More interestingly, the cells also demonstrated excellent stability as they showed negligible degradation of PCE when stored at 85˚C for 500 hours in the dark under nitrogen atmosphere. These results indicate that the newly developed polymers are promising materials for PSCs in the practical use.A critical issue in polymer-based solar cells (PSCs) is to improve the power conversion efficiency (PCE) as well as the stability. Here, we describe the development of new semiconducting polymers consisting of thiophene, thiazolothiazole and naphthobisthiadiazole in the polymer backbone. The polymers had good solubility and thus solution-processability, appropriate electronic structure with narrow band gaps of ~1.57 eV and low-lying HOMO energy levels of ~−5.40 eV, and highly ordered structure with the favorable face-on backbone orientation. Solar cells based on the polymers and PC Bulk heterojunction (BHJ) solar cells composed of semiconducting polymers as p-type and fullerene derivatives as n-type materials (PSCs) are of great interest as flexible and large-area renewable energy sources that can be produced by solution-processes1245679101112JSC). They also need to have a low-lying highest occupied molecular orbital (HOMO) energy level to maximize the open-circuit voltage (VOC) that is proportional to the energy difference between HOMO of the semiconducting polymer and the lowest unoccupied molecular orbital (LUMO) of the fullerene derivative15171819Important structural features desired for semiconducting polymers are well-ordered π–π stacking structure and “face-on” backbone orientation, where the backbone plane lies flat on a substrate, which facilitates the charge carrier transport along the film thicknessVOCs of ~0.9 V in solar cells, their relatively wide band gap of 1.8 eV, i.e., absorption range of up to 675 nm, limited JSCs to ~13 mA cm–2. Thus, for the further improvement of PCE the band gap of PTzBTs must be narrowed while maintaining or even lowering the HOMO energy level. In order to realize such electronic structure, a more electron-poor unit, i.e., a stronger A unit, should be introduced into the backbone.Recently, we have reported that a D-A copolymer system based on alkylthiophenes and thiazolothiazole (TzTz)212223242527In this work, we report on a D-A polymer system incorporating naphthobisthiadiazole (NTz)6142930311 (1a-1b) and the brominated NTz monomers with BO and the 2-hexyldecyl group (HD) on the thiophene rings as R2 (2a-2b) (Scheme S1). Thus, PTzNTz with four different side chain combinations were synthesized . The solubility was significantly different between PTzNTz-EHBO and others; whereas PTzNTz-EHBO was soluble in hot chlorobenzene (CB), or o-dichlorobenzene (DCB), PTzNTz-EHHD, −BOBO, and −BOHD was soluble even in chloroform (CF), CB and DCB below ca. 40 °C. Interestingly, the solubility of PTzNTzs was improved compared with PTzBTs despite the fact that NTz with a more π–extended ring than TzTz was introduced into the backbone. For example, in order to prepare a 5 g/L solution in CB, whereas PTzBT-BOHD required heating at around 100 °C, PTzNTz-BOHD, with the same side chain combination, did not require heating. The number-average molecular weight (Mn), evaluated by GPC at 140, of PTzNTz-EHBO, -EHHD, -BOBO and -BOHD were about 30–50 kDa with the polydispersity index (PDI) of around 2 (All polymers were synthesized via the Stille coupling reaction using the stannylated TzTz monomers with the 2-ethylhexyl group (EH) or the 2-butyloctyl group (BO) on the thiophene rings as Raround 2 . Thermalaround 2 . This imEHOMO) and the LUMO energy level (ELUMO) of the polymers were evaluated by cyclic voltammetry (EHOMO of −5.40–−5.41 eV and ELUMO of −3.44–−3.46 eV (EHOMOs of PTzNTzs (ca. −5.40 eV) were lower than those of PTzBTs . EHOMO was around −5.30 eV, which was lower by ca. 0.1 eV than that of PTzBTs, which is consistent with the results obtained from CV.The HOMO energy level (tammetry  using th−3.46 eV . As expeλmax) was ca. 680 nm (λedge) was determined to be ca. 790 nm, which corresponds to the optical band gap (Eg) of 1.57–1.58 eV  blend films on the indium tin oxide (ITO)/ZnO substrate are shown in q ≈ 1.65–1.70 Å−1) clearly appeared on the qz axis for PTzNTz-EHBO and -BOBO, indicating that they formed crystalline domains with a favorable face-on orientation. The π–π stacking distances (dπ) of these polymers were 3.69 and 3.72 Å, respectively , even though NTz is a more π–extended fused ring and thus is expected to enhance the π–π stacking. This can be explained by the difference in the placement of the alkyl groups. In PTzBTs, all the alkyl groups on the thiophene rings point toward TzTz. In PTzNTzs, on the other hand, whereas the alkyl groups on the thiophene rings neighboring TzTz point toward TzTz, those neighboring NTz point outward from NTz. The less regularly placed alkyl groups as such in PTzNTzs compared to the regularly placed alkyl groups in PTzBTs could somehow weaken the π–π interaction. The difference of crystallinity between PTzNTzs and PTzBTs apparently correlates to the difference of solubility. In addition, in particular, when the alkyl groups are longer than those of PTzNTz-EHBO, the interaction becomes weaker and in turn the solubility becomes significantly higher.The ordering structure of the polymers was investigated by the X-ray diffraction studiesectively . In the ectively , indicat71BM blend films fabricated from CB solutions  was used for the film fabrication as the solvent additive71BM, during which time the polymers crystallize as typically seen for many polymers71BM). The improved ordering structures are consistent with the solar cell performances as shown below.In the polymer/PColutions , only PT71BM blend films by the atomic force microscopy (AFM)  . In the py (AFM) , whereasnd films , on the x/Ag, were used to investigate the photovoltaic properties of the polymers. PC71BM was used as the n-type material, and the active layer was fabricated from the CB solution. The optimal polymer to PC71BM weight ratio was 1:1.5 for all polymers. The current density (J)-voltage (V) curves and the external quantum efficiency (EQE) spectra of the cells under 1 sun of simulated AM 1.5G solar irradiation (100 mW/cm2) are displayed in VOC of 0.84–0.85 V. Although EHOMO of PTzNTzs was lower than that of PTzBT by 0.1 eV, VOCs of the PTzNTzs cells were slightly decreased by 0.03–0.04 V. The origin of the energetic loss is yet unknown. The PTzNTz-EHBO cells gave relatively high JSC of ~16.0 mA cm–2 compared to that of the PTzBT cells (~13 mA cm–2)JSC of around 2–4 mA cm–2, which is consistent with the EQE spectra. It is found that the fill factor (FF) was higher for the cell using PTzNTz-EHBO (0.67) than those using the other polymers (<0.6). The higher JSC and FF of the PTzNTz-EHBO cell than the others agrees well with the results of the 2D GIXD and AFM studies, as the PTzNTz-EHBO blend film showed a higher crystalline structure and a better phase separated structure than the others when fabricated from CB solutions. As a result, PTzNTz-EHBO cells exhibited PCEs of up to 9.0% , and those of the other polymer cells were 1.2–2.1%.Solar cells with an inverted structure, ITO/ZnO/active layer/MoOJSC to ~16.3 and ~15.6 mA cm–2, respectively, which were similar to the value of the cells using PTzNTz-EHBO. The increase in JSC was consistent with the increase in EQE 37C and FF , althougx, was replaced with WOx, the thermal stability of the cells were greatly improved  were synthesized and characterized, and their photovoltaic characteristics were investigated. The solubility of PTzNTzs were greatly improved from that of PTzBTs. PTzNTzs had both narrow band gaps (ca. 1.57 eV) and low-lying HOMO levels (ca. −5.40 eV) compared to thiophene-thiazolothiazole polymers (PTzBTs) that have been reported before. PTzNTz-EHBO with the shortest side chain combination exhibited the best PCE of as high as 9.0% in the inverted solar cells among the polymers synthesized here. Although the cells based on PTzNTz-BOBO and -EHHD with the longer side chains exhibited very low PCEs of ~2%, the use of DIO at the time of fabrication as the solvent additive greatly improved their PCEs to ~8.8%. The difference of the solar cell behavior between these polymers with different side chain combination is likely attributed to the difference in crystallinity. Interestingly, the PTzNTzs cells demonstrated excellent stability as they showed negligible degradation after 500 hour-storage at 85 °C under Nd]thiazole (1a)d]thiazole (1b)c:5,6-c´]bisthiadiazole (2a)3)4 (PPh3 = triphenylphosphine) was purchased from Tokyo Chemical Industry, Co., Ltd. (TCI), and used as purchased. Toluene was purified by a Glass Contour Solvent System prior to use. Polymerization was carried out with a microwave reactor, Biotage Initiator. Molecular weight of the polymers was evaluated by a high-temperature GPC (140 °C), TOSOH HLC-8121GPC/HT, using DCB as the eluent and polystyrene standard.2,5-Bis(3-(2-ethylhexyl-5-(trimethylstannyl)thiophen-2-yl)thiazolo[5,4-3)4 , and CB (5 mL) were added. Then the tube was purged with argon and sealed. The reaction tube was set into a microwave reactor and heated to 180 °C for 40 min. After cooling to room temperature, the reaction solution was poured into 200 mL of methanol containing 5 mL of hydrochloric acid, and stirred for 5 hours. Then the precipitated solid was subjected to the sequential Soxhlet extraction with methanol and hexane, to remove low molecular weight fractions. The residue was then extracted with chloroform for PTzNTz-EHHD, - BOBO, and - BOHD, and CB for PTzNTz-EHBO, and reprecipitated in 200 mL of methanol to yield dark green solid . Anal. Calcd for C70H92N6S8 (PTzNTz-EHBO): C, 65.99; H, 7.28; N, 6.60. Found: C, 65.65; H, 7.15; N, 6.31. Calcd for C78H108N6S8 (-EHHD): C, 67.58; H, 7.85; N, 6.06. Found: C, 67.64; H, 7.80; N, 5.93. Calcd for C78H108N6S8 (-BOBO): C, 67.58; H, 7.85; N, 6.06. Found: C, 67.62; H, 7.74; N, 5.98. Calcd for C86H124N6S8 (-BOHD): C, 68.93; H, 8.34; N, 5.61. Found: C, 68.55; H, 8.20; N, 5.45.To a reaction tube equipped with a stirring bar, the stannylated TzTz monomer (0.10 mmol), the dibrominated NTz monomer (0.10 mmol), Pd connected to the glove box, and the MoOx layer (7.5 nm) or WOx layer (15 nm) was deposited, followed by the deposition of the Ag layer (100 nm). The active area of the cells was 0.16 cm2. J–V characteristics of the cells were measured using a Keithley 2400 source-measure unit in nitrogen atmosphere under 1 sun (AM1.5 G) conditions using a solar simulator . The light intensity was calibrated with a reference PV cell . EQE spectra were measured with a Spectral Response Measuring System . More than 10 different devices were made and measured to collect the photovoltaic properties. For thermal stability tests, cells were stored on a hotplate at 85 °C in the glovebox. J–V characteristics were measured after 1, 2, 6, 24, 100, 200 and 500 hours.Patterned ITO substrates (purchased from Atsugi Micro) were first pre-cleaned sequentially by sonicating in a detergent bath, de-ionized water, acetone, and isopropanol at rt, and in boiled isopropanol each for 10 min, and then baked at 120 °C for 10 min in air. The substrates were then subjected to a UV/ozone treatment at rt for 20 min. ZnO layer was prepared by spin-coating  a precursor solution prepared from zinc acetate dehydrate (0.5 g) and ethanolamine (0.14 mL) in 5 mL of 2-methoxyethanol. ZnO substrates were immediately baked at 200 °C for 30 min in air, and then rinsed with acetone, isopropanol and boiled isopropanol for 10 min. The photoactive layer was deposited in a glove box  by spin coating. CB solution containing 5~7 g/L of the polymer sample with respective amount of PCHow to cite this article: Saito, M. et al. Highly Efficient and Stable Solar Cells Based on Thiazolothiazole and Naphthobisthiadiazole Copolymers. Sci. Rep.5, 14202; doi: 10.1038/srep14202 (2015).


Leishmania “siamensis” and to L. martiniquensis explaining a recent interest for the sand fly fauna where Sergentomyia gemmea and Se. barraudi have been considered possible vectors in the country.Since 1996, there are emerging autochthonous cases of leishmaniasis in Thailand due to Field studies were undertaken in a cave of Phitsanulok Province, Thailand. Phlebotomine sandflies have been studied morphologically and some have been processed for molecular biology (sequencing of cytB rDNA).Sergentomyia: Se. phadangensis n. sp., is described. The association of the male and female is supported by the homology of the sequences of cytochrome b rDNA.A new species of sand fly, belonging to the genus The description of a new species in Thailand is of importance in view of the existence of autochthonous leishmaniases. Leishmania “siamensis” and, surprisingly, to L. martiniquensis [Chinius, three Idiophlebotomus, eight Phlebotomus, one Grassomyia and 13 Sergentomyia [Nemapalpus vietnamensis not considered to be a sand fly [As it is an area which has long been considered to be free of leishmaniasis, there are few data related to Phlebotomine sand flies in South-Eastern Asia. However, the first autochthonous South-Eastern Asian case of leishmaniasis was discovered in Thailand in 1996, followed by a total of 13 cases due to iquensis . This nesand fly  and someDuring field work carried out over one year in the Phadang limestone cave, the most abundant species caught - new to Science - is described in the present paper.Field studies were undertaken between February 2010 and January 2011 in Phadang cave in Noen Maprang, Phitsanulok Province, located in the lower northern part of Thailand at 16°30'58.8"N, 100°40'00.5"E. This limestone cave, situated in a mountainous area, is found in the foothills of a mountain and consists of two asymmetrical caverns. It is estimated to be 15–80 m wide, 80–100 m long, and 1–20 m high, and lies at 108 meters a.s.l. The cave may be wet or dry depending on the season and the amount of sunlight it receives. It is accessible to travelers, but is home to many bats.in toto in Hoyer’s medium and others for the application of molecular biology techniques. Regarding the latter: head, thorax and genitalia were cut off in a drop of ethanol, cleared in boiling Marc-André solution and, after dehydration, mounted on slides in Canada Balsam. The anterior part of the abdomen of each specimen was dried and stored in a vial at −20 °C, before DNA extraction.Phlebotomine sand flies were collected using CDC miniature light traps usually installed overnight from 6 p.m. to 6 a.m. Specimens collected were stored in 96 % ethanol. Some of them were mounted camera lucida. Morphological terminology is that commonly used for Old World sandflies [The specimens were observed under a BX53 microscope equipped with a video camera and measured using Stream motion software . The usual keys for the identification of the sand flies from the indo-chinese region have been consulted –10. Drawandflies , 10–13 aandflies . Abbreviandflies . All meaWe decided to sequence a fragment of cytochrome b (Cyt b) which is the most used molecular marker for Phlebotomine sand fly systematic studies . Genomic2, 50 mM KCl, 0.01 % Triton X 100, 200 μM dNTP each base, and 1.25 units of 5 prime Taq polymerase . The cycle begins with an initial denaturation step at 94 °C for 3 min and finishes with a final extension at 68 °C for 10 min. PCRs were done with the following temperature profile: 5 cycles with 30 sec 94 °C, 40 sec 40 °C, 1 min 68 °C and 35 cycles with 30 sec 94 °C, 30 sec 44 °C, 1 min 68 °C.All the mtDNA and rDNA amplifications were performed in a 50 μl volume using 5 μl of extracted DNA solution and 50 pmol of each of the primers N1N-PDR and C3B-PDR . The PCRAmplicons were analyzed by electrophoresis in 1.5 % agarose gel containing ethidium bromide. Direct sequencing in both directions was performed using the primers used for DNA amplification.The correction of sequences was done using Pregap and Gap softwares included in the Staden Package . ConsensThe sequences analysed in the present study have been deposited in Genbank under numbers KT266691 to KT266700. All of them are similar (100 % homology).Sergentomyia França & Parrot, 1920.Genus Sergentomyia phadangensis n. sp. Polseela, Depaquit & Apiwathnasorn.Species The holotype (labelled THA4), and 15 paratypes specimens labelled P1, P2, P3, P4, P5, THA6, THA8, THA9, THA11, THA12, THA13, THA14, THA15, THA16 and THA19 have been examined. The labels refer to the lab where the specimens have been mounted .*HeadInterocular suture complete.Pharynx narrow. Discrete armature including some little teeth oriented backward.Cibarium with a very important curved armature consisting of 6 to 8 strong and pointed teeth on each side and a few much smaller median and central ones. All these teeth are darkly pigmented in brown. Sometimes some accessory teeth in the upper and lateral parts of the cibarium. Pigment patch: wide, less pigmented than the teeth, triangular with rounded angles.Palpal formula: 1, 2, , 5. About forty Newstead's scales club-like in a patch on the basal face of the third segment.Antennal formula: 2/III-XV. Relatively long ascoids, withour spur, not reaching the next articulation. AIII shorter than AIV + AV.AIII shorter than the labrum.Labial furca closed.*ThoraxNo setae on the mesanepisternum .No pigmentation of mesonotum, pronotum, paratergite, anepisternum, metanotum, posnotum and pleura.*Spermathecae: smooth and wide. No limit between the body and individual duct. Presence of a common basal duct.Furca with a long and thin anterior part.Nine paratypes labelled P27, THA31, THA32, THA34, THA36, THA37, THA38, THA39 and THA40 have been examined. The labels refer to the lab where the specimens have been mounted *HeadInter-ocular suture complete.Cibarium with an important curved armature consisting of three to five large pointed teeth on each side and twelve to twenty denticles along the curve and sometimes outside this line. The latter are observable using the phase contrast option of the microscope.No trace of a pigmented area could be seen.Pharynx quite narrow, with a discrete armature composed of small teeth oriented backward.Palpal formula: 1, 2, , 5. A few Newstead's scales on the third palpal segment.Antennal formula: 1/III-XV. AIII shorter than AIV + AV.Labial furca closed.*ThoraxNo setae on the mesanepisternum .No pigmentation of mesonotum, pronotum, paratergite, anepisternum, metanotum, posnotum and pleura.*Genital ArmatureCoxite with a few internal setae not grouped in a tuft.Style narrow with four terminal spines. The accessory spine is implanted distally .Single paramere, curved, with a rounded top.Surstyle (=epandrium) shorter than the coxite.Aedeagus straight, finger-like.Type-locality: Phadang cave located in the lower northern part of Thailand at 16°30'58.8"N, 100°40'00.5"E, 108 meters above sea level.The holotype  and 10 paratypes  have been deposited in the National Science Museum of Pathumthani (Thailand). Fourteen paratypes  have been deposited in the Museum national d’Histoire naturelle of Paris (France).Sergentomyia phadangensis n. sp. is related to the Phadang cave where it has been caught.Etymology: the name Sergentomyia phadangensis is: urn:lsid:zoobank.org:act:425142C7-380D-46B0-8777-33469075C4DC.In accordance with section 8.5 of the International Code of Zoological Nomenclature, details of the new species have been submitted to ZooBank. The life science identifier (LSID) related to record is urn:lsid:zoobank.org:pub:8920F0C8-79BD-47AA-B367-E052C38023F9. The LSID for the new name Sergentomyia were little studied as they were not regarded as vectors of any human leishmaniasis agent. However, some of them have recently been presented as possible vectors of Leishmania responsible for human infection [For a long time, the species of the genus nfection , in Thainfection .Sergentomyia is the genus with the greatest known diversity, surpassing the number of species of all other Old World genera  taken together [Sergentomyia share the following characters: a mesanepisternum without setae, abdominal tergites 2–6 usually carrying all or most recumbent hairs, a usual 1/III–XV antennal formula in males and 2/III–XV in females with some exceptions, a cibarium with an armature of teeth and/or denticles more developed in females than in males (apart from exceptions), a single paramere, a style with four terminal spines  and an accessory spine.together , 24. TheSe. phadangensis n. sp. exhibits the characters qualifying it for inclusion in the genus Sergentomyia.The species Sergentomyia is subdivided into eight subgenera: Sergentomyia França & Parrot, 1920; Neophlebotomus França & Parrot, 1920; Sintonius Nitzulescu, 1931; Parrotomyia Theodor, 1948; Rondanomyia Theodor, 1948; Capensomyia Davidson; Vattieromyia, Depaquit, Léger & Robert, 2008, and Trouilletomyia Depaquit, Léger & Randrianambinintsoa, 2014. Moreover, there are a lot of unclassified species within the genus Sergentomyia [Mainly based on the spermathecal morphology, the genus entomyia .Se. phadangensis n. sp. exhibits wide and smooth spermathecae and can therefore be included in the subgenus Sergentomyia.Se. dentata (Figs. Se. dentata is wide with a Se. antennata-like proeminent armature whereas that of Se. phadangensis n. sp. is narrow with a Se. schwetzi-like discret armature (Fig. Se. dentata in Thailand [Se. dentata is mainly distributed in the Middle-East and the Eastern Mediterranean basin [This new species can be easily differentiated from all the other species recorded in South-Eastern Asia by its highly developed cibarial armature in both females and males. The teeth are bigger and darker than those of the most closely related species, ta Figs.  and 5. Mure Fig. . The recThailand  should ban basin .Fig. 4CiL. “siamensis” and L. martiniquensis. The local transmission of these parasites is not explained, despite the possible role of two Sergentomyia species: Se. gemmea and Se. barraudi.The description of a new species in Thailand is of importance taking into account the existence of autochthonous leishmaniases caused by


Some errors have been noted in the above-mentioned paper subsequent to publication and so the following corrections should be highlighted.Treatment Patterns, the sentence “Overall, the mean duration of treatment was 152.6 days, and there were no major differences between the two treatment groups, including the duration of add-on HCTZ treatment” should read “Overall, the mean duration of treatment was 151.2 days, and there were no major differences between the two treatment groups, including the duration of add-on HCTZ treatment.” It should be clarified that 152.6 days was the mean duration of the double-blind treatment phase.On page 1091 under the heading Fig. 3, the p value for the FAS group should read p = 0.0005, and not p < 0.0001.On page 1092 in Safety/Tolerability, the sentence “The most common adverse event was peripheral edema, which was reported in 17.8% of OLM/AML patients and 18.1% of PER/AML recipients” should read “The most common adverse event was peripheral edema, which was reported in 18.4% of OLM/AML patients and 17.8% of PER/AML recipients.”On page 1095 under the heading Table 2, the values in the row showing the frequencies of peripheral edema which are currently: 43 (17.8) in the OLM/AML column, 88 (18.1) in the PER/AML column, and 45 (18.4) in the total column; should read: 45 (18.4), 43 (17.8), and 88 (18.1), respectively.On page 1095 in


High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g−1 lignin at pH 8 and 55°C for 24 hours, about 642.83 ± 14.45 ppm hydroxybenzoic acid, 70.19 ± 3.31 ppm syringaldehyde, 22.80 ± 1.04 ppm vanillin, 27.06 ± 1.20 ppm p-coumaric acid, and 50.19 ± 2.23 ppm ferulic acid were produced. In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration  and reaction time  on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield (333.17 ± 49.44 ppm hydroxybenzoic acid, 5.67 ± 0.25 ppm Palm oil production is expanding to fulfill the growing worldwide needs for multiple uses of this commodity. However, the rapid growth of oil palm industry may have a negative impact on the environmental and social aspects. Indeed, sustainability of the supply chain of this commodity is a global challenge . NonethePretreatment technology is a crucial step in lignocellulose biorefinery. A variety of pretreatment methods has been proposed with the aim to alter or remove the structural and compositional impediments to the hydrolysis of cellulose/hemicellulose. Delignification during pretreatment has been proven to improve enzymatic digestibility of biomass. However, pretreatment usually yields large quantity of black liquor that needs to be discharged at the end of the process , 6. Dire p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). Future use of lignin depends on the possibilities of either degrading it into low molecular weight aromatics or as a multifunctional macromolecule. Lignin conversion into diverse value-added products played an important role in the economic viability of biorefinery. The methodologies of lignin conversion can be divided into three groups: (1) hydrothermal, (2) chemical, and (3) enzymatic [β-ether linkage of lignin was also cleaved rapidly, leads to monolignols and further its methyl ester formation [β-aryl ether bonds. Until now, none of the chemical methods can be used commercially for the production of value-added compounds due to environmental concerns [Lignin is the most abundant amorphous aromatic polymer in lignocellulose. It is comprised of three main phenylpropane units, namely,nzymatic , 11. Hydormation . The majormation . For checoncerns . In spitconcerns . Therefo p-hydroxybenzaldehyde, and syringaldehyde) and hydroxycinnamic/phenolic acids  through either chemical or enzymatic methods. All these aromatic compounds possess significant uses in food and pharmaceutical and nutraceutical applications [The types of product which are generated through lignin degradation/oxidation strongly depend on the applied methodologies and parameters . Study –17 reporications –21.Therefore, the main objective of this study was to effectively convert lignin fragment present in the black liquor after alkaline pretreatment of empty fruit bunch fiber (OPEFBF) into a series of novel monomeric aromatic compounds. The NaOH-lignin was initially recovered and then subjected to chemical (nitrobenzene and oxygen) and enzymatic (cutinase and MnP) oxidation/degradation. The effects of sodium hydroxide's concentration and time on the yield of monomeric aromatic compounds from chemical oxidation of lignin were investigated. Besides, reaction condition of cutinase in hydrolyzing NaOH-lignin was optimized for pH, temperature, lignin concentration, enzyme concentration, and time. The performance of each method was evaluated based on the types and yields of the monomeric aromatic compounds produced.The oil palm empty fruit bunch fiber (OPEFBF) provided by Seri Ulu Langat Sdn. Bhd., Dengkil, Selangor, Malaysia, was used as the substrate for this study. The fresh OPEFBF was washed with tap water and sun-dried until the moisture content reached approximately 10-11%. The dried OPEFBF was cut into the size of 5–8 cm and kept in sealed plastic bag at ~4°C until further usage. p-nitrophenyl butyrate (PNPB), hydrogen peroxide, manganese sulphate, disodium succinate, disodium malate, 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) diammonium salt , dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, and sodium chloride . Phenolic compounds used as a reference standard were hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic acid, vanillin, syringic acid, syringaldehyde, p-coumaric acid, and ferulic acid . All chemicals used were of analytical grade.Chemicals used included sodium hydroxide (NaOH), nitrobenzene, chloroform, ethyl acetate, HPLC grade methanol, dimethyl sulfoxide (DMSO),Fusarium solani pisi) and manganese peroxidase (MnP) (Phanerochaete chrysosporium) were determined according to the modified protocol proposed by [µL 50 mM PNPB solution and 100 µL Novozym 51032, in a final volume of 1 mL of 0.1 M sodium phosphate containing 0.9% sodium chloride at pH 7.2, was prepared. The enzyme activity of Novozym 51032 was determined by monitoring the increase of absorbance at 400 nm, with no-enzyme mixture as a blank. For MnP activity determination, 20 µL MnP was added to mixture containing 1 mM manganese sulphate and 72 µM ABTS diammonium salt in 1 mL of 50 mM succinate-10 Mm malate buffer at pH 4.5. About 2.5 µL of 20 mM hydrogen peroxide was added to the mixture to initiate the reaction. The activity of MnP was determined by monitoring the increased absorbance at 415 nm as a function of time. The protein concentration of both enzymes was determined through Bradford method by using Bio-Rad Quick Start Bradford assay kit . Bovine serum albumin (BSA) was used as the reference standard. About 1 mL of Bio-Rad 1x reagent was mixed with 20 µL of BSA at a series of concentrations/enzyme solutions. The absorbance of the mixtures was read at wavelength 595 nm by using VersaMax ELISA microplate reader and analysed with SoftMax Pro software . Specific activity of Novozym 51032 and MnP was found at about 673.1 U mg−1 and 31.3 U g−1, respectively.Enzymes used in this study were Novozym 51032  and manganese peroxidase . The activities of Novozym 51032 . The recovered lignin fragment was washed with distilled water and oven-dried at 105°C overnight. The dried lignin fragment was crushed and ground into fine powder using mortar and pestle [OPEFBF delignification was conducted by soaking the OPEFBF in 50 g Ld pestle , 25. TheNitrobenzene oxidation of lignin was conducted with 2% wt. lignin in 10 mL of 2, 5, and 10% wt. NaOH solutions. About 0.5 mL nitrobenzene was added to the lignin solution, while mixture without nitrobenzene was fixed as experimental control. The oxidation reaction was carried out in an autoclave at 130°C for 30, 90, and 180 minutes. After oxidation, 30 mL chloroform was added to the mixture to remove excess nitrobenzene and its reduction products. Chloroform layer was then discharged by centrifugation at 3000 rpm for 3 minutes. The oxidation products extraction was initiated by adding concentrated sulphuric acid to the reaction mixture (lower layer) to achieve pH 2. The extraction was conducted thrice with an equal volume of ethyl acetate. The ethyl acetate was then evaporated in a rotary evaporator. The dried oxidation products were resolubilized in methanol and subjected to HPLC analysis .For direct oxygen oxidation, 2% wt. lignin was dissolved in 10 mL 2, 5, and 10% wt. NaOH solutions separately. The reaction was conducted in a 100 mL 2-neck round-bottom flask. A condenser was connected to the flask and oxygen gas was bubbled into the lignin solution continuously throughout the entire reaction period. The reaction mixture was heated to 100°C and maintained for 30, 90, and 180 minutes. Oxygen oxidation was carried out under atmospheric pressure . Experim−1 lignin at 55°C with 150 rpm agitation for 24 hours [−1 lignin), time , and temperature  on the efficiency of enzymatic catalysis were carried out.Due to the insolubility of lignin in aqueous buffer solution, lignin dissolution was conducted prior to the enzymatic hydrolysis. Initially, 0.2 g lignin was dissolved in 1 mL DMSO. To study the effect of pH on the enzyme catalytic activity, about 10 mL of 25 mM phosphate buffer at pH 7, 8, 10, and 12 was added to the solution. Lignin hydrolysis by Novozym 51032 was carried out with 2.5 mL cutinase g24 hours . Once th−1 lignin in 50 mM succinate-10 mM malate buffer pH 4.5 was prepared. The enzymatic oxidation reaction was conducted at 30°C with 200 rpm agitation for 48 hours. The reaction was initiated by the addition of 20 mM hydrogen peroxide. Once the oxidation was completed, enzyme inactivation was carried out by heating the reaction mixture to 80°C for 5 minutes. The lignin fragment was then separated from the liquor by centrifugation at 3000 rpm for 3 minutes. The liquor and the recovered lignin were subjected to HPLC and FTIR analysis, respectively.Enzymatic oxidation of lignin by MnP was conducted according to the modified procedure suggested in . A reactµm particle size was used, and the sample was eluted using 0.1% acetic acid (solvent A) and methanol (solvent B). The mobile phase gradient was fixed at 80 : 20 (A : B) from 0 to 24 min, 60 : 40 (A : B) from 24 to 27 min, 20 : 80 (A : B) from 27 to 36 min, and 80 : 20 (A : B) from 36 to 40 min. The mobile phase was filtered through a 47 mm nylon membrane with a pore size of 0.45 µm and degassed. The compounds separation was conducted at a constant flow rate of 1 mL min and 10 µL injection volume. Aromatic compounds identification was carried out by using reference standards comprising of hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic acid, vanillin, syringic acid, syringaldehyde, p-coumaric acid, and ferulic acid. Caffeine was used as an internal standard for quantification. All of the standards and samples were filtered through a 0.22 µm nylon membrane prior to analysis. Data acquisition was performed with Breeze software  [The monomeric aromatic compounds identification and quantification were performed by HPLC equipped with an ultraviolet detector at wavelength 280 nm . A 250 × 4.6 mm HPLC column Gemini C18  with 5  p-hydroxybenzaldehyde, vanillic acid, vanillin, syringic acid, syringaldehyde, p-coumaric acid, and ferulic acid. These eight compounds also have been identified in the study of [ p-hydroxybenzaldehyde, vanillic acid, vanillin, syringic acid, and syringaldehyde. Besides, the results of  p-coumaric acid and ferulic acid, were increased, followed by the increased time. The yields of p-coumaric acid and ferulic acid were found to be decreased when oxidation reaction prolonged. In spite of that, the increase of NaOH concentration also was found to be increase the yield of all oxidation products. However, in the absence of nitrobenzene, the effect of NaOH concentration on the yield of oxidation products was not apparent. Based on the results obtained, the highest yield of all oxidation products was attained at 10% wt. NaOH solution but at different optimum time. The yields of hydroxybenzoic acid (333.17 ± 49.44 ppm with the recovery of 16.66 ± 2.47 mg g−1 lignin), p-coumaric acid (15.21 ± 2.19 ppm with the recovery of 0.76 ± 0.11 mg g−1 lignin), and ferulic acid (44.75 ± 3.40 ppm with the recovery of 2.24 ± 0.17 mg g−1 lignin) were achieved maximum after 30 minutes of reaction, while the maximum yield of p-hydroxybenzaldehyde (5.67 ± 0.25 ppm with the recovery of 0.28 ± 0.01 mg g−1 lignin), vanillic acid (25.57 ± 1.64 ppm with the recovery of 1.28 ± 0.08 mg g−1 lignin), and syringaldehyde (815.26 ± 41.77 ppm with the recovery of 40.76 ± 2.09 mg g−1 lignin) was achieved at 120 minutes reaction. The longest reaction time (180 min) needed to achieve the maximum yield was of syringic acid (75.44 ± 6.71 ppm with the recovery of 3.77 ± 0.34 mg g−1 lignin) and vanillin (168.68 ± 23.23 ppm with the recovery of 8.43 ± 1.16 mg g−1 lignin). No further yield improvement was observed on hydroxybenzoic acid, when the oxidation time was extended. Nevertheless, the extended oxidation reaction up to 180 min caused the yield of p-coumaric acid and ferulic acid diminished. However, the yields of p-hydroxybenzaldehyde, vanillic acid, and syringaldehyde were found to be constant, and even the oxidation had been extended to 180 min.study of , 30 afte p-hydroxybenzaldehyde, vanillin, and syringaldehyde were the oxidized products of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomer unit of lignin, respectively [ p-hydroxyphenyl units. Therefore, the highest yield of syringaldehyde was originated from the oxidation of the majority syringyl units of OPEFBF lignin. However, hydroxybenzoic acid was found to be the second abundant product, rather than the expected vanillin. The work in [ p-hydroxybenzoic acid, while the conversion of p-coumaric acid into p-hydroxybenzaldehyde and hydroxybenzoic acid was also suggested by [α-Cβ bond to form benzaldehyde and its derivatives. Therefore, in the medium of strong alkalinity and the presence of nitrobenzene as oxidant, free p-coumaric acid tends to convert into p-hydroxybenzaldehyde and then hydroxybenzoic acid [ p-hydroxybenzaldehyde and hydroxybenzoic acid along with the p-coumaric acid reduction during the oxidation reaction.Among these compounds,ectively . Accordiectively . The worectively , 31 repo work in  revealedested by . Therefooic acid . This al p-hydroxybenzaldehyde and vanillin was mainly due to the conversion of p-coumaric acid and ferulic acid, respectively. Nonetheless, drastic increment of vanillin yield also could be due to the oxidative cleavage of guaiacyl units in the lignin network. In addition, alkalinity also had been proven to play a significant role during nitrobenzene oxidation. Alkaline nitrobenzene oxidation induced alkaline hydrolysis of interunit ether bonds to depolymerize and solubilize lignin. Once the aldehyde groups are formed, base-catalyzed hydrolysis of ether bonds can occur at higher rate, particularly at high NaOH concentration [α-Cβ bond of the phenylpropane units. High concentration of hydroxide ion in the presence of oxidant has been proven to accelerate the rate of retroaldol cleavage. Mechanism of nitrobenzene oxidation of lignin was proposed by [Under alkaline condition, ester linkages joining phenolic acid are easily cleaved, even at room temperature. However, phenolic benzyl aryl ethers only cleaved at high temperature and with the presence of oxidants . Therefontration . The ratposed by , 34. p-coumaric acid and ferulic acid into their corresponding aldehydes and then its carboxylic acids by Cannizzaro reaction. Therefore, extended oxidation time was associated with the increase of p-hydroxybenzaldehyde, vanillin, hydroxybenzoic acid, and vanillic acid yields. This finding was in accordance with the results shown by the study of [According to , prolongstudy of , 36. Besstudy of . Thus, aDifferent optimum time for optimum yield of different oxidation products indicated that oxidation time has direct influence on the products formed during nitrobenzene oxidation. Different molecular structures of different molecules give rise to different susceptibilities to oxidative degradation during nitrobenzene oxidation in the formation of aromatic compounds . The rea p-hydroxybenzaldehyde, p-coumaric acid, and ferulic acid in the reaction medium of 10% wt. NaOH were not significant (p > 0.05), neither was the presence of oxygen. Therefore, such finding suggested that the effect of oxygen in oxidation is more significant at low NaOH concentration. In general, based on the results obtained, low yield of oxidation products was found in reaction with high NaOH concentrations. On the other hand, the yields of oxidation products had improved with the extension of reaction time, except for hydroxybenzoic acid, p-coumaric acid, and ferulic acid. p-hydroxybenzaldehyde (5.06 ± 0.05 ppm with the recovery of 0.25 ± 0.01 mg g−1 lignin), vanillic acid (25.17 ± 1.16 ppm with the recovery of 1.26 ± 0.06 mg g−1 lignin), vanillin (25.94 ± 2.25 ppm with the recovery of 1.30 ± 0.11 mg g−1 lignin), and syringic acid (41.52 ± 0.64 ppm with the recovery of 2.08 ± 0.03 mg g−1 lignin). However, only the shortest reaction time (30 min) was required for the optimum production of p-coumaric acid (21.53 ± 1.10 ppm with the recovery of 1.08 ± 0.06 mg g−1 lignin) and ferulic acid (40.33 ± 2.59 ppm with the recovery of 2.02 ± 0.13 mg g−1 lignin) at 2% wt. NaOH, while hydroxybenzoic acid (328.02 ± 39.55 ppm with the recovery of 16.40 ± 1.98 mg g−1 lignin) and syringaldehyde (78.43 ± 0.88 ppm with the recovery of 3.92 ± 0.04 mg g−1 lignin) at 5% wt. NaOH. The yields of these oxidation products were either reduced or kept constant, if the oxidation time was extended up to 180 min. This finding suggested the interactive effect of NaOH concentration and reaction time in getting the optimum yield of each compound. Different optimum reaction conditions were identified for the production of distinct compound. Study [For maximum recoveries of all potential aromatic compounds, direct oxygen oxidation was conducted at 2% wt. NaOH for 180 minutes to produce maximum yield ofd. Study  proposedThe highest yield of hydroxybenzoic acid after oxygen oxidation was believed to be due to the release of esterified hydroxybenzoic acid from the lignin network. Under mild oxidation environment, as designed in this experiment, the cleavages of ester and some ether bonds in the lignin network became predominant. The rate of oxidative cleavage of lignin phenylpropane units was minimal under mild reaction condition. Therefore, syringaldehyde and vanillin, which are the common aromatic aldehydes of lignin oxidation, did not appear as the major products in this experiment , 26.−1 by the increase of NaOH concentration from 80 to 120 g L−1 at 160°C. Therefore, oxidation at lower temperature might further reduce the rate of oxygen absorption in the reaction at high NaOH concentration. Low oxygen absorption in the medium will reduce the rate of initiation and thus the yields of oxidation products. Nevertheless, verification on this hypothesis is needed in further study.In addition, discovery of high rate oxygen oxidation at low NaOH concentration in this study has been found to be contradicting with the results from the previous study. According to , the eff p-coumaric acid and ferulic acid at prolonged reaction time probably was the main cause for the decrease of these compounds [ p-coumaric acid and ferulic acid in the formation of aldehydes and then carboxylic acid [Furthermore, subsequent oxidation ofompounds , 17. In lic acid  demonstr p-coumaric acid, and ferulic acid. The highest yield was achieved by hydroxybenzoic acid (642.83 ± 14.45 ppm), followed by syringaldehyde (70.19 ± 3.31 ppm) and ferulic acid (50.19 ± 2.23 ppm) and then p-coumaric acid (27.06 ± 1.20 ppm) and eventually vanillin (22.80 ± 1.04 ppm). These optimum yields were achieved by hydrolyzed lignin with cutinase at pH 8 and 55°C for 24 hours with 8% wt. lignin and 0.625 mL cutinase g−1 lignin. Under these reaction conditions, about 8.04 ± 0.18 mg hydroxybenzoic acid, 0.88 ± 0.04 mg syringaldehyde, 0.63 ± 0.02 mg ferulic acid, 0.34 ± 0.02 mg p-coumaric acid, and 0.29 ± 0.01 mg vanillin have been successfully recovered from each gram of lignin through this catalysis.Cutinase is a serine esterase (cutin hydrolase), particularly acting on the carboxylic ester bonds. Due to the fact that its active site cleft is partly covered by 2 disulfide bridges formed by amino acid chains (hydrophilic nature) and not buried under surface loop (hydrophobic nature), interfacial activation is unnecessary for its catalysis, as it is easily accessible to solvent and substrate. Moreover, oxyanion hole in cutinase structure also allow catalytic activities towards a broad variety of esters, from soluble to insoluble types . Until n p-coumaric acid, and ferulic acid were esterified components in the lignin network. Thus, the finding suggested that the cleavage of ester bonds within the lignin network by cutinase was the leading factor in the production of these compounds. Because cutinase is not an oxidative enzyme, syringaldehyde might emerge from syringyl alcohol units, which was released after lignin network deconstruction by the action of cutinase. Low yield of vanillin was most likely formed by the oxidation of the released free ferulic acid. Slow rate oxidation may happen under atmospheric oxygen condition. Nonetheless, further study on the mechanism of cutinase toward lignin is necessary for clear and definite understanding in this catalysis.It was known that hydroxybenzoic acid, p-coumaric acid (7.72 ± 0.20 ppm), and ferulic acid (14.06 ± 0.13 ppm) were produced. Besides, the results also clearly indicated that the products yield and recovery increased with the increase of pH to an optimum. After that, increase in pH will be accompanied by decrease in yield. Increase of pH from 7 to 10 had improved the products yield and recovery significantly (p < 0.05). Increase to pH 12 had caused adverse effects on both yield and recovery. This result was in accordance with the study of [γ-position of the propane chain. Eventually, vanillin is formed as a result of oxidation reaction in the γ-position, followed by retroaldol reaction of substituted coniferyl aldehyde [Based on the results shown in study of . Cutinasstudy of  indicatealdehyde . In addialdehyde . p-coumaric acid, and 56.95 ± 2.52 ppm of ferulic acid were successfully produced at this condition. However, high substrate content was associated with lower compounds recovery. The products recoveries were found to be reduced, while the substrate content increased from 2% wt. to 10% wt. According to [In addition, the results in rding to , the ratrding to  proposedrding to . Ultimat−1 lignin, the yields of hydroxybenzoic acid, syringaldehyde, p-coumaric acid, and ferulic acid were diminished from 642.83 ± 14.45 ppm, 70.19 ± 3.31 ppm, 27.06 ± 1.20 ppm, and 50.19 ± 1.23 ppm to 542.94 ± 27.88 ppm, 54.94 ± 4.67 ppm, 19.69 ± 0.25 ppm, and 46.93 ± 2.81 ppm, respectively. However, vanillin yield was not significantly affected by the increase of enzyme concentration. Vanillin yields from hydrolysis with 0.625 and 2.5 mL cutinase g−1 lignin were 22.80 ± 1.04 ppm and 20.77 ± 1.12 ppm, respectively. Moreover, the results also demonstrated that the extended reaction time from 24 to 72 hours had reduced the yield of syringaldehyde (from 70.19 ± 3.31 to 34.03 ± 2.01 ppm), p-coumaric acid (from 27.06 ± 1.20 to 23.76 ± 1.63 ppm), and ferulic acid (from 50.19 ± 2.23 to 43.39 ± 1.10 ppm), while it increased the yield of vanillin (from 22.80 ± 1.04 to 32.64 ± 2.35 ppm). Nevertheless, the yield hydroxybenzoic acid was unaffected, even at the prolonged reaction time. There was 642.83 ± 14.45 and 649.62 ± 21.35 ppm of hydroxybenzoic acid produced at 24 and 72 hours reaction, respectively. According to study [−1 lignin) and shortest reaction time (24 hours) were found to be adequate in achieving maximum recovery of aromatic compounds from lignin hydrolysis. High vanillin yield was probably due to the conversion of free ferulic acid after a prolonged reaction time. This suggestion was supported with the results as shown, where the decrease of ferulic acid correlates very well with the increase of vanillin.Furthermore, the results in Tables  p-coumaric acid, and 46.94 ± 1.59 ppm of ferulic acid were produced. The release of aromatic compounds during hydrolysis was in similar manner to the effect of pH. The yields of compounds and recoveries were lower when the hydrolysis was performed at the lowest (40°C) and highest (65°C) temperature. According to a study [−1 lignin) and syringaldehyde  yield and recovery were achieved at the highest temperature (65°C). High yield of both compounds can be attributed to the release of appropriate phenolics from lignin after the cutinase reaction, then followed by slow oxidation under high temperature (65°C) and alkalinity (pH 8).Temperature is the most critical factor affecting the performance and stability of enzyme catalysis process. Based on the results obtained, it was distinctly indicated that 55°C was the optimum temperature for cutinase catalysis. At this optimum temperature, about 557.80 ± 12.76 ppm of hydroxybenzoic acid, 53.94 ± 1.20 ppm of syringaldehyde, 21.68 ± 0.50 ppm of vanillin, 22.81 ± 1.44 ppm of a study , cutinas basidiomycetous fungi. It is an extracellular ligninolytic enzyme containing a heme protein and a highly specific Mn2+ binding site. During MnP-catalyzed oxidation, the chelated Mn3+ ion, but not MnP itself, acts as a low molecular weight mediator and freely diffusible oxidizing species which will ultimately perform oxidative depolymerization of lignin/lignin model compounds [ p-coumaric acid, and ferulic acid were generated at a pronounced level under the designed reaction condition. About 459.48 ± 6.47 ppm of hydroxybenzoic acid, 13.81 ± 0.85 ppm of vanillic acid, 12.48 ± 0.66 ppm of vanillin, 12.42 ± 0.87 ppm of p-coumaric acid, and 21.39 ± 1.45 ppm of ferulic acid were produced from lignin after MnP catalysis. Based on the results obtained, the yield of hydroxybenzoic acid was the highest, followed by ferulic acid and lastly vanillic acid, vanillin, and p-coumaric acid. However, the yields and recoveries of these compounds, except vanillic acid from MnP-catalyzed reaction, were found to be not significantly different (p > 0.05) with the experiment control. The yield of hydroxybenzoic acid, vanillin, p-coumaric acid, and ferulic acid from experiment control (reaction without MnP) were 468.58 ± 16.26, 11.74 ± 0.53, 12.30 ± 0.40, and 19.61 ± 0.58 ppm, respectively. However, the yield of vanillic acid was improved from 10.66 ± 0.81 to 13.81 ± 0.85 ppm in the presence of MnP. Thus, presumably, the release of these compounds was probably due to the oxidation reaction activated by other components in the reaction medium, but not due to the catalytic action of MnP. With this, the role of MnP in the catalysis of lignin deconstruction was found to be insignificant in this study. Such finding can be explained by the study [3+ ion in the oxidation of lignin/lignin model compounds was emphasized in their study. A prominent lignin oxidation reaction was found to occur with the presence of Mn3+ and chelator  without MnP. In spite of that, lower rate of oxidation was also found to take place in the media containing Mn2+, chelator, and H2O2 only. Mechanism of MnP in oxidative catalysis was demonstrated by study [2O2 to ferric ion of MnP, forming iron-peroxide complex. Subsequently, MnP compound I  formed, after the cleavage of peroxide oxygen-oxygen bond by accepting 2 electrons from heme of MnP. A monochelated Mn2+ ion will then act as an electron donor to the reduced MnP compound I into MnP compound II (Fe4+-oxo-porphyrin complex) by cleaving the dioxygen bond hydrolytically. As a result, a water molecule was generated and Mn2+ was oxidized to Mn3+. Similar mechanism was indicated for reduced MnP compound II to generate native MnP. Thus, for each MnP catalytic cycle, two Mn3+ ions and two water molecules were generated. During lignin degradation, reactive phenolic radical was formed by the nonspecific Mn3+ ion attack via hydrogen and one electron abstraction. The generated reactive radicals led to depolymerization of lignin. Besides, reaction system of MnP, chelator, Mn2+, and H2O2 was also found to catalyze Cα-Cβ cleavage, Cα-oxidation, and alkyl-aryl cleavages of phenolic syringyl type β-1 lignin structure. However, only small part of aromatic structures of lignin comprised of phenolic hydroxyl groups. The abundant nonphenolic substructures of lignin are not susceptible to the attack by chelated Mn3+ ion [ p-coumaric acid can also contribute to the yield of hydroxybenzoic acid.Manganese peroxidase (MnP) is a class II peroxidase group inompounds , 45, 46.by study , 23. CatMn3+ ion , 48. MorMn3+ ion . In the 3+ ion generated via autooxidation between H2O2 and MnSO4. Restriction of Mn3+ to attack lignin is the main reason of insignificance of MnP in this catalysis. Furthermore, the success of monomeric aromatic compounds production through lignin hydrolysis by cutinase is a pioneering discovery in this study. Further study in this research is necessary for the improvement of the products' yield.Based on previous results and discussion, it was found that the types and yields of compounds produced from lignin oxidation produced by various methods were different.  p-coumaric acid. This finding suggested that mild oxygen oxidation carried out in this study has preserved the p-coumaric acid from further oxidation. Besides, the highest yield of vanillin, syringic acid, and syringaldehyde from nitrobenzene oxidation also suggested that it was a more severe oxidation method than oxygen oxidation.Overall, nitrobenzene oxidation was proved to be the best method of lignin fragment oxidation, followed by direct oxygen oxidation in generating aromatic compounds from lignin. This finding was supported by study , where n p-coumaric acid, ferulic acid, syringaldehyde, and vanillin. Unexpectedly, cutinase-catalyzed lignin hydrolysis performed better than MnP-catalyzed lignin oxidation. Thus, further study on cutinase reaction on lignin is necessary.Among the tested protocols, nitrobenzene oxidation, followed by direct oxygen oxidation, was the best method in oxidizing lignin for the production of monomeric aromatic compounds. In this study, cutinase-catalyzed lignin hydrolysis was proposed for the first time in the production of hydroxybenzoic acid,


Several studies have analyzed the association of body mass index (BMI) with either the prevalence or incidence of type 2 diabetes (T2D), but no study from Europe or North America has yet analyzed and compared the association of BMI with both incident and prevalent T2D cases.Stratified logistic regression was used to calculate odds ratios (OR), and stratified Cox proportional hazards regression was used to calculate hazard ratios (HR) of the effect of BMI on the prevalence, and incidence of T2D. Wald chi-square statistics were applied when comparing the risk estimates.2) had an OR of 2.83  and obese women (BMI ≥30 kg/m2) had an OR of 12.12  when compared with normal weight women (BMI <25 kg/m2). Among incident T2D cases, overweight women had a HR of 5.01  and obese women had a HR of 15.99  when compared with normal weight women. After stratification by level of physical activity, and adjustment for age, smoking status, and education level, the Wald chi-square statistic for BMI was 180.90 for prevalent T2D cases, and 262.03 for incident T2D cases.Among prevalent T2D cases, overweight women (BMI 25–29.9 kg/mThe predictive effect of BMI was found to be stronger for T2D incidence than T2D prevalence. Type 2 diabetes (T2D) is a chronic disorder of carbohydrate, fat, and protein metabolism. Approximately 60 million people in Europe have diabetes , and 90%The Tehran Lipid and Glucose Study (TLGS) included fasting plasma glucose screening and 2-hour post challenge plasma glucose screening to identify T2D cases in 1999–2001, 2002–2005, and 2005–2008, and found higher odds ratios (OR) for BMI among incident than prevalent T2D cases . HoweverThe NOWAC Study is a prospective nationwide study which started in 1991 , and conOut of the 170,000 women enrolled in the NOWAC Study, 33,919 completed the questionnaires sent in 1998 and 2005 . After exclusion of 2617 participants with missing values, the study sample consisted of 31,302.As T2D typically affects people over 40 years of age , in the Incident T2D cases were defined as participants who reported a T2D diagnosis between 1998 and 2005, and were 40 years of age or over at the time of diagnosis (Table 2). BMI was categorized into three groups: normal weight (BMI <25 kg/m2), overweight (BMI 25–29.9 kg/m2) and obese (BMI ≥30 kg/m2). Both continuous and categorical BMI variables were used in the analyses.Self-reported information on height and weight was used to calculate BMI , and ‘Do you smoke on a daily basis at the moment?’ . Women who answered ‘no’ to the former were categorized as ‘never smokers’. Those who answered ‘yes’ to the former, and ‘no’ to the latter, were categorized as ‘former smokers’, and those who answered ‘yes’ to both questions were categorized as ‘current smokers’.A 10-category scale measured the level of self-reported physical activity in the 1998 questionnaire, the validity of which has been reported . ResponsAll analyses were conducted using SPSS version 18. Means  were calculated for all continuous variables, and the percentage of participants in each category was determined for all categorical variables. General characteristics of the data are presented as means with SDs and frequencies, respectively , and so the categorical variable of BMI was used in the final models instead. In the final model, the estimates of the effects of BMI are presented with 95% confidence intervals (CI). ORs and hazard ratios (HR) are reported. An OR can be interpreted as a relative risk (RR) when the disease prevalence is low [To estimate the predictive effect of BMI on the incidence and prevalence of T2D, stratified logistic and stratified Cox proportional hazards regression were used. To assess the linear trend, the continuous variables  were used. To assess the predictive effect of BMI, the normal weight level was used as a reference in stratified logistic regression and stratified Cox proportional hazards regression analyses. A e is low . Wald chThe NOWAC Study was approved by the Regional Committee for Medical and Health Research Ethics (REK). All women gave written informed consent.2. A higher proportion of incident T2D cases were overweight and obese (combined), compared to prevalent T2D cases. The mean BMI of prevalent T2D cases was slightly higher than that of incident T2D cases (29.8 vs 29.7 kg/m2). The majority of women in the baseline cohort had a normal weight level, while the majority of incident and prevalent T2D cases were obese. Compared with prevalent T2D cases, incident T2D cases were on average younger, had a slightly lower BMI, a slightly higher education level, and a slightly lower level of physical activity , than prevalent T2D (reported in the 1998 questionnaire). Overall, BMI had a stronger predictive effect on T2D incidence than T2D prevalence.The risk of T2D prevalence was weaker than that for incidence, probably because the prevalent cases may have reduced their weight by exercise, physical activity, or diet after diagnosis.Previous validation studies of self-reported height and weight show that participants tend to overestimate their height , while t2 may account for 65%-80% of new diabetes cases [Several studies have adjusted for age , 26–34, es cases , which ies cases , 38. Poses cases , and canes cases .2 than for women with BMI 25.0-29 kg/m2. This is in contrast to our study, although we only used one category [BMI ≥30 kg/m2). However, for women aged 55 years or older, the NHANES III study showed an increased risk of T2D with increasing BMI. In general the association between BMI and T2D prevalence in our study is much higher than in other studies [Previous evidence from large cohort studies suggests that the relationship between BMI and diabetes may not be linear, and the same was observed in our study. In comparison with previous studies, despite differences in the groups of confounders in the model , 31–34,  studies , 31.2 at baseline. Also the proportion of diabetic participants with a BMI of ≥30 kg/m2 in 1995–1997 was similar to that in our study.The HUNT Study  from Nor2, BMI 30–39.9 kg/m2, and BMI .40 kg/m2 were 1.59 (95% CI: 1.46-1.73), 3.44 (95% CI: 3.17-3.74), 7.37 (95% CI: 6.39-8.50) respectively. The model was adjusted for age, education, smoking, sex, and race or ethnicity. Regardless of the use of different cut off points in defining BMI levels, the OR’s are considerably lower as compared to our study.Another study  from the2 for diabetes was not significant, while the OR’s for BMI >30 kg/m2 was 2.55 (95%CI: 1.10-5.92). The model was adjusted for age, waist circumference, use of blood pressure medication, history of high blood glucose, physical activity, and consumption of vegetables and fruits. In comparison, the results from our study show much stronger association of BMI levels with the prediction of type 2 diabetes in the incidence of diabetes.A study from Finland  analyzed2, and BMI ≥30 kg/m2 were 3.22 (95% CI: 2.69-3.87), and 9.06 (95% CI: 7.60-10.8) respectively. The model was adjusted for age, family history of diabetes, alcohol use, smoking status, hormone therapy use, hypertension, high cholesterol, dietary factors, randomized Women’s Health Study treatment groups, and physical activity. The relative risks of developing type 2 diabetes are in line with our research.Women’s Health Study (WHS)  from U.S2, the RR’s of BMI 23.0–24.9 kg/m2, BMI 25.0–29.9 kg/m2, BMI 30.0–34.9 kg/m2, and BMI ≥35.0 kg/m2 were 2.67 (95% CI: 2.13–3.34), 7.59 (95% CI: 6.27–9.19), 20.1 (95% CI: 16.6–24.4), and 38.8 (95% CI: 31.9–47.2) respectively. The model was adjusted for age, time, family history of diabetes, menopausal status, postmenopausal hormone therapy, dietary score, exercise, smoking, and alcohol consumption. The mean BMI was not reported, but the relative risks were substantially larger than our study.The Nurses’ Health Study  from U.SThe Framingham Offspring study from the US analyzed BMI estimates to predict the incidence of diabetes over 7 years of follow-up . DespiteIn conclusion, our study shows that maintaining a normal weight level is beneficial in preventing T2D. Our findings show a stronger predictive effect of BMI on T2D incidence than T2D prevalence. Overall the findings suggest that the majority of T2D cases can be prevented with weight loss.


The polyamorphism in glassy silicon is found to be mainly associated with low-density liquid (LDL).Combining molecular dynamics (MD) simulation and Voronoi polyhedral analyses, we discussed the microstructure evolution in liquid and glassy silicon during cooling by focusing on the fraction of various clusters. Liquid-liquid phase transition (LLPT) is detected in supercooled liquid silicon However, freezing the high-density liquid (HDL) to the glassy state is not achieved as the quenching rate goes up to 10 The medium-range order (MRO) is also argue in glasses with a correlation length extending the first peak in pair coorelation functions (PCF) to a distance up to 1 ~ 2 nm, making the glass different from the liquid.et al reportedSince glasses are in non-equilibrium states, and different configurations could be achieved during structural relaxation2324111316Base on the results in min/gmax,gT is easily identified, and agrees well with the other simulation and experimental resultsP = −0.5 GPa) via viscosities calculation method predicted by Deb et alAbraham parameer, R = g1,n2,n3,n4n>, where in denotes the number of i-edged faces of the Voronoi polyhedrons333410 K/s, (b) Cr3 = 5 × 1011 K/s, (c) Cr6 = 1 × 1013 K/s and (d) Cr8 = 1 × 1014 K/s. Among the high temperature region (above ~1600 K), because of severe thermal motions, the types of the clusters are so diverse that the population is nearly averaged for each cluster. The maxnium fraction is less than 10% and none of them is dominant in HDL. The main structure is eight-fold coordination structure <0, 4, 4, 0> and the number of the types of the Voronoi clusters could reach up to 300–400 or more , significant difference in microstructure can be seen from the In our previous wrok14 K/s), because of the short relaxation time, the main structures in LDL such as six-fold structure <0, 6, 0, 0> and <2, 2, 2, 2> are preserved in the final glass. In contrast, the high coordination structures in HDL always transfer to the 5 or 6 coordination structure, including <2, 3, 0, 0>, <0, 6, 0, 0> and <2, 2, 2, 2>. The HDL structure can not be trapped in the final glass in the present studies.The glassy silicon can be concluded to have three types of amorphous structures: <4,0,0,0>-based glass, <2,3,0,0>-based glass and <4,0,0,0> – <2,3,0,0>- mixture glass. The <2,3,0,0> is the main component in the LDL. The <4,0,0,0> can be transformed from the <2,3,0,0> as an atom is released−15 seconds). The pressure are controlled at 0 GPa via Nose-Hoover barostat. Run lengths range from 0.2 ns (500 000 steps) to 0.0001 ns (100 steps) to adjust the cooling rate from 5 × 1010 K/s to 1 × 1014 K/s. Obviously, the liquid system is hardly to reach the equilibrium and it is under supercooled state.A series of rapid cooling process of liquid silicon is simulated by using LAMMPS code33S.Z. and R.L. designed research and wrote manuscript. S.Z., L.Q. and S.Z. performed the simulations. L.W., X.Z. and M.M. analyzed the results and contributed to discussion. All authors reviewed the manuscript.


Gambierdiscus produce the ciguatoxin precursors responsible for the occurrence of ciguatera toxicity. The prevalence of ciguatera toxins in fish has been linked to the presence and distribution of toxin-producing species in coral reef ecosystems, which is largely determined by the presence of suitable benthic habitat and environmental conditions favorable for growth. Here using single factor experiments, we examined the effects of salinity, irradiance, and temperature on growth of 17 strains of Gambierdiscus representing eight species/phylotypes , most of which were established from either Marakei Island, Republic of Kiribati, or St. Thomas, United States Virgin Island (USVI). Comparable to prior studies, growth rates fell within the range of 0–0.48 divisions day-1. In the salinity and temperature studies, Gambierdiscus responded in a near Gaussian, non-linear manner typical for such studies, with optimal and suboptimal growth occurring in the range of salinities of 25 and 45 and 21.0 and 32.5°C. In the irradiance experiment, no mortality was observed; however, growth rates at 55μmol photons · m-2 · s-1 were lower than those at 110–400μmol photons · m-2 · s-1. At the extremes of the environmental conditions tested, growth rates were highly variable, evidenced by large coefficients of variability. However, significant differences in intraspecific growth rates were typically found only at optimal or near-optimal growth conditions. Polynomial regression analyses showed that maximum growth occurred at salinity and temperature levels of 30.1–38.5 and 23.8–29.2°C, respectively. Gambierdiscus growth patterns varied among species, and within individual species: G. belizeanus, G. caribaeus, G. carpenteri, and G. pacificus generally exhibited a wider range of tolerance to environmental conditions, which may explain their broad geographic distribution. In contrast, G. silvae and Gambierdiscus sp. types 4–5 all displayed a comparatively narrow range of tolerance to temperature, salinity, and irradiance.Benthic dinoflagellates in the genus  Gambierdiscus produce the ciguatoxin precursors responsible for ciguatera toxicity, and their abundance and seasonality has been closely correlated with ciguatera fish poisoning occurrence [Gambierdiscus populations is influenced by many environmental factors; amongst them, regimes of temperature, salinity, and irradiance act as determining factors [Gambierdiscus growth, particularly with respect to species-specific responses, are not well known across the multiple species in this genus.Ciguatera is a common form of phycotoxin-borne seafood disease caused by the consumption of ciguatoxin-contaminated finfish. It is endemic to the tropical and subtropical Caribbean Sea, and the Pacific, Atlantic, and Indian Oceans. Benthic dinoflagellates in the genus currence , 2. Prol factors –7. HowevGambierdiscus growth responses to temperature, salinity, and irradiance. A major hurdle to interpreting these early results is that Gambierdiscus taxonomy was unresolved at that time, and this issue continued until species descriptions were published [G. toxicus or Gambierdiscus sp.; however, many of the strains used could belong to genus of Fukuyoa gen. nov. and any of the 11 species in genus of Gambierdiscus identified today [G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. excentricus, G. pacificus, G. polynesiensis, G. scabrosus/Gambierdiscus sp. type 1, G. silvae/Gambierdiscus sp. ribotype 1, G. toxicus,) and 6 ribotypes . These results could even apply to undescribed Gambierdiscus species, as morphological and phylogenetic details were not provided. Consequently, it is uncertain whether the growth differences among strains used in these studies resulted from intra- or inter-species variability.Several early laboratory studies provided initial data in this regard. Both Bomber et al.  and Mortublished –14. Preved today –20  under laboratory culture conditions [Gambierdiscus spp. occur are generally restricted to salinities of 34–38; however, Gambierdiscus cells have been reported from areas where salinity levels occasionally are outside of this range, such as near river outlets and enclosed water bodies [With respect to salinity, nditions , 23. Ocer bodies , 25.Gambierdiscus [Gambierdiscus, Kibler et al. [-2 · s-1) of surface irradiance supported maximal growth, with optimal growth extending to 75 m depth in the Caribbean. However, these laboratory results still contrast with field observations of Gambierdiscus in habitats exposed to high irradiances, such as sand flats, drifting algae, and detritus [Gambierdiscus spp. were notably different in response to irradiance regimes at the species level [G. carolinianus and G. pacificus were least adapted to high irradiance and experienced mortality at ~300μmol photons · m-2 · s-1. To better understand how irradiance affects Gambierdiscus growth, data from additional species and strains are needed.With regard to irradiance, approximately 10% of full sunlight was previously considered the upper threshold for maximum growth of erdiscus , 4. Usinr et al.  suggestedetritus , 26, 27,Gambierdiscus generally achieves maximum growth at 25–31°C, and cannot survive temperatures below ~15–21°C or over ~31–34°C [Gambierdiscus populations and ciguatera incidence primarily reported from environments with a temperature range of 25–30°C [Gambierdiscus cells at extreme temperatures lower and higher than previously reported, e.g, as low as 14°C [Gambierdiscus species and strains are needed to help interpret these reports, as well as to enhance our understanding of the distribution and seasonality of species within this genus in the context of climate change.Under experimental culture conditions, ~31–34°C , 28, 29. as 14°C , where t as 14°C . Clearly-2 · s-1) of eight Gambierdiscus species/phylotypes: G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus, G. silvae, Gambierdiscus sp. type 4 and Gambierdiscus sp. type 5. Multiple strains were examined for five of the eight species tested. Furthermore, this study represents the first examination of the growth responses of G. silvae, and two Pacific ribotypes (Gambierdiscus sp. type 4 and type 5). The growth responses determined from laboratory experiments were compared with the distribution of Gambierdiscus and ciguatera occurrence in an effort to better understand how these environmental variables influence Gambierdiscus growth and distribution.This study sought to determine the optimal conditions for growth and the tolerances to temperature (16–38°C), salinity (10–60) and irradiance , with the exception of G. carpenteri, which was collected from Halimeda monile at Long Key, Florida Keys . In the Pacific, cells were obtained from Halimeda spp. and coral rubble at Marakei Island, Republic of Kiribati . Macroalgae sample processing, Gambierdiscus isolation, and culture establishment procedures were similar to Xu et al. [-2 · s-1 of light, and 12:12h light: dark photoperiod. Cultures were maintained for 3–12 months to acclimate to laboratory conditions before they were used in the growth experiments. This study included a total of six Gambierdiscus species and two Gambierdiscus ribotypes, the latter of which may represent undescribed species. Details regarding the isolates are listed in In the Caribbean, individual u et al. . IsolateGambierdiscus growth was assessed by in vivo fluorescence using a 10-AU Fluorometer . Previous studies confirmed a linear correlation between in vivo fluorescence (relative fluorescence units or RFU) and cell densities (biomass), in which increasing fluorescence was associated with increasing cell numbers rather than an increase in fluorescence per cell [μ (division · day-1) is the growth rate, and N1 and N0 represent the fluorescence at times t1 and t0, respectively. In all experiments, a total of four sequential transfers were performed: the first transfer was conducted to allow the culture to acclimate to the environmental conditions, and data from the second-fourth transfers were collected to determine the growth rate.per cell –5, 7. Flper cell ,μ  under a constant average temperature of 27°C, and the aforementioned irradiance and photoperiod intervals. Salinity levels ranged from 10–60 , which were created by adding Milli-Q water into natural filtered seawater  to reduce salinity, or adding sea salt evaporated from 0.2 μm filtered natural seawater to increase salinity. This salinity-treated medium was then autoclaved for 45 minutes in Teflon bottles. After autoclaving, the salinity was measured again with a hand-held refractometer and enriched to produce modified K medium where the tris component was omitted. The procedures for determining growth were as described for the temperature experiments. Culture tubes in salinity experiments were placed randomly in the incubator to mitigate micro-environmental differences.-2·s-1 were established using four equidistant shelves in the incubator. Between 0–6 cool white full spectrum fluorescent bulbs were installed in each shelf, and covered by 0–2 layers of nylon window screens to achieve the desired irradiance levels. Irradiance received by the culture tubes was measured using Digital Scalar Irradiance Meter  with probe QSL-100 . Culture tubes were placed in random locations in the incubator to once again minimize micro environmental disparity.Irradiance experiments were performed in an incubator with a constant mean temperature of 27°C. Irradiance levels of 55, 110, 200, and 400μmol photons·m-2 · s-1, under a photoperiod cycle of 12h:12h light: dark. The first transfer was used to acclimate cultures to temperature conditions, and growth rates were calculated from three additional sequential transfers over the culture’s exponential phase of growth.Temperature experiments were conducted in a temperature gradient bar  construcGambierdiscus species in all experiments, coefficient of variation (CV) among conspecific strains was calculated; depending on normality and homoscedasticity of growth rate data, a one-way analysis of variance (ANOVA) , Welch’s ANOVA , Mann-Whitney Test  and Kruskal-Wallis H Test  were performed using SPSS statistics 21 . In addition, to better understand the growth potential of each Gambierdiscus species/strain, growth rate and salinity, and growth rate and temperature were polynomial fitted using OriginPro 9.0.0 . The polynomial equations were further analyzed using Matlab R2013a 8.1.0.604  to calculate the characteristic points where growth maxima, optima, and limitation occurred.To assess growth variability and statistical differences in growth rates within each μm, maximum growth rate; Tm/Sm, temperature/salinity of maximum growth; Topt/Sopt, temperature/salinity of optimum growth range (μ ≥ 0.8 × μm); To/So, the lower temperature/salinity limit for growth, and Tu/Su, the upper temperature/salinity limit for growth. Due to data limitations, polynomial regressive analysis was not performed in the irradiance experiment.The growth characteristics were described by the following parameters similarly defined in Kibler et al. : μm, maxGambierdiscus spp. to different salinity at a constant mean temperature of 27°C are shown in Figs Gambierdiscus growth curves showed a general increase in growth rate with increasing salinity values, followed by a decline at high salinities , G. caribaeus (SH Nov09_3), and G. carolinianus (SH Mar10_12) exhibited low growth at these salinity levels   . High CVd 25–50) .Gambierdiscus growth potential at each salinity, growth rate and salinity were described using the polynomial equation: Y = A + B1X + B2X2 + … + BnXn. Here, X and Y represent salinity and growth rate, respectively. Most strains were fitted to 3rd to 5th order polynomial curves with an R value >0.9 , and the ability to maintain growth or survive at extreme salinities (So and Su). Generally speaking, strains of G. caribaeus and G. carpenteri had a broad Sopt and tolerated extreme salinities, whereas strains of G. belizeanus were tolerant to hypersaline conditions. In contrast, strains of G. carolinianus, G. silvae, and Gambierdiscus sp. types 4–5 were sensitive to extreme salinities.Polynomial curves for salinity experiments exhibited a relatively symmetrical bell shape . In the 0.1–38.5 . The majGambierdiscus strains across the range of irradiances tested (55–400μmol photons · m-2 · s-1) under a constant mean temperature of 27°C and a salinity of 32. Growth responses to varying irradiance were similar for all 17 strains; i.e., growth was slow at 55μmol photons · m-2 · s-1 and generally plateaued in the range of 110–400μmol photons · m-2 · s-1. No obvious decline was observed at 400μmol photons · m-2 · s-1  stopped growing at temperatures ≤ 18.5°C, and strains of 1D0509-16 and 1D0510-22 (Gambierdiscus sp. type 4) died when temperatures exceeded 31°C . Highest variability was reported at temperatures at the upper and lower limits, but Gambierdiscus spp. tested exhibited significant intraspecific variation as well (α<0.05), mainly at mid-range experimental temperatures (The coefficient of variation (CV) was used to assess growth rate variability within species at each temperature . For G. eratures .Gambierdiscus growth potential at each temperature, growth rate and temperature were described using the polynomial equation: Y = A + B1X + B2X2 + … + BnXn (n≤5) where X and Y represent temperature and growth rate, respectively. Major strains were fitted to a 4th to 5th order polynomial equation with an R value >0.85 , BB Apr10_3 and BP May10_1 (G. carolinianus), KML1 (G. carpenteri), 3S0510-19 (G. pacificus), FC May10_9 (G. silvae), 1D0509-16 (Gambierdiscus sp. type 4), and DS0511-03 (Gambierdiscus sp. type 5). In this group, major strains showed a consistent growth plateau over a wide temperature range, e.g., KML1 displayed stable, maximal growth over the temperature range of 21–28.5°C (G. belizeanus), BP Aug08 and SH Nov09_3 (G. caribaeus), SH Mar10_12 (G. carolinianus), 3S0509-27 (G. pacificus), and 1D0510-22 (Gambierdiscus sp. type 4)  .Gambierdiscus growth parameters of μm, Tm, Topt, To, and Tu differed among species, and within individual species; however, strains exhibiting skewed growth curves generally had higher μm and Tm, narrower Topt range, and lower To compared with strains with symmetrical growth curve  showing a symmetrical μ-°C shape, and 0.41 division day-1 in BP Mar10_7 (G. belizeanus) exhibiting a skewed μ-°C shape.th curve . Among tGambierdiscus carpenteri required the highest Tm to realize maximum growth, in comparison with G. silvae, which required the lowest Tm  for a Hawaiian strain [-1 was also possible for a Florida strain, GT600 [Compared with planktonic dinoflagellates, the epibenthic genus on day-1  and growobserved , consistn strain , and undn, GT600 . UnfortuG. belizeanus, G. caribaeus, G. carolinianus, and G. pacificus analyzed during this study were similar to previous reports; however, G. carpenteri exhibited a narrower range of growth than in previous studies  were nonlinear with an approximate Gaussian/bell shape ; again, salinity levels of 34–37 measured during the survey support optimum growth [rized by ), thoughrized by . Salinitm growth .Gambierdiscus survival. For example, below a salinity of 14 in the coastal zone in the Gulf of Mexico [G. caribaeus and G. carpenteri have a good chance of survival or growth.In contrast with the stable or narrow range of salinity in oceanic regions, some coastal locations such as estuaries and bays are affected by freshwater inputs from precipitation and freshwater discharge from land. At these locations, hyposaline conditions may inhibit f Mexico , only G.Gambierdiscus growth. These conditions readily arise in restricted water bodies in the tropical and subtropical areas with high evaporation, poor circulation, and low freshwater input such as tropical lagoons, where salinities can easily exceed 40 [Gambierdiscus isolates from G. belizeanus, G. caribaeus, and G. carpenteri may be able to grow.Besides hyposaline conditions mentioned, hypersaline environments pose another challenge for xceed 40 . In respGambierdiscus and salinity were observed only under extreme salinity conditions . For example, Gambierdiscus was absent from river mouth sites due to low salinity [Gambierdiscus abundance at inshore stations, with Gambierdiscus population maxima co-occurring with peak rainfall [Gambierdiscus populations. Freshwater inputs from precipitation could be advantageous due to nutrient inputs if water salinity remains within a suitable range for growth, but could be disadvantageous when hyposalinity conditions occur that are suboptimal for Gambierdiscus growth.In previous ecological surveys, positive or negative correlations between salinity , 43. Anorainfall . The appGambierdiscus will respond; as benthic organisms, they are distinct from the phytoplanktonic dinoflagellates in that they are generally associated with a benthic macroalgal habitat. Furthermore, one previous salinity shock experiment revealed that growth responses of Gambierdiscus to instantaneous salinity decreases were species-dependent and included a range of responses such as no effect, slowed growth, or mortality [Gambierdiscus species and geographically distinct strains is thus required.Within the context of climate change, precipitation patterns are predicted to occur in which rainfall is less frequent but more intense, and followed by longer dry periods . This neortality . FurtherGambierdiscus strains tested grew at 55–400μmol photons · m-2 · s-1, and no obvious growth inhibition was observed at 400μmol photons · m-2 · s-1. This pattern contrasts with other laboratory studies examining the irradiance response of several Gambierdiscus species. Clones GT600 and GT600A have been shown to be inhibited at irradiances of >232 and >225μmol photons · m-2 · s-1, respectively . SimilaGambierdiscus response to light intensity. The genus Gambierdiscus typically attains optimum growth at ~10% of full sunlight [Gambierdiscus in shallow environments subjected to high light intensities. This study determined that the optimum light intensity for growth was ~4.4–16% of full light (full sunlight = 2500μmol photons · m-2 · s-1), which is higher than reported previously. However, it does not follow ecological observations in which Gambierdiscus cells were detected in the shallow waters of 1–5 m [Gambierdiscus attachment and shields cells from strong light damage [Gambierdiscus spp. exhibited typical characteristics of “shade-adapted” organisms [There is an apparent inconsistency between laboratory findings and field observations of sunlight  or maximsunlight , which asunlight . Interesof 1–5 m , on sparof 1–5 m , on drifof 1–5 m , or floaof 1–5 m . The tolrganisms .Ostreopsis [Gambierdiscus cells tend to produce more mucus at higher irradiance levels. This mucus production causes cell aggregation and a cell complex enveloped by large quantities of mucus protects cells from high light due to self-shading. Besides the physical structures that Gambierdiscus spp. utilize to shade themselves from high irradiance, photoprotection mechanisms observed in other dinoflagellates such as secretion of UV radiation-absorbing compounds and changes in pigment composition should also be considered [Another explanation is that like other benthic dinoflagellates such as in the genus treopsis , Gambiernsidered , 54.Gambierdiscus vertical distribution in the water columns [Gambierdiscus was largely collected from shallow water depths <5 m [G. carolinianus, which was collected from 45.7 m in the northern Gulf of Mexico [-2 · s-1 are sufficient to maintain Gambierdiscus growth, corresponding to >150m depth in tropical waters [Gambierdiscus spp. in deep waters are not likely to experience conditions for optimal growth; however, Gambierdiscus cells inhabiting these ecosystems may serve as source populations for surrounding shallower niches.Irradiance plays an important role in defining  columns . In mostths <5 m , but werths <5 m , 55. Thuf Mexico . These fGambierdiscus growth responses and some environmental parameters such as DIN and phosphate [Gambierdiscus growth is optimal and suboptimal within discrete temperature ranges, which is consistent with many previous observations [G. caribaeus, the symmetric or slightly skewed growth response curve observed in this study is similar to that in Tester et al. [G. caribaeus in Kibler et al. [G. belizeanus, G. carolinianus, and G. pacificus, growth response shapes reported here and those described by Kibler et al. [Although a linear relationship has been observed between hosphate , the grorvations –5, 29. Fr et al.  and KiblG. belizeanus, G. caribaeus, and G. carpenteri may be due in part to their broad tolerance to environmental conditions, especially to temperature. It is not surprising that G. pacificus also exhibited a wide thermal tolerance, as that species is frequently observed and is broadly disturbed in the tropical Pacific. Regarding G. carolinianus, our data agree with previous work indicating that this species has a relatively low maximum temperature for growth (Tm = ~25–27.1°C) and is well-adapted to lower temperatures (To = 15.8°C) [Tm and Tu of G. carolinianus are similar to previous results: 23.8–27.0°C vs. ~25–27.1°C and 30.3–31.6°C vs. 32.5°C, respectively [To in the current study was much higher (18.4–18.6°C vs. 15.8°C) [G. carolinianus originating from St Thomas, USVI may be more sensitive to lower temperatures than the strain isolated from North Carolina. It also implies that differences among strains within each species may reflect geographic origin, and cautions against using one strain to represent the physiological characteristics of a species.Our findings, together with others , 28, 29, 15.8°C) . The temG. silvae and Gambierdiscus types 4 and 5. To date, only Gambierdiscus sp. ribotype 2 and types 2–3 were used in experiments assessing growth response to temperature. Similar to the findings of this study, Gambierdiscus sp. ribotype 2 and type 3 had a narrow Topt, corresponding to 24.5–30.1 and 22–25°C, respectively [Gambierdiscus sp. type 2 showed a Topt of 21–28°C [To our knowledge, this study represents the first characterization of the relationship between temperature and growth for ectively , 29. But 21–28°C . SubsequGambierdiscus used a single strain to represent each species [μm, Tm, Topt, To, and Tu. Although the intraspecific CV of growth rate varied most at either end of the temperature range, statistically significant intraspecific variation was encountered within the Topt range. This agrees with Tindall and Morton [Gambierdiscus and other ciguatera associated dinoflagellates could only be compared near the temperature for optimal growth. Our results are also similar to those documented by Boyd et al. [The majority of growth studies carried out on  species , 7, 28;  species , we examd Morton , who notd et al. , who repd et al. .Gambierdiscus generally has an optimal temperature range of 19–31°C, and sustains growth between 15–34°C . Ths study, ; the higTo indicates the extent to which Gambierdiscus is adapted to low temperatures and helps to define its latitudinal distribution. The growth measurements of 17 Gambierdiscus strains in this study produced To estimates varying from 15.9–20.1°C, which are close to previous laboratory reports, such as 16–19.5°C for Florida Keys isolate GT600 [Gambierdiscus species can survive in a broad range of temperate environments. Along the Pacific coast in Japan, Gambierdiscus cells occurred year-round in locations where the wintertime temperatures drop to as low as ~11°C [Gambierdiscus cultures established from the temperate coasts in Japan survived for at least three months at 10°C [The parameter te GT600  and 15.0te GT600 . Field sas ~11°C , which r at 10°C .To, 15.9°C from BP Aug08 (G. caribaeus), and wintertime Sea Surface Temperature (SST) (http://www.nodc.noaa.gov/about/oceanclimate.html) to predict its possible distribution boundary, this G. caribaeus strain may extend as far north as ~38.0°N, and as far south as ~45.0°S. This predicted range is narrower than the one described in Kibler et al. [Gambierdiscus distribution is attributed to the different To values used; in Kibler et al. [To was 15°C  versus 15.9°C from our study. Clearly, a 1.0°C difference in To results in a marked change in Gambierdiscus’ predicted latitudinal range. However, the boundaries predicted in this study or that of Kibler et al. [Gambierdiscus; i.e. 35°N-37°S [Gambierdiscus populations beyond this range.Using the lowest Tu is another important index for determining how Gambierdiscus responds to high temperature. Estimates of Tu fell within 29.8–35.4°C, a broad range that encompasses previous Tu records, such as 31.1–35.1°C and 25–30°C for multiple isolates in Kibler et al. [Tu range of Gambierdiscus (http://www.nodc.noaa.gov/about/oceanclimate.html), with the warmest areas primarily located in the tropical Caribbean Sea, and the Pacific and Indian Oceans. Within these regions, the Indo-Pacific Warm Pool (IPWP) and West Pacific Warm Pool (WPWP) are two large bodies of seawater whose SST consistently remains above 28.5°C [Tu, Gambierdiscus populations are capable of growth and development, provided that other environmental conditions are suitable for growth. However, once conditions exceed Tu for specific Gambierdiscus species or strains, mortality may occur, possibly altering the composition of Gambierdiscus populations in the system. This may help explain why nations in close proximity to the IPWP have low or negligible ciguatera rates even though they are located within the tropics [Gambierdiscus. Similar conditions exist in Red Sea and Arabian Sea, where rates of ciguatera are also negligible [Gambierdiscus cells have been observed in these areas [Gambierdiscus strains.The temperature parameter r et al. , respecte 28.5°C . If the  tropics , generalgligible . Gambierse areas , 57, whese areas . On the Gambierdiscus abundance is thought to be closely related to ocean warming. If warming waters still satisfy their growth requirement, a positive correlation between temperature and abundance may be expected [Gambierdiscus growth rates with increasing temperatures to argue that there will be range extensions or expansions. Clearly, however, if temperatures exceed thermal tolerance thresholds, Gambierdiscus spp. abundance and ciguatera may decline or disappear in some areas [expected . This hiexpected ) have usme areas , 32, sucTopt and Tu varied from 1.7–3.7°C in this study and 1.2–3.3°C in Kibler et al. [Gamberdiscus strains with a narrow Topt-Tu gap , and narrow gaps between Topt and Tu. However, seawater does not have an infinite capacity to warm, and a phenomena called the tropical thermostat may suppress ocean warming [Gambierdiscus and ciguatera due to rising seawater temperatures is complex. Nevertheless, under conditions of warming, Gambierdiscus populations will likely proliferate in some areas, and possibly decrease in regions where ocean temperatures exceed Tu.The Intergovernomental Panel on Climate Change (IPCC) estimated that by 2100, ocean SSTs will increase by 0.6–2.0°C, with highest increase occuring in tropical and Northern Hemisphere subtropical regions . Given tr et al. , 29, one warming . As a reS1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


A case of disseminated intravascular coagulation (DIC) presenting after surgery for facial trauma associated with multiple facial bone fractures is described. With regard to the oral and maxillofacial region, DIC has been described in the literature following head trauma, infection, and metastatic disease. Until now, only 5 reports have described DIC after surgery for facial injury. DIC secondary to facial injury is thus rare. The patient in this case was young and had no medical history. Preoperative hemorrhage or postoperative septicemia may thus induce DIC. Disseminated intravascular coagulation (DIC) is a dynamic pathologic process in which thrombin forms within the vascular system . DIC is μL and C-reactive protein (CRP) to 12.1 mg/dL and a decrease in platelets to 131 × 103/μL (down from 238 × 103/μL at the time of the accident). CT revealed no abnormalities other than those at the surgical sites. The next day, the patient showed preshock status with a significant decrease in blood pressure, fever over 39.0°C, and transient loss of consciousness. Emergency laboratory tests showed increases in CRP to 23.9 mg/dL, TBIL to 2.1 mg/dL, creatinine to 0.97 mg/dL, lactate to 4.07 mmol/L, prothrombin time to 21.7 s, FDP (fibrin degradation product) to 10.8 μg/mL, and PT-INR  to 1.86, along with a decrease in platelets to 46 × 103/μL  score was 9. From the above results, septic shock was diagnosed and the patient was sent to the intensive care unit (ICU). According to the scoring algorithm criteria established by the Japanese Association for Acute Medicine (JAAM) for DIC [α was administered for the treatment of DIC. After another 7 days, laboratory tests showed a return to nearly normal state, with CRP of 4.09 mg/dL; TBIL of 0.6 mg/dL, Crea of 0.67 mg/dL, WBC of 12,160/μL; platelet count of 175 × 103/μL; prothrombin time of 13.7 s; FDP of 5.3 μg/mL; and PT-INR of 1.17  and plain radiography revealed right pulmonary contusion, fractures of a second rib and the right radius, and airway narrowing. In the maxillofacial region, bilateral condylar and mandibular fractures, LeFort II-type fracture, and blow-out fracture of the orbit were recognized . Airway × 103/μL . The bac of 1.17 . The pat4/μL to 9.8 × 104/μL) between the first visit and the surgery. DIC was possibly triggered in the patient from the large volume of blood loss, consumption of platelets and coagulation factors by acute massive hemorrhage, and a high volume of blood transfusion within a short period [In the oral and maxillofacial region, several reports have described DIC in patients with oral cancer , infectit period , 23. UndThe occurrence of DIC is difficult to predict in cases involving young patients with no underlying disease, as in the present case. Over the last 20 years, no fatal cases of DIC have been reported in association with oral and maxillofacial surgery . Therefo