticle characteristics, such as the size, shape, charge, surface property, and composition [23][24][25][26][27][28] . Unfortunately, the total accumulation of the anti-cancer nanodrugs in the tumor represents a small fraction of total injected dose (1-10%). The majority (40-80%) of the injected nanomedicines end up in the liver and spleen 22 . Moreover, each new modification calls for thorough toxicity, pharmacology, and biomechanics studies before translating to a clinical setting.
Our strategy is to target the RES to temporarily blunt the uptake, i.e., to decrease the toxicities in liver and spleen and to increase the bioavailability of nanodrugs using Intralipid 20%, an FDA-approved fat emulsion used as parenteral nutrition source. The rational for this hypothesis is that the infusion of Intralipid has been reported to inhibit RES function by possibly inhibiting peritoneal clearance and impairing the phagocytic activity of Kupffer cells 29 . Kupffer cells in the liver play an important role in the uptake and metabolism of Intralipid 30 . Our recent findings also support this hypothesis. We have found that, in rodents, Intralipid can reduce RES uptake ~50% and increase blood half-life (t 1/2 ) ~3-fold of nano-and micron-sized superparamagnetic iron-oxide particles 31,32 .
We have carried out this study with an improved Pt anti-cancer nanodrug, DACHPt-incorporated nanoparticles (NP), coated with hyaluronic acid polymer (HA) (DACHPt/HANP). We have found that a single, clinical dose of Intralipid (2 g/kg) can significantly reduce the toxic side effects of our Pt-containing nanodrug in liver, spleen, and kidney. Notably, our findings indicate that Intralipid pre-treatment decreases spleen enlargement, which has been reported as a serious side effect of Abraxane ® . A single dose of Intralipid can decrease Pt accumulation in the liver (by 20.4%), spleen (42.5%), and kidney (39.3%) at 24-hr post nanodrug administration. Consequently, the bioavailability of the Pt-nanodrug increases by 18.7% during the first 5 hr and by 9.4% during 24 hr, respectively.
Intralipid Reduces Toxic Side Effects of Pt-Containing Nanodrug. Intralipid 20% was administered to Sprague Dawley (SD) rats at the clinical dose (2 g/kg) using the clinical route (i.e., intravenously) one hour before i.v. injection of DACHPt/HANP. At 24-and 72-hr post injection of DAHPt/HANP, blood samples were collected to determine serum alanine aminotransferase (ALT) activity and creatinine level to investigate liver and kidney damages. The tissue samples collected at 72-hr post injection were used for histological analysis. The tissue samples from naïve (SD) rats were used as controls.
Pathological Analysis and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay for Apoptotic Cells in Liver. Light microscopic images of hematoxylin/eosin (H & E) stained liver tissue sections are shown in Fig. 1A-C,F-H,K-M. Images of TUNEL stained liver tissue sections are shown in Fig. 1D,E,I,J,N,O. With DACHPt/HANP administration, but no Intralipid pre-treatment, the pathological changes in the liver tissue are characterized by necrosis, as indicated by black arrows in Fig. 1C, which is an example of enlarged view from Fig. 1A,B. Apoptotic cells are observed with TUNEL staining, as indicated by red arrows in Fig. 1D,E, from the liver tissue of this treatment group. An enlarged view of an apoptotic cell is shown as an example in Fig. 1E. These damages are significantly reduced upon Intralipid pre-treatment. The liver tissue sections from the Intralipid pre-treated group are shown in Fig. 1F-J. Very few cell necroses (black arrow in Fig. 1H) and apoptotic cells (red arrows in Fig. 1J) are observed, comparable to the liver tissues of naïve rats (Fig. 1K-O). Spleen Enlargement. Spleen swelling and enlargement are observed from DACHPt/HANP-treated animals, when the animals are sacrificed 72-hr post nanodrug administration ( Fig. 2A left). Intralipid pre-treatment appears to reduce spleen swelling ( Fig. 2A right). The ratio of spleen weight/body weight for a naïve Sprague Dawley (SD) rat is 0.31 ± 0.06 (n = 3). Intralipid treatment does not cause spleen swelling with the ratio of 0.28 ± 0.02 (n = 3). The ratio from a DACHPt/HANP treated SD rat is 0.53 ± 0.08 (n = 3). Upon Intralipid pre-treatment, this ratio reduces to 0.4 ± 0.008 (n = 3). In Fig. 2B, the ratios are shown as the percentage of the normal level.

Changes of DACHPt/HANP Accumulation in Tissues upon Intralipid Pre-Treatment. The Pt
concentration in tissue (spleen, liver, and kidney) and blood of naïve animals or Intralipid along or phosphate-buffered-saline (PBS) treated animals is below 0.01 part per million (ppm).
With the drug being metabolized in the liver, the Pt concentrations reach similar level at 72 hr: 10.1 ± 1.6 and 11.8 ± 3.7 (μ g/g wet weight), without-and with-Intralipid pre-treatment, respectively.

Discussion
After several decades, the research seeking for less toxic Pt drugs and better drug delivery methods, which can decrease the associated side effects and improve the anti-cancer efficacy as well as the quality of life of the patients, still goes on. We have found a new approach to reduce the side effects and increase the bioavailabilities of an anti-cancer Pt-containing nanodrug (DACHPt/HANP), by using an "old" FDA approved agent, Intralipid. Since the approval of cisplatin in 1979, Pt-based drugs, including carboplatin and oxaliplatin (second and third generation), have become the most potent as well as the most widely prescribed anti-cancer drugs 2 . Unfortunately, its continuous use is greatly limited by dose limiting toxicities, partial anti-tumor response in most patients, development of drug resistance, and tumor rela pse 2,3,5,8-10,33 . Nanocarrier-based drug delivery may generate new therapeutic approaches for Pt-drugs. Pt-based nanodrugs are providing encouraging preclinical and clinical results and may facilitate the development of the next generation of Pt chemotherapy 18,19,34 . However, the important questions of how to decrease the RES uptake, which accounts for 40-80% of injected nanodrugs, and how to reduce the toxic side effects caused by this off-target uptake, still need answers. Our studies show that Intralipid pre-treatment can be used to reduce RES uptake and side effects, and improve the bioavailability and clinical applications of Pt-containing nanodrugs. Moreover, we have observed that Intralipid treatment can decrease Pt accumulation in kidney, thus reducing nephrotoxicity of the Pt drug.

Figure 5. Effects of Intralipid pre-treatment on the serum ALT activities (A) and creatinine levels (B)
in DACHPt/HANP treated rats. When the rats are treated with 4 mg Pt/kg of the nanodrug, Intralipid pretreatment group shows significantly lower serum ALT activity and creatinine level (A,B). The group, which is pre-treated with Intralipid followed by the treatment of a higher dosage (6 mg Pt/kg) of DACHPt/HANP, exhibits lower ALT activity (A) and creatinine level (B) than the group, which is treated with 4 mg Pt/kg of the nanodrug, but no Intralipid. * p < 0.001; ** p < 0.05. spleen would cause toxic side effects. For many nanomedicines, the toxicity in the mononuclear phagocyte system is the killer for further development 35 . DACHPt loaded in HANP induces cancer cell apoptosis by causing cross-linking of DNA and DNA-protein. DACHPt-loaded polymeric micelles have been reported to cause liver toxicity 20 . When animals were sacrificed at 72-hr post DACHPt/HANP administration, we observed dramatic swelling and enlargement of the spleen from DACHPt/HANP-treated animals. Pre-treatment with Intralipid 20% (clinical dose, 2 g/kg) can reduce spleen swelling significantly (Fig. 2). Pathological and cell apoptosis analyses reveal that Intralipid can be used to decrease the toxic side effects of our anti-cancer nanodrug in the mononuclear phagocyte system (Figs. 1 and 3). The serum ALT assay also indicates that Intralipid can protect liver from the damage caused by the nanodrug off-target accumulation (Fig. 5A).
In a previous study 31 , we have found that in rodents, Intralipid can reduce RES uptake by ~50% of nano-and micron-sized particles in which MRI contrast agents are loaded. The RES plays an important role in the uptake and metabolism of Intralipid 30,36 . The blood half-life of Intralipid 20% administered by intravenous bolus in rats is 8.7 ± 3.0 min 30,36 . The diameter of the Intralipid particles range from 200 to 1000 nm 37 . As shown in Fig. 6A,B, Intralipid pre-treatment decreases the liver and spleen uptake of the nanodrug by 20.4% and 42.5% at 24-hr post nanodrug administration, respectively.
Interestingly, Intralipid pre-treatment can also decrease the Pt accumulation in the kidney (Fig. 6C1,C2). Nephrotoxicity is one of the most severe side effects of current Pt drugs 2,3,6,8-10 . DACHPt/ HANP nanodrug is designed to increase the concentration and prolong the half-life of DACHPt at tumor sites and to decrease the side effects like nephrotoxicity. Although our Intralipid therapy was originally designed to decrease the RES uptake of the nanodrug, Intralipid pre-treatment could also decrease the Pt drug (DACHPt and/or DACHPt/HANP) accumulation in kidney by 28.7% at 72 hr. Regarding the Pt concentration, we should keep in mind that two components contribute to the Pt concentration: the DAHPt/HANP nanodrug and the DAHPt molecule, which is released from the polymer coating. As a consequence, Intralipid also decreases the nephrotoxicity of the Pt-nanodrug (Figs. 4 and 5B).
This protective effect of Intralipid is so potent that the rats from a higher dosage treatment (6 mg Pt/kg of DACHPt/HANP) exhibit a less hepatocellular and nephrocellular damages (Fig. 5A,B). This indicates that, using Intralipid, the clinicians might be able to give the patients more anti-cancer nanodrugs to kill the tumors with less toxic side effects! Intralipid can change the clearance and increase the bioavailability of the nanodrug. Our results show that a single dose of Intralipid can increase the bioavailability of DACHPt/HANP by 18.7% during the first 5 hr (Fig. 7). It has been reported that after i.v. administration of Intralipid, the circulating ketone bodies increased ~100% in 30 min, which indicates an active metabolism of Intralipid by the liver 30 . This active metabolism might explain the decrease of the effectiveness of Intralipid after 5 hr. To increase and prolong the effectiveness of Intralipid, the administration routes, dosages, and time courses of Intralipid treatment need to be optimized in a future study. Multiple administrations of Intralipid may be necessary.
Moreover, the development of targeted nanomedicine has made an important impact in new drug development in neurology 38 , cardiology 39 , and inflammation 33 . The EPR effect is found not only in cancer, but also in a wide range of inflammatory diseases, such as atherosclerosis [40][41][42] . Thus, our findings for Intralipid pre-treatment could have broad applications besides cancer.

Concluding Remarks
Our study shows that Intralipid can be used to reduce the toxic side effects of Pt-containing anti-cancer nanodrugs in the liver, spleen, and kidney, and also to improve the bioavailability of the nanodrug. Our approach is also a general one applicable to any approved and in-development nanodrugs to improve their bioavailability and to decrease their toxic side effects, without any new modification of the nanodrugs and/or the nanocarriers. Intralipid has been used for over 40 years as a safe source of parenteral nutrition for patients and so can readily translate to clinical use. The outcome of this study has the potential to decrease the toxic side effects of anti-cancer nanodrugs and other nanodrugs, and therefore to reduce human suffering. Also, increasing efficacy could lead to a reduction of the dosage of these expensive drugs: the average cost per dose is US$4,000-6,000. Thus, our findings for the use of Intralipid with nanodrugs can also lead to the reduction of healthcare costs as well as to the improvement of the quality of life for patients who undergo the therapeutic treatment. Preparation and Physical Properties of DACHPt/HANP. DACHPt/HANP was prepared with modified procedures from a previously described method 18 . In brief, DACHPtCl 2 was mixed with silver nitrate ([AgNO 3 ]/[DACHPt] = 2) to form an aqueous complex. The solution was kept in the dark at 25 °C for 24 hr. AgCl precipitates were removed by centrifugation followed by filtration through a 0.22-μ m hydrophilic polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). Subsequently, HA/ Boc-His/PEG graft copolymers, comprising hyaluronic acid (Mw = 16 kD), were added to the aqueous complex of DACHPt at a 0.33 molar ratio of DACHPt to carboxylate groups of the HA modified polymers. The mixture was stirred in the dark for three days at 25 °C. The reaction mixture was sonicated and then purified by ultrafiltration against deionized water to remove uncoordinated DACHPt. The product was filtered through a 0.22-μ m PVDF membrane and lyophilized with 10% trehalose.

Materials and
The particle size and PI of DACHPt/HANP was determined by dynamic light scattering using a ZetaPlus (Brookhaven, Holtsville, NY). Zeta potential was measured by the laser Doppler anemometry (Zeta Plus zeta potential analyzer, Brookhaven Instruments Corporation). TEM images were taken by using a Cryo transmission electron microscope (Cryo-TEM) [JEM-2100 (JEOL, Tokyo, Japan)] operated at 200 kV with attachment of energy dispersive spectroscopy (EDS). A droplet of DACHPt/HANP solution was adsorbed on a cleaned carbon film supported copper grid. After excess sample was removed, phosphotungstic acid (Merck) was used as negative stain reagent to improve the image contrast. TEM grid was dried in the contamination-free environment and reserved in the electronic dry cabinet for further TEM analysis.
Encapsulation Efficiency of DACHPt in DACHPt/HANP. In order to determine the encapsulation efficiency of DACHPt in the nanocomplex, the amount of Pt were quantified by inductively coupled plasma-optical emission spectrometry (ICP-OES) in preparation processes. Encapsulation efficiency (EE %) was calculated using below formula: where W P is the total amount of Pt after purification by passing through a 0.22 μ m filter and W T is the total quantity of Pt determined before purification.
Experimental Design. Male SD rats, with body weights between 250 and 280 g, were used. Intralipid 20% was administered by intravenous injection at a clinical dose of 2 g/kg. PBS was administered to control animals. After one hr, DACHPt/HANP (2 mg Pt/kg for bioavailability and biodistribution studies, n = 14 for Intralipid pre-treatment group and n = 14 for control group; 4 mg Pt/kg for toxicity studies, n = 3 for Intralipid pre-treatment group and n = 3 for control group; 6 mg Pt/kg for another toxicity study to test the serum ALT activity and creatinine level, n = 3 for Intralipid pre-treatment group) was injected intravenously. Blood samples were collected at different time points to determine the bioavailability of DACHPt/HANP. At 5-, 24-, and 72-hr post injection of DACHPt/HANP, tissues (liver, spleen, and kidney) were collected for the Pt-level determination. The tissue samples collected at 72-hr post injection were used for histological analysis.

Blood
Bioavailability. An indwelling jugular vein catheter was used for repeated blood samplings.
Blood samples (100 μ L) were collected at different time points to determine the changes of bioavailability of DACHPt/HANP upon Intralipid treatment. Blood was sampled after DACHPt/HANP injection at 1, 5, 10, 20, 45, and 60 min, 3, 5, 24, 28, 48, 52, and 72 hr. The blood samples were decomposed in HNO 3 (0.5 mL) at 60 °C overnight and re-dissolved in 0.5 mL of 2 N HCl 18,20,43 . Suitable dilutions were prepared using 5% HCl to reach a final Pt concentration in the range of 0.02 to 1 part per million (ppm). Samples were analyzed for Pt concentration by inductively coupled plasma-mass spectrometry (ICP-MS) [NexION 300X (PerkinElmer, Waltham, MA)], with modified procedures from our previous studies 31,32 . 194 Pt, 195 Pt, and 196 Pt isotopes were analyzed and similar results were obtained from the measurement of these three isotopes. The Pt concentrations shown in this manuscript were calculated from the measurements of 194 Pt. Bioavailability was calculated by the area under the curve (AUC), namely the integral of the concentration-time curve, using the trapezoidal rule with the use of KaleidaGraph 4.1 (Synergy Software, Reading, PA).
Pt Levels in Tissues. The wet weight of each tissue sample was recorded. Tissue homogenate (0.5 mL) was decomposed in HNO 3 (1 mL) at 60 °C overnight. The rest of the tissue was fixed in 4% paraformaldehyde for histological analyses. The HNO 3 -digested samples were evaporated and then re-dissolved in 0.5 mL of 2 N HCl 43 . The Pt concentrations in the solution were analyzed by ICP-MS as described above.
Pathological Analysis and TUNEL Assay. Histological examinations and TUNEL assays were performed by the Transplantation Pathology Laboratory of the University of Pittsburgh Medical Center (Pittsburgh, PA). Paraffin-embedded 5-μ m sections were stained with hematoxylin/eosin (H & E), or performed TUNEL staining. For histopathological diagnosis, slides were examined by light microscopy and photomicrographs were taken using a Moticam 2300 camera mounted on an Olympus Provis microscope with Mtic Images Plus 2.0 software.
ALT Activity Assay and Creatinine Colorimetric Assay. The activity of ALT in serum was measured by using the ALT Activity Assay Kit purchased from Sigma-Aldrich, according to the supplier's protocol. Serum creatinine level was measured by using the Creatinine Colorimetric/Fluorometric Assay Kit purchased from BioVision. Statistical Analysis. Statistical analysis was carried out with Student's t test. A p value < 0.05 was considered statistically significant.


extfieldsofstudy: Biology Medicine

text:
Orchestration of ErbB3 signaling through heterointeractions and homointeractions

ErbB receptors form homodimers and heterodimers between family members. To model ErbB2/ErbB3 signaling, single-particle tracking data are used to create a simulation space with overlapping receptor domains. Stochastic modeling of receptor dimerization and phosphorylation reveals the complexity of ErbB2-3 interactions.

Domain Reconstruction Algorithm: Overview
One of the major undertakings of this algorithm is to extract static spatial data from dynamic trajectory data. To accomplish this task, we devised a ranking system that sorts all recorded points of an individual trajectory into two groups: confined (slow moving) or free (unobstructed). The confined points are what would be considered to be in a confinement zone, while the free points are considered to be "free" on the membrane.

Trajectory Analysis
To rank the points in the SPT trajectories, we first calculate the corresponding jump size (displacement) for time steps corresponding to a selected number of frame intervals (1,2,5,10,20,50,100,200). The time step is applied moving forward through the trajectory as well as backwards through the trajectory. Covering this range of step sizes accounts for "holes" that may be in the trajectory data due to experimental conditions such as blinking of the quantum dots or quantum dots temporarily moving out of the focal plane. These jump sizes are calculated for each trajectory in a set of comparable SPT files, then aggregated by step size.
Once the jump size distributions have been compiled, the individual points from each trajectory are ranked. For every point, the relative rank is calculated by comparing the point's jump size to all the other points' jump sizes for a specific step size. The jump sizes for a specific step size are sorted in order from smallest to largest. The rank of a specific point is where it falls in that order, and is converted into a relative (percentage) rank. This is repeated across all points for each step size.
Some points may not have a score for all 16 step sizes due to the aforementioned holes in the trajectories. To account for this, a weighted average is used to determine the overall rank ( score) for each point. First, for each point, the forward and backward ranks for a step size are combined. The combined ranks for each step for that point are then averaged together; this is the final rank for that point.

Cluster Analysis
We rely on distance based hierarchic clustering. This approach had been widely used in the literature, ranging from ecology and genomics to receptors on the cell membrane (Espinoza et al, 2012). Our method is a modified version of that developed by Espinoza and coworkers for TEM images of receptors labeled with gold nano-particles.
Given N points {P 1 ,P 2 ,…,P N } in a plane, with coordinates {(x 1 ,y 1 ),…(x N ,y N )}, we want to partition them into mutually exclusive groups or clusters in a way that reflects their proximity or similarity to each other. This is not a clearly defined notion and the appropriate clustering method should be ultimately determined by the experimental context. Here, we use the "slow" points identified from jump size distributions as indicators of an underlying physical structure (such as lipid rafts); therefore the notion of proximity defined by physical distance to the closest members of the cluster is more appropriate considering, for instance, an average distance to the entire cluster (this is the idea underlying K-means clustering).
We construct clusters by comparing the distance between points to a reference length L, sometimes called a "scale". Two points A, B are in the same cluster if their distance ; we denote this relationship by A ~L B. We extend the relationship by transitivity: if A ~L B and A ~L C, then B ~L C. It is easy to see that the procedure will induce a partition of the set of points. (The ~L relation is symmetric and transitive, therefore it is an equivalence relation in the mathematical sense; the clusters are the corresponding equivalence classes.) The partition into clusters is unique for a given set of points and length scale L. Any two points in a cluster can be linked by a connected path consisting of line segments of length ≤ L connecting points from the same cluster ( Figure S1A).

Contour Drawing
In order to build a geometric area (shape or footprint) around a given cluster, we start with the union of the circles of radius L/2 centered on all members of the cluster. We assume the cluster resulted from hierarchic clustering with distance parameter L, so any member of the cluster must be reached from any other member through a sequence of segments connecting cluster points, such that no individual segment is long than L. The connection graph, shown in Figure S1A, is constructed by putting an edge between all pairs of points with distance ≤ L.
To straighten the boundary of the region defined this way, we extend the area by adding rectangles of height L along the contour of the connection graph (double lines Figure S1C). The reconstructed region is the reunion of the inside of the contour graph, and the circles and rectangles around the vertices and edges of the contour (shaded, respectively grey areas on Figure S1D).
The contour graph or "tight contour" for a cluster of points is defined by the sequence of boundary points and the segments that connect them. The list is constructed by adding new points to the contour, based on the existing points and a reference direction. As the contour is built, it circles around the points in a counter-clockwise direction, so that all the interior points are to the left hand side. When the process is finished, the last point in the contour is identical to the first one.  c. Order the remaining candidates by the clockwise angle from the reference direction to the segment connecting the current point to the candidate; choose the candidate with the smallest angle and add it to the list. d. Set reference direction to point from the newly added point back to the previously added point 3. The process terminates when the same segment is added to the contour. The same point may be visited twice, in opposite directions. Upon successful termination, the last point in the list is the same as the first one. NOTE: The contour defined this way is not unique, but the algorithm always returns a contour that is a refinement of the convex hull of the points, is not self-intersecting, and contains no edges longer than L.

Contour Inflation
The tight contour or contour graph defines the reconstructed region. This contour is often lacking an "inside", and can have sections that are just a single chain of points. The rationale of padding is to represent the area "of influence" of each point in the cluster -roughly, the set of geometric locations that are closer to this cluster than to any points that are not part of the cluster.
The padding adds two types of elements to the core contour and its interior: 1. A rectangle of height L/2 on the outer side of each edge of the tight contour graph 2. A sector of a circle of radius L/2 at each vertex with a positive (convex) angle The contour inflation algorithm constructs a second contour (the outer or padded contour) that encloses the first one. Similarly to the tight contour, the padded contour defined as a polygonal line; the vertices of the polyline are all auxiliary points, and are not elements of the cluster.
The contour inflation algorithm proceeds along the inner contour, and builds the outer contour parallel to it, adding one or more points to account for each vertex of the inner contour. If the angle at the respective vertex is positive (convex), we add the corners of the padding rectangles and points on the arc of the padding circle centered on the vertex; if the angle is negative (concave), then we only add one point, namely the intersection of the two padding rectangles. The perimeter and area of the final shape are given by the length and enclosed area (if any) of the tight contour, plus easily calculable additions due to the padding procedure. Below we describe the algorithm we employed. This algorithm also relies on a reference direction, which corresponds to the segment preceding the current vertex (with the direction defined by that of the algorithm -counterclockwise in our case).
Contour Inflation Algorithm 1. Start at a point on the tight contour. Moving counter-clockwise along the contour, set the reference direction to that of the incoming edge in the counter-clockwise direction, and the current direction to the outgoing edge. 2. The elements of the new contour that are added to represent the current vertex are set based on the angle between the incoming and outgoing directions. a. If the angle is positive (left turn, convex vertex), then add a sector of a circle of radius L/2, centered on the current vertex, and delimited by the radii perpendicular to the incoming and the outgoing edges. The sector is discretized as a sequence of points starting with the end the radius orthogonal to the incoming edge and ending at the other one. At least one intermediate point is added at the end of the radius that is the symmetry axis of the sector. b. If the angle is negative (right turn, concave vertex), we only add the point where the two adjacent rectangles intersect. This point is also on the symmetry axis (bisector) of the angle on the inner contour. 3. The algorithm proceeds along the vertices of the inner contour, adding points to the outer contour for each of them. The process terminates when the starting vertex is reached, and is about to be passed in the same direction as in the first iteration.
A Figure S4. Dimer type distribution after 120 seconds, where ErbB2 is limiting. A BioNetGen version of our model was run for 120 seconds with a ratio of 1:20 ErbB2:ErbB3 receptors. Due to the low level of ErbB2 receptors, ErbB3 receptors are largely sequestered in homodimers. Figure S5. An amino acid substitution of E933Q results from a single point mutation in one of two ErbB3 alleles in the widely-used SKBR3 breast cancer cell line. Figure S2. Compiled ranks for all the points from SPT data for ErbB2 and ErbB3. A) The biomodal distribution for both species is independent of QD label pairs. Here, the QD labels are switched by comparison to Figure 2B in main text (erbB2 QD-655; erbB3 QD-585). B) and C). Plots of characteristic length studies. The average domain perimeter was calculated over a range of characteristic lengths for ErbB2 (B) and ErbB3 (C). The minimum of the average domain perimeter was used to determine the optimal characteristic length that defines the maximum distance. Figure S3. Effects of ErbB inhibitors on ErbB kinase activity. (A) Results of an in vitro kinase assay using immunoprecipitated ErbB3 isolated from CHO stably transfected with HA ErbB2 + ErbB3-mCitrine; cells were incubated with or without 2 min stimulation with 12 nM HRG. Isolated protein was incubated with 10 µM inhibitors (afatinib, sapitinib, or lapatinib) for 15 minutes at 30 o C prior to the addition of ATP. Phosphorylation of the EAY substrate was detected using a pan-phospho-tyrosine antibody, PY20-HRP and EAB substrate. (B) Results of an in vitro kinase assay on immunoprecipitated ErbB2 isolated from resting CHO HA ErbB2 cells. Inhibitors were added as in A.
. Figure S6. Characterization of the ErbB3 E933Q mutation. A.) State-dependent mean squared displacement of ErbB3wt or ErbB3E933Q receptors was calculated from single particle tracking data. Although ErbB3E933Q receptors have a faster mean squared displacement over all, they show similar slowed diffusion when pairs are interacting especially with ligand. B.) Normalized ErbB3 PY1289 phosphorylation levels after 2 minute stimulation followed by lapatinib treatment (10 µM) in CHO ErbB3 wt versus CHO ErbB3 E933Q were plotted over time. As in Figure 3, phosphorylation levels of both receptors were set to 1 for the two minute time point after HRG stimulation. Points were fit to a one-phase exponential decay curve to determine the dephosphorylation half-life. Figure S7. Simulation results with higher levels of ErbB2 alone show increased ErbB2 phosphorylation indicating that overexpression of ErbB2 leads to the formation of active ErbB2 homodimers. ErbB2 is able to overcome the low on-rate for homodimer formation at high density.
A B


Inflammation-related cytokine gene polymorphisms in Behçet’s disease

Behçet’s disease (BD) is a complex, multisystemic inflammatory disorder of unclear etiology. Single nucleotide polymorphisms in tumor necrosis factor (TNF) and interleukin (IL)-10 genes have been implicated in susceptibility to BD with inconsistent results in several ethnic populations. The aim of this case-control study was to evaluate the association of TNF-α (−308G/A), TNF-β (+252A/G), and IL-10 (−1082G/A, −819C/T, and −592 C/A) polymorphisms with susceptibility of BD in Saudi patients. Molecular genotyping of TNF-α, TNF-β, and IL-10 gene polymorphisms was performed to analyze the alleles and genotypes distribution in 272 Saudi subjects, including BD patients (61) and healthy controls (211). The frequencies of allele A and genotype GA of TNF-α (−308G/A) were significantly higher, whereas those of allele G and genotypes GG were significantly lower in BD patients than controls, indicating that A allele and GA genotype are susceptible, while G allele and GG genotype may be refractory to BD. The distribution of frequencies of alleles and genotype of TNF-β (+252A/G) promoter polymorphism was not significantly different between BD patients and healthy controls. Genotypes 1082GG, −819TT, and 592AA of IL-10 polymorphisms are significantly associated with susceptibility risk of BD, while genotypes 1082AA, 1082GA, 819CC, 819CT, 592CC, and 592CA are resistant to BD. This study indicates that TNF-α (−308G/A) and IL-10 (−1082G/A, −819C/T, and −592C/A) polymorphisms are associated with risk of BD susceptibility in Saudi patients. However, larger scale studies in Saudi population as well as in other ethnicities are needed to confirm this association.

Three polymorphisms -1082 A/G (rs1800896), -819 T/C(rs1800871), and -592 A/C (rs1800872) in the promoter region of the IL-10 gene are correlated to the expression level of IL-10. Similarly, several polymorphisms within the promoter region of TNF-α and the intron 1 polymorphism of TNF-β, in particular, have been associated with altered levels of circulating TNF-α. 26,27 TNF-α (-308G/A) polymorphism (rs1800629) affects a consensus sequence for the binding site of transcription factor AP-2 and leads to a less common allele-A (allele 2), which has been associated with increased TNF-α production in vitro 28 and higher rate of TNF-α transcription than wild-type allele G (allele 1). Allele A has been shown to produce six-to sevenfold higher levels of TNF-α transcription. 17,29,30 TNF-β, which is closely linked to TNF-α, has also been shown to contribute to the susceptibility of several inflammatory/autoimmune diseases. [31][32][33][34][35] Higher level of TNF-β is produced by the γδ T-cells of BD patients than by those of healthy controls. 36,37 A polymorphism TNF-β (+252A/G) (rs909253) has been reported to consist of a guanine (TNF-β +252G) on one allele and an adenine (TNF-β +252A) on the alternate allele. The presence of G at this position defines the mutant allele known as TNF-β*1 (allele-1) which is the less frequent allele in white subjects and is associated with higher TNF-α and TNF-β production. 38,39 The candidate gene approach has been useful in identifying susceptibility and severity genes in BD. 40 Besides HLA-B51 molecules, SNPs in TNF and IL-10 genes have been implicated in susceptibility to BD. 24,[41][42][43] However, the results are inconsistent and variations are found among several ethnic populations. So in this study, the association of five polymorphisms in TNF-α, -β, and IL-10 genes with BD susceptibility risk in Saudi patients was evaluated.

Methods subjects
A total of 272 Saudi subjects visiting Prince Sultan Military Medical City (PSMMC), Riyadh, Saudi Arabia, were recruited for this study. Sixty-one unrelated patients with BD, ranging from 20 to 64 years, and 211 unrelated healthy matched voluntary Saudi blood donors, age ranging from 20 to 60 years, were studied for polymorphisms in TNF-α, -β, and IL-10 genes. Patients with any other inflammatory/autoimmune diseases were excluded from the study. Power was calculated online (http://www.stat.ubc.ca/~rollin/stats/ssize/ caco.html).
This study was approved by the research and ethical committee of PSMMC, and written informed consent was obtained from each subject before recruitment. All patients were diagnosed according to the diagnostic criteria prepared by the international study group for BD. 44 Detailed information including demographics, disease duration, treatment duration, and clinical features were obtained from a review of medical records and an interview at the time of enrollment. We evaluated the clinical features such as oral ulcers, genital ulcers, skin lesions (including papulopustular and erythema nodosum-like), pathergy response, ocular inflammation, gastrointestinal lesions, articular manifestations, vascular lesions (including deep vein thrombosis, superficial thrombophlebitis, and aneurysm), central nervous system involvement, and epididymitis. Active or inactive form of BD was determined by clinical parameters. 44

PcR amplification
Genomic DNA was extracted from the peripheral blood of BD patients and controls using QIAamp ® DNA mini kit (Qiagen, Valencia, CA, USA). TNF-α, TNF-β, and IL-10 genes were amplified using amplification refractory mutation systems (ARMS)-polymerase chain reaction (PCR) methodology 45 to detect polymorphisms at position -308 of TNF-α, +252 in Intron1 of TNF-β, and at positions -1082, -819, and -592 of IL-10 genes. PCR amplification was carried out using Ready-To-Go™ PCR Beads (GE Healthcare UK Ltd, Little Chalfont, UK). PCR consisted of ten temperature cycles of denaturation for 15 seconds at 94°C, annealing for 50 seconds at 65°C, and extension for 40 seconds at 72°C. This was followed by 25 cycles of denaturation for 20 seconds at 94°C, annealing for 50 seconds at 59°C, and extension for 50 seconds at 72°C. Final extension was performed at 72°C for 7 minutes. A positive control was included in the PCR assay by amplification of the human growth hormone gene. The amplified product for various samples were separated on a 1.5% agarose gel, stained with ethidium bromide, and then photographed. For quality control, 25% of the random blind samples were repeated for genotyping and positive and negative controls were also used in the PCR. The differences in allele/genotype frequencies between patients and controls were analyzed by the Fisher's exact test. P-values #0.05 were considered significant. The strength of the association of disease with respect to a particular allele/ genotype is expressed by odds ratio, interpreted as relative risk (RR) following the Woolf 's method as out lined by Schallreuter et al. 46 It was calculated only for those alleles/ genotypes which were increased or decreased in BD patients as compared to control group. The RR was calculated for all the subjects using the formula given below: Preventive fraction (PF) indicates the hypothetical protective effect of one specific allele/genotype for the disease. PF was calculated for negative association only, where RR ,1 using the following formula: 47 Values ,1.0 indicate the protective effect of the allele/genotype against the manifestation of disease.

Results
Demographic data and clinical characteristics of patients with BD are presented in Tables 1 and 2. The number of controls per case was 3.4, which yielded a power of 90%. The most frequently found clinical symptoms in BD patients were oral, genital, ocular, musculoskeletal, and cutaneous followed by gastrointestinal, nervous, renal, vascular, and pulmonary symptoms, whereas cardiovascular symptoms were absent in our BD patients. Among BD patients, the male to female ratio was 43:18 (2.4:1). There was no significant difference in clinical manifestation or prognosis comparing men to women in our study.
The genotype and allele frequencies of TNF-α (-308G/A) and promoter polymorphism are presented in Table 3. For quality control, 25% of the random blind samples were repeated and genotyping results were compared with 100% success rate.
The frequency of heterozygous genotype GA was significantly higher in BD patients than controls (P,0.01), whereas the frequency distribution of homozygous genotypes GG and AA was significantly lower in BD than controls (P,0.05). The frequency of allele A was significantly higher in BD patients than control subjects (P,0.01). On the other hand, allele G was significantly lower in BD patients as compared to controls (P,0.01).
The distribution of frequencies of alleles and genotype of TNF-β (+252A/G) promoter polymorphism did not differ   Table 3). The frequencies of allele A and genotypes GA and AA were slightly higher, whereas those of allele G and genotype GG were lower in BD patients than control subjects. The frequency of -1082GG genotype was found to be significantly higher (P=0.04) in BD patients as compared to controls subjects. On the contrary, the frequency of heterozygous genotype GA was significantly lower (P=0.03) in patients as compared to control subjects. The frequency of homozygous AA genotype was higher in patients compared to controls, but the difference was not statistically significant ( Table 4).
The frequencies of -819CC and -819CT genotypes do not differ significantly between BD patients and controls, while the frequency of -819TT genotype was significantly higher in BD patients as compared to controls (P=0.04). Similarly, the frequency of homozygous -592AA genotype was significantly higher in BD patients as compared to controls (P=0.04), while CC and CA genotypes frequencies varied insignificantly in patient and control groups (Table 4).

Discussion
Clinical manifestations in our BD patients were almost similar to those reported earlier from Saudi BD patients by Al-Dalaan et al. 48 Men were more affected than women. The male preponderance has also been reported in the Middle Eastern countries earlier by Kaklamani et al. 49 Influence of sex on BD is well-known, and several reports have suggested a more severe course of disease among males. 50 However, recently Davatchi et al 51 found no strong association between the male sex and major organ involvement, except for vascular lesions. In contrast, females were more affected with BD than males in Japan and Korea. 52 In our study, although no significant difference in clinical manifestation or prognosis between men and women was noticed, however, results should be interpreted with caution as the number of male and female patients is very low.
Significantly higher frequencies of allele A and genotype GA of TNF-α (308) polymorphism in patient group than controls (P,0.001) indicated that allele A and genotype GA are associated with the BD susceptibility risk in Saudi patients. The RR in GA carriers for the disease was very high (RR =43.236). On the other hand, the homozygous genotypes GG and AA might be protective for BD as their frequencies were significantly lower in patient groups than controls (P,0.001 and P=0.04, respectively). Our results are in agreement with a recent meta-analysis which showed higher frequency of allele A in Asian BD population. 42 Our results clearly suggested that A allele and GA genotype confer susceptibility to BD and might be responsible for higher levels of TNF-α in BD patients as TNF-α (308) allele A has been associated with increased TNF-α expression. 53 Moreover, patients with ocular manifestation have statistically significant higher serum levels of TNF-α than those without ocular manifestations.  Significantly higher TNF-α level has been reported in BD patients with active disease compared to those with inactive disease. 14,54 Contrary to these, the G allele and GG genotype of TNF-α (308) polymorphism have been associated with increased risk of BD in Koreans, 55 while no association of TNF-α (308) polymorphism with BD was reported in Turkish, 22,56,57 Korean, 58 Tunisian, 23 Lebanes, 59 Iranian, 60 and Moroccan population. 25 Though several individual studies from various geographical regions or ethnicities showed no association between TNF-α (308) polymorphism and BD, when pooled together in meta-analysis, a significant association of TNF-α polymorphism with BD was observed. 42,43 Zhang et al 43 indicted that the TNF-α (-308G/A) and TNF-α (-857T/C) polymorphisms are linked with BD only in Asians, while the TNF-α (-238A/G) and TNF-α (-1031C/T) polymorphisms are associated with BD in Caucasians. The other meta-analysis of 12 studies by Liang et al 42 using the fixed-effect model also showed an association of the TNF-α (-308G/A) polymorphism with the susceptibility to BD. The discrepancy between the individual studies and meta-analysis has been explained by the facts that the sample size of individual studies is relatively small and that the allelic frequencies of genes often differ substantially in different ethnic groups as shown in various reports. 42,43 In our study, the frequency of TNF-α (308) AA genotype was zero in Saudi BD patients. These results are in agreement with several other findings which also reported the absence of AA genotype in Lebanese, Iranian, and Moroccan BD patients. 25,59,60 Absence of AA genotype in BD patients indicates that AA genotype might be protective for BD. Similarly, lower frequency of GG genotype in Saudi BD suggests its protective role in the Saudi cohort.
Our results of TNF-β (+252A/G) polymorphism showed no significant association with BD in Saudi patients. Other available reports also indicated that there is no association of TNF-β polymorphism with BD in Korean and Tunisian populations. 23,58 In contrast, Verity et al 61 reported a higher TNF-β*2 allele (A allele) frequency among BD cases compared to controls in Palestinian and Jordanian populations. They found strong linkage disequilibrium between HLA-B*51 and allele A of TNF-β (+252A/G) and suggested that both the alleles contribute to disease risk and that their coexpression leads to severe blinding in BD patients. Mizuki et al 62 also reported significantly lower frequency of TNF-β1 (G allele) homozygote in Japanese ocular BD patients than controls. These variations in the association between TNF-β (+252) polymorphism and BD may reflect the heterogeneity in the genetic susceptibility to this disorder.
Our results indicated that genotype-1082GG of IL-10 (-1082G/A) is susceptible to BD, while genotype AA and GA are resistant to BD. Similar to our results, Talaat et al 63 also reported increased frequency of genotype-1082GG and decreased frequency of genotype GA in Egyptian BD patients as compared to controls. However, Dilek et al 64 found lower frequency of AA genotype in Turkish BD patients compared to the control group, but a higher frequency of -1082GA genotype. Contrary to this, Wallace et al 20 reported from two populations (UK and Middle Eastern cohort) that the -1082AA genotype was weakly associated with BD when all patients were analyzed as a group, but not in the UK or Middle Eastern cohorts of patients alone compared to local controls.
Our results also showed that -819TT and 592 AA genotypes of IL-10 are significantly associated with susceptibility risk of BD in Saudi patients. IL-10 (-819T/A) polymorphism has been indicated to be significantly associated with BD. 42 Wu et al 65 reported that -819 T/A polymorphism is associated with BD in Chinese Han population. An association with -819T was also reported in UK and Middle Eastern BD patients, and this was because of an association in the UK but not Middle Eastern patients. 20 Another meta-analysis with results from Turkish and Korean cohorts also showed genome-wide significant associations with -819T of IL10. 19 A meta-analysis showed that IL-10 (-1082G/A, -819C/T, -592C/A) polymorphisms are significantly associated with BD, 42  Recently, emphasis has been given to address and clarify the functional relevance of the different genes found to be associated with BD susceptibility and the potential interactions between genes located within and outside the major histocompatibility complex region. 66 Our results, suggesting an association of allele A and genotype GA of TNF-α (308) with BD in Saudi patients, indicate the functional relevance of the allele A as it is linked with higher levels of TNF-α reported in active BD patients as compared to controls. 14,54 The proinflammatory cytokines, including TNF-α, are potent inducers of inflammation, and elevated levels of these cytokines are frequently associated with the activation of macrophages, thereby influencing the severity of inflammatory responses. These cytokines induce local inflammatory responses and also exert systemic effects. The overexpression of these cytokines may be responsible for the pathogenesis of recurrent BD. 55 TNF-α has been suggested to be directly responsible for activation of T-cells and neutrophils, the main pathogenic changes in BD. 67  (1082GG genotype) of IL-10, an anti-inflammatory cytokine, may not suppress the activity of TNF-α, resulting in increased inflammatory responses in BD patients. IL-10 limits the secretion of proinflammatory cytokines, such as TNF-α and IL-12. 68 It is possible that the pathophysiology of a deficiency in the IL-10 involves undue and prolonged activation of mononuclear cells, resulting in an augmented efflux of inflammatory cytokines including TNF-α. IL-10 is a multifunctional cytokine with profound involvement in diverse areas of the human immune system. 69 These results might have prognostic value for future clinical observations. Further, TNF-α (-308) polymorphism may serve as a guideline in determining the response to anti-TNF-α therapy, as patients with GG genotype are better responders to anti-TNF-α treatment than those with AA or GA. 70,71 However, such associations need further validation and investigation in more patients with BD, as they may have implications for the development of novel therapies as suggested by Xavier et al. 72 The main weakness of the study is the small sample size, while the strengths of the study lies in the fact that it is the first report from Saudi patients with BD, showing association of five polymorphisms in IL-10 and TNF-α and -β genes. The errors in genotyping both cases or controls have been avoided carefully by using a standard protocol with positive and negative controls. Statistical analysis was performed to get P-values, RR, etiologic fraction and preventive fraction, and power.

Conclusion
This study, though based on a small number of cases, clearly shows that TNF-α (-308) and IL-10 (-1082, -819, and -592) polymorphisms are significantly associated with BD susceptibility. This is the first report showing association of TNF-α and IL-10 polymorphisms with BD in Saudi Arabian patients.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
A single acute hepatotoxic dose of CCl4 causes oxidative stress in the rat brain

Carbon tetrachloride (CCl4), a hepatotoxic agent is widely used to study the toxic mechanisms in experimental animals. We have investigated whether oxidative stress is induced in the brain at a single hepatotoxic dosage (1 ml/kg bw) of CCl4. Increased lipid peroxidation (LPO), protein carbonyls (PC) content and glutathione (GSH) depletion were observed in the brain regions of rats treated with CCl4 which was higher than that of liver. A drastic reduction in the activity of glutathione-S-transferase (GST) was seen in the brain regions which was higher than that of liver. Similarly, activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), NADH- and NADPH-dehydrogenase were reduced in the brain regions similar to that of liver. Higher induction of oxidative stress in the brain compared to that of liver implies vulnerability of the brain for CCl4 neurotoxicity. Our study shows that a single hepatotoxic dose of CCl4 is equally neurotoxic to rats.

Introduction
Oxidative stress (OS) results as a consequence of imbalance between ROS and cellular antioxidant defenses. ROS is implicated in the etiology of many disease conditions including cardiovascular diseases, neurodegenerative disorders, cancer and aging [1][2][3]. Major source of free radicals in vivo is autoxidation of flavin thiols, activity of electron transport chain, oxidases, cyclooxygenases, peroxidases etc. [4]. Environmental sources of OS include xenobiotics, organic solvents, pesticides, tobacco smoke, anesthetics, drugs and radiation [5].
Carbon tetrachloride (CCl 4 ) is widely used to induce hepatotoxicity in experimental animals. CCl 4 hepatotoxicity is characterized by hepatocellular necrosis with fat deposition. At acute toxic doses of CCl4, when hepatocellular necrosis exceeds the regenerative capacity of the liver, fatal liver failure often ensues. High doses of CCl 4 results in nonspecific toxicity, including central nervous system depression and respiratory failure resulting in death [6]. The free radicals generated from CCl 4 and the parent molecule by itself, damage endoplasmic reticulum (ER), which leads to accumulation of lipids, reduced protein synthesis and mixed function oxidases activity [7]. CCl 4 belongs to the class of hepatotoxins, which act after metabolic activation. It is metabolized in the endoplasmic reticulum by cytochrome p 450 enzymes (mostly CYP2E1) to the highly reactive trichloromethyl radical (CCl 3 • ). CCl 3 • rapidly reacts with oxygen to form the highly reactive trichloromethylperoxyl radical (CCl 3 OO • ), which rapidly reacts with lipids to form lipid peroxidation products [8]. Polyunsaturated fatty acids or PUFA of the ER and mitochondria are more susceptible to oxidation by the free radicals. The free radical mediated lipid peroxidation is one of the main mechanisms of hepatic injury by CCl 4 [7]. The brain is highly vulnerable to OS than other organs of the body in view of the unusually high rate of oxygen consumption, being rich in PUFA, and low levels of antioxidant enzymes coupled with high amount of non-haem iron [9,10]. Neurotoxic compounds induce OS by inducing lipid peroxidation and altering the antioxidant defenses in the brain [11][12][13]. In view of this, we hypothesized that brain could be a vulnerable target organ for the action of CCl 4 . Although the hepatotoxicity of CCl 4 is well understood, reports on the effects of CCl 4 , on the brain are sketchy [14]. In this study, we have investigated whether a single sublethal, hepatotoxic dose of CCl 4 causes OS in the brain of the laboratory rat.

Animals and treatment
Ninety days old adult male Wistar rats (200-230 g) were divided into two groups of four each. CCl 4 was dissolved in sunflower oil. To the control group, only the vehicle (sunflower oil) was administered. A single dose of CCl 4 (1 ml/kg bw), the dose at which hepatotoxicity occurs [15] was administered by oral gavage and, animals were sacrificed by ether anesthesia after 24 h. Liver and brain were removed and the brain dissected on ice to get the different regions, viz, forebrain, midbrain, and hindbrain, which were processed immediately for biochemical assays. The institute animal ethical guidelines were followed for the animal experiments.

Lipid peroxidation
Lipid peroxidation (LPO) in tissue homogenate was measured by estimating the formation of thiobarbituric acid reactive substances (TBARS) [16]. Tissue homogenate (10% w/v in 1.15% KCl) was mixed with 1.5 ml 20% TCA and 1.5 ml 1.34% TBA and, boiled for 30 min, cooled, which was followed by the addition of 2.5 ml butanol and centrifuged at 2000 rpm for 5 min. The upper layer was read at 535 nm. TBARS as malondialdehyde (MDA) content of the sample was calculated using the molar extinction coefficient 1.43 × 10 −3 M −1 cm −1 and expressed as nmol of MDA formed/mg protein.

Protein carbonyls
Protein carbonyls were estimated by preparing 10% w/v tissue homogenates in 20 mM Tris-HCl buffer (pH 7.4), centrifuged at 3000 rpm for 20 min at 4 • C and 1 ml of the supernatant was precipitated with an equal volume of 20% TCA and centrifuged. The pellet was suspended in 10 mM DNPH and allowed to stand at room temperature for 60 min with occasional vertexing. This was followed by adding 0.5 ml 20% TCA to the mixture and centrifuged, the pellet obtained was washed three times with ethanol:ethylacetate (1:1) and solubilized in 2% SDS (in 20 mM Tris-HCl, 0.1 M NaCl, pH 7.4). Protein carbonyl (PC) content was measured by the method of Levine et al. [17]. The absorbance was read at 360 nm in UV-vis spectrophotometer and the carbonyl content was calculated using a molar extinction coefficient of 22,000 M −1 CM −1 and expressed as g/mg protein.

Glutathione
Glutathione (GSH) content was estimated by the method of Ellman et al. [18], 10% tissue homogenates were prepared in 5% w/v TCA, centrifuged at 2000 rpm for 10 min and supernatant (GSH) was mixed with 10 mM DTNB in 0.1 M phosphate buffer (pH 8.0). The mixture was incubated for 10 min at room temperature and the color was read at 412 nm. Glutathione content was calculated by a standard curve and expressed as g/mg protein.

Antioxidant enzymes
Tissues were homogenized (10% w/v) in ice cold Tris buffer (50 mM, pH 7.4) and centrifuged at 3000 rpm for 20 min at 4 • C. The supernatant was used for biochemical assays.
Glutathione-S-transferase (GST) activity was assayed by the method of Warholm et al. [19] with CDNB as the substrate. The reaction mixture contained 20 mM GSH and the enzyme (supernatant) in 0.1 M phosphate buffer (pH 7.4). The reaction was started by adding 30 mM CDNB and change in the absorbance at 344 nm was monitored in spectrophotometer. The enzyme activity was expressed as nmol CDNB conjugate/min/mg protein.
Glutathione peroxidase (GPx) activity was assayed by the indirectcoupled oxidation method using glutathione reductase [20]. Cumenehydroperoxide (1 mM) and glutathione (0.25 mM) were used as substrates and coupled oxidation of NADPH by glutathione reductase (0.25 U) in Tris buffer (50 mM, pH 7.6) was monitored at 340 nm.
Glutathione reductase (GR) activity was measured by the method of Calberg and Mannervick [21]. The reaction mixture contained 20 mM GSSG and 2 mM NADPH in 100 mM phosphate buffer (pH 7.4). The reaction was started by adding the enzyme and the absorbance change was measured at 340 nm for 3 min.
Superoxide dismutase (SOD) activity was measured using pyrogallol (2 mM) autooxidation as described by Marklund and Marklund [23]. The reaction mixture contained pyrogallol in 0.1 M Tris buffer (pH 8.2) and the enzyme. The reaction was started by adding the substrate and the absorbance was read at 420 nm for 3 min at an interval of 1 min.
Catalase (CAT) activity was assayed by the method of Aebi [22]. The reaction mixture contained 3% H 2 O 2 in 0.05 M phosphate buffer (pH 7.0). The reaction was started by the addition of 100 l of enzyme and the change in absorbance was read at 240 nm for 3 min and activity was expressed as n mole H 2 O 2 /min/mg protein.
NADH-dehydrogenase activity was assayed in a reaction mixture containing 1 mM NADH, 1 mM NBT and enzyme supernatant in 0.02 M Tris buffer (pH 7.4) for 20 min at 37 • C, followed by the addition of 1% Triton X-100. The color developed due to reduction of tetrazolium (formazan) was read at 560 nm.
NADPH-dehydrogenase activity was assayed by the method of Dawson et al. [24]. The reaction mixture containing the enzyme, NADPH (0.5 mM), EDTA (5 mM), NBT (0.5 mM), in 50 mM Tris buffer (pH 7.4) was incubated for 15 min at 37 • C and the color was read at 560 nm as described above.

Protein estimation
Protein estimation was determined by the method of Lowry et al. [25] using bovine serum albumin as the standard.

Oxidative stress markers
Marked increase in lipid peroxidation was seen in all the brain regions of CCl 4 treated rats, being highest in the forebrain (159%) followed by hindbrain (105%) and midbrain (62%), whereas liver showed 63% increase (Fig. 1). Significant increase in protein carbonyl content was observed in the brain regions of CCl 4 treated rats which was higher than that of liver. Reduction of GSH content was seen in the brain regions of CCl 4 treated rats which was highest in the hindbrain (58%) followed by midbrain (46%) and forebrain (40%). GSH depletion in the liver was 31% which was lower than that of brain. Overall, brain showed higher oxidative stress than liver (Fig. 1).

Antioxidant enzymes
Effect of CCl 4 on the antioxidant enzymes was marked and showed distinct regional variation in the brain. A marked reduction in the activity of GST was observed in all the brain regions compared to that of liver in CCl 4 treated rats (Fig. 2). Forebrain CCl4-induced oxidative stress in the rat brain in comparison with the liver. GST: Glutathione-S-transferase; GPx: Glutathione peroxidase; GR: Glutathione reductase. Each bar represents the mean ± S.E, (n = 4). Data were analyzed by Independent Sample T test using SPSS version 14, (*p < 0.05, ** p < 0.01, ***p < 0.005). The numbers above the dark bar represent the percentage difference between control and treated groups. and midbrain showed almost 90% reductions in the enzyme activity while hindbrain showed 80% reduction, whereas, in the liver, it was 45% reduction. GPx and GR activity were reduced by about 50% in all the brain regions, which was comparable to that of liver (Fig. 2).
SOD activity in the liver was decreased by 75%, while in the brain regions, it ranged from 50% to 67%. (Fig. 3). Catalase activity was also markedly decreased from 60% (midbrain) to 42% (forebrain), in the brain of CCl 4 treated rats, whereas, in the liver, the reduction was 54% (Fig. 3). NADH-dehydrogenase showed significant reduction in the activity in hindbrain (67%) followed by forebrain (45%) of CCl 4 treated rats whereas, liver showed 52% reduction (Fig. 3). NADPHdehydrogenase, on the other hand, was not significantly affected in the brain in contrast to that of liver which showed 42% reduction in CCl 4 treated rats (Fig. 3).  Fig. 3. CCl4-induced oxidative stress in the rat brain in comparison with the liver. SOD: Superoxide dismutase; CAT: Catalase; NADH-dehydrogenase; NADPH-dehydrogenase. Each bar represents the mean ± S.E, (n = 4). Data were analyzed by Independent Sample T test using SPSS version 14, (*p < 0.05, ** p < 0.01, ***p < 0.005). The numbers above the dark bar represent the percentage difference between control and treated groups.

Discussion
CCl 4, being lipophilic, easily crosses cell membranes and gets distributed in tissues. CCl 4 is rapidly taken up by both liver and brain, but its toxic effects on the brain are poorly understood compared to that of liver [14]. CCl 4 exerts its toxic effects by generation of free radical CCl 3 • which leads to membrane lipid peroxidation [6]. Hepatic injury by a single dose of CCl 4 (1 ml/kg bw) has been reported by us and others which involves induction of OS (15). In this study, we have shown that the dose at which CCl 4 causes hepatotoxicity causes OS in the brain. Our results show a marked increase in TBARS, an index of LPO, in the brain of treated rats which was higher than that of liver. Free radical mediated LPO is one of the main pathogenic mechanisms of hepatic injury by CCl 4 , and therefore, a similar mechanism could cause neurotoxic injury to the brain. Higher LPO in the brain could be attributed to higher lipid content especially in the white matter which is rich in myelin with lower PUFA than grey matter [26]. The midbrain is relatively heavily myelinated, and therefore, is expected to be more resilient to OS [27]. Reactive oxygen species is constantly produced in cells which is scavenged by cellular the antioxidant enzyme defenses during normal cell metabolism [28]. A delicate balance exists between the rate of formation of H 2 O 2 via dismutation of superoxide by SOD activity and the rate of its removal by catalase and GPx. Any impairment in this pathway will affect the activities of other enzymes in the cascade [29,30]. Induction of antioxidant enzyme activities enhances metabolic detoxification and antioxidant defenses of the tissue, while their reduction results in OS [31,32]. In our study, CCl 4 administration to rats led to reduced antioxidant capacity of the brain as evident from the decreased activity of all the antioxidant enzymes. Many neurotoxic chemicals including ethanol cause OS in the brain [33].
LPO is often the consequence of lowered antioxidant defenses. Our results show increased PC content in the brain which indicates oxidative modifications of proteins, with functional consequences including loss of enzyme activity [34,35]. Oxidative modifications to proteins such as glutathione synthetase and mitochondrial aconitase in the brain, resulting in the loss of enzymatic activity have been reported in ischemia-induced brain damage and Alzheimer's disease [36].
Glutathione, a cytosolic tripeptide, ubiquitously present in all cell types at millimolar concentration is the major non-enzymatic regulator of intracellular redox homeostasis [37]. Our result shows that CCl 4 administration led to marked depletion of GSH level in the brain which was higher than that of liver. GSH depletion predisposes the brain to OS due to compromised redox balance. GSH also serves as a substrate for GPx and GST, primarily involved in the detoxification of toxic electrophilic metabolites via formation of GSH-conjugate [38]. During the enzymatic reaction catalyzed by GPx, GSH is oxidized to GSSG which is reduced back to GSH by GR. GR, therefore plays a central role in maintaining the cellular GSH level. Reduction in GR activity by CCl 4 in the brain, as seen in our results, affects the regeneration of GSH, thus accentuating the effects of GSH depletion [28]. GST plays an important role in protecting cells against ROS mediated injury by detoxification of lipid hydro peroxides formed due to oxidative damage [39]. Our results show drastic reduction in the activity of GST in all the brain regions by CCl 4 which could compromise the biochemical antioxidant defenses of the brain. This is significant particularly since the effect of CCl 4 on the hepatic GST is relatively lessmarked compared to that of brain. Marked depletion of GSH and its dependent enzymes in the brain regions by CCl 4 indicates a major deleterious effect on the brain. NAD(P)H dehydrogenases belongs to a class of enzymes that transfer electrons from NAD(P)H to small acceptor molecules such as quinines. NAD(P)H dehydrogenases are important in the redox balance but their precise role in cell injury and tissue damage is not clear. However, these dehydrogenases may be involved in the antioxidant defense system in relation to protection of cells against electrophilic toxicity [40]. Our results show that CCl 4 administration decreased the activity of NADH dehydrogenase in the brain suggesting altered redox status of the cells.
Our results are consistent with earlier observations on the regional differences in the antioxidant enzymes in the brain regions and add to the evidence that the vulnerability to OS of the brain is region-specific [10,13,41]. Regional variation in the response, the brain regions to CCl 4 implies its differential distribution in the brain and/or its metabolism. Another contributing factor for the regional differences in the OS could also involve differences in the neurotransmitter profile [42,43]. Our study has demonstrated that CCl 4 induces OS in the brain regions by marked alteration of antioxidant balance in both enzymatic and non-enzymatic status. Significant reduction of antioxidant enzymes, along with increased LPO and PC content by a single dose of CCl 4 could be considered a deleterious effect of CCl 4 on the brain. The overall OS response to CCl 4 in the brain was higher than that of liver. Although CCl 4 is well known as a hepatotoxic agent, our results show that it is equally a neurotoxic chemical that causes oxidative damage to the brain. Further studies are needed to ascertain the consequences of CCl 4 action on the brain with regard to cellular damage and neurotoxic consequences.


extfieldsofstudy:
Computer Science
Psychology

text:
Auditory Synaesthesia and Near Synonyms: A Corpus-Based Analysis of sheng1 and yin1 in Mandarin Chinese

This paper explores the nature of linguistic synaesthesia in the auditory domain through a corpus-based lexical semantic study of near synonyms. It has been established that the near synonyms 聲 sheng “sound” and 音 yin “sound” in Mandarin Chinese have different semantic functions in representing auditory production and auditory perception respectively. Thus, our study is devoted to testing whether linguistic synaesthesia is sensitive to this semantic dichotomy of cognition in particular, and to examining the relationship between linguistic synaesthesia and cognitive modelling in general. Based on the corpus, we find that the near synonyms exhibit both similarities and differences on synaesthesia. The similarities lie in that both聲 and音 are productive recipients of synaesthetic transfers, and vision acts as the source domain most frequently. Besides, the differences exist in selective constraints for聲 and音 with synaesthetic modifiers as well as syntactic functions of the whole combinations. We propose that the similarities can be explained by the cognitive characteristics of the sound, while the differences are determined by the influence of the semantic dichotomy of production/perception on synaesthesia. Therefore, linguistic synaesthesia is not a random association, but can be motivated and predicted by cognition.

Introduction
Synaesthesia is a phenomenon of one sensation connecting to another, which has been studied in two distinct disciplines. One is neuroscience, which characterizes synaesthesia as a neural disorder (Cytowic, 1993), and the other one is linguistics that widely describes synaesthesia as a metaphor (Williams, 1976;Geeraerts, 2010). This paper is focused on the linguistic synaesthesia.
Synaesthesia occurs commonly and naturally in languages (Huang, 2015), such as "sweet voice" in English and 高音 gao-yin "high pitch" in Chinese, of which "sweet" and 高 gao "high" are normally perceived through gustation and vision respectively, while "voice" and 音 yin "sound" both belong to the auditory domain, therefore, the whole combinations exhibit an association of different sensations, namely synaesthesia.
In terms of research on linguistic synaesthesia, most previous studies are concentrated on the directionality of synaesthetic transfers, that is, which sensation usually acts as the source domain and which sensation is the target domain. For instance, Ullmann (1957) studied creative usages of synaesthesia on 2000 examples from poems in the 19th century, and proposed a tendency of hierarchical distribution for synaesthesia. He concluded that synaesthetic transfers are usually from much "lower domains" (such as touch and taste) to much "higher domains" (such as vision and hearing) 1 , and the acoustic field emerges as the main recipient. Williams (1976) also claimed that the diachronic meaning change of synaesthetic adjectives in ordinary English obeys a strict rule that can be universal for all human languages, as shown in Figure1. In response to Williams (1976), Zhao and Huang (2015) carried out a study on synaesthesia in the Chinese language and demonstrated that the hierarchy proposed based on English is not applicable to Chinese. Similarly , Strik Lievers (2015) also pointed out that the "directionality principle" reflects the frequency of association types, rather than representing universal constraints on synaesthetic transfers. Thus, linguistic synaesthesia should be investigated specifically for different languages in depth. Unfortunately, fine-grained studies on synaesthesia in Chinese are still scarce.
Given the gap on synaesthesia in Chinese, we think that it could be a good way to start off with examining synaesthesia in near synonyms, because the interaction between synaesthesia and subtle semantic differences in near synonyms could provide much deeper and more fine-grained clues for synaesthesia. The benefits of studies on near synonyms to explain and predict linguistic phenomena have been widely recognized. For example, Chief et al. (2000) suggested that some semantic features obtained by comparing near synonyms can be useful on the prediction of syntactic performances. Similarly, Hong and Huang (2004), Hong and Huang (2005) also pointed that semantic/cognitive features embedded in perceptual near synonyms can influence their usages in the language.
As previous studies show that the auditory domain is the main target of synaesthetic transfers (Ullmann, 1957;Williams, 1976), the near synonyms, 聲 sheng "sound" and 音 yin "sound" in Mandarin Chinese, should be good candidates for our research, which have been established to represent different semantic/cognitive focuses on the sound. Specifically speaking, 聲 is concentrated on the auditory production, while 音 is focused on the auditory perception, although both can refer to the sound (Hong and Huang, 2004;Hong and Huang, 2005). Therefore, through studying the synaesthetic usages of this pair of near synonyms, not only can we test whether synaesthesia is sensitive to the semantic dichotomy of production and perception, but also we can examine the relationship between synaesthesia and cognition.
The present paper is organized as follows: we will introduce the methods of collecting and annotating data in Section 2, and figure out the similarities as well as differences between 聲 and 音 on synaesthesia in Section 3, which will be followed by some explanations in Section 4. In the last Section, we will summarize our main findings and propose our future work.

Methodology: Corpus Selection and
Data Annotation

Corpus Selection and Measurement Criteria
In order to make our study more tenable, we attempt to exhaust the data as possible as we can, and hence rely on the corpus for data collection. We select four widely-used huge corpora as our data sources, of which Sinica Corpus 2 (Chen et al., 1996)  tagging information (Chen et al., 1996), which we think can facilitate our research. Besides, usages in newspapers are normally wellestablished and the controversial issues about grammaticality for some specific examples could be decreased to a degree, and we hence select three journal corpora as the complement (Refer to Hong and Huang, 2006 for the introduction of two GigaWord corpora).
Regarding the data from different corpora, we set some measurement criteria to make them comparable. To be specific, we depend on the statistical information, which includes the frequency information of examples per million word tokens in their respective corpora and also percentage information of each example in its source corpus. For instance, 大聲 da-sheng "loudly" occurs 407 times in Sinica Corpus (4.0 edition) as a "stative and intransitive verb" 6 and word tokens in the corpus is around 10 million, so its calculated frequency is 40.7 and the percentage is 0.00407% for the analysis in this paper. For some complementary examples, which do not appear in Sinica Corpus, we also utilize the criterion to calculate its frequency in their relevant corpora. 7 For example, the calculated frequency of 長聲 chang-sheng "in prolonged voice" is 0.01 and hence its percentage is 0.000001%, because it occurs 10 times in the journal corpus of BCC with around 1000 million word tokens.

Data Collection and Annotation
We exhaust all the examples with two characters in the modifier-head relation in Sinica Corpus, whose heads are either 聲 or 音. If the modifier does not belong to audition but can be classified into any of four other sensations, including touch, gustation, olfaction and vision, we think that this example involves synaesthesia.
In terms of determining the sensory domain of modifiers, we refer to its original meaning in 說文 解字 shuo-wen-jie-zi and 漢典 han-dian 8 . Take the meaning of 雜 za "mixed" in 說文解字 for example, it is paraphrased as 五彩相會 wu-cai-xiang-hui "five colors mixing together", therefore, we classify it into the visual domain.
Moreover, we divide some sensory domains further into several sub-domains. For instance, we distinguish vision into size, dimension and so on.
All the synaesthetic examples we find in Sinica Corpus are words 9 with the information of part of speech, so we calculate the frequency of each example according to different part of speech labels. For some examples that do not appear in Sinica Corpus, we annotate them manually with the part of speech information compared to Sinica Corpus, and utilize the measurement rule mentioned above to obtain their frequencies and percentages for discussion. If one complementary example has different frequencies and percentages in additional corpora, we take the most frequent one and the respective corpus into consideration.
Therefore, our data is summarized as follows in Table 1 and Table 2, with the part of speech 10 , frequency and percentage information 11 .
3 Similarity and Difference on Synaesthesia

Synaesthetic Similarity for the Near Synonyms
As shown in Table 1 and Table 2, we can see that there are some similarities on synaesthesia between the near synonyms. General speaking, as 聲 and 音 both belong to the auditory sensation, other modalities can transfer frequently to describe these two near synonyms, of which morphemes from both visual and tactile domains can be used to modify 聲 and 音. Therefore, hearing is also a productive target domain of synaesthetic transfers in Mandarin Chinese. This is in line with the observation for English (Ullmann, 1957;Williams, 1976). 9 We do not plan to involve the controversial issue about how to determine a sequence is a word or not, for the discrimination will not influence the discussion below.

TASTE
taste 甜聲/D/0.001/0.0000001% "in a sweet voice" from the visual domain. Similarly, 高 gao "high" and 低 di "low" modify 音 most frequently with the same frequency, which are both from vision. If we calculate the ratio of all the frequencies of morphemes from vision to those from touch, the ration for 聲 is around 5 times and that for 音 is about 10 times. It seems that vision is much easier to transfer into hearing than touch in Mandarin Chinese, which has not been mentioned in previous research yet.
In summary, the similarities on synaesthesia between 聲 and 音 exist in two aspects. The first one is that both 聲 and 音 can be productive recipients of synaesthetic transfers, which can be described by visual words and tactile words. The other similar property is concerned with the priority of vision to touch when modifying these two synonyms. In other words, vision is used more frequently to characterize 聲 and 音 than touch.

Synaesthetic Difference for the Near Synonyms
Besides similarities, there are also two differences on synaesthesia for 聲 and 音, one of which is reflected in selective constraints and the other exists in the syntactic function of the whole combinations.

Selective Constraints
As we see in Table 1 and Table 2, there are more domains and more modifiers that can be used for 聲 than 音. 聲 can be naturally described by gustation, but 音 cannot. There are totally 25 morphemes modifying 聲, including 14 from vision, 10 from touch and 1 from taste, while only 16 morphemes modifying 音, including 10 from vision and 6 from touch. In terms of the frequencies of words, words containing 聲 occur more frequently in corpora than those containing 音. Therefore, 聲 seems to be much easier and more common to be described than 音 through synaesthesia. Interestingly, there is another difference between 聲 and 音 on synaesthesia about the selection constraint. That can be instantiated in the symmetry of modifiers selection for sub-domains. Polar items in gradable antonymous relations (Lyons, 1977) in each sub-domain are usually selected for synaesthetic usages, such as 大 da "big" and 小 xiao "small" for the size and 清 qing "clear" and 濁 zhuo "turbid" for the transparency. For some subdomains, there exists the symmetry between modifiers selection. For examples, in terms of the dimension domain for 聲, the modifiers selection is symmetrical, because the polar items 高 gao "high" and 低 di "low" are both selected and there is no other in-between item on the gradable axis that has been selected. However, in terms of the dimension domain for 音, the selection is asymmetrical, for 中 zhong "middle" is also used besides the polar items 高 and 低. In this way, we can calculate the symmetry of modifiers selection for 聲 and 音 in each sub-domain, which is shown in Table 3. Thus, the whole symmetry ratio for 聲 is 50%, while that for 音 is only 38%. Therefore, there is a noteworthy difference on selective constraints for the near synonyms on synaesthesia. Specifically speaking, 聲 receives more synaesthetic modifications than 音, and also employs higher symmetry on modifiers selection in each sub-domain.

Syntactic Function
Another difference on synaesthesia between 聲 and 音 is related to the whole syntactic function of the synaesthetic combinations. All the examples containing 音 refer to the entity, that is, a specific kind of Sub-domains 聲 sheng1 "sound" 音 yin1 "sound" size sound, such as 長音 chang-yin "prolonged sound". However, most examples containing 聲 is concerned with the event, such as 大聲 da-sheng "loudly", for 大聲 can only be used to modify a predicate (e.g., 大聲唱歌 da-sheng-chang-ge "sing loudly") or express a state (e.g., 他說話很大聲 ta-shuo-hua-henda-sheng "his talking is loud"), whereas it cannot denote an entity (e.g., *一個大聲 yi-ge-da-sheng "a loud sound"). As Table 1 and Table 2 shows, only 4 adjective morphemes exclusively form a noun when combining with 聲. In terms of these 4 exceptional examples, we can see that two of them are terminological words in some specific areas, namely 美聲 mei-sheng "bel canto" and 平聲 ping-sheng "level tone". In addition, the other two are used in a very low frequency, and 短聲 duan-sheng "short sound" as well as 雜聲 za-sheng "noise" neither occur in Sinica Corpus, with the calculated frequencies of only 0.013 and 0.014 respectively in the journal corpus of BCC. Therefore, the synaesthetic combinations of 聲 and 音 have different syntactic functions, of which combinations with 聲 are normally related to an event, whereas combinations with 音 always refer to an entity.
In conclusion, although 聲 and 音 are near synonyms and both can denote the sound; there are some noteworthy differences on synaesthesia. 聲 is much easier to be described through synaesthesia than 音, and also employs higher symmetry on synaesthetic selection of modifiers. Moreover, the combinations of 聲 or 音 have different syntactic functions, of which combinations with 聲 are normally related to an event, while combinations with 音 always refer to an entity.

Production and Perception in Sound
As a pair of near synonyms, 聲 and 音 have both similar properties and different characteristics on synaesthesia in Mandarin Chinese, which can be explained by combining the framework of Generative Lexicon (Pustejovsky, 1995) and the semantic/cognitive dichotomy of production and perception in the sound (Hong and Huang, 2004).
As Pustejovsky (1995) proposed that lexical items can be represented in Lexical Inheritance Structure and some semantic information encoded in lexical items are inherited from upper concepts, which determines their performances in languages. Both 聲 and 音 are in the auditory domain, which can denote the sound, thus, both of them can inherit common semantic information from the upper concept, namely sound. Therefore, we think that the similarities on synaesthesia between 聲 and 音 are determined by the whole synaesthetic characteristics for hearing, that is, hearing is a productive recipient of synaesthetic transfers and is much easier to be the target domain of vision than touch. 12 In terms of synaesthetic differences between 聲 and 音, the semantic/cognitive dichotomy of production and perception really play a role. Hong and Huang (2004) suggested that in the whole transmission process of sounds it should include the starting point, the process and the ending point, of which some concepts focus on the starting point, namely the production of the sound, such as 聲, and some concepts concentrate on the ending point, namely perception and evaluation of the sound, such as 音. According to this proposal, the selective differences on synaesthesia between 聲 and 音 can be reasonably explained. From the cognitive point of view (Gibbs, 2006), embodied action can influence conceptualization in languages. Compared with the ending point of the sound, humans can impose much more active actions on the starting point, whereas the perception and evaluation can be received much more passively. Therefore, the production of the sound is conceptualized and characterized more frequently than the perception through synaesthesia. Also, we speculate that the volition may result in the higher symmetry on sound production for 聲, which needs further research.
The difference on syntactic functions of synaesthetic combinations for 聲 and 音 can be explained by the combination of Generative Lexicon and the distinction between production and perception. In Generative Lexicon, qualia structure of a lexical item, including formal role, constitutive role, telic role and agentive role, can predict their performances in languages (Pustejovsky, 1995). As 聲 focuses on the production of the sound, the salient qualia role in its semantic information is the agentive role, which is usually related to an event or predicate that brings something into being. Conversely, synaesthetic modifiers always contribute to the formal role of 音, for 音 focuses on perception and evaluation of the sound.

Conclusion
This paper is devoted to a fine-grained study on the interaction between synaesthesia and the near synonyms 聲 and 音 of the auditory domain in Mandarin Chinese. We take a corpus-based approach and at the same time employ a combination method of qualitative and quantitative analyses. Eventually, some interesting findings are obtained.
In particular, 聲 and 音, as a pair of near synonyms in the auditory domain, have both similar properties and different characteristics on synaesthesia. In terms of similarities, both 聲 and 音 can be productive recipients of synaesthetic transfers and both can be described by vision and touch. Moreover, vision is observed to act as the most predominant source domain for these near synonyms. In addition, the differences on synaesthesia between 聲 and 音 are also apparent. 聲 receives much more synaesthetic modifications than 音, and also has a higher symmetry on synaesthetic modifiers selection. On the other hand, the combinations of synaesthetic modifications with 聲 are normally related to an event (i.e., production of the sound), while those with 音 always refer to an entity (namely the perception or evaluation of the sound).
The similarities and differences mentioned above can be explained by semantic information and cognitive motivations. Both 聲 and 音 can denote the sound, so they can inherit common semantic information from the sound, which result in the similarity on synaesthesia. However, the different cognitive focuses on the sound, namely the distinction between production and perception, make 聲 and 音 have different synaesthetic performances.
In general, we can conclude that auditory synaesthesia is sensitive to the cognitive dichotomy of production/perception in Mandarin Chinese. In other words, synaesthesia can be predicted and determined by the cognitive modelling. Therefore, besides the transfer tendency recognized by previous studies, linguistic synaesthesia exhibit another regularity, that is, it can be determined by cognitive motivations. Therefore, linguistic synaesthesia is not a kind of random associations of different sensations, but of many principles and regularities, which deserves much more attention and deeper studies.
In the following study, we will expand the research scope and test more interactions between synaesthesia and the cognitive modelling, through which we hope that our studies could contribute to bridging the research on synaesthesia in linguistics and that in neuroscience eventually.