extfieldsofstudy: Chemistry Medicine

text:
Enhancement of Cell Adhesion, Cell Growth, Wound Healing, and Oxidative Protection by Gelatins Extracted from Extrusion-Pretreated Tilapia (Oreochromis sp.) Fish Scale

Gelatin has been broadly utilized in the food, pharmaceutical, photographic, cosmetic and packaging industries, and there is also huge potential for novel applications of gelatin in the fields of biotechnology and biomedicine. In the present study, we extracted gelatin from fish processing waste, i.e., scale of tilapia, by a combined method of extrusion-pretreatment and hot water extraction. The extrusion-pretreatment process increases the extraction yield of gelatin. Three gelatins (FS2: preconditioning with double-distilled water (ddH2O) before extrusion; FS12: preconditioning with citric acid solution before extrusion; FS14: preconditioning with acetic acid solution before extrusion) were obtained and all of them enhanced cell adhesion, cell growth, and wound healing in HaCaT cells and protected HaCaT cells from H2O2-induced cellular damage. Among FS2, FS12, and FS14, FS12 exhibited the most pronounced enhancement of cell adhesion, cell growth, and wound healing in HaCaT cells, and thus it may have potential as an effective natural raw material in cell therapies for cutaneous wounds and for reducing H2O2-induced oxidative damage of cells. In additional experiments, it was found that phosphorylations of Akt and mTOR are involved in the signaling pathway activated by FS2, FS12, and FS14 in HaCaT cells.

Introduction
Collagen is found in the extracellular matrix (ECM) of all eukaryotic tissues and one of its main functions is to provide physical protection of tissues [1]. At least 28 types of collagen have been identified, which are referred to as types I-XXVIII, and they are commonly distributed in skin, bones, cartilage, blood vessels, tendons, cornea, ligaments, and other vertebrate organs [2]. Collagen has a right-handed triple superhelical rod that consists of three almost identical polypeptide chains and it can 500×, and 1000×). It can be seen clearly that the gelatin surface mainly displayed a lamellar structure. In most cases, the morphological appearance was shriveled and wrinkled, which is indicative of possessing film-forming characteristics. Although different preconditioning processes were adopted for FS2, FS12, and FS14, the morphologies of these gelatins were not significantly altered. To evaluate the color difference among the extracted gelatins, the Hunterlab L, a, and b values and whiteness for FS2, FS12, and FS14 were measured. The data presented in Table 1 show that FS2 (with ddH 2 O preconditioning) had the largest L value and whiteness, followed by FS14 (with acetic acid preconditioning), and then FS12 (with citric acid preconditioning). These results indicate that the acid preconditioning process may accelerate the browning reaction of fish scale extrudate and cause the extracted gelatins to become darker. In general, color does not apparently influence the functional properties of gelatin. However, gelatin with a whiter appearance is more appealing to consumers. It was reported that the whiteness of fish gelatins from shortfin scad and sin croaker were 78.74 and 83.64, respectively [25]. We found that the whiteness values in FS2 (82.8 ± 0.0), FS12 (76.5 ± 0.6), and FS14 (79.1 ± 0.4) were similar to previously reported values for sin croaker and shortfin scad gelatins. In addition, the whiteness of FS2 was higher than those of FS12 and FS14. The pH values of FS2, FS12, and FS14 solutions were 7.75 ± 0.04, 7.20 ± 0.00, and 6.20 ± 0.00, respectively (Table 1). In general, FS12 and FS14 had lower pH values than that of FS2, possibly due to the acid preconditioning process of FS12 and FS14. Choi and Regenstein (2000) revealed that the gel strength of gelatin was affected by the pH value of gelatin solution. Gelatins showed a maximum gelatin strength at around pH 8, and the gel strengths of gelatins decreased gradually for pH values above or below pH 8 [26]. We have previously shown that the gel strengths (Bloom values) of FS2, FS12, and FS14 were 260.3 ± 1.7, 185.0 ± 5.4, and 157.0 ± 5.1 g, respectively [24]. Thus, our results showing FS2 (pH = 7.75 ± 0.04) had the highest gel strength, followed by FS12 (pH = 7.20 ± 0.00), and then FS14 (pH = 6.20 ± 0.00), are consistent with Choi and Regenstein's (2000) findings [26]. Taken together, the morphologies of lyophilized extracted gelatins were not obviously influenced by various preconditioning processes. However, the acid preconditioning processes decreased the L value and whiteness of gelatins, decreased the pH values of gelatin solutions, and decreased the gel strengths of gelatins. Since FS2, FS12, and FS14 exhibit different physicochemical properties, their biological functions warrant further examination. In most cases, the morphological appearance was shriveled and wrinkled, which is indicative of possessing film-forming characteristics. Although different preconditioning processes were adopted for FS2, FS12, and FS14, the morphologies of these gelatins were not significantly altered. To evaluate the color difference among the extracted gelatins, the Hunterlab L, a, and b values and whiteness for FS2, FS12, and FS14 were measured. The data presented in Table 1 show that FS2 (with ddH2O preconditioning) had the largest L value and whiteness, followed by FS14 (with acetic acid preconditioning), and then FS12 (with citric acid preconditioning). These results indicate that the acid preconditioning process may accelerate the browning reaction of fish scale extrudate and cause the extracted gelatins to become darker. In general, color does not apparently influence the functional properties of gelatin. However, gelatin with a whiter appearance is more appealing to consumers. It was reported that the whiteness of fish gelatins from shortfin scad and sin croaker were 78.74 and 83.64, respectively [25]. We found that the whiteness values in FS2 (82.8 ± 0.0), FS12 (76.5 ± 0.6), and FS14 (79.1 ± 0.4) were similar to previously reported values for sin croaker and shortfin scad gelatins. In addition, the whiteness of FS2 was higher than those of FS12 and FS14. The pH values of FS2, FS12, and FS14 solutions were 7.75 ± 0.04, 7.20 ± 0.00, and 6.20 ± 0.00, respectively (Table 1). In general, FS12 and FS14 had lower pH values than that of FS2, possibly due to the acid preconditioning process of FS12 and FS14. Choi and Regenstein (2000) revealed that the gel strength of gelatin was affected by the pH value of gelatin solution. Gelatins showed a maximum gelatin strength at around pH 8, and the gel strengths of gelatins decreased gradually for pH values above or below pH 8 [26]. We have previously shown that the gel strengths (Bloom values) of FS2, FS12, and FS14 were 260.3 ± 1.7, 185.0 ± 5.4, and 157.0 ± 5.1 g, respectively [24]. Thus, our results showing FS2 (pH = 7.75 ± 0.04) had the highest gel strength, followed by FS12 (pH = 7.20 ± 0.00), and then FS14 (pH = 6.20 ± 0.00), are consistent with Choi and Regenstein's (2000) findings [26]. Taken together, the morphologies of lyophilized extracted gelatins were not obviously influenced by various preconditioning processes. However, the acid preconditioning processes decreased the L value and whiteness of gelatins, decreased the pH values of gelatin solutions, and decreased the gel strengths of gelatins. Since FS2, FS12, and FS14 exhibit different physicochemical properties, their biological functions warrant further examination.    1 7.75 ± 0.04 c 7.20 ± 0.00 b 6.20 ± 0.00 a 1 pH value was measured using FS2, FS12, and FS14 solutions, respectively. 2 The data are represented as means ± standard deviation (SD) (n = 3); different superscript letters in the same row indicate significantly different (p < 0.05).

Enhancement of Cell Adhesion, Cell Growth, and Wound Healing in HaCaT Cells by FS2, FS12, and FS14
In order to evaluate the promotive effects of FS2, FS12, and FS14 on cell adhesion and cell growth, a human keratinocyte cell line HaCaT was adopted as a cell model in the present study. HaCaT keratinocytes are regarded as a good in vitro model of the skin epidermal layer [27], and can be used to assess the therapeutic effects of compounds on tissue regeneration [28]. HaCaT cells can also be utilized in in vitro skin wound healing models [29], and can be used to assess heavy metal-induced human skin damage [30], as well as the oxidative protective effects of various compounds [31]. For cell culture, cells are necessary to adhere onto the surface of a plate that is being used. It has long been known that cell attachment importantly influences the cell proliferation, migration, differentiation, and even the assembly of individual cells into the three-dimensional tissues of animals [32]. HaCaT cells were seeded onto plates pre-coated with different concentrations of FS2, FS12, and FS14, and cultured for 45 min. The cell adhesive activity of the HaCaT cells was then evaluated by crystal violet assay. As shown in Figure 2A,B, the percentages of adhesive cell number in the three gelatin pre-coated samples were increased as compared to the non-coated control. The maximum increment percentage of adhesive cell number was found to be 166% ± 6% at the concentration of 1000 ng/cm 2 on FS12 pre-coated plate. In general, FS12 had the most cell adhesive activity as compared to FS2 and FS14 at the concentrations tested. Previous studies revealed that collagen, the major structural protein found in the ECM of many tissues, is rich in arginine-glycine-aspartic acid (RGD), a cell adhesion motif that is capable of promoting cell adhesion and proliferation [33]. Gelatin is a soluble form of collagen and also possesses RGD, making it highly effective for cell adhesion [34]. We speculate that the content of RGD or exposure to RGD is the main factor that accounts for the enhancement of cell adhesion by FS2, FS12, and FS14. Once the enhancing effect of FS2, FS12, and FS14 on cell adhesion had been verified, we further examined the promotive effects of FS2, FS12, and FS14 on cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 3A, all gelatins FS2, FS12, and FS14 showed cell growth enhancement effects at concentrations from 25 to 200 µg/mL. The percentages of cell growth were dose-dependently increased in FS2-, FS12-, and FS14-treated samples at concentrations from 25 to 75 µg/mL, but they decreased in FS2-, FS12-, and FS14-treated samples at concentrations from 100 to 200 µg/mL. Therefore, excess gelatin may inhibit rather than promote cell growth. The maximum increment percentages of cell growth were found at a concentration of 75 µg/mL for FS2, FS12, and FS14 samples. Thus, the concentration of 75 µg/mL for FS2, FS12, and FS14 was utilized for cell cycle analysis. As shown in Figure 3B, all gelatin extracts FS2-, FS12-, and FS14-treated HaCaT cells exhibited a decline in number of cells in the G 0 /G 1 phase and an increase in the proportion of cells in the G 2 /M phase, as compared to the control. These findings indicate that the increased proliferation rate was attributed to an increased rate of entry into the S phase and further indicated that a large population of cells were in mitosis. Wound healing is a synergic effect among integrin, ECM, and growth factors, and which is probably reactioned as a key regulatory factor for the epidermal proliferation and cell cycle progression [35]. Wound healing involves several cellular and biochemical processes; the first phase is the activation of the inflammatory processes, while the second phase is characterized by angiogenesis and granulation tissue formation, finally leading to re-epithelialization [36]. The wound healing enhancement of FS2, FS12, and FS14 in HaCaT cells was analyzed by scrape wound healing assays. As shown in Figure 4A,B, all gelatin samples FS2, FS12, and FS14 showed wound-healing effects on HaCaT cells dose-dependently. After 12 h treatment and at a concentration of 150 µg/mL, the extent of wound closure reached 40.9% ± 1.3% for FS2, 55.1% ± 0.5% for FS12, and 53.1% ± 3.3% for FS14. After 24 h treatment and at a concentration of 150 µg/mL, the extent of wound closure was 82.6% ± 1.6% for FS2, 88.0% ± 0.8% for FS12, and 83.9% ± 1.8% for FS14. In general, FS12 exhibited the greatest enhancement of wound healing, followed by FS14, and then FS2. These data also clearly show that FS2, FS12, and FS14 have the potential for use in cell therapies to treat cutaneous wounds. Furthermore, the most abundant gelatin sources are pig skin (46%), bovine hide (29.4%), and pork and cattle bones (23.1%) [37]. Thus, in future studies, it may be worthwhile to compare the biological functions of our fish scale gelatins with those of commercial gelatins. In summary, all gelatin samples FS2, FS12, and FS14 enhanced cell adhesion, cell growth, and wound healing in HaCaT cells. Among the gelatin samples tested, FS12 exhibited pronounced enhancement of cell adhesion, cell growth, and wound healing, and thus it is recommended as an effective natural raw material for biotechnological and biomedical applications.
Molecules 2018, 23, x FOR PEER REVIEW 5 of 16 [36]. The wound healing enhancement of FS2, FS12, and FS14 in HaCaT cells was analyzed by scrape wound healing assays. As shown in Figure 4A,B, all gelatin samples FS2, FS12, and FS14 showed wound-healing effects on HaCaT cells dose-dependently. After 12 h treatment and at a concentration of 150 μg/mL, the extent of wound closure reached 40.9% ± 1.3% for FS2, 55.1% ± 0.5% for FS12, and 53.1% ± 3.3% for FS14. After 24 h treatment and at a concentration of 150 μg/mL, the extent of wound closure was 82.6% ± 1.6% for FS2, 88.0% ± 0.8% for FS12, and 83.9% ± 1.8% for FS14. In general, FS12 exhibited the greatest enhancement of wound healing, followed by FS14, and then FS2. These data also clearly show that FS2, FS12, and FS14 have the potential for use in cell therapies to treat cutaneous wounds. Furthermore, the most abundant gelatin sources are pig skin (46%), bovine hide (29.4%), and pork and cattle bones (23.1%) [37]. Thus, in future studies, it may be worthwhile to compare the biological functions of our fish scale gelatins with those of commercial gelatins. In summary, all gelatin samples FS2, FS12, and FS14 enhanced cell adhesion, cell growth, and wound healing in HaCaT cells. Among the gelatin samples tested, FS12 exhibited pronounced enhancement of cell adhesion, cell growth, and wound healing, and thus it is recommended as an effective natural raw material for biotechnological and biomedical applications.

Oxidative Protection Effect of FS2, FS12, and FS14 on HaCaT Cells
In biological systems, many factors may result in inactivation of cellular antioxidant molecules. The inactivation of cellular antioxidant molecules can result in an increase in reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions, and hydrogen peroxide [38]. ROS generate damage to lipids (lipid peroxidation) [39], proteins, and DNA [40]. Human skin, particularly the epidermis, is directly and continuously exposed to a number of chemical and physical environmental stresses. Excess exposure to these stresses may cause erythema, hyperpigmentation, hyperplasia, immune suppression, photoaging, and skin cancer [41][42][43]. It was also found that excessive ROS production and/or their ineffective elimination is implicated in many cutaneous pathological processes [43,44]. In the present study, HaCaT cells were utilized in an in vitro cell model to investigate the protective effect of FS2, FS12, and FS14 against oxidation in human skin. The treatment of HaCaT cells with various concentrations of H2O2 dose-dependently decreased the cell viability. At a concentration of 150 μM H2O2, cell viability was about 50.7% ± 1.6% of the control ( Figure 5A). Thus, 150 μM H2O2 was used to induce cellular cytotoxicity in HaCaT cells. The protective effects of FS2, FS12, and FS14 against oxidation are shown in Figure 5B. Pretreatment of HaCaT cells with FS2, FS12, or FS14 at concentrations of 25 to 150 μg/mL for 24 h gradually attenuated H2O2-induced cellular cytotoxicity ( Figure 5B). The greatest improvement in cell viability for FS2 was from 54.0% ± 0.6% to 77.6% ± 0.6% at a concentration of 150 μg/mL; for FS12 it was from 51.3% ± 3.3% to 79.3 ± 2.2% at a concentration of 125 μg/mL; and for FS14 it was from 54.5% ± 2.8% to 74.5% ± 3.1% at a concentration of 75 μg/mL. In general, all gelatins FS2, FS12, and FS14 exhibited obvious protection against oxidation in HaCaT cells and thus they have potential as natural and effective agents to eliminate ROS-induced cutaneous pathological impairments.

Oxidative Protection Effect of FS2, FS12, and FS14 on HaCaT Cells
In biological systems, many factors may result in inactivation of cellular antioxidant molecules. The inactivation of cellular antioxidant molecules can result in an increase in reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions, and hydrogen peroxide [38]. ROS generate damage to lipids (lipid peroxidation) [39], proteins, and DNA [40]. Human skin, particularly the epidermis, is directly and continuously exposed to a number of chemical and physical environmental stresses. Excess exposure to these stresses may cause erythema, hyperpigmentation, hyperplasia, immune suppression, photoaging, and skin cancer [41][42][43]. It was also found that excessive ROS production and/or their ineffective elimination is implicated in many cutaneous pathological processes [43,44]. In the present study, HaCaT cells were utilized in an in vitro cell model to investigate the protective effect of FS2, FS12, and FS14 against oxidation in human skin. The treatment of HaCaT cells with various concentrations of H 2 O 2 dose-dependently decreased the cell viability. At a concentration of 150 µM H 2 O 2, cell viability was about 50.7% ± 1.6% of the control ( Figure 5A). Thus, 150 µM H 2 O 2 was used to induce cellular cytotoxicity in HaCaT cells. The protective effects of FS2, FS12, and FS14 against oxidation are shown in Figure 5B. Pretreatment of HaCaT cells with FS2, FS12, or FS14 at concentrations of 25 to 150 µg/mL for 24 h gradually attenuated H 2 O 2 -induced cellular cytotoxicity ( Figure 5B). The greatest improvement in cell viability for FS2 was from 54.0% ± 0.6% to 77.6% ± 0.6% at a concentration of 150 µg/mL; for FS12 it was from 51.3% ± 3.3% to 79.3 ± 2.2% at a concentration of 125 µg/mL; and for FS14 it was from 54.5% ± 2.8% to 74.5% ± 3.1% at a concentration of 75 µg/mL. In general, all gelatins FS2, FS12, and FS14 exhibited obvious protection against oxidation in HaCaT cells and thus they have potential as natural and effective agents to eliminate ROS-induced cutaneous pathological impairments.

Phosphorylation of Akt and mTOR is Involved in the Signaling Pathway of FS2, FS12, and FS14 in HaCaT Cells
The frequent activation of the PI3K/Akt/mTOR pathway has been shown to be involved in cell growth, cell survival, development of cancer, and wound healing [45,46]. We thus evaluated the expressions of phosphorylated Akt (p-Akt), total Akt (Akt1), and phosphorylated mTOR (p-mTOR) in HaCaT cells by flow cytometry. As shown in Figure 6A, it was found that gelatin extracts FS2, FS12, and FS14 induced elevated levels of p-Akt as compared to the control at concentrations of 75 and 150 μg/mL. In addition, FS12 showed the most elevated level of p-Akt among the extracted gelatins tested. This observation is compatible with the findings that FS12 showed relatively high levels of cell adhesive activity (Figure 2) and wound healing (Figure 4), indicating that expression of p-Akt may be positively linked to cell adhesion and wound healing in HaCaT cells after gelatin treatments. In addition, the expression levels of Akt1 were not obviously changed with respect to different treatments ( Figure 6B), which served as a control. Moreover, as shown in Figure 6C, we also found that gelatin extracts FS2, FS12, and FS14 induced elevated levels of p-mTOR as compared to the control at concentrations of 75 and 150 μg/mL. These results indicate that the expression of p-mTOR is also involved in FS2-, FS12-, and FS14-induced cellular functions. In summary, the gelatin extracts FS2, FS12, and FS14 promote the cellular functions of HaCaT cells by modulating the Akt/mTOR pathway. Further in vivo elucidation of the molecular mechanism and signaling cascade underlying the cell adhesion, cell growth, and wound healing of FS2, FS12 (in particular), and FS14 is required.

Phosphorylation of Akt and mTOR is Involved in the Signaling Pathway of FS2, FS12, and FS14 in HaCaT Cells
The frequent activation of the PI3K/Akt/mTOR pathway has been shown to be involved in cell growth, cell survival, development of cancer, and wound healing [45,46]. We thus evaluated the expressions of phosphorylated Akt (p-Akt), total Akt (Akt1), and phosphorylated mTOR (p-mTOR) in HaCaT cells by flow cytometry. As shown in Figure 6A, it was found that gelatin extracts FS2, FS12, and FS14 induced elevated levels of p-Akt as compared to the control at concentrations of 75 and 150 µg/mL. In addition, FS12 showed the most elevated level of p-Akt among the extracted gelatins tested. This observation is compatible with the findings that FS12 showed relatively high levels of cell adhesive activity ( Figure 2) and wound healing (Figure 4), indicating that expression of p-Akt may be positively linked to cell adhesion and wound healing in HaCaT cells after gelatin treatments. In addition, the expression levels of Akt1 were not obviously changed with respect to different treatments ( Figure 6B), which served as a control. Moreover, as shown in Figure 6C, we also found that gelatin extracts FS2, FS12, and FS14 induced elevated levels of p-mTOR as compared to the control at concentrations of 75 and 150 µg/mL. These results indicate that the expression of p-mTOR is also involved in FS2-, FS12-, and FS14-induced cellular functions. In summary, the gelatin extracts FS2, FS12, and FS14 promote the cellular functions of HaCaT cells by modulating the Akt/mTOR pathway. Further in vivo elucidation of the molecular mechanism and signaling cascade underlying the cell adhesion, cell growth, and wound healing of FS2, FS12 (in particular), and FS14 is required.

Materials and Chemicals
Fresh TFS was obtained from a fishery factory in Pingtung, Taiwan. TFS was mixed with 0.1 N NaOH to remove non-collagenous proteins, and the mixture was washed with tap water several times until pH reaches 7.0. After drying at 50 • C, the sample was milled into powder (less than 20 mesh) and stored in aluminum foil bags at room temperature until use. Acetic acid and citric acid were purchased from Nihon Shiyaku Industrial, Ltd. (Tokyo, Japan). MTT, dimethyl sulfoxide (DMSO), crystal violet, propidium iodide (PI), and hydrogen peroxide solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trypsin/ethylenediaminetetraacetic acid (EDTA), Dulbecco's modified Eagle medium (DMEM), trypan blue, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco Laboratories (Grand Island, NY, USA). All other chemicals used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

Extrusion-Cooking Procedure
The extrusion-cooking procedure was performed according to the method described previously [24]. After the extrusion-cooking process, extrudates were collected, ground into fine particles, sealed in aluminum foil bags, and stored at 4 • C for further extraction experiments.

Extraction of Gelatin from Fish Scale Extrudate
TFS extrudate powders were soaked in ddH 2 O with a sample ratio of 1:10 (w/v) and shaken in a water bath at 50 • C for 1 h. The mixtures, after centrifuged at 10,200× g for 10 min, the supernatants were collected and lyophilized. Three gelatins, namely FS2 (preconditioning with ddH 2 O), FS12 (preconditioning with 1.26% citric acid solution), and FS14 (preconditioning with 9.37% acetic acid solution) were obtained.

Scanning Electron Microscope (SEM) Examination
Lyophilized gelatin powders were coated with gold using a sputter coater at ambient temperature. Then the surfaces of gelatins were examined with a JEOL JSM-5300 scanning electron microscope (SEM, Peabody, MA, USA) at 5 kV.

Color Analysis
The gelatin samples (about 15 g) were used for the determination of color. Tristimulus color values, namely L (lightness), a (red-green), and b (yellow-blue) values, were recorded using a SA-2000 spectrophotometer (Nippon Denshoku Industries Co., Ltd., Tokyo, Japan). The whiteness was calculated using the following equation:

Measurement of pH
Gelatin solution (6.67%, w/v) was prepared by dissolving gelatin powder in ddH 2 O and then the pH values of gelatin solution were recorded using a SP-2100 pH meter (Suntex, Taipei, Taiwan).

Cell Culture
Human keratinocyte-derived (HaCaT) cells were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). HaCaT cells were grown in DMEM supplemented with heat-inactivated FBS (10%; v/v), streptomycin (100 U/mL), and penicillin (0.1 mg/mL), in a humidified atmosphere of 5% CO 2 at 37 • C. The cells were subcultured every 2-3 days, following trypsinization and seeded in a 10-cm dish at a density of 1 × 10 5 cells/mL.

Gelatin Coating and Cell Adhesion Analysis
Gelatin coating was performed according to a previously described method [47] with some modification. Briefly, different concentrations of gelatin solutions (gelatin powder dissolved in ddH 2 O) were added to the plates and incubated overnight at 37 • C, rinsed twice with ddH 2 O to remove uncoated gelatin, and air dried. Cells (2 × 10 5 cells/mL) were then seeded in triplicate in 600 µL of serum-free DMEM medium in a 24-well plate with and without gelatin precoating. After incubation for 45 min at 37 • C, the medium was discarded and washed with phosphate-buffered saline (PBS) to remove the non-adherent cells. Then some wells were fixed with 95% ethanol, stained with 0.5% crystal violet and photographed. The other wells were quantified by adding 30% acetic acid to dissolve crystal violet and the spectrophotometric absorbance was measured at 630 nm by a PowerWave 340 enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek Instruments, Winooski, VT, USA).

MTT Assay
The number of viable HaCaT cells was determined by MTT colorimetric assay. Exponentially growing cells (2 × 10 5 cells/mL) were seeded in triplicate in a 24-well plate in DMEM with 10% FBS and incubated for 24 h. Treatment of cells with different compounds was carried out for 24 h at 37 • C. After treatment, MTT solution at concentration of 0.5 mg/mL was added onto wells and incubated for 4 h. Then, the DMSO solution was added to produce formazan dyes which represent viable cells.
Cell viability values at absorbance of 570 nm were determined by a PowerWave 340 ELISA plate reader (Bio-Tek Instruments, Winooski, VT, USA).

Cell Cycle Analysis
Cells were plated at 1 × 10 6 cells/mL in 10-cm dish with DMEM growth medium and incubated for 24 h at 37 • C, with 5% CO 2 in a humidified atmosphere. Cells were treated with different gelatin samples at a final concentration of 75 µg/mL for 24 h. The cell cycle analysis procedure was performed according to the method described previously [48].

Scraped Wound Healing Assay
HaCaT cells were seeded in a 10-cm dish with complete medium and grown to confluence. Then, the monolayer was scraped using a 1000 µL micropipette tip and washed with PBS to remove the floating cells. Medium with 10% FBS and gelatins at different concentrations were added to cultures. Photomicrographs were taken at 0 h, 12 h, and 24 h and some representative fields were photographed. The remaining wound area was calculated using PhotoImpact and AutoCAD software and the migration distance of the cells was estimated based on that calculation.

Oxidative Protection Assay
H 2 O 2 has been widely used as an oxidative stress inducer in in vitro or in vivo models [49]. An investigation of oxidative protection was performed according to the method described by Rahimifard and colleagues [50] with some modification. In brief, HaCaT cells (2 × 10 5 cells/mL in a 24-well plate) were incubated for 24 h at 37 • C, with 5% CO 2 in a humidified atmosphere. The cells were then incubated with culture medium and different concentrations (0, 25, 50, 75, 100, 125, and 150 µg/mL) of FS2, FS12, or FS14 for 24 h at 37 • C and 5% CO 2 in a humidified atmosphere. Thereafter, the cells were exposed to 150 µM H 2 O 2 for 24 h at 37 • C. Finally, an assessment of the cells' viability was performed using the MTT assay.

Analyses for Phosphorylated Akt and mTOR
Cells were seeded in a 6 cm dish at 4 × 10 4 cells/mL in 5 mL DMEM growth medium and incubated for 24 h at 37 • C, with 5% CO 2 in a humidified atmosphere. Afterwards, cells were treated with different gelatin samples at a final concentration of 75 or 150 µg/mL for 6 h.

Statistical Analysis
Values are represented as means ± standard deviation (SD). Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) and one-way analysis of variance (ANOVA) and Duncan's multiple range test were employed to test the significance. A p value which is less than 0.05 is considered statistically significant.

Conclusions
In this paper, we extracted gelatins from TFS using the combined methods of extrusion-pretreatment and hot water extraction. The extrusion process facilitated the extraction of gelatin from fish scale by hot water. All extracted gelatins (FS2, FS12, and FS14) enhanced cell adhesion, cell growth, and wound healing in HaCaT cells and protected HaCaT cells from H 2 O 2 -induced cellular damage. Among FS2, FS12, and FS14, FS12 showed the most pronounced enhancement of cell adhesion, cell growth, and wound healing in HaCaT cells. Additional experiments revealed that phosphorylations of Akt and mTOR are involved in the FS2-, FS12-, and FS14-induced signaling pathway in HaCaT Cells. Taken together, all of the studied gelatins FS2, FS12, and FS14 have potential for use as natural and effective agents in cell therapies for treating cutaneous wounds and eliminating ROS-induced cutaneous pathological impairments. Future in vivo studies on the therapeutic usage of gelatins, especially FS12, in the treatment of cutaneous pathology are required.


s2fieldsofstudy: Biology Chemistry

text:
Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial–approved membrane fusion inhibitor

Host cell infection with HIV-1 requires fusion of viral and cell membranes. Sifuvirtide (SFT) is a peptide-based HIV-1 fusion inhibitor approved for phase III clinical trials in China. Here, we focused on characterizing HIV-1 variants highly resistant to SFT to gain insight into the molecular resistance mechanism. Three primary substitutions (V38A, A47I, and Q52R) located at the inhibitor-binding site of HIV-1's envelope protein (Env) and one secondary substitution (N126K) located at the C-terminal heptad repeat region of the viral protein gp41, which is part of the envelope, conferred high SFT resistance and cross-resistance to the anti-HIV-1 drug T20 and the template peptide C34. Interestingly, SFT's resistance profile could be dramatically improved with an M–T hook structure–modified SFT (MTSFT) and with short-peptide inhibitors that mainly target the gp41 pocket (2P23 and its lipid derivative LP-19). We found that the V38A and Q52R substitutions reduce the binding stabilities of SFT, C34, and MTSFT, but they had no effect on the binding of 2P23 and LP-19; in sharp contrast, the A47I substitution enhanced fusion inhibitor binding. Furthermore, the primary resistance substitutions impaired Env-mediated membrane fusion and cell entry and changed the conformation of the gp41 core structure. Importantly, whereas the V38A and Q52R substitutions disrupted the N-terminal helix of gp41, a single A47I substitution greatly enhanced its thermostability. Taken together, our results provide crucial structural insights into the mechanism of HIV-1 resistance to gp41-dependent fusion inhibitors, which may inform the development of additional anti-HIV drugs.

showed greatly increased anti-HIV activity and genetic barrier to inducing drug resistance (25).
Among the newly designed fusion inhibitor peptides, SFT was engineered on the basis of a C34-based gp41 core structure, in which the charged glutamic acid and lysine residues were introduced into the solvent-accessible site while the residues responsible for the NHR binding were maintained unchanged (Fig. 1). Further, a serine residue was added to the N terminus of SFT to increase its helical stability, and Glu 119 was substituted by threonine to enhance its binding with the NHR pocket site. We previously determined the crystal structure of SFT bound to a target mimic peptide, which verified an electronically constrained ␣-helical peptide with significantly improved binding stability (26). As anticipated, SFT showed greatly increased activities in inhibiting both diverse subtypes of WT and T20resistant strains (19,26). In the phase I clinical trials, SFT demonstrated its good safety and pharmacokinetic profiles, especially with a dramatically prolonged half-life (19). In an advanced stage, SFT has been evaluated in clinical phase II trials and approved for clinical phase II trials in China, and thus it will hopefully become the next fusion inhibitor for clinical application. In this study, we focused on characterizing SFT-induced HIV-1 variants to gain new insights into the genetic pathways and mechanisms of HIV-1 resistance to newly developed fusion inhibitors. Several new mutations were identified as conferring high resistance to SFT and cross-resistance to T20 and C34. The mechanisms underlying the resistance were further characterized from multiple aspects, including the binding stability of inhibitors, the functionality of viral Env glycoproteins, the conformation of gp41 core structure, and the structural changes of the NHR helices. The data have provided critical information for the structure-function relationship of HIV-1 Env and gp41-dependent fusion inhibitors and will definitely aid in the development of new anti-HIV drugs.

Identification of SFT-induced resistance mutations
To characterize the structure-activity relationship of newly designed HIV-1 fusion inhibitors, we previously performed the The negatively and positively charged residues introduced for potential formation of salt bridges in SFT are indicated with solid black lines. B, the interaction between the NHR and CHR of gp41. In the current model, the CHR sequence of gp41 packs back to the NHR sequence to form a hairpin-like structure. Three hairpins associate with each other to form a 6-HB conformation. The dashed lines between the NHR and CHR indicate the interaction between the residues located at the e, g and a, d positions in the NHR and CHR, respectively. The NHR-and CHR-derived peptides are depicted as lines to express their sequences and binding sites.

HIV-1 resistance to sifuvirtide
in vitro selection of escape HIV-1 variants to SFT, in which the drug concentration was raised from 0.2 to 9,600 nM after 38 generations of virus passage over 9 months (25). To identify the genetic pathway of SFT-induced resistance, the entire env genes of resistant viruses were amplified by PCR and cloned for DNA sequencing. As shown in Fig. 2, two Env clones (M1 and M2) carry a single V38A mutation; one Env clone (M3) carries a single A47I mutation; 12 Env clones (M4 -M15) carry V38A/ A47I/N126K mutations, and three Env clones (M16 -M18) carry V38A/A47I/Q52R/N126K quaternary mutations. Therefore, four mutant viruses were finally identified from the pool of SFT-resistant HIV-1 variants. The V38A, A47I, and Q52R located within the inhibitor-binding site of gp41 might determine the resistance as primary mutations, whereas the N126K in the CHR served as a secondary mutation. After analyzing all of the cloned Env sequences, no consistent substitutions that apparently caused the resistance were identified in the other sites of gp41 or in gp120 sequence (Fig. S1), and we thus focused on the mutations on the NHR and CHR sequences of gp41.

Resistance profile of HIV-1 mutants to SFT
To identify the mutations responsible for SFT-induced resistance, we generated a panel of HIV-1 NL4-3 Env mutants carrying the amino acid substitutions either singly or in combination ( Table 1). The corresponding pseudoviruses were then produced and used in a single cycle infection assay to determine the inhibitory activity of SFT. Among the single mutations, V38A, A47I, and Q52R conferred 7.93-, 4.58-, and 39.49-fold resistance to SFT, respectively, whereas N126K had minor effect on the inducer (1.7-fold). Apparently, the mutant viruses carrying the combined mutations resulted in dramatically increased -fold changes in resistance to SFT, as shown by the mutants with V38A/A47I (41.61-fold), V38A/A47I/N126K (81.2-fold), V38A/A47I/Q52R (1107.19-fold), and V38A/A47I/Q52R/ N126K (1316.01-fold). Therefore, the mutant virus with V38A/ A47I/Q52R/N126K mutations might finally dominate the resistance phenotype to SFT.

Cross-resistance of HIV-1 mutants to newly designed fusion inhibitors that contain the M-T hook structure
We previously demonstrated that adding an M-T hook structure to the CHR-derived peptide fusion inhibitors

HIV-1 resistance to sifuvirtide
could greatly enhance their binding and antiviral activities as well as the genetic barriers to resistance (24,25,(27)(28)(29)(30)(31). Herein, it was interesting to know the cross-resistance profile of SFT-induced HIV-1 mutants to the MTSFT. As shown in Table 1, the single mutations (V38A, A47I, Q52R, and N126K) conferred little resistance to MTSFT, whereas the combined mutations (V38A/A47I, V38A/A47I/Q52R, V38A/ A47I/N126K, and V38A/A47I/Q52R/N126K) resulted in the resistance with dramatically decreased -fold changes relative to that of SFT. In particular, the single Q52R mutation could result in ϳ40-fold resistance to SFT, but it had no obvious effect on the inhibitory potency of MTSFT, verifying the importance of the M-T hook structure in overcoming the resistance.
Afterward, we also characterized the cross-resistance of SFT-induced mutants to the short-peptide fusion inhibitor 2P23 and its lipid derivative LP-19. As illustrated in Fig. 1, 2P23 is a 23-mer peptide with the M-T hook residues at its N terminus, and LP-19 has been further developed by conjugating a C16 fatty acid group to the C terminus of 2P23. Both inhibitors mainly target the gp41 pocket rather than the T20-resistant site and possess potent inhibitory activities against HIV-1, HIV-2, and simian immunodeficiency virus (SIV) (23,32). Impressively, we found that all of the mutant viruses with single or multiple mutations had no or minor resistance to 2P23 and LP-19.

Effects of the resistance mutations on the binding stability of fusion inhibitors
To understand the mechanisms underlying the SFT-selected resistance, we sought to determine the effects of the resistance mutations on the binding stability of inhibitors. To this end, the NHR-derived peptide N36 with a WT sequence (N36 wt ) or its mutants with single or multiple amino acid substitutions (N36 V38A , N36 A47I , N36 Q52R , N36 V38A/A47I , and N36 V38A/A47I/Q52R ) were synthesized as target surrogates, and the interactions between SFT and target mimic peptides were initially measured by CD spectroscopy. As shown in Fig. 3 and Table 2, CD spectra of all of the peptide pairs displayed typical double minima at 208 and 222 nm, which indicated that SFT interacted with each of the N36 peptides to form 6-HB structures. The V38A and Q52R mutations resulted in decreased ␣-helical contents of the 6-HBs, but the A47I mutation might enhance the ␣-helicity. Defined as the midpoint of the thermal unfolding transition (T m ), the thermostability of 6-HBs demon-strated the binding stability of SFT. It was found that V38A and Q52R severely reduced the T m value, whereas A47I conversely increased it.
We also examined the helical interaction and binding stability of all of the other inhibitors by CD spectroscopy (Table  2). Consistently, the V38A and Q52R mutations reduced the ␣-helical content and T m value of the 6-HBs formed by C34, T20, and MTSFT, whereas the A47I mutation did enhance their ␣-helicity and thermostability. In sharp contrast, V38A and Q52R had no apparent effect on the interactions of 2P23 and LP-19, as shown by the ␣-helical contents and T m values of their 6-HBs, whereas A47I still enhanced the binding stability of inhibitors. As shown by N36 with combined mutations, the A47I mutation could counteract the negative effects of the V38A and Q52R mutations for the binding of inhibitors.

Table 1 Resistance profiles of HIV-1 mutants to SFT and the first-generation fusion inhibitors
The experiment was performed in triplicate and repeated three times. Data are expressed as means Ϯ S.D. -Fold change in the IC 50 was determined relative to the WT level. The t test was performed to judge the significance of the difference between the WT and mutants, and p Ͻ 0.05 values are indicated in boldface type.

HIV-1 resistance to sifuvirtide Effects of SFT-induced mutations on Env-mediated cell fusion and virus entry
We next focused on investigating the effects of SFT-induced resistance mutations on the functionality of viral Env glycoprotein by two approaches. First, the infectivity of HIV-1 NL4-3 pseudoviruses carrying single or combined mutations was determined by a single-cycle infection assay, in which the cell entry efficiency of WT virus was normalized to 100% and the relative virus entry of other mutants was calculated accordingly. As shown in Fig. 4A, all of the pseudoviruses with the Env carrying single resistance mutations (V38A, A47I, or Q52R) or combined mutations (V38A/A47I or V38A/A47I/Q52R) showed markedly decreased cell entry efficiency. Apparently, the Q52R mutation dramatically impaired the functionality of viral Env, and the secondary N126K mutation exhibited a compensatory effect. Second, we measured Env-mediated cell-cell fusion by a dual split protein (DSP)-based fusion assay. It was found that single or combined mutations of V38A and A47I had minor effect on the Env's fusion activity, but Q52R resulted in a dramatic reduction. Consistently, N126K mutation efficiently displayed a compensatory role to the V38A/A47I and V38A/ A47I/Q52R mutants (Fig. 4B). The fusion capacity of diverse Envs was also measured at different time points, and the results supported the observation above (Fig. 4C).

The resistance phenotypes were not associated with the expression and processing of viral Env glycoprotein
It was critical to know whether the resistance mutations affected the expression and processing of the Env glycoproteins, thereby resulting in the phenotypes of viral infectivity and Table 2 Effects of SFT-induced mutations on the ␣-helicity and thermostability of 6-HB structure The ␣-helicity and thermostability of 6-HBs were measured by CD spectroscopy, and the experiments were repeated at least two times to verify the results. NA, not applicable for calculation due to too low (T20) or too high (LP-19) T m values. Ϫ24,881 75 NA

Figure 4. Effects of SFT-induced mutations on the functionality of HIV-1 Env.
A, the WT and mutant HIV-1 NL4-3 pseudoviruses were normalized to a fixed amount by p24 antigen, and their relative infectivity was determined in TZM-bl cells using a single-cycle infection assay. Forty-eight hours after infection, the luciferase activity was measured and corrected for background. The luciferase activity of WT HIV-1 NL4-3 was treated as 100%, and the relative activities of other mutant viruses were calculated accordingly. B, relative fusion activity of WT and mutant HIV-1 NL4-3 Envs was determined by a DSP assay. The Env-transfected HEK293T cells were used as effector cells, and U87-CD4-CXCR4 cells were used as target cells. Similarly, the luciferase activity of WT Env was treated as 100%, and the relative activities of other mutant Envs were calculated accordingly. C, fusion activity of the WT and mutant HIV-1 Envs was determined at different time points by a DSP assay. For both viral entry and fusion, data were derived from the results of three independent experiments and are expressed as mean and S.D. (error bars). t test was performed to compare the WT and mutants; * and **, p Ͻ 0.05 and p Ͻ 0.01, respectively.

HIV-1 resistance to sifuvirtide
resistance. To this aim, we first applied the human anti-gp120 antibody VRC01 and anti-gp41 antibody 10E8 in a capture ELISA-based method, as both antibodies recognize the highly conserved epitopes and possess broadly reactive virus-neutralizing activity, and their reactivity can refer to the expression profile of gp160/gp120/gp41 in the culture media and lysates of transfected cells. As shown in Fig. 5, VRC01 and 10E8 reacted equivalently with the lysates of HEK293T expressing WT and mutant Env glycoproteins that theoretically comprise gp160, gp120, and gp41. Concordantly, VRC01 detected the same level of secreted gp120 proteins in the cell culture supernatants. Further, the expression and processing of the Env glycoproteins were characterized by a Western blotting assay, in which the human anti-HIV polyclonal antibody HIV-IG or mAb HY54 was used to probe the cleaved and uncleaved Env glycoproteins (Fig. 6). The results verified that the resistant mutants had no obvious changes in the protein expression and processing (Fig.  S2). As expected, N126K could abolish the glycosylation site in gp41 (33). We also detected the WT and mutant Env glycoproteins expressed on the surface of transfected cells by flow cytometry and immunofluorescence assays. As shown in Fig. 7 and Fig. S3, no significant differences were observed either. Combined, the data suggested that the observed resistance phenotypes were not determined by the expression profile of viral Env glycoprotein.

The resistance mutations might change the conformation of gp41 core structure
The 6-HB conformation was initially determined by the NHR-derived peptide N36 and the CHR-derived peptide C34 (4), thus representing the core structure of gp41 that plays an essential role in viral fusion and entry. Here, we were intrigued to know whether SFT-selected mutations affected the functionality of viral Env through changing the conformation of gp41 core structure. We also applied two approaches that are routinely used in our laboratory. First, we used a native PAGE (N-PAGE)-based method to visualize the 6-HB complexes formed between diverse N36 and C34 peptides. As shown in Fig. 8, N36 and its mutants showed no band in the native gel because they could migrate up and off the gel due to their net positive charges, whereas the negatively charged C34 and its N126K mutant (C34 N126K ) showed specific bands. Whereas N36 or its mutants were mixed with C34 or C34 N126K , there existed specific bands corresponding to the 6-HB complexes. Densitometric analysis suggested that all the resistance mutations had little effect on the formation of 6-HBs (Fig. S4).
Subsequently, three 6-HB conformation-specific monoclonal antibodies (NC-1, 17C8, and 2G8) were used as probes in an ELISA-based assay (34 -36). As shown in Fig. 9, whereas the V38A mutation might not significantly affect the reactivity of 6-HB with three antibodies, the A47I mutation resulted in a 6-HB with largely increased reactivity, and the Q52R mutationbearing 6-HB only displayed marginal reaction. The binding  HIV-1 resistance to sifuvirtide epitopes of three antibodies have not been finely characterized, but previous studies suggested that NC-1 might mainly target the gp41 NHR (37,38), whereas the critical binding residues of 17C8 and 2G8 were localized at the N-terminal portion of the NHR and the C-terminal portion of the CHR (39). Thus, the reaction patterns of diverse 6-HBs with three antibodies implied the increased or decreased exposure of antigenic epitopes, which might correlate with the structural changes of 6-HB core.

The resistance mutations changed the ␣-helicity and stability of NHR helix
Based on the results above, we sought to delineate the effects of the resistance mutations on the ␣-helicity and thermostability of N36 peptide in the absence of inhibitors, with the intention to infer the structural properties of the viral NHR helices. As measured by CD spectroscopy (Fig. 10, A and B), the WT N36 showed an ␣-helical content of 79% and a T m value of 56°C. In sharp contrast, N36 with the V38A mutation only displayed 18% ␣-helicity, and its T m could not be defined; N36 with A47I displayed 66% ␣-helicity, but its T m increased to 62°C; N36 with Q52R displayed 40% ␣-helicity with a T m of 46°C. Interestingly, N36 with V38A/A47I double mutations showed 66% ␣-helicity with a T m of 60°C, and N36 with V38A/ A47I/Q52R triple mutations had 47% ␣-helicity with a T m of 49°C, which suggested that the A47I mutation could counteract the disruptive effects by the V38A and Q52R mutations.
We recently reported the selection and characterization of HIV-1 variants resistant to the CHR-derived fusion inhibitors MTSC22EK and SC29EK, which also identified key resistance mutations in the inhibitor-binding NHR helices of gp41 (34 -36). To conceptualize that the resistance mutations can directly change the conformation and stability of the gp41 NHR helices, thereby critically determining the binding affinity of inhibitors

HIV-1 resistance to sifuvirtide
and HIV-1 infectivity, we back-checked the effects of MTSC22EK-and SC29EK-induced resistance mutations on the ␣-helical structure of N36 itself by CD spectroscopy. In general, the single or combined resistance mutations could markedly reduce the ␣-helicity and thermal stability of N36 (Fig. 10, C-F). For example, the combination of two MTSC22EK-induced mutations, E49K and L57R, resulted in the ␣-helical content and T m value not applicable for calculation. As an exception, a single N43K mutation rather increased the ␣-helical content of N36 but it did not affect its T m value.

The secondary N126K mutation stabilized the 6-HB structure of SFT-resistant mutants
The N126K substitution in the CHR of gp41 easily emerges in the drug-resistance selection, and it has been considered a secondary mutation that can compensate for the fusion kinetics of resistant viruses. Again, our above data indicated that the N126K mutation exhibited obvious compensatory roles to the fusion and entry capacity of the V38A/A47I and V38A/A47I/ Q52R mutants. Therefore, we sought to further characterize its effect on the binding of C34 with diverse target surrogates, from which we could infer the interactions between the NHR and CHR helices of mutant viruses. As determined by CD spectroscopy, all of the 6-HBs formed between the peptides C34 N126K and N36 or its mutants exhibited relatively increased T m values, which indicated their enhanced ␣-helical thermostability (Fig.  11). For instance, whereas the N36 V38A -C34 -based 6-HB had a T m of 54°C, the N36 V38A -C34 N126K -based 6-HB had a T m of 59°C, and whereas the N36 Q52R -C34 -based 6-HB had a T m of 56°C, the N36 Q52R -C34 N126K -based 6-HB had a T m of 60°C. It should be noted that the A47I mutation-mediated 6-HB  . Effects of SFT-induced mutations on the conformation of gp41 core structure. The reactivity of 6-HBs formed by C34 and N36 or its mutants with conformation-dependent mAbs NC-1 (A), 17C8 (B), and 2G8 (C) was tested by ELISA. The peptide mixture was used to coat the plate wells at 10 g/ml, and the final concentration of a tested mAb was 5 g/ml. Data were derived from three independent experiments and are expressed as mean and S.D. (error bars). A t test was performed to compare the WT and mutants; * and **, p Ͻ 0.05 and p Ͻ 0.01, respectively.

HIV-1 resistance to sifuvirtide
thermostability could be further enhanced by the N126K mutation (Fig. 11C).

Structural basis of SFT-induced resistance
We previously determined the crystal structure of SFT in complex with the target peptide N36 (Protein Data Bank accession number 3VIE), which provided a structural basis for the mechanism of action of SFT (26). Herein, we analyzed the resistance mutations based on the atomic interactions of the 6-HB structure formed by N36 and SFT. Clearly, the residue Val 38 is located at the e position of a helical wheel model, and its hydrophobic side chain interacts with a hydrophobic patch at the terminal portion of SFT, which is formed by the side chains of Asn 145 , Glu 146 , and Leu 149 (Fig. 12A). Because the alanine side chain is much smaller than a valine side chain, the V38A mutation could significantly reduce the area of the interaction interface between V38A and the hydrophobic patch; hence, it accounted for the loss of binding affinity of SFT. In the helical wheel, the residue Ala 47 stays at a g position, where its side chain is located near a hydrophobic patch in the middle region of SFT constituted by the side chains of Ile 131 (distance 4.6 Å), Ile 134 (distance 3.8 Å), and Leu 135 (distance 4.4 Å) (Fig. 12B). Due to the small side chain of Ala 47 , there is a gap between Ala 47 and the hydrophobic patch of SFT. Replacing Ala 47 by isoleu-cine increased the size of the side chain and filled the space between the N36 trimer and SFT; thus, the A47I mutation serves to increase the interfacial area between the NHR trimer and SFT. Because isoleucine has a stable ␤ branched side chain and it is deeply buried inside the hydrophobic core of 6-HB, the A47I mutation may also contribute to the stabilization of the NHR trimer itself, which is consistent with the elevated T m values of 6-HB or the isolated N36 bearing A47I determined by CD spectroscopy. The side chain of residue Gln 52 , which is located at the e position in the CHR helix, mediates several specific hydrogen bonds with both SFT and the adjacent NHR simultaneously (Fig. 12C). Whereas the N⑀2 atom of Gln 52 donates a hydrogen bond (distance, 3.0 Å; angle, 149º) to the OH group of residue Tyr 132 of SFT, the O⑀1 atom of Gln 52 accepts another hydrogen bond (distance, 2.5 Å; angle, 160º) from the N⑀2 atom of Gln 51 on the adjacent NHR helix. Additionally, the O⑀1 atom of Gln 52 accepts the third hydrogen bond from the C␦1 atom (distance, 2.7 Å; angle, 152º) of Ile 131 . Therefore, Gln 52 not only directly interacts with SFT but also stabilizes NHR trimeric coiled-coil; thus, replacing Gln 52 with an arginine could result in the loss of binding affinity with SFT as well as the destabilization of the NHR trimer itself.

HIV-1 resistance to sifuvirtide
From the virus perspective, we analyzed the resistance mutations on the crystal 6-HB structure of C34/N36 (Protein Data Bank accession number 1AIK), which contains the native sequences of gp41 core. As anticipated, the V38A mutation has similar effects on the interaction of N36 and C34, as C34 and SFT share a hydrophobic patch formed by the side chains of Asn 145 , Glu 146 , and Leu 149 . Clearly, the side chain of Val 38 interacts with the side chain of Ile 37 to form a hydrophobic layer (Fig.  12D), whereas three Ile 37 residues on N36 helices interact with each other, which critically determines the conformation and stability of the inner NHR helices; thus, the V38A mutation would dramatically destroy the helical structure of N36 itself. As compared with that of the N36 -SFT complex, the space and distance between Ala 47 on N36 helices and the hydrophobic patch formed by Ile 131 (distance 5.3 Å), Leu 134 (distance 4.3 Å), and Ile 135 (distance 5.2 Å) on C34 helices are much larger (Fig.  12E); thus, it is conceivable that the A47I mutation would enhance the helical interactions of N36 and C34 more efficiently, consistent with the difference in the increased T m values of N36 A47I -SFT (2°C) and N36 A47I -C34 (9°C). Similar to the structure of N36 -SFT, the side chain Gln 52 can form hydrogen bonds with Gln 51 on another N36 and Ile 131 on C34, but it does not contact with His 132 (corresponding to Tyr 132 on SFT) due to a far distance (Fig. 12F). However, the Q52R mutation might not only disrupt the critical hydrogen bond network in the middle region of 6-HB but also introduce an electrostatic repulsion with His-132.
To gain insights into the mechanism of the secondary N126K mutation-mediated compensation for the 6-HB stability and viral infectivity, we also analyzed its potential effects on the atomic interactions of viral 6-HB as mimicked by N36 and C34 peptides. As shown in Fig. 12G, Asn 126 is located at the c position in the CHR helix, and its site chain faces the outside of the binding surface of C34; thus, it is difficult to define the function of Asn 126 in the interhelical interaction of 6-HB structure; however, we can see that a water molecule links Asn 126 and Glu 119 together via hydrogen bonds, which would stabilize the N-terminal PBD of C34 and thus facilitate its interactions with the deep pocket on the NHR helices. Conceivably, the relatively longer and positively charged side chain of lysine might disrupt the water-mediated connection but introduce salt bridges or hydrogen bonds with the negatively charged residues Glu 119 and/or Glu 123 , which can strengthen the intra-and interhelical interactions. We expect a crystal structure for the N36 -C34 N126K complex that can delineate the effect of the N126K mutation in detail.

Discussion
In this study, we dedicated our efforts to exploring the genetic pathway and underlying mechanisms of HIV-1 resistance to the peptide fusion inhibitor SFT. Three primary mutations (V38A, A47I, and Q52R) on the NHR helix and one secondary mutation (N126K) on the CHR helix of gp41 were identified as conferring high resistance to SFT alone or in combination, with a mutant virus with V38A/A47I/Q52R/N126K possibly dominating the resistance. The cross-resistance profiles of SFT-induced mutants to the drug T20, the template C34, and three newly designed fusion inhibitors (MTSFT, 2P23, and LP-19) were also characterized. Impressively, the single and combined SFT-induced mutations did confer cross-resistance to T20 and C34, but they were largely tolerated by the M-T hook structure-modified SFT and short-peptide inhibitors mainly targeting the gp41 pocket site. In the underlying mechanisms, the V38A and Q52R mutations could reduce the binding stability of SFT, C34, and MTSFT, but they had no obvious effects on the binding of 2P23 and LP-19, and the A47I mutation, in sharp contrast, enhanced the binding of inhibitors. Furthermore, the results show that the primary resistance mutations could impair the functionality of viral Env to mediate cell entry/fusion and change the conformation of the gp41 core as well as its internal NHR helices, whereas the secondary N126K mutation did display compensatory roles in both the virus entry and the 6-HB stability. Therefore, the present studies have provided important information for understanding the mechanism of HIV-1 resistance to gp41-dependent fusion inhibitors and would help in the development of new anti-HIV drugs.
The peptide drug T20 remains the only membrane fusion inhibitor available for treatment of viral infection, which has shown effectiveness as a salvage therapy for HIV/AIDS patients who failed to respond to antiretroviral therapeutics that include reverse transcriptase inhibitors and protease inhibitors; however, T20 behaves with relatively weak antiviral activity and a low genetic barrier to inducing drug resistance. The resistance profile of T20 was initially characterized by in vitro selection of escaping HIV-1 variants, which revealed that the 36 GIV 38 motif in the inhibitor-binding site was a hot spot (18). Later, a number of clinical studies demonstrated that T20-resistant mutations appeared predominately within the amino acid Gly 36 -Leu 45 stretch on the NHR of gp41, such as G36D/V/S, I37T, V38A/ E/M, Q40H, N43D/K, and L54M mutations (13)(14)(15)(16)(17)(18). There have been tremendous efforts to develop next-generation fusion inhibitors that can overcome the drawbacks of T20, resulting in a group of new peptides with significantly improved pharmaceutical profiles (19 -24, 28). Meanwhile, considerable effort was also devoted to the selection and characterization of HIV-1 variants resistant to newly designed fusion inhibitors, which delineated their resistance evolution pathways and underling mechanisms (34, 35, 40 -47). Previously, Liu et al. (42) identified a panel of SFT-induced resistance mutations largely overlapping with the T20-resistant sites, including I37T, V38A/M, Q41H/K/R, and N43K. As noticed, the concentration of SFT was escalated to ϳ1,000 nM after 9 -15 generations of in vitro virus passage, and the most resistant HIV-1 variants with combined mutations (e.g. I37T/V38A and I37T/N43K/N126K) displayed ϳ100-fold increased resistance to SFT (42). In fact, our research project was originally focused on comparing the genetic resistance barriers of the M-T hook structuremodified peptide inhibitors rather than identifying the resistance mutations in detail (25); however, we had been intensely curious about the HIV-1 variants that could survive in the presence of ϳ10,000 nM SFT inhibitor. Therefore, the present study was performed and surprisingly found a different panel of SFTresistant mutations, including two sites (A47I and Q52R) that had not been defined previously. In brief, three primary mutations (V38A, A47I, and Q52R) located at the inhibitor-binding site and one secondary mutation (N126K) located at the CHR of gp41, corresponding to four HIV-1 variants, contributed to the resistance phenotype. Two mutant viruses carried a single V38A or A47I mutation, whereas two mutant viruses evolved with combined mutations (V38A/A47I/N126K and V38A/ A47I/Q52R/N126K). From the sequence characterization, one might speculate a genetic resistance pathway with the following scenario. A single V38A or A47I mutation emerged first, which resulted in a modest resistance (5-8-fold); subsequently, the V38A/A47I double mutations emerged, thus conferring a significantly increased resistance (ϳ80-fold). It might require a prolonged selection, but the appearance of Q52R mutation on the background of V38A/A47I could dramatically boost the

HIV-1 resistance to sifuvirtide
resistance phenotype (Ͼ1,000-fold). Apparently, the mutant virus with two or three combined mutations (V38A/A47I or V38A/A47I/Q52R) was accompanied by the second N126K mutation, which also contributed to the resistance levels. Therefore, it is conceivable that the mutant virus with V38A/ A47I/Q52R/N126K would finally dominate the resistance.
In this study, we found that SFT-induced primary mutations, either singly or in combination, mediated considerable crossresistance to the drug T20 and the template peptide C34, except for the A47I mutation, which could result in an increased susceptibility to T20 (ϳ3-fold). Interestingly, all of the HIV-1 mutants with a single mutation (V38A, A47I, or Q52R) had no obvious resistance to the M-T hook structure-modified SFT, and all of the HIV-1 mutants with combined mutations (V38A/ A47I, V38A/A47I/Q52R, V38A/A47I/N126K, and V38A/A47I/ Q52R/N126K) displayed dramatically decreased resistance -fold changes (Table 1). Actually, we are still wondering why the single Q52R mutation could render a ϳ40-fold resistance to SFT but did not affect the inhibitory activity of MTSFT. More impressively, both the single and combined mutations did not confer significant resistance levels to the short-peptide fusion inhibitors 2P23 and LP-19, which also contain the M-T hook structure and mainly target the gp41 pocket site. It is worth noting that Q52R did confer a mild resistance to 2P23, but this phenotype could be mitigated by its combination with other mutations and by a 2P23-based lipopeptide . Taken together, the presented data have further verified that both the M-T hook structure and lipid conjugation are important strategies to design HIV-1 fusion inhibitors possessing markedly improved antiviral activity against WT and mutant viruses, especially for short peptides that target the gp41 pocket rather than the T20-and SFT-resistant sites. It will be interesting to select and characterize HIV-1 mutants resistant to MTSFT, 2P23, and LP-19, which would definitely help in our understanding of the resistance mechanisms of diverse HIV-1 fusion inhibitors.
Previous studies have described multifaceted mechanisms of HIV-1 resistance to peptide fusion inhibitors, including small amino acid-mediated reduced contact, large amino acidmediated steric obstruction, acidic amino acid-mediated electrostatic repulsion, basic amino acid-mediated electrostatic attraction, and disruption of hydrogen bonds and hydrophobic contacts (48). It was also proposed that kinetically restricted entry inhibitors have reduced sensitivity to affinity-disrupting resistance mutations (49,50). Here, we investigated the underlying mechanisms of SFT-induced resistance mutations from two aspects: their effects on the binding stability of inhibitors and on the functionality of viral Env glycoprotein. Clearly, the V38A and Q52R mutations reduced the binding stability of SFT, C34, and MTSFT but not that of 2P23 and LP-19; however, the A47I mutation could further stabilize the binding of each inhibitor. Based on the crystal structures of SFT and C34 bound to a target surrogate, we can see that the residue Val 38 makes critical contacts with the residues Asn 145 , Glu 146 , and Leu 149 of SFT. Thus, the V38A mutation would destroy the interhelical interactions through a mechanism of small amino acid-mediated reduced contact; the residue Ala 47 has hydrophobic interactions with Ile 131 , Ile 134 , and Leu 135 on SFT; the A47I mutation would introduce an extended side chain that enhances the interhelical interactions; the residue Gln 52 critically stabilizes the NHR helices via a hydrogen bond network and interacts simultaneously with Tyr 132 and Ile 131 on SFT; and the Q52R mutation would disrupt both the intra-and interhelical interactions, with a mechanism of large amino acidmediated steric obstruction or basic amino acid-mediated electrostatic repulsion/attraction. From a viral perspective, the resistance mutations could severely impair the ␣-helicity and stability of viral NHR helices and/or 6-HB conformation, which might correlate with the functionality of the Env glycoprotein to mediate viral cell fusion and entry. It is conceivable from an evolutionary angle that the mutant virus would like to find a balance among the mutations, resistance, and viral fitness.
In conclusion, our studies have demonstrated the underlying mechanism of HIV-1 resistance to SFT and verified the importance of the M-T hook structure in overcoming the resistance. Finally, there are several points that should be addressed or considered in future studies. First, can the primary resistance mutations induced by CHR-derived fusion inhibitors universally result in a structural change in the NHR region of gp41? In other words, do the changes in the conformation and stability of NHR essentially determine the resistance by interfering with the binding of inhibitors and the interaction of viral NHR and CHR helices? We are in the process of examining the effects of T20-, C34-, and SC34EK-induced resistance mutations, which may help to generalize a proof of concept; Second, can some of the resistance mutations in the NHR region also play compensatory roles in viral infectivity? To this point, the A47I and E49K mutations did behave like the secondary N126K mutation that can enhance the NHR-CHR interactions. Third, how do we explain the fact that whereas the A47I mutation enhances the binding of SFT, it still mediates the resistance? Now, one may speculate that this mutation can enhance the interaction of viral NHR and CHR helices more efficiently, thus outcompeting the binding of inhibitor. When enhancing the binding of T20 more efficiently, the A47I mutation does increase the sensitivity of drug (Table 1). Fourth, many mutations in the NHR and CHR of gp41 were previously reported to affect the expression and processing profiles of viral Env glycoproteins, but our studies showed that the fusion inhibitor peptide-induced resistance mutations had no such effects. Thus, it will be interesting to clarify this point as a common phenomenon or a misleading coincidence. Fifth, it is critical to characterize whether fusion inhibitor-selected NHR mutations impact the structure and function of HIV-1 Rev response element, as described by previous studies (51,52). Additionally, as the fitness of the resistant viruses to SFT seems to be very poor compared with the parental virus, we are wondering if these viruses can survive in vivo.

Cell lines and culture conditions
HEK293T cells were purchased from the American type culture collection (ATCC) (Manassas, VA); TZM-bl indicator cells stably expressing CD4 and CCR5 along with endogenously expressed CXCR4 were obtained from John C. Kappes and Xiaoyun Wu through the AIDS Reagent Program, Division of HIV-1 resistance to sifuvirtide AIDS, NIAID, National Institutes of Health (17). Both cells were cultured in complete growth medium that consisted of Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin-streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1ϫ minimum Eagle's medium nonessential amino acids (Gibco/Invitrogen) and were maintained at 37°C in 5% CO 2 .

Synthesis of fusion inhibitor and target mimic peptides
A panel of fusion inhibitor peptides (SFT, T20, C34, MTSFT, 2P23, and LP-19) and target mimic peptides (N36, N36 V38A , N36 A47I , N36 Q52R , N36 V38A/A47I , and N36 V38A/A47I/Q52R ) were synthesized with a standard solid-phase Fmoc (N-(9-fluorenyl) methoxycarbonyl) method as described previously (30). All peptides were protected by N-terminal acetylation and C-terminal amidation. To synthesize LP-19, the template peptide 2P23 contains a lysine residue at its C terminus with a Dde side-chain protecting group, enabling the conjugation of a C16 fatty acid that requires a special deprotection step in a solution of 2% hydrazinehydrate/N,N-dimethylformamide. The peptides were purified to a purity of Ͼ95% by reversed-phase HPLC and characterized for correct amino acid composition by MS. Concentrations of the peptides were measured by UV absorbance and a theoretically calculated molar extinction coefficient based on tryptophan and tyrosine residues.

Generation of HIV-1 Env mutants
A panel of HIV-1 NL4-3 Env-based mutants was generated by site-directed mutagenesis as described previously (53). In brief, two primers contained the desired mutation and occupied the same starting and ending positions on opposite strands of plasmid. DNA synthesis was performed by PCR in a 50-l reaction volume using 1 ng of denatured plasmid template, a 50 pM concentration of upper and lower primers, and 5 units of the highfidelity polymerase PrimeStar (TaKaRa, Dalian, China). PCR amplification was performed for one cycle of denaturation at 98°C for 5 min, followed by 18 cycles of 98°C for 15 s and 68°C for 15 min, with a final extension at 72°C for 10 min. The amplicons were digested with DpnI at 37°C for 1 h, and DpnI-resistant molecules were recovered by transforming Escherichia coli strain DH5␣ to antibiotic resistance. The introduced mutations were confirmed by DNA sequencing.

Single-cycle infection assay
The backbone plasmid pSG3 ⌬env that encodes an Env-defective, luciferase-expressing HIV-1 genome was obtained from John C. Kappes and Xiaoyun Wu through the AIDS Reagent Program, Division of AIDS, NIAID, National Institutes of Health (17). HIV-1 pseudoviruses were generated as described previously (53). In brief, 293T cells were cotransfected with pSG3 ⌬env and a plasmid expressing Env glycoprotein. Supernatants were harvested 48 h after transfection, and tissue culture ID 50 values were determined in TZM-bl cells. Peptides were prepared in graded concentrations, mixed with 100 tissue culture ID 50 of viruses, and then incubated for 1 h at room temperature. The mixture was added to TZM-bl cells (10 4 cells/ well) containing DEAE (final concentration 15 l/ml) and incubated 48 h at 37°C. The luciferase activity was measured using luciferase assay reagents and a luminescence counter (Promega, Madison, WI).

DSP-based cell fusion assay
A DSP-based cell-cell fusion assay was performed to determine the fusogenic activity of viral Env as described previously (54,55). The plasmids DSP 1-7 and DSP 8 -11 were kindly provided by Zene Matsuda at the Institute of Medical Science of the University of Tokyo (Tokyo, Japan) Briefly, a total of 1.5 ϫ 10 4 293T cells (effector cells) were seeded on a 96-well plate, and a total of 8 ϫ 10 4 U87-CXCR4 cells (target cells) were seeded on a 24-well plate. On the following day, 293T cells were transfected with a mixture of an Env-expressing plasmid and a DSP 1-7 plasmid, and U87-CXCR4 cells were transfected with a DSP 8 -11 plasmid. Twenty-four hours post-transfection, the target cells were resuspended in 300 l of prewarmed culture medium containing EnduRen live-cell substrate (Promega) at a final concentration of 17 ng/l and then transferred to each well of the effector cells with equal volume. The cell mixture was spun down, and luciferase activity was measured by a luminescence counter (Promega).

CD spectroscopy
CD spectroscopy was conducted to determine the ␣-helicity and thermostability of the isolated or combined peptides as described previously (22). CD spectra were acquired on a Jasco spectropolarimeter (model J-815) using a 1-nm bandwidth with a 1-nm step resolution from 195 to 260 nm at room temperature and corrected by subtraction of a solvent blank. The ␣-helical content was calculated from the CD signal by dividing the mean residue ellipticity ([]) at 222 nm by the value expected for 100% helix formation (Ϫ33,000 degrees cm Ϫ2 dmol Ϫ1 ). Thermal denaturation was performed by monitoring the ellipticity change at 222 nm from 20 to 98°C at a rate of 1.2°C/min.

Detection of viral Env glycoprotein by capture ELISA
A capture ELISA was conducted to determine the effects of the introduced mutations on the expression and processing of viral Env glycoprotein, as described previously (35). Briefly, the wells of an ELISA plate were coated with a sheep anti-gp120 antibody (D7324) at 10 g/ml and blocked by 3% BSA. Cell lysates or culture supernatants (50 l) of Env-transfected cells were added to the wells and incubated at 37°C for 1 h. After extensive washes, 50 l of human anti-gp120 mAb VRC01 or anti-gp41 mAb 10E8 was added at 10 g/ml and incubated at 37°C for 1 h. The bound antibodies were detected by horseradish peroxidase-conjugated goat anti-human IgG. The reaction was visualized by adding 3,3,5,5-tetramethylbenzidine, and the A 450 was measured.

Detection of 6-HBs by conformation-specific antibodies
To detect the 6-HBs formed by N36 and C34 peptides, three 6-HB conformation-specific mAbs (NC-1, 17C8, and 2G8), which react with the N36 and C34 complex but not the isolated peptides, were applied in an ELISA as described previously (34 -36). NC-1 was kindly provided by Shibo Jiang (Lindsley F. Kimball Research Institute of the New York Blood Center, New York) (56); 17C8 and 2G8 were kindly provided by Yinghua

HIV-1 resistance to sifuvirtide
Chen (School of Life Sciences of Tsinghua University, Beijing, China) (39). Briefly, the isolated or mixed peptides were coated to the ELISA wells at 10 g/ml and blocked with 3% BSA. After washing, the anti-6-HB mAb diluted at 5 g/ml was added to the wells and incubated at 37°C for 1 h. After washes, the bound antibodies were detected by horseradish peroxidase-conjugated anti-mouse IgG, the reaction was visualized by the addition of 3,3,5,5-tetramethylbenzidine, and the A 450 was measured.

Western blotting
To verify the expression and processing profile of viral Env glycoprotein by Western blotting, the lysates or culture supernatants of transfected cells were centrifuged at 20,000 ϫ g at 4°C for 15 min to remove insoluble materials. Equal amounts of total proteins were separated by 10% SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk solution in Tris-buffered saline (TBS, pH 7.4) at room temperature for 1 h, the membrane was incubated with the human anti-HIV polyclonal antibody HIV-IG (obtained through the AIDS Reagent Program, Division of AIDS, NIAID, National Institutes of Health) or human anti-gp120 mAb HY54 (which was generated in our laboratory) overnight at 4°C. The following day, the membrane was washed three times in TBS-T and then incubated with IRDye 800CW goat anti-human IgG at room temperature for 2 h. The membrane was then scanned using the Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE). Band intensities were analyzed using ImageJ software (National Institutes of Health).

Flow cytometry assay
The WT or mutant Env glycoproteins expressed on the surface of transfected cells were detected by flow cytometry. Briefly, HEK293T cells were transiently transfected with a specific plasmid and harvested at 36 h after transfection. After two washes with PBS, the human anti-gp120 mAb VRC01 was added to the cells and incubated at 4°C for 1 h. Then cells were washed twice and incubated with Alexa Fluor 488 anti-human IgG (Life Technologies, Inc.) at 4°C for 1 h. After three washes, cells were resuspended in PBS and analyzed by a FACSCalibur (BD Biosciences).

Immunofluorescence assay
An Env-encoding plasmid was transiently expressed in HEK293T cells by transfection. After 36 h, the cells were immobilized by 4% paraformaldehyde, and then 1% BSA was added to block nonspecific binding of the antibodies. After washing twice with PBS, VRC01 was added to the cells at a final concentration of 20 g/ml and incubated at 4°C overnight. After three washes, bound antibodies were detected by Alexa Fluor 488 anti-human IgG and observed under an immunofluorescence microscope.

N-PAGE
N-PAGE was conducted to determine the interactions between N36 and C34 with native or mutant sequences as described previously (22,57). Briefly, N36 was mixed with C34 with the final concentration of each peptide at 40 M and then incubated at 37°C for 30 min. The sample was added with Trisglycine native sample buffer at a ratio of 1:1 and then loaded onto 10 ϫ 1.0-mm Tris-glycine gels (20%) at 25 l/well. Gel electrophoresis was carried out with 100-V constant voltage at 4°C for 3 h. The gel was then stained with Coomassie Blue and imaged with a Bio-Rad imaging system.