Aspergillus niger and Acinetobacter sp., were applied to evaluate their ability to decompose both inorganic phosphates and lecithin. A. niger and Acinetobacter sp. could solubilize calcium phosphates by secreting various organic acids, e.g., oxalic and formic acids. The fungus, A. niger, shows significantly higher ability of solubilizing these inorganic phosphates than Acinetobacter sp., primarily due to its secretion of abundant oxalic acid. However, the bacterium, Acinetobacter sp., could secrete more acid phosphatase than A. niger for lecithin decomposition, i.e., 9300 vs. 8500 μmol L−1 h−1. Moreover, after addition of CaCl2, the released P from lecithin was transformed to stable chlorapatite in the medium. To the contrast, Ca cations inclined to form calcium oxalate  after the incubation of A. niger, as it induced relatively acidic environment after breaking down lecithin. Therefore, this work sheds light on the bright future of applying bacteria and Ca cations in OP pollutant management.Organophosphorus compounds (OP) are stable P source in nature, and can increase eutrophication risk in waterbodies. Lecithin was the most difficult OP to be broken down. In this study, two typical phosphate-solubilizing microorganisms,  OP compounds are widely distributed in soil and appear as various forms, such as inositol phosphates, phospholipids, and nucleic acids2. However, most OP compounds in soil exist as insoluble forms, and only a small fraction (<1%) can be directly used by crops3. OP species, usually derived from the decomposition of aquatic organisms, increase the risk of eutrophication5. The efficient use of P and the remediation of P-induced eutrophication require a better understanding of biogeochemistry of OP compounds.Phosphorus (P) derived from organophosphorus (OP) compounds constitutes 30 to 90% of the total P content in soil6, including two major types: phosphodiesterase (PDE) and phosphomonoesterase (PME)7. Many physical and chemical techniques have been applied to oxidise or break down phospholipids, including UV-LED/TiO2 photocatalytic/photoelectrocatalytic inactivation, acid-based hydrolysis, and graphene oxide-catalytic hydrolysis10. Soluble P originated from orthophosphates could also react with Pb2+, Ca2+, Mg2+, or Fe3+ cations to form minerals such as hydroxylapatite, struvite, or zwieselite, respectively16. However, the potential pathways of OP decomposition and P mineralization regulated by microorganisms are still poorly known.It is critical that phospholipids be transformed into soluble P, as OP compounds are more resistant to decay than are phytates and nucleic acids. The degradation of phospholipids to inorganic phosphate is regulated by phosphatases18. PSMs contribute to P release from both inorganic and organic matters. Solubilisation of (inorganic) phosphate involves the secretion of low molecular weight organic acids (LMWOAs) by PSMs20. The two major categories of PSMs are phosphate-solubilizing bacteria (PSB) and phosphate-solubilizing fungi (PSF)23. PSB include Acinetobacter sp., Pseudomonas aeruginosa, Alcaligenes faecalis, Sphingobium sp., Pantoea agglomerans, Rahnella aquatilis, Enterobacter sp., Burkholderia cepacia, and Bacillus sp.28. PSF also possess a remarkable ability to solubilise phosphate, including Aspergillus niger, Penicillium, Curvularia, and Trichoderma30. PSF usually have higher phosphate-solubilizing ability than PSB32. A. niger and Acinetobacter sp. function as the representative PSF and PSB, respectively34. Both A. niger and Acinetobacter synthesize and secrete phosphatase and phytase under P-deficient conditions, in addition to their secretion of organic acids36. However, few studies have examined the differences in the abilities to solubilize inorganic phosphates and break down OP compounds between PSB and PSF.Phosphate-solubilizing microorganisms (PSMs) are plant growth-promoting microorganisms that have traditionally been used to increase crop yields and improve nutrient levels in soil37. Lecithin also functions as a biogenic mobilizing agent that is commonly used to wash sand contaminated with fuel oil38. Both natural and anthropogenic processes increase levels of lecithin. The excessive accumulation of lecithin in soil and water could stimulate the overgrowth of algae and increase the risk of eutrophication40. Lecithin is decomposed only under extreme physical/chemical conditions, e.g., high temperature, extreme pH, dehydration, freezing, chilling, and high mineral concentrations42. Therefore, it is necessary to develop eco-friendly strategies to control lecithin levels.Lecithin, a model phospholipid, has amphiphilic and surface-active characteristics. It is commonly used in food, cosmetics, and pharmaceutical industries as a co-surfactant or emulsionAcinetobacter (PSB) and A. niger (PSF) to break down lecithin. We also investigated the mineralization of the released P from lecithin. To identify the formation and composition of secondary minerals, we performed scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses.In this study, we compared the abilities of A. niger increased the soluble P concentration in tricalcium phosphate (TCP)-amended PVK medium to 770.5 mg L−1 after incubation  than when TCP was used as a P source. In addition, the pH of the medium decreased to 3.1 (Table Acinetobacter sp. was less able to solubilize inorganic phosphates (from both TCP and FAp) than A. niger. In TCP medium, Acinetobacter sp. increased the soluble P concentration to 402.3 mg L−1 after five-days incubation. Accordingly, the pH of the medium pH decreased to 4.2, which was higher than that of medium containing A. niger  after five-days incubation  after four-days incubation  detected on the fourth day of incubation  was detected on the fourth day of culture  and Acinetobacter . There was also a significant positive correlation between acid phosphatase activity and soluble P concentration in Acinetobacter ; however, this correlation was not significant in A. niger.Pearson’s correlation analysis revealed that the soluble P concentration was positively association with alkaline phosphatase activity for both A. niger and Acinetobacter cultures after incubation in lecithin, followed by the addition of CaCl2  was comparable to that of A. niger when using lecithin as a model OP compound .es Table . Howevernd Table . FurtherA. niger and Acinetobacter sp. secreted LMWOAs such as oxalic and formic acids, which solubilized inorganic phosphates at varying degrees  is a strongly acidic LMWOA49. Additionally, oxalic acid can form secondary precipitates with the Ca2+ released from TCP and FAp, i.e., calcium oxalate. This results in lower concentrations of oxalic acid in A. niger. By contrast, the ability of Acinetobacter sp. to solubilize inorganic phosphate is primarily mediated by weak formic acid.ees Fig. . These r51. Acinetobacter exhibited higher acid phosphatase activity than A. niger during the 3 days in the middle of the incubation period  for acid phosphatase. Therefore, the contrast of the secretion of acid phosphatase might cause such difference of P release. Alkaline phosphatase is also highly efficient in OP decomposition, as reported in studies regarding sludge, but more active in alkaline environment52. Therefore, even the alkaline phosphatase could contribute to lecithin breakdown, but cannot exert its ideal function in this study.Phosphatase is an important extracellular enzyme in OP breakdown and plant P acquisitioniod Fig. . Acinetoger Fig. . Meanwhion Table . In addi54. Here, chlorapatite, a variety of apatite, formed more readily in the supernatant from Acinetobacter sp. than from A. niger when cultured in the presence of CaCl2  and photoelectrocatalytic (PEC) technology, were usually high-cost9. In addition, the removal techniques of inorganic P, such as P adsorptions by bauxite residue and ferric chloride, could induce side effects to aquatic organisms58. The typical PSB, Acinetobacter, is a beneficial plant growth-promoting microorganism, and widely present in water bodies and soil62. Moreover, Acinetobacter sp. usually display a faster rate of reproduction than PSF44. Therefore, PSB could be a potential sustainable “biomaterial” in decomposing OP. Previous studies have proposed that there were several eco-friendly methods to remove the inorganic P by adding Mg2+ and Ca2+ to form struvite and hydroxylapatite56. A combination of PSB and CaCl2 successfully decreased the risk of eutrophication caused by OP in water bodies, and the OP degradation efficiency was about 16.5%, which is comparable to that of PC and PEC techniques9. Furthermore, Ca2+ and Cl− are common elements in soil systems, which will not cause serious environmental pollution64.Many OP removal techniques, such as UV-LED/TiOAcinetobacter (PSB) has a better potential for OP removal than A. niger (PSF). A. niger and Acinetobacter sp. were equally able to decompose OP (~30 mg L−1) after incubation. The ability of A. niger to solubilise TCP and RP was 1.9- and 4.6-fold greater than that of Acinetobacter, respectively, as it secretes a variety of LMWOAs. However, calcium oxalate was detected in the supernatant of medium inoculated with A. niger after the addition of CaCl2 because the environment was weakly acidic . However, stable chlorapatite can be formed in the presence of Ca2+ and Cl− in a neutral solution with addition of Acinetobacter. These findings deepen our understanding of OP decomposition using PSMs.This work demonstrated that Acinetobacter sp. (CGMCC No. 13078) was isolated from cow manure provided by Qinbang Organic Fertilizer Company in Nanjing City, Jiangsu Province, China. A. niger (CGMCC No. 15994) was isolated from fluvo-aquic soil collected from fallow farmland in the upper 20 cm of soil in Nanjing, Jiangsu Province, China  in April, 2017. Pikovskaya (PVK) medium  was used to determine the ability of A. niger and Acinetobacter sp. to solubilize inorganic phosphates66. The main components of PVK medium are 10.0 g/L glucose, 0.5 g/L (NH4)SO4, 0.03 g/L MnSO4·4H2O, 0.3 g/L KCl, 0.3 g/L MgSO4·7H2O, 0.03 g/L FeSO4·7H2O, 0.3 g/L NaCl, and 10 g/L P source. The levels of soluble P released by the two PSMs in PVK medium amended with 10 g tricalcium phosphate (TCP)  or 10 g fluorapatite (FAp)  as a P source after five-days incubation  medium. Acinetobacter sp. suspensions were collected by centrifugation at 16,099.2 g (4 °C) for 2 min. A. niger spore suspensions were collected by washing the spores on the surface of spore-forming PDA medium repeatedly with sterile water and filtering them through six types of gauze into an aseptic flask, according to the modified methods of Ren et al.67. Bacterial cells and fungal spores were adjusted to approximately 1 × 108 CFU per mL using a haemocytometer.18 chromatographic column  was used with a temperature of 30 °C. The mobile phase was 2.5% (NH4)H2PO4 (pH adjusted to 2.5 with phosphoric acid) with the chromatographic reagent methyl alcohol at the ratio of 99:1. The flow rate was controlled at 0.5 mL min−1, and the wavelength of detector was 210 nm.To detect the LMWOAs secreted by PSMs, high-performance liquid chromatography  was used. For each sample, 2 mL of medium was collected from cultures in inorganic phosphate (for both TCP and FAp-amended PVK medium) after five-days of incubation. All samples were filtered through a 0.22-μm membrane prior to analysis. An RP-C−1). A shaking culture experiment was then conducted using both PSMs. First, 1% suspensions (OD600 = 0.5) were inoculated into 50 mL lecithin-amended medium in triplicate. The cultures were shaken at 180 rpm at 30 °C for 5 days. During the incubation, 3-mL samples were collected daily under aseptic conditions and used to measure medium pH and soluble P concentration. The pH of the medium was then measured by a pH meter . The soluble P concentration was measured using the molybdate blue method and quantified spectrophotometrically at an absorbance wavelength of 880 nm.The ability of the PSMs to break down lecithin was quantified in the PVK medium amended with lecithin  and 1 mL 25 mM DPP for 1 h at 37 °C. Subsequently, 1 mL 0.5 M CaCl2 and 4 ml 0.5 M NaOH were added to terminate the reaction. The concentration of DPP was measured spectrophotometrically based on absorbance at 510 nm.To detect acid and alkaline phosphatase secreted by the PSMs during incubation, enzyme activity was analyzed using the disodium phenyl phosphate method (DPP)2  was added to the supernatant at a Ca/P molar ratio of 100:1.Lecithin medium inoculated with the PSMs and incubated for 5 days was centrifuged at 16,099.2 g (4 °C) for 10 min to remove the microorganisms and insoluble residues. The supernatant was collected into a new flask. A rotary evaporator was used to concentrate the supernatant and increase the concentration of soluble P. CaClTo achieve complete precipitation, the supernatant was shaken at 180 rpm at 30 °C for 1 h. The precipitates were collected by filtering through a 0.22-μm membrane and washed twice with sterile water to remove the liquid residue. To obtain dry powered samples, the precipitates were lyophilised at −40 °C for 24 h.69.Duncan’s multiple range test was used to analyse the differences between treatments. Two-way ANOVA and Fisher’s test were used to evaluate the pH and soluble P concentration of the medium after different periods of culture with different PSMs. All differences were considered to be significant at P < 0.05. All statistical analysis was conducted using R software version 3.3.2Supplementary materials


High body mass index (BMI) was significantly associated with hypertension. The purpose of this study is to investigate the association between BMI and hypertension in people in northeast China. Our study was a cross-sectional study conducted from June to August 2012. According to multistage, stratified cluster sampling, a total of 21435 inhabitants aged between 18 and 79 years in Jilin Province were selected randomly. The prevalence of hypertension was 35.66% overall. After adjusting for potential confounders, the multivariable-adjusted odds ratios for the BMI- hypertension association for overweight and obesity were 2.503  and 4.259 . The results of multivariable restricted cubic spline regression analysis showed that there was a non-linear relationship between the continuous change of BMI and hypertension (P < 0.001) after adjusting the confounding factors of different genders and age groups, which indicated that there was an adjusted dose-response association between continuous BMI and hypertension. In 2012, noncommunicable diseases contributed to 68% of worldwide death, and as achronic non-communicable disease, hypertension has become an enormous public health problem due to its high prevalence and low rate of control1. The prevalence of hypertension is on the rise across the African continent, and in South Africa, some populations reported rates as high as 46%2.Hypertension is a multifactorial disease associated with modifiable and non-modifiable risk factors, among which, modifiable factors are obesity, excessive sodium intake, physical exercise in activity and others, non-modifiable factors are age, sex, ethnicity, and genetics and so on6.Approximately 320 million people accounting for nearly 30% of the population 18years old and older have hypertension in China, and among them, <5% have their blood pressure under control6.In China, the problem of hypertension challenges the population health because of its high prevalence among adults7, whereas salt intake is a major source of sodium in the general population8. A higher sodium intake can make blood pressure rise, which be defined as sodium sensitivity9. Genetic aspect, for example, angiotensin converting enzyme (ACE) gene polymorphisms, as a key element in therenin-angiotensin-aldosterone system, is also associated with hypertension10.A high-salt diet is clearly associated with hypertension11. In China, from 1980 to 2013, the combined age-standardized prevalence of overweight and obesity in men and women over the age of 20 was 28.3% and 27.4%, respectively12. Several studies have reported that there is a link between blood pressure increase and weight gain13. Data from NHANES showed that the prevalence of hypertension in obese people is significantly higher than that in the general population14. A prospective analysis also showed that the high prevalence of hypertension in obese patients(>60%) account for 78% of incident of hypertension in men and 64% in women15.In worldwide, the prevalence of overweight and obesity constantly rising and has became a global pandemic. In the United States, the 2011–2012 National Health and Nutrition Examination Survey displayed that approximately 16.9% of youth and 34.9% of adults were diagnosed with obesity17. Obesity has been shown to be associated with a variety of diseases, such as cancer18, hypertension, dyslipidemia, diabetes and cardiovascular disease (CVD)21. There are few studies to quantify the relationship between BMI levels and hypertension, especially using some intuitive methods such as restricted cubic splines (RCS)22. Moreover, nobody has quantified the relationship between BMI levels and hypertension based on the population of Northeast China15.Although the classification system of obesity is not exactly identical, body mass index(BMI) is the most commonly used measure, dividing obesity into overweight and various gradesThe purpose of this study is to analyse the relationship between BMI and hypertension in people in northeast China. In our study, we used RCS function in dose-response analysis to adjust potential confounding factors to quantify the association between BMI and hypertension. In this way, our study can provide information for people’s health promotion.24. The Ethics Committee of Jilin University School of Public Health approved the study (Reference Number: 2012-R-011). Before participating in this survey, all participants had provided informed consent.The study which was carried out in Jilin Province was a community-based, cross-sectional study. It starting from June 2012 and ended in August 2012. Research object of the study was the people aged between 18 and 79 years old, and the people must have lived in Jilin Province for more than 6 months. Multistage stratified cluster sampling was used to select research object from cities representative of the distribution of the Jilin population. The detailed sampling procedure had described previously22. Hypertension was defined as systolic ≥ 140 mmHg and/or diastolic ≥ 90 mmHg following the Chinese Hypertension Prevention Guide (2010 Revised Edition).According to WHO criteria(1999): Through glucoseoxidase method, FPG 7.0 mmol/L and/or 2hPG11.1 mmol/L were regarded as diabetes. Abnormal blood lipids (TC ≥ 5.18 mmol/L or TG ≥ 1.70 mmol/L or HDL-C < 1.04 mmol/L or LDL-C ≥ 3.37 mmol/L) was diagnosed as hyperlipidemia based on the criteria of the “Chinese Guidelines on Prevention and Treatment of Dyslipidemia in Adults”.BMI was defined as the ratio of body weight (kilograms) to the square of height (meters). Based on the Chinese criteria of weight for adults, BMI < 18.5, 18.5 ≤ BMI < 24.0, 24.0 ≤ BMI < 28.0,BMI ≥ 28.0 were respectively defined as underweight, normal, overweight and obeseOccupations were classified into 3 groups: mental labor, manual labor, and retirees or unemployed. Mental labor was defined as the person who was managers, administrators, or technicians. The people who worked in the manufacturing industry, agriculture, or service industry were classified into manual labor. According to average monthly household, household incomes were classified into6 categories: below 500 yuan, 500 to <1000 yuan, 1000 to <2000 yuan, 2000 to <3000 yuan, 3000 to <5000 yuan, ≥2000 yuan. Smoking was defined as never, ever , or current. Drinking habits provided two options–“0” and “1”. “0” was defined as no smoking, and “1” was defined as smoking. Exercise habits were classified into 3 categories based on exercise frequency. The exercise frequency of ≥3times a week was defined as “often exercise”, once or twice a week was defined as “sometimes exercise”, the others were defined as “never or rare exercise”.2, 22 kg/m2, 25 kg/m2, 27 kg/m222. Stata 12.0  was used to carry out the above-mentioned dose-response analyses. A significance level of P < 0.05 (2-tailed tests) was used.Demographics are presented as numbers and frequency distributions for categorical variables as appropriate. Univariate logistic regression analyses were used to determined significant differences between hypertension and nohypertension individuals. The association between BMI and hypertension was investigated by using unconditional multivariable logistic regression models and models which adjusted for age, gender, education level, and other variables, and to evaluate the potential confounding effects among risk factor variables. Multivariable-adjusted odds ratios(ORs) and their 95% confidence intervals (CIs) in 3 different logistic regression models were calculated by the SPSS statistical package, version 19.0  independently. RCS were used to detect the possible nonlinear dependency of the relationship between the risk of hypertension and BMI levels, using 4 knots at prespecified locations according to the percentiles of the distribution of BMI, 18 kg/m2 vs 25.58 ± 3.65 kg/m2, P < 0.001).A total of 21,435 samples participated in our study, screened for data and eventually included in the study for 20,839 by deleting the missing height/weight values. Among the 20,839 participants, the prevalence of hypertension was35.66% , with a number of 3,836 for male and 3,595 for female. The mean age was (47.27 ± 13.34) years. Compared with hypertension participants, the no hypertension individuals had a lower BMI . After adjustment for age and gender, the adjusted odds ratios showed a consistent association between BMI classification and hypertension across the model. In a fully adjusted model, the impact of BMI was slightly diminished when adjusting for residential areas, education, marital status, occupation, household income, smoking and drinking status, exercise. Although the association of BMI classification with hypertension prevalence was reduced with sequential adjustments, BMI showed a significant and clear gradient from the lowest underweight to the highest obesity level.Table Based on the stratification of gender and age, we used RCS model with 4knots to simulate the relationship between BMI and the risk for hypertension. Significant nonlinear dose-response association was showed in the relationship between BMI and the risk of hypertension . And dose-response relationship analysis showed that with the continuous change of BMI, the association strength of hypertension increased nonlinearly.2 of BMI as a reference, the ORs and 95%CIs of the 4knots of BMI were 0.37(0.31–0.45) for18kg/m2, 0.81(0.77–0.84) for 22 kg/m2, 1.46(1.36–1.55) for 25 kg/m2, 1.91(1.78–2.06) for 27 kg/m2. In females, ORs and 95%CIs of the 4 points of BMI at 18 kg/m2, 22 kg/m2, 25 kg/m2, 27 kg/m2 were 0.34(0.28–0.41), 0.80(0.76–0.83), 1.46(1.36–1.55), and 1.86(1.73–2.01). Figures In males, with 23 kg/me 0.370.3–0.45 for2 and 22 kg/m2 were, whereas 25 kg/m2 and 27 kg/m2 were 0.27(0.21–0.36), 0.73(0.69–0.77), 1.65(1.51–1.81), and 2.14(1.95–2.36). For middle-aged adults (45–59 years old) and older adults (65–79 years old), the ORs (95%CIs) for hypertension risk were for BMI at 18 kg/m2were 0.38(0.30–0.47) and 0.43(0.35–0.53), at 22 kg/m2 were 0.81(0.77–0.85) and 0.85(0.80–0.90), where as at 25 kg/m2 were 1.43(1.34–1.53) and 1.32(1.21–1.44), at 27 kg/m2 were 1.87(1.73–2.02) and 1.59(1.43–1.78). All age groups are referenced to BMI 23 kg/m2.Three groups adjusted forpotential confounders that were tailored to the specific population.Similar relationships between hypertension risk and BMI were found in different age groups when using 4 knots (all P value of nonlinear < 0.001; Fig. In the population-based study of Jilin Province in Northeastern China, our study reported the prevalence of hypertension was 35.66% including previously diagnosed hypertension and newly diagnosed hypertension. Regardless of gender or age, after adjusting for confounding factors, continuous changes in BMI are significantly associated with the risk of hypertension. And dose-response relationship analysis showed that with the continuous change of BMI, the association strength of hypertension increased nonlinearly.25. China’s chronic disease monitoring in 2010 reported that the national prevalence of hypertension was 33.5%, which was lower than the rate of 35.66% reported by our study26. Our rate was higher than the rate of 17.2% displayed by the monitoring results of Kunshan city in the same year22, but not reached the rate of 38.6% of Jiangsu Province27.On a national scale in China, findings based on different backgrounds’ survey in the past 30 years basically showed an upward trend in the prevalence of hypertension19 showed that the increasing of BMIs can make blood pressure increase, which indicated that there was not only a strong relationship between BMI and hypertension, but also was an association between the continuous variables of BMI and blood pressure. Our study and the study22 conducted in kunshan city confirmed that there was nonlinear dose-response relationship between BMI and the risk of hypertension.As early as 1996, study28. The pathophysiological explanation of obesity predisposing hypertensionis elevated cardiac output, which perhaps attribute to excess intravascular volume and reduced cardiac contractility29. There was evidence suggested that in obesity individual, nutritional status alteration, gut microbiota, sunlight exposure and physical activity increase played an important role in the hypertension or not30. Further research investigating dose-response association between BMI and hypertension would be a significant step to decrease the social burden of hypertension.Although the mechanism of how obesity caused hypertension was not yet clear, epidemiological evidence had highlighted that there was a consistent correlation between obesity and hypertension, and obesity predisposes hypertensionOur outcomes demonstrated that dose-response relationship exists between BMI and hypertension, and increased BMI is an independent and adjusted dose-dependent risk factor for hypertension among overweight and obesity participants in China.


Lysyl oxidase has emerged as an important enzyme in cancer metastasis. Its activity has been reported to become upregulated in several types of cancer, and blocking its activity has been shown to limit the metastatic potential of various cancers. The small-molecules phenylhydrazine and β-aminopropionitrile are known to inhibit lysyl oxidase; however, issues of stability, toxicity, and poorly defined mechanisms limit their potential use in medical applications. The experiments presented herein evaluate three other families of hydrazine-derived compounds – hydrazides, alkyl hydrazines, and semicarbazides – as irreversible inhibitors of lysyl oxidase including determining the kinetic parameters and comparing the inhibition selectivities for lysyl oxidase against the topaquinone-containing diamine oxidase from lentil seedlings. The results suggest that the hydrazide group may be a useful core functionality that can be developed into potent and selective inhibitors of lysyl oxidase and eventually find application in cancer metastasis research. Lysyl oxidase (LOX) is an enzyme whose activity is associated with multiple diseases including metastatic cancer, stiffening of connective tissueFor many types of cancer, increased LOX activity promotes metastasis of cancer cells and progression of tumor growth. For example, El-Haibi et al. have shown that stimulating breast cancer cells to increase LOX synthesis activates those cells into a metastatic phenotypeBlocking LOX's enzymatic function can reduce migration of cancer cells. Using siRNA to knock down LOX expression can significantly reduce the hypoxia-induced invasiveness of nonsmall-cell lung cancer cellsPichia pastoris amine oxidaseLOX performs its enzymatic function, converting primary amines into aldehydes, by using a quinone cofactor and a redox-active copper ion. Specifically, the quinone cofactor is lysyl tyrosylquinone (LTQ) , which i14C-phenylhydrazinein vivo applications. Other types of hydrazine-containing organic molecules that are more stable than phenylhydrazine, such as alkyl hydrazines, hydrazides, and semicarbazides  were synthesized and compared to phenylhydrazine, including analyses of their kinetic parameters for irreversible inhibition. These comparisons of inhibition parameters and selectivity provide a basis for deciding which of these “core” inhibitor structures are best suited as starting points for development of more complex molecules that could act as potent and selective inhibitors of LOX and also have favorable pharmacokinetic properties. Such inhibitors would be important not only as potential drug candidates, but also as chemical tools to study LOX-related biological mechanisms that are involved in cancer metathesis and other diseases.We set out to evaluate the effectiveness and specificity of various organic hydrazine derivatives to help provide a basis for developing new selective irreversible inhibitors of LOX. These studies directly compare four families of organic hydrazine compounds as inhibitors of LOX and also of lentil seedling diamine oxidase (LSDAO), which was chosen as a representative topaquinone-containing amine oxidase with a somewhat restrictive substrate-binding channel for comparison. Representative members of the alkyl hydrazine, hydrazide, and semicarbazide families (Lens esculenta) were blended and extracted with phosphate buffer . Ammonium sulfate  was used to precipitate contaminants, and then additional ammonium sulfate  was used to precipitate the LSDAO. The crude precipitate was redissolved, dialyzed against buffer A, applied to a DEAE column, and eluted with buffer A. Active fractions were adjusted to pH 6.5, applied to a CM resin column, and eluted with 5 mM phosphate buffer, pH 6.5. Active fractions were concentrated and the buffer was changed to 10 mM phosphate, pH 7.0 before the protein was applied to a Sephacryl S-300 column and eluted with the same buffer. The concentration of the LSDAO dimer was determined from its absorption at 278 nm in 0.1 M phosphate buffer5 M−1 cm−1. The enzyme had very high activity (more than 1 μkat/mg protein). Analysis by a 7.5% SDS PAGE gel showed a single band.The LSDAO extraction process was carried out at 4 °C according to the method of Floris et al.4 M−1 cm −1. SDS-PAGE and mass spectrometric analysis indicated that the isolated LOX was mostly pure, but also included a small amount (∼5%) of a 22 kDa protein impurity that has been identified as tyrosine-rich acidic matrix protein, TRAMP, which is unreactive under the activity assay conditionsLOX was isolated from bovine aorta at 4 °C using slight modifications of previously published proceduresex = 568 and λem = 581 nm). The assay was carried out in boric acid buffer  with 1.2 M urea for LOX or in phosphate buffer  for LSDAO. The final reagent concentrations in the assay mixture were 10 mM cadaverine, 10 μM Amplex UltraRed , and 1 U/mL horse radish peroxidase . The activity assay is adopted from the method described by Palamakumbura et al.−1) were plotted as –1/slope against 1/[inhibitor concentration] to produce a Kitz–Wilson plot. Lines of best fit (linear regression), slopes, y-intercepts, and their associated standard errors were calculated using Graphpad Prism 5.0b. The kinetic parameters KI − 1 and k2 were calculated such that k2 (min− 1) = 1/(y-intercept) and KI − 1 (μM) = slope/(y intercept). Results are tabulated in the Supporting Information.The enzyme activity, which is proportional to the rate of increase in fluorescence, was measured for each experiment. Residual activity was calculated by dividing each rate by a maximum rate determined in the absence of inhibitor such that [residual activity] = ([rate with inhibitor] – [baseline rate])/([maximum rate] – [baseline rate]). Baseline rates, which were typically <2% of the maximum rates, were determined in the absence of enzyme and inhibitor. The residual activities from replicate experiments were averaged. For each inhibitor concentration, log was plotted against inhibition time. Linear lines of best fit forced through the origin were calculated for each data set using Graphpad Prism 5.0b . For each inhibitor, the slopes of these lines , hexanoic acid , EDC hydrochloride , HOBt hydrate , and DCM  were added into a flask. The mixture was stirred (16 h), diluted with DCM (20 mL), washed with NaHCO3 (15 mL), sodium chloride , and NaHSO4 , and dried with sodium sulfate. Volatiles were removed under reduced pressure, and the crude product was purified by column chromatography  to yield pure carbamate 8 . 1H NMR  δ 7.60 , 6.64 , 2.20 , 1.76–1.57 , 1.46 , 1.39–1.20 , . 13C NMR  δ 172.74, 155.68, 81.79, 34.01, 31.32, 28.10, 24.91, 22.30, 13.89. IR : 3314, 3255, 2968, 2934, 2875, 1738, 1673, 1647, 1494, 1365, 1239, 1158. TLC (2:1) hexane/EtOAc, permanganate, fR = 0.31. MS calculated for [C11H22N2O3Na]+, requires m/z = 253.15, found m/z = 253.2 (ESI).Hydrazide 3 hydrochloride. Boc carbazate 8  was dissolved in dioxane (0.5 mL) and diluted with a solution of hydrochloric acid . The flask was capped with a rubber septum, and the mixture was stirred (19 h). Volatiles were removed in a stream of nitrogen gas and then under reduced pressure to yield pure acyl hydrazide 3 hydrochloride . 1H NMR  δ 11.01 , 10.70–10.20 , 2.22 , 1.53 , 1.36–1.11 , 0.86 . 13C NMR  δ 171.72, 32.86, 30.72, 24.45, 21.90, 13.95. IR : 3383, 2957, 2930, 2892, 1699, 1526, 1367, 1222, 1185. MS calculated for [C6H14N2OH]+ requires m/z = 131.12, found m/z = 131.2 (ESI).Carbamate 11. A flame-dried flask was capped with a rubber septum and maintained under a nitrogen atmosphere. Into the flask were added bis(Boc)hydrazine , DMF , 1-bromoheptane , and cesium carbonate . The reaction was stirred under a nitrogen atmosphere (19 h), diluted with brine (20 mL), extracted with EtOAc (2 × 20 mL), and dried with sodium sulfate. Volatiles were removed under reduced pressure, and the crude material was purified by column chromatography  to yield pure carbamate 11 . Based on the NMR signals, this compound may exist as interconverting rotational isomers . 1H NMR  δ 6.38–6.01 , 3.41 , 1.61–1.49 , 1.46 , 1.45 , 1.36–1.19 , 0.87 . 13C NMR  δ 155.30, 80.76, 49.42 (brm), 31.72, 28.93, 28.13, 27.41, 26.59, 22.51, 13.98. IR : 3278, 2979, 2920, 2857, 1698, 1514, 1404, 1364, 1287, 1148, 756. TLC (4:1) hexane/EtOAc, permanganate, fR = 0.63. MS calculated for [C17H34N2O4Na]+  requires m/z = 353.24, found m/z 353.3 (ESI).Hydrazine 4 Trifluoroacetate. Carbamate 11 , DCM (1.5 mL), and trifluoroacetic acid (0.5 mL) were added into a vial. The reaction was stirred (6 h), and volatiles were removed under reduced pressure (toluene azeotrope) to yield pure hydrazine 4 trifluoroacetate . 1H NMR  δ 2.88 , 1.64–1.42 , 1.31–1.11 , 0.86 . 13C NMR  δ 158.89 , 116.94 , 50.63, 31.13, 28.37, 26.04, 24.66, 22.09, 13.96. IR (neat cm− 1): 2957, 2928, 2859, 1684, 1668, 1203, 1140. MS calculated for [C7H18N2H]+, requires m/z = 131.15, found m/z = 131.2 (ESI).Boc-Semicarbazide 14. n-Butylamine , DCM (10 mL), and a sodium bicarbonate solution  were added into a flask. The mixture was cooled in an ice bath (0 °C). Triphosgene  and dry DCM (5 mL) were added into a flask. The solution was cooled to 0 °C and then added into the amine solution. The mixture was stirred (30 min) and then allowed to settle. The organic phase was isolated, dried with sodium sulfate, and returned to the ice bath. tert-Butyl carbazate  was added. The mixture was stirred , washed with sodium bicarbonate  and sodium bisulfate , and dried with sodium sulfate. Volatiles were removed under reduced pressure, and the crude material was purified by column chromatography  to yield pure Boc-semicarbazide 14 . Based on the 13C NMR, this compound appears to exist as a mixture of interconverting rotational isomers . 1H NMR  δ 6.32 , 6.21 , 5.40–5.23 , 3.24 , 1.66–1.23 , 1.48 , 0.91 . 13C NMR  δ 159.22, 156.55, 81.38, 39.57, 31.97, 27.99, 19.76, 13.63. IR : 3315, 2962, 2933, 1721, 1661, 1552, 1368, 1244, 1158, 909, 731. MS calculated for [C10H21N3O3Na]+ requires m/z = 254.15, found m/z = 254.1 (ESI). TLC (2:1) DCM/EtOAc, permanganate, fR = 0.30.Semicarbazide 5 Hydrochloride. Boc-semicarbazide 14 , dioxane (0.7 mL) and a solution of hydrogen chloride  were added into a vial. The vial was capped with a rubber septum, and the mixture was stirred (5 h). Volatiles were removed in a stream of nitrogen gas and then under reduced pressure to yield pure semicarbazide 5 hydrochloride . 1H NMR  δ 9.98–9.63 , 8.63 , 7.02 , 3.04 , 1.47–1.18 , 0.86 . 13C NMR  δ 157.27, 31.70, 19.47, 13.76. . IR : 3299, 2862, 2663, 1682, 1557, 1493, 1282, 647. MS calculated for [C5H13N3OH]+ requires m/z = 132.11, found m/z = 132.2 (ESI).3–5, which are representative members of the hydrazide, alkyl hydrazine, and semicarbazide families, each connect a hydrazine-containing core functional group to a simple aliphatic chain (2N–NH2), which is a potent inhibitor itself. Hydrazide 3 was synthesized by a carbodiimide-promoted amide bond forming reaction between hexanoic acid7. Alkyl hydrazine 4 was formed by alkylating bis(Boc)hydrazine 10 under basic conditions. And finally, semicarbazide 5 was synthesized by trapping isocyanate 13 with mono(Boc)-protected hydrazine 7.Compounds ic chain . To syntic chain  that can50 = 6 μM) is somewhat more potent than the other three hydrazine-based inhibitors (IC50 = 20–100 μM), although the four IC50 values only span a 17-fold range. Of the three new inhibitors, hydrazide 3 is the most potent, followed by alkyl hydrazine 4 and then by semicarbazide 5. In contrast, the inhibitions of LSDAO  and 2k (the rate constant for irreversible reaction)   can be cKI) shows two major trends  It may be that the inhibitors with additional polar and hydrogen-bonding groups near their hydrazine unit are less well matched to a hydrophobic active site, especially for LSDAO, which is known to have its active site buried in a hydrophobic cleftThe results from the kinetics analyses of the three synthetic hydrazines as irreversible inhibitors of LOX and LSDAO enable specific comparisons of their kinetic parameters to be made. For the inhibition of LOX with phenylhydrazine, data previously reported by Kagan and coworkers are also included in this comparisonr trends . First, −1 and have overlapping error ranges  will have a larger percent of their molecules protonated and therefore unable to react compared to phenylhydrazine (pKa of conjugate acid = 5), hydrazides (pKa of conjugate acids = 3–4), or semicarbazides (pKa of conjugate acids = 3–4). For the reactions with LSDAO, the active site – which is believed to be located in a hydrophobic channelKa values and negate this difference by only allowing nonprotonated inhibitors into the active site.Comparing the rate constants for the irreversible step, which are calculated as the reciprocal of the y-intercept, shows that most of the values are at least 0.2 minr ranges . Some of50 value of 0.2 μM and full inhibition at 10 μM have been reported for a 2-h incubationKI = 6 μM and 2 k= 0.16 min−1 (pH 8.0 and 37 °C), although the authors note that the data clearly diverge from linearity at later time points, which is perhaps due to the more complex mechanism of inhibition.BAPN is generally considered to be the most potent and selective inhibitor for LOX. Its selectivity is believed to relate to the fact that it likely modifies a specific (currently unidentified) active-site residue near the lysyl tyrosylquinone that is not present in other amine oxidasesin vivo2NC(O)NHNH2 with no organic scaffold) is nearly as effective as BAPN at inhibiting rat lysyl oxidase in vivoIn vitro experiments showed an IC50 value of 30 μM (inhibition time not specified) for semicarbazide3 and alkyl inhibitor 4 have IC50 values of 20 and 40 μM, respectively, which are not too different from that measured for phenylhydrazine, 6 μM, or from the other hydrazine inhibitors mentioned above. Semicarbazide 5, which has an IC50 of 100 μM, does not perform as well as the other compounds, perhaps due to the extra N–H unit being poorly matched to a hydrophobic region of the enzyme's active site. While these new inhibitors are not as potent or selective as BAPN, due to their straightforward mechanisms of inhibition, the ease with which they can be incorporated into larger molecules, and the precedent of hydrazine groups in FDA-approved drugs, we are optimistic that they have the potential to be able to be developed into inhibitors with strong potency and high selectivity.Previous reports have utilized phenylhydrazine to inhibit LOX; however, much less is known about other families of hydrazine-containing organic compounds as LOX inhibitors. For hydrazides, it has been reported that the hydrazide of isonicotinic acid can partially inhibit LOX in chick embryos 3 shows properties that may be more advantageous. Using LSDAO as an example “off-target” enzyme suggests that phenylhydrazine, whose IC50 value for LSDAO is 600-fold more potent than that for LOX, may be difficult to develop into an inhibitor that is selective for LOX. Hydrazide 3, which has only a 10-fold difference between the two enzymes, might be a better place to start, even though it has 3-fold lower affinity for LOX than phenylhydrazine does.The comparisons between LOX and LSDAO presented herein provide useful insights into how these simple organic hydrazine structures could be applied as warhead groups in larger molecules designed to selectively target LOX. Although phenylhydrazine is the most potent of the four hydrazine compounds at inhibiting LOX, hydrazide 3 also suggests that the hydrazide core will be an excellent place to begin developing inhibitors that selectively target LOX. The rate constants for hydrazide 3 to bind LOX and LSDAO (0.4 and 0.7 min−1 with overlapping error bars) are similar to those seen for some FDA-approved covalent inhibitors such as the anticancer drug Afatinib (2 k= 0.14 min−1)The kinetics analysis of hydrazide In conclusion, the data reported herein compares four families of hydrazine-based inhibitors for their abilities to inhibit quinone-dependent amine oxidases. Representative members of the hydrazide, alkyl hydrazine, and semicarbazide families were synthesized and compared to phenylhydrazine as inhibitors of both LOX and LSDAO. Phenylhydrazine and the alkyl hydrazine inhibit LOX but are much more potent inhibitors of LSDAO. The hydrazide and semicarbazide inhibitors are much less potent inhibitors of LSDAO suggesting that they have more promise for development as selective inhibitors of LOX. Of these two, the hydrazide is five times as potent an inhibitor of LOX and has only a slight preference for LSDAO, which makes it a more promising core functionality to develop into inhibitors that selectively target lysyl oxidase.


The proposed method showed good linearity in the concentration range of 0.2–20.0 µg/L with the determination coefficient greater than 0.995. The detection and quantification limits ranged from 0.06 to 0.13 µg/L and 0.18 to 0.40 µg/L, respectively, which are sufficient to meet the regulatory detection limits as set by most environmental regulations. The accuracy and precision of the method was also good, where the recoveries ranged from 82.5 to 95.4%, and intra- and interday precision was within 7.2% and 14.5%, respectively. The optimized method has been applied to the determination of SCCPs in ten freshwater samples of three different types.Short-chain chlorinated paraffins (SCCPs) are a new type of persistent organic pollutants. In this work, a simple and effective method involving headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF-MS) was developed and optimized for the determination of trace SCCPs in water samples. The key parameters related to extraction and separation efficiency were systematically optimized. The SCCP congener groups were best resolved using an Rxi-5Sil MS (30 m × 0.25 mm × 0.25  Because of their excellent physicochemical properties, SCCPs have been widely used in metalworking applications and polyvinyl chloride processing and used as plasticizers or flame retardants in paints, adhesives, sealants, leather fat liquor, lubricants, plastics, rubber textiles, and polymeric materials since the 1930s + (m/z 278) is very low. Although the fragment ion at m/z 91 was not the most abundant ion, this pentacyclic positive ion [C4H8Cl]+ was considered specific as it was only generated by the rearrangement of polychlorinated n-alkanes during the mass fracture process, and thus, it was selected as the quantitative ion. The other fragment ions at m/z 67, 75, and 103, although they have higher abundance than [C4H8Cl]+, can also be produced by other chlorinated compounds, so they were not selected as quantitative ions.Under the abovementioned optimized conditions, the separation of SCCP congener groups was achieved. According to the distribution rules of SCCPs –26, the µm PDMS), one semipolar (65 µm PDMS/DBV), and one highly polar (85 µm PA) fibers, were investigated in terms of extraction capacity and reproducibility. Here, the extraction capacity was assessed by the peak area ratio, which was calculated by summing all the peak areas of SCCPs and dividing by the internal standard peak area; the reproducibility of the fibers was evaluated by the RSD values of peak area ratios. Each experiment was performed in triplicate with 10 mL blank water spiked at 10.0 µg/L with standard mixture of SCCPs .Initially, three different SPME fibers, including one nonpolar  [µg/L mixed CPs standard solutions  were submitted to the analysis under the optimized conditions.Since MCCPs and LCCPs may also be present in the sample of SCCPs and their presence may interfere with SCCPs analysis, it is necessary to study their influence on SCCPs. To study their effect, 10 mL Milli-Q water spiked with 10 As can be seen from µg/L, C10–13, 55.5% chlorination in acetone). Results showed that the total peak areas of SCCPs in two kinds of samples were basically same, and the RSD values of SCCPs peak areas were less than 0.9%, indicating that the presence of MCCPs and LCCPs would not interfere with the subsequent quantification of SCCPs.Moreover, the samples were compared with those containing only SCCPs  [µg/L, such as 2.4 µg/L for water in the Canadian Environmental Protection Act [µg/L  for surface water in European Water Framework Directive [The LODs and LOQs ranged from 0.06 to 0.13 Q water) , 14, butQ water) , mainly irective , so the µg/L) in five replicates. The recoveries ranged from 82.5% to 104.2% in three types of water samples, demonstrating the good accuracy of the method.The accuracy of the method was evaluated by recovery experiments using the spiked sample at three levels  and river samples (2.98–5.07 µg/L). The concentration of SCCPs in the detected river and lake samples (#3–#10) was generally greater than the values reported in the freshwater of Britain [µg/L) [To confirm the applicability of this method, the proposed method was applied to ten real water samples, including two underground water samples, four lake water samples, and four river water samples. All samples were measured in duplicate.  Britain , Spain [ Britain , Japan [ Britain , and Can Britain . Two lakles 2.98–.07 µg/L.ples #3–#0 was genn [µg/L) . Consideµg/L) and LOQs (0.18–0.40 µg/L) were relatively low and could meet the requirement of quality detection on SCCPs in water samples as mentioned in most international conventions and regulations. Besides, the interference from MCCPs and LCCPs can be excluded in the initial extraction step because these compounds cannot be enriched on the SPME fiber. Based on these observations, we believe that this method can be used for the routine analysis of SCCPs in water samples.The present study is the first report which propose a green, sensitive, and reliable method for the determination of SCCPs in water samples by online HS-SPME and GC × GC-TOF-MS. Compared to the existing analytical methods, this method not only simplifies the operation process saving both time and labor but also yields good accuracy, precision, and sensitivity. The LODs (0.06–0.13


N-vinylcaprolactam P1, P2, P3, P4, and P5 were synthesized via the surfactant free precipitation polymerization (SFPP) at 70 °C in the presence of cationic initiator 2,2’-azobis[2-methylpropionamidine] dihydrochloride (AMPA). The influence of various concentrations of initiator AMPA on particle size, aggregation and lower critical temperature solution (LCST) was investigated by dynamic light scattering (DLS) measurement. The conductivity was measured in the course of the synthesis and during temperature decrease of the reaction mixtures. The polymers were characterized by Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR), 1H NMR, and thermogravimetric analysis. Thermal parameters of the degradations process were investigated using thermogravimetric analysis (TGA/DTA) under non-isothermal conditions in N2 atmosphere. The samples were characterized by powder X-ray diffraction analysis (PXRD).The hydrodynamic diameter (HD), polydispersity index (PDI) and zeta potential (ZP) were measured in aqueous dispersions of the synthesized polymers in temperature 18–45 °C. HD and PDI values at 18 °C were 137.23 ± 67.65 nm (PDI = 0.53 ± 0.18), 83.40 ± 74.46 nm (PDI = 0.35 ± 0.08), 22.11 ± 0.29 nm (PDI = 0.45 ± 0.05), 29.27 ± 0.50 nm (PDI = 0.41 ± 0.04), 39.18 ± 0.57 nm (PDI = 0.38 ± 0.01) for P1, P2, P3, P4, and P5, respectively. The aqueous solutions of the obtained polymers at 18–45 °C had a positive charge. ZP’s for P1, P2, P3, P4, and P5 polymers at 18 °C were 11.64 ± 4.27 mV, 12.71 ± 3.56 mV, 3.24 ± 0.10 mV, 0.77 ± 0.28 mV, 1.78 ± 0.56 mV respectively. The LCST range was between 32 and 38 °C. We conclude that the concentration of initiator affects the size of obtained polymeric spheres and theirs LCST.The thermosensitive polymers of  Despite continuous advances in pharmacotherapy, there are still many factors that limit effective pharmacological treatment, including drug instability in pH conditions of the gastrointestinal tract, low bioavailability, short half-life time, low drug selectivity, and adverse reactions in healthy cells, e.g., in the treatment of cancer . Thus, mSensitive nanopolymers have significant potential for medical applications due to their specific behavior, depending on the external conditions. The physical and chemical properties are modified as a response to a change in the environmental stimulus, such as temperature, pH, magnetic or electric field, light, mechanical stress, and the presence of a given chemical substance ,9,10,11.N-isopropylacrylamide (PNIPAAm), however, it may exert side effects. Another compound—N-vinylcaprolactam (NVCL)—presents similar as NIPAAm characteristics, and is contemporarily studied. The N-vinylcaprolactam was initially characterized in 1957  dihydrochloride (AMPA) varied. A more detailed description of the synthesis procedure had been reported in The aqueous polymer solutions, purified by dialysis method, were freeze-dried. From 100 mL of cleaned solutions of polymers P1, P2, P3, P4, and P5 were obtained about 0.06166, 0.07973, 0.11637, 0.13174 and 0.18569 g of solid product, respectively.−1, increased rapidly after introducing to the system the respective amount of initiator. The average values were 3200, 2200, 1200, 630, and 320 μS cm−1 for P1, P2, P3, P4, and P5, respectively. As expected, the conductivity of the initiator was proportional to its concentration. Addition of the initiator to the reactant mixtures was marked as point (a) in −1 for systems P1, P2, P3, P4, and P5, respectively. The conductivity gradually increased until a plateau phase.The electrolytic conductivity tests were carried out in the course of the polymerization reactions and after synthesis during the cooling of the reaction mixtures. The readings were carried out in one-second time intervals for P1, P2, P4, and P5 and in 30-s intervals for P3. The conductivity profile of aqueous dispersions of P1, P2, P3, P4, and P5 polymers in the course of the polymerization reaction is presented in The conductivity in function of temperature cf.  and of t−1, respectively, which remained transitionally constant, and then gradually decreased to 3167 μS cm−1 for polymer P1, 2124 μS cm−1 for polymer P2, and 562.2 μS cm−1 for polymer P4. In P3 and P5 systems shortly before the expected phase transition, at temperatures 36.5 and 34.8 °C, respectively, the conductivity profile shifted from a slight to a rapid decrease. Further reduction of the temperature resulted in a sharp increase of conductivity from 1088 μS cm−1 in 36.0 °C to 1091 μS cm−1 in 33.0 °C, and from 292.2 μS cm−1 in 32.6 °C to 294.1 μS cm−1 in 30.7 °C for polymers P3, and P5, respectively. In the next stage the conductivity regularly decreased to 1085 and 293 μS cm−1 at 24 °C. Only the solutions of polymer P1 and P2 reached the plateau phase at temperatures 27.7 and 26.2 °C, respectively.The conductivity systematically decreased with the decrease of temperature. Moreover, at temperatures 37.5, 36.5, 36, 34.9, 32.6 °C for systems P1, P2, P3, P4, and P5, respectively, there were variations in the profile of conductivity. At the above-mentioned temperatures, the P1, P2, and P4 systems reached conductivity values of 3184, 2134, and 565.3 μS cm−1 for P1, P2, P3, P4, and P5, respectively. The deflection was visibly marked for polymeric systems P3 and P5. The presented aberrations were followed by regular exponential decay.According to the data in N-vinylcaprolactam (NVCL), initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AMPA), and synthesized polymers P1, P2, P3, P4, and P5 are presented in −1 due to carbonyl peak C=O, at 1652 cm−1 attributed to double bond C=C, at 3108 and 992 cm−1 corresponded to the =CH and =CH2 vinyl peaks, respectively, at 1484 cm−1 C–N stretching vibration, and at 970 cm−1 –CH2 twisting vibrations originating from the seven-membered ring [−1 related to C–H stretching [−1 characteristic for the primary amines, estimated as asymmetric N–H stretch at 3185 cm−1 and symmetric N–H stretch at 3068 cm−1. The peak at 1676 cm−1 can be attributed to C=N stretching vibration. The positions at 1213 and 1065 cm−1 corresponded to C–N stretching vibrations. The peak at 1361 cm−1 was due to presence of methyl group –CH3. The spectra of polymers exhibited broad band at 3600–3000 cm−1 belonged to the O–H stretching. The peaks at 2922 and 2851 cm−1 corresponded to the aliphatic C–H stretching vibrations. The strong band at 1612 cm−1 can involve of bending vibration C=O. The peaks at 973, 1440, and 1479 cm−1 can be attributed to the twisting vibrations of –CH2, stretching vibration of –CH2, and CN bond, respectively [The attenuated total reflection Fourier-transformed infrared spectroscopy (ATR–FTIR) spectra of red ring , furtherretching . In the ectively .1H NMR spectra of monomer NVCL, initiator AMPA, and five synthesized polymers P1, P2, P3, P4, and P5 are given in 1H-NMR spectrum of NVCL six groups of equivalent protons were marked in the spectrum as a, b, c, d, e, and f  of synthesized cationic polymers was investigated in the temperature range of 18–45 °C in unbuffered, aqueous solutions A–E. The −1 are shown in The characteristic TG and DTG curves of the thermal decomposition of nanospheric polymers P1, P2, P3, P4, and P5 obtained at a heating rate of 10 °C minTOnset), temperature of maximum weight loss rate (Tm), extrapolated temperature at which the degradation process ended (TEndset), the temperature at which 0.5 wt% loss occurred (T0.5wt%), and amount of residue at for all samples determined by thermogravimetric analysis are summarized in The TG plots represent the percentage mass loss of tested polymeric samples in the temperature range of from 29 to 800 °C. In all cases TG curves presented a similar course of thermal degradation. In the temperature range of 29–100 °C there was a gradual ca. 5% mass loss, then, up to 350 °C, no weight loss was observed. At temperatures above 350 °C there was a significant ca. 92% weight loss, finishing at about 495 °C. Then the mass of samples was stable until ca. 800 °C, and accounted for ca. 3% of the initial mass. In the DTG plots two peaks were always observed: one at about 52 °C and the second around 437 °C. The characteristic thermal decomposition parameters, e.g., extrapolated onset temperature of decomposition . The shrinking of particles may cause the ejection of charged chemical groups existing inside the sphere on the surface and increase their density. The above considerations are reflected in the observations of turbidity, which is visible at the beginning of the increase in conductivity value  and C–N (1479 cm−1) stretching vibration from groups in polymer chain. A slight change in the position of the peaks responsible for the presence of CO, CN and CH stretching vibrations from 1622 to 1612 cm−1, from 1484 to 1479 cm−1, and from 2930 to 2922 cm−1, respectively, on the spectra of polymers relative to the monomer spectrum may also indicate the occurrence of polymerization. The shifts may due to the conformational changes in the polymeric spheres within the side groups and interactions between atoms have changed. The broad band appearing in the range 3700–3000 cm−1 on the spectra of polymers indicates the presence of hydrogen bonds in the obtained products. The peaks at 970 and 973 cm−1 in the monomer spectrum and polymer spectrum respectively are related to the –CH2 twisting vibrations in the caprolactam ring. Peaks that correspond to the –C–N, –C–C–C–O groups may appear at this wavelength. Signals from the –CO group can be excluded because carbon connected with oxygen by a single bond is not present in both the monomer and polymer molecule of N-vinylcaprolactam. The ring breathing or deformation vibration may occur in these region but vibrations from –C–C group are not interpretatively useful. Based on the data from the spectral database, it was found that in the infrared spectrum of cycloheptane at 950 cm−1 occurs a strong peak [−1 in spectrum of NVCL and 973 cm−1 in spectrum of poly-NVCL mainly due to twisting vibrations of CH2 group in the seven-membered ring.ATR-FTIR spectra of polymers synthesized under different amount of initiator are identical, the absorption bands from polymers P1, P2, P3, P4, and P5 overlap. Therefore, given the fact that for each compound a unique spectrum is created, one can be sure that the same substance was formed during the five syntheses performed. A comparison of the spectra of substrates and products allowed the determination if the polymerization occurred. The analysis of the spectrum of the monomer and spectra of the obtained polymers shows that the peaks at 3108, 1652, 992 cmong peak . This co1H NMR spectra of the NVCL, AMPA and five polymers P1, P2, P3, P4, and P5 in DMSO-d6 solution were measured as additional confirmation that the polymerization process was successful and to support the structure and composition of prepared polymers. Analyzing the 1H-NMR spectrum of NVCL the peaks originate from the protons of vinyl group were found in the range δ = 4.27–7.17 ppm  as illustrated in The absence of sharp peaks on the all polymers’ patterns indicates that the obtained products are not crystalline and generally exhibit amorphousness. Upon decreasing the initiator content, there was a gradual and progressive increase of the peaks’ intensities, which may be due to the effect of the amount of initiator on the molecular structure of the polymer. The obtained results of the crystalline form of commercial NVCL correspond very well with the data from the literature . The basN-vinylcaprolactam  and 2,2′-azobis(2-methylpropionamidine) dihydrochloride  were obtained from commercial and industrial suppliers and were used without further purification. NMR solvent—dimethyl sulfoxide-d6  was purchased from Sigma-Aldrich CHEMIE GmbH, Sternheim, Germany. Dialysis tubing cellulose membrane  was received from Sigma-Aldrich CHEMIE GmbH, Sternheim, Germany. Deionized water fulfilled requirements of PN-EN ISO 3696:1999 for analytical laboratories; it was filtered in an HLP 20 device equipped with microfiltration capsule 0.22 μm  and was applied in all following procedures.−1, T = 25 °C). The reaction set was equipped in reflux condenser, temperature probe, conductivity cell (K = 1 cm−1), and magnetic stirrer (250 rpm). After nitrogen bubbling for ca. 30 min, an appropriate amount of the dry sample of free radical cationic initiator—AMPA, was introduced to the reaction vessel. Next, after ca. 10 min, the monomer (NVCL) dissolved in 100 mL of deionized water at room temperature with stirring in a beaker was added. The total volume of the reaction mixture was 1000 mL. The reaction was carried out over 6hours in with respective mixing and continuous nitrogen flow. The temperature of reaction mixture was kept constant of 70 ± 1 °C throughout the course of the reaction. −1. On average, the purification procedure involved six 24-hour cycles for every sample. Cleaned aqueous solutions of polymeric product (100 mL) were frozen, and lyophilized by Alpha 1–2 LD  two times for 26 h, and stored dry.Five derivatives of N-vinylcaprolactam,  were synthesized via the surfactant free precipitation polymerization (SFPP). First, the 2000-mL four-necked round bottom flask reaction vessel was filled with 900 mL deionized water, which is a solvent for all reactants, and heated to 70 °C  and temperature sensor Pt-1000A (0–100 ± 0.35 °C). Measuring accuracy of the device up to 19.999 mS cm−1 was ±0.1% and from 20.000 mS cm−1 was ±0.25%. The conductivity and temperature sensors were placed into the reaction vessel and remained in permanent contact with the reaction mixture. Conductivity measurements during synthesis were performed at 70 °C manually and, during the cooling procedure, with an automatic compensation of temperature.The conductivity of the aqueous solution of the reaction system in the course of the synthesis and during cooling procedure was measured using a model CC-505 laboratory conductometer  equipped with an EC-60 conductivity sensor with a glass housing and constant cell of −1 over the wavenumber ranging from 4000 to 400 cm−1. The background spectra using a blank ATR were recorded before prior to applying the sample to the crystal and was automatically subtracted from measured spectrum of sample. The sample was applied directly on the area of the monolithic diamond crystal cell and compressed using a pressure arm. Before each measurement ATR was cleaned using methanol-soaked tissue paper.The transmission and multiple attenuated total reflection infrared spectra of lyophilized samples were measured using a Nicolet iS50 FT-IR spectrometer  with DTGS (deuterated triglycinesulphate) detector. Each spectrum was the Fourier transformation of 32 scans per sample collected at a resolution of 4 cm1H NMR spectra were recorded in NMR solvent deuterated dimetylsulfoxide, , at 299.15 K using a Bruker spectrometer with the working frequency of 300 MHz . Coupling constants (J) are in Hertz (Hz) and chemical shifts (δ) are expressed in ppm. Analyzed by NMR, samples were prepared by dissolving about 10 mg of each compound in 7 mL of DMSO-d6.The The hydrodynamic diameters of the nanopolymers aqueous dispersion were measured on a Zetasizer Nano device from Malvern Instruments  by the dynamic light scattering method (DLS). The Zetasizer system was equipped with standard-red He–Ne laser, output power of 4 mW, and wavelength of λ = 633 nm, as a light source. The light intensity was adjusted during the measurement sequence by automatically setting the attenuator for the laser beam. The scattered light was detected at an angle of 173°, the backscattering setting. The DLS measurements were carried out in a temperature-controlled cell over a temperature range of 18–45 °C in one degree steps using a polyacrylic disposable DTS-0012 cuvette . The cuvette was filled with 1 mL of polymer solution, purified by dialysis and not diluted. The measurements rounds were carried out five times at each temperature to give an average hydrodynamic diameter and size distribution. The number of replicates conducted in one round was adapted automatically in the range of 10–100 measurements. At each new temperature samples were allowed to equilibrate for 120 s. The polydispersity index (PDI) was calculated as a parameter of the particle size distributions using Malvern Instrument software based on the Stokes–Einstein equation for spheres. The size and PDI parameters were calculated according to the definition in the ISO 13321:1996E and ISO 22412:2008 ,39,40.The zeta potential (ZP) was measured by analyzing the electrophoretic mobility of synthesized polymers nanoparticles in aqueous dispersion. The measurements were carried out by using a Zetasizer Nano device from Malvern Instruments  in type DTS-1070 U-shaped cuvettes  and applying the Doppler effect . ZP values were calculated from the Smoluchowski model approximation to Henry’s equation (f(Ka) = 1.5). The electrophoretic mobility was measured as a function of temperature in the range of 18–45 °C in one degree steps and with an equilibration time of 120 s. At each temperature measurement rounds were executed five times.−1) under an N2 atmosphere (with a flow rate of 50 mL min−1). The lyophilized amorphous samples form of approximately 5 mg were loaded into a corundum crucible and gently pressed. The samples were not grated beforehand. During the heating the weight loss of samples as a function of temperature and time was recorded automatically. To determine the mass change and DTG curves the Netzsch Proteus 7.1.0 analysis software  was used.The thermal stability of the synthesized polymeric nanospheres was analyzed by using a TG 209 F1 Libra instrument with automatic sample changer (ASC), . The measurements were carried out under non-isothermal heating conditions in a temperature range of 29–800 °C (heating rate 10 °C min1.2 radiation (1.5418 Å). All samples were previously crushed in a mortar and then measured at 295 K in ambient atmosphere under constant conditions of 30 kV, 10 mA. The PXRD diffractograms were measured at 2θ, in the Bragg–Brentano (θ/2θ) horizontal geometry between 5° and 70° with 0.02° increments, and a recording time of 1.0 s/step. The variable rotation was 15 min−1, with a divergence slit 0.6 mm in size.The X-ray powder diffraction (PXRD) patterns were measured on an X-ray diffractometer on a Bruker D2 PHASER diffractometer  with a LynxEYE detector using Ni-filtered CuKα1H NMR confirm that the polymerization reaction of NVCL has occurred. The polymer solutions were also characterized by measurements of ZP and the polydispersity index depending on temperature. The results of PDI measurements showed a relatively low degree of polydispersity for polymers P1, P2, P3, and P4 in the entire temperature range whereas, in the case of the P5 polymer, after initiating the phase transformation the polydispersity index was close to unity. The recorded ZP values indicated the positive charge on the surface of the poly-N-vinylcaprolactam particles and stability at higher temperatures. The solid products of synthesis were also subjected to PXRD and DSC tests and as a result the amorphity of the poly-NVCL was confirmed and the characteristic thermal decomposition parameters were determined. Purified by dialysis, aqueous suspensions of the obtained polymers, have a pH in the range of 7.43–6.85, which makes them suitable for applications as drug carriers.Surfactant-free precipitation polymerization in an aqueous environment at 70 °C degrees is an effective method of synthesis the polymer of N-vinylcaprolactam. In these studies we conducted five syntheses that differed only in the initial concentration of the initiator. As a result of the polymerization process five thermosensitive poly-NVCLs: P1, P2, P3, P4, and P5, strongly aggregating after exceeding the lower critical solubility temperature, were obtained. The hydrodynamic measurements of the particles confirmed that products were of nano-size. The conductometric measurements during synthesis allowed monitoring of the polymerization process. The particle size measurement versus temperature by the dynamic light scattering method was a suitable method to determine the phase transition temperature of thermally-sensitive poly-NVCL. Furthermore, the conductometric measurements of the reaction mixture after the synthesis and during the cooling can also be used to determine the value of LCST. The phase transition temperature was determined for the all synthesized polymers and it has been found that the changes of the initial concentration of the initiator caused changes in the LCST value. The ATR-FTIR and


Escherichia coli coexpressing AAR and ADO was highest for AAR from Synechococcus elongatus PCC 7942 (7942AAR), which has high substrate affinity for 18-carbon fatty acyl-ACP, resulting in production of mainly heptadecene. In contrast, the hydrocarbon yield was lowest for AAR from Synechococcus sp. PCC 7336 (7336AAR), which has a high specificity for 16-carbon substrates, leading to production of mainly pentadecane. However, even the most productive AAR (7942AAR) still showed low activity; thus, residues within AAR that are nonconserved, but may still be important in hydrocarbon production need to be identified to engineer enzymes with improved hydrocarbon yields. Moreover, AAR mutants that favor shorter alkane production will be useful for producing diesel fuels with decreased freezing temperatures. Here, we aimed to identify such residues and design a highly productive and specific enzyme for hydrocarbon biosynthesis in E. coli.Acyl-(acyl carrier protein (ACP)) reductase (AAR) is a key enzyme for hydrocarbon biosynthesis in cyanobacteria, reducing fatty acyl-ACPs to aldehydes, which are then converted into hydrocarbons by aldehyde-deformylating oxygenase (ADO). Previously, we compared AARs from various cyanobacteria and found that hydrocarbon yield in E. coli coexpressing ADO. Moreover, by combining these mutations, we successfully created 7336AAR mutants with ~ 70-fold increased hydrocarbon production, especially for pentadecane, when compared with that of wild-type 7336AAR. Strikingly, the hydrocarbon yield was higher in the multiple mutants of 7336AAR than in 7942AAR.We introduced single amino acid substitutions into the least productive AAR (7336AAR) to make its amino acid sequence similar to that of the most productive enzyme (7942AAR). From the analysis of 41 mutants, we identified 6 mutations that increased either the activity or amount of soluble AAR, leading to a hydrocarbon yield improvement in E. coli, are more highly effective in hydrocarbon production, especially for pentadecane, than wild-type AARs. Our results provide a series of highly productive AARs with different substrate specificities, enabling the production of a variety of hydrocarbons in E. coli that may be used as biofuels.We successfully designed AAR mutants that, when coexpressed with ADO in  A mer (HCOOH) , 17. Escynthesis , 18.E. coli coexpressing ADO with AARs derived from eight representative cyanobacteria [Synechococcus elongatus PCC 7942 (7942AAR) and lowest for the AAR from Synechococcus sp. PCC 7336 (7336AAR). The hydrocarbon yield was dependent on both the activity and amount of soluble AAR protein. Our results showed that 7942AAR had the highest activity, while both the activity and amount of the soluble form were low for 7336AAR. However, even the activity of 7942AAR was low, with a turnover rate of 0.51 min−1 [Previously, we analyzed the amount of hydrocarbon produced in bacteria . We foun51 min−1 . Thus, i51 min−1 .E. coli coexpressing ADO with either 7942AAR or 7336AAR produced mainly heptadecene (C17:1) or pentadecane (C15:0), respectively. Because shorter alkanes have lower freezing temperatures and because alkanes have higher cetane numbers than alkenes [E. coli.Interestingly, the substrate specificity of AAR depends on the habitats of the derived cyanobacteria . We show alkenes , 23, AARE. coli, we introduced single amino acid substitutions into the least productive AAR (7336AAR) to make its amino acid sequence more similar to that of the most productive AAR (7942AAR). We identified six mutations that are responsible for improving either the activity or amount of soluble AAR, leading to an increase in hydrocarbon yield in E. coli coexpressing ADO. Furthermore, by combining these mutations, we succeeded in creating 7336AAR mutants with increased hydrocarbon production, especially for pentadecane, in E. coli.In this study, to identify AAR residues that are essential for improving hydrocarbon production in E. coli [Nostoc punctiforme PCC 73102 (73102AAR), Gloeobacter violaceus PCC 7421 (7421AAR), and Synechocystis sp. PCC 6803 (6803AAR), in ascending order [Based on multiple sequence alignment of the AARs Fig. , we desiE. coli . Hydrocang order .Fig. 1AmWhen the amino acid sequences of the AARs were compared, only nine amino acids of 7336AAR differed from those of other AARs  . In total, 41 mutations were designed, with each mutant 7336AAR having a single amino acid substitution, and their hydrocarbon yields were assessed by a hydrocarbon production assay.Nostoc punctiforme PCC 73102 (73102ADO) in E. coli and attempted to measure the amount of hydrocarbon produced, amount of soluble AAR, AAR activity, and AAR substrate specificity , while the amount of soluble AAR protein in E. coli was quantified by western blotting. AAR activity was calculated as the total amount of hydrocarbon divided by the amount of soluble AAR [E. coli [We coexpressed each of the 41 single mutants of 7336AAR with ADO from uble AAR . Note thuble AAR , 19, and[E. coli .The results showed that the amount of soluble wild-type 7336AAR was too low to be quantified by western blotting  was calculated as a ratio of the amount of soluble protein to the protein expression level , heptadecene (C17:1), and heptadecane (C17:0) [ C17:0) , 21, 24.7:0 , 21,E. coli cells. To clarify the degree of contribution of these two factors to hydrocarbon production, we performed a correlation analysis with the double mutant data   Fig. d. Moreov97) Fig. h, i. A nThe above results showed that the S298A single mutant increased the hydrocarbon yield ~ sixfold compared with that of wild-type 7336AAR nes catalyzed by ADO can be a rate-limiting step in the overall process of hydrocarbon biosynthesis in els Fig. . We introli Fig. a. These oli Fig. c, and 5bE. coli, the hydrocarbon yield was only ~ 2% that achieved with 7942AAR  at residues 162–168 , 30. ThuE. coli coexpressing ADO. Moreover, by combining these mutations, hydrocarbon production increased ~ 70-fold compared with that of wild-type 7336AAR, with a hydrocarbon yield higher than that of 7942AAR. Thus, we succeeded in developing AARs that are highly productive , even more so than their wild-type counterparts. Our results will provide a series of highly active AARs with different substrate specificities, enabling the production of a variety of hydrocarbons in E. coli that can be used as biofuels. The present findings will also be useful for improving the activity of existing enzymes, such as 7942AAR, by introducing various mutations at the six residues identified to determine hydrocarbon yield.In this study, we introduced single amino acid substitutions into the least productive AAR (7336AAR), which has high specificity for 16-carbon fatty acyl-ACP/CoA, to make its amino acid sequence more similar to that of the most productive AAR (7942AAR), which has high affinity for an 18-carbon substrate. By analyzing 41 mutants of 7336AAR, we identified mutations that are responsible for improving either the activity (L20R and S200E) or the amount of soluble AAR , leading to an increase in the amount of hydrocarbon produced, especially for pentadecane, in Plasmids containing 7336AAR and 73102ADO were previously constructed based on the pETDuet-1 coexpression vector  . All AARE. coli by coexpression of AAR and ADO as described previously [E. coli BL21 (DE3) pLysS competent cells were transformed with the plasmid described above and inoculated onto a 2 × YT agarose plate containing carbenicillin (50 μg/mL) and chloramphenicol (34 μg/mL). The colonies on the plate were seeded into M9 minimal liquid medium containing ampicillin (50 μg/mL) and chloramphenicol (34 μg/mL) and precultured at 37 °C overnight. The preculture was seeded at an optical density at 600 nm of 0.1 into M9 minimal medium or M9-rich medium containing 100 µM ammonium iron (II) sulfate and 1 mM isopropyl-β-d-1-thiogalactoside . M9-rich medium was composed of 6.4 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/L NH4Cl, 0.5 g/L (NH4)2SO4, 5 g/L casamino acids , 10 g/L glucose, 0.2 g/L MgCl2·6H2O, 10 mL/L BME vitamins 100 × solution , 10 mL/L trace mineral solution, 50 µg/mL ampicillin, and 34 µg/mL chloramphenicol [2, 2 g/L Na2MoO4·2H2O, 1.9 g/L CuSO4·5H2O, 0.5 g/L H3BO3, and 2 g/L ZnCl2 with the addition of HCl to maintain the pH at 4.7. The culture was incubated in a 96-deep-well plate at 37 or 30 °C for 16 h. The cell culture was sonicated, and 500 µL of the cell lysate was centrifuged to separate the supernatant and pellet fractions, both of which were used to quantify the amounts of soluble and insoluble AAR and ADO by western blotting. Next, 800 µL of the cell lysate was mixed with an equal amount of ethyl acetate by vortexing. The organic phase was separated from the aqueous phase by centrifugation and was subjected to GC–MS analysis using a Shimadzu gas chromatograph mass spectrometer GCMS-QP2010 Ultra  as described previously [Alk(a/e)nes were produced in eviously , 24, 31.phenicol . The traE. coli, which was quantified by western blotting. The measurements were taken three times, and the means ± standard errors are shown.AAR activity was calculated as the total amount of hydrocarbons  produced in the culture divided by the amount of soluble AAR expressed in The amounts of soluble and insoluble AAR were measured by western blotting as described previously . In brieAdditional file 1: Figure S1. Hydrocarbon production using single mutants of 7336AAR. a, b Relative amount of total hydrocarbon (a) and pentadecane (b) produced in E. coli coexpressing 73102ADO and a single mutant of 7336AAR. The data are shown in descending order of total hydrocarbon yield. c Fractions of pentadecane, heptadecene, and heptadecane relative to total hydrocarbon yield. In all panels, a horizontal dotted line shows the value for wild-type 7336AAR, which was used as a control (denoted by * and “WT”). In panels a and b, the values are normalized to those of the wild-type enzyme. All measurements were taken in triplicate, and the mean ± standard error is shown.Additional file 2: Figure S2. Western blotting of the supernatant and pellet fractions of the E. coli cell lysates. The E. coli cell culture expressing wild-type (WT) or the S298A mutant of 7336AAR with 73102ADO was sonicated and centrifuged to separate the supernatant (S) and pellet (P) fractions. The bands for 7336AAR (38.8 kDa) and 73102ADO (27.4 kDa) are indicated by arrows. Marker denotes the lane with molecular weight markers (37 and 29 kDa). The lanes labeled pETDuet-1 show the results for E. coli transformed with an empty pETDuet-1 plasmid containing neither AAR nor ADO.Additional file 3: Figure S3. Correlation analysis of the 7336AAR double mutants. a Relative amount of total hydrocarbon plotted against the relative amount of insoluble AAR. b, c Solubility is plotted against the relative amount of insoluble AAR (b) and relative protein expression level of AAR (c). d, e, f Relative activity of AAR plotted against the relative amount of insoluble AAR (d), solubility of AAR (e), and relative protein expression level of AAR (f). In each panel, a red continuous line indicates a linear regression obtained using all data, and the corresponding correlation coefficient, r, and p values are shown in red. A blue broken line indicates a linear regression obtained without using the data for N13Q, V60I, and K110D. The data points for the N13Q, L20R, V60I, K110D, and S200E mutants are indicated.Additional file 4: Figure S4. Hydrocarbon production using the 7336AAR multiple mutants. A weakened T7 promoter was used to reduce protein expression levels. a, b Relative amount of total hydrocarbon (a) and pentadecane (b) produced in E. coli coexpressing 73102ADO and a multiple mutant of 7336AAR. c Relative activity of AAR. d Fractions of pentadecane, heptadecene, and heptadecane relative to the total amount of hydrocarbon. e Relative amount of soluble AAR in E. coli. f Relative amount of insoluble AAR in E. coli. g Solubility of AAR. h Relative protein expression level of AAR. In all panels, the order of the data is the same as that in Fig. a-c, e, f, and h, the values are normalized to those of the S298A control. All measurements were taken in triplicate, and the mean ± standard error is shown.Additional file 5: Figure S5. Comparison of 7942AAR, wild-type 7336AAR, S298A single mutant, and the three most productive multiple mutants of 7336AAR using M9 minimal medium. a, b Absolute amount of total hydrocarbon (a) and pentadecane (b). c Relative activity of AAR. d Relative amount of soluble AAR in E. coli. In all panels, the values are normalized to those of the S298A single mutant, which was used as a control (denoted by * and “298 (Control)”), and a horizontal dotted line shows the value for the S298A control.Additional file 6: Figure S6. Comparison of 7942AAR, the S298A single mutant, and the three most productive multiple mutants of 7336AAR using M9-rich medium. a, b Absolute amount of total hydrocarbon (a) and pentadecane (b). c Relative activity of AAR. d Relative amount of soluble AAR in E. coli. In all panels, the values are normalized to those of the S298A single mutant, which was used as a control (denoted by * and “298 (Control)”), and a horizontal dotted line shows the value for the S298A control.Additional file 7: Figure S7. Helical propensity of residues 289–304 of AAR predicted by the AGADIR server [


P < .001). Male , white , being unmarried , the elderly  were independently associated with higher risk of suicide mortality. Surgery  was also relative with higher risk of suicide. Our study observed significant decrease in suicide mortality among NSCLC patients in US over past decades. Older age, male sex, unmarried status, and surgery were risk factors of committing suicide. Clinicians should be aware of these high‐risk groups.Lung cancer patients have an increased risk for committing suicide. But no comprehensive study about the suicide issues among non‐small‐cell lung cancer (NSCLC) patients has been published. We aimed to estimate the trend of suicide rate and identify the high‐risk group of NSCLC patients. Patients diagnosed with primary NSCLC were identified from Surveillance, Epidemiology, and End Results (SEER) database (1973‐2013). Suicide mortality rate (SMR) were calculated. Multivariable logistic regression was employed to find out independent risk factors for suicide. Among 495 889 NSCLC patients, 694 (0.14%) of them died from suicide. The suicide mortality rates have significantly decreased (before 1993: 0.21%, 1994‐2003: 0.16%, after 2004: 0.09%,  There is significant decrease in suicide mortality among NSCLC patients in US over past decades. Older age, male sex, unmarried status, surgery were risk factors of committing suicide. SEER database encompasses about 28% of United State population and collect cases diagnosed between 1973 and 2013.We extracted data of patients diagnosed with primary NSCLC from SEER database(1973‐2013) using the SEER*Stat software , using International Classification of Disease for Oncology, Third Edition (ICD‐O‐3), morphology codes: 8012/3, 8046/3, 8070/3, 8140/3, 8240/3, 8250/3, 8560/3 and 9053/3; and site codes: C33.9, C34.0, C34.1, C34.2, C34.3, C34.8 and C34.9.Patients whose cause of death variable coded as “Suicide and self‐inflicted injury” were identified. We obtained the demographic and clinicopathological data from the SEER database, including age, sex, race, marital status, year at diagnosis, state, tumor site, grade, histologic type, stage, surgery, cause of death, survival time, vital status and radiation. Patients were divided into 3 groups according to age at diagnosis . Race were sorted by white, black and others. We classified patients as married or unmarried. Year of diagnosis were separated into 3 groups . We classified the tumor site as upper lung, middle lung, lower lung, and bronchus/others. Grade of tumor were categorized into I/II, III/IV, and unknown groups. Surgery and radiation were both classified as performed, not performed, and unknown. Disease stage for the analysis was coded based on the variable “SEER Historic Stage A.” We give a value of 0.5 months to those who didn't survive for a full month after diagnosis, because SEER record their survival time in months.http://www.r-project.org). Statistical significance was set at two‐sided P < .05.Univariate analysis using chi‐square test was used to compare patients committed suicide with those died from other causes. Multivariable logistic regression was employed to find out independent risk factors for suicide. All statistical analyses were performed using R version 3.4.2 software  of them died from suicide. Among all patients, 207 306 (41.8%) of them are female while 288 583 (58.2%) of them are male. Among those patients committed suicide, 77 (11.1%) of them are female, and 617 (88.9%) of them are male. In total, 403 288 (81.3%) of them are white, 59 005 (11.9%) of them are black, and 33 596 (6.8%) of them are unknown and other races. As for those suicided patients, 634 (91.4%) of them are white, 25 (3.6%) of them are black, and 35 (5.0%) of them are unknown or other races. In all, 138 210 (27.9%) of them were diagnosed below 60, 237 648 (47.9%) of them were diagnosed between 60 to 75, and 120 031 (24.2%) of them were diagnosed over 75.3.2P < .001) (Table P < .001), Michigan , Washington  show significant drop of suicide mortality rate.The suicide mortality rates between all 3 time intervals were significantly different , white (P < .001), diagnosed between 60 and 75 (P = .034), squamous cell carcinoma (P = .006), surgery (P < .001) and without radiation therapy (P = .018)  Table .P < .001), race , marital status , year at diagnosis , age at diagnosis  were independently associated with higher risk of suicide mortality. As for clinical factors, surgery  was relative to higher risk of suicide , white patients (P < .001), and patients didn't have radiation therapy (P = .115). Concerning the time after diagnosis, the highest suicide mortality rate was found to be the first year after diagnosis (P = .008).Univariate analysis displayed that higher suicide mortality rate was associated with male patients (P < .001), race , marital status  were independently correlated to higher risk of suicide mortality. As for clinical factors, radiation  were relative to higher risk of suicide. Finally, time elapsed from cancer diagnosis was also relative with higher rate of suicide mortality (P = .008), with the first year of diagnosis taking the highest rate , followed by the second year   is not significantly associated with time after diagnosed. But SMR is observed to be higher within the first year of NSCLC diagnosis among patients diagnosed between 2004 and 2013. It has been reported that cancer patients were at high risk of suicide within the first year of diagnosis and associated demographic and clinical factors were analyzed.While the demographic risk factors of cancer‐related suicide are focused on, psychological and social risk factors are frequently missed. Suicide is a complicated phenomenon that biological, psychological and social risk factors would interact and influence on it. Cancer diagnosis may lead to demoralization of NSCLC patients, such as hopelessness and helplessness, which can lead to suicidal ideation.There are some limitations in our study. Suicide is a complicated phenomenon affected by factors such as economic level and education level. These factors are not included in SEER database. Additionally, SEER database only contains data of US patients, so our study is limited to US patients, and research over the world is still needed. Moreover, details of treatment to NSCLC were not taken in, and we only know whether patients have undergone surgery and radiation or not. Details of treatment such as the time after surgery and chemotherapy may be associated with suicide of cancer patient.5In summary, our study observed a significant decrease in suicide mortality among NSCLC patients in US over past decades. Older age, male sex, unmarried status, and surgery were risk factors of committing suicide. Grade, stage, histologic type and primary site of NSCLC appear not relate to suicide. It can help clinicians identified these NSCLC patients for better support and suicide prevention. Further studies are still needed.All of the authors have no conflicts of interested to declare.