Abstract. We identified scattered coal burning as the largest contributor to ambient volatile organic compounds (VOCs), exceeding traffic-related emissions, during the heating season in Beijing prior to the rigorous emission limitations enacted in 2017. However, scattered coal is underestimated in emission inventories generally, because the activity data are incompletely recorded in official energy statistics. Results of positive matrix factorization (PMF) models confirmed that coal burning was the largest contributor to VOC concentrations prior to the emission limitations of 2017, and a reduction in scattered coal combustion, especially in rural residential sector, was the primary factor in the observed decrease in ambient VOCs and secondary organic aerosol (SOA) formation potential in urban Beijing after 2017. Scattered coal burning was included in a corrected emission inventory and we obtained comparable results between this corrected inventory and PMF analyses, particularly for the non-control period. However, a refined source sub-classification showed that passenger car exhaust, petrochemical manufacturing, gas stations, traffic evaporation, traffic equipment manufacturing, painting, and electronics manufacturing are also contributors to ambient VOCs. These sources should focus on future emission reduction strategies and targets in Beijing. Moreover, in other region with scattered coal-based heating, scattered coal burning is still the key factor to improve the air quality in winter.

Abstract. In order to determine the origins of dissolved organic matter (DOM) occurring in coastal seawater of the Sihwa Lake, South Korea, which is semi-enclosed by a dyke, we measured the stable carbon isotopic ratio of dissolved organic carbon (DOC-δ13C) and optical properties (absorbance and fluorescence) of the DOM in two different seasons (March 2017 and September 2018). The concentrations of DOC were generally higher in lower-salinity waters in both periods, while a significant excess of DOC was observed in 2017 in the same salinity range. The main source of DOC, dependent on salinity, was found to be from marine sediments in the freshwater-seawater mixing zone rather than from terrestrial sources based on the DOC-δ13C values (−20.7±1.2 ‰) and good correlations among DOC, humic-like fluorescent DOM (FDOMH), and NH4+ concentrations. However, the excess DOC observed in 2017 seems to originate from terrestrial sources by direct land-seawater interactions rather than from in-situ biological production, considering the lower DOC-δ13C values (−27.8 ‰ to −22.6 ‰) and higher spectral slope ratio (SR) of light absorbance, without increases in FDOMH and NH4+ concentrations. This terrestrial DOM source could have been exposed to light and bacterial degradation for a long time, resulting in nonfluorescent and low-molecular-weight DOM, as this study area is surrounded by the reclaimed land. Our results suggest that the combination of these biogeochemical tools can be a powerful tracer of coastal DOM sources.

Abstract. Permafrost regions store large amounts of soil organic carbon and nitrogen, which are major sources of greenhouse gas. With climate warming, permafrost regions are thawing, releasing an abundance of greenhouse gases to the atmosphere and contributing to climate warming. Numerous studies have shown the mechanism of nitrous oxide (N2O) emissions from the permafrost region during the growing season. However, little is known about the temporal pattern and drivers of nongrowing season N2O emissions from the permafrost region. In this study, N2O emissions from the permafrost region were investigated from June 2016 to June 2018 using the static opaque chamber method. Our aims were to quantify the seasonal dynamics of nongrowing season N2O emissions and its contribution to the annual budget. The results showed that the N2O emissions ranged from −35.75 to 74.16 μg·m−2·h−1 during the nongrowing season in the permafrost region. The mean N2O emission from the growing season were 1.75–2.86 times greater than that of winter and 1.31–1.53 times greater than that of spring thaw period due to the mean soil temperature of the different specified periods. The nongrowing season N2O emissions ranged from 0.89 to 1.44 kg ha−1, which contributed to 41.96–53.73 % of the annual budget, accounting for almost half of the annual emissions in the permafrost region. The driving factors of N2O emissions were different among during the study period, growing season, and nongrowing season. The N2O emissions from total two-year observation period and nongrowing season were mainly affected by soil temperature, while the N2O emissions from growing season were controlled by soil temperature, water table level, and their interactions. In conclusion, nongrowing season N2O emissions is an important component of annual emissions and cannot be ignored in the permafrost region.

Abstract. The period 36–18 ka was a dynamic phase of the last glacial, with large climate shifts in both hemispheres. Through the bipolar seesaw, the Antarctic Isotope Maxima and Greenland DO events were part of a global concert of large scale climate changes. The interaction between atmospheric processes and Atlantic meridional overturning circulation (AMOC) is crucial for such shifts, controlling upwelling- and carbon cycle dynamics, and generating climate tipping points. Here we report the first temperature and humidity record for the glacial period from the central South Atlantic (SA). The presented data resolves ambiguities about atmospheric circulation shifts during bipolar climate events recorded in polar ice cores. A unique lake sediment sequence from Nightingale Island at 37° S in the SA, covering 36.4–18.6 ka, exhibits continuous impact of the Southern Hemisphere Westerlies (SHW), recording shifts in their position and strength. The SHW displayed high latitudinal and strength-wise variability 36–31 ka locked to the bipolar seesaw, followed by 4 ka of slightly falling temperatures, decreasing humidity and fairly southern westerlies. After 27.5 ka temperatures decreased 3–4 °C, marking the largest hydroclimate change with drier conditions and a variable SHW position. We note that periods with more intense and southerly positioned SHW are correlated with periods of increased CO2 outgassing from the ocean. Changes in the cross-equatorial gradient during large northern temperature changes appear as the driving mechanism for the SHW shifts. Together with coeval shifts of the South Pacific westerlies, it shows that most of the Southern Hemisphere experienced simultaneous atmospheric circulation changes during the latter part of the last glacial.

Abstract. Since decades, microplastics and microglass enter aquatic and terrestrial environments. The complexity of the environmental impact is difficult to capture and consequences on ecosystem components e.g. such as soil microorganisms are virtually unknown. Addressing this issue, we performed an incubation experiment by adding 1 % of five different types of impurities (≤ 100 µm) to an agricultural used soil (Chernozem). Four microplastic types (polypropylene (PP), low density polyethylene (LD-PE), polystyrene (PS) and polyamide12 (PA12)) and microglass were used as treatment variants. After 80 days of incubation at 20 °C, we examined soil microbial community structure by using phospholipid fatty acids (PLFA) as markers for bacteria, fungi and protozoa. The results showed that soil microorganisms were not significantly affected by the presence of microplastic and microglass. However, PLFAs tend to increase in LD-PE (27 %), PP (18 %) and microglass (11 %) treated soil in comparison with untreated soil, whereas PLFAs in PA12 (32 %) and PS (11 %) treated soil decreased. Interestingly, the comparison of PLFA contents between microplastic types revealed significant differences of PA12 (−87 %) and PS (−42 %) compared to LD-PE. Furthermore, bacterial PLFAs showed a much higher variability after microplastic incubation whereby fungi seem to be more unaffected after 80 days of incubation. Same for protozoa, which were more or less unaffected by microplastic treatment showing only minor reduction of the PLFA contents compared to control. In contrast, microglass has obviously an inhibiting effect on protozoa because PLFAs were under the limit of determination. Our study provides hints, that microplastics have, depending on type, contrary effects on soil microbiology and microglass seems to be highly toxic for protozoa.

Objectives. To investigate the metabolic profile in patients with aortic stenosis (AS) after transcatheter aortic valve replacement (TAVR) and explore the potential biomarkers to predict prognosis after TAVR based on metabolomics. Methods and Results. Fifty-nine consecutive AS patients were prospectively recruited. Blood samples from the ascending aorta, coronary sinus, and peripheral vein at before and after TAVR were collected, respectively. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry were performed to analyze the metabolic profile before and after TAVR. Influential metabolites were identified by integrating the univariate test, multivariate analysis, and weighted gene coexpression network analysis (WGCNA) algorithm. PLS-DA analysis revealed a significant extremely early (within 30 minutes after TAVR) alterations of metabolites in the ascending aorta, coronary sinus, and peripheral vein. The early (within 7 days after TAVR) changed metabolites in the peripheral vein were involved in purine metabolism, primary bile acid biosynthesis, glycerolipid metabolism, amino sugar and nucleotide sugar metabolism, one carbon pool by folate and alanine, and the aspartate and glutamate metabolism pathway. We used volcano plots to find that the cardiac-specific changed metabolites were enriched to the sphingolipid metabolism pathway after TAVR. Besides, WGCNA algorithm was performed to reveal that arginine and proline metabolites could reflect left ventricle regression to some extent. Conclusion. This is the first study to reveal systemic and cardiac metabolites changed significantly in patients with AS after TAVR. Some altered metabolites involved in the arginine and proline metabolism pathway in the peripheral vein could predict left ventricle regression, which merited further study.

Malus domestica (Apple) is one of the most widely cultivated cash crops of Nepal. Jumla and Mustang are two major pocket areas for the production of apple. Flavonoids including quercetin and rutin are potent antioxidants present in apples. This study was designed to quantify and compare the presence of quercetin and rutin in different plant parts (peel, leaf, and bark) among various cultivars of Malus domestica from two pocket zones of Nepal. A new HPLC-UV method was developed and validated for the quantification of quercetin and rutin. Polyphenols, flavonoids, and carbohydrate contents were determined by colorimetric methods. 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was carried out to measure in vitro antioxidative activity. Acid hydrolysis of each extract was carried out by the standard method to measure aglycone quercetin content after hydrolysis of its glycosides. The total rutin content ranged from 3.69 ± 1.34 to 374.50 ± 2.35 mg/100g dry extract weight. Before the acidic hydrolysis, the total quercetin content ranged from 2.96 ± 0.13 to 171.05 ± 0.95 mg/100g dry extract weight whereas its amount increased highly after the hydrolysis and it ranged from 80.84 ± 19.65 to 7445.32 ± 29.25 mg/100g dry extract weight. Total polyphenol content ranged from 19.48 ± 0.23 to 123.48 ± 1.84 µg gallic acid equivalent/mg of dry extract weight. Similarly, flavonoid content ranged from 2.21 ± 0.72 µg to 755.54 ± 1.91 µg quercetin equivalent/mg of dry extract weight. Total carbohydrate content ranged from 144.15 ± 3.73 to 484.65 ± 2.63 µg glucose equivalent per 0.5 mg dry extract weight. All the extracts showed the various degrees of antioxidant activity in a dose-dependent manner. Among them, stem bark of the Jonathan Jumla showed potent antioxidant activity with IC50 value of 13.003 µg/mL. The present study provides the information about variation of the phytochemical content among the different cultivars, parts, and geographic locations. Furthermore, it revealed that bark of Malus domestica cultivars had high quercetin and rutin content with high antioxidant activity.

Auricularia auricula is a kind of nutrient-rich edible fungus, which has the reputation of “king of vegetarians.” In this paper, the electronic nose combined with GC-MS technology was used to analyze the volatile components of A. auricula in Heilongjiang, Jilin, Shanghai, and Sichuan provinces to investigate the differences and characteristics of A. auricula in different origins. The results showed that the electronic nose could obviously distinguish the samples from Jilin and Shanghai with a high degree of discrimination, while it was inappropriate to distinguish the samples from Heilongjiang and Sichuan Province. GC-MS was used to further analyze the volatile compounds in A. auricula qualitatively and quantitatively. The results showed that 98 volatile components were detected and 23 of them were common components, including alcohols, aldehydes, acids, esters, hydrocarbons, and other volatile components. The relative content of acetic acid and diethyl azodicarboxylate in A. auricula from the four origins was relatively high. According to the relative odor activity value (ROAV), it was found that the key compounds that caused the aroma difference between different origins were 1-octene-3-ol, cis-3-nonene-1-ol, (E)-2-octenal, (E)-2-nonenal, (E,E)-2,4-nonadienal, and 3-methyl butanal.

Background. In developing countries, the prevalence of bacterial infections is quite rampant due to several factors such as the HIV/AIDS pandemic, lack of hygiene, overcrowding, and resistance to conventional antimicrobials. Hence the use of plant-based antimicrobial agents could provide a low-cost alternative therapy. Rosmarinus officinalis is reputed as a medicinal plant in Ethiopia; however, its antibacterial activity against many of the clinical isolates remains overlooked. Methods. Tender foliage of R. officinalis was collected and extracted in ethanol (EtOH) and evaluated for their antimicrobial activity against ten multidrug-resistant (MDR) clinical isolates, human type culture pathogens, and meat-borne bacterial isolates by employing agar well diffusion assay. Results. EtOH extract of R. officinalis efficiently subdued the growth of all tested MDR clinical isolates in varying degrees. Salmonella sp. and Staphylococcus aureus were found to be the most sensitive clinical isolates. Likewise, it efficiently repressed the growth of meat-borne pathogens, particularly, S. aureus and Salmonella sp. showing its potentiality to be used as a natural antibacterial agent in the meat processing industry. The mechanism of antibiosis of plant extract against meat-borne pathogens is inferred to be bactericidal. Chemical constituents of the crude plant extract were analysed by Gas Chromatography-Mass Spectroscopy (GC-MS), Fourier Transform Infrared (FT-IR), and UV-visible spectroscopy showing genkwanin (26%), camphor (13%), endo-borneol (13%), alpha-terpineol (12%), and hydroxyhydrocaffeic acid (13%) as the major compounds. Conclusion. Overall results of the present study conclude that R. officinalis could be an excellent source of antimicrobial agents for the management of drug-resistant bacteria as well as meat-borne pathogens.

Asarum is a traditional medicine and has been widely used as remedies for inflammatory diseases, toothache, headache, local anesthesia, and aphthous stomatitis in China, Japan, and Korea. Our previous research found that safrole and methyl eugenol were vital compounds that contribute to distinguish the different species and raw Asarum and its processed products apart. The pharmacokinetics of safrole and methyl eugenol after oral administration of Asarum extract has not been reported yet. In this study, a rapid and simple gas chromatography–mass spectroscopy (GC–MS) method that has a complete run time of only 4.5 min was developed and validated for the simultaneous determination and pharmacokinetic study of safrole and methyl eugenol in rat plasma after administration of Asarum extracts. The chromatographic separation was realized on a DB-17 column ( $30\text{m}\times 0.25\text{mm}\times 0.25\mu \text{m}$ ). And detection was carried out under selected ion monitoring (SIM) mode. Plasma samples were pretreated by $n$ -hexane. The pharmacokinetic parameters provided by this study will be beneficial for further developments and clinical applications of Asarum.

Compound-specific stable chlorine isotope analysis (CSIA-Cl) is an important method for identifying sources of organochlorine contaminants and helping assess their quantification of transformation processes. However, the present CSIA-Cl is challenged by either redundant conversion pretreatment or complicated mathematical correction. To overcome the mentioned problems, a novel method has been developed for the CSIA-Cl of eight organochlorine pesticides using gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-qMS) in this study. The instrument parameters, acquisition mode, and required injection amounts were optimized in terms of the precision of GC-NCI-qMS. An ionization energy of 90 eV and emission current of 90 μA were selected, and the precisions for eight organochlorine pesticides were in the range of 0.37‰–2.15‰ in single ion monitoring (SIM) mode when the injected amount was 0.50 mg L−1 (viz. 0.5 ng on column). Furthermore, when standards from Supelco and O2si were calibrated using standards from AccuStandard regarded as external isotope standard, chlorine isotope composition of α-hexachlorocyclohexane (α-HCH) and 2, 2-dichloro−1, 1-bis (4-chlorophenyl) ethylene (p, p′-DDE) in Supelco and O2si was confidently differentiated. The provenance identification method was validated by three organochlorine contaminated groundwater samples and showed a prospect in identifying the source of organochlorine pesticides.

To identify a rice genotype with a wider or specific adaptability, the magnitude and patterns of genotype interaction with the environment are valuable clues. However, such information on the nutraceutical lipid compound in rice is limited. In this study, concentrations of five pigmented rice genotypes with different lipid compound levels were evaluated for α-tocopherol, γ-oryzanol, phytosterols, octacosanol, and squalene contents. The research was conducted in eight environments (four locations and two years). Combined analysis of variance showed that levels of α-tocopherol, γ-oryzanol, octacosanol, and squalene were mainly affected by genotype, accounting for more than 42.57% of the total variance. This suggests that improvement of these traits may be achieved by using conventional rice breeding methods. A large proportion of the variation in campesterol and β-sitosterol contents was contributed by the environment. The effect of genotype by environment was significant for all observed traits, especially for γ-oryzanol, β-sitosterol, and squalene. This complicates identification of superior genotypes for these compounds across all environments. Stability parameters showed rice genotypes with high levels of nutraceutical lipid compounds to be sensitive to changes in environmental conditions; thus, specific adaptation is suitable. Our findings suggest that rice breeders and producers should be testing and selecting rice genotypes possessing high added-value lipid concentrations in specific growing conditions or regions.

The contents of main components in 45 walnut kernels from 5 walnut-planting provinces in China (Yunnan, Shaanxi, Shandong, Hebei, and Sichuan) were determined using colorimetry, high-performance liquid chromatography, and gas chromatography, including flavonoids, vitamin E, trace elements, fatty acids, and amino acids. The levels of flavonoids and vitamin E were higher in walnuts from Yunnan than in walnuts from the other four provinces. The levels of zinc and iron were relatively higher in walnuts from Shandong, and the calcium content in walnuts from Yunnan was much lower. No obvious difference was found in crude fat concentration for nuts from the five provinces, but differences were observed in the crude protein content. Oleic acid was the predominant unsaturated fatty acids in all walnut species; the monounsaturated fatty acid content was the highest in walnuts from Yunnan. The data obtained here provided insight into differences in walnuts resulting from different growing environments and germplasm genetic traits.

To continue releasing San Cristóbal Galápagos tortoises housed in managed-care facilities at the Giant Tortoise Breeding Center of Galápagos National Park (Galapaguera de Cerro Colorado) to the Otoy Ecological Farm, health assessments and physical examinations were conducted. As a part of these wellness examinations, blood was drawn from 11 tortoises to analyze fatty acid concentrations. Fatty acid levels can provide insight into the nutritional profiles, immune status, and reproductive health of vertebrates. To the co-author’s knowledge, there is no current information about fatty acids in this species. It was hypothesized that there would be inherent differences based on the different geographic ranges, diets, sex, and age of turtles. It was noted that the ω-6/ω-3 ratio was higher for the breeding center than for the ecological farm and that overall polyunsaturated fatty acids (PUFAs) did not have any significant differences. The ω-6/ω-3 findings can contribute to a global picture of these fatty acids across taxa, as reptiles are underrepresented in this area of research. Additional results are a resourceful starting point for future investigations into how fatty acids are affected in Galápagos tortoises.

Background Dietary fiber, including inulin, promotes health via fermentation products, such as short-chain fatty acids (SCFAs), produced from the fiber by gut microbiota. SCFAs exert positive physiological effects on energy metabolism, gut immunity, and the nervous system. Most of the commercial inulin is extracted from plant sources such as chicory roots, but it can also be enzymatically synthesized from sucrose using inulin producing enzymes. Studies conducted on rodents fed with a cafeteria diet have suggested that while increasing plasma propionic acid, synthetic inulin modulates glucose and lipid metabolism in the same manner as natural inulin. Therefore, this study aimed to determine the effects of a synthetic inulin, Fuji FF, on energy metabolism, fecal SCFA production, and microbiota profiles in mice fed with a high-fat/high-sucrose diet. Methods Three-week-old male C57BL/6J mice were fed a high-fat/high-sucrose diet containing cellulose or Fuji FF for 12 weeks, and the effects on energy metabolism, SCFA production, and microbiota profiles were evaluated. Results Body weight gain was inhibited by Fuji FF supplementation in high-fat/high-sucrose diet-fed C57BL/6J mice by reducing white adipose tissue weight while increasing energy expenditure, compared with the mice supplemented with cellulose. Fuji FF also elevated levels of acetic, propionic and butyric acids in mouse feces and increased plasma propionic acid levels in mice. Moreover, 16S rRNA gene amplicon sequencing of fecal samples revealed an elevated abundance of Bacteroidetes and a reduced abundance of Firmicutes at the phylum level in mice supplemented with Fuji FF compared to those supplemented with cellulose. Fuji FF also resulted in abundance of the family Bacteroidales S24-7 and reduction of Desulfovibrionaceae in the feces. Conclusion Long term consumption of Fuji FF improved the gut environment in mice by altering the composition of the microbiota and increasing SCFA production, which might be associated with its anti-obesity effects.

Phosphate (P) availability often limits biological nitrogen fixation (BNF) by diazotrophic bacteria. In soil, only 0.1% of the total P is available for plant uptake. P solubilizing bacteria can convert insoluble P to plant-available soluble P (ionic P and low molecular-weight organic P). However, limited information is available about the effects of synergistic application of diazotrophic bacteria and P solubilizing bacteria on the nitrogenase activity of rhizosphere and nifH expression of endosphere. In this study, we investigated the effects of co-inoculation with a diazotrophic bacterium (Paenibacillus beijingensis BJ-18) and a P-solubilizing bacterium (Paenibacillus sp. B1) on wheat growth, plant and soil total N, plant total P, soil available P, soil nitrogenase activity and the relative expression of nifH in plant tissues. Co-inoculation significantly increased plant biomass (length, fresh and dry weight) and plant N content (root: 27%, shoot: 30%) and P content (root: 63%, shoot: 30%). Co-inoculation also significantly increased soil total N (12%), available P (9%) and nitrogenase activity (69%) compared to P. beijingensis BJ-18 inoculation alone. Quantitative real-time PCR analysis showed co-inoculation doubled expression of nifH genes in shoots and roots. Soil nitrogenase activity and nifH expression within plant tissues correlated with P content of soil and plant tissues, which suggests solubilization of P by Paenibacillus sp. B1 increased N fixation in soils and the endosphere. In conclusion, P solubilizing bacteria generally improved soil available P and plant P uptake, and considerably stimulated BNF in the rhizosphere and endosphere of wheat seedlings.

Abstract Background Central nervous system (CNS) infectious diseases are common diseases in emergency rooms and neurology departments. CNS pathogen identification methods are time-consuming and expensive and have low sensitivity and poor specificity. Some studies have shown that bacteria and viruses can produce specific volatile organic compounds (VOCs). The aim of this study is to find potential biomarkers by VOC analysis of cerebrospinal fluid (CSF) in patients with bacterial and viral meningitis/encephalitis (ME). Methods CSF samples from 16 patients with bacterial ME and 42 patients with viral ME were collected, and solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) was used to analyze the metabolites in the CSF. Results There are two substances (Ethylene oxide and Phenol) that were found to be different between the two groups. Ethylene oxide was significantly greater in the group of bacterial ME patients than in the viral ME group of patients (P < 0.05). In addition, phenol was remarkably increased in the group of ME patients compared with the bacterial ME patients (P < 0.05). Conclusions Ethylene oxide and phenol may be potential biomarkers to distinguish bacterial ME and viral ME. VOC analysis of CSF may be used as a supporting tool for clinical diagnosis.

Abstract Background: Combined methylmalonic acidemia and homocystinuria, cobalamin C type (cblC defect) is the most common inborn error of cobalamin metabolism. Even with rapid diagnosis and treatment, the long-term outcome remains poor. A reliable method for the prenatal diagnosis of cblC defect is needed for parental decisions regarding continuation of pregnancies of cblC defect fetuses.Results: The between-day and within-day imprecision of Hcy assay were 1.60%∼5.87% and 1.11%∼4.31%, respectively. For the 248 at-risk fetuses, there were 63 affected fetuses with cblC defect and 185 unaffected fetuses. The levels of Hcy in 63 affected fetuses were significantly higher than those in 185 unaffected fetuses, without overlap between the affected and unaffected groups. Sixteen fetuses displayed inconclusive genetic results of MMACHC variants, in which seven fetuses were determined to be affected with elevated amniotic fluid levels of Hcy, C3, C3/C2 and MMA. The remaining nine fetuses were considered unaffected based on a normal amniotic fluid metabolite profile. The diagnostic sensitivities of Hcy and other characteristic metabolites including propionylcarnitine (C3), ratio of C3 to acetylcarnitine (C2; C3/C2), methylmalonic acid (MMA), and methylcitrate acid (MCA) in amniotic fluid were 100%, 87.50%, 100%, 85.71%, and 28.57%, respectively. The respective specificities were 92.05%, 100%, 100%, 97.73%, and 99.43%. Conclusions: Hcy appears to be another sensitive characteristic metabolite biomarker for the prenatal diagnosis of cblC defect. The combination of Hcy assay with acylcarnitine and organic acid analysis is a fast, sensitive, and reliable prenatal diagnostic biochemical approach. This approach could overcome the challenge of the lack of genetic analysis for families with at-risk cblC defect fetuses.

Abstract Acacia nilotica is an important medicinal plant, found in Africa, Middle East, and Indian subcontinent. Every part of the plant possesses a wide array of biologically-active and therapeutically important compounds and have been used in traditional-system of medicine. We reported the antileishmanial activity of Acacia nilotica (A. nilotica) bark methanolic extract through in vitro assays and dissected the mechanism of its action through in silico studies. Bark methanolic extract exhibited anti-promastigote and anti-amastigote potency with IC50 value of 19.6 + 0.9037 µg/ml and 77.52 + 5.167 µg/ml respectively in time and dose dependent manner. It showed very low cytotoxicity having CC50 value of 432.7 + 7.71 µg/ml on human-macrophage cell line, THP-1. The major constituents identified by GC-MS analysis are 13-docosenoic acid (34.06%), lupeol(20.15 %), 9,12-octadecadienoic acid (9.92 %) and 6-octadecanoic acid (8.43 %) bind effectively with the potential drug-targets of Leishmania donovani (L. donovani ) including sterol 24-c-methytransferase (SMT), trypanothione reductase (TR), pteridine reductase (PTR1) and adenine phosphorybosyl transferase (APRT); suggest the possible mechanism of its antileishmanial action. The highest affinity with all these targets was shown by lupeol. The pharmacokinetic studies, predicted bioactivity scores and acute toxicity studies of major extract constituents support safe antileishmanial drug candidate. This study proved the antileishmanial potential of bark-methanolic extract A. nilotica and its mechanism of action through the inhibition of potential drug targets of L. donovani.

Abstract Background Natural mint flavor is produced by extraction from mint, which is not efficient enough to make it environment friendly process. (−)-Carvone is the monoterpenoid with key flavor of spearmint, and there has been an attempt to produce (−)-carvone by recombinant Escherichia coli. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in E. coli independently, the yield of (−)-carvone was low in the previous study.ResultsWe have found a by-product formation when cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in single cell. These by-products were determined as dihydrocarveol and dihydrocarvone. We hypothesized that the enzymatic kinetics and the expression levels of P450 and CDH are quite different in E. coli. Therefore, two strains independently expressing P450/CPR and CDH were mixed with different mixing ratio, confirming increase in carvone production and decrease in by-product formation when CDH input was reduced. To determine the optimum balance of enzyme expressions, proteome analysis quantification concatemer (QconCAT) method to quantify P450, CPR, and CDH was developed. Using the QconCAT standard protein that was artificially created by concatenating the tryptic peptides, the ratio between P450 and CDH was calculated, and their optimum ratio to maximize (−)-carvone production was shown. Then, a single strain expressing both P450/CPR and CDH was constructed to imitate the superior expression ratio. The upgraded strain showed 15-fold improvement compared to the initial strain, showing 44 ± 6.3 mg/L of (−)-carvone production from 100 mg/L (−)-limonene as a starting substrate.ConclusionsImproved expression balance of P450 and CDH in recombinant E. coli increased the (−)-carvone production using (−)-limonene as the direct substrates by the whole-cell biocatalysis, showing approximately 150 times higher titer than previous report. Our study showed the usefulness of proteome analysis QconCAT method in the strain development for industrial biotechnology field.