Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics.The adaptation of Chikungunya virus (CHIKV) to a new vector, the  A. albopictus mosquitoes, which promoted re-emergence of the virus, was determined to involve an E1-A226V amino acid substitution, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. Here we showed that novel substitution, E2-L210Q identified in Kerala, India in 2009, caused a significant increase in the ability of CHIKV to infect and develop a disseminated infection in A. albopictus. This may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics. Our findings represent some of the first evidence supporting the hypothesis that adaptation of CHIKV (and possible other arboviruses) to new niches is a sequential multistep process that involves selections of at least two adaptive mutations.Since 2004, chikungunya virus (CHIKV) has caused a series of devastating outbreaks in Asia, Africa and Europe that resulted in up to 6.5 million cases of arthritic disease and have been associated with several thousand human deaths. Although the initial step of CHIKV adaptation to  The potential of RNA viruses to emerge into new environments often depends on their ability to efficiently adapt to new hosts. These adaptations sometimes comprise a stepwise process that includes 1) initial viral introduction/establishment in the recipient species, followed by 2) finite adjustment/optimization of the virus replication and transmission strategies for specific environments associated with a new host Alphavirus, genus Togaviridae] emerged in 2004 in Kenya and subsequently spread into many countries in the Indian Ocean basin [hence the name: Indian Ocean lineage (IOL)], causing devastating outbreaks of arthritic disease Aedes (A.) albopictus, which was previously considered only a secondary CHIKV vector A new lineage of Chikungunya virus (CHIKV)  . These din vivo infections, our data further support the conclusion that the E2-L210Q substitution was most likely selected only by A. albopictus.Alternatively, E2-L210Q could have been selected as a result of CHIKV adaptation to a vertebrate host in India, probably humans. Although we did not observe any fitness change associated with this mutation during propagation of CHIKV in Vero cells (derived from African green monkey kidneys), to extend our analysis we repeated competition experiments using the human-derived cell line 293 (embryonic human kidney) because earlier studies showed that CHIKV can infect and replicate in various primary human cell lines including epithelial, endothelial, fibroblast, muscle satellite and macrophages  after oral infection  at the blood meal titers of 3.5 and 2.5 Log10(pfu)/mL, and the oral infectious dose 50% (OID50) value calculated for SL07-226V-210Q was 8.9 times lower (higher infectivity) than that for SL07-226V (10(pfu)/mL) probably reflected the oral dose nearing saturation. By way of comparison, earlier studies, including those using the SL07 strain, determined that the well-established A. albopictus-adaptive substitution E1-A226V mediates a much greater, ∼100-fold decrease in OID50 values To further test the hypothesis that the E2-L210Q substitution affects CHIKV fitness only during initial infection of the L07-226V . The lacA. albopictus midgut cells, we developed a replicon/helper system for the SL07 strain. Sub-genomic replicons of alphaviruses can be packaged into virus-like particles (VLPs) by co-transfection of replicon and helper RNAs into susceptible cells To directly study the effect of E2-L210Q substitution on initial CHIKV infection of Since transfection efficiency of viral RNA is critical in determining the efficiency of VLP production, we switched to BHK-21 cells that have superior RNA susceptibility compared to Vero cells. Earlier, we observed that CHIKV isolates that have not been passaged in rodent-derived cells lines (including SL07) are impaired in their ability to replicate in BHK-21 cells . Therefore, to ensure efficient recovery of CHIKV VLPs from BHK-21 cells, double BHK-adaptive substitutions (nsP1-L407P and nsP3-T348A) were introduced into the SL07 i.c. (see The SL07 replicon expressing green fluorescent protein (GFP) was packaged into VLPs using w.t. SL07 helper (with E2-210L and E1-226A residues) or using a modified helper encoding E2-L210Q and E1-A226V substitutions. The SL07 replicon expressing cherry fluorescent protein (CFP) was packaged into VLPs using a helper encoding E2-210L and E1-226V residues . In addiA. albopictus as shown in  and presented in blood meals to shown in . At 1 anshown in . We founshown in . Similar midguts . InfectiA. albopictus , and was competed against a CFP-expressing replicon packaged using a helper encoding E2-210Q and E1-226A residues (GFP/E2-210Q/E1-226A). The E2-L210Q substitution caused a 2.3–2.4-fold increase in CHIKV infectivity for A. albopictus midgut cells . These results as well as surveillance data indicating that CHIKV was transmitted primarily by A. albopictus in Kerala state of India when the E2-L210Q substitution was first detected A. albopictus mosquitoes mediated by the previously characterized E1-A226V substitution was probably just a beginning of multi-step adaptive process that included the selection of a second (E2-L210Q) and possibly additional, future mutational steps by IOL strains now circulating in urban areas. These mutations, which have no deleterious effect on transmission by A. aegypti, will enable CHIKV to even more efficiently exploit urban transmission in environments populated by A. albopictus, but also to maintain the ability to utilize A. aegypti, which tends to occur in major urban centers A. albopictus raise serious public health concerns that even more efficient transmission may exacerbate the already devastating CHIK epidemics in India and Southeast Asia. Furthermore, the introduction of the E2-L210Q strain into new areas like Italy and France, where autochthonous cases have already occurred A. albopictus thrives. Considering the broad global distribution of A. albopictus, including nearly throughout the Americas, the E2-L210Q substitution may significantly increase the risk of CHIKV becoming endemic in additional locations.In this study we showed that an E2-L210Q substitution recently identified in CHIKV populations of Kerala State, India, when expressed in the background of the initial adaptive E1-226V substitution, confers a selective advantage by increasing initial infection of A. albopictus mosquitoes that were reared from wild-caught larvae collected in Kerala State, suggesting that transovarial transmission (TOT) may also play a role in CHIKV maintenance, especially during dry seasons A. albopictus of CHIKV strains with the E1-A226V substitution Interestingly, Niyas et al. (2010) demonstrated that CHIKV strains with the E2-L210Q substitution can be isolated from adult A. albopictus midgut cells remains unknown. Earlier, we hypothesized that the E2 region around position 211 could be directly involved in interactions with a specific cell surface receptor A. albopictus in concert with the E1-A226V substitution, and that residue E2-211I, which is common among CHIKV strains, blocks this effect. Moreover, using virus overlay protein binding assays (VOPBA) to study CHIKV binding to the proteins associated with the brush border membrane fraction of A. albopictus midguts, we demonstrated that the E2-T211I substitution dramatically alters CHIKV interactions with as yet unidentified proteins (KT unpublished). The recently determined crystal structure of the CHIKV E2 glycoprotein B sheet of the E2 protein, which is exposed on the virion surface on the lateral side of domain B, suggesting that these positions could be involved in interactions with cellular proteins. Substitutions of the aliphatic moieties with polar residues in this region may therefore be directly responsible for changing CHIKV affinities to as yet unidentified receptor(s). Interestingly, positions E2-207, E2-213 and E2-218, which have been shown to be involved in VEEV adaptation to equine and mosquito hosts The molecular mechanism explaining the effects of the E2-L210Q substitution on CHIKV infectivity for A. albopictusA. albopictusA. albopictus cycle, and indicates the need for a more detailed, continuous molecular characterization of CHIKV strains from throughout its distribution.In the study by Niyas et al (2010) that discovered the E2-L210Q substitution in CHIKV strains from Kerala, only limited portions of CHIKV genomes including the nsP2, E2 and E1 genes were sequenced A. albopictus midgut infectivity when expressed in the background of E1-226V, whereas the same substitution caused only a 2.3–2.4-fold increase when expressed in the background of E1-226A  or 10-fold (E1-226V background) weaker effect compared with E1-A226V  that has resulted in a strain circulating in India with the fittest phenotype detected yet for transmission by ApaI marker) were introduced in various combinations into the i.c. of SL07 using conventional PCR-based cloning methods ApaI marker) was introduced into the pRep-GFP construct that allows comparison of the relative RNA quantities in an experimental, mixed infection sample. The helper plasmids were generated by deleting the 373–7270 nt. cDNA fragment from i.c. of SL07 that has mutations of interest at E1-226 and E2-210. Plasmids were propagated using the MC1061 strain of E. coli in 2xYT medium and purified by centrifugation in cesium chloride gradients. Detailed information for all plasmids is available from the authors upon request.The SL07 (SL-CK1) strain of CHIKV was isolated in 2007 from a human in Sri-Lanka (GenBank Acc. No. HM045801.1). This strain belongs to Indian subgroup of the IOL 2, in Minimal Essential Medium  supplemented with 5% fetal bovine serum (FBS). BHK-21(S) [Baby Hamsters Kidney] and 293 (Human Embryonic Kidney) cells were maintained at 37°C with 5% CO2 in MEM-alpha (Invitrogen) supplemented with 10% FBS and 1x MEM vitamin solution (Invitrogen). The Galveston colonies of A. albopictus and A. aegypti mosquitoes were established from the mosquitoes collected in Galveston, TX (USA). Thailand colonies of A. albopictus and A. aegypti mosquitoes were established from mosquito eggs collected in Bangkok, Thailand. All manipulations and handling of mosquitoes were done as described previously Vero cells (African green monkey kidney) were propagated at 37°C, with 5% COin-vitro transcribed RNA into Vero cells. RNA was transcribed from SP6 promoter of the NotI linearized i.c. DNA using the mMESSAGE mMACHINE kit . Ten µg of RNA were electroporated into 107 Vero cells using a BTX-Harvard Apparatus ECM 830 Square Wave Electroporator  and 2mm cuvette at the following conditions: 680V, pulse length 99 µs, 5 pulses, with an interval between the pulses of 200ms. Cells were transferred to a 75 cm2 tissue culture flasks with 14 mL of Leibovitz L-15 (L-15) medium supplemented with 10% FBS and 5% tryptose phosphate broth . At 3 h post electroporation the cell supernatant was replaced with 14 mL of L-15 medium and maintained at 37 °C without CO2. Cell culture supernatants were collected at 24 and 48 h and stored at −80°C.Infectious viruses were generated by electroporation of the 5 electroporated Vero cells was serially ten-fold diluted and cells were allowed to attach to sub-confluent monolayers (1x106 cells/well) of uninfected Vero cells in six-well plates To estimate the specific infectivity of electroporated RNAs, an aliquot containing 1x10in-vitro transcribed replicon and helper RNA were mixed and electroporated into 107 BHK-21(S) cells as described above for Vero cells. Cells were maintained in L-15 medium at 37°C, followed by harvesting supernatants at 30 h post-electroporation. The titer of VLPs was determined by titration on Vero cells as described earlier 6 Vero cells were seeded in six-well plates and, after a 16 h incubation at 37°C, monolayers were infected with 10-fold dilutions of the samples for 1 h at 37°C, followed by adding 2 mL of MEM. After 24 h of incubation at 37°C the numbers of GFP- or CFP-expressing cells were quantified by fluorescent microscopy and titers were expressed as infectious units (i.u.)/mL.To generate CHIKV VLPs expressing residues of interest in E2 and E1 glycoproteins, BHK-21(S) cells were used, which have superior RNA susceptibility compared to Vero cells. To ensure efficient recovery of CHIKV VLPs from BHK-21 cells, all CHIKV replicons were designed to include BHK-adaptive mutations (nsP1-L407P and nsP3-T348A) identified after rescue of w.t. i.c's in BHK-21(S) cells. Ten micrograms of A. albopictus and A. aegypti mosquitoes was analyzed using direct competition experiment as described earlier ApaI marker. Viral mixes were used to prepare infectious blood meals by dilution in an equal volume of the defibrinated sheep blood , then orally presented to 4–5 day old female mosquitoes at 37°C as described previously 4 Vero cells/well. At 3 dpi, supernatant from virus-induced CPE (cytopathic effect)-positive wells was used for RNA extraction followed by RT-PCR with 41855ns-F5 (5̀-ATATCTAGACATGGTGGAC) and 41855ns-R1 (5̀-TATCAAAGGAGGCTATGTC) primers sets using One-Step RT-PCR kit . The PCR products were digested with ApaI restrictase  and separated on 1.5% agarose gels followed by ethidium bromide staining. One PCR band in the digested sample corresponded to disseminated infection for one out of two viruses in the pair; two bands indicated that both viruses disseminated in the same mosquito. Differences in dissemination efficiencies were tested for significance with a one-tailed McNemar test.The role of viral mutations at position E2-210 on CHIKV dissemination in A. albopictus and Vero cells were performed as described above with minor modifications. For the first passage, virus SL07-226V-210Q was mixed with 100-fold excess SL07-226V-Apa to generate infectious blood meals containing 5x105 pfu/mL (combined). The blood meal was used for oral infection of A. albopictus  followed by virus extraction from combined head and leg homogenates derived from 50 individual mosquitoes in 1.5 mL of MEM medium at 10 dpi. Homogenates were filtered and used to infect 75 cm2 flasks of Vero cells. At 2 dpi, cell culture supernatants were diluted 1∶10 in L-15 medium and mixed with equal volumes of defibrinated sheep blood to prepare a blood meal for the second passage. The cycle was repeated a total of 3 times. At 10 dpi of third mosquito passage, heads and legs of individual mosquitoes were processed as described above.Viral competition experiments with serial, alternating CHIKV passaging in A. albopictus, the mosquitoes were exposed to blood meals containing 1∶1 mixes of [SL07-226V-Apa and SL07-226V-210Q] and [SL07-226V and SL07-226V-210Q-Apa]. Depending on the experiment, at 1, 2, 3 and 7 dpi whole mosquito bodies, mosquito carcasses, or mosquito midguts were collected in pools of ten, and were used for RNA extraction using TRIzol . RNA was RT-PCR amplified, followed by ApaI restrictase digestion of amplicons as described above. Gel images were analyzed using TolaLab (version 2.01) and relative fitness for a given virus during competition was determined as the ratio between E2-210L and E2-210Q bands in the sample, divided by the starting ratio of E2-210L and E2-210Q in the blood meal. The results were expressed as an average value of 2 pools of 10 mosquitoes midguts per pool.For CHIKV competition experiments in specific body parts of A. albopictus  using capillary needles as described previously For CHIKV competition experiments in intrathoracically infected mosquitoes, 5 pfu of SL07-226V-Apa and 5 pfu of SL07-226V-210Q in 0.5 µL of L-15 media were directly injected into thoraxes of cold-anesthetized A. albopictus, the SL07-226V and SL07-226V-210Q viruses individually were serially 10-fold diluted, mixed with defibrinated sheep blood and presented orally to A. albopictus (Thailand). At 10 dpi individual mosquitoes were triturated in one mL of MEM and used to infect 5x104 Vero cells in duplicate in 96 well plates. CHIKV was detected by observing virus-induced CPE. The difference in the infection rates between SL07-226V and SL07-226V-210Q was tested for significance with a two-tailed Fishe50) values were calculated using the PriProbit program (version 1.63).To investigate the relationship between the blood meal titers and infection rates in A. albopictus (Thailand) were infected with 1∶1 mixes (based on i.u. titers) of GFP- or CFP-expressing subgenomic replicons packaged into VLPs using CHIKV helpers that differed by substitutions at positions E1-226 and E2-210 Click here for additional data file.Figure S2The effect of the E2-L210Q substitution on CHIKV fitness in Vero cells. Above each figure is a schematic representation of the viruses used in the competition assay. Vero cells were infected at multiplicity of infection of ∼0.1 pfu/cell in triplicate with a 1∶1 mixture of [SL07-226V-Apa and SL07-226V-210Q] (A) and [SL07-226V and SL07-226V-210Q-Apa] (B). At 2 dpi cell culture supernatants were collected for RNA extraction and viral RT-PCR analysis. The relative fitness (RF) within a given competition was determined as the average ratio between E2-210L and E2-210Q bands in the sample (r), divided by the starting ratio of E2-210L and E2-210Q bands in the inoculum (i) used for infection.(TIF)Click here for additional data file.Figure S3The effect of the E2-L210Q substitution on CHIKV fitness in A. aegypti . Above is a schematic representation of the viruses used in the competition assay. Asterisks indicate authentic (w.t.) residues for the SL07 strain at the indicated positions. Graph shows numbers and proportions of mosquitoes containing virus populations expressing leucine (210L), glutamine (210Q) or a mixture of both residues (210L/210Q) in heads and legs of A. aegypti  assayed at 10 dpi. BM indicates combined titers of CHIKV (E2-210L and E2-210Q) in blood meals used for mosquito infection. The difference in number of mosquitoes with E2-210L versus E2-210Q residues was tested for significance with a one-tailed McNemar test.(TIF)Click here for additional data file.Figure S4Effect of the E2-L210Q substitution on replication of CHIKV replicon particles in A. albopictus midguts after oral infection with VLPs. A - schematic representation of VLP production and the experimental design used in the mosquito infectivity study. A. albopictus (Thailand colony) were orally infected with blood meals containing 3x106 i.u./mL of GFP/210Q/226V and 3x106 i.u./mL of CFP/210L/226V VLPs. At 1, 2 and 3 dpi, mosquito midguts were dissected and 2 pools of 5 midguts per pool were used for RNA extraction and RT-PCR analysis (B). Relative fitness (RF) was determined as the average ratio between bands corresponding to VLPs expressing E2-210Q and E2-210L residues in the sample, divided by the initial ratio of E2-210Q and E2-210L bands in the BM used for mosquito infections.(TIF)Click here for additional data file.Figure S5Effect of the E2-L210Q substitution expressed in the background of the E1-226A residue on dissemination of CHIKV in A. albopictus mosquitoes (Thailand). Above is a schematic representation of the viruses used in the competition assay. Asterisks indicate authentic (w.t.) residues for the SL07 CHIKV strain at the indicated positions. A 1∶1 mixture of viruses SL07 and SL07-210Q-Apa was presented orally to A. albopictus and at 10 dpi, the presence of disseminated E2-210L and E2-210Q CHIKV infection was assayed as described in the (TIF)Click here for additional data file.Table S1Recovery of the viruses after electroporation of in vitro transcribed RNA. a – amino acids at positions E1-226. b – amino acids at positions E2-210. c – specific infectivity of in vitro transcribed RNA expressed as pfu/1 µgRNA. d – supernatants of electroporated Vero cells were collected at 48 h. Virus titers were determined by titration on Vero cells and expressed as Log10(pfu)/mL. h – hours post electroporation.(DOC)Click here for additional data file.


One of the most frequently used and efficient suicide gene therapies for prostate cancer is HSV-TK/GCV system, but its application has been limited due to lack of favorable gene vector and the reduction of "bystander effect". We investigated the effect of a novel combination of HSV-TK/GCV fused with Cx43 and gemcitabine using non-viral vector generation 5 polyamidoamine dendrimers (G5-PAMAM-D) on PC-3 cells.RT-PCR and Western blot were used to detect TK and Cx43 expression. Cell viability and proliferation were measured by using MTT assay. Cell apoptosis was detected with double-staining of Annexin V-FITC and propidium iodide (PI) by flow cytometry. Nude mice models were established to evaluate the therapeutic effect in vivo.G5-PAMAM-D efficiently delivered recombinant plasmids into PC-3 cells and HSV-TK and Cx43 could be expressed successfully. With gemcitabine, G5-PAMAM-D mediated HSV-TK and Cx43 expression effectively inhibited prostate cancer PC-3 cell proliferation, leading to more cellular apoptosis and inhibiting PC-3 tumor growth in nude mice models.This study illustrates that this new suicide gene system mediated by G5-PAMAM-D is effective in decreasing PC-3 cell proliferation and inducing cell apoptosis, and inhibiting tumor growth in vivo. In a word, our study could provide a potential approach for gene therapy of prostate cancer. The incidence and mortality of prostate cancer are gradually increasing in the world in recent years . LocalizAdenovirus vectors are the most common viral vectors used for anti-cancer therapy in human clinical studies . AlthougStudy has indicated that "bystander effect" is essential in suicide gene therapy . Increas2 in RPMI1640 medium , supplemented with 10% fetal bovine serum (FBS) , penicillin (100 U/ml) and streptomycin (100 μg/ml). Six-week-old male BALB/c nude mice  were used for the experiments. Mice were maintained under specific pathogen-free, temperature-controlled conditions at the animal facilities of College of Life Science in Naikai University. Mice were handled in accordance with Institutional Animal Welfare Guidelines.Human prostate cancer PC-3 cells were obtained from our TianJin Institute of Urology. PC-3 cells were maintained at 37°C in a humidified atmosphere of 5% COG5-PAMAM-D is synthesized and kindly provided by Key Laboratory of Bioactive Materials of Ministry of Education of Nankai University . PlasmidPC-3 cells were plated until they reached 70-80% confluency, then different mixtures (G5-PAMAM-D mixed with two different plasmids at a 1:3 molar ratio respectively) were added gently to cells covered with serum-free medium. All the cells were divided into 7 groups: Group A, B (G5-PAMAM-D/pCMV-TK-Cx43); Group C, D (G5-PAMAM-D/pCMV-TK); Group E (G5-PAMAM-D); Group F (pCMV-TK-Cx43); Group G (neither vector nor plasmid). Gemcitabine would be added to group A, C and F when MTT assay and apoptosis detection were conducted. After incubation for 6 h at 37°C, the medium was replaced with fresh serum-containing medium. After 48 h of incubation, cells were used for different experiments.Total cell RNAs were isolated 48 h after transfection using Multisource Total RNA Miniprep Kit . The synthesis of cDNA and PCR were performed using one-step RT-PCR system . PCR was run in a 50 μl reaction volume. The PCR specific primer pairs  were 5'-CCGCTCGAGATGGCCTCGTACCCCGGCCATCAACA-3'(forward) and 5'-CGAAAGCTTACCAGAACCACCGTTAGCCTCCCCCATCTCCCGGGCA-3' (reverse) for analysis of TK, 5'-GTCGACATGGGTGACTGG AGCGCCTT-3' (forward) and 5'-GCGGCCGCCTAGATCTCCAGGTCATCAGG-3'(reverse) for analysis of Cx43. The amplified product of TK had 1143 bp and that of Cx43 had 1149 bp. The PCR products of TK and Cx43 were analyzed by 0.8% agarose gel electrophoresis.48 h after transfection, cells were rinsed twice with cold PBS, then homogenized in RIPA cell lysate and centrifuged. The protein concentration of each sample was quantified by the Bradford assay. Fifty micrograms of total cell extract protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% skimmed milk for 1 h at room temperature, the membrane was incubated with goat anti-TK  or rabbit anti-Cx43  antibody overnight at 4°C. After extensive washes, blots were incubated with the dilution (1:2000) horseradish peroxidase conjugated anti-goat or anti-rabbit IgG  for 90 minutes at 37°C. The bands were developed with 3,3'-diaminobenzidine (DAB). GAPDH  was used as an internal control.2 at 37°C), fresh complete medium was replaced. Then 20 μl of 5 mg/ml MTT solution was added to each well. After 4 h of incubation at 37°C, the supernatant was removed and 150 μl of DMSO were added to each well. The absorbance of optical density at 570 nm (A570) was measured using a Microplate Reader. Cell growth inhibition rate was calculated according to the formula (A570 of control cells-A570 of treated cells) × 100%/A570 of control cells and the curve was drawn.3--2,5-diphenyl-tetrazoliumbromide (MTT), GCV, gemcitabine were all purchased from Sigma (USA). 48 h after transfection, 200 μl of the complete medium was replaced with that containing different concentrations of GCV  and gemcitabine (500 μmol/l) was added to Group A, C and F at the same time. After incubation for 6 h . 48 h after transfection, 200 μl of the complete medium containing GCV (50 μg/ml) was administrated and gemcitabine (500 μmol/l) was added to Group A, C and F at the same time. After incubation for 6 h, cells were centrifuged and resuspended with cold binding buffer at concentration 1.0 × 106 cells) in 200 μl of mixture containing PBS and Matrigel matrix basement memberane  at 3:1 ratio of volume was injected subcutaneously into the right armpit regions of mice. When the tumor volume reached approximately 0.1 cm3, usually ten days after cell implantation, the mice were randomly divided into eight groups . The mice were submitted to intratumoral injection three times  with the mixture of G5-PAMAM-D/pCMV-TK-Cx43(Group I and II), the mixture of G5-PAMAM-D/pCMV-TK(Group III and IV), the mixture of G5-PAMAM-D (Group V), the mixture of pCMV-TK-Cx43 (Group VI), PBS (Group VII) and no treatment (Group VIII). 24 hours after the injections of mixtures every time, GCV (100 mg/kg) was administered intraperitonial once a day for 5 consecutive days. Gemcitabine (20 mg/kg) was administrated intraperitonial in Group I, III and VI at day 2, 8 and 14 respectively. The tumor volume was calculated at regular intervals according to the formula V = π/6 × length × width2. After mice were sacrificed, tumors were extracted and weighted. Tumor specimens were used for histological analysis.A suspension of PC-3 cells . The significance of differences between multiple groups was determined by one-way analysis of variance followed by the LSD test. Data was expressed as means ± standard deviation. Statistical significance was set at P < 0.05.To evaluate whether recombinant plasmids can be successfully transferred into PC-3 cells by using G5-PAMAM-D, and whether TK and Cx43 genes can be expressed, total PC-3 cell RNAs and cell proteins were extracted from different groups after transfection. The expressions of TK and Cx43 were detected by RT-PCR and Western blot. As shown in Figure To investigate the effect induced by this new suicide gene system, the level of cell proliferation was assessed by the MTT assay. As shown in the cell growth inhibition rate curve Figure , cell grBased on staining the cells with Annexin V-FITC and PI, flow cytometry was used to quantify the extent of apoptosis. The points of lower right quadrant in Figure To evaluate the effects of the suicide gene system on the PC-3 tumor growth in animal mode, the tumor volume and tumor weight were measured. Average tumor volumes were similar in all mice before therapy. All mice survived during the period of treatment. We found from Figure One of the most frequently used and efficient suicide gene therapy approaches in cancer treatment is HSV-TK/GCV system . There wAn important element of HSV-TK/GCV gene therapy was the"bystander effect", by which a high percentage of tumor cell death can occur, even when a low percentage of cells was transfected . SuicideIn line with the studies in vitro, we established prostate cancer nude mice model with PC-3 cells. After different experimental intervention, the status of tumor growth was observed. The results demonstrated that tumor growth was significantly inhibited by double-enhanced suicide gene system. Histological analysis showed that tumor tissues were damaged after administration of treatment system in our study visually. These experimental results in vivo suggested the system played a significant role of suppressing tumor progression.In a word, our study implied that expression of Cx43 and gemcitabine could enhance the "bystander effect" of HSV-TK/GCV gene therapy. This double-enhanced suicide gene system was effective in inducing cell growth inhibition and apoptosis in vitro and suppressing tumor growth in vivo. It may be a potential approach for gene therapy of prostate cancer and could provide a choice to attempt suicide gene therapy for locally advanced or metastatic prostate cancer. Nonetheless, the effect of this therapy system on prostate cancer needs to be verified in other prostate cancer cell lines. In addition, we will also investigate the inhibitory effects of this system on tumor model in situ. This system will stand a good chance to be used for improving prostate cancer treatment combined with other therapies.This study illustrates that this new suicide gene system mediated by G5-PAMAM-D is effective in decreasing PC-3 cell proliferation and inducing cell apoptosis, and inhibiting tumor growth in vivo.The authors declare that they have no competing interests.YC carried out the experiments, analyzed the data and drafted the manuscript. GW conducted the experiments and participated in the design of the study. DK, ZZ, KY and RL assisted with experiments and help to draft the manuscript. WZ conceived of the study, and participated in its design. YX conceived of the study, participated in its design and revised the manuscript. All authors read and approved the final manuscript.


Schizosaccharomyces pombe. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of “distal pheromone action”, but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of “proximal pheromone action”.Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in  Saccharomyces cerevisiae [Schizosaccharomyces pombe [Saccharomyces yeasts is to guide the mating projection to a cell of the opposite mating type [Mating pheromones in ascomycete fungi play an important role in conjugation . Yeast previsiae ,3 and Sces pombe ,5. One oing type . A partning type . Anothering type . Mating S. pombe, conjugation is induced between two different mating types, h+ (P) and h– (M) [S. pombe is illustrated in map2+ [mfm1+, mfm2+, and mfm3+ [mfm genes are processed by as yet unidentified proteolytic enzymes to produce the same nonapeptide. Comprehensive mutagenesis has demonstrated that the primary sequence of the C-terminal half of M-factor is important for recognition by Map3 [In the fission yeast  h– (M) –11. Matin map2+ . M-cellsn map2+ ,14. M-fan map2+ . Mature d mfm3+ ,16. Prec by Map3 . Both Ma by Map3 .Saccharomyces cerevisiae [A pheromone-stimulated cell forms a projection from the cell tip that is directed toward a cell of the opposite mating type. Such polarized growth is controlled by a cell polarity regulator complex composed of Cdc42 (a Rab family GTPase), Scd1 (GEF for Cdc42) and Scd2 (a scaffold protein) . On solirevisiae ,20. AlthIn nature, fission yeasts are thought to live in a semi-aqueous environment. Because a pheromone gradient is unlikely to be formed in liquid culture, polarized growth of the projection might be controlled by a totally different mechanism. Prior to cell fusion, cell adherence occurs between opposite mating-type cells, resulting in macroscopic cell agglutination , which mM- and P-cells are mutually stimulated by mating pheromones. Notably, M-factor production is induced solely by nitrogen-starvation and does not need stimulation by P-factor, whereas P-factor expression is enhanced by M-factor . These oS. pombe strains used in this study are listed in S. pombe cells were grown on yeast-extract agar medium (YEA) supplemented with adenine sulfate (75 mg/l), uracil (50 mg/l) and leucine (50 mg/l). Antibiotics  were added to YEA medium at a final concentration of 100 µg/ml. Minimal medium SD was used for selection of auxotrophic mutants. Other synthetic media, MM+N and MM−N, were used for overexpression of manipulated genes. Solid media used for mating and sporulation were MEA and SSA. For induction of mating in liquid media, a shift from SSL+N to nitrogen-free medium (SSL−N) was applied [oC for growth and at 28oC for mating, unless stated otherwise.The  applied ,25. Cell6 cells/ml. Cultures were shaken at 30oC overnight. A 3–5 ml batch of SSL−N was then inoculated with the cells at a density of 4×107 cells/ml, and cultured at 28oC with shaking (at 200 rpm).Cells grown on YEA were inoculated into SSL+N liquid medium at a cell density of 2×10Zygotes (%) = (2Z+2A+S/2) × 100/(V+2Z+2A+S/2)The frequency of zygotes including asci was determined by light microscopy. Usually, triplicate samples (at least 250 cells each) were counted. Cell types were classified into four groups: vegetative cells (V), zygotes (Z), asci (A) and free spores (S). The percentage of zygotes was calculated according to the following equation: 600 before sonication). The cell suspension was then subjected to sonication (20 kHz) for 10 sec to disperse cell aggregates completely. Immediately after sonication, the OD600 was determined (OD600 after sonication). The agglutination index (AI) was calculated according to the following equation: AI =OD600 (after sonication) / OD600 (before sonication)The intensity of sexual cell agglutination was photometrically estimated according to Shimoda and Yanagishima . Sexual This value has been shown to be a function of mean size of cell aggregates .natR, kanR and hygR) on the chromosomes  were mixed and cultured in SSL−N medium for 2 days. The cell suspension was diluted and spread on different drug-containing plates. The number of colonies was counted after 3 days of incubation.Mating efficiency was also deterimined by a quantitative assay of genetic recombination. Heterothallic haploid strains carrying drug-resistance markers , was transformed into heterothallic h+ strains, FS114 and FS133. Additionally, a few deletion sets of the map4 promoter were constructed by PCR. The primer sequences are presented in A pDB248’-based multicopy plasmid carrying the ed pTA14  was recoPRO-lacZ was assayed in two transformants (FS135 and FS403) by β-galactosidase activity. To avoid loss of the pTA(map4PRO-lacZ) plasmid, transformants were maintained on SD medium lacking leucine. After permeabilization [Expression of map4lization , β-galaclization .map4-GFP (green fluorescent protein) fusion gene, which was chromosomally integrated at the authentic map4 locus in the map4::ura4+ disruption background [mam3-mCherry fusion gene was integrated at the authentic locus. Fluorescence intensity of cells was analyzed by Image-J software (http://rsbweb.nih.gov/ij/features.html).Expression of Map4 was examined by fluorescence intensity of a ckground . SimilarFluorescent signals of GFP and mCherry were observed under a fluorescence microscope . Digital images obtained by a differential interference contrast microscope were recorded. Microphotographs were analyzed for the frequency of zygotes and prezygotes (paired cells in contact at projection tips). The long axis (L) and the short axis (S) of a cell were measured, and the L/S ratio was calculated as a measure of cell shape.S. pombe in liquid medium starts with sexual cell agglutination in response to mating pheromone secreted by cells of the opposite mating type [mfm1,2,3Δ) mutant, FS55  for 4 hr. The P-factor-less cell population exhibited significant agglutination without exogenous P-factor, and the addition of synthetic P-factor further enhanced the intensity of agglutination  harboring a deletion of the ignaling .map3 or mam2, encoding the M-factor or P-factor receptor, respectively  and P-type (Map4) cells ,23. It hignaling  and thattination . These emap4+ has been reported to be dependent on Ste11, which is a key transcription factor for sexual reproduction [map4+  activity from this construct was not detectable in a ste11Δ mutant (PRO-lacZ) carrying various deletions of the map4 promoter region was then introduced into a heterothallic P-type strain, FS103. For each construct, three independently isolated transformants were cultured in SSL−N medium with or without 200 nM M-factor. After 6 hr of incubation, β-gal activities were assayed (lacZ gene with a full-length map4 promoter (ca. 600-bp) gave a high level of β-gal activity in the presence of M-factor. Complete deletion of the map4 promoter sequence resulted in null activity. Our previous promoter analysis of another M-factor responsive gene, mat 1-Pm+, showed that a TR-box immediately upstream of the TATA-box is critical for expression [map4 promoter, the most proximal TR-box (TR-II) is positioned 10 nucleotides upstream of the putative TATA box. Deletion of the proximal 100-bp stretch encompassing TR-II did not cause a reduction in β-gal activity. Additional deletion of a distal 250-bp sequence including a TR-box (TR-I) reduced the β-gal activity to about 40% of the full-length positive control. Thus, deletion of the putative TR-box elements did not result in a complete loss of map4 expression. These deletion analyses indicated that the two TR box elements are not critical for the expression of Map4.Transcription of oduction . A canonoduction , and we Δ mutant . The plapression . In the ade6-M210 allele, which leads to red-colored colonies on adenine-limited medium. We counted the number of white colonies (M-cells derived from the wild-type strain) and red colonies (M-cells derived from M-factor-less strain) on solid medium. As shown in As described above, homothallic M-factor-less mutant (FS55) cells are unable to agglutinate, but this defect is phenotypically suppressed by exogenous M-factor   or turned red (ade6-), and the white and red colonies were counted  were differentially marked by three drug-resistance markers, mam1+ gene encodes an ABC transporter that is specific to M-factor secretion [mam1Δ mutant secretes no mature M-factor, and thus is sterile [mam1Δ mutants may accumulate these hypothetical intracellular peptides. If these putative intracellular peptides were involved in zygote formation, then mam1Δ mutants  would be able to mate with P-cells in the presence of exogenous M-factor. As shown in A possible explanation for the above results is that mature M-factors or by-product peptides, made by proteolysis of the M-factor precursor protein, play an indispensable role in the mating capability of M-cells. The ecretion –37. A ma sterile . mam1Δ mCells placed on solid medium can sense a pheromone gradient and elongate a mating projection toward the strongest source of the pheromone . The proThe kinetics of mating events was also examined with the M-factor-less mutant (FS55) cultured with or without M-factor. As shown in fus1Δ allele. The fus1Δ mutant shows defects in cell fusion, but forms prezygotes [fus1Δ mutant, as compared with the wild-type strain. For the fus1Δ mutant, the L/S ratio was 1.3 times higher in aggregated cells than in free cells  was incubated in SSL−N liquid medium containing 200 nM synthetic M-factor for 6 hr, when strong cell agglutination occurred. The culture was allowed to stand for a few minutes to allow the cell aggregates to sediment and then the aggregated cells and free dispersed cells were separately prepared. After sonication, cell size was measured to calculate the L/S ratio. As shown in ezygotes . The polee cells , left. Fee cells , right. A wide variety of sex pheromones in animals serve to attract individuals of the opposite sex. Fungi including yeasts also release mating pheromones to enhance the chance of finding cells of the opposite sex . BecauseS. pombe requires both the P-type adhesin glycoprotein Map4 and its M-type counterpart, Mam3 [map4 promoter region  in the absence of the opposite mating-type cells has also been observed. P-type cells exhibit marked tip growth (shmooing) after treatment with high concentrations of exogenous M-factor  or after the accumulation of native M-factor in mating-deficient mutants such as map4Δ . Such shmap4Δ . These rS. pombe [A Rab family small G-protein, Cdc42, is a major polarity regulator in S. pombe . RecentlS. pombe  reportedIn a liquid culture with shaking, a pheromone gradient may not be stably formed, meaning that polarization must be established differently from the mechanisms proposed for mating on solid medium. Our findings suggested that a firm contact between a pair of M- and P-cells is crucial for polarized cell elongation , which is essential for fixing the relative positions of M- and P-cells in liquid culture. Polarity establishment is attained by a more direct action of M-factor secreted by close neighbors . Only M-cells that secrete M-factor can be chosen as a mate by P-cells. Mating steps in liquid culture regulated by these two different modes of pheromone actions are illustrated in Figure S1mam1Δ strain Eg928 (B), and a homothallic map4Δ strain FS71 (C). Experimental procedures were followed as described in The strains included a homothallic wild-type strain L968 (A), a homothallic (TIF)Click here for additional data file.Figure S2PRO-lacZ) was introduced into an h+ wild-type strain, FS85 (A) and the h+ste11Δ mutant, FS114 (B). The transformants were cultured in SSL+N lacking leucine, and then in SSL−N with or without 200 nM M-factor. Samples were taken every 2 hr and subjected to a β-galactosidase assay.The plasmid pTA(map4(TIF)Click here for additional data file.Figure S3oC. Observation was continued for the indicated duration (minutes) under an optical microscope. Frames were taken every 10 min. The arrow indicates a pair of cells, probably comprised of M- and P-type cells. Note that the cells were initially round in shape and protruded a pointy projection from the contact region. Scale bar, 10 µm.Cells of the homothallic wild-type strain (L968) were cultured in SSL−N for 4 hr. Aggregated cells were obtained and placed on an agarose slab gel containing SSL−N at 28(TIF)Click here for additional data file.Table S1(DOCX)Click here for additional data file.


The disease is classified into 2 distinct types according to the nature of auto-antibodies. Disturbances of the calcium-parathyroid hormone-vitamin D axis are frequently associated with chronic liver disease. Patients with AIH have a high prevalence of vitamin D deficiency. Genetic studies have provided the opportunity to determine which proteins link vitamin D to AIH pathology, namely, the major histocompatibility complex class II molecules, vitamin D receptors, toll-like receptors, cytotoxic T lymphocyte antigen-4, cytochrome P450 CYP2D6, regulatory T cells (Tregs) and the forkhead/winged helix transcription factor 3. Vitamin D also exerts its effect on AIH through non-genomic factors, namely, mitogen-activated protein kinase signaling pathways, γδT cells, interferon-gamma nitric oxide synthase, and reactive oxygen stress. In conclusion, vitamin D may have a beneficial role in AIH and improves liver function in concanavalin A-induced mouse AIH. Calcitriol is best used for AIH because it is the active form of a vitamin D The disease is classified into 2 distinct types according to the nature of autoantibodies , 2. Typeatocytes  and contatocytes . Vitamin CYP2C9) . Some st CYP2C9) . However CYP2C9)  demonstr CYP2C9)  demonstr CYP2C9) . A major the VDR . Another the VDR . Both in the VDR . In conc the VDR . TherefoStudies have suggested that several genes in the major histocompatibility complex (MHC) region promote susceptibility to AIH. Located in the MHC region, human leukocyte antigen (HLA) genes have been implicated in AIH susceptibility. The genes of DRB1*0301 and DRB1*0401 are the susceptibility genes for type 1 AIH in Caucasian American, northern European, and Italian patients . The genGenetic studies provide an opportunity to link molecular variations with epidemiological data. DNA sequence variations, such as polymorphisms, exert both modest and subtle biological effect on AIH. Vitamin D exhibits immune-modulatory and anti-proliferative effects through VDR in diseases. Among a variety of VDR gene polymorphisms, BsmI and TaqI have been reported to be associated with autoimmune liver diseases. The FokI polymorphisms of VDR have been linked to AIH in German and Chinese populations , 25. TheToll-like receptors (TLRs) are a group of glycoproteins that function as surface trans-membrane receptors and are involved in the innate immune responses to exogenous pathogenic microorganisms. Substantial evidence exists for an important role of TLRs in the pathogenesis and outcome of AIH. Healthy livers contain low mRNA of TLRs, such as TLR-1, TLR-2, TLR-4, TLR-8, TLR-9, TLR-10, and signaling molecules (MD-2 and My88) compared with other organs . In ConA+ CD25- T cells [Cytotoxic T lymphocyte antigen-4 (CTLA-4) is involved in the regulation of T cells and upregulated during T-cell-mediated immune response. CTLA-4 functions as a negative regulator by binding to the B7 family co-stimulatory molecules on antigen-presenting cells with higher affinity than CD28. The CTLA-4 protein is known to have a role in the pathogenesis of AIH. Mice vaccinated with a plasmid containing the N-terminal region of mouse CTLA-4 and antigenic region of human CYP2D6 developed anti-LKM1 and anti-LC1 antibodies of the IgG2 subclass. In addition, the ALT levels have been correlated with both the presence of anti-LKM1/anti-LC1 antibodies and liver necroinflammation . The mRN T cells .3 into 25OHD, and vitamin D 25-hydroxylase deficiency resulted in vitamin D deficiency [The cytochrome P450 (CYP) superfamily of enzymes is responsible for the oxidation, peroxidation, and/or reduction of vitamins, steroids, and xenobiotics, as well as the metabolism of drugs. CYP2D6, an important member of this superfamily, has been identified as the major auto-antigen in type 2 AIH . CYP2D6 ficiency .+ and the forkhead/winged helix transcription factor 3 (Foxp3) [+CD25high T cells were reduced in numbers, expressed low levels of Foxp3, and were less effective at inhibiting target cell proliferation in patients with AIH compared with healthy controls [+ T cells through direct VDR binding to the Foxp3 gene [+ regulatory T cells.Regulatory T cells (Tregs) are a distinct lymphocyte with inhibitory properties that affect the activation of the immune system. Tregs are characterized by the expression of CD25 (Foxp3) . During controls . The Foxcontrols , 56. Howcontrols , 58. Calxp3 gene . Calcitrxp3 gene . Taken tThe mitogen-activated protein kinase (MAPK) pathways provide a key link between the membrane-bound receptors that receive these cues and changes in the pattern of gene expression. The MAPK pathways include the extracellular signal-regulated kinase (ERK) cascade, stress activated protein kinases/c-jun N-terminal kinase (SAPK/JNK) cascade, and p38 MAPK/RK/HOG cascade . ActivatT lymphocytes recognize foreign antigen by means of their surface receptor. According to the receptor type, T cells may be divided into αβ and γδ subsets. In peripheral blood, γδT cells represent a small number of T cells. However, γδT cells may play important roles in the pathogenesis of AIH. An elevation in the relative and absolute counts of γδT cells in the peripheral and portal areas of patients with AIH has been detected . γδT celMost human T cell clones obtained from healthy donors produce IFN-γ, IL-2, IL-4, and IL-5 upon stimulation with various mitogens. Soluble liver antigen-specific IFN-γ responses were significantly more frequent in AIH patients than controls . IFN-γ iNitric oxide (NO) is involved in host defense reactions and plays a key role in the pathophysiology of vascular disorders. NO production in both sera and liver tissue increased after liver injury induced by delayed-type hypersensitivity to picryl chloride . AnimalsReactive oxygen species (ROS) have been suggested to play a role in AIH. The amount of ROS found in AIH livers was significantly higher than in healthy human livers . MarkersThe relationship between vitamin D and AIH has been discussed. Vitamin D may have a beneficial role in AIH. Genetic studies have provided the opportunity to determine what proteins link vitamin D to AIH pathology. Vitamin D also exerts its effect on AIH through non-genomic mechanisms. Calcitriol is useful in treating AIH, because it is an active form of a vitamin D3 metabolite, and its receptors are present in the sinusoidal endothelial cells, Kupffer cells, and stellate cells of normal livers, and the biliary cell line.


To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an  Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs and contributes to several important diseases, such as myocardial infarction, hypovolemic shock, thromboembolism, and acute kidney injury (AKI) -4. Ischein vitro I/R. To mimic the in vivo renal I/R injury, a renal proximal tubular cell line LLC-PK1 derived from pig kidney were treated with a mitochondrial respiration inhibitor (antimycin A) and a non-metabolizable glucose analog (2-deoxyglucose) to induce ischemia injury followed by reperfusion with growth medium [in vitro I/R injury model.AMPK, a heterotrimeric complex, consisting of a catalytic α-subunit and regulatory β- and γ-subunits with three isoforms, is abundantly expressed in the kidneys . AMPK ish medium ,28. The  Antimycin A, 2-deoxy-D-glucose (2-deoxyglucose), RAD001 (mTOR inhibitor), and 3-methyladenine  were purchased from Sigma-Aldrich . Rapamycin was purchased from Calbiochem . Compound C (AMPK inhibitor) was purchased from Merck .2. NRK-52E cells were purchased from the Bioresource Collection and Research Center . NRK-52E cells were cultured in DMEM  supplemented with 5% FBS and 1% antibiotics  at 37°C under 5% CO2.LLC-PK1 cells, an established renal proximal tubular cell line derived from pig kidney, were purchased from American Type Culture Collection (ATCC) and cultured in growth medium consisting of medium 199  supplemented with 3% fetal bovine serum (FBS) and 1% antibiotics  at 37°C under 5% CO2PO4, 25 mM NaHCO3, 1 mM CaCl2, 1 mM MgCl2, pH =7.4) with antimycin A  and 2-deoxyglucose  for 1.5 h to induce in vitro ischemia injury [in vitro ischemia injury. The reperfusion was achieved in LLC-PK1 cells and NRK-52E cells by washing with KH buffer and then cultured in complete growth medium for various time courses. LLC-PK1 cells were incubated in a Krebs-Henseleit (KH) buffer  and 10 μg/ml of PI (Sigma-Aldrich) for 20 min. Flow cytometric analysis was performed using Becton Dickinson FACSCalibur cytometer with an argon ion laser (488 nm) as the excitation light. Cell Quest version 6.0 software was used for DNA content analysis.in vitro I/R treatment, LLC-PK1 cells were collected in different reperfusion time points and then washed with PBS twice. Following centrifugation, the PBS was discarded and the cells were stained with Annexin V-FITC and PI staining kit (BD Biosciences) for 15 min at room temperature in the dark as previously described [After escribed . Flow cyin vitro I/R treatment, the transfected cells were harvested and stained with Hoechst33258 . For MDC staining, the cells were treated with 50 μM MDC (Sigma-Aldrich) in the medium and incubated at 37°C for 20 min. The localizations of LC3 and autophagosome formations were examined by fluorescence microscopy.The transient transfection in LLC-PK1 cells was performed by the Lipofectamine 2000 reagent (Invitrogen) according to the manufacture's recommendations. The cells were transfected with a control pcDNA6.2 vehicle or a GFP-LC3 fusion protein expression vector (pcDNA6.2-Em GFP-LC3). After Western blotting analysis was performed as previously described . After t5’-CTTGAAATGTGTGCAAATCTAA-3’. Cells were treated with hexadimethrine bromide (10 μg/ml) and lentiviral particles (40 μl) for 24 h, and then replaced with fresh medium. The stable expressing cells were selected using 2 μl/ml puromycin.The shRNAs were purchased from Open Biosystems, Thermo . The LLC-PK1 cells were infected with lentivirus expressing the targeting shRNA for AMPKα1 or scrambled as per the manufacturer’s instructions. The target sequences of AMPKα1 shRNA is presented as P-value is less than 0.05 or 0.01. Data are presented as means ± SDs. The significant difference from the respective controls for each experimental test condition was assessed by one-way analysis of variance (ANOVA) and Student's t-test. The difference is significant if the in vivo I/R reaction [ To evaluate the effects of I/R on renal cell apoptosis, LLC-PK1 cells were treated with antimycin A (12.5 to 100 μM) and 5 mM 2-deoxyglucose to mimic the reaction ,28. As sin vitro I/R injury model. LC3 activation is commonly used to monitor autophagy and the mount of LC3-II is clearly correlated with the number of autophagosomes [To investigate whether autophagy takes a part in I/R-induced renal cell injury, we examined autophagy induction in LLC-PK1 cells in an agosomes . We obseagosomes . MDC posin vitro I/R injury model. A pharmacological inhibitor of autophagy 3MA [in vitro I/R injury model.We next investigated the role of autophagy in an hagy 3MA  was usedin vitro I/R injury model  kinase, p27, and direct activation of autophagic genes ,43. In ain vitro I/R model to mimic the in vivo renal I/R condition [in vitro I/R. To confirm the effect of I/R on LLC-PK1 cells, a rat normal renal tubular cell line NRK-52E was used. We found that AMPK phosphorylation and LC3-II formation are also increased in NRK-52E cells during in vitro I/R. The I/R-increased cell injury could also be enhanced by compound C. However, this chemically induced I/R cell model has its limitations and may not completely represent the in vivo clinical condition of I/R injury. A more direct I/R model may be needed to confirm the findings of the present study in a future study. Accordingly, we have tried to test the in vivo effects of I/R on renal tissues. Our preliminary data showed that AMPK phosphorylation and LC3-II formation are also increased in renal cortex of I/R-treated rats (data not shown). The detailed effects and mechanisms need to be further investigated in the future. LLC-PK1 is known as a renal proximal tubule cell line ,46. Androndition . Resultsin vitro I/R injury model. The present study also provides the evidence that autophagy protects renal tubular cells from I/R injury through an AMPK-down-regulated mTOR signaling pathway, which may be a potential target for therapeutic intervention in the renal I/R injury.In this study, we demonstrated for the first time that an AMPK-regulated autophagy induction pathway plays an important protective role in the I/R-induced renal proximal tubular cell injury in an


Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brain endothelial cell line (hCMEC/D3) were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp) of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp) but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine.30% of epilepsy patients receiving antiepileptic drugs (AEDs) are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven  However the hypothesis that P-gp has a causal role in refractory epilepsy remains unproven, but recent data have confirmed that P-gp protein expression is up-regulated at the epileptic focus in refractory patients Epilepsy affects 50 million people worldwide. Antiepileptic drug (AED) therapy is ineffective in approximately 30% of patients, who continue to have seizures despite regular dosing MDR1 knockout mice st generation transport inhibitors are licensed drugs (e.g. cyclosporine), shown to be high affinity substrates; 2nd generation inhibitors (e.g. PSC-833), which are more specific for P-gp; and 3rd generation inhibitors (e.g. tariquidar), which show the highest specificity for P-gp. However, there are continuing contradictory reports on whether AEDs are substrates for P-gp and this is summarised by two recent reviews MDR1 knockout mice has shown that rodent P-gp affects brain accumulation of phenytoin but not lamotrigine or carbamazepine ABCB1 polymorphisms with refractory epilepsy ABCB1 genotype ABCB1 SNPs and refractory epilepsy P-gp has a wide anatomical distribution and plays an important role in the bioavailability and disposition of many xenobiotics, as shown using in-vitro systems for characterisation of P-gp substrates and found the bidirectional assays to be the method of choice, while the calcein AM and P-gp ATPase assays were also useful in characterising P-gp substrates into different categories There are many diverse assays to determine if a compound is a P-gp substrate with a recent review proposing a decision tree for identifying P-gp substrates in-vitro transport systems.Given the conflicting evidence regarding P-gp-mediated transport of AEDs, the aim of the present work was to use multiple assay systems to investigate whether phenytoin, lamotrigine and carbamazepine interact with P-gp. We present data on these 3 drugs in seven 3H-digoxin and 14C-phenytoin were purchased from PerkinElmer  with specific activity of 23.5Ci/mmol and 53.1mCi/mmol respectfully. 14C-carbamazepine was provided by Ciba  with specific activity of 49mCi/mmol and 14C-lamotrigine was a kind gift from GSK  with specific activity of 52.9mCi/mmol. All other chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated.Tariquidar was synthesised by Dr. Oliver Langer, Medical University of Vienna, Department of Clinical Pharmacology, Austria. 100 cells were grown as previously described ABCB1 and respective wildtype LLC-PK1 cells were kindly provided by Prof. Borst  Caco-2 cells, MDCKII±Pgp, hCMEC/D3 cells, CEM and VBLThe porcine brain microvessel isolation and culture protocol was based on previously published papers Xenopus laevis oocyte expression plasmid (pBluescriptII-KSM) containing either wild-type human ABCB1 cDNA or an ATPase deficient mutant of ABCB1  were utilised. The ATPase deficient mutant encodes for a transporter with an amino acid change (G534D) that has previously been ectopically expressed and shown to be expressed at equivalent levels as the wild type protein and with normal membrane insertion but has a complete loss of drug-stimulated ATPase activity in-vitro transcription kit was utilised following manufacturer’s instructions. As described previously, oocytes were extracted, injected with the cRNA or distilled water (50nl for each) and were maintained for 3 days Cells were equilibrated in transport buffer (HBSS containing 25mM HEPES and 0.1% (w/v) bovine serum albumin at pH 7.4) at 37°C with accumulation assays performed as previously described 2. For transport experiments, cell monolayers were equilibrated in transport buffer and replaced with appropriate solutions of 1.5ml in the apical and 2.6ml in the basal compartment.Caco-2 cells were seeded onto transwell polycarbonate membrane inserts (Corning) with pore size 0.4 µm and membrane diameter of 24 mm and cultured for 21 days. MDCKII±P-gp and LLC-PK1±P-gp were seeded and cultured for 8 days. The minimum trans-endothelial electrical resistance (TEER) acceptable for each cell line was above 100 Ω.cmFor oocyte transport studies involving measurement of efflux, oocytes were injected with a radiolabelled drug in the absence or presence of an P-gp inhibitor to give final intra-oocyte concentrations of 1µM for digoxin or 5µM for phenytoin. An intra-oocyte volume of 500nl was used to calculate the final concentrations 5cells/cm2. At confluence, culture medium was replaced by serum-free medium containing hydrocortisone (550nM), and 8-4-chlorophenylthio-cAMP (250µM) and RO-20-1724 (17.5µM) for 24 hours. This was conducted to elevate intracellular cAMP and thereby encourage differentiation of BBB phenotype including tight junctions. Cell monolayers were used when TEER exceeded 340 Ω.cm2. For permeability assays, DMEM without phenol red with added HEPES (25mM) and 0.1% BSA was pH adjusted to 7.4 and used as transport medium. Cells were incubated at 37°C for 1 hour with radiolabelled compounds and cold compound to give a final concentration of 6µM in the apical compartment for apical-to-basal direction of transport. Samples were taken from both compartments.Apparent permeability (Papp) in cm/s for the compounds studied was calculated as previously described The PBECs were seeded onto rat-tail collagen/fibronectin-coated transwell filter inserts at a density of 1×10Flow cytometric analysis of cell surface P-gp expression was carried out in all cell lines as described previously t-test or by ANOVA followed by Tukey-Kramer test for multiple comparisons using GraphPad Prism 5. The significance values were as follows: * P<0.05, ** P<0.01, *** P<0.001.All data are presented as mean ± standard deviation. To assess statistical significance the samples were compared using a 100 cells were found to have 220 times more membrane P-gp expression than the CEM parental cells (used as baseline). The transfected LCC-PK1-P-gp and MDCK-P-gp cells had P-gp expression 86 and 43 times the baseline value, respectively. Endogenous expression of P-gp in Caco-2 and hCMEC/D3 cells was found to be 4.2 and 11.6 times the expression in CEM cells.The membrane protein expression of P-gp was determined in the cells using an epitope specific antibody to P-gp with relative fluorescence units compared to the low P-gp expressing CEM cells . The VBLin-vitro model of multi-drug resistance were performed. The VBL100 lymphoblastoid cell line has been generated by continuous culture of CEM cells with vinblastine, which has induced multi-drug resistance through over-expression of P-gp 100 cells were assessed in the presence or absence of tariquidar. Tariquidar is a non-competitive inhibitor of P-gp that can inhibit both P-gp and BCRP. The increased expression of P-gp in the drug-resistant VBL100 cells resulted in less accumulation of digoxin (used as a positive control) than in CEM cells, while tariquidar restored accumulation  implying the possible involvement of other endogenous transporters. Interestingly, a recent study utilising the concentration equilibrium approach proposed that P-gp does not transport carbamazepine, but does transport its active metabolite carbamazepine-10,11-epoxide in vivo.No transport of phenytoin, carbamazepine and lamotrigine by human P-gp was previously shown by a conventional bidirectional transcellular permeability method in three cell lines that express P-gp; Caco-2, LLC-PK1- P-gp and MDCKII- P-gp Xenopus laevis oocyte expression system was validated for P-gp protein expression (data not shown) and transport function. This expression system has several advantages for drug transport due to the transient nature of protein expression and low expression of endogenous transporters in oocytes The in-vivo BBB hCMEC/D3 cells are an immortalised human brain endothelial cell line 50 for P-gp inhibition and this might then mask active transport. The three AEDs tested in this study have been described as either non- P-gp inhibitors or weak inhibitors. The concentrations tested are lower than any P-gp inhibitory effect and would therefore be unlikely to yield a false negative result. For example in LLC-PK1-P-gp cells, drug treatment with 100µM of either phenytoin, carbamazepine or lamotrigine showed no inhibition of the uptake of the P-gp substrate A potential issue to consider when investigating interactions with P-gp is that the concentrations tested might be near to or above the IC100 cells showed an increase in carbamazepine accumulation. This suggests an unidentified influx transporter for carbamazepine in these cells and is certainly worthy of further study. An alternative approach to trying to group AEDs with diverse physicochemical properties and structures into one interaction profile might be to consider each AED individually. For example we have recently identified lamotrigine as a substrate for the influx transporter, hOCT1, in human brain endothelial cells and this may explain its good permeability into the brain despite its unfavourable physicochemical properties Another variable to consider is the involvement of additional drug transporters. A study investigating ABCG2 (BCRP) found no interaction (as either substrates or inhibitors) with major AEDs In summary, P-gp does not provide a unifying basis for drug resistance in epilepsy since not all AEDs are substrates. In particular, we found no interaction of lamotrigine or carbamazepine with P-gp. Lamotrigine and carbamazepine do not conform to the properties of P-gp substrates as set out by Giacomini et al. P-gp is over-expressed in patients with refractory epilepsy Figure S1Concentration equilibrium approach in Caco-2 monolayer. Transport of a) 5µM 3H-digoxin or (b) 5µM 3H-digoxin in the presence of 300nM tariquidar or (c) 5µM 14C-phenytoin or (d) 5µM 14C-lamotrigine or (e) 5µM 14C-carbamazepine in Caco-2. Samples were taken at each indicated time point over a 4 hour time course. Data are expressed as mean ±SD (n = 3). * significantly different compared to cells without inhibitor .(TIF)Click here for additional data file.Figure S2Concentration equilibrium approach in LLC-PK1 transfected with human P-gp for the transport of different concentrations of lamotrigine. Transport of a) 20µM 14C-lamotrigine or (b) 40µM 14C-lamotrigine in LLC-PK1±P-gp in the absence or presence of 300nM tariquidar. Samples were taken at each indicated time point over a 6 hour time course. Data are expressed as mean ±SD (n = 3). * significantly different compared to wild type cells. # significantly different (P<0.05) compared to LLC-PK1+P-gp cells in the absence of tariquidar.(TIF)Click here for additional data file.Figure S3Concentration equilibrium approach in MDCKII transfected with human P-gp for the transport of AEDs. Transport of a) 5µM 3H-digoxin or (b) 5µM 14C-phenytoin or (c) 5µM 14C-lamotrigine or (d) 5µM 14C-carbamazepine in MDCKII ±P-gp in the absence or presence of 300nM tariquidar. Samples were taken at each indicated time point over a 6 hour time course. * significantly different compared to wild type cells . # significantly different compared to MDCKII+P-gp cells in the absence of tariquidar .(TIF)Click here for additional data file.


In echocardiography (EC), classification of diastolic dysfunction (DD) is widely accepted and mainly assessed using three criteria (figure1): mitral blood flow , lateral wall velocity  and pulmonary vein flow . With these three characteristic flow and velocity patterns and their ratios E/A, E/E´ and S/D, regular diastolic function can be clearly distinguished from three degrees of DD (I-III°).Cardiovascular magnetic resonance (CMR) has excellent capabilities to assess blood flow and myocardial tissue motion using phase contrast (PC-CMR) imaging but has not been used to classify diastolic function similar to the EC approach.We sought to compare the feasibility of PC-CMR wtih echocardiographic doppler imaging for the assessment of DD using the echocardiographic flow and velocity approach for DD-classification.After acquisition of regular short axis cine SSFP volumetry and 2-3-4chamber views, in 22 patients with various cardiovascular diseases we performed single-slice short-axis PC-CMR  similarly to typical EC locations at the tip of mitral leaflets in diastole on a 1.5T whole body MRI system (Philips Achieva) to generate mitral E-and A-waves, lateral S`-E`-A-velocities, E/A- and E/E`-ratios. PC-CMR for PVF was planned orthogonally to the cine 4-chamber plane 1cm distal from pulmonary vein inflow into the left atrium. Directly after MRI, EC was performed to generate complementary data for MBF, LWV and PVF. After generating all curves and ratios, patients were classified into 4 groups  for both techniques.EC and PC-CMR could be performed in all patients, whereas EC PVF could not be assessed in 4 patients due to reduced flow signals.20/22 patients (91%) were categorized similarly, whereas in 2 cases PC-CMR insignificantly underestimated DDI° and mis-diagnosed them as normal. Mean scan-time for MBF, LWV and PVF was 5.20±1.31 min., mean analysis time was 4.10±1.31 min.For the first time we could show that PC-CMR analysis of DD is feasible and showed excellent agreement with the widely accepted EC method for classification of DD. PC-CMR could offer the potential of a practically and reasonably time-consuming approach for the clinically important assessment of DD omitting sophisticated MRI sequences and dedicated software analysis tools.


Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far.N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom.Systemic host N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on  Defective RNAs (D RNAs), which have internal deletion of viral sequences, have been described for a variety of plant viruses ,2. Some Beet Necrotic Yellow Vein virus (BNYVV), transmitted by zoospores of Polymyxa betae, is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania  were used. The PCR products were purified and cloned into the pMD19-T vector to obtain a full-length cDNA clone named pMDR3 and three D RNAs cDNA clones, pMDRNA3α, pMDRNA3β and pMDRNA3γ . The freshly synthesized transcriptions were mixed with total RNA of T. expansa infected by isolate BN12, the mixture supplemented with an equal volume of inoculation buffer  were rubbed onto T. expansa, C. amaranticolor or N. benthamiana leaves. Local lesions generally appeared at 5-8 days post inoculation, while systemic symptom of N. benthamiana appeared at 12-14 dpi.Infectious plasmids were linearized by T. expansa leaves and systemic infected N.benthamiana were harvested at 7 and 14 dpi, respectively. Total RNA was extracted for RT-PCR detection and Northern hybridization, as described previously [P-labeled cDNA specific for RNA1 (nt5815-6531), RNA2 (nt145-714), and RNA3 (nt445-1102) sequences, respectively.Inoculated eviously . Probes The following primers were used for RT-PCR: for detection of RNA 2, the forward primer BN81  and the reverse primer BN80 , for detection of RNA3, the forward primer BN78  and the reverse primer BN77 , for detection of RNA 4, the forward primer BN209  and the reverse primer BN210 (5'-CTAATCGTGATAAAAGACAAACCA-3'complementary to nt1205 to 1228) and for detection of RNA 5, the forward primer BN225  and the reverse primer BN226 .The authors declare that they have no competing interests.YW carried out most of the experiments and wrote the manuscript. HF anticipated the inoculation and the construction of D form RNA3s. XW and ML provided useful advice. CH, DL and JY conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


Expression of HLA-B*57 and the closely related HLA-B*58:01 are associated with prolonged survival after HIV-1 infection. However, large differences in disease course are observed among HLA-B*57/58:01 patients. Escape mutations in CTL epitopes restricted by these HLA alleles come at a fitness cost and particularly the T242N mutation in the TW10 CTL epitope in Gag has been demonstrated to decrease the viral replication capacity. Additional mutations within or flanking this CTL epitope can partially restore replication fitness of CTL escape variants. Five HLA-B*57/58:01 progressors and 5 HLA-B*57/58:01 long-term nonprogressors (LTNPs) were followed longitudinally and we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. The Sequence Harmony algorithm was used to detect homology in amino acid composition by comparing longitudinal Gag sequences obtained from HIV-1 patients positive and negative for HLA-B*57/58:01 and from HLA-B*57/58:01 progressors and LTNPs. Although virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, these escape mutations were not associated with disease progression. In sequences from HLA-B*57/58:01 progressors, 5 additional mutations in Gag were observed: S126N, L215T, H219Q, M228I and N252H. The combination of these mutations restored the replication fitness of CTL escape HIV-1 variants. Furthermore, we observed a positive correlation between the number of escape and compensatory mutations in Gag and the replication fitness of biological HIV-1 variants isolated from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations. HLA class I alleles are associated with the clinical course of HIV-1 infection. HLA-B*57 and the closely related HLA-B*58:01 are overrepresented in so called long-term nonprogressors (LTNPs) Previously, we studied 5 HLA-B*57/58:01 LTNPs and 5 HLA-B*57/58:01 progressors longitudinally, and observed similar frequencies of HIV-1 Gag-specific CTL responses and dynamics in escape mutations in HLA-B*57/58:01-restricted CTL epitopes Here, we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. Virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, and these escape mutations were not associated with disease progression. In the HLA-B*57/58:01 progressors, 5 additional mutations were observed in the Gag protein that increased the replication rate of the HLA-B*57/58:01 CTL escape variants.To study differences in the Gag protein of virus isolates obtained from HLA-B*57/58:01 progressors and LTNPs that may explain the differences in replication kinetics, we compared Gag sequences (positions 1-364) obtained from various time points during the course of infection  using thFirst, we identified amino acid variation in Gag specific for patients carrying HLA-B*57 or HLA-B*58:01 irrespective of their disease course. Therefore, we compared longitudinally obtained sequences from HLA-B*57/58:01 patients  were placed in the NL4-3.Ba-L molecular clone together with the 6 mutations specifically associated with the presence of HLA-B*57/58:01   and 3. Tet al. reported that the compensatory mutations H219Q, I223V, M228I, G248A and N252H partially restored replication kinetics of NL4-3 virus carrying the T242N escape mutation et al. and our present analysis on the replication fitness of the virus, we placed the described mutations in the NL4-3.Ba-L carrying the HLA-B*57/58:01-specific mutations  and the IW9 epitope (I147L), and a mutation adjacent to the KF11 epitope (S173T). Additionally, two other mutations (V159I and T280V) were observed specifically in viral variants from HLA-B*57/58:01 positive individuals; however, these mutations could not be related to escape from CTL pressure restricted by HLA-B*57/58:01. When comparing viral Gag sequences obtained late in infection from HLA-B*57/58:01 progressors and HLA-B*57/58:01 LTNPs, frequent changes in the amino acid sequence were observed for progressors at 5 positions: S126N, L215T, H219Q, M228I, N252H. An increase in replication kinetics was observed after introduction of two single mutations (L215T and H219Q) in the NL4-3.Ba-L virus containing the HLA-B*57/58:01-specific mutations. Nonetheless, the combination of all mutations associated with HLA-B*57/58:01 disease progression resulted in an even higher increase in replication kinetics. Introduction of a combination of mutations L215T and H219Q did not result in a significant increase in replication kinetics compared to the NL4-3.Ba-L virus containing the HLA-B*57/58:01-specific mutations, indicating that the combination of these two mutations does not account for the increase in replication kinetics observed for the virus containing the HLA-B*57/58:01-specific mutations. These findings suggest that multiple mutations are required to restore viral fitness after escape from HLA-B*57/58:01-restricted CTL responses et al. reported a higher frequency of mutations H219Q, I223V, M228I, G248A and N252H in combination with the HLA-B*57 T242N CTL escape mutation in sequences obtained from HLA-B*57 progressors than seen in sequences from HLA-B*57 non-progressors A previous study by Brockman Our results and previous observations by others The Gag protein is highly conserved and amino acid variation observed in Gag seems to be limited to specific positions, whereas other positions remain unchanged due to structural constraints. As a result, a high degree of conformity was observed in the amino acid positions described here and by previous studies In conclusion, we here identified 5 positions in the Gag region of HIV-1 that are more frequently mutated in sequences from HLA-B*57/58:01 progressors. The combination of these mutations can restore the replication fitness of CTL escape HIV-1 variants, which may explain the differences in disease progression. Furthermore, we observed that the total number of escape and compensatory mutations correlated with the replication fitness of biological HIV-1 isolates obtained from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations.9cells/l per year over a period of at least 10 years .Previously, we reported on 22 participants from the Amsterdam Cohort Studies (ACS) on HIV infection carrying the protective HLA-B*57 or HLA-B*58:01 allele who showed large differences in clinical disease course Two participants (L6 and L7) were heterozygous for the 32 base pair deletion in the CCR5 gene, whereas all other participants had a CCR5 wild type genotype. Typing of the HLA A and B alleles of all participants  did not The ACS has been conducted in accordance with the ethical principles set out in the declaration of Helsinki and written informed consent is obtained prior to data collection. This study was approved by the Amsterdam Medical Center institutional medical ethics committee.Five HLA-B*57/58:01 progressors and five HLA-B*57/58:01 LTNPs were followed longitudinally. Clonal HIV-1 variants were isolated from patient PBMCs that were obtained early after seroconversion or study entry, at a time point as late as possible in the course of infection before the start of therapy and from PBMCs isolated at an in between time point  [25]. Fr5′-CGACGCAGGACTCGGCTTGCTG-3′; HXB2 nucleotides 684−705) and Gag outer reversed  and 2 different inner PCR primer combinations: Gag BssHII fw  or Gag ClaI fw  in combination with Gag p17 rev  and Gag p17 fw  in combination with Gag ApaI rev .Viral DNA was isolated from cryopreserved PBMC that were infected with one clonal HIV-1 variant, using the L6 isolation method gag region were translated and edited with the BioEdit program .PCR products were purified and sequenced with the ABI prism BigDye Terminator sequencing kit  on an ABI 3130 XL DNA sequencer according to the manufacturer's protocol using the same PCR primers that were used for the nested PCR. DNA sequences were analyzed with Seqman software . The nucleotide sequences of the gag sequences obtained from these patients were analyzed with Sequence Harmony (SH) www.ibi.vu.nl/programs/seqharmwww). SH measures the overlap in distribution of amino acid types between two subgroups (A and B), in this case gag sequences obtained from HLA-B*57/58:01 and non- HLA-B*57/58:01 patients or HLA-B*57/58:01 LTNPs and HLA-B*57/58:01 progressors, at a certain position (i) in the sequence alignment as follows:x at position i in the sequence and p, where Np is the number of sequences for patient p in the calculation of the SH score, such that each patient in total has equal weight in the SH analysis.To evaluate amino acid differences in Gag between patients positive and negative for HLA-B*57/58:01 and between HLA-B*57/58:01 positive LTNPs and HLA-B*57/58:01 progressors, the In the SH analyses performed here, a cut-off value of 0.90 was used to determine amino acid positions that are different between two groups of sequences. As the SH values are in a logarithmic scale, one could interpret the 0.90 cut-off as an approximate 10% difference in amino acid distribution. The high cut-off allows for detection of relatively small differences against a background of overall high conservation. Note that conservation overall or within a group of sequences is not explicitly taken into account in the SH analysis. The cut-off of 0.9 for the SH score implies that sites are already of interest when the frequency of occurrence of some amino acid types at a certain position changes. Positions where the difference between the two groups of sequences was located at a minor amino acid for both groups, or where the aberrant amino acid was identical to our NL4-3.Ba-L backbone were excluded from the analysis.corrected p-value of <1e-9. In the discussion of the results, we will for simplicity state p<0.0001 for Z<−10 .Z-scores were calculated based on random SH scores from 100-fold random shuffling of sequence labels ; we assumed a normal distributions of these random SH scores based on previous tests 2 incubator at 37°C.293T cells (ATCC) were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% inactivated fetal calf serum , 100 U/ml penicillin  and 100 µg/ml streptomycin (Gibco) in a humidified 10% CO6 cells/ml in a humidified 10% CO2 incubator at 37°C. PBMC cultures were continued in IMDM supplemented with 10% FCS, 20 U/ml recombinant interleukin 2 , 100 U/ml penicillin and 100 µg/ml streptomycin at a cell density of 1×106/ml in a humidified 10% CO2 incubator at 37°C.PBMC from 10 healthy donors were isolated from buffy coats by Ficoll density centrifugation, pooled and cryopreserved. After thawing, the PBMC pool was stimulated for three days in Iscoves' Modified Dulbecco's Medium (IMDM) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml ciproxin  and 1 µg/ml phytohaemagglutinin  at a cell density of 5×10gag region of the molecular clone NL4-3.Ba-L was removed using restriction enzymes BssHII and ApaI and then cloned into the pGEM T easy vector . Mutations in the gag region were introduced using site directed mutagenesis as described by the manufacturer . The gag insert was sequenced to confirm successful mutagenesis using primers T7 (5′-TAATACGACTCACTATAGGG-3′) and SP6 (5′-GATTTAGGTGACACTATAG-3′).For the construction of HIV-1 variants that contain mutations in the Gag protein, the gag insert was ligated into the full length molecular NL4-3.Ba-L clone using BssHII and ApaI restriction sites. The full-length NL4-3.Ba-L molecular clones were then transfected into 293T cells using the calcium phosphate method gag regions of obtained viruses were sequenced as described above to confirm the introduction of mutations.For the production of replicating NL4-3.Ba-L clones containing the desired mutations, the mutated 6 PHA-stimulated pooled PBMC were inoculated with 100 TCID50 per virus variant for 2 hours at 37°C in a shaking water bath in a total volume of 1.5 ml. Subsequently, the inoculated PBMC were washed with 5 ml of IMDM supplemented with 10% FCS, and cultured in IMDM supplemented with 10% FCS, 20 U/ml rIL2, 5 µg/ml polybrene, 100 U/ml penicillin and 100 µg/ml streptomycin at a cell density of 1×106 per ml in a humidified 10% CO2 incubator at 37°C. On day 5, 8, 11 and 14 after inoculation, 1×106 fresh PHA-stimulated pooled PBMC in 1 ml of IMDM culture medium supplemented with 10% FCS, 20 U/ml rIL2, 5 µg/ml polybrene, 100 U/ml penicillin and 100 µg/ml streptomycin were added to the cultures. Samples for determination of p24 antigen production (75 µl) were harvested every day after inoculation. All samples were tested for p24 antigen production simultaneously at the end of the experiment using an in-house p24 ELISA To determine the viral replication rates of the different HIV-1 molecular clones and biological variants, 2×10gag region of all samples was sequenced as described above to confirm the presence of mutations. The replication rates of all viruses were tested at least in twofold and in 2 independent experiments for which the same pool of healthy donor PBMC was used. P24 values were normalized to the mean p24 values observed for the constructed NL4-3.Ba-L virus carrying mutations associated with the presence of HLA-B*57/58:01. The area under the curve for the replication curves spanning day 2−17 was calculated and normalized for the constructed NL4-3.Ba-L virus carrying mutations associated with the presence of HLA-B*57/58:01. Statistical significance of differences in the p24 production on day 11 and the area under the curve were tested with the unpaired Student's T test. In On the last day of the replication experiments, DNA from every well was isolated using the L6 method To test the correlation between the number of escape and compensatory mutations in Gag with the replication fitness of biological HIV-1 variants obtained from HLA-B*57/58:01 progressors and LTNPs, a Spearman correlation test was performed. P values <0.05 were considered significant. Statistical analyses were preformed using Graphpad Prism version 5 and SPSS version 19.Figure S1Sequence variation in Gag affects viral replication fitness. A. Replication kinetics of constructed NL4-3.Ba-L viral variants containing mutations associated with HLA-B*57/58:01 in the absence or presence of compensatory mutations. The area under the curve (day 2–17) was calculated and normalized mean AUCs were compared using the unpaired Student's T test. Statistical significance compared to the mutant virus carrying the mutations associated with the presence of HLA-B*57/58:01 are denoted in red, and significance compared to the virus carrying all mutations associated with the presence of HLA-B*57/58:01 and with disease progression is shown in blue. Statistical significance is indicated as follows: * p<0.05, ** p<0.01, *** p<0.0001. Error bars represent 2.5–97.5 percentiles. Data from one representative experiment are shown. B. Replication kinetics of constructed NL4-3.Ba-L viral variants containing mutations associated with HLA-B*57/58:01 in the absence or presence of compensatory mutations described by Brockman et al. The area under the curve (day 2–17) was calculated and normalized mean AUCs were compared using the unpaired Student's T test. Statistical significance compared to the mutant virus carrying the mutations associated with the presence of HLA-B*57/58:01 are denoted in red, and significance compared to the virus carrying all mutations associated with the presence of HLA-B*57/58:01 and with disease progression is shown in blue. Statistical significance is indicated as follows: * p<0.05, ** p<0.01, *** p<0.0001.Error bars represent 2.5 – 97.5 percentiles. Data from one representative experiment are shown.(TIF)Click here for additional data file.Table S1Amino acid sequence variation in Gag. A. Sequence variation within Gag located at amino acid positions associated with the presence of HLA-B*57/5801 or disease progression as identified with SH in sequences obtained from LTNPs from various time points during the course of HIV-1 infection. B. Sequence variation within Gag at amino acid positions associated with the presence of HLA- B*57/5801 or disease progression as identified with SH in sequences obtained from progressors from various time points during the course of HIV-1 infection.(DOC)Click here for additional data file.