GSTP1 gene in 500 normal individuals from Delhi. GSTP1 polymorphism was analysed by PCR-RFLP using amplification refractory mutation system (ARMS) assay. Two polymorphic sites in GSTP1  have been analysed simultaneously, which results in four alleles: GSTP1*A , GSTP1*B , GSTP1*C  and GSTP1*D . The GSTP1 allele frequency in Delhi population was 0.663, 0.248, 0.069, and 0.020 for GSTP1*A, GSTP1*B, GSTP1*C, and GSTP1*D respectively. The frequency of Ile105 and Val105 allele was 0.683 and 0.317 respectively and it was calculated for the purpose of comparison with published data where all the four alleles were not analysed. GSTP1 alleles from Delhi population were compared with reported frequencies from all over India, and from other ethnic groups worldwide. This study would provide a basic database for future genetic studies.Glutathione S-transferases (GSTs) belong to a super family of phase II detoxification enzymes, which play an important role in protecting cells from damage caused by endogenous and exogenous compounds by conjugating reactive intermediates with glutathione to produce less reactive water-soluble compounds. In the present study, we determined the frequencies of two polymorphisms in exon 5 and exon 6 of  Glutathione S-transferases (GSTs), found in virtually all eukaryotes, are a multigene family of phase-II metabolic enzymes, which catalyze the conjugation of reduced glutathione with a variety of endogenous and exogenous electrophilic compounds, including several potentially toxic carcinogens and chemotherapeutic drugs , therebyGSTM1, GSTT1 and GSTP1 polymorphisms are extensively studied worldwide. The GSTM1 and GSTT1 genes are located on chromosome 1p13.3, and 22q11.2 respectively. Homozygous deletions of GSTM1 and GSTT1 genes are common and result in a complete loss of enzyme activity. The frequencies of GSTM1 null alleles display race and ethnic variations, being highest in Europeans (42–60%) & Asians (41–63%) compared with that of Africans (16–36%) (GSTT1 null genotypes is somewhat less in Europeans (13.31%) compared with that of Africans (14–57%) and in Asians (35–48%)  . However(35–48%) .S-transferase P1 (GSTP1) gene spanning approximately 2.8 kb is located at 11q13 and contains seven exons  in exon 5 and in second, a C→T transition at nucleotide 341 resulting in replacement of alanine → valine at the amino acid position114  in exon 6. Hence, the human GSTP1 locus comprises of four different alleles: GSTP1*A (wild type Ile 105 → Ala114), GSTP1*B , GSTP1*C  and GSTP1*D   . GSTP1 pVal 114) . GST enzPeripheral blood from 500 normal controls was collected in vials containing EDTA after receiving their informed consent. The controls selected for the study were either normal volunteers from the Institute, or normal healthy individuals visiting with the patients in various hospitals of Delhi. Information on age, sex, smoking and alcohol habits was obtained. The ethical clearance was obtained from our Institute's ethical committee.GSTP1, using two different sets of primers amplifying exon 5 and exon 6. It included a forward Primer upstream of the codon 105 substitution (5′-ACC CCA GGG CTC TAT GGG AA-3′) and two reverse primers; primer ‘A’ (5′-TCA CAT AGT CAT CCT TGC CGG-3′) (Ala 114 specific) and primer ‘B’ (5′-TCA CAT AGT CAT CCT TGC CGA-3) . Two PCRs were performed for each DNA sample. PCRs were performed in 50 μl reaction volume containing 50–100 ng of genomic DNA, 50 mM KCl, 2.5 mM MgCl2, 200 mM Tris (pH 8.4), 200 μM of dNTP each, 1.5 units of Ampli Taq DNA polymerase , forward primer and reverse primers A or B (0.3 μM each). PCR was performed with initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, elongation at 72 °C for 2 min, and a final extension at 72 °C for 7 min. A 998 bp fragment was amplified (An ARMS (amplification refractory mutation system) assay, described by  was perfmplified a.105 (GSTP1*A or GSTP1*D) and fragments of 73, 93, 250, 260, and 322 bp with Val 105 (GSTP1*B or GSTP1*C) and six fragments of 73, 93, 250, 260, 322, and 343 bp in heterozygote individuals . The initial ARMS PCR was used to identify Ala114 or Val 114 genotypes. Therefore, amplification in individuals with Ala 114/Ala 114 was observed with reverse primer A, and with reverse primer B in individuals having Val 114/Val 114, and amplification in heterozygote for Ala 114/Val 114 was observed with both primers A and B. Therefore genotypes in various combinations  were identified that resulted from A, B, C, and D alleles . Of 500 individuals, 97 (19.4%) were smokers, 126 (26.5%) were alcoholics and 43(8.6%) were betel nut/tobacco chewers. Among the smokers, 85 (87.6%) were males and 12 (12.4%) were females.A total of 500 healthy individuals, 274 (54.8%) males: 226 , GSTP1 AB (33.8%) and GSTP1 AC (8.2%) genotypes  was observed (data not shown).The frequency distribution of GSTP1 alleles in Indian and global populations is tabulated in GSTP1*A, GSTP1*B, GSTP1*C and GSTP1*D was calculated (p < 0.001). In Egyptians, both GSTP1*C and GSTP1*D alleles were not observed. GSTP1*D was absent in two Italian studies and rare in other studies also  with a few exceptions  (GSTP1 genotypes did not achieve Hardy–Weinberg equilibrium in the clubbed data of North Indians (p < 0.001), South Indians (p < 0.002) and Central Indians (p < 0.01)  . The fre < 0.01) .GSTP1 alleles in four main world populations namely Africans, Europeans, Asians and Indians, with the present study (N = 1606) in Africans,  in Europeans,  in Asians and  in Indians. Genotype frequencies of GSTP1 Ile105Val polymorphism followed Hardy–Weinberg equilibrium in Europeans (p = 0.62), and Asians (p = 0.77) but not in Africans (p = 0.004) and Indians (p < 0.001). The frequency of Ile105 and Val105 allele was observed to be 0.683 and 0.317 respectively in the present study and the population was in Hardy–Weinberg equilibrium. Highly significant  linkage disequilibrium was observed between Ile105 and Ala114 and between Val105 and Val114.We have calculated and compared the frequency of nt study . The genGSTP1 gene differ in susceptibility to various chemical carcinogens  and African American (0.51) and least frequent among Tanzanians (0.14)  but not from Africans, Europeans and Indians. Similar results were also observed in other studies  . The ranom Japan . Observa studies .GSTP1 Val/Val genotype is uncommon and exists in 5% of Caucasians (GSTP1 enzyme has been reported to be 2–3 times less stable than the Ile105 form (GSTP1 Val/Val genotype had significantly better survival in hepatocellular carcinoma patients (GSTP1 Ile105/Val 105 genotype increase susceptibility to breast cancer in Asian population (GSTP1 Ile105/Val 105 or Ile 105/Ile105 genotype (GSTP1 Ile/Ile were reported to have the lowest risk of prostate cancer (The ucasians . This fo105 form  and poss105 form . Individpatients , but thipatients  and breapatients . In a mepulation , while agenotype . Individe cancer , and thie cancer  and basae cancer . An overe cancer . Thus GSGST alleles would also help in studying the effect of various carcinogens in different populations having various exposures and giving personalised treatment in case of cancer. Understanding the contribution of GST gene polymorphisms and their interactions with other relevant factors may improve screening diagnostic assays for various cancers. Thus, our results signify the impact of ethnicity and provide a basis for future epidemiological and clinical studies.GSTP1 associated risk is probably disease-dependent, and may reflect differences in relevant substrates. Individuals with different combinations of


Aim. To evaluate visual outcome and endothelial cell density (ECD) after Descemet's Membrane Endothelial Keratoplasty (DMEK) in comparison with Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK) in the fellow eye for Fuchs endothelial dystrophy (FED). Design. Single-centre, retrospective case series. Methods. 30 eyes of 30 patients undergoing DMEK, who completed a 1-year follow-up, were compared with 30 fellow eyes which underwent DSAEK for bilateral FED. Main outcome measures studied included Best Corrected Visual Acuity (BCVA) and ECD during a 1-year follow-up period. Results. BCVA improved from 0.78 ± 0.35 logMAR, and 0.73 ± 0.31 logMAR before surgery to 0.22 ± 0.1 logMAR and 0.35 ± 0.12 logMAR 6 months after DMEK and DSEK, respectively (P < 0.001). At one year after surgery, the BCVA was maintained at 0.21 ± 0.12 logMAR and 0.34 ± 0.1 logMAR, respectively, after DMEK and DSAEK. A statistically better visual outcome was observed after DMEK compared to DSAEK (P < 0.05) in fellow eyes. Conclusions. DMEK provided better visual rehabilitation when compared to DSAEK. Nevertheless, there were no significant differences with regard to the ECD within a 1-year follow-up. The last decade has witnessed a revolutionary shift in the treatment of corneal endothelial disease . Commenc2 were used in this study. The corneas were taken from donors aged between 40 and 65 years. Published data by Laaser and associates [The study commenced after obtaining the Institutional Ethics and Scientific Committee approval. Thirty eyes of thirty patients undergoing DMEK who completed a 1-year follow-up, were included in this study. Patients with other ocular comorbidity besides FED were not included. All eyes were pseudophakic with a posterior chamber intraocular lens implanted previously. These eyes were compared with 30 fellow eyes of the same patients who underwent DSAEK earlier. Indication for surgery was bilateral FED. All patients received a graft prepared from a corneoscleral button that had been stored in Cornisol  at 4°C (short-term cultured graft) in coordination with our eye bank. All donor corneas with ECD more than 1800 cells/mmsociates  suggestsThe EDM was stripped from the donor corneal stroma by a technique previously described by Kruse et al. , which iμ head. The stromal side of the corneal lenticule was marked with 3 spots of different sizes to ensure correct orientation in the anterior chamber of the host. After marking the epithelium with 8 mm marker, the patient's EDM was removed under air using an inverted hook (reverse Sinskey's hook) inside the 8 mm marking. The graft was delivered to the patient's eye using the standardized pull-through technique by Busin et al. [The preparation of donor corneal lenticules was performed using the ML7  microkeratome with a 400 n et al. . The graAll patients were started postoperatively on a tapering dose of prednisolone acetate 1% eye drops  over a period of 2 months and Vigamox eye drops  4 times a day for a month. After completion of 2 months, patients were started on Dexoren –S eye drops  4 times a day for the first month and then reduced to 2 times a day. Patients were on a maintenance dose of Lotepred eye drops  twice a day for next 6 months and once a day thereafter. The patients were followed up at 1 and 15 days after surgery and at 1, 3, 6, and 12 months postoperatively. In the eye bank, donor ECD and viability were evaluated in vitro with an inverted light microscope . Postoperatively the endothelium was photographed and evaluated in vivo using a Topcon SP3000p noncontact autofocus specular microscope  at 3, 6, and 12 months. Images of the central corneal window were analyzed and manually corrected and three measurements of ECD were averaged.P < 0.001) at 6 months after surgery. In eyes that underwent DSAEK, the BCVA improved from preoperative values of 0.73 ± 0.31 logMAR to 0.38 ± 0.22 logMAR at 3 months and to 0.35 ± 0.12 logMAR (P < 0.001) at 6 months after surgery (P < 0.05) (P = 0.2) in the two groups. Three eyes in the DMEK group and 3 eyes in the DSAEK group had preexisting cystoid macular edema (CME) which was contributory to the low postoperative visual acuity. Eyes with CME showed a modest improvement in visual acuity after surgery, from preoperative values of 0.02 ± 0.02 logMAR and 0.03 ± 0.12 logMAR, respectively, in the DSAEK and DMEK groups to postoperative values of 0.08 ± 0.12 logMAR and 0.09 ± 0.12 logMAR units. All eyes were pseudophakic with a posterior chamber intraocular lens implanted previously. ECD of donor corneas in DMEK group was 2378 ± 172 cells/mm2 and decreased to 1775 ± 121 cells/mm2 at 6 months after surgery (P < 0.001). ECD of donor corneas in DSAEK group was 2248 ± 142 cells/mm2 and decreased to 1806 ± 141 cells/mm2 at 6 months after surgery (P < 0.001)  in the endothelial cell loss observed between the two groups. The mean preoperative central corneal thickness (CCT) was 552.3 ± 34.1 μ and 561.24 ± 25.1 μ in the DMEK and DSAEK groups, respectively (P = 0.07). The mean thickness of the lenticule implanted in DMEK and DSAEK groups was, respectively, 11.18 ± 1.4 μ and 91.1 ± 10.1 μ. The mean postoperative central corneal graft thickness as measured by AS-OCT was 10.12 ± 1.2 μ and 81.11 ± 11.2 μ in the DMEK and DSAEK groups, respectively, at 6 months. The mean postoperative CCT observed in the DSAEK group of 621.19 ± 44.1 μ was significantly higher than in the DMEK group of 580.12 ± 14.1 μ (P < 0.001) All patients  with mean age of 55.12 ± 9.2 years (range 44–71 years) completed a 1-year follow-up following DMEK and DSAEK. Mean time interval between DSAEK and DMEK surgeries in fellow eyes was 12.1 ± 3.5 months (range 8–14 months). In our study, in eyes that underwent DMEK, the BCVA improved from preoperative values of 0.78 ± 0.35 logMAR to 0.32 ± 0.11 logMAR at 3 months and to 0.22 ± 0.1 logMAR  . 24 eyes< 0.001) , Table 3After DMEK, 3 eyes (10%) showed partial dehiscence of the DM and required air injections in the early postoperative period. After DSAEK, none of the eyes required repeat air injections. In our study, there was loss of two donor corneas while stripping the DM in the DMEK group. No other complications like graft rejection were noted during the follow-up period.μm showed a statistically significant improvement in Best Spectacle Corrected Visual Acuity (BSCVA) compared to thicker grafts. Another data described by Busin, who introduced the ultra-thin DSAEK (UT-DSAEK) with a graft thickness of 73 ± 14 μm, reported better functional outcome when compared to conventional DSAEK. Thus, DMEK, where the donor graft does not have a posterior lamella, is thought to provide a better functional outcome.When DSEK was first introduced by F. W. Price Jr. and M. O. Price in 2005, it was observed that the technique maintained the structural integrity of the cornea and also provided rapid visual recovery for the patients . SubsequIn this study, eyes with DMEK were found to have a better visual rehabilitation when compared to the fellow eyes which underwent DSAEK similar to that observed by Goldich et al. . In our An advantage of DMEK is that the two procedures DMEK and DALK (Deep Anterior Lamellar Keratoplasty) can be combined, wherein a single cornea can be used twice. This procedure is also known as split cornea transplantation. This therefore reduces the need for corneal tissue and thereby and limits shortage of grafts. The cost and time involved in procuring the corneal tissue are also reduced. Data published by Heindl and associates , 44 sugg


Hepatitis C virus (HCV) genotype and subtype are related to disease progression and response to antiviral therapy. Current HCV genotype and subtype distribution data, especially for genotypes 3 and 6, are limited in China. Our purpose was to investigate the current HCV genotype and subtype distributions in chronic hepatitis C patients in China.n = 1012) were enrolled, and demographic information and possible transmission risk factors were collected. Serum samples were subjected to reverse-transcription polymerase chain reaction, followed by direct DNA sequencing and phylogenetic analysis of the NS5B and core/E1 regions to determine HCV genotypes/subtypes. The geographical distributions of HCV genotypes/subtypes were analyzed. Demographic information and transmission risk factors were compared between different HCV genotypes/subtypes.Chronic hepatitis C patients . More genotype 1 and 2 patients underwent blood transfusion than those with genotype 3 . More genotype 3 and 6 patients had a history of intravenous drug use than those with genotypes 1 and 2 .Four genotypes and seven subtypes of HCV were detected in 970 patients. Subtypes 1b, 2a, 3a, 6a, 3b, 6n, and 1a were detected at frequencies of 71.96 %, 19.90 %, 3.20 %, 2.16 %, 1.96 %, 0.41 %, and 0.41 %, respectively. Genotypes 3 and 6 showed an increasingly wide geographic distribution over time. Patients with subtypes 1b and 2a were older than those with 3a, 3b, 6a, and 6n subtypes  contains supplementary material, which is available to authorized users. Hepatitis C virus (HCV) is a leading cause of chronic liver disease and presents a major threat to global public health. Worldwide, more than 185 million people have been infected [HCV can be classified into seven genotypes and at least 67 confirmed subtypes, 20 provisionally assigned subtypes, and 21 unassigned subtypes , 4.In China, the estimated prevalence of HCV is 0.43 % . A previOverall, 1012 chronic hepatitis C patients were enrolled from 23 centers. Patients were located in 24 administrative units from six different geographic regions throughout China. Table Reliable nucleotide sequences of the NS5B and core/E1 region were obtained for 970 samples and used to construct phylogenetic trees. The phylogenetic analysis of both the NS5B and core/E1 regions revealed the presence of genotypes 1, 2, 3, and 6, while no genotype 4, 5, or 7 strains were found in this study  patients. The next most abundant genotypes were 2, 3, and 6, found in 193 (19.90 %), 50 (5.15 %), and 25 (2.58 %) patients, respectively. Evaluations of subtype revealed that the most common was 1b, accounting for 71.96 % (698/970) of cases, followed by 2a . Subtypes 3a, 6a, and 3b were identified in 31 (3.20 %), 21 (2.16 %), and 19 (1.96 %) patients, respectively. The least frequent subtypes were 1a and 6n, with each accounting for 4 (0.41 %) specimens.In phylogenetic analyses of the NS5B and core/E1 regions, the sequences of each subtype were randomly distributed with other corresponding reference sequences derived from the Los Alamos HCV database. No clear geographical clustering of Chinese isolates in this study was detected , southern and western (subtype 5), and northeastern (subtype 4) China. Only 2 subtypes were detected in central China.p < 0.05 in each for all pairwise comparisons of 1b and 2a with 3a, 3b, 6a, and 6n). There were no differences in the mean ages between subtypes 1b and 2a . There was also no difference in the mean ages between patients with subtypes 3a, 3b, 6a, and 6n .Age differences were observed for some HCV subtypes. Patients infected with HCV subtype 1b (49.8 ± 15.0 years) and 2a (51.2 ± 12.9 years) were older than those infected with 3a (36.1 ± 9.8 years), 3b (37.0 ± 9.2 years), 6a (38.2 ± 12.1 years), and 6n  and 2 (p = 0.011) patients underwent blood transfusion compared to patients with genotype 3 infections. More genotype 1 patients underwent surgery than those with genotype 3 (p = 0.003). More genotype 2 patients underwent invasive procedures than those with genotype 1 (p = 0.015). Invasive procedures were performed more frequently in patients with genotype 2 and 3 infections compared to genotype 6 . The history of IDU was more prevalent in genotype 3 and 6 patients than in patients with genotypes 1 or 2 .A comparison of patients’ risk factors for HCV infection and the detected HCV subtypes are shown in Table n = 13) and 2a (n = 3), although these patients were all from different families. The mean age was 50.1 ± 10.9  years and 37.50 % (6/16) of the patients were female. They were from all geographic regions, except Southern China.Family clustering of HCV was reported in 16 patients with subtypes 1b  received blood before 1992; in this period, there was no screening of donated blood and blood products for HCV. These results indicate that subtypes 1b and 2a are more related to blood transfusion than genotype 3, and also partially explain why subtype 1b and 2a patients are generally older than genotype 3 patients when first diagnosed. Previous studies from other countries and regions reported that genotypes 6 and 3 are more frequent among IDU patients than in the general population –19. Our This study reveals the current HCV genotype and subtype distribution among Chinese chronic hepatitis C patients. At least four genotypes and seven subtypes were prevalent nationally. The distribution of HCV genotypes vary by geographic region, while subtypes 1b and 2a remain the most prevalent in China. Genotype 3 and 6 HCV infections are no longer restricted to southern and western China, but are now nationwide, and this altered distribution appears closely related to IDU.From June 2011 to March 2012, treatment-naive chronic hepatitis C patients were randomly enrolled from 23 hospitals in China. HCV infection had been confirmed (anti-HCV antibody and HCV RNA-positive) within 90 days prior to enrollment. Patients who had received anti-HCV therapy were excluded; no other exclusion criteria were applied. Patients were from 24 administrative units, which covered northeast China , northern China , central China , eastern China , southern China (Guangdong Province), and western China . Demographic information  and transmission risk factors , or intravenous drug use (IDU), and family clustering of HCV infections) were collected from each patient with a face-to-face interview based on a questionnaire. Serum specimens were collected and sent to the central laboratory of the Beijing Ditan Hospital for study.This study was approved by the ethics committee of Beijing Ditan Hospital of Capital Medical University. Written informed consent was obtained from all subjects when enrolled.HCV RNA was extracted from 140 μL of each serum sample using the QIAmp Viral RNA Mini kit , followed by one-step RT-PCR assays with the One Step RNA PCR kit . Outer sense and antisense primers derived from the NS5B and CORE/E1 region of the HCV genome were used for RT-PCR as described previously . Nested The sequences of the core/E1 and NS5B regions were aligned and edited with reference sequences of each subtype using the CLUSTALW multiple alignment program in BioEdit software . HCV subt-test was used to compare the mean ages between patients infected with different HCV subtypes. The Pearson Chi-Square test was used to compare the ratio of risk factors between different genotypes. p values < 0.05 were considered statistically significant.Statistical data analyses were performed with SPSS 13.0. The continuous data were expressed as the mean ± standard deviation (SD) or as otherwise stated, and categorical variables in absolute numbers and percentages. The student’s This study was approved by the Ethics Committee of Beijing Ditan Hospital, Capital Medical University.


Titanates, for example, Li4Ti5O12, intercalate lithium at∼1.6 V versus Li+/Li, avoiding problematic lithium plating at the expense of reduced cell voltage. There is interest in 1 V anodes, as this voltage is sufficiently high to avoid lithium plating while not significantly reducing cell potential. The sulfides, LiVS2 and LiTiS2, have been investigated as possible 1 V intercalation electrodes but suffer from capacity fading, large 1st cycle irreversible capacity or polarization. Here we report that the 50/50 solid solution, Lix1+(V0.5Ti0.5)S2, delivers a reversible capacity to store charge of 220 mAhg−1 (at 0.9 V), 99% of theoretical, at a rate of C/2, retaining 205 mAhg−1 at C-rate . Rate capability is excellent with 200 mAhg−1 at 3C. C-rate is discharge in 1 h. Polarization is low, 100 mV at C/2. To the best of our knowledge, the properties/performances of Li(V0.5Ti0.5)S2 exceed all previous 1 V electrodes.Graphite, the dominant anode in rechargeable lithium batteries, operates at ∼0.1 V versus Li Lithium sulfides have been previously investigated as 1 V anodes for Li-ion batteries, but suffered from significant performance issues. Here, the authors report on a 1 V lithium sulfide electrode with noteworthy performance, demonstrating that sulfide-based electrodes may merit further exploration. Li intercalation into LiTiS2 occurs at a lower voltage of 0.5 V, and exhibits very poor capacity retention on cycling, the capacity reducing to only 120 mAhg−1 after just five cycles. LiTiS2 also exhibits a massive 1st cycle irreversible loss of capacity of over 180 mAhg−1 (ref. x1-Tix)S2 and find that the 50/50 composition Li(V0.5Ti0.5)S2 delivers a significant improvement in properties/performance over other compositions, including the end members. A total of 50% replacement of V by Ti is sufficient to lower the potential (by lowering the Fermi level in the d-states) even on deintercalation (charge) below 1 V (0.9 V), but not to the extent of LiTiS2 (where the consequence is massive irreversible capacity loss). The irreversible loss of capacity for Li(V0.5Ti0.5)S2 on the 1st cycle is only 42 mAhg−1 at C/2 (compared with 180 mAhg−1 for LiTiS2). Furthermore, the polarization on the first cycle is half that of LiVS2 at the same rate. Li(V0.5Ti0.5)S2 exhibits a reversible capacity of 220 mAhg−1 at C/2  dropping only marginally to 200 mAhg−1 at 3C. The point of the paper is not to claim that the Li(V0.5Ti0.5)S2 material reported here is a commercially viable anode, with all practical problems solved. Rather it is to show that LiMS2 based electrodes are not limited to the relatively poor performance  reported previously and that sulfide based electrodes may merit further exploration in the future.However, previous work on LiVSx1-Tix)S2 solid solution were synthesized as described in the Methods section, where the electrode fabrication and characterization methods are also described. The powder X-ray diffraction patterns are shown in 2 structure, composed of hexagonal close packed S2− with alternate sheets of octahedral sites occupied by Li and M cations, 0.5Ti0.5)S2 and the blend of 50 wt% LiVS2 and 50 wt% LiTiS2 is shown in +-occupying tetrahedral sites that share faces with Li in octahedral sites, and is therefore energetically unfavourable. Instead, upon intercalation, all the Li+ ions in the octahedral sites in LiMS2 are displaced to the neighboring tetrahedral sites in the alkali metal layers, as there are twice as many tetrahedral as octahedral sites the intercalating Li can be incorporated up to a theoretical maximum of Li2MS2 (ref. The range of compositions across the Li(VMS2 ref. .0.5Ti0.5)S2 was found to be the best performing of all the Li(Vx1-Tix)S2 materials. Materials with more vanadium displayed increased polarization and deintercalation takes place above 1 V, comparisons with Li(V0.6Ti0.4)S2 and LiVS2 are presented in 0.4Ti0.6)S2 and LiTiS2 data in Li(V0.5Ti0.5)S2 are shown in −1 (2nd cycle) with the plateau on deintercalation  being located at 0.9 V. The polarization (separation between discharge and charge plateaus) is small, ∼100 mV (rate=C/2). The load curves are invariant on subsequent cycling, save for a slow reduction in capacity. Concerning the first cycle, there is an initial capacity of 20 mAhg−1 at ∼2.1 V, associated with the M4+/3+ redox couple and consistent with a small degree of lithium deficiency in the as-prepared material, Li0.92(V0.5Ti0.5)S2, see refinement of structure, −1on cycle 1, 2 and LiTiS2 (ref. 0.5Ti0.5) S2.The charge–discharge curves for LiS2 solid solution compared with the stoichiometric LiVS2 and LiTiS2 end members, it is important to ascertain if lithium intercalation into Li(V0.5Ti0.5)S2 operates by the same mechanism. The results below show that the improved properties of Li(V0.5Ti0.5)S2, are obtained despite a similar two-phase intercalation process and all three materials forming a SEI layer due to electrolyte degradation. Considering the structure of Li(V0.5Ti0.5)S2 and its evolution on intercalation/deintercalation and cycling, joint refinements were carried out on the as-prepared material using the powder X-ray diffraction data in 0.92(V0.5Ti0.5)S2 is in good agreement with the capacity associated with the intercalation at 2.1 V (0.08 Li corresponds to 18 mAhg−1). There is no evidence of site exchange between Li and V/Ti, that is, the structure remains layered, and no evidence of V/Ti long-range order. The V/Ti ratio refined to 1:1 within errors. The variation of structure with Li insertion/removal is shown in 0.92(V0.5Ti0.5)S2, accounting for the first 18–20 mAhg−1  Å and c=6.1582(2) Å to a=3.4377(4) Å and c=6.1591(2) Å. There is no change in the diffraction patterns between points B and C. Thereafter, Li intercalation is a 2-phase process, in accord with the plateau in 0.5Ti0.5)S2 is replaced continuously by Li2(V0.5Ti0.5)S2. On lithium extraction the 2-phase process is reversed, associated with the plateau on charge, as seen at point I in 0.5Ti0.5)S2 and Li2(V0.5Ti0.5)S2 and the patterns at A, B, C, H and J by single phases of the two end members. A cyclic voltammogram collected on a 3-electrode cell with the Li(V0.5Ti0.5)S2 as the working electrode is shown in 0.5Ti0.5)S2 is essentially similar to that of LiVS2 (ref. Given the change in properties/performance exhibited by the Li(Va and c lattice parameters for Li1(V0.5Ti0.5)S2 and Li2(V0.5Ti0.5)S2 are respectively a=3.4377(4) Å, c=6.1591(2) Å and a=3.7894(7) Å, c=6.229(2) Å. The volume expansion on intercalation is 23% yet the phase transformation is facile. The expansion along a is associated with the elongation of the (Ti/V)-S bond as formally (Ti/V)3+ is reduced to (Ti/V)2+. As the d-bands in sulfides are relatively wide compared with oxides, the electrons are well delocalized across the (Ti/V)-S2 slabs. The c-axis expansion requires energy to increase the separation of the slabs. The small c-axis expansion, only 1.5%, may be responsible for the facile nature of the 2-phase intercalation reaction that exhibits a small polarization and excellent reversibility. Powder X-ray diffraction (PXRD) data collected on cycling, 0.5Ti0.5)S2 is 20 μm and remains so on cycling. All of these results indicate that the material is stable on cycling.The refined 2 and LiTiS2, where it was inferred only from the electrochemistry−1 of irreversible capacity on the first cycle for Li(V0.5Ti0.5)S2, −1 of capacity between B and C may be assigned to electrolyte reduction, as there is no change in the lattice parameters. Beyond C, the Rietveld refinements of the 2-phase mixtures of Li(V0.5Ti0.5)S2 and Li2(V0.5Ti0.5)S2 along the plateau provide the ratios of the two phases, which in turn gives the amount of lithium and hence charge that has been inserted into the structure. These values are presented in −1 at the beginning of discharge associated with the M4+/3+ redox couple. There is almost no difference in these values across the plateau, indicating that no significant electrolyte reduction occurs between C and G in 0.5Ti0.5)S2 (222 mAhg−1) and the observed capacity on the 2nd cycle, Electrolyte reduction and SEI layer formation have been reported for LiVS0.5Ti0.5)S2 per unit area as in −1 loss observed between those voltages for the electrode containing Super S is associated with electrolyte reduction on the carbon surface. The capacity between 0.93 V and point C, 8 mAhg−1, could be due to reduction of the electrolyte on the surface of Li(V0.5Ti0.5)S2 as it is present in the electrode with and without carbon. It might also arise from a very small amount of intercalation, corresponding to x=0.03, too small to be seen in the X-ray data. The irreversible capacity loss occurring at the lower voltage beyond the plateau, <0.7 V, appears also due to a combination of carbon and Li(V0.5Ti0.5)S2, since it increases when super S is present. Overall, −1 arises from electrolyte reduction on the Li(V0.5Ti0.5)S2 surface that is, 11% of the reversible capacity, the rest is due to reduction on the surface of the super S.It is important to recall that the composite electrodes contain 10% super S carbon, and this will account for some of the observed electrolyte reduction (irreversible capacity). Cells were constructed with the same loading of Li(V0.5Ti0.5)S2 itself, and to reduce significantly the capacity fading on cycling.To explore the SEI layer formation as a function of cycling, alternating current (AC) impedance spectroscopy and SEM (scanning electron microscopy) images were collected from cycles 1 to 100, 0.5Ti0.5)S2 was incorporated as the anode in a full lithium-ion cell with a LiCoO2 cathode. The cell was constructed such that the overall capacity was anode limited, in order to show the performance of the latter, see Methods section. First charge capacity, corresponding to insertion of lithium into Li(V0.5Ti0.5)S2, exhibits a larger irreversible capacity than was observed when the counter electrode was lithium metal (∼75 mAhg−1 as opposed to 42 mAhg−1), see −1 compared with 278 mAhg−1 for the cell with a Li counter electrode, the difference is the same as the additional irreversible capacity (75–42 mAhg−1), consistent with the additional irreversible capacity in −1, just below the theoretical capacity; this reduces to just over 180 mAhg−1 by the 10th cycle, see LiS2. We suggest that the relatively slow rate of reaction in air is likely due to the formation of a thin oxide layer that slows decomposition.Sulfides are used in a variety of applications ranging from pigments270.5Ti0.5)S2, is reported that operates at 0.9 V versus Li+/Li 2 (the voltage of which is >1 V versus Li+/Li) and LiTiS2 (which exhibits a very large irreversible capacity loss on the 1st cycle of 180 mAhg−1). Electrolyte reduction on Li(V0.5Ti0.5)S2 during the first cycle accounts for only 11% of the first cycle capacity. Performance is better than previously reported 1 V anodes, to the best of our knowledge. Future work should focus on reducing the 1st cycle irreversible capacity while forming a robust SEI layer minimizing further electrolyte reduction, by examining lowering the amount of conductive additive in the electrode and investigating electrolyte additives or seeking alternative electrolytes to those optimized for graphite anodes.An intercalation anode, Li(V0.5Ti0.5)S2 was synthesized from Li2S , Ti powder , V powder  and S powder . Appropriate ratios of starting materials were mixed and ground together in an Ar filled MBraun glovebox. The mixture was then placed in a graphite crucible, which was in turn placed in a quartz tube. The quartz tube was subsequently sealed under vacuum. The reactants were heated at 1,050 °C for 72 h before being quenched to room temperature.LiS2 composite electrode, a lithium metal counter electrode and a glass microfiber GF/F separator (WhatmanTM) saturated with electrolyte, a 1 M solution of LiPF6 in ethylene carbonate–dimethyl carbonate 1:1 ((v/v) (BASF)), were constructed. Li-ion cells were constructed similarly with composite LiCoO2 electrodes  replacing the lithium metal counter. Celgard monolayer polypropylene separator was used in addition to the glass microfiber separator. All handling was carried out in an Ar filled MBraun glovebox. Electrochemical measurements were conducted using a Maccor series 4,200 battery tester. Cyclic voltammetry and AC impedance were conducted on 3-electrode cells using a VMP3 electrochemical workstation (Biologic).Composite electrodes were fabricated using the active material, super S carbon and Kynar Flex 2801 (a co-polymer based on polyvinylidene difluoride) binder in a mass ratio of 80:10:10. The mixture was then cast on copper foil using tetrahydrofuran as the solvent. The loading of active material was ∼6–8 mg cmα1 radiation operating in transmission mode with the samples sealed in 0.2 mm capillaries, expect for the experiments on air sensitivity, which were carried out in air. Time-of-flight powder neutron diffraction data were collected on the POLARIS high-intensity; medium resolution instrument at ISIS, Rutherford Appleton Laboratory (UK) with samples sealed in 2 mm quartz capillaries. The structures were refined by the Rietveld method using TOPAS AcademicPowder X-ray diffraction patterns were obtained using a Stoe STADI/P diffractometer employing CuKHow to cite this article: Clark, S. J. et al. Li(V0.5Ti0.5)S2 as a 1 V lithium intercalation electrode. Nat. Commun. 7:10898 doi: 10.1038/ncomms10898 (2016).Supplementary Figures 1-8 and Supplementary Table.


Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype.Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis plants were treated with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate 1-ethyl-3-methylimidazolium acetate ([C2C1im][OAc]) at 10 % wt biomass loading at either 70 °C for 5 h or 140 °C for 3 h. After pretreatment at 140 °C and subsequent saccharification, the relative peak sugar recovery of ~26.7 g sugar per 100 g biomass was not statistically different for the wild type than the peak recovery of ~25.8 g sugar per 100 g biomass for the engineered plants (84 versus 86 % glucose from the starting biomass). Reducing the pretreatment temperature to 70 °C for 5 h resulted in a significant reduction in the peak sugar recovery obtained from the wild type to 16.2 g sugar per 100 g biomass, whereas the engineered lines with reduced lignin content exhibit a higher peak sugar recovery of 27.3 g sugar per 100 g biomass and 79 % glucose recoveries.Engineered Arabidopsis lines generate high sugar yields after pretreatment at 70 °C for 5 h and subsequent saccharification, while the wild type exhibits a reduced sugar yield relative to those obtained after pretreatment at 140 °C. Our results demonstrate that employing cell wall engineering efforts to decrease the recalcitrance of lignocellulosic biomass has the potential to drastically reduce the energy required for effective pretreatment.The engineered The online version of this article (doi:10.1186/s13068-015-0275-2) contains supplementary material, which is available to authorized users. The Arabidopsis was stored at 4 °C in a cold room before use. Arabidopsis was pretreated with [C2C1im][OAc] at both 70 °C for 5 h and 140 °C for 3 h using a previously published protocol [2C1im][OAc] was 10 % (w/w) with 2 g of starting biomass for each replicate.1-ethyl-3-methylimidazolium acetate, [C2C1im][OAc] and water) phases. The recovered solid was lyophilized (Labconco FreeZone(12), Kansas City, MO, USA) and used for analysis.After pretreatment, the samples were thoroughly mixed, and hot water as an anti-solvent was added at 3.5 times the initial total mass (of both biomass and IL) to recover any solubilized biomass. The mixture of IL, water, and biomass was centrifuged to separate the solid (biomass) and liquid  of t Tables , and 4, Arabidopsis samples was determined using the two-step acid hydrolysis procedure of the National Renewable Energy Laboratory (NREL) [OAc] to wet each sample (about 150 μL) and a thermocouple. The slide was placed in a temperature controlled  in-house heater . Samples were started at room temperature and ramped to the specified temperature during imaging to using the high heat setting. Samples, on average, reached the specified temperature (70 or 140 °C) before 30 min and fluctuated ± 5 °C. Autofluorescent images during heating were collected with a Zeiss LSM 710 confocal system mounted on a Zeiss inverted microscope . Images were collected every 20 to 30 min, and select images are shown . Significance is indicated with the following:


The quality of dietary fats is associated with risk of cardiovascular diseases (CVD). We aimed to investigate the association between fatty acids in erythrocyte membrane phospholipids and CVD risk factors in middle-aged Korean adults.Fifty-five middle-aged adults who underwent health examinations were included in this retrospective and cross-sectional study. Anthropometry, serum lipids, clinical parameters, and erythrocyte membrane phospholipid fatty acid data were obtained from a registry.p = 0.042, p = 0.021, or p = 0.008 respectively) compared to low quartile groups. While C16:1n7 and/or C18:1n9 were positively associated with CVD risk factors, the delta 9 desaturase activity index (D9D) (C18:1n9/C18:0) was only significantly increased in high quartile groups for TG and blood pressures . Conversely, TG and blood pressures showed inverse relationships with C20:4n6 or D5D (C20:4n6/C20:3n6). C18:3n6 and/or D6D (C18:3n6/C18:2n6) were positively associated with insulin resistance and diabetic parameters. Particularly high D6D was detected in high quartile groups of FBS and insulin (p = 0.016 and p = 0.042). In linear regression analysis, D9D and/or C14:0 + C16:0 were significant contributors to serum TG and blood pressures. D6D was a contributing factor to FBS.The proportion of C14:0 and C16:0 was greatly elevated in high quartile groups for triglyceride (TG) and systolic and diastolic blood pressure groups (SBP and DBP) ).The online version of this article (doi:10.1186/s12944-015-0135-x) contains supplementary material, which is available to authorized users. Cardiovascular disorders (CVD) is especially prevalent in developing countries, including Korea, due to rapid dietary changes and Western style diets. It is well known that the quality of dietary fats, such as saturated fats, determines the risk of developing CVD . For insThe type of fats in diets affects the composition of fatty acids in human bodies, including in blood . Many stIn many cases, either plasma or serum was used to investigate the composition of fatty acids relationships with CVD –9. Fattyp = 0.014 and p = 0.007, respectively)  are shown in Table The levels of cardiovascular risk factors were investigated for their correlations with the proportions of fatty acids in erythrocyte membrane phospholipids Table . The patTo examine the differences in levels of cardiovascular risk factors among the quantile subgroups of D9D, D5D, D6D, and the sum of C14:0 and C16:0 (C14:0 + C16:0) percentages, fatty acid indices were rank-transformed to generate subgroups as quantile groups. Significant differences among the subgroups of fatty acid indices were found in FBS, insulin, TG, SBP, and DBP Fig. . As concThe relationship between fatty acid indices and FBS, insulin, TG, and blood pressures were examined after adjusting for sex, age, and BMI Table . After ar = 0.437, p = 0.001). Dairy product intake showed a negative association with C20:3n6 and C22:4n6 . The food group for tofu and eggs was positively associated with C20:5n3 . Other food groups, including meat, fish, shellfish, fruit, and rice, did not show any significant associations in the study subjects.There was a positive association between C16:1n7 and the vegetable and seaweed group  often used as markers of fish intake [Based on the criteria for metabolic syndrome, 24.6 % of study subjects were centrally obese, and 22.4 % were obese according to BMI criteria (reference). BMI showed a positive association with C14:0. Waist circumference, another marker for abdominal obesity, was positively associated with C14:0 and C16:0 but negatively correlated with C18:2n6. These findings might indicate high dietary intake of SFAs and low intake of C18:2n6 in the subjects because C18:2n6 is not produced in the body. These results are similar to previous findings suggesting a positive association of obesity-related markers (BMI and waist circumference) with SFAs such as C16:0 and a negative association with C18:2n6 . TherefoAlterations in serum lipid profiles are known risk factors for CVD. Of study subjects, 27 % had hypertriglyceridemia and 21 % had low HDL-C. AI and serum TG levels showed a positive relation with SFAs C14:0 and C16:0 whereas HDL-C was negatively associated with these markers. High saturated and/or animal fat diets increase serum TG levels . BecauseWe also noticed that serum TG had strong positive associations with C16:1n7 and C18:1n9. D9D catalyzes the production of C16:1n7 and C18:1n9 from C16:0 and C18:0, respectively . OverexpOther cardiovascular risk factors, including ApoB, blood pressures, HOMA-IR, and homocysteine, were also positively correlated with C18:1n9. Inconsistent with our findings, an increased incidence of left ventricular hypertrophy and cardiovascular risk factors were shown to be strongly associated with increased SFA and C18:1n9 in serum cholesterol esters .Interestingly, high blood pressures are significantly associated with high D9D and low C24:1n9. Changes in D9D were closely associated with blood TG, which might indirectly influence blood pressures. C24:1n9 levels in erythrocyte membrane fatty acids were highly positively associated with dietary PUFA intake along with C18:3n3 and C18:2n6 . InverseC18:1n9 was positively associated with both serum TG and blood pressures, but C20:1n9 and C24:1n9 had negative associations with these risk factors. These findings suggest that monounsaturation in long chain fatty acids with fewer than 20 carbon atoms (<20 carbon-LCFAs) was likely associated with high TG and high blood pressure while monounsaturation in >20 carbon-LCFAs was inversely related to serum TG and blood pressures.C20:4n6 and C20:5n3 are produced by series action of D6D, elongase and D5D , 22. A hDiabetes-associated risk factors, including FBS, insulin, and HOMA-IR were strongly associated with C14:0 and C18:3n6, a product generated by D6D. D6D also showed positive correlations with FBS, HbA1c, and HOMA-IR. The highest quartile of D6D had the highest levels of FBS. In linear regression analysis, adjusted D6D remained significant for FBS, indicating that D6D might contribute to FBS. Our findings are in line with previous reports showing that insulin resistance was associated with an increase in D6D and C18:3n6 , 26. In Here, we provided evidence that the fatty acid patterns of erythrocyte membrane phospholipids are strongly associated with CVD risk factors in middle aged Koreans. In particular, high serum TG is associated with high D9D and C14:0 + C16:0. FBS was positively associated with D6D. SBP and DBP showed strong positive associations with D9D. Because our study was a retrospective cross-sectional study, we could not infer the cause and effect relationship between fatty acid indices and CVD risk factors. Specific associations found in our study warrant further large scale studies involving a greater number of subjects.All the data from patients were retrieved from a registry upon approval by Chung-Ang University Institutional Review Board (IRB) (IRB number C2014199 (1396)). Patients who underwent a specialized health examination from January 2014 until January 2015 at Chung-Ang University Hospital Examination Centre were initially assessed for eligibility. Inclusion criteria were age between 40 years and 65 years, anthropometry measurements, blood biochemistry, erythrocyte membrane fatty acid composition, and dietary intake assessment. From 62 participants in total, five patients were excluded due to missing measurement data. Chung-Ang University IRB approved all procedures for this research.A body composition analyzer  was used to measure height (cm), weight (kg), body fat percentage (%), and waist circumference (cm). Body mass index (BMI) was measured using the obtained height and weight. Blood pressure was measured from the arms using an automatic blood pressure monitor  after patients had rested for 10 minutes and a steady heart rate was achieved. The values were averaged from two readings of systolic and diastolic blood pressures.Blood tests were carried out after 10 hours of fasting. Three ml of blood obtained from the forearm vein  was collected in a vacuum blood collection bottle. Blood was treated with 3 % EDTA and centrifuged at 4 °C, 3000 rpm for 15 minutes to separate the plasma. Separated plasma was stored at −20 °C until analysis. Blood levels of fasting blood glucose (FBS), HbA1c, total cholesterol , HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), homocysteine, and apolipoprotein B (ApoB) were assessed using an automatic chemistry analyzer . Hypersensitive C reactive peptide (hs-CRP) was measured using COBAS INTEGRA 700  with the particle enhanced immunoturbidometric method. Artherogenic index (AI) was calculated using the formula ‘/HDL-C’ .After centrifugation of blood in an EDTA-treated tube at 4 °C, 3000 rpm for 15 minutes, red blood cells were separated from plasma. Red blood cells were washed three times using phosphate buffered saline (PBS pH7.2). The total volume of red blood cells was doubled by adding PBS to the RBC suspension, and then samples were stored frozen at −20 °C until analysis. As an internal standard to measure lipid weight, 100 μg of heptadecanoic acid (17:0) was added to 200 ml of plasma. The extraction of total lipids from RBC and plasma was carried out using ice-cold chloroform/methanol  containing 0.01 % (w/v) butylated hydroxytoluene (BHT) by the Folch method . The LepTo assess diet quality, a modified version of the mini dietary assessment index for Korean adults (K-MDA) developed by Kim et al.  was usedp <0.05 was considered statistically significant.All data were statistically analyzed using SPSS Version 21.0 . The values were expressed as mean and standard deviation (SD). To compare men and women, the study variables were examined by independent t-test if variables demonstrated normal distribution or by Mann–Whitney U test if variables were not normally distributed. Normal distribution was assessed by the Shapiro-Wilk test. Relationships among BMI, blood pressures, blood chemistry, and proportions of fatty acids were tested using Spearman’s rank correlations. To evaluate the relationships between fatty acid indices and cardiovascular risk factors, D9D, D5D, D6D, and C14:0 + C16:0 values were rank-transformed and classified into categorical variables: Q1 (below 25 % quantile), Q2 (between 25 and 50 %), Q3 (between 50 and 75 %), and Q4 (above 75 % quantile). Differences in the values of cardiovascular risk factors among categorical groups were evaluated using a nonparametric Kruskal-Wallis test followed by a nonparametric two-tailed Mann–Whitney test. Univariate relationships between cardiovascular risk factors and fatty acid indices, including D9D, D5D, D6D, M/S ratio, and C14:0 + C16:0 were tested using simple linear regression analysis adjusted for sex, age, and BMI. In these analyses, D9D, D5D, and D6D were logarithmically transformed. A value of


The coronin family of actin-binding proteins regulate actin branching by inhibiting Arp2/3. We recently reported 2 interactions that were unique to coronin-1C: binding of a Rac1 inhibitor, RCC2, to the unique linker region and Rac1 itself to the propeller domain in a manner that differs from that proposed for other coronins. Through these interactions coronin-1C redistributes Rac1 from the back of the cell to the leading edge for either activation or sequestration by the associated Rac1-inhibitor, RCC2. Here we investigate the relationship between the Rac1- and actin-binding properties of coronin-1C and find that, although actin appears to be involved in the retrafficking of Rac1, signaling by Rac1 lies upstream of the stress fiber-formation, for which the coronins were originally characterized. ECMextracellular matrixPAKp21-activated kinaseCRIBCdc42/Rac-interacting binding domainGEFguanine nucleotide exchange factorDirectional and persistent cell migration is essential for a large number of physiological processes in multicellular organisms. In the embryo, complex patterns of cell migration are involved in proper tissue formation. In the adult, the migration of leukocytes into damaged or infected tissue is crucial for an effective immune response, and migration of keratinocytes and dermal fibroblasts are key aspects of wound healing. In addition, the etiology of a wide variety of diseases features aberrant cell migration, ranging from metastases in cancer to autoimmune syndromes.in vivo migration in vertebrates is exemplified by the fibroblast migration defects, and consequently wound healing defects, caused by disrupting syndecan-4 or Rac1 in mice.Motile cells possess the ability to migrate in all directions, and only in response to chemoattractants such as growth factors and extracellular matrix (ECM) proteins do cells polarize.However, until recently, mechanisms by which cells distinguish between ECM receptor engagement at the front and sides of the cell have been lacking, as have explanations of how Rac1 is redistributed from the sides to the front of a migrating cell, and how the cytoskeleton feeds back into Rac1 regulation. In a recent paper from our laboratory, we identified the actin-binding protein, coronin-1C, as a key regulator of Rac1 trafficking that was necessary for Rac1 activation at the leading edge.Dictyostelium discoideum coronin contains a partially conserved Cdc42- Rac1-interactive binding (CRIB) domain that may also mediate interaction with Rac1.Dictyostelium coronin and human coronin-1C. Furthermore, the Rac1-binding Arg31 is not conserved in human coronin-1A. The different effects of coronin-1C and coronin-1A on Rac1 are manifested at the functional level with coronin-1A delivering Rac1 to the plasma membrane, and coronin-1C facilitating release.To better understand the nature of the coronin-1C/Rac1 interaction, molecular docking was used to generate a model that would allow us to predict key interacting residues. In the absence of a coronin-1C crystal structure, a homology model of this protein was generated from the coronin-1A structureThe interactions of coronin-1C with both Rac1 and Arp2/3 suggest a possibility of coordination between cytoskeletal regulators and the cytoskeleton itself, however the position of the Rac1-binding site raises questions about possible competition with actin binding. Actin binding by the coronin-1C propeller is mediated by Arg28, which is conserved between type I coronins , which is not typically where Rac1 would be expected to localize. GFP-coronin-1C was also detected in small vesicles in the perinuclear region of the cell, where it colocalised with actin puncta . Telomerase-immortalized human fibroblasts were cultured at 37°C in DME, 15% foetal bovine serum, 4.5 g/l glucose, 25 mM HEPES and 2 mM L‑glutamine. Where appropriate, fibroblasts were transfected with pDsRed-Rac1 and GFP-Coro1C using Fugene HD . HeLa cells stably expressing GFP-Rab4, Rab5, Rab7 or Rab11 were a gift from Professor Pete Cullen, University of Bristol, and were cultured at 37°C in DME, 10% foetal bovine serum, 4.5 g/l glucose and 2 mM L‑glutamine.The generation of immortalized MEFs has been described previously.TM modification for enhanced specificity were purchased from Dharmacon (Thermo Fisher Scientific). Sequences targeted the sense strand of mouse Coro1C (CCGUUGAAUUAAUUACGUA) or Rac1 (AGACGGAGCUGUUGGUAAAUU), or human Coro1C (GCACAAGACUGGUCGAAUU). For knockdown, 160 pmol of targeted or control oligo was transfected into a 90% confluent 25-cm2 flask using Dharmafect2 reagent . After 18 hours, the cells were passaged and cultured for 2 days before transfecting again to ensure substantial knockdown. Cells were passaged 18 hours after the second round of transfection and used within 2-3 days. Expression of target proteins in comparison with mock-transfected cells was tested by Western blotting.siRNA duplexes with ON TARGET4 cells per coverslip in DME, 10% FBS, 4.5 g/l glucose, 25 mM HEPES, and then treated with 50 μM EHT1864 if appropriate. Spread cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 0.5% (w/v) TrX diluted in PBS, and blocked with 3% (w/v) BSA in PBS. Fixed cells were stained with phalloidin , mounted in Prolong®Antifade  and photographed on a Leica SP5-II confocal laser scanning microscope using a 100x, NA 1.4 PlanApo objective. Maximum projection images were compiled, bandpass filtered, and analyzed using ImageJ software.13-mm diameter glass coverslips were coated with 10 μg/ml fibronectin  in Dulbecco's PBS containing calcium and magnesium , and blocked with 10 mg/ml heat-denatured BSA for 30 minutes at room temperature. Cells were spread for 4 hours at 1.25 × 10Alignment is measured using the dot product. Each straight segment of the track defines a vector If the vectors are parallel d is then scaled so that for random arrangement of fibers, alignment = 0, for parallel fibers, alignment = 1http://www.fundp.ac.be/urdm/bioinf/esypred/), followed by model building in MODELLER and macromolecular docking with the Rac1-GDP crystal structure  (PDB 1I4D) using the ClusPro sever  (http://cluspro.bu.edu/home.php).The Coro1C homology model was generated using the ESyPred3D server  (5b1-integrin ligand (50K).de novo synthesis of ECM and other syndecan-4 ligands, MEFs were pretreated with 25 μg/ml cycloheximide  for 2 hours and spread for 2 hours in DME, 4.5 g/l glucose, 25 mM HEPES, 25 μg/ml cycloheximide. Where appropriate, spread cells were treated 10 μM Rho Kinase inhibitor, Y27632  or 10 μg/ml recombinant fibronectin polypeptide encompassing type III repeats 12-14 that comprises the syndecan-4 ligand (H/0).Tissue culture-treated plastic dishes  were coated with 20 μg/ml recombinant fibronectin polypeptide encompassing type III repeats 6‑10 that comprises the a


Inclusion of the red blood cells (RBC)/plasma partition ratio (K) and the individual hematocrit levels in the estimation of the theoretical Cplasma from Cdbs of PHT and VPA further improved the identity between the observed and the estimated theoretical Cplasma. Bland-Altman plots indicated that the theoretical and observed Cplasma of PHT and VPA agreed well, and >93.0% of concentrations was within 95% CI (±2SD); and similar agreement (1∶1) was also found between the observed Cdbs and Cplasma of CBZ. As the Cplasma of CBZ, PHT and VPA can be accurately estimated from their Cdbs, DBS can therefore be used for drug monitoring in PWE on any of these AEDs.To facilitate therapeutic monitoring of antiepileptic drugs (AEDs) by healthcare professionals for patients with epilepsy (PWE), we applied a GC-MS assay to measure three AEDs: carbamazepine (CBZ), phenytoin (PHT) and valproic acid (VPA) levels concurrently in one dried blood spot (DBS), and validated the DBS-measured levels to their plasma levels. 169 PWE on either mono- or polytherapy of CBZ, PHT or/and VPA were included. One DBS, containing ∼15 µL of blood, was acquired for the simultaneous measurement of the drug levels using GC-MS. Simple Deming regressions were performed to correlate the DBS levels with the plasma levels determined by the conventional immunoturbimetric assay in clinical practice. Statistical analyses of the results were done using MedCalc Version 12.6.1.0 and SPSS 21. DBS concentrations (C Epilepsy is a neurological disease that requires chronic treatment with antiepileptic drugs (AEDs). To date, the most commonly used AEDs are still carbamazepine (CBZ), phenytoin (PHT) and valproic acid (VPA). These drugs have maximum efficacy and minimum toxicity when their plasma drug levels are within their therapeutic indexes. Hence, routine plasma concentration monitoring is recommended especially during dose adjustments, for compliance check and/or for adverse drug reaction investigation Various biological matrices including cerebrospinal fluid, tear and saliva have been used for TDM Earlier studies on concurrent monitoring of multiple AEDs from one DBS were done mostly with high performance liquid chromatography (HPLC) and included whole blood concentrations of AEDs such as carbamazepine, phenytoin, lamotrigine and barbiturates with limited clinical validation In our population of PWE, CBZ, sodium valproate (VPA) and phenytoin (PHT) are the most popular antiepileptic drugs (AEDs) - used either as mono or polytherapy This study has obtained approval from the SingHealth Institutional Review Board (CIRB No. 2011/269/A). Only PWE who had routine plasma CBZ, VPA and/or PHT, blood and liver biochemistry monitoring on the day of visit were approached for written consent prior to blood sampling.Assuming constant analytical standard deviations, the sample size recommended for method validation was suggested to be at least 41 per AED based on the following information: range ratio = 2, α = 5%, power = 90%, standardized slope deviation of 4 Venous whole blood samples were collected in EDTA tubes. Two drops of blood from the withdrawn blood, ∼30 µL each, were spotted onto 903 cards  and dried at room temperature, 25°C for at least 3 hours. The rest of the whole blood was sent to hospital laboratory for plasma AED quantitations as per routine protocols. To maintain direct comparability with plasma levels, DBS samples were stored at −80°C until the day of analysis. The 3 AEDs were proven to be stable at −20°C and 25°C for at least 10 days at concentrations ranging from 0.5 mg/L to 100 mg/L .The routine plasma AEDs quantifications were done in the hospital laboratory using particle enhanced turbidimetric inhibition immunoassays . The imprecisions in CV%  for low, medium and high concentrations based on 21 data points over a period of typically 10 days are as following: i) Carbamazepine [range 2.0–20.0 mg/L] between run = 9.2% , 7.6% , 5.9%  and within run = 3.9% , 2.7%  ii) Phenytoin [2.5–40 mg/L] between run = 8.0% , 5.8% , 4.5%  and within run = 2.1% , 1.5% , 2.9%  iii) Valproic acid [10.0–150.0 mg/L] between run = 7.8% , 7.5% , 7.6%  and within run = 2.5% , 1.0% , 3.6% .g. 400 µL of supernatant was transferred into 15 mL Kimble glass tube  for evaporation under nitrogen gas for 15 mins at 40°C. After addition of 100 µL of toluene to the dried sample, second drying phase under similar condition was carried out. Subsequently, derivatization was attained using 50 µL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane  incubated at an optimum 70°C for 50 min. Derivatised samples were cooled to room temperature and diluted with 50 µL of heptane before vortexing for 1 min. Finally, 80 µL of mixture was transferred into a 200 µL conical base inert glass insert placed in a 2 mL amber glass vial .Quantitation was based on one 6-mm diameter DBS punch from the centre of the spot, containing approximately 15 µL of blood. AEDs extraction was performed using 500 µL of analytical grade (99%) acetonitrile  and 1 molar sodium hydroxide  at a ratio of 24∶1, v/v with 1 µg/mL 5-(p-methylphenyl)-5-phenylhydantoin (5MP)  as internal standard. The extraction procedure involved 1 min of vortexing and 5 min of sonication. Then, the mixture was centrifuged for 15 min at 6000 The analytical assay was developed and validated with a GC-MS system that comprised of GC 2010 Shimadzu GC coupled to a GCMS-QP2010 Plus quadrupole MS . GCMSsolution (version 2.0), was utilized for data acquisition and peak area computation. DB5 ms (30 m×0.25 mm×0.25 µm) supplied by Agilent Technologies J&W, Inc. was used as the capillary column. Injector temperature was set at 250°C, while ion source at 220°C. Injection volume of 1 µL was subjected to split ratio of 1∶5 and column flow was set at 1.9 mL/min. Column temperature began at 90°C with a 0.2-min isothermal hold. Temperature was then ramped at 4 different rates: i) 10°C/min to 120°C, held for 0.5 min ii) 65°C/min to 285°C, held for 0.5 min iii) 10°C/min to 291°C, held for 0.2 min and iv) 60°C/min to 300°C for a final isothermal hold of 5 min. Selective ion monitoring (SIM) mode was used for detection of the target analytes at their respective retention times and are tabulated in Calibration and quality control standards were prepared in blood and spotted onto the 903 cards at 30 µL each. One 6-mm diameter disc was punched out from each DBS and used for analysis. The assay was validated over a range of 0.5–120 µg/mL for all three AEDs. Accuracy ranged from 100–110% and imprecision was <10%. The calculated limit of detection was 0.05 µg/mL for VPA and approximately 0.07 µg/mL for both PHT and CBZ. The recoveries of analytes were relatively high (75%–97%) and consistent (SD≤8.2%). Although the inconsistency increased to 11% at the lower limit of quantitation for CBZ, it was still within the FDA acceptable lower limit of quantification (<20%) .dbs) and plasma concentrations (Cplasma) determined by the respective methods were directly compared. Theoretical Cplasma was calculated using a formula which accounts for the individual hematocrit values and red blood cell-to-plasma partition (RBC/plasma) ratio dbs, rendering the effect of Hct on computation of theoretical Cplasma to be negligible. Moreover, the K for CBZ had been reported to be approximately equal to 1 DBS concentrations , and 2 PWE were excluded as outliers. Characteristics of PWE within each AED group are tabulated in A total of 181 PWE were recruited but only 165 PWE who provided DBS were included in the final analyses. Fourteen PWE were excluded due to undetectable Cdbs and Cplasma for the three AEDs. Good correlations were demonstrated for all three AEDs, with correlation coefficients of r = 0.8381, 0.9305 and 0.8531 for CBZ, PHT and VPA, respectively. Cdbs and Cplasma of CBZ were almost identical with the slope converging with line of unity. In contrast, Cdbs of PHT and VPA were found consistently lower than their corresponding Cplasma, averaging at 2.8±1.89 µg/mL (29.7±13.59%) and 28.3±12.73 µg/mL (49.5±22.3%), respectively (p<0.005). Moreover, 95% CI for the slopes of PHT and VPA diverge anti-clockwise from the line of unity and did not cross the value 1. This signifies that there was at least a proportional increase in correlation between Cplasma and Cdbs of the two AEDs.plasma, definite improvements in fit between data points from the turbimetric assay and DBS assay were observed. The lines of regression for respective AEDs rotated closer to the line of unity and data points clustered in greater proximity along the identity line, albeit marginal decrease in correlation coefficients for PHT and VPA , phenytoin (PHT) and valproic acid (VPA) under different storage conditions on Day 5 and Day 10. Benchtop represents 25°C while Freezer represents −20°C. QC denotes quality control.(DOCX)Click here for additional data file.Table S2Percentage of mean extraction recovery of analytes along with their respective residual standard deviation (RSD) at different concentrations in spiked blood. The consistent and high recovery (>70%) of the analytes allowed for reliable quantitative studies.(DOCX)Click here for additional data file.


A549 cells when co-exposed with BaP  along with 0.1 µg/ml,0.5 µg/ml and 1 µg/ml of TiO2 NPs, showed significant reduction in the toxic effects of BaP, as measured by Micronucleus Assay, MTT Assay and ROS Assay. In order to explore the mechanism of protection by TiO2 NP against BaP, we performed in silico studies. BaP and other PAHs are known to enter the cell via aromatic hydrocarbon receptor (AHR). TiO2 NP showed a much higher docking score with AHR (12074) as compared to the docking score of BaP with AHR (4600). This indicates a preferential binding of TiO2 NP with the AHR, in case if both the TiO2 NP and BaP are present. Further, we have done the docking of BaP with the TiO2 NP bound AHR-complex (score 4710), and observed that BaP showed strong adsorption on TiO2 NP itself, and not at its original binding site (at AHR). TiO2 NPs thereby prevent the entry of BaP in to the cell via AHR and hence protect cells against the deleterious effects induced by BaP.Polycyclic aromatic hydrocarbons (PAH), like Benzo[alpha]Pyrene (BaP) are known to cause a number of toxic manifestations including lung cancer. As Titanium dioxide Nanoparticles (TiO Human exposure to xenobiotics is almost inevitable. Most of the human cancers are caused due to exposure to xenobiotics including PAHs and hence they are ultimately preventable. PAHs are produced during the combustion processes of organic materials during industrial and other human activities, like processing of coal and crude oil, vehicle traffic and cigarette smoke. PAHs may cause carcinogenesis by damaging the DNA and/or a number of proteins BaP enters the cell via aryl hydrocarbon receptor (AHR) After the enzymatic metabolism, BaP is converted to benzo[a]pyrene-7,8-diol-9,10-epoxides (BPDE), a crucial carcinogenic metabolite of BaP, that reacts primarily with the N2 position of guanine residues and to a minor coverage with the N6 position of adenine residues in DNA 2 for example titanate nanoSheets (TNS) and titanate nano tubes (TNT) have also been synthesized and used as additives for removing harmful compounds from cigarette smoke 2 is a naturally occurring oxide of http://www.absoluteastronomy.com/topics/Titanium titanium, and is biologically inert at lower doses, whereas, at higher doses it may induce slight toxicity and even apoptosis Recently, titanium dioxide nanoparticles have been employed in scavenging the high molecular weight polycyclic aromatic hydrocarbons (PAHs) from the contaminated soils 2 NPs could be exploited in the biological system to safeguard against the deleterious effects of PAHs exposure. We also explored the doses of TiO2 NPs, at which they provide maximum protection. Further, in silico experiments were also performed using bioinformatics tools, to attain insight of mechanism of protection.Considering this, we designed the present study in order to explore whether the discussed property of the TiOMost of the specified chemicals, reagents, diagnostic kits etc were purchased from Sigma Chemical Company Pvt. Ltd. . Cell culture media, PBS, antibiotic-antimycotic were purchased from Hi-Media .2 NPs  without any coating were purchased from Sigma Aldrich . Particles were sterilized by heating to 120°C for 2 h and suspended in phosphate-buffered saline (stock: in 1 µg/ µl PBS). The mean hydrodynamic diameter and zeta potential (ζ) of the TiO2 NPs suspension in complete medium as determined by dynamic light scattering (DLS) measurement was 434.1 nm and −7.83 mV, respectively, as described previously by us Anatase form of TiOA549 cells (lung carcinoma cells) were obtained from the cell bank of NCCS Pune, Maharashtra, India, and were grown in a humidified atmosphere with 5% CO2 at 37°C. The cells were cultured in Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal bovine serum and 1% antibiotic and anti-mycotic solution, according to the standard procedure. Prior to use in the experiments, cell viability was estimated using trypan blue dye exclusion assay following the protocol as described earlier Different doses of BaP  were tested in A-549 cells for the selection of most suitable doses for various assays in our study.2 NPs , ii) highest genotoxic dose i.e. 25 µM of BaP, and iii) co-exposure of BaP (25 µM) and different doses of TiO2 NPs .Three different sets of cells were treated with: i) different doses of TiO2 NPs , ii) highest cytotoxic dose i.e. 75 µM of BaP, and iii) co-exposure of BaP (75 µM) and different doses of TiO2 NPs .Three different sets of cells for each of three different time periods  were treated with: i) different doses of TiO2 NPs , ii) highest ROS producing dose i.e. 50 µM of BaP, and iii) co-exposure of BaP (50 µM) and different doses of TiO2 NPs .Three different sets of cells for each of four different time periods  were treated with: i) different doses of TiOThe genetic damage was assessed by MN assay as described earlier 4) were allowed to adhere for 24 h in 5% CO2 at 37°C and 20% humidity in 96-well culture plates. After the exposure for 6h, 12h and 24h, MTT (5 mg/ml of stock in PBS) was added (10 µl/well in 100 µl of cell suspension), and plates were incubated for another 4h. At the end of incubation period, the reaction mixture was carefully taken out and 200 µl of dimethyl sulfoxide was added to each well, the contents were mixed well by pippeting up and down several times. The plates were kept on rocker shaker for 10 min at room temperature and then read at 550 nm using multiwell microplate Reader . Untreated sets were run under identical conditions and served as basal control.Percentage cell viability was assessed using the 3--2, 5-diphenyl tetrazolium bromide (MTT) assay as described earlier 4 per well) were seeded in 96-well black bottom culture plates and allowed to adhere them for 24h in CO2 incubator at 37°C. The medium was then aspirated and cells were exposed to different conditions as describes for 2h, 6h, 12h and 24h. On the completion of respective exposure periods, cells were incubated with DCFH-DA (10 mM) for 30 min at 37°C. The reaction mixture was then aspirated and replaced by 200 ml of PBS in each well. The plates were kept on rocker shaker for 10 min at room temperature in the dark. Fluorescence intensity was measured using multiwell microplate reader  on excitation wavelength at 485 nm and emission wavelength at 528 nm. The data were expressed as percentage of the unexposed control.ROS generation was assessed in A549 cells using 2',7'-diclorodihydrofluorescein di-acetate  dye as fluorescence agent. ROS generation was performed as described earlier ab-initio modeling to build the 3D structure of AHR as shown in PDB structure of aryl hydrocarbon receptor (AHR Uniprot entry - P35869) is not available in the PDB databank. I-TASSER AHR 3D structure has been submitted in Protein Model Data Base, (PMDB ID- PM0078981) de novo, via the internet (http://molecular-networks.com/products/corina), by program CORINA on server running in Computer-Chemie-Centrum, Universiy of Erlangen-Nurnburg, Germany. It is a rule- and data-base system, that automatically generates 3D atomic coordinates from the constitution of a molecule as expressed by connection table or linear code as shown in The SMILES (Simplified Molecular Input Line Entry Specification) notations of the BaP were obtained from the ZINC database (ZINC ID - 01530818). The 3D-structure of BaP was generated oAfter studying the anatase crystal structure, we found that anatase is the thermodynamically favored phase. According to the anatase lattice parameters, the tetragonal crystal have lengths  as; A = B = 3.782 Å, C = 9.502. Å and angles  as; Alpha  =  beta  =  gamma  = 902 anatase crystal structure.The Accelrys Discovery studio 2.5 program was found to be the most suitable software for the designing of TiO2 anatase by using the anatase lattice parameters, a surface was created and this unit cell was extended in the desired directions (axis) creating a new surface of TiO2 comprising  of 5 unit cells in × direction and 2 unit cells in the Z direction. This gave a surface of dimensions 1.891×0.3782×1.9004 nm3 as shown in After the construction of Unit cell of TiOin silico docking analyses were performed using PatchDock 2 NP. The resultant pdb file obtained after AHR and TiO2 NP docking was used as AHR-TiO2 NP complex, and was docked with BaP by uploading the receptor and molecules in PatchDock Server, an automatic server for molecular docking. Clustering RMSD was chosen as 4.0 Å.All the All the experiments were performed in triplicates and were repeated twice. The final results were expressed as mean of the values obtained from all experiments. The standard error of mean (SEM) was also calculated. Statistical analysis was performed by one-way analysis of variance (ANOVA) using Newman- Keuls test to compare all the groups by graph pad prism3. In all the cases, p<0.05 was considered as significant.Different doses of BaP  were tested in A-549 cells to determine the most suitable dose for each assay in the study.The MN induced were 43.33±3.844, 52±2.646, 47±2.082 & 24.67±2.333 MN/1000 cells at 10 µM, 25 µM, 50 µM and 75 µM BaP concentrations respectively after 24h exposure, against 5±1.528 MN/1000 in control (unexposed) cells as shown in The cell viability, following exposure to 10 µM, 25 µM, 50 µM and 75 µM of BaP concentrations was observed as 85±4.041, 90±2.646, 71±3.215, and 48±2.333% after 6h, 83±2.43, 80±3.44, 68±4.152, and 37.68±3.423% after 12h exposure, and 80±1.01, 75±4.381, 50.2±2.153, and 12.72±2.412% after 24h exposure respectively, against control (unexposed) cells, as shown in ROS generation following exposure to 10 µM, 25 µM, 50 µM and 75 µM of BaP concentrations were observed as 160±5.29, 199±19.35, 230±7.23, and 262±5.044% after 2h, 162±7.42, 212±8.76, 234±8.66, and 230±5.77% after 6h, and 140±5.19, 200±2.88, 245±8.66, and 210±2.88% after 12h of exposure and 130±6.35, 170±5.77, 225±8.56, and 140±2.517% after 24h respectively, against unexposed cells, as shown in 2 NPs  induced slightly higher number of MN  as compared to unexposed control (5±0.5774 MN/1000 cells). The highest genotoxic dose (25 µM) of BaP induced significantly (p<0.05) high numbers of MN (53.33±4.41 MN/1000 cells). Whereas, co-exposure of BaP (25 µM) and different doses of TiO2 NPs  resulted in significant reduction (p<0.05) of MN induction , as compared to BaP exposed cells as shown in Micronucleus assay was performed to analyze genotoxicity in different experimental sets. Different doses of TiO2 NPs exposure resulted in very slight cytotoxicity. The % of viable cell observed after exposure to TiO2 NPs  was 98.64±0.2022, 97.6±0.3711, and 96.73±0.1802 after 6h, 97.45±0.2461, 96.93±0.3169, and 94.83±0.5185 after 12 h, and 95.3±0.6582, 93.77±0.2963, and 92.47±0.2767 after 24h respectively. But, BaP exposure at selected dose of 75 µM showed significant (p<0.05) reduction in cell viability, which as 46.51±1.147% after 6 h exposure, the effect became more intense after 12h (39.12±1.33%) and 24h (10.41±0.494%) exposure. Whereas, co-exposure of BaP (75 µM) with different doses of TiO2 NPs , resulted in significant increase in cell viability  as compared to BaP exposed cells as shown in MTT assay was performed to analyze the cytotoxicity in different experimental sets. TiO2 NPs  was 102.5±2.201, 103.5±2.871, and 103.3±3.808%, respectively after 2h, 104.5±0.2999, 105±0.4533, and 107.5±0.3143% after 6h, and 104.5±0.2999, 105±0.4533, and 107.5±0.3143% after 12h, 104.6±0.8764, 106.5±1.459 and 108.6±0.2267% after 24h, respectively. The exposure to selected dose (50 µM) of BaP induced significantly (p<0.05) higher ROS . Whereas, the co-exposure of BaP (50 µM) with different doses of TiO2 NPs  resulted in significant reduction in ROS generation  as compared to ROS generation by BaP alone at all time periods, as shown in DCFH-DA staining was performed to analyze ROS generation in different experimental sets. The % of ROS observed after exposure to different doses of TiOab initio modeling), as shown in 3D structure model of AHR was generated using I-TASSER online server  of TiO2 NP showed high binding affinity with AHR with a docking score of 12074, as compared to the docking score of BaP with AHR (4600). Docking score of BaP with AHR-TiO2 NP complex was 4710.TiOThe chemical nature of binding site residues of AHR within a radius of 4A° with BaP showed hydrophobic interaction with Pro180, Ser181, Cys183, Gly187, Leu196, Val200, Asn204, Leu259, Pro260, Leu265, Ala269, Thr270, Leu272 and Pro274 residues, as shown in 2 NP showed hydrogen bond interaction with Gln667-NE2: O98 bond length 3.11 Ǻ, Gln667-N: O38 bond length 2.53 Ǻ, Gln 383- NE2: O75 bond length 1.94 Ǻ, Asp144N: O4 bond length 2.91 Å, Ser 682: OG:O19 bond length 3.26 Å, Gln149 NE2: O19 bond length 2.45 Å, Try 696 CZ-OH: O5 bond length 3.08 Å & O8 bond length 3.08 Å and hydrophobic interaction with Tyr 145, Ser 151, Phe 148, Leu 369, Asn 673. Asn 366, Agr 384, Pro 385, Leu 413, Try 719, Phe 406, Glu 488, Pro 665, Gln 671 & Ser 682, as shown in The chemical nature of binding site residues of AHR within a radius of 4A° with TiO2 NP complex was adsorbed at the surface of TiO2 NP, as shown in 2 NP complex within a radius of 4A°, showed the hydrophobic interaction with Gln 666, Try 719, Phe 700, Pro 669, Gln 698, Thr 408, Phe 406, Phe 675, Thr 696.Whereas, the BaP when docked with AHR-TiOThe present study was designed to explore the probability of protective application of nanoparticles against environmental carcinogen induced toxicity. The doses of BaP, the reference carcinogen in the study, were optimized for MN, MTT and ROS generation assays in A549 cells. The BaP exposure caused significant reduction in cell viability, which was dependent on period of exposure and was highest with 75 µM at 24h (10.41±.494%). This effect could be attributed to enhanced production of ROS as a result of BaP exposure which was found to be highest with 50 µM at 12h (246.9±1.178%). Further, ROS generation might have caused the DNA damage which was highest with 25 µM at 24h (53.33±4.41MN/1000 cells).Which is in accordance with the results of past toxicological studies of BaP 2 NPs  along with BaP.In order to evaluate the protective effect of nanoparticles, A549 cells were co-exposed to some non-toxic doses of TiO2 NPs offered protection and raised the viability of A549 cells as compared to viability in only BaP exposed cells as measured by MTT assay after co-exposure. The protective effect was slightly higher with 0.1 µg/ml concentration of TiO2 NPs than 0.5 µg/ml and 1.0 µg/ml TiO2 NPs.At all doses, the TiO2 NPs caused significant lowering in ROS production in BaP exposed A549 cells at all time periods. Again, the effect of 0.1 µg/ml concentration of TiO2 NPs was marginally higher as compared to 0.5 µg/ml and 1 µg/ml TiO2 NPs.Similarly, co-exposure of all three doses of TiO2 NPs caused significant reduction in MN induction by BaP in A549 cells. Again, the effect of 0.1 µg/ml concentration of TiO2 NPs was marginally higher as compared to 0.5 µg/ml and 1 µg/ml of TiO2 NPs.Similar effects were observed in the MN assay also, where co-exposure of all three doses of TiO2 NPs in low dose.All three end points used for study depicted clear cut reduction in the toxicity of BaP, which indicated the protective potential of TiOIn silico approach was applied in order to explore the probable mechanism by which TiO2 NP provided protection against BaP induced toxicity. Previous studies have established that AHR is responsible for the entry and regulation of the enzymatic metabolism of BaP to Benzo[a]pyrene -7,8-diol-9,10-epoxides (BPDE), a crucial carcinogenic metabolite of BaP, which reacts primarily with the N2 position of guanine residues and to a minor coverage with the N6 position of adenine residues in DNA 2 NPs with AHR, revealed that TiO2 NP bind with much higher molecular docking score with AHR (12074) as compared with docking score of BaP with AHR (4600). This establishes a strong possibility of preferential binding of TiO2 NP over BaP with AHR, incase both TiO2 NP and BaP are present together in cell vicinity. Further, to investigate how AHR might respond to BaP when TiO2 NP is already bound to AHR, the BaP was docked with AHR-TiO2 complex. The BaP was adsorbed strongly at the TiO2 NP (score 4710) and not at its original binding site at AHR, as shown in 2 NP towards PAHs and some other chemical carcinogens present in cigarette 2 NP might have caused the shifting in binding position of BaP from AHR to TiO2 bound to AHR.The docking study, performed to determine the binding abilities of BaP and TiO2 NP on AHR as well as adsorption of BaP on TiO2 surface could be an effect of nanosized TiO2 in order to minimize high free surface energy, to accomplish the atomic coordination at the surface and to establish electronic neutrality 2 nanoparticles itself, rendering BaP unavailable to its target molecules, which requires further in depth analysis.Once a bulk substance is brought to nano-size, it loses its surface atomic coordinates thereby increasing free surface energy. The stronger binding of the TiO2 nanoparticles in A549 cells, along with the probable mechanism of TiO2 NPs protection against BaP. To the best of our knowledge, this is the very first study suggesting future prophylactic application of nanoparticles as guardian against the chemical carcinogens at the molecular/cellular level. We are in process of further investigating whether TiO2 NPs are capable of protecting cells against other chemical carcinogens also? And if this process of protection can be further enhanced by modifying the surface chemistry of TiO2 NPs. Also, we are exploring the capacities of other nanoparticles like CNT, Fullerene etc. for their potential to provide protection against chemical carcinogens.The present study clearly describes the attenuation of BaP induced toxicity by TiO


Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general.The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like  Sordaria macrospora, which is a well-established model organism used to study the formation of fungal fruiting bodies, three-dimensional organ-like structures. We analyzed STRIPAK subunit PP2Ac1, catalytic subunit 1 of protein phosphatase PP2A, to study the importance of the catalytic activity of this protein during sexual development. The results of our yeast two-hybrid analysis and tandem affinity purification, followed by mass spectrometry, indicate that PP2Ac1 activity connects STRIPAK with other signaling pathways and thus forms a large interconnected signaling network.The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood. The first fungal STRIPAK was described in  Reversible protein phosphorylations are among the most common posttranslational modifications  that areThe number of phosphatases is similar to the number of kinases. However, significantly fewer phosphatase genes than kinase genes exist, since phosphatase diversity is based on the multimeric nature of most protein phosphatases. For example, major and highly abundant S/T protein phosphatase 2A (PP2A) is a heterotrimeric protein phosphatase. A scaffolding subunit (PP2AA) and a catalytic subunit (PP2Ac) form a core complex, which is joined by a B-type regulatory subunit to complete the functional holoenzyme. The PP2A core complex is well conserved, and mammalian cells contain two isoforms each of PP2AA and PP2Ac . In fung8–Regulatory PP2A subunits mediate substrate specificity and are classified into four families, called B, B′, B′′, and B′′′. Although members of these protein families within a single species show rather low sequence similarity, homologs of a given family are well conserved from yeasts to humans. The B′′′ subunits are the latest members of the regulatory subunits and are referred to as striatins . Striati13–Sordaria macrospora, which comprises, besides PP2AA, the striatin homolog PRO11, the STRIP1/2 homolog PRO22, the SLMAP homolog PRO45, the Mob3 homolog SmMOB3, and the germinal-center kinases SmKIN3 and SmKIN24  analysis and tandem affinity purification (TAP)-mass spectrometry (MS) with PP2Ac1 as bait revealed that PP2Ac1 directly interacts with PRO22 and PP2AA. Our studies indicate that catalytically active PP2Ac1 is a major regulator of fungal sexual development and must be considered a key component of the fungal STRIPAK complex.We previously characterized the STRIPAK complex of the filamentous fungus  SmKIN24 , 16. Pre SmKIN24 , 18. TheS. macrospora genome identified two genes encoding catalytic subunits 1  and 2  of PP2A. Both are highly homologous to their human homologs , the fungus forms vegetative mycelium. Some hyphae differentiate into female gametangia, the ascogonia, which develop into immature fruiting bodies (protoperithecia) and then into pear-shaped fruiting bodies called perithecia. They protect the sexual sporangia (asci), each containing eight ascospores   . The numpp2Ac1 mutant strains. Unlike the wild type, which frequently formed hyphal fusion bridges, the deletion strain was unable to undergo hyphal fusion  and the B-type regulatory subunit of PP2A, CDC55 (SMAC_04241) were identified in two experiments and one experiment, respectively. Furthermore, we detected the scaffold protein for the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway, PRO40 (SMAC_04815), and the CWI pathway components RHO1 (SMAC_06239)  and GTPaOur TAP-MS data gave us an insight into the composition of the protein complex containing PP2Ac1. However, direct interactions within this complex remained elusive. The TAP-MS analysis presented here, together with previous TAP- and AP-MS analyses, emphasizes a putative connection between STRIPAK and the CWI pathway , 25. SimSaccharomyces cerevisiae TOR1) repeats 8 to 12, interacted in the Y2H analysis.Since BD-PP2Ac1 and BD-PRO11 fusion proteins show transactivation, we tested the binding of PP2Ac1 to subfragments of PRO11, which is the homolog of mammalian striatin and thus the regulatory subunit of PP2A in STRIPAK. To determine the interaction sites of PRO22 and PP2AA that mediate binding to PP2Ac1, we used subfragments as displayed in pp2Ac1 strain and slightly but not significantly higher in the Δpro22 and Δpp2Ac1 Δpro22 strains.We identified the CWI pathway components as interaction partners of STRIPAK subunits PP2Ac1 and PRO22. Thus, we analyzed the phosphorylation status of MAK1 in both deletion strains and the double deletion strain with an appropriate antibody. As shown in pp2Ac1 derivative with the highly conserved histidine replaced with a glutamine at position 59 (H59Q). Mammalian homolog proteins harboring the H59Q mutation in the active site were inactive in vitro . We purified glutathione S-transferase (GST)-PP2Ac1, GST-P2Ac1A50G, GST-PP2Ac1H59Q, and GST from Escherichia coli . In the ascomycetes S. macrospora and Neurospora crassa, many of these developmental mutants are impaired in hyphal fusion , and shows reduced vegetative growth rates  complex and controls cell cycle arrest during mating, the FAR complex negatively regulates the TORC2 signaling network  (Stratagene)  . Alterna PJ69-4α  as previ PJ69-4α , 63. The PJ69-4α .S. macrospora strains used in this study are listed in 2O2, or 100 mM dithiothreitol (DTT). For each experiment, three dishes were inoculated with an 8-mm-diameter agar plug and incubated for 2 to 3 days. The growth front was marked every 24 h . E. coli strains were grown in LB liquid culture with ampicillin for selection while shaking at 280 rpm and 37°C until reaching an optical density at 600 nm of 0.5 to 0.6, when overexpression was induced with 100 ng/ml anhydrotetracycline for 1 h at 280 rpm and 37°C. Overexpression of the GST gene was performed with plasmid pGEX-4T-1 as previously described  was generated and analyzed for production of NTAP-PP2Ac1 by Western blotting as previously described . Sexual development and hyphal fusion were documented by differential interference contrast (DIC) microscopy with strains inoculated onto BMM-coated glass slides for 2 to 7 days or after 2 days on cellophane-covered MMS dishes, respectively . Fluores2, and 0.05% (wt/vol) acetylated bovine serum albumin. The reaction was performed three times in triplicate for 60 min at 30°C in the dark. Fluorescence measurements were taken after the addition of 1.9 ml of distilled water. To verify that fluorescence was due to a dephosphorylation reaction by PP2Ac1, we performed two inhibitor experiments, each in triplicate, with 5 nM okadaic acid added to the reaction mixtures or with the GST tag alone.The phosphatase activity of purified recombinant proteins was measured in a phosphatase activity assay with DiFMUP as the substrate , 77. ThiText S1 Supplemental materials and methods. Download Text S1, DOCX file, 0.02 MBDataset S1 TAP-MS with PP2Ac1 as bait. Download Dataset S1, XLSX file, 0.5 MBFigure S1 S. macrospora (sm) PP2Ac1 and -2  and Homo sapiens (hs) PP2AcA and -B . The PP2A, PP4, and PP6 phosphoprotein phosphatase metallophosphatase domain (MPP_PP2A_PP4_PP6) is underlined in green. The yellow and purple boxes contain the active sites and the highly conserved C terminus, respectively. Asterisks indicate the positions of the A50G (blue) and H59Q (orange) amino acid substitutions generated in this study. %ident. is percent identity to the amino acid sequence of PP2Ac1. Download Protein sequence alignment of Figure S1, TIF file, 0.7 MBFigure S2 pp2Ac1 locus in the wild-type and Δpp2Ac1 mutant strains. Oligonucleotides and corresponding PCR fragments are indicated by arrows and gray lines, respectively. Restriction enzyme sites used for hydrolysis prior to Southern blot analysis are indicated, and thick gray lines show the probes used for Southern hybridization. The image is not drawn to scale. (B) Homologous integration at the 5′ flank (5′ HR) and 3′ flank (3′ HR) and the presence of pp2Ac1 were tested in PCR assays with primer pairs KO-4678-1 and d1, KO-4678-2 and d2, and 4678-NTAP-fw and 4678-NTAP-rv, respectively. Wild-type (wt) genomic DNA served as a control, and the negative control (NC) contained no DNA. (C) Southern hybridization with radioactively labeled probes specific for hph and pp2Ac1 after restriction of genomic DNA with BamHI. (D) Quantification of perithecium formation in fertile complemented Δpp2Ac1 mutant strains. Perithecia were counted as described in P > 0.01, respectively. Strains not connected by brackets show significantly different numbers of perithecia (P < 0.01). Download (A) The Figure S2, TIF file, 1.5 MBFigure S3 pp2Ac1 and pro22 loci in the wild-type and Δpp2Ac1 Δpro22 mutant strains. Arrows indicate oligonucleotides for deletion verification. Gray lines show PCR fragments. The image is not drawn to scale. (B) We verified the Δpp2Ac1 Δpro22 strain by PCR analysis with primer pairs KO-4678-1/d1, KO-4678-2/d2, 4678-NTAP-fw/4678-NTAP-rv, pro22_vp1/d1, pro22_vp2/d2, and pro22_vp2/pro22_vp3 to analyze the homologous integration of the 5′ and 3′ flanks of pp2Ac1 and pp2Ac1 and those of pro22 and pro22, respectively. Wild-type (wt) genomic DNA served as a control, and the negative control (NC) contained no DNA. (C) Stress-related growth on SWG medium containing 100 mM DTT, 0.6 M sorbitol, 0.01% H2O2, or 0.6 M KCl in petri dishes stimulating ER, high-sugar-induced osmotic, reactive oxygen species, and high-salt-induced osmotic stress compared to growth on SWG medium. Growth tests were performed in petri dishes in triplicate, and the growth fronts were marked every 24 h for 2 to 3 consecutive days. Error bars indicate standard deviations. Download (A) The Figure S3, TIF file, 2 MBFigure S4 +, not determined. Download Growth control for yeast strains in Y2H analyses. Shown is the growth of the strains shown in Figure S4, TIF file, 1.4 MBFigure S5 pp2Ac1, Δpro22, and Δpp2Ac1 Δpro22 mutants compared to that in the wild type (wt). Western blot analyses of MAK1 phosphorylation levels and tubulin  were done with anti-phospho-p44/42 and anti-tubulin antibodies, as depicted at the top and bottom, respectively. Download Phosphorylation of MAK1 in STRIPAK ΔFigure S5, TIF file, 1 MBTable S1 S. macrospora strains used in this study.Table S1, DOCX file, 0.02 MBTable S2 Oligonucleotides used in this study.Table S2, DOCX file, 0.02 MBTable S3 Plasmids used in this study.Table S3, DOCX file, 0.04 MB