Eriocheir sinensis during reproductive migration. It is however unclear the effects of salinity on the gonadal development of male E. sinensis as well as their physiological responses to salinity during reproductive migration. This study investigated the effects of four salinities  on gonadal development, osmoregulation and metabolism of adult male E. sinensis over a 40-day period. The results showed that elevating salinity promote gonadal development, increase hemolymph osmolality and K+ and Mg2+ concentrations (P < 0.05). The 12 ‰ salinity resulted in the highest contents of taurine and arginine in the hemolymph while the highest contents of threonine, phenylalanine, lysine, ß-alanine, tryptophan, ornithine and total free amino acids were found for 0 ‰ treatment (P < 0.05). A decreasing trend was detected for the Na+/K+-ATPase activity and its mRNA expression level in the posterior gills with salinity (P < 0.05). Total saturated fatty acids in the anterior gills decreased with increasing salinity (P < 0.05); the 0 ‰ treatment had the highest total polyunsaturated fatty acids in the posterior gills while total n-6 polyunsaturated fatty acids increased with salinity (P < 0.05). The hemolymph glucose and uric acid showed a decreasing trend as salinity while an increasing trend was found for the hemolymph triglyceride and high-density lipoprotein cholesterol (P < 0.05). The 12 ‰ treatment had the highest levels of hemolymph malonaldehyde and hepatopancreatic γ-glutamyltranspeptidase (P < 0.05). In conclusion, these results suggested that the brackish water promote gonadal development of male E. sinensis, and increase osmolality and ionic concentrations in hemolymph while reduced the activity of Na+ /K+- ATPase and its mRNA expression in the posterior gills as well as metabolism.As a catadromous species, salinity is a key parameter that affects gonadal development of Chinese mitten crab  Eriocheir sinensis, is a catadromous crustacean species, which distribute widely across East Asia from China to the Korean Peninsula and has a very complex life history: the juvenile E. sinensis spend most of their lives in freshwater, but after puberty molt, adult crabs must migrate downstream to estuaries for reproduction  by an IKA homogenizer . The homogenate was centrifuged at 10, 000×g for 10 min at 4°C, and the supernatant was collected for the later analysis. The activity of Na+/K+-ATPase and total soluble protein in the supernatant were assayed by a spectrophotometer  and commercial assay kits . A unit of ATPase activity was obtained as 1 μmol inorganic phosphate (Pi) produced from the reaction in which ATP was resolved by ATPase in per mg tissue protein per hour (μ mol Pi-1 hr -1 mg protein -1).Approximately 0.2 g of posterior gills was homogenized in 1 mL of ice-cold homogenization buffer  were added to a 5 mL tube and homogenized using an IKA homogenizer . The homogenate of each sample was centrifuged at 10, 000×g for 10 min at 4°C, and the supernatant was collected for the later analysis.The hepatopancreas tissue plus the 5-fold volume (v/w) of icy physiological saline solution [210 mmol LThe total cholesterol (TC), triglyceride (TG), uric acid (UA), urea and glucose (GLU) were determined according to Qiu et al. (2011)  while thP < 0.05), differences among treatments were identified using Duncan’s multiple range tests. For all statistical analysis the SPSS statistical package version 16.0 was used and significant relationship were determined when P < 0.05.Data are presented as mean ± standard error (SE). All data was analyzed using one-way ANOVA after confirmation of normality and homogeneity of variance. If any significant differences were detected (P > 0.05). An increasing trend was detected for the gonadosomatic index (GSI) of adult male E. sinensis with increasing salinity (P < 0.05) while no significant difference was found between 12 ‰ and 18 ‰ treatments (P > 0.05). On the other hand, salinity appeared to have no significant effect on hepatosomatic index (HSI) of the crabs . No significant differences were found for the contents of aspartic acid, β-aminoisobutyric acid and citrulline among all treatments (P > 0.05). Total free amino acids (TFAAs) showed a clear trend of decrease with increasing salinity, and the significant higher TFAAs was found for 0 ‰ treatment compared to the other treatments (P < 0.05).The crabs from salinity 12 ‰ treatment detected the highest levels of taurine, α-aminoadipicacid (α-AAA) and arginine in hemolymph , but not that of the 0 ‰ treatment (P > 0.05). The highest and the lowest mRNA expression level of Na+/K+-ATPase were found for the 0 ‰ and 18 ‰ treatments, respectively (P < 0.05).A decreasing trend was found for the activity of NaP < 0.05), while no significant changes were found for the other SFAs. For monounsaturated fatty acid (MUFA), the percentages of C16:1n7, C18:1n7 and total monounsaturated fatty acids (∑MUFA) firstly increased significantly with increasing salinity to reach a peak at 6 ‰ but then decreased with increasing salinity (P < 0.05). For polyunsaturated fatty acid (PUFA), the 6 ‰ also resulted in the highest percentage of C18:3n3, while the C20:5n3 decreased with increasing salinity (P < 0.05). The C22:6n3 showed an increasing trend with increasing salinity while the highest and the lowest n-3/n-6 ratio was found for the 12 ‰ and 18 ‰ treatments, respectively (P < 0.05).The fatty acids profile in the anterior gill was shown in P < 0.05). For MUFA, 6 ‰ treatment had the highest percentages of C18: 1n9 and ∑MUFA, while the C18:1n7 percentage firstly showed an increasing trend with salinity with a peak detected for the 12 ‰ treatment (P < 0.05) before decreased slightly at 18 ‰ (P > 0.05). For PUFA, the highest percentages of C18:2n6 and C18:3n3 was found for 6 ‰ treatment (P < 0.05), however, the C20: 2n6, C20:5n3, ∑n-3 PUFA and n-3/n-6 ratio decreased with increasing salinity. The percentages of C20:4n6 and ∑n-6 PUFA showed an increasing trend with salinity (P < 0.05); the ∑PUFA firstly decreased and then increased significantly with increasing salinity, and the lowest level was detected for 6 ‰ treatment (P < 0.05).For fatty acid profile in the posterior gill . The perP < 0.05, P < 0.05). Meanwhile, the highest and the lowest activities of γ-GT and ALP were found for the 6 ‰ and 18 ‰ treatments, respectively (P < 0.05). No significant difference was detected for TC, LDL-C, urea, SOD and ACP among all treatments (P > 0.05)  .P < 0.05, P < 0.05) while the activity of γ-GT firstly increased significantly with salinity to reach a peak at 12 ‰ (P < 0.05) but decreased slightly 18 ‰. SOD showed a fluctuating pattern with the highest level detected for the 18 ‰ treatment while the highest activity of ALP was found for the 6 ‰ treatment (P < 0.05). No significant difference was detected for GLU, UA and MDA as well as the activity of ACP among treatments (P > 0.05).The total lipids in hepatopancreas firstly showed an increasing trend with increasing salinity, with a peak detected for the 12 ‰ treatment, before decreased at 18 ‰  the osmolality of brackish water is closer to that in the hemolymph of the male E. sinensis, hence reduced energy consumption on osmoregulation [Past studies on salinity effects on gonadal development of crustaceans have been focused on females of various shrimp species –35, so fgulation , 36, resgulation ; 2) certgulation , 38. Pregulation –40, whicgulation .+/K+-ATPase, and transport proteins [+/K+-ATPase and its mRNA expression level in the posterior gills decreased significantly with increasing salinity, similar results was shown for E. sinensis subjected to short-term salinity changes [2O -1; at 0 ‰, hemolymph osmolality/external osmolality = 608.33 ± 19.54/10.67 ± 0.58 mOsm kg H2O -1, + / K+—ATPase was reduced as salinity increased.Gills play a key role in osmotic and ionic regulation in crustaceans , 42. Preproteins , 26. In  changes –45 as we changes –47. SuchScylla serrata) [E. sinensis decreased significantly with elevated salinity they exposed to. Such a result might be an adaptive response to reduce membrane fluidity and as the result, lower gill Na+/K+-ATPase activity and energy were required to maintain ionic concentrations at higher salinities [Previous studies have shown that crustaceans, including crabs, responded to ambient salinity changes by changing fatty acids compositions in different tissues, particularly in gills , 47–48. serrata) , the perlinities , 49.E. sinensis is a euryhaline species, but is also known as a strong hyper- osmoregulator but a weak hypo-osmoregulator [+ and Mg2+. Previous studies showed that Na+ and Cl- were two most important ions contribute to hemolymph osmolality of crustaceans [+ and Cl- were several orders higher than other ions, and similar results were reported for the mud crabs, Scylla paramamosain [S. serrata [Portunus pelagicus [+ and Mg2+ increased significantly with increasing salinity, which may be explained by 1) K+ and Mg2+ also contributed to hemolymph osmolality although relatively minor; 2) high Mg2+ level in hemolymph could inhibit Na+/K+-ATPase activity [+/K+-ATPase found in the posterior gills under higher salinities.egulator , 45. In staceans –16. In tmamosain  and S. s serrata , and theelagicus . Additioactivity , 52, whiE. sinensis were found to be taurine, glutamine, proline, glycine, alanine and arginine, ranging from 200 to 1200 nmol mL-1. A decreasing trend was found for threonine, proline, phenylalanine, tryptophan, lysine and total free amino acids (TFAAs) with increasing salinity, which is in agreement with a previous study [E. sinensis subjected to long-term higher salinity exposure. However the biochemical pathways leading to changes in FAAs during higher salinity acclimation remain to be studied.In addition to inorganic ions, the hemolymph free amino acids (FAAs) are also known as important contributors to osmoregulation, which play an important role in intracellular osmoregulation in crustaceans although their concentrations are lower than those of inorganic ions –18. The us study . A possius study , 53. TheE. sinensis subjected to different salinities [E. sinensis [The biochemical parameters generally mirror the physiological status of linities , 15. In linities –55. Sinclinities , hence lsinensis , which csinensis . In thissinensis .Marsupenaeus japonicas [E. sinensis in brackish water. Furthermore, the UA as the end product of nitrogen metabolism [E. sinensis had relatively low protein catabolism rate when in brackish water.The cholesterol is not only an important component of cell membranes, but also a precursor for biosynthesis hormones regulating gonadal development and molt in crustaceans , 59. Theaponicas . Such retabolism  decreasetabolism –64 and cExopalaemon carinicauda [E. sinensis.SOD is an important antioxidant enzyme of the antioxidant defense systems in crustaceans, which can scavenge the free radicals and prevents tissue damage , 65. Theinicauda . The resinicauda . Meanwhiinicauda . In thisinicauda . The γ-Ginicauda –69. The E. sinensis [E. sinensis. A similar result was reported for the euryhaline crab Chasmagnathus granulatus [ACP and ALP are two important phosphatases that are crucial for metabolism and the immune system of sinensis . In the anulatus .E. sinensis. The brackish water conditions increased hemolymph osmolality and major ion concentrations while reduced Na+/K+-ATPase activity and its mRNA expression levels, as well as total polyunsaturated fatty acids contents in the posterior gills. Consequently the adult male E. sinensis subjected to salinity of 12 ‰ and 18 ‰ likely had lower energy expenditures on osmoregulation, which could be channeled for gonadal development.The present study demonstrated that brackish water promoted gonadal development of the adult male S1 FigP < 0.05) in hemolymph osmolality among different salinity treatments, while the different capital letters “A, B, C, D” indicated significant differences (P < 0.05) in external osmolality among all different salinity treatments. The superscript ‘**’ on the top of the bars indicated vary significant differences (P < 0.01) on the osmolality between the hemolymph of male crabs and their respectively external media.Different lowercase letters “a, b” on the tops of the bars indicated significant differences ((TIF)Click here for additional data file.


PGE1 pretreatment attenuated IR injury in extrahepatic cholestatic liver probably by suppressing MDA, MPO, TNF-α, IL-1β, ICAM-1, and Bax levels and improving GSH and Bcl-2 levels. In conclusion, PGE1 protects extrahepatic cholestatic liver from IR injury by improving hepatic microcirculation and reducing oxidative stress damage, intrahepatic neutrophil infiltration, and hepatocyte apoptosis.The aim of this study is to explore the hepatoprotective effect of intraportal prostaglandin E1 (PGE1) on liver ischemia reperfusion (IR) injury using an extrahepatic cholestatic model, observing oxidative stress markers, proinflammatory factors, apoptotic marker proteins, and an adhesion molecule. The extrahepatic cholestatic model was induced by common bile duct ligation. After seven days, rats were subjected to ischemia by Pringle maneuver for 15 min, followed by 1, 6, or 24 h of reperfusion. Prostaglandin E1 (PGE group) or normal saline (NS group) was continuously infused from 15 min before liver ischemia to 1 h after reperfusion. After reperfusion, histopathological evaluation of the liver was performed, as were measurements of bilirubin, biochemical enzymes, oxidative stress markers (GSH and MDA), proinflammatory factors (MPO, TNF- Many patients with extrahepatic cholestasis, such as hilar cholangiocarcinoma, need to undergo liver resection for radical resection of lesions, which is an effective means of long-term survival , 2. In oCholestasis can damage sinusoidal endothelial cells, induce hepatic microcirculation dysfunction, and render the liver susceptible to warm IR compared to normal liver –5. IR wiThere are several approaches to prevent or attenuate liver IR injury, like ischemic preconditioning, pharmaceutical pretreatment, antioxidant preconditioning, gene targeting, and others . Drug prMale albino Wistar rats (SPF), weighing 250 to 300 g, were purchased from Central Animal House of Shengjing Hospital, an affiliate of China Medical University. They were housed in individual cages with wood-chip bedding and acclimatized for 7 days before any experimental procedures under standardized laboratory conditions in a temperature-controlled room with 12-hour dark/light cycles. Standard rodent chow and water were provided ad libitum to all the animals. This project followed our institution's criteria for care and use of laboratory animals in research, which conformed to the National Institutes of Health Guidelines.n = 18) control group and the prostaglandin E1 treatment  group. The PGE group was infused a PGE1 solution in a dose of 0.5 μg·kg−1·min−1  dissolved in normal saline at a rate of 2 ml/h using a portable infusion pump. The NS group received normal saline infused at the same speed. Each of the two groups was further evenly divided into three subgroups according to different reperfusion times.Animals underwent bile duct ligation for 7 days and were randomly divided into two groups: the normal saline , direct bilirubin (DBil), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured using standard clinical methods with an autobiochemical analyzer .μm intervals and stained with hematoxylin/eosin for histological examination with light microscopy. Liver damage was analyzed by two pathologists blindly using Suzuki's criteria [Liver tissue specimens of the left lateral lobe were biopsied and fixed in 10% (V/V) buffered formalin and embedded in paraffin. Sections were cut at 4 criteria .g and 3,500g for 10 min at 4°C, and the supernatants were used for determination of total GSH levels and MDA content, respectively. Levels of GSH and MDA were assayed with commercial assay kits, which were purchased from the manufacturer . The absorbance of the samples was assessed with a spectrophotometer at the wavelength of 412 nm and 532 nm, respectively.Liver tissue samples were homogenized in ice-cold phosphate buffer (pH 7.4). The homogenates were centrifuged at 16,000Liver MPO activity was determined in the liver tissue homogenates by methods suggested by the manufacturer and measured using a spectrophotometer at 460 nm. All reagents were purchased from Nanjing Jiancheng Biology Engineering Institute. MPO activity was expressed as units per gram of tissue.g for 10 min at 4°C, and the supernatants were used for determining the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). Levels of TNF-α and IL-1β were assayed with commercial ELISA assay kits as indicated by the manufacturer . The absorbance was assayed with a spectrophotometer at the wavelength of 492 nm.Tissue samples were subsequently homogenized in ice-cold phosphate buffer (pH 7.4). The homogenates were centrifuged at 16,000The expressions of intercellular adhesion molecule-1 (ICAM-1), Bcl-2, and Bax on paraffin-embedded liver tissue sections were examined by using SP method, as previously reported . Five fiP < 0.05 were considered statistically significant.All data were expressed as mean ± standard deviation (SD) and analyzed by Prism software (GraphPad 6). Two-way ANOVA with Sidak's multiple comparisons test was used to evaluate statistical significance. Values of P > 0.05, Figures P < 0.05, Figures In the present study, the serum levels of TBil, DBil, and ALP in the PGE group were decreased at 1, 6, and 24 h, respectively, compared with those in NS group but had no statistical differences   .The results showed that when compared with the NS group, PGE1 significantly lowered the levels of ICAM-1, at 1, 6, and 24 h after reperfusion, respectively. For both groups, ICAM-1 expression reached the peak at 6 h after reperfusion .β, and TNF-α in liver tissue at 1, 6, and 24 h after reperfusion, respectively, when compared to the NS group. The levels of MPO, IL-1β, and TNF-α reached a peak at 6 h after reperfusion and then decreased in both of the two groups . These results show that PGE1 reduces neutrophil infiltration and is consistent with previous studies [α, probably mediated through the inhibition of activated Kupffer cells and of generated ROS [α. TNF-α upregulates ICAM-1 expression, which aids neutrophils to roll, adhere, and migrate during parenchymal infiltration [MPO is a sensitive indicator of infiltrating neutrophils. TNF-trophils , 25. Our studies , 20. PGEated ROS . This cultration . PGE1 maltration , 11.2+ from the endoplasmic reticulum, and interacts with cytochrome c to activate caspase signaling pathways that can produce apoptotic bodies and induce apoptosis [2+, the release of cytochrome c from mitochondria, and other factor-mediated apoptosis [In this study, PGE1 pretreatment reduced the expression of the proapoptotic protein Bax and increased the expression of antiapoptotic protein Bcl-2 after reperfusion, which hinted PGE1 could reduce the apoptosis of liver cells. Bax-dependent mitochondrial outer membrane permeabilization is regulated by the interaction between proapoptotic and antiapoptotic members of Bcl-2 family . After Bpoptosis . Bcl-2, poptosis .In this study, we found that PGE1 preconditioning protects liver IR injury with extrahepatic cholestasis by improving hepatic microcirculation and reducing intrahepatic neutrophil infiltration, oxidative damage, and hepatocyte apoptosis. This study still has some limitations. For example, we have not done researches at the genetic levels and signaling pathways and also have not evaluated the liver function such as bile production after 24 h reperfusion. For the goal to apply in a clinical condition, we also need to perform further research using a large animal model.


Results indicated that longan ECs had a high growth rate in the condition of blue or green light, at intensity ranged from 16 μmol·m-2·s-1 to 64 μmol·m-2·s-1, and duration from 8 h to 16 h. In addition, the contents of total flavonoids, rutin, and epicatechin were determined. Results indicated that flavonoid contents of longan ECs reached the highest value under blue light, at 32 μmol·m-2·s-1 and 12h/d. Blue light promoted the accumulation of epicatechin, but inhibited the synthesis of rutin. Finally, the expressions of flavonoid pathway genes, miRNAs and target genes were analyzed by qPCR. These results indicated that miR393 and its target gene DlTIR1-3, miR394 and its target gene DlAlMT12, and miR395 and its target gene DlAPS1 had a negative regulating relationship under blue light in longan ECs. Furthermore, miR393, miR394, and miR395 acted on target genes, which negatively regulated flavonoid key genes DlFLS and positively regulated key genes DlCHS, DlCHI, DlF3′H, DlDFR, DlLAR, and finally affected the accumulation of flavonoids. The treatment of longan ECs under the blue light at the intensity of 32 μmol·m-2·s-1 for 12 h/d inhibited the expression of miR393, miR394 and miR395, which promoted the expression of target genes and the accumulation of flavonoids and epicatechin, but inhibited the synthesis of rutin.While flavonoid metabolism’s regulation under light conditions by structural genes and transcription factors is understood, the roles of microRNAs (miRNAs) in this pathway have been rarely reported. In this paper, the accurate control of light was firstly enabled through the specially designed plant growth chamber which ensures consistency and accuracy of the cultivation of longan ECs and the repeatability of the experiments. Then, longan ECs were cultured in this chamber for 25 days. The change of growth rate of longan ECs was compared under different light qualities , intensities (16, 32, 64, 128, 256 μmol ·m Dimocarpus longan Lour.), Sapindaceae, is a tropical/subtropical fruit plant, with high nutritional and medicinal values. The medicinal ingredients in longan are mainly secondary metabolites, including flavonoid substances [Longan , chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H) and dihydroxy flavonol reductase (DFR) [Research on the light-controlled molecular regulatory mechanisms involved in flavonoid metabolism has progressed . The mecse (DFR) . The flase (DFR) . In addise (DFR) . The maise (DFR) ,7. The rse (DFR) ,7.Arabidopsis’ miR393 can act on auxin response factors 1 and 9, and thus, regulate glucosinolate synthesis [squamosa promoter-binding protein-like transcription factor (SPL) was described by Gou et al. [While flavonoid metabolism’s regulation under light conditions by structural genes and transcription factors is understood , the rolynthesis . The negu et al. . The funu et al. . Thus, wIn most of the related literatures, the definitions and descriptions of light sources are quite inaccurate, and the illumination inside the growth chambers is generally uneven ,19. Thestransport inhibitor response protein 1 (DlTIR1-3), miR394 and the target gene aluminum-activated malate transporter 12 (DlALMT12), miR395 and the target gene ATP sulfurylase 1 (DlAPS1), and flavonoid biosynthesis related genes DlCHS, DlCHI, flavonol synthase (DlFLS), DlF3′H, DlDFR and leucoanthocyanidin reductase (DlLAR). These studies provided new insights into the molecular mechanisms that allow light to influence plant flavonoid metabolism.Here, we investigated longan ECs cultured using light-control technology. The growth status and flavonoid contents were observed and analyzed. To understand the regulatory mechanism behind miRNAs roles in light-affected flavonoid metabolism in longan ECs, we compared and analyzed their relationships, including miR393 and the target gene Traditional plant growth chambers are often equipped with light sources of unknown or inexact wavelengths that distribute uneven illumination and light intensity levels. Illumination uniformity was achieved through the improvement of plant growth chambers . The cenLongan ECs were obtained using previously published methods ,21. Long-2·s-1 and 12h/d respectively with light qualities setting at blue (457 nm), green (515 nm), white, and red (660 nm); 2) Both light quality and photoperiod were fixed at blue and 12h/d respectively with light intensities setting at 16, 32, 64, 128, and 256 μmol·m-2·s-1; 3) Both light quality and intensity were fixed at blue and 32 μmol·m-2·s-1 respectively with photoperiods setting at 8, 12, 16, 20, and 24h.Longan ECs were put into plant growth chamber and control groups were incubated in dark. Three different kinds of treatments were investigated in the experiments. 1) Both intensity and photoperiod were fixed at 32 μmol·mEqual pieces (0.04 g) of longan ECs with diameters of 5–7 mm were placed onto separate bottles (240ml). There were 30 bottles used in each treatment, which lasted for 25 d at 25 ± 2°C with a relative humidity of 55%–60%. Each treatment was repeated for three times. After the treatments, all of the materials were stored at −80°C for later use.The browned and polluted longan ECs were removed, and 25 bottles of longan ECs of good growth status were chosen. The fresh weights of the longan ECs were determined, and the growth rate of each bottle was calculated. Growth rate = /Starting fresh weight × 100%. Longan ECs were observed using an inverted electron microscope (Leica DM IL).The flavonoid contents were determined using the method of Li  with somThe epicatechin contents were determined using the method of Zuo  with somThe following chromatographic conditions were used. The mobile phase consisted of water containing 0.1% formic acid (A) and methanol (B). The composition of the mobile phase was 5%–25% (B) for 0–12 min, 25%–35% (B) for 12–15 min, and 35%–5% (B) for 15–18 min. The column temperature was set at 30°C. The injection volume was 5 μL, and the flow rate was 0.2 mL/min. All standards and samples were detected by UPLC (ACQUITY UPLC H-Class) at a wavelength of 280 nm.The rutin contents were determined using the method of You  with somThe following chromatographic conditions were used. The assay was performed on a Diamonsil C18  column with an isocratic elution in methanol: 0.1% acetic acid (34:66) at a flow rate of 1.0 mL/min. The column temperature was set at 30°C. All standards and samples were detected by UPLC (ACQUITY UPLC H-Class) at a wavelength of 257 nm.® Premix Ex Taq™ II  on a LightCycler480 Real-time PCR system. EF -1a, elF-4a and DlFSD1a acted as the reference genes, and the relative gene expression levels were evaluated using the method described by Lin et al. [−ΔΔCt method. The quantitative PCR primers’ information is listed in Total RNAs were extracted from longan ECs grown under different light treatments using the TriPure Isolation Reagent . The RNA quantities were determined using an Agilent 2100 bioanalyzer and were checked for denaturation by agarose gel electrophoresis. RNAs were stored at −80°C for later use. Total RNAs from longan ECs were reverse-transcribed and used for qPCR in accordance with previous methods ,27. Exprn et al. . dlo-miRQuantitative results of the gene expression and flavonoid contents analyses were presented in terms of means ± SDs of at least three biological replicates. The effect of different light conditions on the flavonoid contents and the gene expression were analysed by one-way analysis of variance (ANOVA) followed by Duncan’s test using SPSS version 19.0. These pictures were made using the GraphPad Prism 6.0 software.The growth state of longan ECs was affected by light quality. Longan ECs in the dark, and under blue and green light conditions grew well, with a crunchy texture. But the growth status in the white and red light was poor. The texture mixed closely and the longan calli were watery with strong viscosity . Almost -2·s-1 to 64 μmol·m-2·s-1 were thrivinge, but the growth status under 128 μmol·m-2·s-1 to 256 μmol·m-2·s-1 was very poor and the structure was wet and sticky ..CHS and DlCHS, DlCHI and DlDFR reached their peak value under blue light of 32 μmol·m-2·s-1. However, the expression of DlFLS had maximum value under the dark treatment, and the expression of DlFLS was suppressed under blue light treatment  presented negative correlation under blue light of different intensities in longan ECs  presented negative correlation under blue light of different photoperiods in longan ECs  followed by low (175 μmol·m−2·s−1) and high (450 μmol·m−2·s−1) in orchid tissue-cultured seedlings [Garcinia mangostana) fruit [−2·s−1 was adopted to study light effects on longan ECs.Different light qualities can be effectively used on plants under the appropriate optical density, termed photosynthetic photon flux density, conditions . Thus, teedlings . In tropa) fruit , anthocya) fruit . Thus, tThe circadian rhythms of plant cells are affected by photoperiod, which effects the synthesis of secondary metabolites . For theGenes related to light-inducible flavonoid metabolic pathways have been widely reported . HoweverTIR1[TIR1 was a key factor in the plant auxin signal transduction pathway[DlTIR1 promoter contained a lot of light reaction-related components [DlTIR1 in light response. In addition, the fact of miR393 regulated target gene DlTIR1-3 and the model of the negative correlation between their expressions were confirmed by Lai et al [DlTIR1-3, then affected the accumulation of flavonoids under blue light conditions.In previous studies, researchers found that both miR393  and TIR1TIR1 could ren pathway. The synn pathway. Meanwhimponents  and flavai et al . In concDlALMT12 was also proved through psRNAtarget software analysis. As the blue light changes, miR394 and DlALMT12 present a negative correlation. In addition, ALMT have important regulation function in the synthesis of malic acid in the TCA [DlALMT12 and then further influenced the accumulation of flavonoids under blue light.In our studies, the fact of miR394 regulated target gene  the TCA . In high the TCA . In shorAPS1 specifically. The over-expression of miR395 results in the APS1 unable to receive external signal stimulation [DlAPS1 was also proved through psRNAtarget software analysis in longan EC. As the blue light changes, miR395 and DlAPS1 present a negative regulatory correlation. Sulfate assimilation is a key component in both primary and secondary metabolisms. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5’-phosphosulfate (APS) [DlAPS1, then influenced flavonoid synthesis under blue light conditions.It was reported that miR395 could shear mulation . The miRte (APS) . In brieCHI and Csn-miR2593e targets ANR then regulates the accumulation of flavonoids [Arabidopsis miR393 can act on auxin response factors 1 and 9, and thus, regulates glucosinolate synthesis [SPL transcription factor  was described by Gou et al [Arabidopsis [The miRNAs can act on the genes encoding its rate-limiting enzyme, and also act on transcription factors, then regulate the secondary metabolism of plants . Degradoavonoids . Howeveravonoids . Arabidoynthesis . The negou et al . Anotherbidopsis . TherefoThe miRNAs may affect the expression levels of multiple structural genes in the flavonoid pathways, and the effects of miRNAs may be more obvious than those of certain flavonoid-related genes . miRNAs Thus, miR393, miR394, and miR395 played an important role in the flavonoid metabolism of longan ECs under blue light conditions, which needed to be further verification using genetic transformations.DlTIR1-3, miR394 and its target gene DlAlMT12, and miR395 and its target gene DlAPS1 had a negative regulating relationship under blue light in longan ECs. Furthermore, miR393, miR394, and miR395 acted on target genes, which negatively regulated flavonoid key genes DlFLS and positively regulated key genes DlCHS, DlCHI, DlF3′H, DlDFR, DlLAR, and finally affected the accumulation of flavonoids. The miRNAs played an important role in the flavonoids metabolism of longan ECs regulated by blue light, which showed that light regulated the molecular mechanism of flavonoids metabolism in plants.In our study, miR393 and its target gene S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigA, dark treatment; B, blue light treatment; 1, epicatechin.(TIF)Click here for additional data file.S5 FigA, dark treatment; B, blue light treatment; 2, rutin.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.S14 Table(DOCX)Click here for additional data file.


B. pseudomallei. Melioidosis is endemic in Southeast Asia and northern Australia; however, the global distribution of B. pseudomallei and the disease burden of melioidosisis are still poorly understood. Melioidosis is difficult to treat, as B. pseudomallei is intrinsically resistant to many antibiotics and requires a long course of antibiotic treatment. The mortality rates remain high in areas of endemicity, with reoccurrence being common. Therefore, it is imperative to diagnose the disease at an early stage and provide vital clinical care to reduce the mortality rate. With limitations in treatment and lack of a vaccine, it is crucial to study the immune response mechanisms to this infection to get a better understanding of disease susceptibility and pathogenesis. Therefore, this study aimed to analyze the gene expression levels of important cytokines to establish useful correlations for diagnostic and therapeutic purposes.Melioidosis is a life-threatening infectious disease caused by a soil-associated Gram-negative bacterium,  Burkholderia pseudomallei. Understanding the disease susceptibility and pathogenesis is crucial for developing newer diagnostic and therapeutic strategies for this life-threatening infection. In this study, we aimed to analyze the gene expression levels of important cytokines in melioidosis patients and establish useful correlates with disease biomarkers compared to cases of sepsis infection caused by other pathogens and healthy individuals. A Qiagen common human cytokines array profiling the gene expression of 84 important cytokines by real-time quantitative PCR (RT-qPCR) was used. We analyzed 26 melioidosis cases, 5 healthy controls, and 10 cases of sepsis infection caused by other pathogens. Our results showed consistently upregulated expression of interleukins (IL) interleukin-4 (IL-4), interleukin-17 alpha (IL-17A), IL-23A, and IL-24, interferons (IFN) interferon alpha 1 (IFNA1) and interferon beta 1 (IFNB1), tumor necrosis factor (TNF) superfamily 4 (TNFSF4), transforming growth factor (TGF) superfamily, bone morphogenetic proteins 3 and 6 (BMP3 and BMP6), transforming growth factor beta 1 (TGFB1), and other growth factors, including macrophage colony-stimulating factor (M-CSF), C-fos-induced growth factor (FIGF), and platelet-derived growth factor alpha (PDGFA) polypeptide, in melioidosis patients compared to their expression in other sepsis cases, irrespective of comorbidities, duration of fever/clinical symptoms, and antibiotic treatment. Our findings indicate a dominant Th2- and Th17-type-cytokine response, suggesting that their dysregulation at initial stages of infection may play an important role in disease pathogenesis. IL-1A, interleukin-1 beta (IL-1B), and IL-8 were significantly downregulated in septicemic melioidosis patients compared to their expression in other sepsis cases. These differentially expressed genes may serve as biomarkers for melioidosis diagnosis and targets for therapeutic intervention and may help us understand immune response mechanisms.Melioidosis is an underreported infectious disease, caused by the Gram-negative bacterium IMPORTANCE Melioidosis is a life-threatening infectious disease caused by a soil-associated Gram-negative bacterium, B. pseudomallei. Melioidosis is endemic in Southeast Asia and northern Australia; however, the global distribution of B. pseudomallei and the disease burden of melioidosisis are still poorly understood. Melioidosis is difficult to treat, as B. pseudomallei is intrinsically resistant to many antibiotics and requires a long course of antibiotic treatment. The mortality rates remain high in areas of endemicity, with reoccurrence being common. Therefore, it is imperative to diagnose the disease at an early stage and provide vital clinical care to reduce the mortality rate. With limitations in treatment and lack of a vaccine, it is crucial to study the immune response mechanisms to this infection to get a better understanding of disease susceptibility and pathogenesis. Therefore, this study aimed to analyze the gene expression levels of important cytokines to establish useful correlations for diagnostic and therapeutic purposes. Melioidosis is a life-threatening infectious disease that is endemic in Southeast Asia and northern Australia . A recenBurkholderia pseudomallei infection have been studied in several animal models . In this study, we aimed to analyze the gene expression profiles of 84 important human cytokines  of melioidosis cases had associated comorbidities, and diabetes was the most common comorbidity (n = 17) in this study. Out of 23 confirmed cases of melioidosis, 16 were classified as septicemic or bacteremic.A total of 26 cases of melioidosis were analyzed, of which 23 were confirmed cases (culture positive) and 3 were probable cases (high antibody titer positive). A majority , and family with sequence similarity 3, member B (FAM3B), were significantly downregulated in other bacterial sepsis infection cases compared to their expression in healthy controls , IL-1 receptor antagonist (IL-1RN), IL-27, and IL-8 in melioidosis patients compared to their expression in healthy controls . IL-16, nfection –13, 15. 10.1128/mSphere.00121-17.2TABLE S2 n = 26) compared to PBMCs of other sepsis cases (n = 10). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S2, PDF file, 0.1 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of melioidosis patients  compared to PBMCs of healthy controls (n = 5). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S3, PDF file, 0.1 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of melioidosis patients  compared to PBMCs of healthy controls (n = 5). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S4, PDF file, 0.1 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of other sepsis infection cases  compared to its expression in other sepsis infections . IL-17 iesponses . Anti-ILesponses , 20. IL-esponses . IL-27 hesponses , 23. As 10.1128/mSphere.00121-17.5TABLE S5 n = 16) compared to PBMCs of other sepsis cases (n = 10). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S5, PDF file, 0.1 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of septicemic melioidosis cases  compared to PBMCs of other sepsis cases (n = 10) and healthy controls (n = 5). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S6, PDF file, 0.1 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of diabetic melioidosis cases  compared to their expression in other bacterial sepsis cases  compared to their expression in other sepsis cases  and >15 days fever/clinical symptoms duration (n = 21) compared to PBMCs of other sepsis cases (n = 10). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S7, PDF file, 0.2 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of melioidosis cases with ≤15 days of fever/clinical symptoms duration  was downregulated in melioidosis patients compared to its expression in healthy controls . IFNA1 aB. pseudomallei infection during the early stages , which plays a major role in B cell activation and development, proinflammatory cytokines, and lymphotoxin alpha (LTA) were downregulated in the melioidosis cohort compared to their expression in healthy controls , 37.+ T cells and the production of Th2 cytokines like IL-4 (Tumor necrosis factor (ligand) superfamily 14 (TNFSF14), which plays a major role in T cell proliferation, TNFSF4, which is responsible for Th2 cell differentiation, and TNFSF8, implicated in blocking Th1 responses, were upregulated in the melioidosis cohort compared to their expression in other sepsis infection cases  to S8. Uike IL-4 , 40. It ike IL-4 . TNFSF14ike IL-4 . TNFSF8,ike IL-4 . These f10.1128/mSphere.00121-17.8TABLE S8 n = 14) and >15 days of antibiotics treatment (n = 8) compared to PBMCs of other sepsis cases (n = 10). Shading indicates statistically significant differential gene expression at a P value of <0.05. Download TABLE S8, PDF file, 0.2 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of melioidosis cases with ≤15 days of antibiotics treatment , inhibin beta A (INHBA), and transforming growth factor beta 1 (TGFB1) showed significant upregulation in melioidosis patients compared to their expression in healthy individuals . BMP3, BHigh-level expression of BMP3, BMP6, TGFB1, and TGFB2 was observed in the septicemic melioidosis cohort compared to that in other bacterial sepsis cases .TGF-β was upregulated during melioidosis infection, with increased levels being correlated to severe melioidosis in human hosts . Our stuFig. 1 to Tables S6 to BMP3 and BMP6 were consistently upregulated in melioidosis patients compared to their expression in other sepsis cases, irrespective of factors like the duration of clinical symptoms, antibiotic treatment, and comorbidities such as diabetes  to S8. B to Platelet-derived growth factor alpha (PDGFA) polypeptide was significantly upregulated in melioidosis patients compared to its expression in healthy individuals . DownregPDGF is an important growth factor that plays a crucial role in blood vessel formation (angiogenesis) and regulates cell growth and differentiation. THPO stimulates the production and differentiation of megakaryocytes, thus regulating platelet production. FIGF plays an active role in angiogenesis and vascular endothelial cell growth . IncreasDownregulated expression of leukemia inhibitory factor (LIF), an IL-6 class cytokine that inhibits cell differentiation, and a similar cytokine, oncostatin M (OSM), was observed in the early-acute-phase melioidosis cases  compared to their expression in other sepsis cases  to S8. S to n = 5), we could not see any statistically significant differential expression of some of the inflammatory response genes involved in early innate immune responses. However, our results showed consistently upregulated expression of interleukins IL-4, IL-17A, IL-23A, and IL-24, interferons IFNA1 and IFNB1, TNF superfamily 4 (TNFSF4 or OX40L), TGF superfamily, BMP3, BMP6, TGFB1, and other growth factors, including CSF1, FIGF, and PDGFA, in melioidosis patients compared to their expression in other sepsis cases, irrespective of comorbidities, the duration of fever/clinical symptoms, and antibiotic treatment  . This maAs diabetes was seen as a major comorbidity in our experimental cohort, we analyzed our data to see if there was any significant differential expression between diabetic melioidosis cases and nondiabetic melioidosis cases. The gene expression patterns in these two groups were comparable, and we could not find any statistically significant differential expression, indicating that the differential expression was largely due to melioidosis infection (see 10.1128/mSphere.00121-17.9TABLE S9 n = 17) compared to PBMCs of nondiabetic melioidosis cases (n = 9). Download TABLE S9, PDF file, 0.1 MB.Relative expression ratios of 84 investigated gene targets in PBMCs of diabetic melioidosis cases , United States Army Medical Research and Material Command (USAMRMC). Patients fitting the clinical case definition of melioidosis, i.e., febrile illness for more than 5 days, pneumonia, septic arthritis, skin lesions, septicemia, and lung, soft tissue, or deep abscess, were recruited for initial screening for melioidosis. Blood, pus, and other patient specimens were collected for bacterial cultures, and serum samples were collected for indirect hemagglutination (IHA) antibody testing. Any positive bacterial cultures were further screened and confirmed as B. pseudomallei and/or had high antibody titers (>640) by the IHA test were recruited for our study and classified as positive cases of melioidosis. Culture- and PCR-positive samples were considered confirmed cases of melioidosis. Samples with an antibody titer of >640 by IHA testing were considered probable cases of melioidosis. At the time of recruitment, all melioidosis patients were undergoing antibacterial treatment.Patients who were culture positive for B. pseudomallei as negative controls for our gene expression profiling study  who were negative for ng study .B. pseudomallei was done at the admitting hospital using conventional culture techniques for blood, sputum, pus, and other specimens. Bacterial isolates that were oxidase-positive, gentamicin-resistant, and Gram-negative bacilli were forwarded to the reference laboratory in Colombo, where they were subcultured to establish pure growth and maintained at −70°C in 15% brain heart infusion (BHI)-glycerol for subsequent definitive tests. Bacteria were resuscitated by subculture onto 5% blood agar and incubated for 24 h at 37°C to give single-colony growth for all subsequent tests.Primary isolation of B. pseudomallei grown on blood agar from a patient’s sample was resuspended in ultrapure water. The suspension was heated at 95°C for 10 min and centrifuged at 13,500 × g to pellet the cell debris. The supernatant was used as the template for all subsequent PCR assays. A real-time PCR assay was done for gene targets of the lpxO, YLF, and BTFC gene clusters using primers and methods described previously  for lymphocyte purification. The lymphocytes were purified using the Ficoll fractionation method according to the manufacturer’s instructions, lysed with RLT buffer , homogenized, and stored at −80°C for total RNA extraction.Total RNA was extracted from the stored cell lysate samples using the Qiagen RNeasy minikit  according to the manufacturer’s protocol. RNA extracted from 0.6 million PBMCs was used for cDNA synthesis as the standard for all samples analyzed by real-time quantitative PCR (RT-qPCR). cDNA was synthesized using the Qiagen first-strand kit  according to the manufacturer’s recommendations. The synthesized cDNA samples were stored at −20°C until further use.2 Profiler PCR array  was used for this study. The PCR protocol and thermal profile recommended by the manufacturer were followed. PBMCs from 26 melioidosis cases (identified as confirmed or probable cases), 10 other bacterial sepsis cases , and 5 healthy negative controls were analyzed by RT-qPCR.The Qiagen human common cytokines RTCT) method  method  accordin


To evaluate the efficacy and safety of stereotactic body radiation therapy (SBRT) in patients with small hepatocellular carcinoma(sHCC) who were ineligible for surgery or ablation therapies.From March 2011 to December 2012, 28 cases with sHCC which were ineligible or refused surgical resection, transplantation or local ablation were treated with CyberKnife SBRT. Median size of tumors was 2.1 cm (range:1.1–3.0 cm), a dose of 10-15Gy per faction was given over 3–6 consecutive days, resulting in a total dose of 35-60Gy.The median follow-up period was 36 months, with the response rate of complete response (CR) in 17 cases, partial response (PR) in 8 cases, stable disease (SD) in 2 cases and progressive disease (PD) in one case. Overall response rate was 89.28%. Overall survival rates in 1, 2 and 3 years were 92.86, 85.71 and 78.57%, respectively. Local control rates in 1, 2 and 3 years were 96.43, 92.86 and 89.28%, respectively. No grade ≥ 3 hepatic toxicity was observed.CyberKnife treatment was a safe and effective option for sHCC, which had shown good local control, high overall survival rates and low toxicity. CyberKnife SBRT could be served as an alternative treatment for patients with sHCC which is unsuitable for surgical treatment or local ablation. Hepatocelluar carcinoma (HCC) is the most common primary liver tumor and the fifth common malignant disease globally . The annCyberKnife SBRT, which stands for the image guided precision radiation therapy, is the new type of stereotactic radiosurgery. The most important features of CyberKnife treatment are online position correction, respiratory synchronous tracking, real-time tracking and image guided radiotherapy, etc. Respiratory synchronous tracking system guides the accelerator to track the tumor movement, which can control the precision within 1 mm, realizing the precise therapy. Meanwhile, it enhances the dose of tumor region, reduces the dose of normal tissues nearby, and increases the therapeutic gain ratio. The aim of our study is to evaluate the curative effect and security of CyberKnife SBRT in treating sHCC in our hospital.Between March 2011 and December 2012, 28 sHCC patients were treated with CyberKnife SBRT. All patients met the criteria as follows: (a)The max diameter of the tumors ≤3 cm; (b) Without portal vein tumor thrombus and lymphatic metastasis; (c)Without extrahepatic metastasis; (d)unfeasible, difficult or refusing to undergo surgery or percutaneous ablative therapies; (e)Child-Pugh Classification(CPC) A or B; (f) eastern cooperative oncology group(ECOG) score 0 or 1; (g)Single tumor.The clinical diagnosis was based on the result of imaging according to the international guidelines on the management of HCC . The chaBaseline characteristics of 28 patients were listed below Table . Twenty-Fiducial implantation: 27 patients were implanted 3 to 5 fiducials 7 days prior to receiving CyberKnife treatment. The fiducials were 6 cm from tumors in the vicinity. The implantation of fiducials was to provide target tracking during CyberKnife treatment. Twenty seven patients with the implanted fiducials adopted respiratory tracking and fiducial tracking techniques simultaneously, while one patient adopted X-sight spine tracking techniques.o; slice thickness of 1 mm; voltage of 120 KV tube current of 400 mA; scan length of 20 cm; pixel size of 512 × 512. The scan is head first with dorsal decubitus.CT localization: CT scan was undergone 1 week after fiducial implantation. In addition to benchmark image as plain CT scans, the auxiliary images were chosen based on patients’ conditions, and these images are enhanced CT scans, MRI, positron emission tomography-computed tomography(PET-CT) and hepatic arteriography, etc. The acquired parameters of CT images are as follows: tilted angel of 0CyberKnife treatment plan design: Radiation therapists delineated the gross target volume (GTV) and organs at risk . Planning target volume (PTV) expands 3-5 mm of GTV. All plans were designed by G4 CyberKnife MultiPlan (Version 4.0.2). A total dose of 35-60Gy was delivered in 3–6 fractions. The treatment plan enclosed PTV with 72–80% isodose line of maximum dose quated to the prescribed dose. In addition, normal tissues dose was within normal radiotherapy tolerance dose (TG-101 report). CyberKnife treatment plan was transferred into treatment control system. The treatment will be started after data verification.Follow-up period was defined from the last treatment to patient’s loss or death. Follow-up was done every 3 months in the first year, then 3 or 6 months thereafter. A review of each patient’s prior medical history, physical examination, complete blood counts, biochemical profiles, tumor markers, liver function and imaging examinations such as enhanced abdominal CT scans or MRI were performed at each follow-up.Short-term therapeutic evaluation was based on Modified Response Evaluation Criteria in Solid Tumors (mRECIST) . CompletP value < 0.05 was defined as the threshold of statistical significance. The overall survival and local control rates were estimated using the Kaplan-Meier method. Factors significantly associated with OS and LC were identified by multivariate analysis using a stepwise Cox model, with calculation of hazard ratios (HRs) and 95% confidence intervals (CIs).SPSS 19.0 was used for statistical analysis. A All 28 patients completed the treatments. After 3–6 months of SBRT, there were 17 patients with CR (60.71%), 8 patients with PR (28.57%), 2 patients with SD (7.14%) and 1 patient with PD (3.58%). The response rate was (CR + PR)/28 × 100% = 89.28%, and the disease control rate was (CR + PR + SD)/ 28 × 100% = 96.42%.The median follow-up was 36 months (3-53 months). By October 2015, among the enrolled 28 patients, 6 patients died, in which 2 patients died of upper gastrointestinal bleeding, 1 hepatic encephalopathy, 1 hepatorenal syndrome, 1 hepatic failure and 1 obstruction of biliary tract. The overall survival rates of 1-year, 2-year and 3-year were 92.86, 85.71 and 78.57%, respectively. The local control rates of 1-year, 2-year and 3-year were 96.43, 92.86 and 89.28%, respectively  was prescribed to treat aleucocytosis. However, conservative method was applied to thrombocytopenia, and the platelet elevated gradually after the radiotherapy. None severe gastrointestinal complication happened, such as haemorrhage and perforation.P>0.05; Table P = 0.774, LC P = 0.415) as well.To investigate the risk factors for overall survival rate and local control rate, clinical data, including sex, age, type of chronic hepatitis, cirrhosis, AFP, CPC and any other characteristics were analyzed. The results were all with no statistical significance  after radiotherapy. Among the recurrent patients, 9 patients received TACE, 1 patient adopted RFA, 3 patients were retreated with CyberKnife SBRT.For patients with single lesion, especially sHCC, resection is the most effective treatment, with which the 5-year overall survival rate may run up to 60–70% , 10. SadCyberKnife SBRT, featured with accurate high-dose radiotherapy, was first applied to recurrent squamous cell carcinoma of head and neck patients who have no resection options or not suitable for surgery . Blomgre3, and median total doses were 45Gy. The result indicated local control rates at 1-year and 2-year were 84 and 74.6%. In the meanwhile, total doses, tumor diameter and tumor volume were considered as the prognostic factors [Yoon SM and his coworkers reported 93 sHCC patients treated with SBRT, whose 1-year and 3-year OS was 86 and 53.8%, respectively, tumor diameter less than 6 cm, tumor number less than three, single fraction dose 10-20Gy, total doses 30-60Gy. They considered SBRT could be served as an alternative option for the patients unsuitable for other radical treatments . In our  factors . We analGrade I and II hematotoxicity and hepatotoxicity were the main toxicity in our study, which was characterized with low-grade aminopherase elevation, bilirubin elevation, thrombocytopenia and aleucocytosis, and most of them could return to normal gradually after radiotherapy. Only one patient with hepatitis B liver cirrhosis suffered liver failure after 3 months of radiotherapy, who did not have liver enlargement, massive ascites and viral replication. For liver cirrhosis patients, the causes of liver function damage were complex and hard to figure out. Whereas for patients whose tumor was controlled well after radiotherapy and liver function failure wasn’t caused by tumor progress, they were more likely to suffer from serious liver injury with RILD. RILD was the major obstacles of HCC radiotherapy, and the incidence rate of liver radiotherapy was 5–10% when the dose of whole liver achieved 30-35Gy. The main inducing factors of RILD are the basic liver function, the liver cirrhosis and the PTV range . Jung J CyberKnife SBRT is proved to be effective, with its main advantages being synchronous respiratory tracking, high dose delivery and low fraction . Moreove


Functionalized pentacene derivatives continue to provide unique materials for organic semiconductor applications. Although oligomers and polymers based on pentacene building blocks remain quite rare, recent synthetic achievements have provided a number of examples with varied structural motifs. This review highlights recent work in this area and, when possible, contrasts the properties of defined-length pentacene oligomers to those of mono- and polymeric systems. Over the past few years, significantly improved optoelectronic properties have been achieved by conjugating chromophores such as thiophenes ,2 and anOrganic materials can provide numerous advantages over their inorganic counterparts, including ease of tunability of the HOMO and LUMO levels through chemical functionalization, compatibility with flexible substrates, as well as lower manufacturing costs. Today the field of organic semiconductors seems on the doorstep of revolutionizing applications in the areas of field effect transistors , light e1, 1 is both benchtop stable and highly soluble in common organic solvents, which allows for easy processing, including purification and solution deposition of thin films for device fabrication. Additionally, the size of the trialkylsilyl groups allows for control of the solid-state packing .,30.29,30afford 3 . From pe6–13 were then obtained in good yield from 4 by simple esterification of a bis-acid chloride , however, no aggregation is observed by UV–vis spectroscopy in CH2Cl2. Furthermore, no change in the qualitative shape or absorption maxima (±1 nm) is found for the UV–vis spectra of dimers 6–13 and trimers 16–17 compared to monomeric pentacenes 3–5. Molar absorptivities (ε) increase monotonically as a function of the number of pentacene units: dimers 6–13 have roughly twice the ε values of monomer 5, while trimers 16–17 have ε values three times that of 5 (comparison made at λmax = 645 nm). Fluorescence studies using an excitation wavelength (λexc) of 551 nm show that the emission maximum, located at 652 nm, is identical for all dimers and trimers. Fluorescence quantum yield (ΦF) studies show that dimers 6–13 all show quantum yields ΦF = 0.08–0.11, whereas the values for trimers 16–17 (ΦF = 0.06) are about half that of the dimers. Thermal analysis by differential scanning calorimetry (DSC) shows that all dimers and trimers begin to decompose somewhere in the range of 360–380 °C, and TGA analysis reveals no significant weight loss below 370 °C. Charge transport properties have unfortunately not been reported for these oligomeric materials.Solution-state aggregation is observed for oligomers 18 and 19, synthesized by reacting diol 5 with the acid chloride of either glutaric or adipic acid, respectively (Mw) or number-average molecular weight (Mn) were reported for these polymers, MALDI MS analysis showed macromolecules of over m/z 17,000  for 18 and m/z 15,000  for 19. In addition to the generalized structure in In addition to the oligomers described above, Tykwinski and Lehnherr reported non-conjugated pentacene-based polymers ectively  29,30].,30.18 an18 and 19 are qualitatively equivalent  to those of the corresponding mono-, di- and trimers (vide supra). These polymers are soluble in solvents such as CH2Cl2, CHCl3, and THF and can be handled in the presence of air and water without any noticeable decomposition. Thin films cast from solution exhibit only a slight red-shift in λmax (<10 nm) in comparison to their solution-state UV–vis absorptions. DSC and TGA analysis of polymers 18–19 show thermal stabilities comparable to dimers 6–13 and trimers 16–17. UV–vis absorption and emission characteristics for Dendrimers are branched, 3-dimensional, defined-length oligomers and have been synthetically targeted for a range of applications, such as biomedical , catalys20 and 21  in 86% yield  in 76% yield.In 2009, Tykwinski and Lehnherr reported the synthesis and properties of pentacene-based dendrimers 0 and 21  37]. Th. Th20 an6% yield . A mild 20 and 21 show significant solubility in solvents such as CH2Cl2, CHCl3, and THF. Similar to their linearly arranged cousins (oligomers 6–13 and 16–17), no qualitative change in the UV–vis absorption profile for the dendrimers is found in comparison to monomer 5  for the most intense absorption (309 nm) is approximately three times larger than that of 20 , as expected based on the number of pentacene chromophores in each molecule. Solution-state fluorescence reveals a small Stokes shift of only 213 cm–1 for both dendrimers, suggesting minimal molecular rearrangement upon photoexcitation. Although the fluorescence spectra of dendrimers 20 and 21 appear similar to that of monomeric building block 5  to ΦF = 0.04 (20) to ΦF = 0.03 (21). This trend is analogous to that observed in pentacene-based oligomers 6–13 and 16–17 (vide supra). Both onomer 5 a. The mo18–19 and dendrimers 20–21 by solution spin-casting. A specific element of this study examined photocurrent yield . Thiquinone .27) and its benzothiophene analog (28) . Th. Th48–49entacene , likely 48 onto ITO. EL devices had the following configuration: ITO/m-MTDATA/NPB/Alq3:48 or 49 (5%)/LiF/Al -N-phenylamino)triphenylamine; NPB = N,N'-bis(1-naphthyl)-N,N'-diphenyl-1,1'-biphenyl-4,4'-diamine). Devices using pentacene 48 as a dopant showed red EL spectrum at 636 nm with 0.03 cd/A efficiency, while those using pentacene 49 showed dramatically better performances of 0.21 cd/A. These values are still both lower than those of devices with 6,13-diphenylpentacene (23) using 0.55 mol% pentacene:Alq3 which have 1.2 cd/A performance as reported by Kafafi and coworkers  cycloaddition of diynes . or. or52  to –5.14 eV for (63) , which is consistent with increasing the effective conjugation length (61) to –3.35 eV (63). The overall effect is a reduced electrochemical band gap, from 2.08 eV (61) to 1.79 eV (63), as the conjugation length increases with the length of the oligothiophene side chain. Analysis of the thermal stability (determined via TGA) as well as photostability of these hybrids affords no real trends as a function of the number of thiophenes in the side chain, although the alkylated thiophenes (61–63) are consistently more stable than their unalkylated counterparts. Increasing the number of thiophene units in the side chain results in a progressive decrease in solubility, although the alkylated derivatives have improved solubility compared to the alkyl-free derivatives. Solid-state packing of 62, determined by X-ray crystallography, reveals a 1-dimensional slipped-stack arrangement with interplanar distance of 3.41 Å between the pentacenes.In 2009, Zhang and coworkers have reported the synthesis of pentacenes substituted with oligothiophenes at the 6,13-positions , as welln length . As for 64 (64 was accomplished by carrying out an in situ desilylation of 65 followed by Sonogashira-type coupling reaction with bromide 66 to afford 64. Pentacene 64 had UV–vis absorptions in the Q-band region at 555 nm and 823 nm (λmax) in THF, with Soret bands at 421 and 493 nm. The band gap was determined from the UV–vis absorption edge to be 1.41 eV, which compared well with the electrochemically determined band gap of 1.40 eV measured in CH2Cl2.In 2005, Therien and coworkers reported the synthesis of conjugated bis[(porphinato)zinc(II)] compounds having a variety of aromatic chromophores as a π-spacer, one of which was a pentacene, namely compound 64  77]. Th. Th64  reveals Soret bands at 429 nm and 482 nm and an extremely broadened Q-band in the 500–800 nm region (λmax = 751 nm). Intramolecular charge transfer is tentatively assigned for the lowest absorption band because it is significantly red-shifted in comparison to the absorption spectra of the components. Pentacene 67 emits at λmax,em = 778 (in THF) using λexc = 429 nm. Electrochemical study of 67 reveals that oxidation is irreversible , while quasi-reversible reduction is observed at –0.85 V and –1.10 V vs. standard calomel electrode (SCE). The first reduction potential of 67 is closer to that of pentacene precursor 65 (0.92 V vs. SCE) than the reduction of model porphyrin 69 (–1.23 V vs. SCE) suggesting the reduction is occurring at the pentacene moiety. The photovoltaic properties of 67 have been studied, and, despite having a broad absorption spectrum able to capture a significant portion of the solar output spectrum, devices based on 67 have lower overall efficiencies (0.10%) than related materials in which the pentacene moiety is formally replaced with an anthracene moiety . The poor performance of sensitized solar cells based on pentacene 67 has been attributed to rapid non-radiative relaxation from the singlet excited state.Very recently, Lin and coworkers have synthesized conjugated pentacene-porphyrin derivative  Therien , startin partner . UV–vis 67 just described above  to compete effectively against the addition of a second acetylide into the highly soluble lithiated alkoxide intermediate. With 82 in hand, the subsequent addition of two or more equivalents of the second acetylide  resulted unsymmetrical diol 83. The addition of the two different nucleophiles to 6,13-pentacenequinone can be combined into a one-pot procedure, providing 83 (with R = Ph) in comparable yields (84% yield for the 1-step synthesis of 83vs. 77% for the 2-step route) . Ke. Ke73–81p route) . Unsymmecene see . It shoute scope ,30,79,8084 , which is more red-shifted than any of the unsymmetrical derivatives 73–81.Using a stepwise approach, Tykwinski and Lehnherr synthesized polarized pentacene 84  79]. Ad. Ad84 (71) typically have a very narrow absorption region in the ultra-violet region (centered around ca. 310 nm) in addition to absorption bands in the visible region of 525–660 nm; they are quite transparent in the region of 350–525 nm . Th. Th100 (d regime .104) to produce 3,3',9,9',10,10'-hexabromo-2,2'-bipentacene . The mechanism was postulated to proceed via formation of pentacene radical 107 (via loss of Br•) followed by bimolecular recombination of 107. Little is known about dimer 105 other than its constitution as established by MALDI-TOF MS and a tentative λmax value of ca. 688 nm (in toluene).In 2009, Neckers reported the photochemical dimerization of 2,3,9,10-tetrabromopentacene  108a (peripentacene) and 109 (trisperipentacene) as determined by laser desorption FTMS  remain, and this would result in a molecular mass increase of m/z 2 (108b), 4 (108c), or 6 (108d). The offset between the two pentacene moieties is not necessary, and these signals may be due to unfused sites such as in structure 108e; a number of isomers can be imagined. No experimental evidence has conclusively determined the exact structures related to these signals, but a theoretical computational study has been carried out . T. T108a (ried out . Likewis110 and 111 provides random copolymers 112 and 113 depending on the relative mole ratio of the monomers used (112 has a weight-average molecular weight (Mw) of 86,000 g·mol–1 and polydispersity (PDI) of 1.7, while increasing the pentacene content results in polymer 113 with a lower MW of 61,000 g·mol–1 and smaller PDI of 1.6. Thin films of polymer 113 have an absorption λmax at 623 nm and emission at λmax,em = 625 nm (excitation at 365 nm). Polymer light emitting diodes (PLEDs) have been constructed using the following arrangement: ITO/PEDOT/polymer/Ca/Al. PLEDs with polymer 113 show a turn-on voltage of 30 V, a maximum photoluminescence of 240 cd/m2, and photometric efficiencies of 0.11 cd/A when operated at 100 cd/m2. The turn-on and operating voltages for 113 are higher than those of the homopolymer (polyfluorene). The incorporation of the pentacene into the polymer is believed to create electron- or hole-carrier traps, which likely cause a decrease in the carrier mobility. By decreasing the content of pentacene in the polymer, i.e. going from 113 to 112, turn-on voltages are reduced to 11 V and maximum luminance and photometric efficiencies improved to 1020 cd/m2 and 0.32 cd/A, respectively. It is noted that the PLEDs had a half-life of less than 10 h, which is insufficient for practical applications.In 2001, Tokito and coworkers have reported the first pentacene polymers . Ni[0]-mers used . Polymer114–115 [116, 117 was used as a regio-mixture of 2,9- and 2,10-dibromopentacene to form copolymers 114–116 via a Sonogashira-type coupling with 1,4-diethynylbenzenes. Similarly, polymer 116 was formed from dibromides 117 via Suzuki coupling with fluorene diboronic ester 118, followed by endcapping of the polymer with phenyl groups.In 2007, Okamoto and Bao reported conjugated polymers 114–115 , which w115 116, . Dibromo114 has a number-average molecular weight (Mn) = 1.13 × 104 g·mol–1 and PDI = 2.21, while polymer 115 has Mn = 2.39 × 104 g·mol–1 and PDI = 2.42, while polymer 116 has Mn = 3.64 × 104 g·mol–1 and PDI = 3.21. Polymer 115 shows greater solubility (>5.0 mg/mL in chlorinated solvents) compared to 114 (~0.3 mg/mL) which is likely due to the increased branching of the alkoxy group on the aryl spacer. Polymer 116 is soluble in halogenated benzenes, such as o-dichlorobenzene (o-DCB) with solubility of ~5.0 mg/mL. DSC analysis of polymers 114–115 shows no thermal transitions between 25 and 350 °C, while TGA data for polymers 114–116 reveals thermally induced weight loss occurs around 400 °C. Polymer 115 has better solution-state photochemical stability in comparison to 6,13-bis(triisopropylsilylethynyl)pentacene (1) as observed by monitoring the decrease in absorption at λmax in the UV–vis spectra over time. This is attributed to the slightly lower energy HOMO (by 0.04 eV) in polymer 115 compared 1, making the polymer harder to oxidize (116 and PCBM has been attempted, but result in "poor performance" [60 and its derivatives which would destroy the pentacene chromophore [Polymer  oxidize . Fabricaormance" . This maomophore ,110,111.An array of functionalized pentacenes has been synthesized over the past decade, some of which have provided useful devices. Pentacene oligomers and polymers, on the other hand, are still a relatively unexplored research area with much promise for interesting properties. Much the same as functionalization of monomeric pentacene-based materials has afforded the ability to vary electronic properties and control solid-state organization, oligomers and polymers offer many of the same opportunities. The incorporation of pentacene and related chromophores into an oligo- or polymer framework is, however, a very young field and many discoveries await the hands and imagination of talented synthetic chemists.


Cyperus rotundus rhizome along with another 11 previously identified rhizome essential oil constituents and α-humulene, which lacks the only carbonyl group present in ZER, as well as binary mixtures of ZER and seven active compounds were tested for repellency to male B. germanica. The results were compared to N,N-diethyl-3-methylbenzamide (deet). In filter-paper choice tests, ZER was the most repellent compound, and α-humulene was ineffective, which indicates that the α,β-unsaturated carbonyl group of ZER is a prerequisite component for repellency. At 81.5 μg cm−2, enhanced repellency was produced by binary mixtures of ZER and 1,8-cineole, (+)-dihydrocarvone or (R)-(+)-limonene . These mixtures were very effective against male B. germanica within 24 h and were more repellent than a single compound or deet alone. The optimum ZER content was determined to be more than 50%. In Ebeling choice box tests at 652.4 μg cm−2, these compounds and deet resulted in complete repellency to intact male B. germanica, while they exhibited 35–47% repellency to antennectomized male one. Mixtures formulated from the active constituents of the C. rotundus rhizome could be useful as potential repellents for controlling B. germanica.The compounds 1,8-cineole and zerumbone (ZER) from the  Blattella germanica L. (Blattodea: Blattellidae), is one of the major sources of potent allergens in sensitive populations3. Approximately 43% of the United States (US) population (6–59 years old) is allergic to at least one common indoor allergen, and 26% are sensitive to B. germanica4. Cockroach allergens are associated with feces, saliva, secretions and fragments of their body parts5. The prevalence and severity of allergic diseases, such as asthma and rhinitis, caused by cockroaches are increasing, especially among children2, and the diseases are some of the most serious global public health problems8. In addition, cockroach exuviae can support large populations of the European house dust mite, which is an important producer of various allergens that lead to exacerbated cases of bronchial asthma9.The German cockroach, 9. The most widely used insect repellent products are currently based on N,N-diethyl-3-methylbenzamide (deet)10, which continues to be an effective compound. However, deet has many problems, such as having an unpleasant odor and causing damage to certain plastics and synthetic rubber as well as causing medical issues such as central nervous system depression, urticaria and potential encephalopathic toxicity11. In addition, the number of approved insecticides and repellents may be reduced in the near future in the US by the US Environmental Protection Agency as re-registration occurs under the 1996 Food Quality and Protection Act12. The removal of conventional insecticide or repellent products from markets due to the increase in insecticide resistance or welfare concerns will have a serious impact on the proliferation of B. germanica14. Therefore, there is a pressing need for the development of eco-friendly alternatives for the control of B. germanica, particularly alternatives with repellency because many insecticides are difficult to apply to inaccessible places, do not reach deep, insecticide-free harborages and cannot be applied to sensitive environments9.Cockroach repellents can be used as a preventive tool in the transport and storage of merchandise, protecting sensitive areas such as kitchens, children’s nurseries and hospitals, and reaching inaccessible cockroach hiding places, such as crevices, electrical ducts, plumbing ducts and cabinet voids, as well as for protecting sensitive electronic equipment, communications equipment and food industry facilities18. Many efforts have been focused on plants as potential sources of commercial repellents, in part, because certain plant preparations meet the criteria of minimum-risk repellents19. Previous studies have shown that the methanol extract and essential oil from the rhizome of coco-grass, Cyperus rotundus L. , possessed good repellency against male B. germanica. Limited information is available concerning the potential uses of the C. rotundus rhizome and its constituents for the control of B. germanica, despite the insecticidal properties of the plant20.Biorepellents and botanical insecticides derived from plants may provide potential alternatives for household and medically important pest insects because plants are a potential source of bioactive secondary metabolites that are perceived by the general public as relatively safe and pose fewer risks to the environment with minimal impacts on human healthC. rotundus rhizomes, and another 11 previously identified constituents without insecticidal activity from the rhizome essential oil20 had repellent activity against male B. germanica compared to deet, a positive control, in a filter-paper choice bioassay. Deet is registered as an insect repellent in South Korea21. The repellency of binary mixtures of ZER and seven active compounds was also evaluated. Finally, the repellency of α-humulene was examined to discern whether the α,β-unsaturated carbonyl group of ZER is essential for repellant activity. The relationship between repellency and boiling points (BPs) of the test compounds is also discussed.In this study, our aim was to assess whether the two monoterpenoids, 1,8-cineole and zerumbone (ZER), which were extracted from C. rotundus rhizome were tested against male B. germanica using a filter-paper choice assay  and nuclear magnetic resonance (NMR) spectroscopy. The two repellent compounds were 1,8-cineole (1) and zerumbone (2)  was identified based on the following evidence: colorless oil; EI-MS (70 eV), m/z (% relative intensity): 154 [M]+ (98), 139 (68), 126 (18), 108 (93), 93 (49), 81 (100), 71 (80), 69 (61), 55 (53), 53 (21); 1H NMR : δ 1.46–1.95 , 1.46–1.55 , 1.33 , 1.14 , 0.92 ; 13C NMR : δ 75.8 (C-8), 72.0 (C-1), 34.3 (C-4), 32.5 , 29.3 , 27.8 (C-7), 23.8 ; and distortionless enhancement by polarization transfer (DEPT) spectra. Zerumbone (2) was characterized as follows: pale yellow crystal; EI-MS (70 eV), m/z (% relative intensity): 218 [M]+ (85), 203 (9), 189 (10), 175 (7), 163 (21), 150 (28), 135 (100), 121 (17), 107 (85), 96 (61), 91 (30), 67 (24), 53 (23); 1H NMR : δ 6.00 , 5.86 , 5.20 , 2.40–2.50 , 2.31–2.40 , 2.25–2.31 , 2.15–2.25 , 1.80 , 1.67 , 1.46 , 1.11 , 0.97 ; 13C NMR : δ 207.0 (C-8), 163.4 (C-10), 151.4 (C-6), 139.0 (C-7), 137.8 (C-3), 128.2 (C-9), 126.2 (C-2), 43.4 (C-1), 40.5 (C-4), 39.0 (C-11), 29.9 (C-14), 25.6 (C-5), 24.6 (C-15), 15.5 (C-12), 11.9 (C-13); and DEPT spectra. The interpretations of the proton and carbon signals of compounds 1 and 2 were largely consistent with the findings of Liu et al.22 and Dai et al.23, respectively.Filter-paper choice bioassay-guided fractionation of the B. germanica was evaluated using a filter-paper choice assay -Dihydrocarvone, (R)-(+)-limonene and (1S)-(−)-verbenone resulted in 86, 84 and 83% repellency, respectively. All these compounds were significantly more potent repellents than deet at 163.1 μg cm−2. The repellency of (S)-(−)-limonene, β-caryophyllene and (−)-(E)-pinocarveol did not differ significantly from deet, while the repellency of (−)-caryophyllene oxide was significantly lower than deet. (E)-carveol, (−)-α-copaene, α-humulene, (1R)-(−)-myrtenal and (1R)-(−)-myrtenol were almost ineffective (<5% repellency).The repellency of the 14 compounds and deet, which was used as a positive control, against male ay Table . The res−2 against male B. germanica was evaluated using Ebeling choice boxes (Table E)-Pinocarveol resulted in complete repellency at the 8 h post-treatment interval. (+)-Dihydrocarvone, β-caryophyllene and (1S)-(−)-verbenone produced complete repellency at 4 h, while (R)-(+)-limonene, (1S)-(−)-limonene and 1,8-cineole showed 85–70% repellency. Overall, all these compounds were less potent repellents than deet.The repellency of the eight selected active compounds and deet at 652.4 μg cmes Table . The repR)-(+)-limonene at the 70:30, 50:50 and 30:70 ratios was between 94 and 90%, between 91 and 85% and between 82 and 79% at 24 h post-treatment, respectively. The mixtures with the 70:30 ratio were significantly more effective than either the corresponding compound or deet alone. The repellency of the other four binary mixtures at the 70:30, 50:50 and 30:70 ratios was between 83 and 80%, between 79 and 73% and between 73 and 68%, respectively.The differences in repellency after the application of the seven binary mixtures were significant Table . The repB. germanica resulted in a diminished response to the five tested compounds and deet at 652.4 μg cm−2 in the Ebeling choice boxes (Table P < 0.0001) in the repellency of ZER to intact and antennectomized male B. germanica. Similar differences in the response of male cockroaches to (+)-dihydrocarvone, β-caryophyllene, (1S)-(−)-verbenone, 1,8-cineole and deet in intact versus antennectomized treatments were also observed.Antennectomy of male C. rotundus rhizome along with 11 previously identified constituents and binary mixtures of ZER and seven active compounds against male B. germanica was assessed. The repellency of these materials was compared to deet, which is a currently available synthetic repellent, to determine whether they would be suitable for future commercial cockroach repellents. Historically, C. rotundus has been widely used as an analgesic, a sedative, an antiplasmodic agent, an antimalarial and a treatment for stomach disorders25.In this study, the repellency of 1,8-cineole and ZER from the 28. Repellent constituents derived from plants against cockroaches include flavonoids29, phenylpropanoids30 and terpenoids33. For example, the monoterpenoids, E,Z- and Z,E-nepetalactone as well as pulegol, isopulegol and pulegone work against B. germanica, and the phenylpropanoids, safrole and isosafrole, work against Periplaneta americana (L.) (Blattodea: Blattidae) and have been reported as repellent compounds32. In the current study, the repellent constituents of the C. rotundus rhizome were determined to be the sesquiterpenoid ZER and the monoterpenoid 1,8-cineole. Of the tested compounds, ZER was the most potent repellent. Potent repellency was also produced by β-caryophyllene, (+)-dihydrocarvone, (R)-(+)-limonene, (S)-(−)-limonene, (−)-(E)-pinocarveol and (1S)-(−)-verbenone. This study is the first report of the repellency of ZER and pinocarveol against B. germanica, although 1,8-cineole has been reported to have repellency against B. germanica nymphs34 as well as contact and fumigant toxicity and ovicidal and feeding-deterrent activity against Tribolium castanaeum (Herbst) (Coleoptera: Tenebrionidae)35 and fumigant toxicity against three major stored-grain insects36.Many plant extracts and essential oils produce repellency to different insect species37 and some are insect repellents38. Because of their high volatility, the essential oils and their constituents are usually effective against arthropods only for a relatively short period, which is typically less than 2 h40. However, available information on B. germanica repellency is limited because most previous studies that investigated repellency used a bioassay with a short time period, which was mainly 5 to 10 min. The compounds 1,8-cineole and α-pinene have been reported to have 92 and 82% repellency, respectively, against B. germanica nymphs after a 2 h post-treatment at a concentration of 5 ppm33. However, Appel et al.41 reported that mint oil extract at the surface concentration of 2 mL of mint oil to 457.5 cm2 was extremely repellent (approximately 100%) to adult males of B. germanica and P. americana for 14 days.Volatile constituents derived from most plant extracts and essential oils consist of alkanes, alcohols, aldehydes and terpenoids (particularly monoterpenoids)E)-pinocarveol and ZER were very effective against male B. germanica for 8–10 h, while (+)-dihydrocarvone, β-caryophyllene and (1S)-(−)-verbenone were very effective for 4 h. Nevertheless, these compounds were less potent repellents than deet. The differences in repellency could be attributed to the differences in the quantitative losses because of the different volatility of the compounds, as described by Brown and Hebert42. The ability of a chemical vapor to function as a repellent has been reported to be related to its BP, and BPs between 230 and 260 °C at atmospheric pressure were found to be the most desirable for an effective repellent42. The BPs of (E)-carveol and (−)-α-copaene coincide with the optimal range but were less effective than either ZER with higher BPs, compounds with lower BPs or compounds with similar BPs. Thus, other factors, such as structural characteristics , rather than BP appear to be associated with repellency of these compounds against male B. germanica. For example, β-caryophyllene was more effective than caryophyllene oxide. Interestingly, α-humulene, which lacks only the α,β-unsaturated carbonyl group present in ZER, was virtually ineffective. This finding indicates that the α,β-unsaturated carbonyl group of ZER is a prerequisite component for repellency to B. germanica. Peterson et al.31 studied the repellency of catnip, Nepeta cataria L. , as well as its essential oil constituents, against male B. germanica. They reported that the iridoid monoterpenoid, E,Z-nepetalactone, was significantly more active than the Z,E-isomer. In addition, ZER, (+)-dihydrocarvone, β-caryophyllene, (1S)-(−)-verbenone, 1,8-cineole and deet produced complete repellency against intact male B. germanica, while these compounds exhibited 35–47% repellency against antennectomized male B. germanica. These results indicate that antennae are partly responsible for the perception of the tested compounds. Detailed tests are needed to fully understand their modes of repellent action.In the current study with Ebeling choice box tests, (−)-, expect for equivalent or near-equivalent mixtures, which had significantly lower repellency43. In the current study, repellency against male B. germanica produced by the binary mixtures of ZER and 1,8-cineole, (+)-dihydrocarvone or (R)-(+)-limonene at three mixture ratios were more pronounced compared to repellency produced by the corresponding compound and deet alone. The improved effectiveness of repellency could be attributed to the lower evaporation rate and/or better persistence of ZER in the combined presence of another compound, as described by Khan et al.44 and Tuetun et al.45. The optimum ZER content was determined to be more than 50% according to our laboratory results. This original finding indicates that binary mixtures could be promising repellant products that are novel and effective against B. germanica. However, these terpenoids are volatile, and the volatility problem may be alleviated by special formulations, such as microencapsulation, that can reduce volatile loss, as described by Peterson et al.31.It is well known that repellency against various insect species was more pronounced in binary mixtures of phytochemicals compared to the corresponding single compounds. For example, a significantly enhanced repellency was produced through binary mixtures of C. rotundus rhizome-derived products containing active compounds, especially zerumbone and 1,8-cineole, could be useful as repellents in the control of B. germanica populations in sensitive environments in which conventional insecticides would be inappropriate, as long as special formulations  that facilitate the slow release of active compounds can be selected or developed. Further research is needed for the practical applications of plant-derived preparations as novel cockroach repellent products to establish their safety profiles in humans. In addition, their effects on non-target organisms and the environment need to be established. Finally, detailed tests are needed to understand how to improve repellency potency and stability for eventual commercial development.In conclusion, 1H and 13C NMR spectra were recorded in CD3OD on an Avance 400 WB spectrometer  at 400 and 100 MHz, respectively, using tetramethylsilane as an internal standard. The chemical shifts are given in δ (ppm). The DEPT spectra were acquired using Bruker software. The mass spectra were obtained on a GCMS-QP 2010 Ultra gas chromatograph-mass spectrometer . Silica gel 60 (0.063–0.2 mm)  was used for column chromatography. Merck pre-coated silica gel plates (Kieselgel 60 F254) were used for analytical thin layer chromatography (TLC). A Spectra System P 2000 high-performance liquid chromatograph (HPLC)  was used to isolate the active compounds.The C. rotundus rhizome essential oil21 are listed in Table Two constituents, 1,8-cineole and ZER, were identified in this study, and another 11 previously identified constituents from the B. germanica (susceptible KSS strain)20 were maintained in the laboratory without exposure to any known insecticide. Cockroaches were reared in glass jars (43-cm diameter × 30 cm) containing Purina calf food pellets , distilled water and a cardboard shelter at 27 ± 2 °C and 60 ± 5% relative humidity (RH) under a 12:12 h light:dark cycle. Because male B. germanica are more sensitive than females to olfactory stimuli46, adult males were used for the repellency bioassays.The stock cultures of C. rotundus was purchased from the Boeun medicinal herb shop . A voucher specimen (CR–01) was deposited in the Research Institute of Agriculture and Life Sciences at Seoul National University.The rhizome of C. rotundus were pulverized, extracted with methanol (3 × 3 L) at room temperature for 2 days and filtered. The combined filtrate was concentrated by rotary evaporation at 40 °C to yield approximately 152 g of a dark greenish tar. The extract (100 g) was sequentially partitioned into hexane- (16.4 g), chloroform- (45.3 g), ethyl acetate- (10.7 g) and water-soluble (27.6 g) portions for the subsequent bioassays. The organic solvent-soluble portions were concentrated under vacuum at 40 °C, and the water-soluble portion was concentrated at 50 °C. To isolate the active constituents, 244.6 μg cm−2 of each C. rotundus rhizome-derived fraction were tested in a filter-paper choice bioassay, as described by Petterson et al.31.Air-dried rhizomes (600 g) of Rf values on the TLC plates were pooled. The spots were detected by spraying the plate with 10% sulfuric acid and then heating the samples on a hot plate. Active fractions 1–10 (H1) were obtained. Fraction H1 was re-chromatographed on a 5.5 × 70 cm silica gel (600 g) column by elution with a gradient of hexane and ethyl acetate  and a final elution with methanol (2 L) to provide 25 fractions (each approximately 200 mL). Fractions 1–5 (H11) was re-chromatographed on a silica gel column by elution with a gradient of hexane and ethyl acetate  and a final elution with methanol (2 L) to provide 25 fractions (each approximately 200 mL). Active fractions 1–5 (H111) were obtained. Fraction H111 was re-chromatographed on a silica gel column by elution with a gradient of hexane and ethyl acetate  and a final elution with methanol (2 L) to provide 25 fractions (each approximately 200 mL). A preparative HPLC was used to separate the constituents from active fractions 6–15 (H1112). The column was a 19 mm i.d. × 300 mm μPorasil  with a mobile phase of hexane and ethyl acetate (97:3 by volume) at a flow rate of 2.0 mL min–1. Chromatographic separation was monitored using a UV detector at 265 nm. Finally, two active constituents 1 (510 mg) and 2 (224 mg), were isolated at retention times of 10.4 and 13.3 min, respectively.The hexane-soluble fraction 15 g) was the most biologically active fraction . Each test was conducted between 20:00 to 23:00 h with overhead florescent lighting at 27 ± 2 °C and 60 ± 5% RH in 5-min test periods, as described previously31. A 12.5-cm diameter Whatman no. 2 filter paper  was cut in half. One side was treated with different amounts  of each test compound in 1 mL methanol, and the other side was treated with 1 mL methanol. Deet served as the positive control and was used in a similar manner. After drying the samples in a fume hood for 1 min, each treated paper was placed on the bottom section of a disposable Petri dish (15-cm diameter × 2 cm). The lid of the dish had a 1 cm hole cut in the center for introducing the cockroach directly into the center of the arena. One insect at a time was introduced. The hole was then blocked by using a small piece of tissue paper to prevent the cockroaches from escaping. Immediately after the introduction of the insect, the number of seconds it spent on the treated or untreated side within 300 s was timed with two stopwatches. If a compound produced ≥40% repellency at a given concentration, further bioassays were conducted. Filter papers and cockroaches were used once then discarded. Each trial was repeated 10 times.A filter-paper choice test described previously by Petterson −2 at the 24 h post-treatment, which was based on the preliminary test results. Deet served as the positive control. All treatments were replicated 10 times.To determine an effective mixture ratio for ZER and another seven active compounds, the repellency of binary mixtures at five tested ratios  was tested at a concentration of 81.5 μg cmB. germanica was tested in Ebeling choice boxes47 as described previously by Appel et al.41, with a slight modification. A choice test was carried out using a series of two connected acrylic boxes (each 5.5 × 11 × 11 cm). The top side of each compartment of the choice box had nine holes (0.5-mm diameter) on the surface to permit airflow. Except for the top side of the dark compartment of the choice box, the inner surface of other five walls was evenly painted with 652.4 μg cm−2 of the test compounds in 2 mL of methanol using a brush, and the amount was based on the preliminary test results. Food and water were placed on the bottom section of the dark compartment of the choice box. Deet served as a positive control and was similarly formulated. Negative controls  consisted of 2 mL methanol only. Treatments were allocated randomly to the choice boxes. After drying the samples in a fume hood for 3 min, 20 adult male B. germanica were released into the untreated compartment of the choice box and were allowed to enter the treated compartment for 24 h. Cockroaches were able to move freely between the dark (treated) and the lighted (untreated) compartments through two holes (1-cm diameter) in the partition separating the sides. Choice boxes had the same environmental conditions as those used for colony maintenance. Banks of white fluorescent lights were 1.6 m above the choice boxes and produced a light intensity in the untreated compartment of 300–350 lux using an INS Digital Lux Meter . The number of B. germanica in each compartment was recorded at every 15 min interval until 24 h post-treatment. Six replicates were used for each treatment in a completely randomized design.The residual repellency of the eight selected compounds to male B. germanica, a razor blade was used to remove the antennae at the scape, as described by Petterson et al.31. The cockroaches were allowed to recover from the procedure for 24 h before they were exposed to 652.4 μg cm−2 of each compound and deet in 2 mL methanol according to the choice box method above.For the tests that used antennectomized male Tu − Tt)/Tn] × 100, where Tu is the number of seconds a cockroach spent on the untreated side, Tt is the number of seconds a cockroach spent on the treated side, and Tn is the total number of seconds (300)31; % repellency for the Ebeling choice box assay = 100 − [(Ta/Tb) × 100], where Ta is the number of cockroaches in the treated group and Tb is the number of total cockroaches that were tested41. The percentages of repellency were transformed to arcsine square-root values for analysis of variance (ANOVA). The Bonferroni multiple-comparison method was used to test for significant differences among treatments48. A Student’s t-test was used to test for significant differences between the two treatment methods48. A compound with <10% repellency was considered to be ineffective. Means ± standard errors (SEs) of untransformed data are reported.The repellent index was calculated according to the following formula: % repellency for filter-paper choice assay = [(Supplementary Information


Pseudomonas aeruginosa bacteriophages belonging, respectively, to the Lit1virus genus of the Podoviridae family and the Pbunavirus genus of the Myoviridae family. Their genomes are 72,778 bp and 65,712 bp long, containing 94 and 93 predicted open reading frames, respectively.vB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A are  Pseudomonas aeruginosa, as the host strain, two virulent bacteriophages were isolated in Orsay, France, in the year 2014. Phage vB_PaeP_PcyII-10_P3P1 (P3P1) was found in compost and produced large clear plaques, whereas phage vB_PaeM_PcyII-10_PII10A (PII10A) was isolated from wastewater and formed small clear plaques. PII10A and P3P1 could be propagated on nine and 10, respectively, out of 27 tested clinical strains representative of the P. aeruginosa diversity 86. Heterogeneity in the number of Cs reduced the sequencing quality at this position. The PII10A genome was 65,712 bp long with a GC content of 55.5%. Automatic ORF detection and annotation were performed with RAST, and the obtained annotations were confronted to function predictions made using Phagonaute, a website allowing the display of distant protein homologies detected with HHsearch  under the accession numbers LT594787 and LT594786, respectively, within BioProject PRJEB14592, together with the raw sequence reads. The versions described in this paper are the second versions.The complete sequences of