s2fieldsofstudy: Chemistry Biology

text:
Protective Effect of Salidroside from Rhodiolae Radix on Diabetes-Induced Oxidative Stress in Mice

It has been confirmed that diabetes mellitus (DM) carries increased oxidative stress. This study evaluated the effects of salidroside from Rhodiolae Radix on diabetes-induced oxidative stress in mice. After induction of diabetes, diabetic mice were administered daily doses of 50, 100 and 200 mg/kg salidroside for 28 days. Body weights, fasting blood glucose (FBG), serum insulin, TC (total cholesterol), TG (triglyceride), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were measured. Results showed that salidroside possessed hypoglycemic activity and protective effects against diabetes-induced oxidative stress, which could significantly reduce FBG, TC, TG and MDA levels, and at same time increase serum insulin levels, SOD, GPx and CAT activities. Therefore, salidroside should be considered as a candidate for future studies on diabetes.

Introduction
Diabetes mellitus (DM) is a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. Such a deficiency results in increased concentrations of glucose in the blood, which in turn damage many of the body's systems, in particular the blood vessels and nerves [1]. Chronic hyperglycemia during diabetes causes glycation of body proteins that in turn leads to secondary complications affecting eyes, kidneys, nerves and arteries [2]. There are two types of diabetes, namely type 1 and type 2. Type 1, insulin-dependent diabetes mellitus (IDDM), in which the body does not produce any insulin, most often occurs in children and young adults. Type 1 diabetes accounts for 5-10% of diabetes. Type 2, noninsulin-dependent diabetes mellitus (NIDDM), in which the body does not produce enough, or properly use, insulin, is the most common form of the disease, accounting for 90-95% of diabetes [3]. The world prevalence of diabetes among adults (aged 20-79 years) in 2010 will be 6.4%, affecting 285 million adults, and will increase to 7.7%, and 439 million adults by 2030. Between 2010 and 2030, there will be a 69% increase in numbers of adults with diabetes in developing countries and a 20% increase in developed countries [4,5]. In recent years, many studies have confirmed that DM carries increased oxidative stress [6]. Hyperglycemia can increase oxidative stress due to increased mitochondrial production of the superoxide anion nonenzymatic glycation of proteins and glucose autoxidation [7][8][9]. Free fatty acid (FFA) levels, which are elevated in diabetes and insulin resistance, may also contribute to the increased production of reactive oxygen species (ROS) due to increased mitochondrial uncoupling and ß-oxidation [10,11] Several studies have shown that antioxidants could be useful in preventing or attenuating the adverse effects of chronic hyperglycemia [12][13][14].
The modern drugs, including insulin and other oral hypoglycemic agents such as biguanides, sulphonylureas, α-glycosidase inhibitors, control the blood glucose level as long as they are regularly administered but they may also produce a number of undesirable effect [15][16][17][18]. Hence there is a need to search for newer anti-diabetic agents that have high therapeutic efficacy with minimum side effects. Over the past few decades, Traditional Chinese Medicine has been confirmed to be effective and promising in the therapy of diabetes and its complications with few undesirable effects compared with the modern drugs [19][20][21][22]. Based on a large number of chemical and pharmacological research works, numerous bioactive compounds for diabetes have been found in Chinese medicinal plants [3]. The roots of Rhodiola sachalinensis A. BOR. (Gao-shan-hong-jing-tian in Chinese), Rhodiolae Radix, have been widely used as a hemostatic, antitussive, tonic, and endermic liniment for burns and contusions in Traditional Chinese Medicine [23][24][25]. Moreover, in animal models, Rhodiolae Radix extract has been reported to have a very good effectiveness in the control of blood glucose [26,27]. Salidroside, 2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside, a compound with a phenol glycoside chemical structure (Figure 1), is isolated from the Rhodiolae Radix as one of its main active ingredients and is thought responsible for all the documented pharmacological effects of the medicinal plant. Previous studies demonstrated that salidroside has important bioactivities including anti-fatigue, anti-inflammatory, anticancer, antioxidant, anti-aging, hepatoprotective effects, etc. [28][29][30][31][32]. Besides, Tong et al. indicated that salidroside has a significant effect on glucose, fat and amino acid metabolism and can improve the acrobic capacity. Another study also proves that salidroside could stimulate glucose uptake in differentiated L6 rat myoblast cells [33]. However, few studies have examined the hypoglycemic activity of salidroside. The present study was undertaken to evaluate the effects of salidroside on diabetes-induced oxidative stress in mice.  Table 1 shows the effect of salidroside administration on body weight in mice. Before the experiment, body weights between groups were not significantly different, but after 7 days, body weighs of normal controls (NC) were significantly increased when compared with the diabetic groups (P < 0.05). However, body weight changes were not significant between the diabetic control (DC), low dose salidroside (50 mg/kg) treatment (DLT), middle dose salidroside (100 mg/kg) treatment (DMT) and high dose salidroside (200 mg/kg) treatment (DHT) groups. After 28 days, NC group had an average increased body weight of 27.7%. When compared with DC group, body weights were significantly increased (P < 0.05) in DLT, DMT and DHT groups. The pancreas is the primary organ involved in sensing the organism's dietary and energetic states via glucose concentration in the blood and in response to elevated blood glucose, insulin is secreted [34]. Alloxan, induces "chemical diabetes" in a wide variety of animal species by damaging the insulin secreting cells of the pancreas. This damages a large number of β-cells, resulting in a decrease in endogenous insulin release, which paves the ways for the decreased use of glucose by the tissues [35]. Alloxan-induced diabetes is characterized by severe loss in body weights and similar results also were observed in the present study. This loss of body weights could be due to, dehydration and catabolism of fats and protein [36]. Previous reports show that protein synthesis is decreased in all tissues due to decreased production of ATP and absolute or relative deficiency of insulin [37]. In this study, the diabetic control group showed significant loss of body weight and the salidroside treatment groups showed significant prevention of this loss in body weights, which might be as a result of its ability to reduce hyperglycemia. It is very meaningful that the prevention effects could be applied to clinical diabetics who lose weight. Figure 2 shows the effect of salidroside administration on fasting blood glucose levels in mice. FBG levels of NC group maintained constant during the experimental period and were significantly decreased when compared with the diabetic groups before experiment (P < 0.05). After 14 days, FBG levels of DLT, DMT and DHT groups were significantly decreased when compared with DC group (P < 0.05) in a dose-dependent manner and the strongest effect was seen with 200 mg/kg dose. After 28 days, FBG levels changes were not significant between the NC and DHT groups. It is known that effective control of the blood glucose level is a key step in preventing or reversing diabetic complications and improving the quality of life in diabetic patients [38][39][40]. The hypoglycemic activity of salidroside in alloxan-induced diabetic mice has been indicated here by estimation of fasting blood glucose levels, as the important basal parameter for monitoring of diabetes. The possible mechanism of this hypoglycemic activity may be by potentiation of pancreatic secretion of insulin from β-cell of islets or due to enhanced transport of blood glucose to peripheral tissue [41]. Therefore, the effect of salidroside on serum insulin levels in alloxan-induced diabetic mice was investigated. Figure 3 shows the effect of salidroside administration on serum insulin levels in mice. Serum insulin levels of DC group were significantly decreased when compared with the NC groups (P < 0.05). After 28 days, DLT, DMT and DHT groups showed a dose-dependent increase in serum insulin levels compared with the DC group (P < 0.05) and the strongest effect was seen with 200 mg/kg dose. Results of the current study showed that serum insulin levels have been increased significantly in diabetic mice after the salidroside supplementation. The increment of serum insulin levels might be due to the renewal of beta cells in the pancreas or the recovery of partially destroyed β-cells [42,43]. Figure 4 shows the effect of salidroside administration on blood lipid levels in mice. TC and TG levels of DC group were significantly increased when compared with the NC groups (P < 0.05). After 28 days, TC and TG levels of DLT, DMT and DHT groups were significantly decreased when compared with the DC groups (P < 0.05). Diabetes is frequently accompanied by multiple metabolic abnormalities, including dyslipidemia, which increases the risk for cardiovascular disease. Reduction in TG and TC through dietary or drug therapy has been found beneficial in preventing diabetic complications as well as improving lipid metabolism in diabetic patients [44][45][46]. In this study, TG and TC levels have been decreased significantly in diabetic mice after the salidroside supplementation. These effects might be due to low activity of cholesterol biosynthesis enzymes and/or low level of lipolysis, which are under the control of insulin [47]. Table 2 and Table 3 show the effect of salidroside administration on antioxidant activities enzymes and MDA levels in kidney and liver in mice. There appears to be a significant increase (P < 0.05) in the MDA levels associated with the diminution of the SOD, GPx and CAT activities of DC group in comparison with NC group. After 28 days, SOD, GPx and CAT activities of DLT, DMT and DHT groups were higher and MDA levels were lower than that of DC group (P < 0.05).

Effect of Salidroside Administration on Antioxidant Enzymes Activities and MDA Levels in Kidney and Liver in Mice
It has been confirmed that diabetes mellitus carries increased oxidative stress. Diabetics and experimental animal models exhibit high oxidative stress due to persistent and chronic hyperglycemia, which depletes the activity of antioxidative defense system resulting in elevated levels of oxygen free radicals [48][49][50].  Attack by high levels of free radicals and simultaneous decrease of the expression of antioxidant enzymes, may enhance membranes' susceptibility to lipid peroxidation and lead to pancreatic β-cell dysfunction, as well as other cellular organelle damage [51,52]. The increase in lipid peroxide levels is one of the most important contributing factors in the development of diabetes-related complications [35]. Thus, the ideal antidiabetic drug should have hypoglycaemic effects. Besides, it may be better to have some antioxidative properties too.
Earlier studies had reported a decrease in the activities of antioxidant enzymes (SOD, CAT and GPx) in the kidney and liver in diabetic animal [53,54]. Similar results also were observed in the present study and the administration of salidroside significant increased SOD, GPx and CAT activities in the kidney and liver of diabetic mice. Lipid peroxidation products such as MDA are generated under high levels of un-scavenged free radicals and may bring about protein damage and inactivation membrane bound enzymes, so they play an important role in pancreatic damage associated with diabetes [52]. In this study, MDA levels have been decreased significantly in the kidney and liver in diabetic mice after the salidroside supplementation. This means that the salidroside may reduce reactive oxygen free radicals or improve the activities of antioxidant enzymes, prevent (or reduce) lipid peroxidation. Therefore, salidroside showed in our study it had a direct and indirect preventive and protective effect in diabetes by decreasing oxidative stress.

Chemicals and Reagents
Salidroside (99% purity) was purchased from Chengdu Sikehua Biotechnology Co., Ltd. (Chengdu, China). A glucose analyzer (GT-1640) and glucose check strips were purchased from Arkray Inc. (Japan). Alloxan was purchased from Sigma Chemical Co. (USA). Reagents for TC (total cholesterol) and TG (triglyceride) were purchased from Beijing Chengxinde Biochemistry Reagent Co. (Beijing, China). Reagents for superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Biochemistry Reagent Co. (Nanjing, China). Reagents for serum insulin were purchased from Beijing Beifang Pharmaceutical Co. (Beijing, China). All of the other chemicals and reagents were standard commercially available biochemical quality. Water was purified with a Milli-Q purification system and was used to prepare all solutions.

Animals
Male mice of original Kun-ming strain, weighing 18-22 g, were used for the study. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese National Institutes of Health. The animals were obtained from the Animal Department of the Beijing Institute of Traditional Medical and Pharmaceutical Sciences (Beijing, China) and were housed in a room maintained at 23 ± 2 °C with relative air humidity of 45% to 55% on a 13-hour light/11-hour dark cycle. Mice were provided a standard laboratory chow and water ad libitum. The approval of this experiment was obtained from the Institutional Animal Ethics Committee of Yanshan University (Qinghuangdao, China).

Preparation of Diabetic Mice
The mice were adapted to diet and environment for one week before the experiment began. After a 24-hour fasting, the mice were induced with a single injection of 4% alloxan prepared freshly at a dose of 200 mg/kg bw. Diabetes was confirmed by the determination of tail vein blood glucose levels on the third day after administration of alloxan. Mice having blood glucose levels greater than 11.1 mmol/L were considered diabetic and were used for the study [55].

Experimental Design
After confirmation of the diabetic state, normal and alloxan-induced diabetic mice were randomly divided into five groups of ten per group.
Group I: normal control (NC), normal mice were allowed to free access to a normal diet and treated daily with distilled water (5 mL/kg) by gavage using a 20 gauge feeding needle for 28 days.
Group II: diabetic control (DC), diabetic mice were allowed to free access to a normal diet and treated daily with distilled water (5 mL/kg) by gavage using a 20 gauge feeding needle for 28 days.
Group III: low dose salidroside (50 mg/kg) treatment (DLT), diabetic mice were put on a normal diet and treated daily with 50 mg/kg of salidroside by gavage using a 20 gauge feeding needle for 28 days.
Group IV: middle dose salidroside (100 mg/kg) treatment (DMT), diabetic mice were put on a normal diet and treated daily with 100 mg/kg of salidroside by gavage using a 20 gauge feeding needle for 28 days.
Group V: high dose salidroside (200 mg/kg) treatment (DHT), diabetic mice were put on a normal diet and treated daily with 200 mg/kg of salidroside by gavage using a 20 gauge feeding needle for 28 days.
This selection of doses was based on preliminary experiments, wherein, doses of 50-200 mg/kg was tried and confirmed to be suitable and effective in test mice. Fasting blood glucose (FBG) levels were measured for once every week. Blood was collected from tip of the tail vein (starting from 9:00 a.m.) after a 12-to 14-hour overnight fast. At the same time, body weights of mice were measured using a balance. On the last day of experiment, the mice were deprived of food overnight and sacrificed by cervical dislocation. Blood was collected in polystyrene tubes without the anticoagulant. Serum was immediately separated by centrifugation at 3,000 rpm at room temperature for 10 min. Samples were stored at −20 °C for the assay of TC, TG and serum insulin.
The kidney and liver were carefully removed and homogenized in ice-cold 0.15 M Tris-KCl buffer (pH 7.4) to yield a 10% (w/v) homogenate. The latter was next subjected to high-speed centrifugation for 30 min at 4 °C. The resulting supernatant was used as such for assaying GPx, SOD, CAT activities and MDA levels.

Statistical Analysis
All results are expressed as mean ± S.D. for ten mice in each group. To determine the effect of treatment, data were analyzed using one-way ANOVA repeated measures. p-values of less than 0.05 were regarded as significant. Significant values were assessed with Duncan's multiple range test. Data were analyzed using the statistical package "SPSS 12.0 for Windows".

Conclusions
In conclusion, the present investigation showed that salidroside possess hypoglycemic activity and exert protective effects in experimental diabetes, possibly by reducing diabetes-induced oxidative stress and hence protects organism from oxidative damage and dyslipidemia. This may be attributed to its antioxidative potential. Therefore, salidroside should be considered as a candidate for future studies on diabetes.


extfieldsofstudy: Medicine Chemistry

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Thermodynamic study of benzocaine insertion into different lipid bilayers

Thermodynamic study of benzocaine insertion into different lipid bilayers J. J. López Cascales,1,a) S. D. Oliveira Costa,1 and R. D. Porasso2 1Universidad Politécnica de Cartagena, Grupo de Bioinformática y Macromoléculas (BIOMAC), Aulario II, Campus de Alfonso XIII, 30203 Cartagena, Murcia, Spain 2Instituto de Matemática Aplicada San Luis (IMASL) Departamento de Física, Universidad Nacional de San Luis/CONICET, D5700HHW, San Luis, Argentina

I. INTRODUCTION
The existence of certain drugs with anesthetic properties has been known for over a century since the pioneering studies of Freud 1 with cocaine. Some of these anesthetic molecules are widely used in clinical anesthesiology for pain relief and during surgical operations. However, the molecular mechanism of these drugs is still a subject of debate, although there is a broad consensus on the role played by sodium channels. [2][3][4][5] There is also a tendency to concentrate studies on specific anesthetic-protein interactions, with less attention paid to the effects of the anesthetic on the protein environment, e.g., the lipids that surround the protein membranes.
In this sense, several authors 6,7 have suggested a way of action based on a ligand-receptor binding mechanism, in which the anesthetics could bind to the protein forming the sodium channels, which would block ion transduction. However, alterations in the lipid constituents of the membrane could also influence the microstructure and function of these ion channels. The requirement of proteins to bind with specific lipids in annular clefts or interactions with certain phospholipid head groups means that any displacement of these lipids due to the presence of anesthetics could also alter the protein functions. In this regard, studies based on gramicidine 8 have demonstrated that an anesthetic dissolved into the membrane induces changes in the overall dynamics of the protein, altering its function in the process. In addition, a more complex and sophisticated understanding at molecular level is required since the cellular membrane is a raft of lipids a) Author to whom correspondence should be addressed. Electronic mail: javier.lopez@upct.es.
of different compositions that incorporate certain membrane proteins. In this context, even when the argument for a lipid site action might be revived based on recent developments and research, 9 almost a century ago Meyer and Overton 10, 11 associated the potency of anesthetics with their solubility in a lipid bilayer, at a time in which the lipid bilayer structure had not even been described. It has been suggested that the solubility of anaesthetics in a lipid bilayer may cause a disturbance in membrane fluidity 12-17 that could affect the protein structure of sodium channel formation, and as a consequence of this perturbance, a conformational change in the ion channel could take place, resulting in the blockage of ion transduction. 18 Thus, rather than considering the cell membrane as a homogeneous entity, it should be considered as a structure with different domains formed by different components, in which lipid rafts of different composition are spread over the membrane, and certain protein functions might be moderated by specific lipids. 19 Thus, perturbations in a part of the membrane bilayer could affect other specific regions of membrane proteins. Therefore, studies on alterations in membrane protein signaling should look at complex processes rather than a mere alteration in the binding site of a membrane protein.
In summary, it would seem essential to characterize in the first instance the thermodynamic parameters associated with the insertion process of a local anesthetic into the bilayer according to its lipid composition, with the aim of providing insight into this process at molecular level. For this purpose we used benzocaine, a local anesthetic widely used in topical applications, which exists only in its neutral form under physiological conditions. 14, 20 The lipid bilayer was formed by DPPC (dipalmitoylphosphatidylcholine) and DPPS (dipalmitoylphosphatidylserine), where DPPC and DPPS correspond to an uncharged (neutral) and charged (negative) phospholipid in physiological conditions, respectively. Different lipid bilayers were generated by varying the lipid percentage that constitutes the membrane.
To provide insight into the process of insertion of the benzocaine into a lipid bilayer, molecular dynamics simulation was used. Based on previous studies of molecular insertions into lipid bilayers, 21,22 the study of the thermodynamic insertion of benzocaine into a lipid bilayer was carried out with atomic resolution. Information regarding the spontaneity of the insertion ( G) and the thermodynamic parameters involved in this process ( H and S) is provided.

A. Setting up the simulation conditions
The lipid bilayer was assembled from 72 lipids (36 lipids per leaflet). Further information on how the three-dimensional computational box were generated, may be consulted in previous works. [23][24][25] Assuming that asymmetric models of lipid bilayers 25 mimics much better a biological plasma membrane than the models of symmetric lipid bilayer; however, with the aim of studying the thermodynamic properties involved in the benzocaine insertion into binary lipid bilayers of different compositions, symmetric bilayers of different DPPC/DPPS ratios were simulated. This substitution was made on both sides of the bilayer so that the bilayer symmetry was maintained in all simulations. Thus, the parameter χ was defined as follows: where χ represents the fraction of DPPS with respect the total number of lipids that form the membrane. In this respect, χ = 0 corresponds to a pure membrane of DPPC, and χ = 1 represents a membrane constituted only by DPPS. Thus, the following membranes were assembled as a function of the DPPC:DPPS composition: 72:0, 60:12, 48:24, 24:48, and 0:72, corresponding to χ = 0, 0.2, 0.3, 0.7, and 1, respectively. The system corresponding to χ = 0 contained 2517 molecules of the single point charge (SPC) water model 26 in the simulation box. In the case of χ = 0, to balance the negative charge associated with each DPPS lipid, the corresponding sodium ions Na + were introduced by substitution of water molecules, to balance the charge associated to the DPPS. Thus, 12, 24, 48, and 72 sodium ions were introduced into the system to balance the number of DPPS in the bilayer. As regards the anesthetic, two benzocaine molecules were introduced into the system at different positions along the z-axis perpendicular to the lipid surface. A scheme of the benzocaine molecule used in this work is shown in Figure 1. The force field parameters and electrostatic charge distribution of the DPPC and DPPS were the same as those used by Egberts et al. 27 and Cascales et al. 28 in previous simulations of pure bilayers of DPPC and DPPS in their liquid crystalline state. The charge distribution and force field parameters for benzocaine were taken from a previous article. 22 The MD simulations were carried out using the GROMACS-3.3.3 package. The system was coupled to a weak temperature and pressure coupling bath algorithm proposed by Berendsen,29 with coupling constants of 0.1 ps and 1 ps for the temperature and pressure, respectively. A time step of 2 fs was used as integrator time to generate the trajectories. The electrostatic interactions were evaluated using the fast particle-mesh Ewald method 30,31 and the Lennard-Jones potential was used for the long range interactions, considering a cut-off of 1 nm. Finally, all the bond lengths were constrained using LINCS. 32 All the simulations were performed at 350 K and 1 atm. This temperature was chosen above the transition temperature of 314 K and 326 K for the DPPC and DPPS, respectively, 33,34 to ensure that the bilayers were always in their liquid crystalline phase. In addition, this temperature provided a window of temperatures to avoid any change in the lipid phase, within the range of temperatures (between 345 K and 355 K) used to calculate the thermodynamic properties of the system.

B. Free energy profile
The umbrella sampling method 35 was used to evaluate the free energy profile associated with benzocaine insertion into a lipid bilayer. The relationship between the free energy profile and the potential mean force is depicted in the following equation: where G b (z) represents the free energy profile along the zaxis perpendicular to the bilayer, R the constant of the gases, T the temperature, C eq b (z) the concentration profile of the benzocaine along the z-axis, C * b the concentration of benzocaine in the solution (aqueous solution in our case), and P MF (z) the potential of mean force computed with respect to a reaction coordinate equal to the z coordinate of the center of mass of the benzocaine molecule, using the umbrella algorithm and W H AM method (weight histogram analysis method). 36 Since an accurate profile of C eq b (z) could not be obtained during the simulation time, a biasing potential must be applied to the benzocaine. In this respect, 36 independent simulations of 50 ns each were performed, for each of the five systems studied in this work, which amount a total of 9000 ns of simulation time. In each of the 36 simulations for any of the systems studied, the benzocaine was restrained at a given depth in the bilayer by a harmonic potential on the z-coordinate perpendicular to the membrane plane, leaving it to move freely on the membrane plane. The force constant of this harmonic spring was 3000 kJ mol −1 nm −2 for all the restrained positions of the benzocaine across the membrane. Thus, benzocaine was shifted by 0.1 nm between consecutive simulation windows. As in previous works, 21, 22, 37 two benzocaine molecules were introduced into the system to save computing time, with a separation of 3.5 nm along the z-axis to ensure the absence of relations between both molecules. Figure 2 depicts four snapshots of the system corresponding to χ = 0, where one can appreciate the benzocaine molecules placed at different positions along the z-axis.

A. Surface area and deuterium order parameters
In order to validate the simulation parameters and force field used in this work, two different properties were studied, the surface area per lipid and the deuterium order parameter for the lipids forming the bilayer in the absence of benzocaine. Figure 3 shows the average running surface area per lipid for three of the five bilayers simulated in this work, in the absence of benzocaine, as a function of their lipid compositions, χ . From Figure 3, an equilibration time of 10 ns was estimated, for all the bilayers studied in this work. Table I depicts the average surface area per lipid calculated from the last 40 ns of the trajectory length.
Unfortunately, only the surface area corresponding to pure lipid bilayers (formed exclusively by DPPC or DPPS) can be compared with experimental data. In the case of a pure DPPC bilayer, corresponding to χ = 0, a surface area per lipid of 0.683 ± 0.006nm 2 was measured, which is in good agreement with the experimental data of 0.70 nm 2 proposed by Phillips et al. 38 and Janiak et al. 39 for a DPPC bilayer at 350 K. In the case of the DPPS bilayer, corresponding to χ = 1, a surface area of 0.531 ± 0.005 nm 2 was calculated, which agrees with the experimental data of DPPS in its liquid crystalline phase, which range from 0.45 to 0.55 nm 2 . 34,40 In general, the surface area per lipid was seen to decrease as χ increased (in other words, with the increasing content of DPPS in the lipid bilayer) in our simulations.
Another property widely used to validate simulations involves the deuterium order parameter of the methylene groups along the lipid hydrocarbon tails, −S CD . Experimentally, −S CD can be calculated from H-NMR experiments of specific deuterated methylene groups along the hydrocarbon lipid tails with the axis normal to the lipid bilayer. 17,41,42 By simulation, the order parameters may be calculated from the methylene hydrogen orientation with respect the z-axis normal to where x, y, z are the local coordinates of the system, θ a is the angle made by the molecular axis with the bilayer normal, and δ ab is the Kronecker delta. From simulation, the order parameter −S CD can be determined using the relation proposed by Egberts and Berendsen, 43 where S xx and S yy are the terms of the order parameter tensor of Eq. (3).
As it was explained in previous works, 23, 25 a procedure was implemented in the S CD calculations to circumvent the fact that hydrogens are not explicitly considered in the unit atoms that form the lipid molecules in our simulations.
Thus, Fig. 4 shows the deuterium order parameters of three of the systems studied in this work. From this figure, one can appreciate the good agreement between simulation and experimental data for pure membranes of DPPC and DPPS (the only two cases for which experimental data are available), and the enhancement of the order parameters of the lipid tails with increasing χ , accompanied by shrinking of the lipid surface with increasing DPPS content.
In this regard, to explore the effect of benzocaine on the order parameter of the lipids that form the bilayer, Fig. 5 depicts the variation of the order parameters of a pure bilayer  of DPPC with 2 and 10 benzocaine molecules that can move freely inside the bilayer, corresponding to benzocaine concentrations of 4.7 and 23.3 g/cm 3 , respectively. Thus, Fig. 5 shows a slight increasing in the disorder at the end of the lipid tails with the increasing of the benzocaine (BZCA) concentration (although this effect is small to be significant considering the error bars of our simulations), in good agreement with the experimental data of Kuroda et al., 42 where they reported how the disorder increased beyond the carbon 8 in presence of benzocaine. In line with these results, almost no variation in the surface area per lipid and thickness of the lipid bilayer was measured from our simulations. Figure 6 depicts the free energy profile associated with the insertion of benzocaine into a lipid bilayer as a function of the lipid composition, χ . Note the existence of a minimum in the free energy profile located in the hydrocarbon region within the lipid bilayer. These values correspond to   Figure 6 shows how increases in the DPPS content (higher values of χ ) lead to a widening of the free energy profile along the z-axis. This phenomenon is associated with the increasing thickness of the lipid bilayer as a result of the increase in the order parameters of the lipids, as was discussed above. Figure 7 shows how the maximum in free energy associated with benzocaine insertion into a lipid bilayer fits an exponential function, which can be expressed in its linear form as follows: ln( G max )/(kJ mol −1 ) = −2.93χ + 1.48 (5) or expressed in its exponential form as follows:

B. Free energy profile, G(z)
expressed G in both equations in kJ/mol. This result confirm that an increase in the DPPS content of the lipid bilayer facilitates the insertion of the benzocaine into the lipid bilayer by weakening the barrier effect of the free energy located at the lipid/solution interface. This result may be related with the experimental evidence that the activity of certain local anesthetics is modulated by the DPPS composition of the cellular membrane (as corresponds to most of the cells of the central nervous system compared with other eukariotic cells). Thus, Baenziger et al. 44 reported how the presence of charged lipids in the membrane enhances the drug activity by increasing the partitioning of tetracaine into the lipid bilayer, which is in a good agreement with our findings.
With the aim of validating the free energy profiles obtained by the umbrella sampling method, Fig. 8 shows the free energy profile G(z) across the lipid bilayer, and the normalized distribution of probability of two free benzocaine molecules in the interior of a lipid bilayer of χ = 0.3. This figure underlines the consistency of the results obtained using two different computational techniques, where the minimum of the free energy coincides with the maximum probability of finding benzocaine in the interior of the lipid bilayer. Analogous results were obtained for the rest of systems studied in this work.

C. Enthalpy and entropy associated with the insertion process
One aspect of undoubted interest in any thermodynamic study is related with the identification of the enthalpy and entropy contribution to the free energy of the process. In this respect, from classical thermodynamics it is known that entropy S, enthalpy H, and the free energy G are related by the following expression: where H and S correspond to the variation in enthalpy (energy) and entropy (disorder) associated with the thermodynamic process. Thus, experimentally, the variation in entropy associated with a certain thermodynamic process can be obtained from the difference in free energy measured at two different temperatures, as follows: This differential equation can be solved numerically as follows: Thus, from the free energy profile of G at two different temperatures, the entropy associated with the insertion of benzocaine into the bilayer can be estimated. Once S has been calculated (from Eq. (7)), the enthalpic contribution to the free energy can be estimated as well. In our case, additional free energy profiles corresponding to χ = 0,0.3 and 1 were obtained at temperatures of 345 K, 350 K, and 355 K. To reduce the error bars, the trajectory lengths were extended to 80 ns each, which amounted to a total of 17 280 ns of extra simulation time. Figure 9 shows the enthalpy and entropy profiles associated with benzocaine insertion into the lipid bilayer, corresponding to the membranes of composition χ = 0 (a pure DPPC bilayer), χ = 0.3 (a mixture of DPPS + DPPC), and χ = 1 (a pure DPPS bilayer).
Unfortunately, even when the simulation trajectories were lengthened to 80 ns each (compared with the previous simulations of 50 ns), the error bars of S and H at the lipid/solution interfaces show long error bars, which limits the accuracy of our results. However, the error bars were smaller within the bilayer for both enthalpy and entropy. From the results depicted in Fig. 9, and based on the fact that benzocaine insertion into the membrane is a spontaneous process, it is observed how entropy is the driving force of the process, considering that benzocaine insertion into the bilayer is an endothermic process.
Thus, the shape associated with the entropy from bulk solution to the interior of the lipid bilayer may be explained as follows: 1. In bulk solution, the benzocaine dipole presents a homogeneous orientational distribution function, as evidenced in a previous work. 22 However, in the vicinity of the lipid/water interface, this distribution is strongly perturbed due to the fact that the benzocaine dipole preferably points toward the bilayer surface. This perturbation in the orientational distribution function of benzocaine is enhanced by the presence of DPPS in the bilayer. As a consequence, increasing the anisotropy of the benzocaine dipole orientation by increasing the DPPS content in the lipid bilayer should lead to a diminution in the entropy (or disorder), which is in good accordance with our simulation data. 2. Within the bilayer, an increase in entropy is expected with increasing disorder of the membrane (chaos increases, i.e., the entropy). Thus, from the results of the deuterium order parameters (see Sec. III A), an increase in the DPPS fraction in the lipid bilayer increases the order of the lipid tails (i.e., decreased disorder). Thus, an increase in order in the interior of the lipid bilayer correlates with a diminution in the entropy of the same as reflected in Fig. 9.
Unfortunately, as far as we know, no experimental data are available to make a straight comparison with our simulation results. However, Lozano, and Martínez 45 studied the transference process associated with cetoprofene from aqueous solution to the interior of a DPPC liposome. These authors proposed the following thermodynamic data for this process: G= −32.72±0.09 kJ/mol, H = 56.2 ± 1.5 kJ/mol, and S = 0.298 ± 0.008 kJ/mol.K, results that are in good agreement with our simulation data for a pure bilayer of DPPC with G = −24 ± 0.14 kJ/mol, H = 73.82 ± 0.02 kJ/mol, and S = 0.29 ± 0.01 kJ/mol.K.
Thus, considering the H and S profiles depicted above, the free energy profile G(z) can be obtained for a range of T in the absence of phase transition. Figure 10 shows the free energy profile in the range 340-360 K, for a DPPC bilayer (χ = 0). From this figure, it can be observed how an increase the temperature produces an increase in the free energy in the middle of the lipid bilayer, as might be expected from an entropic process. However, almost no variation in maximum free energy was observed at the lipid-solution interface in the range of temperatures studied. An analogous trend was shown by the other bilayers of different χ (not included in this work). This enhancement in the free energy with temperature is in a good agreement with the experimental results provided by Matsuki et al.,20 where an increase in the partition constant for benzocaine between a water/DPPC bilayer was measured.

IV. CONCLUSIONS
MD simulations of benzocaine insertion into lipid bilayers of different composition pointed to the spontaneity of this process, regardless of the lipid composition of the bilayer, with values ranging from −24 to −28 kJ/mol, for χ = 0 (pure DPPC) and χ = 1 (pure DPPS). However, even though this is a spontaneous process (from a thermodynamic point of view), as it penetrates into the bilayer, benzocaine must pass through a barrier of free energy that depends exponentially on the DPPS content of the bilayer. Thus, an increase in the DPPS fraction of the lipid bilayer facilitates the insertion of the benzocaine into the bilayer, an observation that could be related with the activity of certain drugs that depend on the lipid composition of the cell membrane.
As regards the nature and spontaneity of benzocaine insertion, it was seen how this insertion process is mainly dominated by the entropic nature of the process. Thus, as the order of the lipid bilayer associated with the presence of DPPS increases (higher χ values), so the entropic contribution to the insertion process diminishes.


extfieldsofstudy: Biology Medicine

text:
Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution

The R. prowazekii 3-ketoacyl-(acyl-carrier-protein) reductase is similar to those from other prokaryotic pathogens but differs significantly from the mammalian orthologue, strengthening its case as a potential drug target.

Introduction
Epidemic typhus has afflicted humans for a long time, with the first written record going back to 1083 AD (Szybalski, 1999). The obligate bacterial parasite Rickettsia prowazekii is responsible for the disease, which is transmitted by various arthropod vectors, most commonly body lice (Pediculus humanus humanus) or flying squirrel fleas (Orchopeas howardii) and lice (Neohematopinus sciuropteri), through their saliva or feces. Epidemic typhus flourishes in areas with overcrowding or poor hygiene, such as jails or refugee camps. Although louse control has succeeded in suppressing typhus in the modern era, recent outbreaks in the USA (Reynolds et al., 2003), Africa (Mokrani et al., 2004) and Europe (Tarasevich et al., 1998) have re-established R. prowazekii as a re-emerging disease threat; its potential use in bioterrorism (Radulovic & Azad, 2002) places it in category B of the Biodefense Category A, B and C Priority Pathogens of the National Institute of Allergy and Infectious Diseases.
The genome of R. prowazekii, a member of the Gram-negative alphaproteobacteria, is not large at just over 1.1 million base pairs, with only 834 open reading frames found (Andersson et al., 1998). This reduced proteome is representative of the dependence of Rickettsia on its host for many biosynthetic functions and leaves fewer innate enzyme targets for antipathogenic targeting. One well conserved pathway that it shares with other bacterial pathogens is for fatty-acid biosynthesis as a primary source of cell-membrane lipids.
In R. prowazekii fatty-acid biosynthesis occurs via the type II fattyacid synthase pathway, which is thought to be descendant from an algal plastid (Wilson, 2002) and consists of four to eight individual enzymes that start with acetyl coenzyme A and malonyl-(acyl-carrierprotein) to create elongated fatty acids for the lipid bilayer (Fig. 1). In contrast, human fatty-acid biosynthesis is performed by a single large multifunctional peptide, the multienzyme type I fatty-acid synthase, which passes the substrate from one domain to the next. 3-Ketoacyl-(acyl-carrier-protein) [3-ketoacyl-(ACP)] reductase, gene name fabG, is the third enzyme in the type II pathway and catalyzes the chemical reaction ð3RÞ-3-hydroxyacyl-ðacyl-carrier-proteinÞ þ NADP þ ! 3-oxoacyl-ðacyl-carrier-proteinÞ þ NADH þ H þ : While many structures of 3-ketoacyl-(ACP) reductase have been solved, those from Escherichia coli (Price et al., 2001), Mycobacterium tuberculosis (Cohen-Gonsaud et al., 2002) and Burkholderia pseudomallei (PDB entry 3ftp; Seattle Structural Genomics Center for Infectious Disease, unpublished work) to name only a few, only five structures in total have been solved for Rickettsia and the solution of the structure of an essential pathway enzyme in this bacterial parasite holds promise for drug development against typhus.

Protein expression and purification
FabG from R. prowazekii strain Madrid E (NCBI NP_221114; fabG gene RP762; UniProt P50941; Pfam ID PF00106; EC 1.1.1.100) spanning the full-length protein from residues 1 to 241 was cloned from genomic DNA into a pAVA0421 vector using ligationindependent cloning (Aslanidis & de Jong, 1990) to produce a construct with an uncleaved N-terminal hexahistidine-affinity tag followed by the human rhinovirus 3C protease-cleavage sequence (sequence MAHHHHHHMGTLEAQTQGPGS-ORF). R. prowazekii FabG was expressed in E. coli using BL21(DE3)R3 Rosetta cells and autoinduction medium in a LEX bioreactor as follows. A 3 ml starter culture of LB broth with ampicillin was grown for $18 h at 310 K. ZYP-5052 auto-induction medium was freshly prepared as per Studier's published protocols (Studier, 2005). Ampicillin was added to 2 l sterile auto-induction medium, which was inoculated with all of the overnight culture and then placed into the LEX bioreactor. The culture was grown for $24 h at 298 K; the temperature was reduced to 288 K and the culture was grown for a further $60 h. To harvest, the cultured medium was centrifuged at 4000g for 20 min at 277 K. The cell paste was flash-frozen in liquid nitrogen and stored at 193 K.
The frozen cells were resuspended in lysis buffer (25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 0.025% sodium azide, 0.5% CHAPS, 10 mM MgCl 2 , 1 mM TCEP, 250 ng ml À1 AEBSF and 0.05 mg ml À1 lysozyme). The resuspended cell pellet was disrupted on ice for 30 min with a Virtis sonicator (408912; set at 100 W power with alternating cycles of 15 s pulse on and 15 s pulse off). The cell debris was incubated with 20 ml Benzonase nuclease (EMD Chemicals; 25 units ml À1 ) at room temperature for 45 min and clarified by centrifugation on a Sorvall SLA-1500 at 14 000 rev min À1 for 75 min at 277 K. The protein was purified from the clarified cell lysate by immobilized metal-affinity chromatography on an Ni-NTA HisTrap FF 5 ml column (GE Healthcare) equilibrated with binding buffer (25 mM HEPES pH 7.0, 0.5 M NaCl, 5% glycerol, 30 mM imidazole, 0.025% sodium azide, 1 mM TCEP) at 277 K. The recombinant protein was eluted with 250 mM imidazole. The protein was collected in the flowthrough and was further resolved by size-exclusion chromatography (SEC) using a HiLoad 26/60 Superdex 200 column (GE Healthcare) at 277 K. Pure fractions collected in SEC buffer (25 mM HEPES pH 7.0, 500 mM NaCl, 2 mM DTT, 0.025% sodium azide and 5% glycerol) as a single peak were pooled and the protein was concentrated to 25.35 mg ml À1 , as determined via A 280 including compensation for the absorption coefficient, with good solubility. The 2.5 ml sample was flash-frozen and stored at 193 K prior to crystallography. structural communications Figure 1 Fatty-acid biosynthetic pathway for R. prowazekii. The R. prowazekii fatty-acid biosynthesis map from the KEGG PATHWAY database (Kanehisa & Goto, 2000) is shown with FabG highlighted in red.

Crystallization and X-ray data collection
For the FabG target at a protein concentration of 23.4 mg ml À1 in SEC buffer, two sparse-matrix screens were set up, JCSG+ (Emerald BioSystems) and PACT (Molecular Dimensions), following the strategy of Newman et al. (2005). 0.4 ml protein solution was mixed with 0.4 ml well solution and equilibrated at 289 K against 100 ml reservoir solution using 96-well Compact Jr crystallization plates from Emerald BioSystems. Crystals suitable for diffraction studies were obtained within two weeks from JCSG+ screen condition D3: 100 mM sodium/potassium phosphate pH 6.2, 50% PEG 200, 200 mM NaCl. The crystals, which were approximately 0.1-0.15 mm in each direction, did not need additional cryoprotection and were flashfrozen by plunging them into liquid nitrogen.
A native data set was collected to 2.25 Å resolution in-house at Cu K wavelength using a Rigaku SuperBright FR-E+ rotatinganode X-ray generator equipped with Osmic VariMax HF optics and a Saturn 944+ CCD detector ( Table 1). Because of the rather long c axis (183 Å ) and the rather small in-house detector (94.4 mm), the data set was collected with fine ' slicing (0.3 ). The diffraction data were reduced with XDS/XSCALE (Kabsch, 2010) to 2.25 Å resolution. The crystals belonged to the primitive tetragonal space group P4 1 2 1 2. The data-collection and refinement statistics are summarized in Tables 1 and 2.

Structure solution and refinement
Using the BLAST (Altschul et al., 1997) homology-search server at NCBI, Brucella melitensis glucose/ribitol dehydrogenase (PDB entry 3emk; Seattle Structural Genomics Center for Infectious Disease, unpublished work) was found to be the closest homolog with a known structure at the time, with 54% sequence identity. The monomer of PDB entry 3emk was modified with the CCP4 program   jF obs j À jF calc j = P hkl jF obs j. The free R factor was calculated using 5% of the reflections omitted from the refinement (Winn et al., 2011).

Figure 2
Crystal structure of R. prowazekii 3-ketoacyl-(ACP) reductase (FabG). FabG from R. prowazekii crystallizes with two molecules per asymmetric unit, which are shown as ribbon diagrams with purple helices and yellow strands. The active-site residues (Ser143, Tyr156 and Arg160) are grouped near the loop connecting 5 and 5. Secondary-structure elements are labeled from both chains. A tetramer is generated by crystallographic symmetry. Molecules A 0 and B 0 are shown as ribbon diagrams with blue helices and yellow strands. The figure was generated with CCP4mg (McNicholas et al., 2011).

Figure 3
Structural superimpositions of monomeric units for FabG. In this figure, the monomers of FabG from R. prowazekii (PDB entry 3f9i; purple helices, yellow strands), from E. coli (PDB entry 1i01; magenta), from M. tuberculosis (PDB entry 1uzm; light blue) and the NADP + -bound structure from V. cholerae (PDB entry 3rsk, pink) are superimposed. There is a significant structural variability between these structures in the proximity of the NADP + -binding site.
CHAINSAW (Stein, 2008;Winn et al., 2011), which deleted four residues (Glu77-Gly80) and mutated 107 residues to their last common atom. Based on a packing density V M (Matthews, 1968) of 2.24 Å 3 Da À1 , corresponding to 45% solvent, two molecules were expected to be present in the asymmetric unit. Molecular replacement was performed with the CCP4 program Phaser (McCoy et al., 2007) using the modified model of 3emk and data to 3 Å resolution. Phaser could position two molecules in the asymmetric unit with rotation/translation Z scores of 5.4/17.7 and 5.4/26.7, respectively, and an R factor of 0.52. The initial model was improved with ARP/wARP (Langer et al., 2008), which built 367 residues in 13 chains with R work = 0.259 and R free = 0.307. This model was then extended and refined in iterative cycles of REFMAC5 (Murshudov et al., 1997) and Coot (Emsley et al., 2010). In REFMAC5 no data-cutoff criteria were used and NCS restraints were applied given the limited resolution of the data.
The final model for FabG contained two protein chains spanning residues Met1-Val241 for either chain, with gaps at Gly85-Asp99, Gly138-Gly141 and Lys182-Leu189 in chain A and Thr87-Asp99 and Ile139-Gly141 in chain B. The two protomers are very similar and superimposed with an r.m.s.d. of 0.44 Å . 129 water molecules were located. In refining this structure the inclusion of TLS terms did not significantly improve the refinement; hence, no anisotropic displacement factors were deposited. The final structure was validated with Coot and MolProbity (Chen et al., 2010) and deposited in the PDB with the identifier 3f9i.

Overall structure
The structure of R. prowazekii FabG (Fig. 2) is very similar to those reported for E. coli FabG and other bacterial FabGs; the protomer assumes a Rossmann-fold structure with a seven -stranded twisted parallel -sheet flanked by four -helices on either side and two motifs at the core (Fig. 2). R. prowazekii FabG crystallizes with two protomers per asymmetric unit. A tetramer is generated by crystallographic symmetry. According to PISA analysis (Krissinel & Henrick, 1997) there are two types of interface: a larger interface exists between AB and A 0 B 0 comprising 1690 Å 2 with an estimated binding energy of À126 kJ mol À1 and a smaller interface exists between AB 0 and A 0 B comprising 845 Å 2 with an estimated binding energy of À71 kJ mol À1 . For FabGs tetramers are typically observed. It is therefore assumed that R. prowazekii FabG is in a tetrameric state.

Comparison with other bacterial 3-ketoacyl-(ACP) reductases
The R. prowazekii Alignment of solved structures of bacterial 3-ketoacyl-(ACP) reductases. Multiple sequence and secondary-structure alignment of bacterial orthologs. Numbering along the top is based on the alignment consensus. Predicted -helices, -strands, coils and turns are shown as pink cylinders, yellow arrows, grey corkscrews and blue curved arrows, respectively. Sequences are taken from the PDB. From top to bottom: E. coli, 1i01 (Price et al., 2001);M. tuberculosis, 1uzm (Cohen-Gonsaud et al., 2002); Pseudomonas aeruginosa, 2b4q (Miller et al., 2006) I. Shumilin, W. Anderson & W. Minor, unpublished work) over 208 residues, as shown in Fig. 3, whereas the r.m.s.d. with Sus scrofa mammalian FAS (PDB entry 2vz8; Maier et al., 2008), used here as a homologue for the unsolved human FAS structure, is 2.5 Å over 80%. Although there are closer orthologs to Rickettsia FabG than the three aforementioned bacterial structures, these four structures represent prominent bacterial pathogens and the overall structural similarity is useful to note. Furthermore, a multiple sequence alignment (Fig. 4) of eight bacterial orthologs of 3-ketoacyl-(ACP) reductase ranges from 30 to 54% sequence identity over 93-95% sequence coverage. Of these eight orthologs, only one is not an NIAID Class A-C pathogen. Based on these high overall correspondences in sequence and secondary and tertiary structure, conclusions drawn from the other bacterial structures may also be applicable to the Rickettsia protein.
FabG has been shown to have negative cooperative binding of NADPH (Price et al., 2001) and this effect is enhanced by the presence of acyl carrier protein (ACP). NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG. Thus, unlike other members of the short-chain dehydrogenase/ reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad (Ser143, Tyr156 and Arg160) and alters the affinity of the other substrate-binding sites in the tetrameric enzyme (Price et al., 2001). This behavior could explain why the pocket around the NADP + in Fig. 3 differs significantly among the four bacterial targets and serves as a caveat that a given bacterial FabG antidote may not immediately be effective against Rickettsia.
While a number of antimicrobial options have been identified for targeting most of the enzymes in the FAS II pathway, FabG is still relatively unaddressed, in part owing to the conformational change that it undergoes. Epigallocatechin gallate (EGCG), along with other related plant polyphenols, has been shown to be a mixed-type inhibitor of E. coli FabG, although inhibiting fatty-acid synthesis was not the only target of EGCG . Similar findings have been generated against the eukaryotic Plasmodium falciparum ortholog (Tasdemir et al., 2006). Additionally, plant polyphenols have not been considered to be effective against Gram-negative bacteria owing to their effective permeability barrier as well as the action of efflux pumps; deactivating these pumps in conjunction with plant polyphenol administration has been shown to be effective (Tegos et al., 2002). Nonetheless, Zhang and coworkers concluded that the flexible substrate-binding pocket, while a challenge to model for druggability (Price et al., 2004), offers the prospect of utilizing a more potent slow-binding inhibitor . In contrast, Poncet-Montange and coworkers inferred from an inactivated mutant form of the M. tuberculosis FabG1 that drug-target screening could be among ligands that freeze the enzyme in its inactive form (Poncet-Montange et al., 2007).

Conclusion
The SSGCID has solved a 2.25 Å resolution structure of R. prowazekii FabG, a 3-ketoacyl-(acyl-carrier-protein) reductase, which corresponds well to the solved structures for other notable bacterial pathogens while differing from mammalian analogs. For Rickettsia in particular this is a significant target for drug development as the organism has a minimal proteome with very few essential biosynthetic pathways maintained. Owing to a significant conformational change during substrate binding, it has been difficult to identify suitable inhibitors for this essential and highly conserved enzyme.


extfieldsofstudy:
Medicine
Materials Science

text:
A label-free biosensor based on silver nanoparticles array for clinical detection of serum p53 in head and neck squamous cell carcinoma

By using triangular silver (Ag) nanoparticle array, a localized surface plasmon resonance (LSPR) nanosensor was fabricated and shown to sense serum p53 protein in vitro, which is involved in head and neck squamous cell carcinoma (HNSCC). The nanosensor consists of a triangular Ag nanoparticles array with single particle dimension of 120 nm in-plane width and 45 nm out-of-plane height. When examined using LSPR nanobiosensor, the results indicated significant difference in LSPR shifts (Δλmax) between HNSCC patient and control. Although there is need for precise quantification and large-scale prospective, this report shows that the LSPR nanobiosensor provides a promising platform with attractive advantages for serological diagnosis or molecular diagnosis in tumor, such as HNSCC. This is the first clinical application of the LSPR nanosensor in HNSCC.

Introduction
p53 protein is a 53-kDa nuclear tumor suppressor protein present in humans and is encoded by the TP53 gene. When tumors develop, point mutations at the TP53 gene can lead to overexpression of p53 proteins, which contribute to continuous cell division and canceration. Overexpression of p53 has been reported in 60% of laryngeal carcinomas, 37% of hypopharyngeal carcinomas, and 52% of tongue carcinomas. [1][2][3] With the mortality and disintegration of tumor cells, p53 protein released from cancer cells will enter into the circulation. The serum p53 protein level in carcinoma of colon, lung, and pancreas was significantly increased compared with normal controls. [4][5][6] It was reported that 68 out of 75 patients (91%) with head and neck squamous cell carcinoma (HNSCC) had detectable serum p53 protein in the preoperative blood. 7 The detection of serum p53 protein may play an important role in serological diagnosis of tumor, including HNSCC. The serum p53 protein level can be examined mainly by enzyme-linked immunosorbent assay (ELISA). ELISA has advantages of sensitivity, reproducibility, and stability. Its applications are constrained for high-titer anti-p53 antibody interference of some noncancer patients and it is a time-consuming, complicated and inconvenient procedure. To address these considerations, sensitive, easy to use, and cost-effective portable biosensors that are capable of providing continuous monitoring and rapid detection of serum p53 protein need to be developed.
The localized surface plasmon resonance (LSPR)-based nanobiosensor is a new type of optical biosensor technique that combines nanotechnology with optical biosensor technology. LSPR is one of the specific characteristics of metallic or metalized nanostructured materials, such as noble metal nanoparticles. It can be excited when the incident photon frequency is resonant with the collective oscillation of the conduction electrons. 8 Transmission peaks of LSPR-related spectra are sensitive to the electric medium on the surface of noble metals. It demonstrates that the applicability of this biosensor can be more defined in a wide range of fields, such as medical, food safety, environmental monitoring, and drug screening. [9][10][11] The aim of the present study is to apply the developed LSPR biosensor based on the triangular silver (Ag) nanoparticles for the detection of p53 protein levels in samples from HNSCC patients. This is the first case report of the LSPR sensor that responds to serum p53 protein of a cancer patient and a healthy control.
Absolute ethanol and phosphate-buffered saline (PBS; 10 mM, pH 7.4) were purchased from Beijing Biosynthesis Biotechnology Company (Beijing, China). The buffer used in the experiments was prepared using double-distilled water.

Patients and sample
Two serum samples from one HNSCC patient and one healthy control were collected from West China Hospital of Stomatology, Sichuan University, Chengdu, People's Republic of China, in 2009. Informed consent documents were signed prior to the study.
The HNSCC patient was diagnosed by biopsy preoperatively and had no prior radiotherapy or chemotherapy. One venous blood sample was obtained from the patient before operation and another from a healthy volunteer as healthy control.
Once blood was collected, it was allowed to stand at 37°C, for 2 h, and then was centrifuged at 800 g for 10 min. The serum was collected and stored at −80°C until analysis.

Preparation and functionalization of the LsPr-based nanobiosensor
The sensor chip in this work was produced using nanosphere lithography (NSL) technology. K9 glass substrates (Juke Co., Chengdu, China) were cleaned by piranha solution (1:3 30% H 2 O 2 /H 2 SO 4 ) at 80°C for 30 min. Then they were rinsed with copious amounts of second distilled water and sonicated for 60 min in 5:1:1 H 2 O/NH 4 OH/30% H 2 O 2 . After 7 µL of nanosphere solution (Duke Scientific, Palo Alto, CA) was spin coated onto the substrate, 50 nm Ag was deposited by a thermal evaporation system (C-Vac400-I; C-Vac Inc., Chengdu, China). Finally, the Ag nanostructures on the substrate for the experiments were formed after the nanospheres were removed by ultrasonic in ethanol.
We measured the surface modality of the samples by using a scanning electron microscope (S-4800; Hitachi, Tokyo, Japan). The triangular Ag nanoparticles have dimensions of 120 nm in-plane widths, 400 nm period of the nanoparticles array as measured by SEM, and 45 nm outof-plane heights as measured by a sidestep apparatus. Functionalization of the sensor chip is a multistep process that provides the surface for specific detection applications. First, a self-assembled monolayer of 2 mM 1-OT/1 mM 11-MUA acid was formed onto the surface by incubation in an ethanoic solution for 24 h. After thoroughly rinsing and drying, the biochip was then incubated in 20 mM EDC/10 mM S-NHS/2 µg/mL monoclonal mouse anti-p53 solution for 12 h in the dark to link the antibody to the surface of nanoparticles. EDC, a zero-length coupling agent, can couple the amine groups on the antibody to the carboxyl groups on 11-MUA. This process is facilitated  Figure 2.
The serum samples were then rinsed using 0.01 M PBS buffer, and 40 µL of diluted serums from human serum were spotted onto the surface of this functionalized biochip for 150 min (Figure 3).

spectroscopy and observation
The integrated LSPR sensor used in this study is our homebuilt system (Figure 4). The peak wavelength of LSPR extinction spectrum (λ max ) excited by the Ag triangular nanoparticles array was measured and recorded using a UVvisible spectroscope (Sciencetech 9005; Sciencetech, Ottawa, Canada). Practical measurements of the UV-visible extinction spectra were carried out using a fiber-coupled spectrometer (USB4000; Ocean Optics, Dunedin, FL) and a CCD detector (Koan Electro-Optics Co., Shanghai, China).
The measuring procedure was divided into three steps: 1) collecting the reference light without sensor chip, 2) collecting the background noise to increase the accuracy, and 3) measuring the transmission from the sensor chip without any analysis. The sensor's extinction spectra can be caculated by software. With these processes, the sensor's extinction spectra can be easily obtained by in computer, and we can record and compare the data of different sensor chips. The shift toward longer wavelengths is referred to as a redshift and is denoted as (+), whereas the shift toward shorter wavelengths is referred to as a blueshift and is denoted as (−). The system resolution of this biosensor is about 5 nm. When the wavelength shift of the absorbance peak is less than + 5 nm, it indicates that the biosensor cannot sense the medium at the nanoparticles-solution interface.

Results
LSPR spectra of the Ag nanosensors in each processing step are shown in Figures 5 and 6, with the incidence wavelength ranging from 400 to 700 nm. The resulting extinction spectrum of the bare Ag nanoparticles is depicted in Figure 5A, where the LSPR λ max was measured to be 588.2 nm ( Figure 5A). The LSPR λ max of Ag nanoparticles after modification with 11-MUA/1-OT was 623.5 nm ( Figure 5B). The LSPR λ max shift corresponding to this step was a 35.3-nm redshift. After p53 antibody attachment, the λ max of modified Ag nanoparticles chip was 673.84 nm ( Figure 5C). There is an additional 50.34-nm redshift from the second peak. The LSPR shifts from 673.84 to 676.08 nm during the serum incubation for the control serum, and then ∆λ max was a 2.24-nm shift (Figure 5D). Redshift volume of 2.24 nm is less than 5 nm, which demonstrates that LSPR sensor chip shows no response to control serum.
The measured extinction spectrum of serum from the HNSCC patient is shown in Figure 6. The λ max of the biochip   Figure 6C, D). There was a 17.67-nm shift, which is a significant shift to demonstrate the p53 level change in the sample. The peak shift for the HNSCC patient is more than 5 nm, which is larger than that of the control. These results suggest that the LSPR nanobiosensor can respond to p53 protein in HNSCC patient's serum and illustrate the difference of serum p53 level of the HNSCC patient and healthy control.

Discussion
The optical characteristics of the nanochip were indexed by the wavelength shift of the absorbance peak, namely ∆λ max . 8  The biochip after incubation with control serum sample, λ max = 676.08 nm. All extinction measurements were collected at room temperature.

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Biosensor for clinical detection of serum p53 in HNsCC shift using our LSPR-based nanochip. We found that the extinction spectroscopy varies with each step of surface functionalization for the Ag nanoparticles. In addition, the results showed a significant difference in LSPR shifts (∆λ max ) between the HNSCC patient and control. LSPR sensor chip showed no response to control serum, whereas it showed obvious response to HNSCC serum. This suggested a different concentration of p53 protein level between two groups. Chow et al demonstrated that the mean serum p53 protein concentration of the HNSCC patients was 59.45 pg/mL, which was significantly higher than that of the healthy donors (16.4 pg/mL) when examined using ELISA. 7 This result is similar to our experimental results. Moreover, our LSPR method provides free-labeling process, which cannot be obtained using ELISA.
These results indicate the future prospects of LSPR sensor chip application in tumor serological diagnosis. However, further investigation is necessary to evaluate the feasibility of clinical application of this LSPR biosensor system. A large-sample clinical random case-control study with a long follow-up period is in preparation.
This experiment is the first case report of the LSPR sensor, which responded to serum p53 protein of a HNSCC patient and a healthy control. The triangular Ag nanoparticles have dimensions of 120 nm in-plane width, 45 nm out-of-plane height, and 400-nm period of the nanoparticles array. The size of Ag triangle is different from that used in published research. Ag nanoparticles can produce stronger localized surface plasmon than gold nanoparticles, although they are more easily oxidized. In addition, the LSPR peak of Ag particles is also easier to detect. 12 The chip can be preserved in a vacuum plastic bag as long as 1 month. In a normal environment at room temperature, the storage period is 1 week. Meanwhile, the regeneration can be achieved by removing the Ag nanoparticles on the substrate, which is advantageous because the substrate can be thoroughly cleaned so as to improve the fabrication quality of the chips for future experimental use.
All the extinction measurements were carried out at room temperature, and all the samples were exposed in ordinary room conditions with dust and vapor, which illustrates that our Ag particle-based sensor can be used in an open space without any sterilization process. The real-time LSPR-shift assay had a similar response to a commercial surface plasmon resonance instrument, but it demonstrated less interference from bulk refractive index. In addition, LSPR possesses greater spatial resolution, both lateral and normal, when compared with SPR.

Conclusion
For the f irst time, we have proposed a LSPR-based nanobiosensor for the detection of p53 protein from an HNSCC patient and a control. We discovered dramatic differences in LSPR shifts (∆λ max ) between the HNSCC The biochip after incubation with HNsCC serum sample, λ max = 689.08 nm. All extinction measurements were collected at room temperature.

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Zhou et al patient and control. Further large-scale prospective study is worthwhile to evaluate the value of our nanobiosensor for serological diagnosis or molecular diagnosis in tumors, such as HNSCC. However, we anticipate that this technology will be a promising candidate for point-of-care diagnosis and therefore promote the development of personalized medicine in a simple and rapid format.


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Combined directed ortho-zincation and palladium-catalyzed strategies: Synthesis of 4,n-dimethoxy-substituted benzo[b]furans

A new route to regioselectively dialkoxy-functionalized benzo[b]furan derivatives has been developed from 3-halo-2-iodoanisoles bearing an additional methoxy group, which have been accessed through an ortho-zincation/iodination reaction. Two palladium-catalyzed processes, namely a Sonogashira coupling followed by a tandem hydroxylation/cyclization sequence, give rise to new and interesting dimethoxy-substituted benzo[b]furans.

Introduction
The directed ortho-metallation (DoM) reaction has been widely used as a powerful and efficient method for regioselective functionalization of aromatic compounds and different directing groups have been used to facilitate the deprotonation reaction [1][2][3][4][5]. Various strong bases such as alkyl lithiums and their derivatives (for instance, TMEDA-activated complexes [6] and heavier alkali metal tert-butoxide-complexed alkyl lithium reagents, known as superbases and introduced by Schlosser [7]), as well as lithium dialkylamides, have usually been employed to perform deprotonative metallations. Whereas the use of these strong bases has several limitations regarding the presence of certain functional groups (mainly carboxylic acid derivatives and halogens), the introduction in the last years of new organometallic "ate" complexes [8,9], which combine an alkali metal with either magnesium, zinc, aluminium, or copper, has allowed more selective metallation reactions. The milder reaction conditions required make these deprotonation reactions tolerant to the coexistence of a wider range of functional groups. In this field, the work of Kondo and Uchiyama is remarkable as they described highly chemo-and regioselective deprotonative zincation [10][11][12], alumination [13], and cupration [14] reactions of some functionalized aromatic and heteroaromatic compounds, as well as of meta-functionalized haloaromatics. In particular, the alkali metal mediated zincation reactions have turned out to be very useful processes and the structures and reaction pathways of TMP-zincates (TMP = 2,2,6,6-tetramethylpiperidine) have been studied in detail [15][16][17][18][19].
On the other hand, benzo[b]furan is a basic skeleton found in a variety of significant natural products [20], and some derivatives are also used as organic materials, due to their optical and electronic properties [21]. Thus, many synthetic efforts have been devoted to the synthesis of this type of compound [22,23]. In particular, several benzo[b]furan derivatives with oxygenbearing substituents, such as hydroxy, or alkoxy, at the benzene moiety are known to be biologically active compounds [24][25][26][27][28] ( Figure 1). Among the various approaches developed for the synthesis of the benzofuran ring system, the cyclodehydration of α-aryloxy ketones [29], the Claisen rearrangement of an allyl aryl ether followed by Pd-catalyzed intramolecular oxidative cyclization [30], and the tandem Sonogashira coupling/heterocyclization of 2-halophenols with terminal alkynes [31], are some of the most used. However, their application to the synthesis of 4-substituted benzo[b]furans is especially challenging, because the meta-substituted starting materials tend to cyclize at the less hindered ortho-position, leading to the formation of 6-substituted heterocycles or a mixture of 6-and 4-substituted ones [32].
The DoM strategy, when linked with different cross-coupling Pd-catalyzed reactions, could provide a superior approach for the construction of polysubstituted aromatic and heteroaromatic compounds [33][34][35][36][37][38]. In this context, we studied the o-lithiation of 3-halophenols and the resulting 2,3-difunctionalized phenol derivatives were applied to the synthesis of 4-functionalized benzo[b]furans [39], 4-or 7-alkoxyindoles [40], and 7-oxy-substituted benzo[b]thiophenes [41] by employing Pd-catalyzed cross-coupling reactions or halocyclization processes. Following our interest in the development of strategies for the synthesis of functionalized benzo[b]furan derivatives [39], we envisaged that 4,n-dimethoxybenzo[b]furans could be regioselectively synthesized from 3-halo-2iodoanisoles bearing an additional methoxy group, by combining two palladium-catalyzed processes, that is a selective Sonogashira coupling and a tandem hydroxylation/ heterocyclization reaction. The required o-dihaloanisole derivatives could be prepared by a selective ortho-metallation reaction and subsequent electrophilic quenching with iodine (Scheme 1).
Thus, we decided to test this deprotonative metallation on the commercially available dimethoxyhalobenzene derivatives 3  with the same t-Bu 2 Zn(TMP)Li, easily prepared by reaction of preformed di-tert-butylzinc with lithium tetramethylpiperidide. Under these reaction conditions the zincation reactions took place regioselectively and, after treatment with iodine, afforded the corresponding 3-halo-2-iodoanisole derivatives 4 in good yields (Table 1). It is interesting to note that substrates 3c and 3d bearing the two methoxy groups in a meta relationship selectively undergo metalation at the position between the methoxy and the halide groups, irrespective of the nature of the halogen atom (chloro or bromo) ( Table 1, entries 3 and 4).
Having an efficient protocol for the synthesis of dimethoxyhaloiodobenzene derivatives 4, and with the retrosynthetic analysis outlined in Scheme 1 in mind, we tackled the selective introduction of an alkynyl moiety at the iodinated position. The presence of two different halogen atoms in compounds 4 implies that a selective Sonogashira coupling reaction should occur ( Table 2). This has been achieved in two different ways.
Considering the steric hindrance of the required position (o,odisubstituted) we employed a copper-and solvent-free methodology for the Sonogashira coupling that uses tetrabutylammo- By either of these procedures, A or B, o-alkynylhaloarenes 5-7 were prepared usually in high yields from the corresponding dimethoxyhaloiodobenzenes 4 ( Table 2). Starting from chloroiodo derivatives 4a,c and e good yields were obtained for the corresponding coupled products 5-7 by using the Pd/TBAF method A, with no significant side-products (Table 2, entries  1,2,7,8 and 14). However, under these conditions brominecontaining compounds 4b,d and f afforded variable amounts of dialkynylation products in some cases, and so the Pd/Cu catalyzed procedure B gave rise to better selectivities and yields of the desired alkynes 5-7 (Table 2, entries 4,6,10,12,16 and 18).
According to our retrosynthetic analysis (Scheme 1), the final step to achieve the benzofuran derivatives should be the incorporation of the hydroxy group followed by in situ heterocyclization. In recent years, the direct hydroxylation of aryl halides has been developed by several groups by using palladium-or copper-catalyzed protocols. Whereas the reactions under copper catalysis work well for aryl iodides [45][46][47][48], the palladium-catalyzed hydroxylation also takes place with aryl bromides and chlorides [49][50][51]. Thus, in our case we employed the Pd-catalyzed Buchwald protocol [49] in an attempt to synthesise the desired dimethoxybenzo[b]furan derivatives. Thus, reaction of the previously prepared o-haloaryl alkynes 5-7 with KOH, in the presence of catalytic amounts of Pd 2 (dba) 3 (Table 3, method C). In general, slightly better results were obtained starting from aryl bromides instead of aryl chlorides. In addition, we also observed better yields for the corresponding benzofuran derivatives 9 (Table 3, entries 5-9) derived from the starting o-alkynylhalobenzene derivatives 6, bearing the two methoxy groups in a 3,5-relationship relative to the halide. Moreover, because of the extended reaction times needed for complete consumption of the starting materials, we developed an alternative protocol under microwave irradiation that shortens the time required for the coupling to a few minutes (method D), and the final products were obtained in similar yields to those obtained by the conventional coupling method C.

Conclusion
We have developed a synthetic route to access regioselectively functionalized 4,n-dimethoxybenzo[b]furans through combined directed ortho-metallation (DoM)/palladium-catalyzed reactions. The deprotonative ortho-zincation of meta-functionalized haloanisoles bearing an additional methoxy group, followed by electrophilic quenching with iodine allows the regioselective and straightforward synthesis of highly functionalized dihalodimethoxybenzene derivatives. A subsequent selective Sonogashira coupling with terminal alkynes, followed by direct hydroxylation with KOH of the resulting o-haloaryl alkyne and in situ heterocyclization, afforded the benzo[b]furan derivatives.
In addition, we have developed a new procedure for the Pd-catalyzed hydroxylation reaction that allows the coupling to take place within in a few minutes.