MX, have been viewed as typical ionic compounds, characterized by 1:1 ratio necessary for charge balance between M+ and X−. It was proposed that group I elements like Cs can be oxidized further under high pressure. Here we perform a comprehensive study for the CsF-F system at pressures up to 100 GPa, and find extremely versatile chemistry. A series of CsFn (n ≥ 1) compounds are predicted to be stable already at ambient pressure. Under pressure, 5p electrons of Cs atoms become active, with growing tendency to form Cs (III) and (V) valence states at fluorine-rich conditions. Although Cs (II) and (IV) are not energetically favoured, the interplay between two mechanisms  allows CsF2 and CsF4 compounds to be stable under pressure. The estimated defluorination temperatures of CsFn  compounds at atmospheric pressure , are attractive for fluorine storage applications.Alkali halides  Yet, some ionic compounds do not strictly obey this rule. For instance, MgO2 can be prepared at very high oxygen fugacities, in which anions form the peroxide group [O2]2−In general, for a given ionic compound Asn compounds. According to Miao's calculation, CsF2 becomes stable at 5 GPa, CsF3 at 15 GPa, and CsF5 at 50 GPa. Considering [CsF2]− and [CsF5] are isoelectronic to the well-known molecular XeF2  results in an unexpected variety of stable CsFn compounds under moderate pressure.It appears that increasing pressure promotes the formation of increasing oxidation states. Our recent work has discovered that Xe will form stable oxides under high pressure, in which Xe exhibits the oxidation states of +2, +4 and +65]− Ref. , this pin (n ≥ 1) to be stable, with CsF being stable at all pressures. Thus we focused our search on the CsF-F system at pressures 0, 30, 50, 75, 100 GPa. As shown in 2, CsF3, CsF5, which are thermodynamically stable already at ambient pressure. As pressure increases, CsF2 becomes unstable above 19 GPa, while CsF4 appears on the convex hull  at pressures in the range between 17–80 GPa. CsF3 and CsF5 are stable in the entire pressure range up to 100 GPa. However, all of the stable compounds undergo a series of phase transitions, with dramatic changes of the electronic structure.We have performed variable-composition structure searches using the USPEX code89103 phases. At ambient pressure, we find CsF3 adopts a rhombohedral structure . Bader analysis also supports this conclusion: there is a charge transfer of 0.950 e from Cs to F3 species, very close to the value in CsF (0.928 e), but the charge distribution within F3 is not even: two end F atoms have the charge of -0.406 e, while the central atom only has -0.138 e. Trihalide anions [X3]−  are well known, but the [F3]− species has only been experimentally found as CsF3 complexes in argon matrix122 . At 27 GPa, CsF3 undergoes a phase transition to a monoclinic phase C2/c. More interestingly, this structural transition coincides with a striking increase in Cs's Bader charge, as shown in e, far beyond the +1 formal charge of alkali elements under ambient conditions, suggesting Cs+ has been further oxidized. Our previous study has shown that Xe under high pressure can be oxidized to +2, +4 and +6 states. Cs3+ is isoelectronic to Xe2+. This phase transition can be interpreted as a transition from Cs+[F3]− to [CsF2]+[F]−. This is also evidenced by the dramatic change of Cs-F distance. In the C2/c phase, there are two types of F atoms (F1 and F2), each Cs is surrounded by 2 F1 and 4 F2. At 50 GPa, the calculated Cs-F1 distance is 2.01 Å, while the Cs-F2 distance is 2.58 Å, appearing to be consistent with a Jahn-Teller distortion related to the open-shell Cs3+ configuration. However, compared with Cs-F bond length (2.69 Å) in ionic CsF, we conclude that Cs-F2 interaction is very close to a typical ionic Cs-F bond, while each individual Cs-F1 bond has much stronger interaction . Therefore, it can be viewed as [CsF2]+[F]− complex. A similar discussion can be also found in Miao's workC2/m structure is less stable than the C2/c structure found here.Let us first look at CsF3, CsF5 is also stable in the entire investigated pressure range between 0–100 GPa. At 0 GPa, we found a monoclinic P21 phase is stable against decomposition to any other stable compositions . P21-CsF5 can be described as packing of Cs+ and [F5]− species. [F5]− ion has a V-shape and F-F bond lengths are 1.617, 1.953, 1.858, 1.617 Å, and F-F-F bond angle at central F atom is 98.592°. Bader analysis in 5 group has charge -0.958 e. The hypothetical pentafluoride anion [F5]− has also been proposed by Riedel5]− groups and C2/c symmetry becomes stable. Around 21 GPa, C2/c phase transforms to another monoclinic C2/m phase, which can be represented as [CsF2] [F3]. The [CsF2] unit is very similar to the one in C2/c-CsF3 (Bader charge is 0.820e), indicating that Cs achieves the +3 oxidation state. At the same time, [F3] is a typical polyfluoride anion with Bader charge -0.820e; thus the whole structure can be viewed as [CsF2]+[F3]−. At 47 GPa, consistent with Miao's results5 molecules. We again plot the variation of Cs's Bader charge in stable CsF5 compounds with pressure. Indeed, analysis indicates a two-step oxidation of Cs +1 → +3 → +5, coinciding with the transition sequence .Similar to CsF3 and CsF5 are stable alongside the known compound CsF in the whole investigated pressure range (0–100 GPa). Unlike the recently discovered exotic sodium chlorides3]−, [F5]−). Note that this is different from the previous study2 and CsF4 can be stable at intermediate pressure ranges.Our results show CsFI4/mmm) XeF2-like molecular structure was proposed to be stable at 5-20 GPa2 crystal to be unstable against decomposition to CsF3 and CsF at all pressures. A class of CsF2 compounds, however, has been found to be stable at low pressures in our prediction. At 0 GPa, another I4/mmm CsF2 phase is found to be stable. As shown in + and [F4]2− ions. The calculated Bader charges are 0.924 e for [Cs], and -1.848 e for [F4], suggesting the formation of [Cs]+ and [F4]2−. Therefore, [Cs]2+ is not favoured by energy, but CsF2 can be stabilized due to the formation of the [F4]2− anion. [F4]2− has not been observed by chemists so far, except that Riedel et al4]−. Yet our comprehensive structural search suggests that [F4]2− based CsF2 should be stable. Tetraiodide anion [I4]2− is knownI4/mmm-CsF2 would undergo a phase transition to an orthorhombic phase (Pbam) at 2.8 GPa, which also contains Cs+ and [F4]2− ions. Above 19 GPa, CsF2 is no longer stable as there is a dramatic change in the valence of Cs from (I) to (III). At around 20 GPa, CsF4 becomes stable in a monoclinic form (C2/m) , and the structure has space group P-1. As shown in 2]+[F4]2− ([F4]2− anions appear again!). Miao investigated the possibility of CsF4 molecule structurally similar to XeF4, which is contradictory to chemical intuition . Indeed, our results suggest that Cs4+ based compound is energetically unfavored. But we found that the most stable CsF4 structure above 57 GPa shows the oxidation state higher than +3. As shown in 2]+[F]− · [CsF5]0. Therefore, half of Cs atoms (in [CsF2]) have III valence, while the other half of Cs (in [CsF5]) have V valence state. The resulting structure, crystallizing in P-1 symmetry, is stable up to 79 GPa between C2/c-CsF3 and Fddd-CsF5. We note that there also exists a stable phase of XeF3 that can be represented as [XeF2]· [XeF4]Previously, a tetragonal (m (C2/m) . One canLight halogens, fluorine (F) and chlorine (Cl), at normal conditions are highly reactive and toxic gases. For chemical industry and laboratory use, this presents great inconvenience. Their storage in the gaseous form (even as liquefied gases) is very inefficient, and compressed gas tanks may explode, presenting great dangers. At normal conditions, the volume of 22.4 litres (L) of pure fluorine gas weighs just 36 grams (g), illustrating the dismal inefficiency of storage in this form. To the best of our knowledge, no effective and safe fluorine storage materials are known. Both F and Cl have a huge range of industrial applications, which would benefit from such storage materials, especially if they can achieve high storage capacity, stability and reversibility.n  compounds can be stable at zero temperature and ambient pressure. One mole of CsF5  contains 2 moles of F2 gas . The reaction CsF5 = CsF + 2F2(gas), is thermodynamically unfavourable at zero temperature , but will be favourable on increasing temperatures, due to the higher entropy of the F2 gas (202.8 J/(mol·K) at standard conditions)3 can be thermally decomposed, and then again be synthesized at lower temperatures at nearly room temperature window. CsF5, having the highest F content, can be used for fluorine storage at low temperature conditions. Thermodynamic stability of the predicted polyfluorides at atmospheric pressure means that there will be ways to “recharge” them with fluorine after defluorination, and such reversibility is a strong advantage of the proposed fluorine storage materials.In this work, we found that a series of CsFn phases show extremely rich chemistry. At ambient pressure, novel and unexpected compounds CsF2, CsF3, CsF5 are thermodynamically stable because of the formation of polyfluoride anions of [F3]−, [F4]2−, [F5]−. Our results confirm the previously proposed polyfluoride anions , and suggest a new ion (F42−). Under high pressure, 5p electrons of Cs atoms can become chemically active, making Cs3+ and Cs5+ energetically favourable. Although our prediction found Cs2+ and Cs4+ states are far from being stable, stochiometric compounds CsF2 and CsF4 can become stable at 0–20 and 15–80 GPa, respectively, but these contain Cs+, Cs3+, Cs5+, [F4]2−, [F3]−. As shown in +, [CsF2]+), polyfluoride anions , and neutral molecular species (CsF5). Our hope is that this report will stimulate further experimental studies and serve as a guide for the design of fluorine storage materials.We have presented a comprehensive study of possible stable compounds in the CsF-F binary system under pressure. CsFxCsF + yF]). During the initialization, USPEX samples the whole range of compositions of interest randomly and sparsely. Chemistry-preserving constraints in the variation operators are lifted and replaced by the block correction scheme which ensures that a child structure is within the desired area of compositional space, and a special “chemical transmutation” operator is introduced. Stable compositions are determined using the convex hull construction: a compound is thermodynamically stable if the enthalpy of its decomposition into any other compounds is positive. Structure prediction was done in conjunction with ab initio structure relaxations based on density functional theory (DFT) within the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation (GGA)π × 0.06 Å−1. For post-processing, the selected low-enthalpy structures were treated by using hard PAW potential of F (F_h), using a energy cut off of 1000 eV. Such calculations provide an excellent description of the known structures (CsF and F2) and their energetics. To ensure that the obtained structures are dynamically stable, we calculated phonon frequencies throughout the Brillouin zone using the finite-displacement approach as implemented in the Phonopy code20Searches for the stable compounds and structures were performed using an evolutionary algorithm, as implemented in the USPEX code89101Q.Z. designed the project and carried out structure prediction and electronic structure calculations. Q.-F.Z performed phonon calculations. Q.Z. and A.R.O analysed the data and wrote the paper.Supplementary Online Materials: Formation of Stoichiometric CsFn Compounds


In this study we used computational methods to design novel C5a1 receptor ligands. However, functional screening in human monocyte-derived macrophages using the xCELLigence label-free platform demonstrated altered specificity of our ligands. No agonist/antagonist activity was observed at C5a1, but we instead saw that the ligands were able to partially agonize the closely related complement receptor C3a receptor. This was verified in the presence of C3a receptor antagonist SB 290157 and in a stable cell line expressing either C5a1 or C3a receptor alone. C3a agonism has been suggested to be a potential treatment of acute neutrophil-driven traumatic pathologies, and may have great potential as a therapeutic avenue in this arena.The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury. Complement C3a and C5a are key mediators produced by this cascade, and their dysregulation has been linked to a plethora of inflammatory and autoimmune diseases. Consequently, this has stimulated interest in the development of ligands for the receptors for these complement peptides, C3a receptor, and C5a Two maj1 (C5aR/CD88)  , such astermined . Increastermined .1, has been targeted for drug design  using label-free methods that provided cell activation read-outs. Several hits were identified and, based on the activation profiles, these hits appeared to be C3a agonists. Screening of the peptides in the presence of C3a receptor and C5a1 antagonists confirmed C3a receptor as the target. Thus modification to the C-terminal sequence conferred C3a agonist activity on C5a peptides.A two-stage de novo protein design framework previously described  was appl22.1The computational design framework  with acetylated N-termini at >95% purity. Purity was confirmed with HPLC. The C-terminus was unblocked in all peptides.2.37 in Iscove׳s Modified Dulbecco׳s Medium (IMDM) (Invitrogen Life Technologies) containing l-glutamine supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin and 50 μg/ml streptomycin. Human macrophage colony-stimulating factor (M-CSF) (Peprotech) was added to media to allow cells to differentiate into human monocyte-derived macrophages (HMDM). Over the 7 day differentiation process, cells were incubated at 37 °C and 5% CO2.Human monocytes were isolated from blood donations to the Australian Red Cross Blood Service . The isolation of mononuclear cells was as described previously by 2.42 for 2 h prior to ligand addition. Ligands were prepared at a final DMSO concentration of 0.5% (or less) in serum-free IMDM. Antagonists PMX53 and SB 290157 were incubated for 1 h prior to agonist addition, at concentration ranges known to cause substantial inhibition of receptor activation in these cells. After agonist addition, measurements were taken continuously for ~1.5 h at 37 °C. The RTCA software was used for data analysis, with statistical analysis using GraphPad Prism 5.0c .HMDM were seeded overnight at 30,000 cells/well in 384-well E-Plates (Roche). Approximately 24 h later, HMDM media were exchanged for serum-free IMDM and equilibrated at 37 °C and 5% CO2.51 or human C3a receptor  and the3.2P<0.001), peptides 20, 31, 47, 48 and 54. Peptide 49 was identified as a potential hit from independent data (data not shown) despite not evoking a response in the initial screen and so was retested at the full dose range along with the five identified hits  at 10 mM, were screened at a single dose of 8 μM on HMDM using an impedance based label-free system, the xCELLigence; DMSO at <0.5% was found to have no effects in this assay. hits see B. Intere3.31 antagonists, SB 290157 and PMX53, respectively , the activation obtained was 5–10% of the maximum activation achieved by a high dose of C5a , which have been shown to express the C5aing tool . From thing tool A.1, as highlighted in 1 differ significantly, where the evolution of the signal peaks at ~1 min after peptide addition for C3a receptor and ~4 min after peptide addition for C5a1. The intensity of the signal varies too, where a cell index of 1.98 and 4.53 is observed for C3a receptor and C5a1, respectively. As the receptors for both C3a and C5a are expressed in HMDM, it is entirely feasible these peptides were activating C3a receptor instead of C5a1. To discriminate whether the signal observed for peptides 20, 31, 47, 49 and 54 on HMDM was attributable to C3a or C5a, a C3a receptor antagonist, SB 290157, and C5a1 antagonist, PMX53, was employed.Interestingly, the receptor signaling profile generated by these hits more closely resembled that of C3a than C5a1 antagonist, PMX53 , was shown to be an antagonist but also had partial agonist behavior , had receptor affinity in the low nanomolar range (d-Ala-Arg), although this peptide too shows promiscuity for C3a receptor (1, we identified novel sequences that agonize the C3a receptor.Although these peptides were designed using the C-terminal domain of C5a, they demonstrated activity at C3a receptor, but not C5a agonist  and a ch agonist . In factagonists . This mabehavior . Finch ear range . Work byar range  also repreceptor . In thisRecent evidence  has showSystematic and sequential amino acid replacements and truncations of the active peptide sequences may provide further insight into the key residues critical for the agonist activity observed at the C3a receptor. This knowledge will be key in optimizing the potencies of these peptides further, in the hope of developing them as therapeutics .1 hits, and hits for C3a receptor indicates promiscuity of C3a receptor and C5a1 ligand binding sites. Presumably the smaller size of these peptides enabled binding to C3a receptor. These C3a receptor agonist hits could be extended towards acute therapies to limit neutrophil mobilization in disease (In conclusion, our screen identified several potent small peptide C3a receptor agonists. The lack of C5a disease  or used


Cytochrome P 450 1A1 (CYP1A1), Glutathione -S -transferase M1 (GSTM1) and T1 (GSTT1) polymorphisms from India are meagre, and reports from Andhra Pradesh are lacking.Lung cancer is one of the most preventable causes of death globally both in developed and developing countries. Although it is well established that smokers develop lung cancer, there are some smokers who are free from the disease risk. The predisposition to lung cancer is attributed to genetic polymorphisms in xenobiotic metabolizing genes. Reports on assessment of xenobiotic metabolizing genes like CYP1A1, GSTM1 and GSTT1 in NSCLC patients and healthy individuals specific to population of Andhra Pradesh, a South Indian state was attempted by multiplex PCR and RFLP, and this is the first study which tried to correlate oxidative stress with the polymorphisms in xenobiotic metabolizing genes. Results showed that CYP1A1 m1 ‘CC’ genotype was significantly associated with lung cancer susceptibility with a 2.3-fold risk, CYP1A1 m2 ‘AG’ gene polymorphisms with 8.8-fold risk and GSTT1 (−/−) genotype demonstrated a twofold risk of disease susceptibility.Assessment of polymorphisms in GSTT1 and super oxide dismutase activity; CYP1A1 m1, m2 and GSTT1 with glutathione peroxidase activity; CYP1A1 m1 and GSTM1 with melondialdehyde levels; and CYP1A1 m1 and GSTT1 with 8-oxo-7,8-dihydro-2′-deoxyguanosine. A higher risk of lung cancer seems to be associated with combined gene polymorphisms of phase I and phase II enzymes than that ascribed to single gene polymorphism.A combined role of genetic polymorphisms and smoking status can be attributed for the cause of lung cancer. Further, the association between oxidative stress and genetic polymorphisms showed a correlation between The online version of this article (doi:10.1186/s40001-016-0209-x) contains supplementary material, which is available to authorized users. In Phase II, the modified xenobiotics are conjugated to polar compounds facilitated by enzymes such as glutathione S-transferases [) which are divided into five classes , and catalyse the conjugation of highly reactive PAHs to soluble glutathiones [GSTM1 preferentially detoxifies carcinogens (epoxides and hydroxylated derivatives) derived from tobacco, whereas GSTT1 causes the biotransformation of many toxins such as butadiene and ethylene oxides (ingredients of tobacco smoke) [Xenobiotic metabolism is the process of detoxification of endogenous or exogenous carcinogens/poisons and occurs in two phases. In Phase I, csferases . Among tsferases . The phaathiones . Among tCYP1A1, GSTM1 and GSTT1 have been projected as the potential modulators of cancer susceptibility [CYP1A1 and GSTM1 polymorphisms in lung cancer was reported [GSTT1 deficiency was demonstrated (GSTT1 null) not to increase the risk of lung cancer [CYP1A1 and GSTM1 gene polymorphisms were found to vary among different ethnic populations [CYP1A1 2A and CYP1A1 2C genetic polymorphisms are common, whereas in Caucasians, the variation in CYP1A1 2C is rare [GSTM1 null type is more common in Asians than in Caucasians [CYP1A1 polymorphism, tobacco smoking and lung cancer was found to be high in Japanese and Chinese populations, whereas the same was not observed in Caucasians [GSTM1 null genotype with squamous cell and small cell carcinomas in Asians was found to be significant [GSTM1 null genotype with CYP1A1 polymorphisms augmented lung cancer risk [Among all the cancers, carcinoma of the lung is responsible for the high death rate throughout the world . Althougtibility . Althougreported , 12. Howg cancer , 14. Theulations , 16. Amo is rare . Similarucasians . Null geucasians , 19–22. nificant , 24. Furcer risk , 26.CYP1A1 gene polymorphism in 100 patients with lung cancer was assessed, and a 2.68-fold risk was observed for CYP1A1 2C allele and in the presence of a single copy of the variant CYP1A1 (CYP1A1 * 1/2A) and for null GSTT1 genes, a threefold increased risk of lung cancer was demonstrated [CYP1B1 and GSTM1 polymorphisms in the population [CYP2E1 polymorphisms in six ethnic groups of South Indian population were demonstrated [CYP1A1 MspI homozygous variant allele and GSTT1 null deletion frequency were significantly higher in smoking-induced lung cancer patients compared with other populations [CYP450 and GST gene polymorphisms and the risk of lung cancer include (1) limited number of subjects from different areas which are not representative of the entire population; (2) subjects exposed to different environmental conditions; and (3) different gene polymorphisms being evaluated. Besides ethnic background, life style and dietary habits also contribute to the increased risk of lung cancer. The dietary habits, environmental factors and tobacco consumption vary between the Northern and Southern regions of India. Tobacco consumption is rampant in both North and South Indian populations [In the Indian context, studies on association of lung cancer and genetic polymorphisms are limited. In a North Indian cohort, the risk of nstrated . Anotherpulation . CYP2E1 nstrated . A studyulations . The Souulations . AlthougBesides genetic factors, biochemical markers such as oxidative stress and antioxidant responses were also implicated in the development of lung cancer which showed changes in the oxidant and antioxidant statuses in the peripheral lymphocytes of non-small cell lung cancer (NSCLC) patients . Howevern = 250) were enroled from the general population of the same geographical region. Routine medical check-up was conducted, and history of illness was recorded by a health practitioner. Those who appeared apparently healthy without any history of cancer or other chronic diseases were considered as normal. The co-morbid conditions among controls included 8 (3.2 %) of them being diabetic, 11 (4.4 %) being hypertensive, and none having hypothyroidism. Study subjects who were used to smoking at the time of diagnosis were considered as smokers and those who had smoked at least 100 cigarettes in their life time were considered as ex-smokers. Among the NSCLC smokers, 51 (48.11 %) and 47 (44.34 %) consumed cigarettes and bidis, respectively, and 8 (7.55 %) consumed both. In the case of NSCLC patients who are ex-smokers, the cigarette and bidi consumers were 26 (61.9 %) and 15 (35.72 %), and 1 (2.38 %) consumed both bidi as well as cigarette. Pack years were computed as the number of cigarettes smoked per day multiplied by the duration of smoking in years, and the average tobacco consumption was expressed in pack years. Among the control smokers, 48 (76.19 %) consumed cigarettes, while 15 (23.81 %) were bidi smokers. In case of ex-smokers, 8 (66.66 %) were cigarette smokers, while 4 (33.34 %) were bidi smokers.The subjects of the present study included 246 newly diagnosed and previously untreated NSCLC patients referred to Indo-American cancer Hospital from various regions of Andhra Pradesh, India during the period June 2008–2012. 98.2 % of the patients and all the control subjects included in our study were natives of Andhra Pradesh. All the patients were rated as positive for NSCLC by histological analyses and were classified using revised lung cancer staging system . The co-The study was carried out with the approval of Institutional Ethics Committees of Indo-American Cancer Hospital and Institute of Genetics and Hospital for Genetic Diseases. Educated and informed consent was obtained from all the subjects of the study. A standard questionnaire was used to document the socio-demographical characteristics such as age, sex, lifestyle , occupational exposure , history of smoking, number of cigarettes per day and duration of smoking.2 ml of whole blood was collected in vacutainers (BD Biosciences) containing ethylenediamine tetra acetic acid (EDTA) for DNA isolation, and 3 ml was collected in heparinized vacutainers for the assessment of oxidative stress markers from healthy controls and NSCLC patients. Genomic DNA was isolated  from 300 μl of whole blood and was stored in −80 °C until further use.CYP1A1 m1 and m2 genes (rs4646903 and rs1048943) was carried out as described earlier [2, 1 U Taq DNA polymerase with 1× reaction buffer . The PCR cycle consisted of 1 min at 94 °C, 1 min at 61 °C (for CYP1A1 m1)/63 °C (for CYP1A1 m2) and 1 min at 72 °C with initial denaturation of 5 min at 94 °C and final extension of 10 min at 72 °C. The PCR amplicons generated for m1 (340 bp) and m2 (204 bp) were subjected to restriction digestion. Msp1 and BsrD1 restriction enzymes were used to detect polymorphisms in the CYP1A1 m1 and m2, respectively. The reaction mixtures were incubated at 37 °C for 12 h, electrophoresed on 3.0 % agarose gel and stained with ethidium bromide  for visualization. All the sampling experiments were done in duplicates. Restriction digestion was repeated in cases which were unclear. Positive samples were included in each run of PCR as well as restriction digestion to ensure that the samples were properly digested.Genotyping for  earlier . The priGSTM1 and GSTT1 gene deletions (rs4025935 and rs71748309) were analysed simultaneously by multiplex PCR [GSTM1 deletion, the primers used were GSTM1 F (5′-GAACTCCCTGAAAAGCTAAAGC-3′) and GSTM1 R (5′-GTTGGGCTCAAATATACGGTGG-3′). For GSTT1, the primers used were GSTT1 F (5′-TTCCTTACTGGTCCTCACATCTC-3′) and GSTT1R (5′-TCACCGGATCATGGCCAGCA-3′). The PCR amplicons were electrophoresed on a 4 % agarose gel, stained with ethidium bromide, and the results were documented using a gel documentation system (Bio-Rad). The presence of GSTM1 and that of GSTT1 genes were indicated by the resulting 215- and 480-bp PCR amplicons, respectively. A DNA sample with GSTM1 and GSTT1 alleles present was run as a positive control in each run. As an internal control, human albumin gene (HAB) was amplified (350 bp) using the primers HAB F (5′-CAACTTCATCCACGTTCACC-3′) and HAB R (5′-GAAGAGCCAAGGACAG GTAC-3′) for the authentication of multiplex PCR.The 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels in the urine samples of healthy controls and NSCLC patients were measured in 125 patients and 100 controls using commercially available kits . Lipid peroxidation products were measured in the plasma of 246 patients and 250 controls as described earlier . Red celt test. ANOVA  was used for comparisons among the three or more groups. Multiple regression analysis was done to investigate the associations of the independent variables. Pearson correlation analysis was used for testing relationships between genotypes in patients and controls. The results were considered to be significant at p values of less than 0.05 (indicated by *). The differences in the distribution of genotype frequencies were calculated using the χ2 test. Genotype frequencies were checked for deviation from Hardy–Weinberg equilibrium and were not significantly different from those predicted. Odds ratios and 95 % confidence interval (95 % CI) were calculated to assess the relationship between CYP1A1 and GST gene polymorphisms.The data were analysed using the SPSS 15.0 program . The significance of the differences between controls and patients end point means were analysed using Student’s n = 246) who had no previous history of diagnosis and healthy controls (n = 250) included in this study are given in Table p ≤ 0.001).The general characteristic features of NSCLC patients , 200 and 140 bp for homozygous minor (CC), respectively  with 2.25-fold risk of disease susceptibility.The homozygous major (TT), heterozygous (TC) and homozygous minor (CC) genotype frequencies of CYP1A1 m2 homozygous major (AA), heterozygous (AG) and homozygous minor (GG) genotypes in healthy controls were 78.4, 13.6 and 8 %, whereas the same were 29.67, 58.14 and 12.19 % in NSCLC patients, respectively  with an estimated 8.8-fold risk of developing lung cancer in individuals with this genotype.The frequencies of p = 0.02, χ2 = 4.79, OR 12.48, CI 0.69–224.5) in patients demonstrated a 12-fold risk of developing lung cancer compared to the controls. The combination of ‘CC/AG’  showed a 6.9-fold risk of susceptibility to lung cancer, while heterozygous m1/m2 ‘TC/AG’  presented with a five fold risk.Healthy controls displayed higher percentage of homozygous major (TT/AA) genotype combination (44.8 %) followed by the combination of homo/hetero , and hetero/homo , among all SNP combinations  (Table ts Table . GSTT1 g2) Table .Table 4GGSTM1 and GSTT1 polymorphisms Wild/Wild, Wild/Null, Null/Wild and Null/Null in healthy control were 66.80, 10.40, 23.60 and 1.6 %, respectively, whereas in NSCLC patients the frequencies were 59.34, 14.63, 21.95 and 4.06 %, respectively. It was clear that the GSTM1 Wild/GSTT1 Wild genotype followed by GSTM1 Null/GSTT1 Wild combinations were more predominant in both healthy controls and NSCLC patients  and GSTM1 Null/GSTT1 Null  combinations, indicating a 1.97- and 2.6-fold risk of disease susceptibility, respectively   and CYP1A1 m2 (A/G)  genotypes had an increased lung cancer. Lung cancer patients who smoked and having CYP1A1 m1 T/C, C/C and CYP1A1 m2 A/G, G/G and GSTT1 null (−/−) genotypes were at higher risk compared to the controls  wild and GSTT1 (+/+) wild were the predominant genotypes ; CYP1A1 m2 A/G + GSTM1 null (−/−); and CYP1A1 m2 G/G + GSTT1 wild (+/+) with estimated risks of sixfold, sixfold and 10.5-fold, respectively  showed a 19-fold risk; CYP1A1 m1 T/C + CYP1A1 m2 G/G + GSTM1 wild (+/+) showed 11.6-fold risk and CYP1A1 m1 G/G + CYP1A1 m2 A/G + GSTM1 wild (+/+) showed a 10.5-fold risk of disease susceptibility  vs null (−/−); GPx activities between CYP1A1 m1 T/T vs T/C and T/T vs C/C; CYP1A1 m2 A/A vs A/G and A/A vs G/G genotypes and GSTT1 wild vs null genotypes. Mean MDA levels were significantly different with respect to CYP1A1 m1 and GSTM1 genotypes. The difference in 8-OHdG levels between the genotypes was significant only for CYP1A1 m2 gene and GSTT1 gene polymorphisms. The difference between the genotypes of different genes for SOD, GPx, MDA and 8-OHdG levels were not significant in the control group.The impact of ty Table , Glutathty Table , MDA  was reported [CYP1A1, GSTM1 and GSTT1 polymorphisms with the risk of NSCLC in the population of Andhra Pradesh.Xenobiotic metabolising enzymes expedites purging of a variety of toxic substances, thereby gaining prominence in the pathophysiology of cancer. Hence, gene polymorphisms in the enzymes that are intricate in the metabolism of carcinogens may regulate an individual’s predisposition to cancer including lung cancer . Besidesflicting . In the reported , suggestCYP1A1 m1 homozygous minor genotype (C/C) was recorded among NSCLC patients. Association of lung cancer risk with homozygosity of CYP1A1 variant alleles was reported in Chilean and Caucasian populations [CYP1A1 polymorphism with lung cancer risk was reported [CYP1A1 m2 heterozygous ‘AG’ genotype. Similar observations (higher frequency of CYP1A1 m2 (A/G) allele) were reported in lung cancer patients from Korea [CYP1A1 m2 (G/G) allele frequency was demonstrated to be lower in Caucasians than Japanese [CYP1A1 m2 genotypes were on the higher side in Chilean lung cancer patients [CYP1A1 plays an important role in the aetiology of lung cancer in the population of Andhra Pradesh state.In the present study, a high frequency of ulations –41. Likereported , 42, 43.om Korea . On the Japanese . Heterozpatients . The higpatients . ResultsGSTM1 wild and null genotypes were detected, respectively, in 73.98 and 26.1 % of lung cancer patients. A similar trend was observed in the healthy controls . Similarly, GSTM1 null genotype was not associated with the increased risk of lung cancer, and the proportions of the NSCLC patients and healthy controls exhibiting GSTM1 null genotype were apparently equal. Similar trends were observed in the South and North Indian population cohorts [GSTM1 null or deletion genotype was reported to be prevalent in about 50 % of Caucasians, 33 % of African Americans and 45 % of Japanese [GSTT1 null genotype was high in lung cancer patients compared to the controls, which is consistent [GSTT1 deletion polymorphisms was reported in 13–28 % of Caucasians [GSTM1 and GSTT1 were found to be 22.4 % and 17.6 %, respectively, in the South Indian population; 54 % and 13 %, respectively, in the East Indian population [GSTT1 polymorphism rather than GSTM1 genotype.In the current study,  cohorts , 46. On Japanese  lung cannsistent , 41, 48 nsistent , 49 withucasians . Similarpulation ; 41 % anCYP1A1m1 T/C, C/C or CYP1A1m2 A/G, G/G genotypes and GSTT1 null genes were at higher risk of disease susceptibility to lung cancer. A threefold risk of lung cancer associated with CYP1A1 m1 genotype was reported [CYP1A1 m1 allele was demonstrated. In the present study, no risk of lung cancer was associated with GSTM1 null genotype in smokers and non-smokers. The association between GSTM1 genotype and cumulative smoking is controversial [CYP1A1 polymorphisms rather than GSTM1 polymorphisms and smoking contribute to the higher risk of lung cancer. Our results are in accordance with another study from North India where the relative risk for the carriers of variant CYP1A1 genotypes was high [A majority of the patients included in our study were bidi smokers (made of crude particles of dried tobacco leaves wrapped in a tendu or temburni leaf and rich in tar and nicotines); and bidi smoking is known to generate stronger carcinogen load than cigarette . Our datreported . Furtherreported , 42 and reported , 53 withoversial . Strongeoversial . Resultswas high . The disCYP1A1 m2 and GSTM1) or three  genotypes had a profound effect on susceptibility to lung cancer up to 14-fold depending on the genotypic interaction. Correlations between lung cancer risk and combinations of CYP1A1, GSTM1 and GSTT1 is of particular interest since these genotypes suggest that alterations in the action of phases I and II enzymes lead to defective metabolism of xenobiotic compounds, thereby potentiating the cancer risk. It was suggested that individuals having polymorphisms in more than one of these genes are at higher risk than having for only one gene [CYP1A1 and GSTM1 null genotypes and increased lung cancer risk was evidenced in summarized data of 46 studies of Chinese populations [CYP1A1 and GSTM1 genes [CYP1A1, GSTM1, GSTP1 and GSTT1 polymorphisms and their association to lung cancer in a cohort of North Indian population was reported [GSTM1 null, GSTT1 null, CYP1A1 homozygous major genotype combination and a 3.5-fold increased risk, although not statistically significant, in individuals possessing the GSTM1 null, GSTT1 null, CYP1A1 homozygous minor genotype combination [CYP1A1, GSTM1 and GSTT1 genetic polymorphism with NSCLC.Analysing multiple gene interactions provide better understanding to assess the risks associated with lung cancer risk. In our study, the combinations of two  results in no functional enzymatic activity, thereby failing to detoxify several xenobiotics including tobacco smoke constituents and finally leading to increased generation of ROS and lowered GPx activity and The role of oxidative stress in the pathophysiology of a variety of cancers including lung cancer was well documented . Genetic studies –65 demon studies –68. It iThe limitations of our study are that the healthy controls and NSCLC patients were in the ratio of 1:1, and some of the NSCLC patients had co-morbid conditions. Further studies with large sample size can provide concrete data on the combined effect of genetic polymorphisms and NSCLC. The effect of co-morbid conditions to the contribution of gene polymorphisms observed cannot be ruled out.In conclusion, we report that in the population of Andhra Pradesh, the South Indian state, a higher risk of lung cancer was associated with combined gene polymorphisms of phase I and phase II enzymes, than with a single susceptible gene. Risk assessment of NSCLC can be related to gene polymorphisms and oxidant status. This finding may have an important implication for the prevention of smoking and occupational exposures in susceptible individuals.


Immunological status is differentiated with age, influencing treatment and outcome . The aiThe study comprised 16 polytrauma patients admitted to the ICU, aged 78 ± 8 (Group I) and 16 with arterial disease, aged 74 ± 5 (Group II). Ten milliliters of peripheral blood were collected from each patient, divided into two tubes with/without anticoagulant. Diluted 1:10 whole-blood samples were stimulated with 500 pg/ml LPS, at 37°C, for 4 hours. Serum and cell culture supernatants (CCSP) were removed and stored at -70°C. TNFα and IL-6 were measured in serum and CCSP by ELISA.P < 0.001 and IL-6, P < 0.001). Ex vivo cytokine release showed the opposite direction. There was a significantly lower TNFα and IL-6 release for Group I  compared with Group II. TNFα ex vivo release from the samples of Group II was >300 pg/ml. See Figures Serum proinflammatory cytokines were significantly elevated after severe trauma against control group (TNFα, Older patients showed adequate immunological response, considering the limit of 300 pg/ml. The incidence of severe trauma was involved in the downregulation of immune activity and should be considered. Group I patients do not have the opportunity to precondition their immune status. Group II patients can better compensate operative therapies.


Many cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks.We report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay, based on the substrate competitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks and an increase in permissive marks at the promoter and transcription start site of re-expressed genes, while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3. The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2.We have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.The online version of this article (doi:10.1186/s13148-015-0118-9) contains supplementary material, which is available to authorized users. EZH2 along with EED and SUZ12 are the indispensible core components of the Polycomb Repressive Complex (PRC2) responsible for maintenance of the repressive epigenetic mark H3K27me3: trimethylation of lysine 27 of histone 3 , 5 and sEHMT2  and the highly homologous EHMT1  are HKMTs partly responsible for mono- and di-methylation of lysine nine of histone 3 ; repressive chromatin marks found on the promoter regions of genes that are often aberrantly silenced in cancer . EHMT2 iIn addition to its role in methylating H3K9, EHMT2 has been shown to be able to methylate H3K27 , 27. It EZH2 and EHMT2 expression on epigenetic regulation at select target genes, compared to knockdown of either gene alone  had limited effects on mRNA levels of these target genes. However, double knockdown of EZH2 and EHMT2 had dramatic effects on the level of SPINK1 mRNA, a gene which was not upregulated by silencing of EZH2 or EHMT2 individually. Thus, for at least certain genes, dual reduction in EZH2 and EHMT2 levels is necessary to observe marked changes in target gene expression 48 h following knockdown.SiRNA knockdown in the MDA-MB-231 breast tumour cell line was used to examine the effect of combined inhibition of one Fig. . Knockdot manner . KnockdoKRT17, FBX032 and SPINK1 and the H3K27 demethylase JMJD3 .Next, the effects on cell viability of GSK343 and UNC0638 used alone or in combination were examined Fig. . Treatme343 Fig. . This isBoth EZH2 and EHMT1/2 belong to the SET-domain superfamily , the catA compound library based on the selective BIX-01294 EHMT2 inhibitor was synthesised and characterised analogously to previously reported methods , 25, 32.To further evaluate the three hit compounds identified, we treated MDA-MB-231 cells for 48 and 72 h at various concentrations of compounds and examined gene expression effects Fig. . All hitChromatin immunoprecipitation (ChIP) experiments were carried out on treated MDA-MB-231 cells to verify that the detected gene upregulation is indeed due to chromatin remodelling Fig. . We testp = 4.53x10−43) and the validation array (p = 1.99x10−49). GSK343 and UNC0638 also both showed a significant upregulation of EZH2 target genes  or UNC0638, (p = 1.7x10−4).Agilent microarrays were used to perform gene expression profiling in MDA-MB-231 breast cancer cells after 24 h of treatment with the hit compound HKMTI-1-005, the EZH2 inhibitor GSK343  and EHMTnes Fig.  though tThe same enrichment tests were repeated using target gene sets identified in an EZH2 siRNA knockdown study in another breast cancer cell line, MCF-7 . Almost The microarray data showed a clear upregulation of the levels of SPINK1 mRNA  but not the selective EZH2 (GSK343) or EHMT2 (UNC0638) inhibitor compound . Consistent with induction of apoptosis-related genes, hit compound HKMTI-1-005 induces apoptosis in MDA-MB-231 cells in a dose-dependent manner, as measured by Caspase 3/7 activity  database . The BenHMT2 UNC038 inhibiThe IC50 of the dual inhibitor compounds HKMT-1-005 in cell viability (MTT assay) or clonogenic growth assays of MDA-MB-231 cells compared to the starting point for the chemical library BIX-01294, as well as UNC0638 and GSK343, are shown in Table The anti-proliferative effect of HKMTI-1-005 on a small panel of breast cancer cell lines was determined, with IC50 values in the range 2–10 μM Table . All of 3H]S-adenosyl-L-methionine (SAM) to a biotinylated-H3 (1–25) peptide substrate, mediated by EHMT2. A comparable PRC2 enzymatic assay was employed here to assess biochemical inhibitory activity of our hits against EZH2. A trimeric PRC2 complex (EZH2:EED:SUZ12) was employed in this assay, along with a biotinylated-H3 (21–44) peptide substrate. This revealed HKMTI-1-005, HKMTI-1-011 and HKMTI-1-022 to have PRC2 IC50 values of 24, 12 and 16 μM, respectively, under these assay conditions , further confirming a substrate competitive inhibitory mode for this chemotype.Perhaps more importantly, in accordance with our design rationale, mechanism of inhibition studies on representative hit HKMTI-1-005 revealed it to have a well-defined, peptide substrate competitive mechanism of action  and 1 DNA methyl-transferase (DNMT)  cell assay based on the re-expression of epigenetically silenced genes, we report the identification of hit compounds that phenocopy the effects of dual EZH2/EHMT2 pharmacological inhibition and dual SiRNA gene knockdown.The recently reported specific EZH2 inhibitors are all co-factor competitive, the majority of which have converged to a common chemotype , media was removed and 1.5 ml of TRIzol (Invitrogen) was added directly to lyse cells and RNA was isolated according to the manufacturer’s instructions. Reverse transcription was done using the SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instructions. Each measurement was done in triplicate, and the List of Primers can be found in Additional file 5 cells/6 cm well and treated for 48 h with siRNAs given in Additional file SiRNA experiments were carried out on the MDA-MB-231 cell line using Qiagen reagents, according to the manufacturer’s instructions. In brief, cells were seeded at a density of 1 × 10ChIP was accomplished using Dynabeads Protein A (Invitrogen) according to , except 2 incubator. The MTT-product was solubilised with 100 μl DMSO, and for 1 h, incubated in the dark at room temperature. The optical density was read at 570 nm with PHERAstar.For clonogenic assays, cells were treated with compound for 48 h, media were removed and replaced with fresh media for a further 12 days to measure colony formation. For cell viability assays, lymphoma cells were plated at 20,000 cells in 200 μl per well in U-bottom 96 well plates in RPMI medium +20 % FCS. Cells were re-suspended 48 h later, diluted 10-fold in PBS + propidium iodide (PI) and the concentration of PI negative cells was counted using an Attune flow cytometer with autosampler. Breast cancer cells were seeded at a density of 10,000 cells/well in a sterile 96 clear-well plate with 150 μl of DMEM (+10 % FCS and 2 mM L-Glutamine). Each compound treatment was performed in triplicate for 72 h at concentrations of 100 nM and 1, 5, 10 and 50 μM in 100 μl of full-medium. After 72 h, 20 μl of MTT solution  was added to the medium and incubated for 4 h at 37 °C in a CO5 were treated with HKMTI-1-005 (1–7.5uM) for 48 h. Following lysis in Triton Extraction Buffer , 1/1000 protease inhibitor), nuclei were re-suspended in 0.2 N HCL at a density of 4 x 107 nuclei per millilitre and incubated overnight at 4 °C to acid-extract the histones, before being centrifuged at 6500g for 10 min at 4 °C. Protein concentration was determined using the Bradford assay. H3K27me3, H3K9me3, H3K9me2, H3K9me and total H3 protein expression levels in the histone extract samples were determined using Western blot analysis using H3K27me3 , H3K9me3 , H3K9me (1:1000) and H3  antibodies. After washing, the membrane was incubated with a horseradish peroxidase-labelled secondary antibody . The membrane was incubated for 1 min with 5 mL of Pierce ECL Western blotting substrate (Thermo Scientific). Images were captured using Konica Minolta SRX101A Tabletop X-Ray film processor.MDA-MB-231 cells seeded in 6 well plates at a cell density of 3 x 10Agilent 80k two-colour microarrays were used to profile gene expression changes induced by treatment with drug compounds in MDA-MB-231 cells, both at 24 and 48 h. In the initial microarray experiment, three replicates were used for each drug, time combination, and in the validation study, four replicates were used. A separate untreated control sample was used for comparison with each replicate. Sample labelling, array hybridization and scanning were performed by Oxford Gene Technologies, according to manufacturer’s instructions. Feature extracted files were imported into GeneSpring (Agilent), and data was normalised to produce log2 ratios of treated/untreated for each replicate of each drug, time combination.t statistics for differential expression across the replicates for each drug treatment. After multiple testing adjustments by the Benjamini-Hochberg method, p < 0.1 was used to denote significant differential expression in the initial microarray experiment and p < 0.05 in the validation experiment.Normalised log2 gene expression ratios were analysed using LIMMA  to obtailimma. The same method was used to evaluate systematic up- or downregulation of pathways as annotated in ConsensusPathDB [A list of EZH2 targets in MDA-MB-231 cells were taken from . StatistusPathDB . FurtherusPathDB  for explA meta-analysis of 18 microarray experiments were carried out as described in ‘Supplementary Methods’, resulting in the list of consensus EZH2 target genes given in Additional file


Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.A new PLA Depending on the molecular taxonomy, intracellular and secretory PLA2s are currently classified in six to twelve groups [2s are enzymes of 13–18 kDa with 5–8 disulfide bonds whose activity requires millimolar Ca2+ concentration [Phospholipase A2 groups show highly conserved molecular regions and similar three-dimensional structure [ Bothrops sp. envenomation is often due to the PLA2 activity [ in vitro experiments and are frequently associated with PLA2 of bothropic venoms [Despite the variety of the local and systemic pathophysiological effect, such as myotoxicity, neurotoxicity, anticoagulation, hemolysis, hypotension, platelet aggregation inhibition, and bactericidal and proinflammatory activities, PLAtructure . The majactivity –5. Althoc venoms –14.B. pauloensis is found in humid and cool regions in the central and Southwest of the state of São Paulo [ Bothrops snakes, 18% (52 cases) were caused by B. pauloensis [2 from the venom of B. pauloensis. We also investigated whether this isolated PLA2 possesses neurotoxic activity.ão Paulo , 16 and ão Paulo . From thVenom was purchased from Sigma Chemical Co. .Solvents (HPLC grade), 4-nitro-3-octanoyloxy-benzoic acid, sequencing grade bovine pancreatic trypsin and other reagents were also obtained from Sigma Chemical Co. .2 from B. pauloensis venom was purified by reverse phase HPLC, according to the method described by Ponce-Soto et al. [μL of buffer A (0.1% TFA) and centrifuged at 4500 g; the supernatant was then applied to a μ-Bondapak C18 column , previously equilibrated in buffer A for 15 min. The protein elution was then conducted using a linear gradient  of buffer B (66.5% acetonitrile in buffer A) at a constant flow rate of 1.0 mL/min. The chromatographic run was monitored at 280 nm of absorbance. The purity and PLA2 activity were monitored according to Sections Bp-13 PLAo et al. , with mi2, under reducing and nonreducing conditions [α-lactoalbumin: 14 .Tricine SDS-PAGE in a discontinuous gel and buffer system was used to estimate the molecular mass of Bp-13 PLAnditions . The use2 sample (30 μg) was hydrolyzed at 105°C for 24 hours, in 6 M HCl (Pierce sequencing grade) containing 1% phenol (w/v). Hydrolyzates were reacted with 20 μL of derivatized solution  for one hour at room temperature. Afterwards, PTC-amino acids were identified and quantified by HPLC, by comparing their retention times and peak areas with those from a standard amino acid mixture (Sigma-Aldrich).Amino acid analysis was performed on a Pico-Tag Analyzer (Waters Systems), as described by Heinrikson and Meredith , with mi2 was analyzed by MALDI-TOF mass spectrometry using a Voyager-DE PRO MALDI-TOF apparatus  equipped with a pulsed nitrogen laser . The amount of 1 μL of sample in 0.1% TFA was mixed with 2 μL of sinapinic acid matrix . The matrix was prepared with 30% acetonitrile and 0.1% TFA and its mass analyzed under the following conditions: 25 kV accelerating voltage, the laser fixed at 2890 μJ/cm2, 300 ns delay, and linear analysis mode [Molecular mass of intact native and alkylated Bp-13 PLAsis mode .2, alkylated tryptic peptides were fractionated by RP-HPLC, manually collected, lyophilized, and resuspended in 80% H2O, 20% acetonitrile and 0.1% TFA. One peptide was introduced separately into the mass spectrometer source using a syringe pump at a 500 nl/min flow rate. Before performing a tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) was acquired for each HPLC fraction over the mass range of 400–2000 m/z, aiming to select the ion of interest. Subsequently, these ions were fragmented in the collision cell (TOF MS/MS mode). Different collision energies were used depending on the mass and charge state of the ions. The resulting product-ion spectra were acquired with the TOF analyzer and deconvoluted using the MassLynx-MaxEnt 3 algorithm (Waters). Singly charged spectra were manually processed using the PepSeq application included in MassLynx.For de novo sequencing of N-terminal, the first 52 amino acids from Bp-13 PLA2 activity was measured using the assay described by Cho and Kézdy [μL of buffer , 20 μL of synthetic chromogenic substrate 4-nitro-3- (octanoyloxy) benzoic acid 3 mM, 20 μL of water, and 20 μL of PLA2 fractions (1 mg/mL) or whole venom (1 mg/mL) in a final volume of 260 μL. After adding the samples, the mixture was incubated for up to 40 min at 37°C, absorbance reading at intervals of 10 min. The enzyme activity, expressed as the initial velocity of the reaction (V0), was calculated based on the increase of absorbance after 20 min.PLAnd Kézdy  and Holznd Kézdy  modified2 were determined by incubating the four reaction buffers with different pH ranging from 4 to 10 and at different temperatures, respectively. The effect of substrate concentration  on enzyme activity was determined by measuring the increase of absorbance after 20 min in optimum pH and temperature. The effect of different concentration of Ca2+ on Bp-13 PLA2 enzymatic activity was tested by preincubating the enzyme with different ion concentrations  at 37°C for 30 minutes prior to standard experiment. Also, the effects of different divalent ions  were tested in presence (1 mM) or absence of Ca2+. Finally the effect of urea (4 M) on the enzymatic activity was tested by preincubating Bp-13 for 30 minutes at 37°C.The pH and optimal temperature of PLAAll assays were done in triplicate and the absorbance at 425 nm was measured using a VersaMax 190 multiwell plate reader .2. Mice and young chick were sacrificed by halothane inhalation. The hemidiaphragm (PND) and extensor digitorum longus (EDL) muscles isolated from mice and biventer cervicis muscle (BC) isolated from chick were mounted according to Bülbring [Male Swiss mice (Mus musculus) weighing 20–30 g and 8-day-old young chick (Hy Line W36) were used for twitch-tension studies in presence of Bp-13 PLABülbring  and GinsBülbring , respect2-5% CO2) Tyrode solution, whose composition in mM was NaCl 137, KCl 2.7, CaCl2 1.8, MgCl2 0.49, NaH2PO4 0.42, NaHCO3 11.9, and glucose 11.1, pH 7.4, 37°C. A supramaximal pulse  delivered by a Grass S48 electronic stimulator  was applied through electrodes placed around the motor phrenic nerve (PND) and tendon (EDL). For both preparations, the isometric muscle tension was recorded using a force-displacement transducer Load Cell BG 50 g  coupled to a physiograph . The PND preparations were allowed to stabilize for at least 20 min before the addition of Bp-13 toxin at different concentrations: 0.71 μM (10 μg/mL), 1.42 μM (20 μg/mL), 3.56 μM (50 μg/mL), and 7.12 μM (100 μg/mL). The EDL preparations were incubated with Bp-13 toxin at 3.56 μM.The PND and EDL mammal preparations were suspended under a constant resting tension of 5 g/cm and 0.5 g/cm, respectively, in a 5 mL (for PND) and 3.5 mL (for EDL) organ bath containing aerated (95% OμM) were incubated with Bp-13 toxin at 1.42 μM and 3.56 μM concentrations and the twitch response was recorded under direct muscle stimulation at supramaximal pulses of 70 V, 0.1 Hz at 2 ms duration. The effect of divalent ions, Mg2+, Mn2+, Sr2+, and Cd2+ (10 mM), on the Bp-13 PLA2 activity was done by replacement of Ca2+ (1.8 mM) in the nutritive Tyrode solution; replacement of Ca2+ by Sr2+ (4 mM) was also assayed. The effect of temperature (5 to 60°C) during 20 min was read at 425 nm.Some curarized PND preparations , aerated with carbogen (95% O2-5% CO2) at 37°C. A bipolar platinum ring electrode was placed around the muscle tendon, within which run the motor nerve trunk. Field stimulation using a Grass S48 stimulator set at 0.1 Hz, 0.2 ms, and 4–6 V was applied and the muscle contractions and contractures were recorded isometrically via force-displacement transducer coupled to a physiograph. The muscle responsiveness to exogenously applied acetylcholine  and KCl (13.4 mM) was recorded in the absence of field stimulation both prior to toxin addition and at the end of the experiment (120 min). The BC preparation was stabilized for at least 15 min before addition of Bp-13 at concentration of 3.56 (50 μg/mL) and 7.12 μM (100 μg/mL). The results were compared with control BC preparations incubated with Krebs solution alone.The biventer cervicis (BC) preparations were suspended in a 5 mL organ bath containing Krebs solution  in the presence of low Ca2+ concentration (1 mM) decreased enzyme's activity; the substitution of Ca2+ by Mn2+, Mg2+, Sr2+, or Cd2+ (10 mM) in the absence of Ca2+ (0 mM) also reduced PLA2 activity , 3.56 μM (n = 6), and 1.42 μM (n = 3) of Bp-13 PLA2 was 18 ± 1 min, 28 ± 3 min, and 120 ± 4 min, respectively (P < 0.05); Bp13 at 0.71 μM concentration induced a 25% paralysis only after 120 ± 2 min relative to control (P < 0.05)  in the nutritive Tyrode solution in the absence of Ca2+ or presence of 1 mM Ca2+ showed significant loss of the catalytic activity of the toxin indicating that these divalent ions cannot replace the Ca2+ for the development of the PLA2 catalytic activity. The replacement of 1.8 mM Ca2+ by 4 mM Sr2+ in the Tyrode solution prevented the blocking effect of Bp13 PLA2 (3.56 μM) since the twitch-tension response showed an amplitude of 83.7 ± 14% after 120 min incubation which was not different from baseline of the control preparations (Assays to study the neuromuscular activity of Bp-13 PLA < 0.05) . The catarations . The fin2 showed that the optimal enzymatic activity occurred around 38°C (μM) and under direct electrical stimulation showed that Bp-13  was able to cause a significant contracture followed by blockade of the contractile response  on the catalytic activity of Bp-13 PLAund 38°C , insert.μM Bp13 PLA2 concentration was 120 min ± 2 min (2+ (1.8 mM) was replaced by Sr2+ (4 mM). The Bp-13 (3.56 μM) addition caused a significant blockade of the twitch tension which achieved 90% after 80 min of toxin addition in the normal Tyrode solution. The replacement of 1.8 mM Ca2+ by 4 mM Sr2+ also prevented completely such neuromuscular blockade induced by the toxin (3.56 μM) which was sustained until the end of observation . Similarly, the lowering of temperature to 24°C prevented the blockade of twitch tension in the EDL preparation (not shown).In the EDL preparations, the time needed for 50% paralysis at a 3.56  ± 2 min . As disp2 than the mammalian preparations. Bp-13 PLA2 (3.56 and 7.12 μM) induced an irreversible but mild decrease of the twitch tension of 21 ± 6% and 28 ± 2% after 120 min, respectively (μM) was significant just when Bp-13 PLA2 concentration was set at 7.12 μM (P > 0.05). In contrast, Bp-13 PLA2 regardless of the concentration, 3.56 or 7.12 μM, did not interfere in the KCl (13.4 mM) induced contracture  [2 NeuTX-I and NeuTX-II) [2) [ B. pauloensis snake crude venom. Interestingly, another Asp49 PLA2, the Bp13, of the same Bothrops species venom was now isolated and characterized biochemically and pharmacologically. Such a diversity of PLA2 isoforms in the venom of a same species evidences the necessity of developing efficient methodologies to purify and identify different isoforms in venom fractions otherwise considered homogeneous. The RP-HPLC was more suitable to purify Bp-13 PLA2 than other conventional methods previously described for other toxins of the same venom, since it required just a single chromatographic step [The presence of Bp-10 and Bp-11 (K49 PLAd Bnsp7) , Bp-14 aeuTX-II) , and Bp-hic step , 22, 27.2 was confirmed by rechromatography on an analytical RP-HPLC μ-Bondapak C18 column. SDS-PAGE showed the monomeric nature of Bp-13 and a relative molecular mass of ~14 kDa and it was confirmed by MALDI-TOF mass spectrometry with a molecular mass of 14035.628 Da. MALDI-TOF/MS has a precision in measuring protein molecular mass of 0.1% Da; thus, this characteristic allows us to demonstrate that Bp13 is another PLA2 isoform present in the Bothrops pauloensis venom, as was for other bothropic PLA2 isoforms [The purity of Bp-13 PLAisoforms , 27–32.2 suggests that this PLA2 is a basic protein because it possesses more basic residues  than acid residues , 14 half- Cys, and also because this protein showed high content of residues Tyr, Pro, Gly, and Lys which was a composition featured by other catalytic active bothropic myotoxins such as the 6-1 PLA2 and 6-2 PLA2 isoforms from B. jararacussu orthe BaTX, a basic PLA2 from B. alternatus [The amino acid composition of Bp-13 PLAternatus , 27.2, a conserved N-terminal helix region forms a hydrophobic channel involving L2, Q4, F5, and I9. Conversely, the level of identity between PLA2s is very high in Ca2+-loop sequence (residues 24–34 YGCXCGXGGRG) and in the active site (residues 42–54 DRCCFVHDCCYXK) [2 catalysis. The N-terminal amino acid sequence of the first 52 residues from Bp-13 PLA2 shows these regions highly conserved and directly linked to catalytic activity.In Asp49 PLAHDCCYXK) , 33–35. 2 herein analyzed showed the presence of some important mutations in N-terminal sequence (up to the 52nd residue). Thus, Bp-13 PLA2 shows K7 -> Q7, N13 -> T13, F20 -> Y20, R34 -> G34, and F46 -> Y46, which are strategic positions for expression of the catalytic activity. The presence of K7 in Bp-13 PLA2 shows that this residue can contribute to keep hydrophobic cavity conformation of the N-terminal region. The N-terminal channel present in PLA2 enzymes is highly conserved and provides access to the lipid substrate to the PLA2 catalytic site. Also, Bp-13 shows at position 13 the lack of Thr residue usually found in other PLA2s enzymes; however, both BnpTX-I (A13 -> T13) and NeuTx-I (A13 -> T13) also showed mutation in this position for polar residue, suggesting that this position is not as well conserved and that it could be a structural feature for the PLA2 of B. pauloensis. The same was observed for the position 16, but in this particular case this position conserved its hydrophobic nature. Despite the change, Bp-13 still maintains catalytic activity and indicates that these residues have no important role in Bp-13 activity [Bp-13 PLAactivity , 36. Crotalus PLA2 shows classic Michaelis-Menten behavior. However, PLA2 activity of Bp-13 from B. pauloensis shows a discrete allosteric-like behavior, and this activity is enhanced by the presence of even low Ca2+ concentrations. The PLA2 from C. durissus terrificus venom exhibits a typical PLA2 activity, since it hydrolyzes synthetic substrates at position 2 and preferentially attacks substrates in their micellar state [2 exhibits allosteric behavior with a Vmax of 11.6 nmol/min and a Km of 11.8 mM.Breithaupt  reportedar state , 37. Sim2 showed a sigmoidal kinetic behavior; this phenomenon was observed for other Crotalinae PLA2s [2, SDS-PAGE without reducing agents showed a weak band at ~28 kDa [However, at low concentrations of the synthetic substrate, Bp-13 PLAae PLA2s , 38–40, 2 was resistant to heat and acid like PLA2s from Crotalus d. cascavella [ C. d. collilineatus, and Lachesis muta muta [2 from C. mitchelli pyrrhus [ C. d. terrificus [ B. neuwiedi [Bp-13 PLAscavella , 41, C. uta muta  venoms;  pyrrhus , C. d. trrificus , and B. neuwiedi  snake ve2, the activity of Bp-13 PLA2 was shown to be completely Ca2+-dependent [2 with other divalent ions  in the presence of 1 mM Ca2+ or in Ca2+ absence reduced or abolished the enzymatic activity.Pharmacologically, as for other types of PLAependent . The coi2+ replacement by 4 mM Sr2+ abolished the neuromuscular blockade. It is well known that calcium ions are essential cofactors for the enzymatic activity of both toxic and nontoxic phospholipases A2 [2+, can bind to the same site of the Ca2+, allowing neuromuscular transmission; nevertheless, this ion does not substitute Ca2+ in the catalysis processes [2+-dependent indicates that the enzymatic activity might contribute for such effect as for neuwieditoxin I and neuwieditoxin II, an Asp49 PLA2 from B. pauloensis [2 from B. marajoensis venom, also an Asp49 PLA2. Likewise, a high catalytic activity was found in neuwieditoxin-I and -II Asp49 PLA2s, whose neuromuscular blockade was also temperature-dependent [2+ and the need of temperature equal or above 30°C for enzymatic activity of Bp-13 identify the toxin as a typical Asp49 PLA2.The Capases A2 . Severalrocesses –50. Thusuloensis . Also, tuloensis  with Bmaependent . Both th in vitro [Bothropic envenomation does not cause neurotoxic clinical signs, but some experimental studies have shown that the venom of several species causes neuromuscular blockadein vitro , 51, 52 in vitro , 53. B. pauloensis crude venom causes partial blockade of directly evoked muscular contractions in BC preparations; in 2007, the authors suggested that neuwieditoxin-I and -II (Asp49 PLA2s) from B. pauloensis venom were probably responsible for the venom presynaptic neurotoxicity in vitro. However, herein it was shown that BC preparations showed low responsiveness to the Bp-13 (less than 30% of neuromuscular blockade) when compared to the crude venom. This could mean that this new Asp49 PLA2 from B. neuwiedi venom has preponderantly a muscular action, thus differing from the presynaptic neurotoxic action of neuwieditoxin-I and -II Asp49 PLA2s referred by Borja-Oliveira et al. [Borja-Oliveira et al.  reporteda et al. . Bothrops pauloensis snake venom, as a new member of the Asp49 PLA2 family. It is suggested that the Bp-13 PLA2 catalytic activity may contribute to the neuromuscular effect already reported for the crude venom on rodent preparations. It is suggested that the main effect of the Bp-13 toxin seems to be on the fiber sarcolemma, with prominence in the mice preparation. In BC preparations, the Bp-13 PLA2 showed little neuromuscular effect. This study is an additional contribution for understanding the toxic effect caused by the Bothrops pauloensis crude venom.Taken together, these results identify Bp-13 isolated from


We also investigated influence of polymorphisms of CYP2E1 and CYP1A1m2 genes on SCE frequency. Our results showed that there was significant increase in frequencies of SCE among the mutant genotypes of CYP2E1 and CYP1A1m2 as compared to wild genotypes. Our study indicated that long term exposure of toluene can increase genotoxic risk in paint workers. This study investigated genetic damage in paint workers mainly exposed to toluene as it is a major solvent used in paint thinners. Sister chromatid exchange (SCE) assay was used as biomarker of genotoxicity. Blood samples were collected from 30 paint workers and 30 control subjects matched with respect to age and other confounding factors except for exposure to toluene. SCE frequency was found to be significantly higher in paint workers (4.81 ± 0.92) as compared to control individuals (1.73 ± 0.54) ( Organic solvents, a class of chemical genotoxic agents, are mainly used in petrol pumps , dry cleaning , as paint thinners , and so forth. The exposure of organic solvents can lead to euphoria and hallucinations while high doses may produce life-threatening effects such as convulsions and coma. The long term exposure may lead to cancer risk [ CYP2E1 and CYP1A1 genes are mainly involved in metabolism of toluene. CYP2E1 gene forms 2 alleles c1 and c2. The genotype c1/c1 was defined as type A, genotype c1/c2 as type B, and homozygote c2/c2 as type C or CYP2E1∗5B [ CYP2E1 genetic polymorphism varies significantly among different ethnic groups [ CYP1A1 gene is characterized by several polymorphisms, one of which located in exon 7 and consists of an amino acidic exchange (CYP1A1∗2B or CYP1A1 m2). The variants of the CYP1A1 gene have been extensively studied, especially to evaluate their possible role in DNA damage and cancer promotion [Various kinds of biomarkers have been applied for biological monitoring of exposed population. The examples of such biomarkers include cytogenetic changes , somatic mutations, and changes in tumor suppressor genes. Sister chromatid exchanges (SCEs) are interchanges of DNA replication products between sister chromatids at apparently homologous loci, suggested to represent homologous recombination repair of DNA double strand breaks , 3. BiomP2E1∗5B . The CYPc groups . The CYPromotion .The study was carried out on paint workers (30) and control group consisting of healthy individuals (30) matched with respect to age and other confounding factors except for exposure to toluene. Participants were informed of the objectives of the study. They were asked to sign an informed consent form and each participant was personally interviewed by a standard questionnaire having information related to age, personal medical history, and occupational history of exposure to paint thinners. The research protocol was approved by the Ethical Committee of Kurukshetra University, Kurukshetra, Haryana, India.To assess the toluene exposure in exposed population, the levels of hippuric acid in urine samples of exposed and control subjects were analyzed by colorimetric method .A total of 5 mL venous blood was collected from each subject in two separate vacutainer tubes containing sodium heparin and dipotassium ethylenediaminetetraacetic acid (EDTA) for lymphocyte culture setup and DNA extraction, respectively. The samples were brought to laboratory in a well-insulated icebox. All exposed individuals have minimum 2 years of working exposure. Blood samples of subjects were collected after finishing of their work shift.Short term peripheral blood lymphocytes (PBL) cultures were set up using earlier studied techniques of Moorhead et al.  with minμg/mL), and 2% phytohaemagglutinin. 5-Bromo-2-deoxyuridine in final concentration of 10 μg/mL culture was added after 24 h of incubation. The cultures were incubated for another 48 h at 37°C and ±5% CO2. Two drops of colchicines in a final concentration of 0.2 μg/mL was added 45 min prior to the harvesting. The cells were harvested by centrifugation and then treated with hypotonic solution (0.075 M KCl) and fixed in methanol : acetic acid (3 : 1). From a suspension of fixed cells, slides were prepared by the air drying method and stained with Hoechst 33258 and 4% Giemsa stain solution following the method of Perry and Wolff [For sister chromatid exchange (SCE) analysis, whole heparinized blood (0.4 mL) was added to 5 mL of RPMI 1640 culture medium containing 1% L-glutamine, 20% fetal bovine serum, penicillin (100 IU/mL), streptomycin  410 bp, heterozygote (c1/c2) 410, 290, and 120 bps, and homozygous mutant (c2/c2) 290, 120 bps. The digested PCR products were separated by 2.5% agarose gel electrophoresis and were detected by ethidium bromide staining.The 5-flanking polymorphic site of thei et al. . The pri CYP1A1m2 polymorphism (204 bp). The restriction enzyme BsrDI was used to digest PCR product (204 bp) to identify mutant genotypes as later ones generate 149 and 55 bp (for the m2 site) fragments. The restricted products were analyzed by electrophoresis in 3% agarose gel containing ethidium bromide.The primers 5′-TTC CAC CCG TTG CAG CAG GAT AGC C-3′ and 5′-CTG TCT CCC TCT GGT TAC AGG AAG-3′  were usep < 0.05 was considered as the significant level for the statistical analyses.Statistical analysis was performed using SPSS software package . All tests were performed in duplicate and results were expressed as means ± SD. Multivariate ANOVA test was used with post hoc analysis for the comparison of frequencies in multiple subgroups among studied population. The demographic characteristics of studied population have been summarized in p < 0.05) (Mean concentration of hippuric acid in urine samples of exposed subjects (0.43 ± 0.017 mg/mL) was significantly higher than that of control subjects (0.17 ± 0.014 mg/mL) ( < 0.05) .In paint workers mean frequency of SCE was found to be significantly higher (4.81 ± 0.92) than control population (1.73 ± 0.54) .Workers having >6 years of exposure duration had significant high SCE frequency (5.46 ± 0.72) compared to individuals having <6 years of exposure (4.32 ± 0.73) .Individuals being >40 years were found to have significant higher SCE frequency in exposed and control population , respectively . There wCYP1A1m2 homozygous mutants showed significant difference in SCE frequency in exposed (5.61 ± 0.66) and control group (2.36 ± 0.00). CYP2E1 homozygous mutants (mt/mt) in exposed subjects (6.11 ± 0.08) and heterozygous mutants (wt/mt) in control subjects (1.88 ± 0.45) showed high SCE frequency  SCE values were found. In a study among 25 male public building painters aged between 18 and 62 years in Mexico, increased SCE levels in lymphocytes were found when compared to the same number of sex- and age-matched unexposed controls [p < 0.05) [ CYP2E1 and CYP1A1m2 as compared to wild genotypes. Similar to our results, Kezic et al. [ CYP1A1, CYP2E1, EPHX1, GSTM1, GSTT1, and GSTP1 enzymes in a group of male CSE  patients (N = 97) and controls (N = 214). Comparing patients and controls, higher frequencies of the variant ∗5B allele of the CYP2E1 gene and of the variant GSTP1∗C allele were found.In present study we found that exposed population has significantly high SCE frequency compared to that of control. Popp et al.  investigcontrols . Further < 0.05) . Contrad < 0.05)  studied c et al.  studied  CYP2E1 and CYP1A1m2 gene modulate the genotoxic effects in paint workers. The determination of CYP2E1 and CYP1A1m2 genotypes status could also be helpful in providing baseline data as an individual marker of susceptibility in exposed population. It is recommended that occupational paint workers are required to train regularly and always be given appropriate personal protective equipment.In our study, we have found that


Lycium barbarum polysaccharides (LBPs) against oxidative stress–induced apoptosis and senescence in human lens epithelial cells.We aimed to investigate the protective effect of 2O2) for 24 h with or without pretreatment with LBPs. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, intracellular reactive oxygen species (ROS), and the loss of mitochondria membrane potential (Δψm) were detected by flow cytometric analyses. Expression levels of Bcl-2 and Bax proteins were measured by western blot analysis. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were quantized using commercial enzymatic kits according to the manufacturer's instructions. To study senescence, SRA01/04 cells were pre-incubated with LBPs and all cells were then exposed to 100 µM H2O2 for 96 h. Cellular senescence was assessed by morphologic examination and senescence-associated β-galactosidase  staining.To study apoptosis, SRA01/04 cells, a human lens epithelial cell lines, were exposed to 200 µM hydrogen peroxide (H2O2-induced cell apoptosis, the generation of ROS, the loss of Δψm, and the levels of MDA. LBPs also inhibited H2O2-induced downregulated Bcl-2 and upregulated Bax proteins and increased the levels of SOD and GSH enzyme activity. Moreover, LBPs significantly attenuated H2O2-induced cellular senescence.LBPs significantly reduced H2O2-induced apoptosis by modulating the generation of ROS, loss of Δψm, Bcl-2 family, and antioxidant enzyme activity and attenuating cellular senescence.These findings suggested that LBPs protect human lens epithelial cells from H Although surgery has proved effective for cataracts, it is associated with high cost and inevitable risks; therefore, cataracts remain the main cause of vision loss and blindness worldwide Lycium barbarum is a well-known traditional Chinese herbal medicine that has multiple pharmacological and biological functions, including neuroprotection L. barbarum polysaccharides (LBPs) extracted from L. barbarum fruits, are believed to be the main component responsible for these biological activities 2O2-induced breaks in the DNA in mouse testicular 2O2 on apoptosis, senescence, cell viability, the generation of ROS, mitochondrial membrane potential (Δψm), pro-apoptotic proteins, and the level of antioxidant enzymes in human lens epithelial cells was assessed in vitro.In the current study, the ability of LBPs to protect against the adverse effects of HLycium barbarum was purchased from Ning Xia Huizu Autonomous Region, People's Republic of China. Polysaccharides from Lycium barbarum was prepared by the method of Yu 2 at 37°C. When grown to 80–85% confluence, the cells were either treated with 200 µM H2O2  for 24 h or pre-incubated with different concentrations of LBPs for 24 h and then treated with 200 µM H2O2. At the indicated time points, the cells were collected for different assays.The SV40 T-antigen-transformed human lens epithelial cell line 4 cells/well) were seeded into 96-well plates with five replicates for each group. The next day, the cells were pretreated with different concentrations of LBPs  for 24 h and exposed to 200 µM H2O2. After incubating for 24 h, the cells were inoculated with 10 µL of CCK-8 solution  and incubated again for 2 h at 37°C, after which absorbance was measured at an optical density of 450 nm.The cells  staining  was used to quantify the amount of cell apoptosis. Briefly, SRA01/04 cells were grown on a six-well plate at 2×102O2. Four hours later, the cells were harvested and incubated with DCFH-DA  for 30 min at 37°C in the dark. After incubation, the ROS generation was determined using the BD FACSAriaflow cytometry system.The intracellular ROS levels were determined using the 2′7′-dichlorofluorescin diacetate (DCFH-DA) assay kit . In brief, SRA01/04 cells were pretreated with or without 400 mg/L LBPs for 24 h and then subjected to 200 µM HΔψm was evaluated using the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) assay kit . The cells were incubated with 200 µM H2O2 for 24 h or pretreated with 400 mg/L LBPs for 24 h before treatment with H2O2. Then, JC-1 dye  was added. After incubating for 20 min at 37°C, the Δψm of cells was examined using the BD FACSAria flow cytometry system. Mitochondrial depolarization was assessed by a decrease in the intensity ratio of the red/green fluorescence.2O2 for 24 h. At the end of the treatment, the cells were harvested and sonicated with phosphate buffer (pH 6.8) containing 1.0 mM phenylmethylsulfonyl fluoride to obtain cell homogenates. The homogenates were centrifuged at 4,000 rpm at 4°C for 10 min. The supernatants were used for measuring cellular MDA, SOD, and GSH using the commercially available assay kits . The MDA level was calculated by evaluating the thiobarbituric acid reacting substances at a wavelength of 532 nm using the Infinite M200 microplate reader . SOD activity was examined using the xanthine oxidase method as previously described SRA01/04 cells were pretreated with LBPs or its vehicle control for 24 h and incubated with 200 µM H2 at 37°C. When grown to 70% confluence, the cells were treated with 100 µM H2O2 for 96 h or pretreated with 400 mg/L LBPs for 24 h followed by the addition of 100 µM H2O2. At established time points, SRA01/04 cells were washed with PBS, fixed in 4% paraformaldehyde for 3–5 min at room temperature, and rinsed with PBS. The cells were then incubated with freshly prepared SA-β-gal stain solution  overnight at 37°C. Total and blue-stained cells were counted in 10 fields at a magnification of 40 times, and the SA-β-gal-positive cells were expressed as a percentage of total cells.SRA01/04 cells were grown in DMEM medium supplemented with 10% FBS in humidified 5% CO2O2 for 96 h. At the end of treatment, SRA01/04 cells were collected and washed with PBS, fixed in 70% ethanol at 4°C, and treated with 10 mg/mL RNase for 30 min at 37°C. Finally, cells were stained with 50 µg/mL PI and analyzed using the BD FACSAria flow cytometry system.PI staining kit  and flow cytometry were used to measure cell cycle distribution. The SRA01/04 cells were cultured in six-well plates and pretreated with LBPs or its vehicle control for 24 h and then incubated with 200 µM HAfter all treatments, cultured cells were washed with cold PBS and then lysed in a RIPA buffer  containing a Protease Inhibitor Cocktail  and centrifuged at 12000 g for 20 min at 4°C. The supernatant protein concentration was measured using a BCA assay kit  according to the manufacturer's instructions, after which 50 µg protein from the cells were subjected to 8–10% SDS-PAGE and transferred into a polyvinylidene difluoride membrane. The blot was incubated with antibody against Bcl-2, Bax , and GADPH . The enzyme used was horseradish peroxidase–conjugated secondary antibody . Signals were visualized with enhanced chemiluminescence (ECL)  and quantitated using Image J. The ratio of the expression of target proteins was determined after normalizing the individual GADPH levels. Each experiment was repeated three times.Data were analyzed using the SPSS 16.0 . Values were presented as mean ± S.D. For comparison of the different groups, analysis of variance (ANOVA) with the post-hoc least significant difference (LSD) test was used. p<0.05 was considered statistically significant.2O2 as the working concentration according to our preliminary experiment (data not shown). SRA01/04 cells were incubated with multiple concentrations of LBPs  and showed no significant cytotoxicity; however, cell cytotoxicity was detected at a concentration of 1600 mg/L LBPs. In addition, LBPs promote SRA01/04 cell growth in a dose-dependent manner at a concentration of 200–400 mg/L (P<0.05) (We selected 200 µM H(P<0.05) .2O2-induced cell damage in SRA01/04 cells, the viability of SRA01/04 cells after 200 µM H2O2 incubation for 24 h, with or without LBP pretreatment at different concentrations, was detected. As shown in 2O2 for 24 h reduced cell viability to 54.3±6.1%, and LBP pretreatment at a concentration of 200–400 mg/L for 24 h prevented this reduction in cell viability. LBPs exerted maximum protection at 400 mg/L.To confirm LBPs protection against H2O2-induced cell apoptosis was examined. Based on the results from CCK-8 assay, the SRA01/04 cells were incubated with or without 400 mg/L LBPs and then subjected to 200 µM H2O2 for 24 h. As observed under an inverted microscope, a high portion of cells demonstrated apoptosis-like changes include blebbing of the plasma membrane, rounding and shrinkage of cells before the plasma membrane disruption in the H2O2 –alone group; however, the proportion of apoptosis-like cells decreased in the group that was pretreated with LBPs (2O2. The rate of cell apoptosis was 13.8±1.25% (n = 3) in the LBP + H2O2 group, much lower than that in the H2O2 group (25.9±1.5%) (n = 3)  .2O2 for 4 h resulted in the generation of ROS with approximately a two-fold increase compared to that in the control cells; however, pretreatment with 400 mg/L LBPs significantly decreased the levels of ROS in H2O2-exposed cells.DCFH-DA was used to measure the level of ROS production. As shown in 2O2-treated and LBP + H2O2–treated cells  was assessed using JC-1. Less than 4% of the control cells showed mitochondrial depolarization. After H2O2 treatment, the percentage of cells that displayed mitochondrial depolarization increased in both but the levels were much lower in the LBP + H2O2–treated cells . LBPs significantly alleviated the damage caused by H2O2 and reduced the ratio of SA-β-gal–positive cells to 29±4.2% in the SRA01/04 cells.To investigate the effect of LBP on SRA01/04 cell senescence, premature senescence was induced in SRA01/04 cells by long-term treatment with low doses of Hnescence . SA-β-ga2O2 or pretreated with LBP. Flow cytometric analysis revealed that the cell cycle of H2O2-treated SRA01/04 cells was arrested (P<0.01) in the G0/G1 phase; however, in the LBP-treated cells, this action was significantly attenuated in the G0/G1 phase of the cell cycle (P<0.01)  . These o2O2-induced SRA01/04 cell damage. The results showed that treatment with LBPs effectively protected SRA01/04 cells, reduced the generation of ROS and loss of Δψm, modulated the expression of Bcl-2 and Bax proteins, and increased antioxidant enzyme activity after H2O2-induced oxidative stress.Many studies have demonstrated that antioxidants, such as vitamins C and E, and the carotenoid xanthophylls zeaxanthin and lutein might protect against oxidative stress in lens epithelial cells L. barbarum, have a complicated role in the life and death of cells. Ho et al. Oxidative stress–induced apoptosis in lens epithelial cells plays an important role in cataract formation, and its prevention is of therapeutic interest. LBPs, isolated from the aqueous extracts of 2O2) oxidative stress conditions. Under the inverted microscope, SRA01/04 cells exposed to H2O2 exhibited apoptotic-like signs; however, in the presence of LBPs, the proportions of apoptotic cells were significantly decreased. Moreover, flow cytometry analysis showed that LBPs markedly reduced apoptosis in H2O2-treated cells. These indicated that LBPs have a protective effect on cells by inhibiting H2O2-induced cell apoptosis.In another study 2O2-treated cells showed an increase in intracellular ROS and a loss of Δψm and unregulated the expression of Bax and downregulated the expression of Bcl-2; however, the generation of ROS was inhibited by pretreatment with LBPs. We determined that LBPs protect SRA01/04 cells against H2O2-induced apoptosis by stabilizing Δψm and increasing the expression of Bcl-2, and increasing the ratio of Bcl-2 to Bax in lens epithelial cells exposed to H2O2.Apoptosis is a physiological process of cell death that plays a key role in a variety of biologic systems, which has been recognized as providing an important molecular basis for cataracts 2O2, these natural antioxidant defense systems are destroyed, and enough ROS might be generated to exceed their self-scavenging ability, resulting in oxidative stress and leading to protein modification and degradation, DNA and mitochondrial damage, and eventual cell death 2O2 significantly accelerated lipid peroxidation and decreased the levels of SOD and GSH enzymes; however, treatment with LBPs significantly reversed elevated MDA levels and enhanced SOD and GSH activity. The data suggested that treatment with LBPs significantly inhibited H2O2-induced lipid peroxidation, which might be related to the increased activity of antioxidant enzymes.Oxidative stress results from an imbalance between ROS production and elimination. Under normal conditions, the lens contains high levels of reduced GSH Drosophila2O2 treatment caused a morphological change in the cells, positive SA-β-gal staining, and G0/G1 phase cell cycle arrest, which suggest that a low dose of H2O2 treatment could induce cell senescence in SRA01/04 cells; however, LBP pretreatment dramatically reduced cellular senescence markers under chronic oxidative stress in these cells, which is consistent with a protective effect and might help explain part of the mechanism by which the life span of the cells is extended.It has been recognized that accumulation of oxidative stress will contribute to the aging processes In conclusion, our results demonstrated that LBPs have the potential to protect human lens epithelial cells from oxidative-stress damage by modulating the generation of ROS, loss of Δψm, the Bcl-2 family, and antioxidant enzyme activity and by attenuating cell senescence; therefore, LBPs are a potent antioxidant and anti-aging agent that could be exploited as a potential implication in the prevention of cataractogeneis.


Ruegeria halocynthiae MOLA R1/13b is an alphaproteobacterium isolated from the Mediterranean sea sponge Crambe crambe. We report here the genome sequence and its annotation, revealing the presence of quorum-sensing genes. This is the first report of the full genome of a Ruegeria halocynthiae strain. Ruegeria halocynthiae MOLA R1/13b  was isolated in 1:5 marine R2A agar (in 75% seawater) at 20°C, from the sponge Crambe crambe collected on 20 January 2010 from a 12-m depth at the Bay of Banyuls . A major step in our isolation protocol is that sponge homogenates were acclimated for 24 h at 16°C with marine R2A added at a 1:200 final dilution, before strain isolation. Based on its 16S rRNA gene sequences, the strain is phylogenetically related to Ruegeria halocynthiae MA1-6 (99% sequence identity to 16SrRNA genes) and belongs to the Rhodobacteraceae (Roseobacter clade).de novo assembled with NGEN v11 . The genome was annotated using Prokka 1.7  at 25°C over 48 h. DNA was extracted using a cetyltrimethylammonium bromide (CTAB)-based method (Ruegeria halocynthiae MOLA R1/13b is 4,320,292 bp, with a G+C content of 58.3%, including 4,205 coding sequences, 2 rRNAs, and 53 tRNAs. The genome annotation revealed the presence of genes involved in quorum sensing. Two autoinducer type 1 synthases homologous to luxI and 6 corresponding luxR receptors (Ruegeria halocynthiae. Thus, this draft genome reinforces previous observations suggesting that marine bacteria are able to communicate using quorum sensing in sponge microenvironments where these cells can be found at high concentrations (The sequence draft genome sequence of JQEZ00000000.The whole-genome shotgun project has been deposited at DDJB/EMBL/GenBank under the accession no.