Topotactic solution synthesis yields nanostructured tin chalcogenides, SnSx1–Sex with controllable composition; spark plasma sintered SnS0.1Se0.9 achieves ZT ≈ 1.16 at 923 K via microstructural texture tuning. via “soft chemical” organic-free solution syntheses to yield layered pseudo-ternary tin chalcogenides on a 10 g-scale. SnS undergoes a topotactic transformation to form a series of S-substituted tin selenide (SnSe) nano/micro-plates with tuneable chalcogenide composition. SnS0.1Se0.9 nanoplates were spark plasma sintered into phase-pure, textured, dense pellets, the ZT of which has been significantly enhanced to ≈1.16 from ≈0.74 at 923 K via microstructure texturing control. These approaches provide versatile, scalable and low-cost routes to p-type layered tin chalcogenides with controllable composition and competitive thermoelectric performance.Anion exchange has been performed with nanoplates of tin sulfide (SnS)  Main group metal chalcogenides (MCs) are excellent candidates for thermoelectrics,Chemical transformations, including ion exchange, topotactic and pseudomorphic reactions, represent a versatile and effective means to produce new materials with control over crystal structure, composition and morphological complexity.vice versa, thus offering opportunities to refrigerate and to harvest electricity from waste heat via the Peltier and Seebeck effects, respectively.ZT values of 2.6 and 2.3 along the b and c crystallographic directions, respectively at 923 K.ZT values cannot yet emulate those in the single crystalline material.x1–Sx (0 ≤ x ≤ 1),x1–PbxSeZT to 1.2 at 773 K x1–PbxSe).Thermoelectric materials can be utilised to convert thermal energy directly into electricity and e.g. >10 g SnS and SnS0.1Se0.9, respectively; Fig. S1 and S2In this study, we demonstrate how the combination of two organic-free aqueous solution strategies (anion exchange following direct precipitation) can be utilised to synthesise LTC nano/micro-plates with tuneable chalcogenide composition  Å, b = 3.9877(2) Å and c = 4.3242(2) Å. Scanning electron microscopy (SEM) images (h00) planes  images  reveal t) images  can be i) images  demonstr) images  and S4d†σ), which rises from ca. 760 S m–1 at 323 K to ca. 2250 S m–1 at 523 K, gradually decreases to ca. 1475 S m–1 at 723 K and once again increases to ca. 1960 S m–1 at 773 K (e.g. ∼5–32 S m–1 at 523 K) synthesised by mechanical alloyinge.g. ∼590 S m–1 at 523 K)e.g. ∼530 S m–1 at 523 K).σ values of SnS pellets can be attributed to the organic-free surfaces of the plates, the orientation of the plates, high crystallinity and the high degree of densification  values for SnS pellets indicate p-type conducting behaviour  (e.g. ∼0.01–0.10 mW m–1 K–2 at 573 K).κ) for SnS reduces from ≈1.918 W m–1 K–1 at 323 K to ≈0.786 W m–1 K–1 at 773 K (κL) is the main contributor  fabricated by various methods,Thermoelectric measurements were performed on SnS pellets perpendicular to the pressing direction . The pelat 773 K . These vehaviour . The Seet 773 K)  that excat 773 K , to whictributor . The ZT at 773 K . The ZT via an organic-free anion exchange that involved injecting a NaHSe solution into the SnS suspension, followed by boiling for 2 h  (eqn (1) and (2)).To demonstrate the utility of the organic-free strategy in obtaining a range of thermoelectrics with prescribed microstructure and tailored composition, we focused on the synthesis of substituted Sn materials, conscious that thermoelectric properties should improve at higher Se concentration.h Fig. S2. SamplesKsp) of SnSe in water as compared to that of SnS.θ  Å, b = 4.1507(2) Å, c = 4.4334(2) Å) with an anion site occupancy of 0.91(1) : 0.09(1) Se : S, corresponding to a composition close to SnS0.1Se0.9 and includes 1.4(1) wt% of SnS as a secondary phase. SAED patterns along the [100] zone axis (0.1Se0.9 {011} plane spacings) identify preferred orientation along the 100 direction. Hence both the morphology and crystal structure before and after anion exchange are essentially unaltered and the process is topotactic . PXD shows that the product consists of both SnSx1–Sex (x ≈ 0.9) and SnS  by either hot pressing or spark plasma sintering (SPS). Moreover, the overall Se : S ratio remains constant and the distribution of S and Se becomes more even across each plate . On decreasing the Se : Sn molar ratio (and x) from 0.9 through 0.8 to 0.5, the SnSx1–Sex products accordingly contain more sulphide together with rising phase fractions of SnS  which follows Vegard's law  ratio increases from ∼0.47, through ∼0.77, ∼0.85, ∼0.89, ∼0.92 to ∼0.93  and two-step (denoted SnS0.1Se0.9-2) SPS processes. The two-step SPS process can give rise to the superplastic flow of unconstrained samples in the transverse direction (i.e. perpendicular to the pressing direction), in turn producing textured samples.h00) plane  reflections. In fact, the orientation degree (F) for (h00), estimated by the Lotgering methodca. 0.25 and ca. 0.46, for SnS0.1Se0.9-1 and SnS0.1Se0.9-2, respectively, demonstrating the greater texturing in the latter sample. SEM images of the cleavage surfaces , while those in SnS0.1Se0.9-1 are more directionally disordered, which is consistent with PXD results. EDS . As with SnS samples, SnS0.1Se0.9-1 and SnS0.1Se0.9-2 demonstrate a combination of clean particle surfaces, pronounced plate orientation, high crystallinity and high density. Coupled with the reduced bandgap engendered by Se doping, the high σ values observed can be plausibly rationalised. That SnS0.1Se0.9-2 exhibits higher conductivity is likely due to the enhanced (h00) texturing induced by the two-step SPS process. Electrical conductivity within the Sn–Se layers (in the bc plane) of SnSe is expected to be much higher than that perpendicular to the layers .0.1Se0.9-2 (evidenced as more strongly textured) is approximately 20.6% higher than that of SnS0.1Se0.9-1  for SnS0.1Se0.9-1 and SnS0.1Se0.9-2 (ca. 0.50 mW m–1 K–2) at 473 K and (of ca. 0.63 mW m–1 K–2) at 873 K. The S2σ vs. T behaviour hence broadly follows the temperature variation of σ, although the power factor for SnS0.1Se0.9-2 is marginally inferior to SnS0.1Se0.9-1 above 573 K. Interestingly SnS0.1Se0.9 and SnS show very similar trends in the variation of σ, S and S2σ with temperature  materials .Both SnS0.2Se0.8  and ≈0.4.87I0.03 ), and maκ) of the pellets .x1–Sex and so the difference in κ can be attributed to the different degrees of texturing in SnS0.1Se0.9-1 and SnS0.1Se0.9-2. The thermal conductivity along the a-axis (perpendicular to the Sn–Se planes) is much lower than that along b or c.κ in the pellets is the lattice thermal conductivity (e.g. κL ≈ 0.427 W m–1 K–1 for SnS0.1Se0.9-1 and κL ≈ 0.620 W m–1 K–1 for SnS0.1Se0.9-2 at 773 K) ,κL could be driven further towards this theoretical minimum. It is also interesting to note that SnS , has a higher κL than SnS0.1Se0.9 across the whole measurement temperature range   and the Fig. S26e.ZT could be calculated for the pellets using the above electrical and thermal transport data ZT ≈ 0.5–1.2)e.g. ≈0.6 for Ag-doped SnSe at 750 K,via anion exchange. Further systematic study of the thermoelectric performance of SnSx1–Sex as a function of x should reveal the optimum materials and processing parameters in the system. Moreover, it may be possible to offset loss of performance with decreasing x by co-doping with appropriate low toxicity, Earth-abundant metals. Although homogeneity of the materials is realised in SPS treated pellets, we note that the extent of anion exchange during synthesis will be governed primarily by reaction kinetics. Both the kinetics and thermodynamics (Ksp) could be modified by replacing water with another solvent, but given the economic and environmental advantages in using aqueous chemistry, modifying reaction temperature  would be the obvious parameter(s) to investigate towards achieving complete topotactic conversion  further as described above  and electrical conductivity, σ  to yield a transparent Na2SnO2 solution that was then heated to boil. 40 ml of Na2S(aq) (0.5 mol L–1) was promptly injected into the boiling solution, which was then further boiled for 2 h. The reaction was terminated at this point for the synthesis of SnS, while for the synthesis of SnSx1–Sex materials such as SnS0.1Se0.9, used as an example here, 40 ml of freshly prepared NaHSe(aq) (0.25 mol L–1) was promptly injected into the SnS suspension that was boiled for another 2 h and then cooled to room temperature. Heating and cooling of the solution were performed under Ar(g) on a Schlenk line. The products were washed with DIW and ethanol and dried at 50 °C for 12 h. Scaled-up syntheses of SnS and SnS0.1Se0.9 were performed with eight-fold and six-fold precursor concentrations, respectively. SnS pellets (density of 5.17 g cm–3) were sintered in a graphite die (diameter: 15 mm) under vacuum by SPS . SnS0.1Se0.9 pellets (density of 5.95 g cm–3) were sintered in a graphite die under vacuum by SPS in either a 1-step  or 2-step  process.63150 mmol NaOH and 10 mmol SnCl1 radiation, λ = 1.5406 Å). Rietveld refinement was performed against PXD data using the GSAS and EXPGUI software packages.S) and electrical conductivity (σ) of pellets were measured simultaneously using a Linseis LSR-3 instrument from 300–923 K. The thermal conductivity, κ of the pellets was calculated by κ = DCpρ, where D, Cp and ρ are the thermal diffusivity coefficient, specific heat capacity and density, respectively. D of the SnS0.1Se0.9 pellets was measured using a Netzsch LFA 457 from 300–923 K  was estimated by the Wiedemann–Franz law  was calculated by subtracting κe from κ. ZT was calculated via ZT = S2σT/κ. Hall measurements were performed on a nanometrics HL5500 Hall system using a van der Pauw configuration.PXD data were collected with a PANalytical X'pert Pro MPD diffractometer in Bragg–Brentano geometry (Cu KαFig. S27a; Cp of SFig. S27b was calcThere are no conflicts to declare.Supplementary informationClick here for additional data file.


Tetracera indica Merr. (Family: Dilleniaceae), known to the Malay as ‘Mempelas paya’, is one of the medicinal plants used in the treatment of diabetes in Malaysia. However, no proper scientific study has been carried out to verify the traditional claim of T. indica as an antidiabetic agent. Hence, the aims of the present study were to determine the in vitro antidiabetic potential of the T. indica stems ethanol extract, subfractions and isolated compounds.T. indica stems were subjected to cytotoxicity test using MTT viability assay on 3T3-L1 pre-adipocytes. Then, the test groups were subjected to the in vitro antidiabetic investigation using 3T3-L1 pre-adipocytes and differentiated adipocytes to determine the insulin-like and insulin sensitizing activities. Rosiglitazone was used as a standard antidiabetic agent. All compounds were also subjected to fluorescence glucose (2-NBDG) uptake test on differentiated adipocytes. Test solutions were introduced to the cells in different safe concentrations as well as in different adipogenic cocktails, which were modified by the addition of compounds to be investigated and in the presence or absence of insulin. Isolation of bioactive compounds from the most effective subfraction (ethyl acetate) was performed through repeated silica gel and sephadex LH-20 column chromatographies and their structures were elucidated through 1H–and 13C–NMR spectroscopy.The ethanol extract and its subfractions, and isolated compounds from T. indica stems ethanol extract. Wogonin, norwogonin and techtochrysin induced significant (P < 0.05) adipogenesis like insulin and enhanced adipogenesis like rosiglitazone. Wogonin and norwogonin also exhibited significant (P < 0.05) glucose uptake activity.Four monoflavonoids, namely, wogonin, norwogonin, quercetin and techtochrysin were isolated from the T. indica stems possess antidiabetic potential revealing insulin-like and insulin-sensitizing effects which were significant among the compounds. This also rationalizes the traditional use of T. indica in the management of diabetes in Malaysia.The present study demonstrated that the flavonoids isolated from the  Diabetes mellitus (DM) is a very old disease which was reported by the ancient Egyptians more than 300 years ago. Diabetes is a metabolic disorder which is caused by partial or complete absence of insulin or insulin resistance. At present time, occurrence of diabetes has become a major global health issue because of its alarming prevalence all over the world. It is projected that about 20% of the world’s population is liable to become diabetic over the next 10 years. According to the Center for Disease Control and Prevention , 29.1 million people in the United States of America have diabetes, which is 9.3% of their total population. World’s major pharmaceutical industries are in a race to fight against the chronic complications of diabetes and are coming up with different forms of insulin and insulin mimetics  glucose uptake test which proves its potential of being an antidiabetic agent -2-deoxy-d-glucose (2-NBDG) was obtained from Molecular Probes by Life Technologies, USA. Black microplates were collected from SPL Life Science Limited, Korea and 96 well flat bottom sterile microplates were bought from Greiner Bio-one Cellstar. Phosphate Buffered Saline (PBS) was purchased from GIBCO by Life Technologies, Invitrogen, USA. 3-isobutyl-1-methylxanthine (IBMX) was purchased from EMD Millipore Corp., USA. Dexamethasone was bought from Calbiochem, EMD Chemicals, Inc. San Diego, CA.2. The cells were incubated in humidified atmosphere with 5% CO2 supply at 37 °C. The media was changed after every two days and the cells were subcultured upon 80% confluence.3T3-L1 mouse pre-adipocyte cells were cultured according to the protocol provided by the ATCC, Rockville, MD, USA. The growth media was used as DMEM containing 4.5 g/L D-glucose and L-Glutamine with no sodium pyruvate and it was completed by adding 1% penicillin-streptomycin and 5% Fetal Bovine Serum (FBS). The cells were cultured in Greiner-bio CellStar flask of 25 and 75 cm5 cells) were seeded in 96-well plates and allowed to get confluent for 48 h. Subsequently, they were treated with extract, fractions and isolated compounds (dose: 0.78–100 μg/ml) for 48 h. Later, the cells were treated with MTT at 5 mg/ml. Every well was treated with 20 μL of MTT and incubated for 4 h. Finally, 100 μL of DMSO was transferred to every well to dissolve the water-insoluble purple formazan crystals  X 100.Insulin is a potent adipogenic hormone that triggers an induction of a series of transcription factors governing differentiation of pre-adipocytes into mature adipocytes. The transformation of the pre-adipocytes to mature adipocytes is known as adipogenesis or differentiation. The adipogenesis was induced according to the protocol reported previously , 15, 16.Three different concentrations of wogonin , norwogonin  and techtochrysin  were determined according to the safety of the cells initially confirmed through MTT assay. The 3T3-L1 pre-adipocytes were incubated for 2 days in the differentiation media. On ‘day 2’, the media was changed to insulin media (IM) (viz. 10 μg/mL insulin in DMEM) for 2 days. Finally, on day 4, the DMEM was applied and was changed in every 2 days for 6–8 days.26H24N4O) is a lysochrome (fat-soluble dye) diazo dye generally used to stain lipids and neutral triglycerides on frozen sections. It is red powder with maximum absorption observed at 518 nm. After adipogenesis, the cells were fixed with 10% formalin in phosphate-buffered saline (PBS) for 1 h at room temperature. Afterwards, the cells were washed 3 times with PBS and stained with freshly prepared Oil Red O (3 parts of Oil Red O of 0.6% in 2 parts of deionized water) from the stock solution for 1 h. Cells were again washed with distilled water and approximately 1 mL isopropanol was further added. Oil Red O staining was extracted by isopropanol after 5 min. Finally, the absorbance was measured using microplate reader at 520 nm : δ 6.835 , δ 6.349 , δ 8.136 , δ 7.653 , δ 3.983 , 12.591 ; 13C–NMR : δ 157.34 (C-2), δ 105.18 (C-3), δ 182.42 (C-4), δ 157.15 (C-5), δ 99.00 (C-6), δ 163.59 (C-7), δ 127.93 (C-8), δ 149.91 (C-9), δ 104.48 (C-10), δ 131.42 (C-1′), δ 129.25 (C-2′), δ 126.37 (C-3′), δ 131.95 (C-4′), δ 126.37 (C-5′), δ 129.25 (C-6′), δ 61.07 (C-8a); ESI-MS m/z 284 [M]+; 283 [M-H]−; 285 [M + H]+ corresponding to (C16H12O5) : δ 6.783 , 6.365 , 7.635 , 8.165 , 12.356 ; 13C–NMR : δ 153.17 (C-2), 104.98 (C-3), 184.54 (C-4), 154.54 (C-5), 98.65 (C-6), 163.62 (C-7), 131.51 (C-8), 145.67 (C-9), 104.21 (C-10), 124.88 (C-1′), 129.00 , 126.56 , 131.75 (C-4′); ESI-MS m/z 270 [M + H]+; 269 [M-1H]− corresponding to C15H10O5 : δ 6.260 , 6.516 , 6.997 , 7.700 , 7.819 , 12.45 ; 13C–NMR : 175.60 (C-4), 164.07 (C-7), 161.18 (C-5), 156.88 (C-9), 147.36 (C-4′), 146.00 (C-2), 144.86 (C-3′), 135.81 (C-3), 122.84 (C-1′), 120.56 (C-6′), 115.33 (C-5′), 114.78 (C-2′), 103.17 (C-10), 98.17 (C-6), 93.56 (C-8); ESI-MS m/z 301 [M-2H]−, 303 [M + H]+ corresponding to C15H10O7 : δ 6.68 , 6.45 , 6.60 , 7.97 , 7.58 , 3.90 ; 13C–NMR : 162.72, 108.73, 180.23, 163.32, 97.87, 165.57, 96.66, 161.58, 108.50, 132.68, 130.37, 127.35, 132.88, 127.35, 130.37, 56.64; ESI-MS m/z 268 [M + H]+, 267 [M-1H]− corresponding to C16H12O4 [MHQ-4 (Techtochrysin) (5-hydroxy-7-methoxyflavone): Light greenish yellow crystal; mp: 295 °C; U.VC16H12O4 .50 (concentration at which sample kills 50% of the cells) was considered the safe concentration  for the adipocyte cells in order to assess antidiabetic evaluation of test substances. The concentrations at which more than 50% cells found to be alive were selected for an in vitro antidiabetic evaluation. Eight concentrations of each sample (0.78 to 100 μg/mL) had been used to check whether it were safe to the adipocytes cells or not. Results suggested that the T. indica stems ethanol extract inhibited 18.60% cells at its highest concentration (100 μg/mL) which was found to be highly significant (**p < 0.005) in comparison to the control group. So, the ethanol extract was considered safe for the cells at its highest concentration (viz. 100 μg/mL). Similarly, both fractions (hexane and ethyl acetate) were also found to be safe at their highest concentrations viz. 35.27% and 21.40%, respectively, and both varied from the control significantly (**p < 0.005 inhibition)  showed IC50 at 25 μg/mL (51.97% inhibition) and techtochrysin (MHQ-4) showed IC50 at 50 μg/mL (49.40% inhibition). However, the remaining two phenolic compounds (norwogonin and quercetin) were found to be safe up to 100 μg/mL. The percent inhibitions at their highest concentrations of norwogonin (MHQ-2) and quercetin (MHQ-3) were found to be 22.75% and 25.48%, respectively. So, it was concluded that these compounds up to 100 μg/mL can be safely used to evaluate bioactivity due to their safe profile at the tested concentration through MTT assay.Phenolic compounds isolated from the ethyl acetate fraction of T. indica’s stems ethanol extract, its sub-fractions and isolated phenolic compounds from the bioactive fraction were evaluated in order to check whether they can induce adipogenesis in the presence and absence of insulin. Rosiglitazone was taken as positive control which was technically used as a marker for insulin-sensitizing activity of the samples. The results showed that the T. indica stems ethanol (95%) extract, ethyl acetate fraction and three of the four phenolic compounds  showed effects in a dose-dependent manner similar to insulin- like activity and also insulin sensitizing activity. However, non-polar fraction (hexane fraction) and quercetin (MHQ-3) showed no effect on stimulating adipogenesis without insulin. Moreover, both did not exert any insulin sensitizing effect in comparison to rosiglitazone . The results in our study show an increase stimulation of the 2-NBDG uptake in the absence of insulin. It was found out that all three phenolic compounds exerted a dose-dependent effect as the activity was found to increase with the increased concentrations of all three phenolic compounds. Interestingly, it was also found out that 2-NBDG uptake by wogonin was higher than the insulin (control) at its highest concentration i.e. 50 μg/mL. Norwogonin also exhibited 2-NBDG uptake activity higher than the insulin by showing higher fluorescence absorbance at its highest concentration i.e. 100 μg/mL. In the same experiment, rosiglitazone was taken as a positive control to determine insulin-sensitizing effect on 3T3-L1 adipocytes. The results suggested that all three phenolic compounds exert insulin sensitizing effect in a dose-dependent manner. Fluorescence absorbance of the wogonin was slightly less than the rosiglitazone at its highest concentration i.e. 50 μg/mL, however, norwogonin showed more 2-NBDG uptake than the rosiglitazone at its highest concentration i.e. 100 μg/mL. Moreover, techtochrysin also showed 2-NBDG uptake on matured adipocytes, which was, however, less than the wogonin and norwogonin at its highest concentration i.e. 50 μg/mL. Techtochrysin exhibited concentration-dependent 2-NBDG uptake by showing a slightly higher absorbance (0.58) than the insulin at its highest concentration in fluorescence microplate reader. However, techtochrysin did not display any significant insulin sensitizing activity when it was compared with the absorbance of the rosiglitazone Fig. .Fig. 6FlAdipocytes are the major site of insulin action and, thereby, play an important role in glucose metabolism as well as in the regulation of whole-body glucose homeostasis. The widespread epidemics of obesity and type-2 diabetes mellitus (T2DM) suggest that both conditions are closely interconnected. The increased incidence of extreme visceral obesity and obesity-related cardiovascular risk factors are closely related to the rising prevalence of cardiovascular diseases and T2DM , 13. TheT. indica stems ethanol extract, its sub-fractions and isolated compounds from the bioactive fraction in order to check whether they can induce adipogenesis in the presence or absence of insulin. Rosiglitazone, a thiazolidinediones drug, was taken as positive control which is an insulin-sensitizing agent that acts by improving the sensitivity of peripheral tissues to insulin through activating PPAR-γ receptor [T. indica stems ethanol extract, ethyl acetate fraction and three monoflavonoids isolated from the ethyl acetate fraction viz. wogonin, norwogonin and techtochrysin showed effects in a dose dependent manner similar to insulin-like- and insulin sensitizing activities. However, non-polar fraction (hexane fraction) showed no effect on stimulating adipogenesis in the absence of insulin. Moreover, the hexane fraction also did not show any insulin sensitizing effect in comparison to rosiglitazone. Stems ethanol extract, ethyl acetate fraction and three monoflavonoids  at their highest concentration showed absorbance almost similar to control (insulin) and also exhibited similar activity like rosiglitazone.3T3-L1 pre-adipocytes after undergoing the differentiation to adipocytes serve as excellent in vitro models and are considered valuable tools in understanding the glucose metabolism. In recent research studies, 3T3-L1 pre-adipocytes have been extensively used for the adipogenesis. The pre-adipocytes are adherent fibroblast-like in shape and they form confluent monolayer. Pre-adipocytes are differentiated into adipocytes through an inducer known as adipogenic cocktail which includes insulin, phosphodiesterase inhibitor methylisobutylxanthine (IBMX), fetal bovine serum (FBS) and the synthetic glucocorticoid dexamethasone . Our stureceptor , 39. ThePPARγ ligands can affect the adipocyte differentiation and are reported to have an effect on glucose uptake in 3T3-L1 adipocytes . Hence, MTT viability test helps to determine the safe concentration of the tested compounds used in the bioactivity experiments on pre-adipocytes. Hence, in our research study, a total of 8 different concentrations (doses) were initially evaluated and finally 3 safe doses were selected to evaluate their bioactivities in the experiments. All groups were compared with the untreated control group to check the significant differences. It was observed that the insulin control and rosiglitazone control groups were significantly different from the untreated control group. As the mechanism of action for insulin sensitizer is to increase the insulin activity, we treated rosiglitazone along with insulin to observe the insulin-sensitizing activity. Moreover, the treatment groups without insulin were compared with the insulin groups to understand the insulin-like activity of the groups whereas the groups containing the insulin and sample (compounds/extracts) were compared to rosiglitazone group to check the insulin-sensitizing activity. It was observed that the effect of adipogenesis was manifested in a dose dependent manner. The ethanol extract, ethyl acetate fraction, wogonin, norwogonin and techtochrysin showed insulin like activity. Moreover, ethanol extract, ethyl acetate fraction, wogonin, norwogonin and techtochrysin exhibited activity similar to insulin at their highest dose. The groups also showed same trend when they were compared with rosiglitazone-insulin combination. In contrast, however, the hexane fraction and quercetin showed no significant adipogenesis when they were evaluated without insulin. Additionally, no insulin sensitizing activity was observed with the hexane fraction and quercetin either.T. indica stems ethanol extract, ethyl acetate fraction, wogonin, norwogonin and techtochrysin exert insulin sensitizing activity in the same manner as rosiglitazone does and therefore are potential antidiabetic substances.Our study showed an increase stimulation of the 2-NBDG uptake in the absence of insulin. We can see a dose-dependent increase for all the three phenolic compounds. All three compounds  exerted a dose-dependent activity as the activity was found to increase with the increased concentration of all compounds. Interestingly, it was also observed that, 2-NBDG uptake by wogonin was higher than the insulin control at its highest concentration (50 μg/mL). Same results were manifested by the norwogonin as well. The fluorescence absorbance at the highest concentration i.e., 100 μg/mL was found to be much higher than the insulin. In the same experiment, rosiglitazone was taken as positive control to determine insulin-sensitizing effect on 3T3-L1 adipocytes. The results suggested insulin sensitizing effect on a dose dependent manner for wogonin and norwogonin. Fluorescence absorbance of the wogonin was slightly less than the rosiglitazone at its highest concentration (50 μg/mL), however, norwogonin showed more 2-NBDG uptake than the rosiglitazone at its highest concentration (100 μg/mL). This suggests strong antidiabetic potential for both the monoflavonoid aglycones. Moreover, techtochrysin also exhibited 2-NBDG uptake on matured adipocytes, however, less significant than the wogonin and norwogonin at its highest concentration i.e. 50 μg/mL. Hence, it can be summarised that T. indica stems ethanol extract possess antidiabetic effect on 3T3-L1 adipocytes. Wogonin and norwogonin were found to reveal significant insulin-like and insulin-sensitizing activities at their safe concentrations during adipogenesis. Furthermore, both monoflavonoids displayed significant increase in stimulating 2-NBDG in the absence of insulin. Norwogonin showed higher 2-NBDG uptake activity than the rosiglitazone. This clearly proves insulin-sensitizing ability of these two monoflavonoids. Our study suggests antidiabetic potential of the isolated compounds of T. indica in terms of insulin-like and insulin-sensitizing effects. However, we still suggest further in depth research study on the isolated compounds that might lead to the discovery of efficacious antidiabetic drugs.The present study has shown that wogonin, norwogonin and techtochrysin isolated from


The etiology of Kawasaki disease (KD) remains unknown. However, many studies have suggested that specific genetic factors and/or some infectious agents underlie the onset of KD. Previous studies have suggested that human adenovirus (HAdV) is one of the triggering pathogens of KD. Here, we report monozygotic twin boys who sequentially developed KD in conjunction with acute HAdV type 3 (HAdV-3) infection.The patients were four-year-old monozygotic twin boys. The elder brother developed a high fever and was diagnosed with HAdV infection with an immunochromatographic kit for HAdV (IC-kit). He was transferred to our institute after persistent fever for 7 days. On admission, he already fulfilled all the diagnostic criteria for KD. His laboratory data were as follows: WBC, 9700/μl; CRP, 2.42 mg/dl; IFN-γ, 99.8 pg/ml; and TNF-α, 10.9 pg/ml. He received intravenous immunoglobulin (IVIG) and aspirin and responded well, with no coronary artery abnormalities. The younger brother, who was also IC-kit-positive, was hospitalized on the same day as his elder brother after persistent fever for 3 days. His data on admission were as follows: WBC, 12,600/μl; CRP, 5.54 mg/dl; IFN-γ, 105.0 pg/ml; and TNF-α, 33.6 pg/ml. Although he developed all of the typical KD symptoms by day 4, his fever subsided spontaneously on day 6 without IVIG or aspirin. However, he developed a dilation of the coronary artery in the region of the left circumflex artery bifurcation on day 10. His coronary artery dilation had resolved 3 months after onset. HAdV-3 DNA was detected with PCR in stool samples from both patients, and HAdV3 was isolated from the younger brother’s stool sample. Serum neutralizing antibodies to AdV3 were also significantly elevated in both patients, suggesting seroconversion.There have been few reports of the simultaneous development of KD in monozygotic twins. Notably, both twins had an acute HAdV-3 infection immediately before they developed KD. These cases strongly suggest that KD was triggered by HAdV-3 infection, and they indicate that specific immune responses to some pathogens (such as HAdV-3), arising from genetic susceptibility, play a critical role in the pathogenesis of KD. Kawasaki disease (KD) is an acute systemic vasculitis syndrome, first reported by Dr. Tomisaku Kawasaki in 1967 , 3. PrevThe patients were four-year-old monozygotic twin boys who had previously been healthy and had no family history of KD.®adeno, TAUNS Laboratories Inc. Shizuoka, Japan). His laboratory data were: white blood cells (WBC), 9700/μL ; hematocrit, 38.8%; platelet count, 35.7 × 104/μL; albumin, 4.1 g/dL; total bilirubin, 0.5 mg/dL; sodium, 130 mEq/L; aspartate aminotransferase (AST), 31 IU/L; alanine aminotransferase (ALT), 16 IU/L; and C-reactive protein, (CRP) 2.4 mg/dL. His serum cytokine profile was: granulocyte-colony stimulating factor (G-CSF), 384.0 pg/mL; interferon-γ (IFN-γ), 99.8 pg/mL; interleukin-6 (IL-6), 43.8 pg/mL; IL-8, 36.9 pg/mL; IL-18, 1200.6 pg/mL; tumor necrosis factor (TNF-α), 10.9 pg/mL; soluble tumor necrosis factor receptor 1 (sTNFR-1), 1106.9 pg/mL; and sTNFR-2, 10,013.4 pg/mL ; hematocrit, 36.8%; platelet count, 39.2 × 10Fecal samples were collected from both patients during the acute phase of the disease. These samples were tested by PCR assays, and HAdV-3 genomes were detected in the samples from both patients. Additionally, HAdV-3 was directly isolated from the younger brother’s fecal sample. Both patients were negative for serum neutralizing antibodies against HAdV-3 upon admission, but these antibodies were significantly elevated in both patients 2 weeks after their admission, suggesting that they underwent seroconversion. The patients’ father and elder sister also developed clinical symptoms consistent with acute HAdV infection around the time that the twins were infected.Here, we have presented a case of 4-year-old monozygotic twins who sequentially developed KD in conjunction with HAdV-3 infection. There have been few case reports of monozygotic twins who developed KD simultaneously, and only two of these reports identified the trigger of KD Table  5–10].T–10.T5–10ITPKC) and caspase 3 (CAPS3) significantly increase susceptibility to KD [It has recently been presumed that the etiology of KD combines genetic susceptibility and specific infection, both of which are essential for KD development. Patients with KD seem to have some genetic predisposition, and ethnic differences in the morbidity and familial aggregation of KD have been reported . Some futy to KD , 12, 13.Staphylococcus, Bacillus cereus, and Yersinia have all been reported as triggering pathogens of KD. In general, KD most commonly develops in infants, toddlers, and young children; adult patients are rarely reported [HAdV, group A streptococcus, reported . Most chHAdV infection itself could be one of the differential diagnoses of KD. HAdV infection also shows KD-like symptoms such as conjunctival injection, red cracked lips, and cervical lymphadenopathy. However, skin erythema and swollen red palms and soles followed by desquamation are distinctive features of KD. Additionally, a coronary lesion can allow the definitive diagnosis of KD. In our case, the younger brother’s echocardiography revealed a dilation of the left circumflex artery, so we definitively diagnosed him with KD.Several previous reports show that HAdV was detected by PCR in samples from the respiratory tract of patients with KD –17. HoweOur report contributes further evidence that a specific response to a pathogen such as HAdV, combined with genetic susceptibility, plays an essential role in the development of KD.


The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses.Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus  in vitro and in ferrets is described. This A/H5N6 virus possessed high polymerase activity, mediated by the E627K substitution in the PB2 protein, which corresponds to only one biological trait out of the three that were previously shown to confer airborne transmissibility to A/H5N1 viruses between ferrets. This might explain its lack of airborne transmission between ferrets. After intranasal inoculation, A/H5N6 virus replicated to high titers in the respiratory tracts of ferrets and was excreted for at least 6 days. Moreover, A/H5N6 virus caused severe pneumonia in ferrets upon intratracheal inoculation. Thus, A/H5N6 virus causes a more severe disease in ferrets than previously investigated clade 2.3.4.4 viruses, but our results demonstrate that the risk from airborne spread is currently low.Since their emergence in 1997, A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage have diversified in multiple genetic and antigenic clades upon continued circulation in poultry in several countries in Eurasia and Africa. Since 2009, reassortant viruses carrying clade 2.3.4.4 hemagglutinin (HA) and internal and neuraminidase (NA) genes of influenza A viruses of different avian origin have been detected, yielding various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8. Previous studies reported on the low pathogenicity and lack of airborne transmission of A/H5N2 and A/H5N8 viruses in the ferret model. However, although A/H5N6 viruses are the only clade 2.3.4.4 viruses that crossed the species barrier and infected humans, the risk they pose for human health remains poorly characterized. Here, the characterization of A/H5N6 A/Guangzhou/39715/2014 virus IMPORTANCE Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus in vitro and in ferrets. The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses. Highly pathogenic avian influenza (HPAI) A/H5 viruses of the A/goose/Guangdong/1/1996 (GsGd) lineage first emerged in Hong Kong in 1997 . MoreoveIn contrast to A/H5N8 viruses, which spread worldwide in a global fashion via wild migratory birds , the dis6–A/H5N6 viruses are the only clade 2.3.4.4 viruses that have crossed the species barrier and infected humans. The first human cases of infection with the two circulating lineages of A/H5N6 viruses were both reported in China , 10. As The global spread via wild birds and complex reassortment history of clade 2.3.4.4 viruses required a detailed molecular and phenotypic characterization, especially of the newly emerged A/H5N6 viruses. Several research groups previously reported on the low pathogenicity and lack of airborne transmissibility in the ferret model of the Korean, European, and North American A/H5N8 viruses and North American A/H5N2 viruses, probably partially due to the presence of internal genes originating from wild bird LPAI viruses –16. In c–In vitro characterization of phenotypes that have been associated with airborne (respiratory droplets and aerosols) transmission of A/H5N1 viruses, such as receptor binding preference, thermostability, pH stability, and polymerase activity was performed. The potential of A/H5N6 GZ/14 to transmit via the airborne route between ferrets was assessed, as well as its pathogenicity in the ferret model. While our study suggests that the public health risk posed by this A/H5N6 virus is low, the A/H5N6 GZ/14 showed a high polymerase activity mediated by the E627K substitution in PB2, replicated to higher titers in the respiratory tracts of ferrets and was more pathogenic than a clade 2.3.4.4 A/H5N8 virus  virus, which belongs to genotype G1 and caused one of the first human cases of A/H5N6 infection in 2014 . In vitrVibrio cholerae neuraminidase [VCNA]), and either α2,3-SA or α2,6-SA were subsequently rebuilt using specific sialyltransferases. Both control viruses, the avian A/H5N1 virus A/Indonesia/5/2005 (IN/05) and the human A/H3N2 virus A/Netherlands/213/2003 (NL/03), displayed the expected receptor attachment pattern by binding exclusively to α2,3-SA and α2,6-SA, respectively  and α2,6-linked sialic acids , respectively. In order to assess the receptor specificity of A/H5N6 GZ/14, a resialylated red blood cell assay was performed. Briefly, SA were stripped off turkey red blood cells (TRBCs) upon sialidase treatment  as an internal control, and luciferase (firefly) as a reported gene. The experiment was performed at 33°C and 37°C. Polymerase activities were expressed as a percentage of the polymerase activity of the human A/H1N1 virus, HK/98, which was set at 100% for both temperatures. At 33°C, the polymerase activity of the A/H5N6 GZ/14 was higher than that of other viruses tested, including A/H5N1 IN/05 and A/H5N8 ck/NL/14  and the nucleoprotein (NP) of different GsGd viruses, human A/H1N1 virus or avian A/H5N2 virus, luciferase (ck/NL/14 . At 37°Cck/NL/14 –22. In o5.25 50% tissue culture infective doses (TCID50)/ml, and long-term, until 7 days postinoculation (dpi)  . Upon ex6 TCID50 of virus. Daily, the animals were monitored for body weight and clinical signs. Nose and throat swabs were also collected daily. At 3 and 6 dpi, three animals were sacrificed, and necropsies were performed. Data from this experiment were compared to historical data on intranasal challenge of ferrets with A/H5N8 ck/NL/14 and A/H5N1 IN/05  . This boThe shedding of A/H5N6 GZ/14 from the respiratory tracts of ferrets, as measured by virus titration of throat and nose swab samples, was high and prolonged, similar to that of A/H5N1 IN/05, and longer and higher in titers than those of A/H5N8 ck/NL/14 .8.3 and 106.8 TCID50/g of tissue, respectively) (7.1 TCID50/g of tissue) in the lung of one animal. A/H5N6 GZ/14 was also detected at 3 dpi in extrarespiratory tissues such as the olfactory bulb (mean titers of 104.2 TCID50/g of tissue). At 6 dpi, A/H5N6 GZ/14 was detected only in the lung of one animal, which was the animal that had lost the most weight (18%)   and D. Mht (18%) . The lowht (18%) .At 3 dpi, high titers in the nasal turbinates correlated with the presence of virus antigen in the caudal respiratory epithelium and more abundantly in the olfactory epithelium in the nose of all ferrets ; Table 2At 6 dpi, virus antigen distribution in the nasal epithelium (respiratory and olfactory) was similar to that of animals euthanized at 3 dpi albeit with fewer positive cells . Virus a6 TCID50 of virus. At 1 dpi, all animals were alert, but they were playful only after stimulation (activity status [AS] of 1). At 2 dpi, in the morning, one ferret was found dead, while the two other ferrets were less active than the day before (AS of 2). Later in the afternoon, these two ferrets were neither alert nor playful when stimulated (AS of 3). One animal presented with severe dyspnea and succumbed to the infection shortly after. The other ferret was very weak, demonstrated swollen conjunctiva and mild dyspnea and was euthanized because of its poor condition. The animal that died at 1 dpi had lost 6% of its starting body weight, and the other two ferrets had lost 12% and 10% of their starting body weights.Most humans infected with an HPAI A/H5 GsGd virus developed severe pneumonia resulting in acute respiratory distress syndrome. Differences in virus tropism and pathogenicity after different routes of inoculation of ferrets have already been reported , and it 7.2 TCID50/g of tissue)  .Gross pathological analysis revealed a severe diffuse to coalescing pulmonary consolidation affecting 90 to 100% of the lungs of all animals, characterized by dark red and depressed lung parenchyma. The RLWs of these ferrets were between 1.3% and 1.6% . The ferTo study the antigenic relationship between A/H5N6 GZ/14 and other clade 2.3.4.4 viruses and the neutralization of A/H5N6 GZ/14 by postinfection antisera raised against available candidate vaccine viruses (CVVs), we performed a hemagglutination inhibition (HI) assay . Apart fin vitro and in vivo characterization of the clade 2.3.4.4 A/H5N6 GZ/14 virus in order to evaluate the risk for humans. Sequence analysis of A/H5N6 GZ/14 virus revealed a few amino acid substitutions that have previously been described as mammalian adaptation markers for other A/H5 viruses of the GsGd lineage: 94N   , 22, 27.ct (URT) , 29: a cUsing a resialylated TRBC assay, A/H5N6 GZ/14, as well as A/H5N8 ck/NL/14, was shown to display a typical pattern of avian influenza viruses by binding exclusively to α2,3-SA. These data are in accordance with previous studies, in which A/H5N6 GZ/14 and another human A/H5N6 isolate, A/Sichuan/26221/2014, were found to bind only to α2,3-SA , 31. IntHA acid stability has been described as an important host range factor, associated with adaptation of avian and swine influenza viruses to the human host, airborne transmission of avian viruses, and pandemic potential , 34–37. 34–Using a minireplicon assay, we demonstrated that A/H5N6 GZ/14 possessed a high polymerase activity, which was mediated by E627K in PB2. Out of the 16 A/H5N6 human isolates, seven isolates carry the PB2-E627K, and one isolate, A/Sichuan/26221/2014, carries the PB2-D701N substitution, which has also been associated with increased replication and transmission in mammalian hosts , 41. Acqin vitro assays, we predicted that A/H5N6 GZ/14 would not be transmissible via the airborne route, which was confirmed using the ferret model. Ferrets are used as animal models to study airborne transmission of influenza viruses, as it was shown that human influenza viruses transmit via the airborne route between ferrets, while avian influenza viruses do not, corresponding to what is also observed in humans  (Lonza), 1.5 mg/ml sodium bicarbonate (NaHCO3) (Lonza), 10 mM HEPES (Lonza) and 1× nonessential amino acids (NEAA) (Lonza). 293T cells were cultured in Dulbecco modified Eagle’s medium (DMEM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 U/ml STR, 2 mM l-Glu, 1 mM NaHCO3, and 1× NEAA. Vero cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 mg/ml STR, and 2 mM l-Glu.Madin-Darby canine kidney (MDCK) cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Greiner), 100 U/ml penicillin (PEN) (Lonza), 100 U/ml streptomycin (STR) (Lonza), 2 mM in vivo experiments, a full recombinant A/H5N6 GZ/14 virus was used. For in vitro experiments , recombinant viruses containing six gene segments of A/Puerto Rico/8/1934 (PR/34), the HA without the multibasic cleavage site (MBCS) and the NA of A/H5N6 GZ/14 or A/H5N8 A/chicken/Netherlands/EMC-3/2014 (ck/NL/14) were used. Control viruses for thermostability and resialylated red blood cell assays were recombinant viruses containing seven gene segments of PR/34 and the corresponding HA without the MBCS (if applicable). Prototypic viruses used in hemagglutination inhibition assay were recombinant viruses containing six gene segments of PR/34, the HA without the MBCS, and the NA of the corresponding virus with the exception of the A/goose/Eastern China/1112/2011 and A/gyrfalcon/Washington/41088-6/2014, which was a recombinant virus carrying seven genes of PR/34 and the corresponding HA. Virus stocks were propagated up to three times in MDCK cells and titrated in MDCK cells as described below. The sequences of the virus stocks used for every experiment were checked using Sanger sequencing. Virus names were abbreviated as follows: A/Indonesia/5/2005 (IN/05), A/Netherlands/213/2003 (NL/03), A/Mallard/Sweden/49/2002 (ml/SW/02), A/Hong Kong/156/1997 (HK/97), A/Vietnam/1194/2004 (VN/04), A/turkey/Turkey/1/2005 (tk/TK/05), A/Anhui/1/2005 (AN/05), A/gyrfalcon/Washington/41088-6/2014 (gy/WAS/14), and A/goose/Eastern China/1112/2011 (gs/EC/11).All eight gene segments of a human virus isolate, A/H5N6 A/Guangzhou/39715/2014 (GZ/14), were amplified from copy DNA by PCR using specific primers and cloned in a modified version of the bidirectional reverse genetics plasmid pHW2000 . Recombil-Glu, 1.5 mg/ml NaHCO3, 10 mM HEPES, 1× NEAA, and 20 μg/ml trypsin . Three days after inoculation, supernatants of cell cultures were tested for agglutinating activity using TRBCs as an indicator of virus replication. Infectious virus titers were calculated from 4 replicates each of the homogenized tissue samples, nose swabs, and throat swabs and from 10 replicates of the virus stocks by the method of Reed and Muench  1 h after inoculation and cultured in infection medium, consisting of EMEM supplemented with 100 U/ml PEN, 100 U/ml STR, 2 mM d Muench .Ferrets were housed and experiments were conducted in strict compliance with European guidelines  and Dutch legislation . All animal experiments were approved by the independent animal experimentation ethical review committee “stichting dier experimenten commissie (DEC) consult” (Erasmus MC permit number EMC4028). Research projects involving laboratory animals can be executed only if they are approved by the animal experiments committee (DEC). The DEC considers the application and pays careful attention to the effects of the intervention on the animal and its discomfort and weighs this against the social and scientific benefit to humans or animals. The researcher is required to keep the effects of the intervention to a minimum, based on the three R’s .6 TCID50 of virus distributed into the two nostrils (250 µl per nostril). Each donor ferret was then placed in a transmission cage. One day after inoculation, one naive recipient ferret was placed opposite of each donor ferret. Each transmission pair was housed in a separate transmission cage designed to prevent direct contact but to allow airflow from the donor ferret to the recipient ferret. Nose and throat swabs were collected at 1, 3, 5, and 7 days postinoculation (dpi) from donor ferrets and at 1, 3, 5, 7, and 9 days postexposure (dpe) from the recipient ferrets and subjected to endpoint titration in MDCK cells. Donor ferrets were euthanized at 7 dpi and recipient ferrets were euthanized at 14 dpe to allow assessment of seroconversion.Four ferrets were inoculated intranasally with a total dose of 106 TCID50 of A/H5N6 GZ/14 by applying 250 μl of virus suspension dropwise to each nostril. Clinical scores in all groups were assessed every day. Activity status was scored as follows: 0, alert and playful; 1, alert and playful only when stimulated; 2, alert but not playful when stimulated; 3, neither alert nor playful when stimulated. Dyspnea was characterized by open-mouth breathing with exaggerated abdominal movement  were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE) for examination by light microscopy.Six ferrets were inoculated intranasally with a total dose of 10movement . Body wemovement . Tissues6 TCID50. The experimental design and procedures were similar to those performed for the intranasal inoculation.In a second experiment, three ferrets were inoculated intratracheally with 3 ml of virus dilution containing 10For detection of influenza A virus antigen, sequential slides of all tissues were stained with a primary antibody against the influenza A virus nucleoprotein (NP) as described previously . In eachVibrio cholerae NA (VCNA) (Roche) in 8 mM calcium chloride (CaCl2) at 37°C for 1 h. After two washing steps with PBS, VCNA-treated TRBCs were resuspended in PBS containing 1% bovine serum albumin (BSA) to a final concentration of 0.5% TRBCs. Removal of SA was confirmed by observation of a complete loss of hemagglutination of the TRBCs by control influenza A viruses. Subsequently, resialylation was performed using 0.5 mU of α2,3-N-sialyltransferase (Sigma) or 25 mU of α2,6-N-sialyltransferase (Sigma) and 1.5 mM CMP-sialic acid (Merck) at 37°C for 2 h in a final volume of 75 μl to produce α2,3-TRBCs and α2,6-TRBCs, respectively. After two washing steps, the TRBCs were resuspended in PBS containing 1% BSA to a final concentration of 0.5% TRBCs. Resialylation was confirmed by hemagglutination of viruses with known receptor specificity: A/H5N1 IN/05 and A/H3N2 NL/13. The receptor specificity of A/H5N8 ck/NL/14 and A/H5N6 GZ/14 was tested by performing a standard hemagglutination assay with the modified TRBCs.All sialic acids (SA) were removed from the surfaces of TRBCs by incubating 1% TRBCs in PBS with 50 mU of l-Glu. The next day, the cells were fixed using 80% ice-cold acetone, washed, and stained using a 20% Giemsa solution . Visual inspection of the cell cultures for the presence of syncytia (multinucleated cells) was used to determine the pH threshold required for fusion.Influenza virus HA-induced cell fusion was tested in Vero-118 cells transfected with 5 μg of HA cloned in pCAGGs expression plasmid using Xtremegene transfection reagent according to the manufacturer’s instructions (Roche). One day after transfection, the cells were harvested using trypsin-EDTA and plated in 24-well plates. The next morning, the cells were washed, and medium was replaced with IMDM (Lonza) containing 10 μg/ml of trypsin. After 1 h, the cells were washed with PBS and exposed to PBS at pH 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, or 5.9 for 10 min at 37°C. Subsequently, the PBS was replaced by IMDM supplemented with 10% FBS, 100 U/ml PEN, 100 mg/ml STR, and 2 mM In short, viruses were diluted to 128 HA units (HAU)/25 μl using PBS. The samples were incubated in a thermal cycler for 30 min at temperatures of 50°C, 51.7°C, 54.3°C, 56°C, 58.5°C, and 60°C. Subsequently, the HA titer was determined by performing a hemagglutination assay using TRBCs.Renilla gene under the cytomegalovirus [CMV] promoter) together with the mix of PB2, PB1, PA, and NP plasmids, cloned in a bidirectional reverse genetics plasmid, in quantities of 125, 125, 125, and 250 ng, respectively. The transfected cells were incubated at 33°C or 37°C. After 24-h incubation, the supernatants were discarded, and the cell extracts were prepared in 100 μl of lysis buffer. The luciferase levels were assayed with luciferase assay system (Promega) and detected by a luminometer. Polymerase complexes of control viruses were included in the assay: a human H1N1 virus A/Hong Kong/54/1998 (HK/98), two human A/H5N1 viruses A/Vietnam/1203/2004 (VN/1203/04) and A/Indonesia/5/05 (IN/05), and an avian virus A/H5N2 A/mallard/Netherlands/3/1999 (ml/NL/99).293T cell monolayers were transfected with 125 ng of luciferase reporter plasmid (firefly open reading frame flanked by the noncoding regions of the M segment under control of a PolI promoter) and 12.5 ng of an internal control plasmid . Although all experiments are conducted in closed class 3 cabinets and isolators, special personal protective equipment, including laboratory suits, gloves, and FFP3 facemasks, is used. Air released from the class 3 units is filtered by high-efficiency particulate air (HEPA) filters and then leaves via the facility ventilation system, again via HEPA filters. Only authorized personnel that have received the appropriate training can access the ABSL3+ facility. For animal handling in the facilities, personnel always work in pairs. The facility is secured by procedures recognized as appropriate by the institutional biosafety officers and facility management at Erasmus MC and Dutch and U.S. government inspectors.All experiments were conducted within the enhanced animal biosafety level 3 (ABSL3+) facility of Erasmus MC. The ABSL3+ facility consists of a negative-pressure laboratory (−30 Pa) in which all


Here we report a new approach to create highly permeable and selective nanofiltration membranes coated with microporous poly-p-xylylene nanofilms fabricated through a dry chemical vapor deposition process by using [2.2]paracyclophanes derivatives on ultrafiltration membranes. The introduction of crosslinking points into rigid poly-p-xylylenes enhanced microporosity and mechanical strength due to insufficient packing and depression of structural relaxation among polymer chains in three-dimensional networks. Crosslinked nanofilms with thicknesses down to 50 nm showed outstanding permeability for water and alcohols at a pressure difference of 0.5 MPa and exhibited higher rejection ratios for water-soluble organic dyes than non-crosslinked nanofilms. Poly-p-xylylene nanofilms also showed an excellent blocking property for non-polar organic solvent permeation through specific interaction of hydrophilic pores with organic solvents.Poly- To enhance its performance, high permeability and selectivity are required. Conventional polymer separation materials have a performance trade-off between the permeability and the selectivity. The target small molecules can penetrate selectively by passing through pores in the dense packing of rigid polymer chains. However, the dense packing of rigid polymers results in a poor permeability. To overcome this trade-off, the polymer has been designed at the molecular level, and the separation layer has been made thinner to provide sufficient permeability and high selectivity for membranes6.Membrane separation process has been widely used in various application fields including water purification, gas separation, and processing in the pharmaceutical and food industries owing to its high-energy efficiency9. The inefficient packing among contorted and rigid MOP chains creates intrinsic voids of less than 2 nm diameter, and thin MOP layers provide both the permeability and selectivity to support scalable separation processes15. Recently, Livingston et al. succeeded in fabricating defect-free crosslinked-polyester nanofilms on porous supports made by the interfacial polymerization16. The crosslinked nanofilms with controlled microporosity showed a much better separation performance in organic solvents than films made from non-contorted planar monomers. The crosslinking of contorted polymers in the nanofilms enabled the permeability of organic solvents to be enhanced while maintaining the membrane selectivity. Although the interfacial polymerization has been used as a simple and controllable technique to prepare the crosslinked polymer films in reverse osmosis desalination and nanofiltration (NF)4, the ester or amide linkages in these condensation polymers can be broken by hydrolysis and oxidation. Therefore, clean and chemically stable MOP nanofilms that have high permeability and selectivity are still a challenge to fabricate.Microporous organic polymers (MOPs) are a notable candidate for highly permeable and selective membranesp-xylylenes (Parylene), in which benzene rings are connected by ethylene linkers, have been widely used as protective coatings of printed circuits and medical devices, barrier layers of metal surfaces to prevent corrosion, and lubricants17. This polymer can be prepared by using a chemical vapor deposition (CVD) process of low-molecular-weight [2.2]paracyclophanes18. In this process, two p-xylylene monomers are formed by vacuum vapor pyrolysis of [2.2]paracyclophane above 550 °C, and the reactive monomers polymerize into pure poly-p-xylylenes on the surface of targets at room temperature in a vacuum ambient. This self-initiated process enables uniform poly-p-xylylene films to form on the target surfaces without voids or pinholes. Conformal coating of various devices with poly-p-xylylene films exhibits outstanding barrier properties for gasses, water, and organic solvents owing to dense packing of rigid polymer chains. If well-defined pore structures can be created in poly-p-xylylene nanofilms fabricated by the CVD dry process, microporous nanofilms are potentially useful materials for reliable and clean membranes that have selective separation functions.All-carbon-structural and high-molecular-weight poly-p-xylylenes without using wet processes. We used the CVD process with [2.2]paracyclophane derivatives to prepare uniform crosslinked poly-p-xylylene nanofilms less than 50 nm thick on ultrafiltration (UF) supports19. The polymerization of [2.2]paracyclophanes that have polar substituents can create interconnected hydrophilic micropores within the nanofilms. We measured the permeance of water and alcohols as well as the retention of various dye molecules. The crosslinked network of poly-p-xylylenes diminishes the formation of crystalline domains among rigid polymers, and enables the rapid permeance for organic solvents by enhancing of microporosity. In addition, the nanofilms exhibit an excellent separation performance for solutes.Here, we report a new versatile approach to fabricate microporous nanofilms on the basis of poly-1 and 2 that have one or two nitriles  or organic solvents  at room temperature. The film thickness deposited on Si wafers is proportional to the initial amount of 1 or 2  annihilation lifetime spectroscopy (PALS) , which is generated by exposing an insulating material to a positron source 22Na, correlates with the average free volume size in materials. The o-Ps lifetimes of 1.74 and 1.66 ns for 3 and 4 correspond to the average diameters of 0.52 and 0.50 nm. These suggest that the CVD polymerization of 1 and 2 can create free volumes less than 1 nm in diameter within poly-p-xylylene nanofilms.Since polar nitrile groups can form hydrogen bonds with water molecules, we designed and synthesized two [2.2]paracyclophanes s Figure 20. Synthr 2 Fig. . Figure LS) Fig. 21,22. Thp-xylylene films 3 and 4 were characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analyses. The FT-IR spectrum of 4 on the Si wafer showed two characteristic bands at 2220 and 1730 cm−1 assigned to C≡N and C = O stretching vibrations  and 4 (83.3 ± 1.5°) were less than 90°, and 4 has a lower contact angle than 3 . From these results, nitrile groups in 1 or 2 were partially oxidized into carboxylic acid during vacuum vapor pyrolysis process, and the films 3 and 4 exhibit hydrophilicity because of the introduction of polar nitrile and carboxylic acid as a substituent of poly-p-xylylenes.The chemical compositions of the poly-ons Fig. , whereasn 3 Fig. . From t3 showed a peak at 2θ = 19.5° with a shoulder peak at 2θ = 17.0°  in the (020) plane and a hexagonal crystal structure (β phase) in the (400) plane, respectively24. The other film 4 displayed a single reflection peak at 2θ = 22.4°. Both films 3 and 4 were a semi-crystalline material containing crystalline domains made from the packing of rigid polymer chains. The packing of polymer chains in 3 and 4 is anticipated to obstruct the permeability of small molecules through poly-p-xylylene nanofilms.The wide-angle X-ray scattering (XRD) pattern of .0° Fig. . These tp-xylylene chains, we designed the crosslinked polymer network by using a [2.2]paracyclophane dimer 5 nickel(0) (Ni(cod)2) in 76% yield. In accordance with the simulated optimized structure of 5, two [2.2]paracyclophane units in 5 were connected with a twisted biphenyl bridge, and the torsional angle between two units is estimated to be 63°  by using the dead-end membrane filtration system. No permeation of water could be detected for the UF membrane with hydrophobic Parylene C nanofilm over a period of one week at a pressure difference of 0.5 MPa. In contrast, water permeation of a membrane with 41-nm-thick nanofilm 3 could be readily observed at the same pressure difference, and the average water permeance was 1.3 ± 0.3 L m−2 hr−1 MPa−1 ) to measure permeability for water and organic solvents Fig. . The scalms Fig. . The peress Fig. 26, and wMPa Fig. . Nanofilp-xylylene nanofilms, four water-soluble dyes that had different molecular sizes and charges were used as probes27. The rejection ratios (R) of four dyes were determined in accordance with the equation (Cf-Cp)/Cf × 100, where Cf and Cp are the dye concentrations of the feed and permeate sides of the dead-end membrane filtration system 2 + (1 − a/r)4] × 100, where R is the rejection ratio, a is the radius of the dye, and r is the radius of the free volume28. The r for 3 and 4 were estimated to be less than 0.6 nm. These r values were almost double of the free volume sizes for dried samples determined by PALS, suggesting the enlargement of free volume size by filling with water. The molecular weight cut-off (MWCO) for 3 and 4 were 1500 g/mol, determined from the rejection measurements of polyethylene glycols with different molecular weights 29.The membranes coated with crosslinked nanofilms al Table . This ca7. Use of solvent-resistant nanofiltation membranes for non-aqueous mediums holds strong potential for food, refining, and pharmaceutical industries30. Methanol achieved the highest permeance of 54.5 ± 5.0 L m−2 hr−1 MPa−1, and the permeance of alcohols decreased as the molecular size increased 5. On the other hand, n-hexane did not pass through this membrane, suggesting that the nanofilm 7 rejected the penetration of non-polar n-hexane. When n-hexane was dispersed into water as an emulsion by using sodium lauryl sulfate (SLS), the filtrate that passed through the membrane coated with 7 did not contain n-hexane and SLS as monitored by FT-IR . The nanofilm 7 can separate water from the mixtures of alkanes and water in the presence of surfactants .We also investigated the permeation properties of organic solvents  for the membranes coated with a 41-nm-thick nanofilm -IR Fig. . The na29 and the dye rejection ratio was decreased by reducing of the film thickness. These were mainly due to the packing of linear polymer chains and the fluctuation of polymer chains. To achieve high water permeance while keeping selectivity high, we introduced the crosslinking points within poly-p-xylylene nanofilms by using twisted [2.2]paracyclophane dimer 5. The introduction of twisted crosslinking points into poly-p-xylylenes led to enhanced microporosity as well as improved mechanical strength. The membranes coated with thinner crosslinked MOP nanofilms offer outstanding performance in terms of nanofiltration in water and alcohols. Moreover, we found the blocking property of non-polar organic solvent permeation through the nanofilms, suggesting the specific interaction of hydrophilic pores with organic solvents. The nanofilms efficiently recovered water from an oil-water emulsion. Efficient membrane separation of oil and water can contribute to minimamize chemical processes in oil recovery and water purification31. Since free volume size can be tuned by modifying substituents in [2.2]paracyclophane, robust and clean nanofilms fabricated by the CVD process have a great potential to realize efficiently separate membranes for various target molecules.We have demonstrated the deposition of defect-free MOP nanofilms onto UF membranes fabricated by a dry CVD polymerization process of [2.2]paracyclophanes that have polar substituents. Although the ultrathin nanofilms worked as selective layers for aqueous solution that had a sharp molecular size cutoff in accordance with their free volume sizes, their water permeance was much lower than those of previously reported membranes3 and 4 were analyzed by positron annihilation lifetime spectroscopy system (Fuji Imvac. PALS-200A). The pore size in the nanofilms was obtained from the annihilation lifetime-positron count curve according to the reported method32. The Vickers hardness (HV) values of a 50 nm-thick deposited on Si wafers were determined by dynamic ultra microhardness tester (Shimazu DUH-211) with Vickers indenter.Surface morphologies of nanofilms were characterized using atomic force microscopy (AFM) images in non-contact mode by a JEOL JSPM-5400 system and field-emission scanning electron microscope (FE-SEM) images recorded in a Hitachi S-5000 FE-SEM with Pt sputter coating. IR spectra were obtained on a Shimazu IR Prestige-21 with DuraSample IR II. The crystalline structure of the samples was analyzed using wide-angle X-ray diffraction (Rigaku XRD-DSC) with Cu Kα radiation. The chemical compositions of nanofilms were analyzed using X-ray photoelectron spectroscopy . The free volume sizes of dry nanofilms 1: 4-Bromo[2.2]paracyclophane was synthesized by standard iron-catalyzed bromination of [2.2]cyclophane in 76% yield33. The mixture of 4-bromo[2.2]paracyclophane  and CuCN  in 10 ml 1-methyl-2-pyrrolidone (NMP) was heated at 220 °C with stirring for 20 h under N2. After cooling, the reaction mixture was poured into 10 ml of 25 wt% aqueous ammonia solution and stirred for one night. The solid in the mixture was dissolved in CH2Cl2 and the organic layer was washed with 20 ml water for three times. The organic layer was dried with MgSO4 and evaporated. The product was purified by column chromatography  and sublimination to give a white solid. Yield 0.10 g (50%). 1H-NMR : δ = 6.90 , 6.75 , 6.52 , 6.57 , 6.52 , 6.48 , 3.54–3.47 , 3.32–3.26 , 3.16–2.99 . 13C-NMR : δ = 143.2, 139.9, 138.5, 138.0, 136.1, 135.7, 133.5, 132.5, 131.8, 131.6, 129.9, 117.9, 113.8, 34.3, 34.0, 33.4, 33.2. FT-IR: 2220 cm−1 (-CN). ESI-TOF HRMS (APCI): m/z 234.1215 (M + H), calcd. for C17H15N: m/z 233.1204.2 was synthesized from 4, 4′-dibromo[2.2]paracyclophane by the literature method34. Yield 47%. 1H-NMR : δ = 7.05 , 6.81 , 6.75 , 3.60–3.53 , 3.34–3.06 . 13C-NMR : δ = 144.2, 141.4, 137.0, 132.7, 118.8, 116.1, 35.2, 33.4. FT-IR: 2218 cm−1 (-CN). ESI-TOF HRMS (APCI): m/z 259.1150 (M + H), calcd. for C18H14N2: m/z 258.1157.535: The mixture of 4-bromo[2.2]paracyclophane , Ni(cod)2 , 1,5-cyclooctadiene , and 2,2′-bipyridyl  in 180 ml dry THF was heated at 60 °C with stirring for 40 h under N2. After cooled down to room temperature, the mixture was extracted with EtOAc, washed with brine, dried over with MgSO4, and concentrated under reduced pressure. The crude product was purified by column chromatography  to afford 5 as a white powder. Yield 2.63 g (76%). 1H-NMR : δ = 6.42–6.64 , 6.27–6.36 , 2.68–3.46 . 13C-NMR : δ = 140.4, 140.1, 139.9, 139.7, 139.5, 139.0, 138.2, 136.8, 135.5, 134.1, 133.3, 133.2, 133.0, 132.9, 132.8, 132.5, 132.2, 132.1, 130.9, 130.1, 36.0, 35.9, 35.3, 35.0. ESI-TOF HRMS (APCI): m/z 415.2450 (M + H), calcd. for C32H30: m/z 414.2348.p-xylylene nanofilms were deposited onto UF membranes by using a Parylene deposition equipment . [2.2]Paracyclophanes 1 and 2 were sublimed at 175 °C under vacuum pressure and pyrolyzed at 650 °C to create diradical reactive intermediate. The gaseous intermediate was then fed into a deposition chamber kept at room temperature and the intermediate could efficiently polymerize on the surfaces of UF membranes ) and Si wafer to form nanofilms 3 and 4. Crosslinked nanofilms 6 and 7 were prepared from the mixed powders of 1 or 2 with 5 (1 or 2: 5 = 2: 1 w/w) under the same CVD condition of 3 and 4. The film thicknesses of nanofilms on Si wafer were determined by using a surface profiler .Poly-2 and stirring speed was 500 r.p.m. Permeate sample for solvent permeance measurements were collected intervals of 10 min and samples for rejection evaluations were taken after 2 hr. The solvent permeance  was determined by the equation J = V/(A x t x p), where V is the permeate volume, A is the effective membrane area, t is the unit time, and p is the applied pressure. The solute rejection tests were carried out using solutions ([dye] = 10 μM) containing one of the following dyes in water: Rose Bengal (1017 g/mol); Rhodamine 6 G (479 g/mol); Methylene Blue (319 g/mol); and Methyl Orange (327 g/mol)  at 20 °C and 0.5 MPa. Data on water permeance and dye rejection performance was the average value by using five membranes coated with nanofilms, which were fabricated by five independent processes under the same conditions. The measurements were done after initial compaction phase until a permeance reached a steady state. The effective membrane area was 4.1 cmol) Fig. . Dye conSupporting Information


Cellvibrio sp. PR1 is a xylanolytic and agarolytic bacterium isolated from the Pearl River. Strain PR1 is closely related to Cellvibrio fibrivorans and C. ostraviensis (identity > 98%). The xylanase and agarase contents of strain PR1 reach up to 15.4 and 25.9 U/mL, respectively. The major cellular fatty acids consisted of C16:0 (36.7%), C18:0 (8.8%), C20:0 (6.8%), C15:0 iso 2-OH or/and C16:1ω7c (17.4%), and C18:1ω7c or/and C18:1ω6c (6.7%). A total of 251 CAZyme modules  were identified from 3,730 predicted proteins. Genomic analysis suggested that strain PR1 has a complete xylan-hydrolyzing  and agar-hydrolyzing enzyme system (2 β-agarases and 2 α-neoagarooligosaccharide hydrolases). In addition, the main metabolic pathways of xylose, arabinose, and galactose are established in the genome-wide analysis. This study shows that strain PR1 contains a large number of glycoside hydrolases. Sugars released from polysaccharides are usually used for the production of value-added products, such as biofuels , 2, antiellulose ; meanwhiellulose . Highly  Cellvibrio from the Pseudomonadaceae family was first discussed by Blackall et al. in 1986 [ Cellvibrio ostraviensis, C. vulgaris, C. mixtus, C. fibrivorans, C. gandavensis, C. japonicus, C. fulvus, and C. diazotrophicus, have been identified to date . Bacteria from the genus Cellvibrio are usually Gram-negative and aerobic, and these bacteria are known cellulose, xylan, starch, and chitin degraders [ C. japonicus degrades all of the major plant cell wall polysaccharides  by the activities of approximately 130 possible glycoside hydrolases [ C. mixtus J3-8 is a xylanolytic bacterium with no cellulolytic activity and has a large number of genes that are not annotated [ Cellvibrio sp. PR1 was isolated from a water sample from the Pearl River. Xylanolytic and agarolytic activities are observed in strain PR1. Based on the bacterial-algal interactions between strain PR1 and microalgae, the microalgal growths were enhanced by coculturing with strain PR1 and by using xylan or xylose as feedstock [The genusegraders . For exannotated . A cyanoeedstock . To bett Cellvibrio sp. PR1 was isolated from a freshwater sample collected from the Pearl River (23°8′N and 113°17′E). The strain was deposited in the China General Microbiological Culture Collection Center  and the NITE Biological Resource Center  under the accession numbers CGMCC 1.14955 and NBRC 110968, respectively.Strain−1 NaNO3, 0.04 g L−1 K2HPO4, 0.075 g L−1 MgSO4·7H2O, 0.036 g L−1 CaCl2·2H2O, 1 mg of glucose, and 1 ml of trace element solution. The trace element solution contains the following: 2.86 g L−1 H3BO3, 1.86 g L−1 MnCl2·4H2O, 0.22 g L−1 ZnSO4·7H2O, 0.39 g L−1 Na2MoO4·2H2O, 0.08 g L−1 CuSO4·5H2O, 0.05 g L−1 Co(NO3)2·6H2O, 6 g L−1 citric acid, 6 g L−1 ferric ammonium citrate, and 1 g L−1 EDTANa2. pH was adjusted to 7.2. Xylan , cellulose , starch , and agarose  were used as the primary carbon source for testing xylanase, cellulase, amylase, and agarase activities, respectively.The medium used for strain PR1 cultivation contains the following: 1.5 g L2S production was studied by growing the strain in a tube that contains culture medium supplemented with 5 g/L sodium thiosulfate and detected by using a filter-paper strip saturated with lead acetate [The cells of strain PR1 growing in exponential phase were collected and used for the examination of cell morphology by transmission electron microscopy  at 80 kV. NaCl tolerance was tested in culture medium with additional salt concentration ranging from 0% to 5% (w/v). Growth was tested at different temperatures  and pH levels (4–10). The pHs were adjusted by using disodium hydrogen phosphate-citrate buffer solution (for pH 4–8) and borax-sodium hydroxide buffer solution (for pH 9-10). Anaerobic growth was determined at 30°C in an anaerobic chamber for 7 days. Gram staining was performed following the standard Gram procedure. Motility was examined on a semisolid culture medium supplemented with 0.5% (w/v) of agar. Catalase and oxidase activities were investigated following the procedures by Barrow and Feltham . H2S pro acetate . Starch  acetate . The sub Escherichia coli K-12 as a reference. Cellular fatty acids were extracted according to the MIDI protocol [The genomic DNA of strain PR1 was isolated and purified by using a TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit . The genomic DNA GC content was determined by HPLC according to the method of Mesbah et al. , with Esprotocol  and idenThe complete 16S rDNA sequence of strain PR1 was obtained from its genomic sequence by using the DNAMAN program and was deposited in the GenBank under the accession number KT149658. The 16S rRNA gene sequence of strain PR1 and related sequences were aligned by using CLUSTAL X  using the-value < 0.00001. Two genomes from the closely related species that include Cellvibrio sp. BR (GenBank accession number AICM00000000.1) and C. japonicus Ueda107 (GenBank accession number CP000934) were selected for the comparative analysis of functional genes that involve carbohydrate metabolism. Gene-encoding glycoside hydrolases (GHs), carbohydrate esterases (CEs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate-binding modules (CBMs), and auxiliary activities (AAs) were identified by using the Carbohydrate-Active EnZyme (CAZy) database [E-value cutoff of 1e − 20; the annotation was confirmed only when the two database searches yielded positive results; the positive CAZymes were assigned to an EC number.The genome of strain PR1 was sequenced by using Illumina HiSeq 2000 sequencer; the methods of data processing and assembly were presented in our previous work . The gendatabase  and dbCA−1 of each carbon source. The cultivation was maintained at 30°C on a rotary shaker with a speed of 150 rpm. Fermented broths were collected after 24 hours' cultivation. The supernatant from culture medium was collected by centrifugation at 12,000 ×g and 4°C for 10 min; 1 ml of supernatant was added to the phosphate buffer solution  and modified with 1.0% (w/v) of the related substrate for 60 min. The reducing sugar was measured by using the 3,5-dinitrosalicylic acid (DNS) method [μmol of reducing sugar  per min. The protein concentrations were determined by the Lowry method and BSA as the standard. The optimal temperatures of the enzyme activities were studied by subjecting reactions to different temperatures ranging from 30°C to 50°C in a PBS (pH 7.0). The NaCl tolerance of related enzyme was tested under extra NaCl concentrations ranging from 0 to 5% (w/v), and the influence of pH on enzyme activity was tested under different pHs (4–11); the pHs (pH 3–8) were adjusted by disodium hydrogen phosphate-citrate buffer solution, the pHs (pH 9-10) were adjusted by borax-sodium hydroxide buffer solution, and the pH 11 was adjusted by sodium phosphate-disodium hydrogen phosphate buffer solution.The activity assays of xylanase and agarase were performed by using the fermentation broth that was cultured in the medium with xylan and agarose as the primary carbon source. The fermentation was performed in a 1000 mL Erlenmeyer flask containing 300 mL of culture media and supplied with 1 g L) method . One uniμm long and 0.5 μm wide. The cells have single polar flagella with a length of approximately 2-3 μm , N-acetyl D-glucosamine (except for the C. ostraviensis LMG 19434), and chitin (the same as the C. fulvus LMG 2847 and C. vulgaris NCIMB 8633) for growth. Different from the other five Cellvibrio species, strain PR1 utilizes agar and carboxymethyl cellulose for the growth and shows β-galactosidase and α-fucosidase activities, but leucine arylamidase activity was not observed. Moreover, acid phosphatase, β-glucosidase, α-galactosidase, and β-Glucuronidase activities were observed in strain PR1, but valine arylamidase and naphthol-AS-BI-phosphohydrolase activities were not detected.Strain PR1 was able to grow at temperatures of 15–45°C (optimum at 30–37°C), additional salt of 0–2.5% NaCl (optimum at 0-1%), and pH of 6–10 (optimum at pH 7-8). The strain is unable to grow under an anaerobic condition. Results show that the strain has a motility capability. It was found that strain PR1 was positive for catalase, oxidase, and starch hydrolysis activities and negative for H15:0 iso 2-OH or/and C16:1ω7c (summed in feature 3), and C18:1ω7c or/and C18:1ω6c (summarized in feature 8). Different from the other five species, saturated C16:0 was the most abundant fatty acid in strain PR1 that accounted for 36.74% of the total fatty acids. The C18:1ω9c, C19:0, C20:0, and summarized features 2 and 8 that were not detected in the other five strains were found in strain PR1. These fatty acids accounted for 21.8% of the total fatty acids. Additionally, the C12:0 2-OH and unsaturated C18:1ω7c that are abundant in other Cellvibrio species were not identified in strain PR1.The fatty acid compositions of strain PR1 and related strains are summarized in The extracellular xylanase and agarase activities of strain PR1 were assessed after 48 h incubation at 30°C . The optThe optimal agarase activity was determined to be 26.0 U/mL under the condition of 40°C, pH 7, and 0.5% extra NaCl . It was  C. japonicus Ueda107, Cellvibrio sp. BR Cellvibrio sp. OA-2007, Cellvibrio sp. PSBB023, C. mixtus, and Cellulomonas gilvus were summarized in https://doi.org/10.1155/2017/6304248).The genomic features and CAZyme families of strain PR1,β-glucosidases and 12 endoglucanase-encoding genes, but a lack of cellobiohydrolase-encoding gene in the genome was observed .Strain PR1 has an incomplete cellulose hydrolysis system, which has 4 observed . The samβ-xylanases, 16 GH43 β-xylosidases, 17 α-arabinofuranosidases, 9 acetyl xylan esterases, 2 α-glucuronidases, and 2 ferulic acid esterases in strain PR1.Xylanase, xylosidase, arabinofuranosidase, acetyl xylan esterase, glucuronidase, and ferulic acid esterase are essential to complete xylan hydrolysis. The xylanase attacks the backbone of xylan and generates oligosaccharides; the xylosidase releases xylose from the oligosaccharides along with the activities of the other four enzymes by attacking the side chains . There aα-Agarase attacks α-1,3-glucosidic bonds to generate agarooligosaccharides, which are hydrolyzed to galactose by the β-1,4-3,6-anhydro-L-galacopyranose hydrolase. (2) β-Agarase attacks α-1,4-glucosidic bonds to generate neoagarooligosaccharides, which are hydrolyzed to galactose by the α-1,3-L-neoagarooligosaccharide hydrolase. The strain PR1 contains 3 GH50 and 1 GH86 β-agarases along with 2 GH117 α-neoagarooligosaccharide hydrolases, whereas only the Cellvibrio sp. BR contains 2 GH50 and 1 GH86 β-agarases along with 1 GH117 α-neoagarooligosaccharide hydrolase (The complete hydrolysis of agar/agarose includes two kinds of pathways. (1) ydrolase .Xylan and agarose are hydrolyzed to xylose, arabinose, galactose, and so forth, in vitro by extracellular enzymes. These monosaccharides are assimilated by cells and take part in energy generation and substance transformation. The main metabolic pathways of xylose, arabinose, and galactose in strain PR1 are proposed on a genome-wide analysis. As shown in  C. japonicus, Cellvibrio sp. BR, Cellvibrio sp. OA-2007, Cellvibrio sp. PSBB023, C. mixtus, and Cellulomonas gilvus were also analyzed. C. fibrivorans R-4079 and C. ostraviensis LMG 19434 were the closely related strains of strain PR1, and the comparative phenotypes and genomes on these strains were also studied; however, only the genome information of C. japonicus Ueda107, Cellvibrio sp. BR, Cellvibrio sp. OA-2007, Cellvibrio sp. PSBB023, and C. mixtus J3-8 is available in public databases. This work is still helpful for the investigation of strain PR1 characteristics in a natural ecosystem.In this work, we comparatively studied the phenotypes and extracellular enzyme activities of strain PR1 and the genomic features and carbohydrate hydrolysis pathways of Cellvibrio are known to degrade cellulose, xylan, starch, chitin, and so forth; the members of Cellvibrio species, including C. ostraviensis, C. fibrivorans, C. fulvus, C. vulgaris, C. mixtus, C. gandavensis, C. japonicus, and C. diazotrophicus, were isolated from soils, rhizospheres, and giant snails [ Cellvibrio was first isolated from freshwater and has xylanolytic and agarolytic activities. β-Agarases were also observed in Cellvibrio strains that were isolated from wastewater treatment plants [Bacteria from the genust snails , 22, 23.t plants  and sedit plants . C. mixtus J3-8 was the abundance of GH11 xylanases; the xylanases located in GH11 are considerably more active than GH10 xylanase for its high substrate specificity, great stability and plasticity, and small protein sizes. In strain PR1, 2 GH10 and 2 GH11 xylanases were identified. However, the RNA-Seq results suggested the GH10 xylanase-encoding genes (FPKM: 1265 for PRGL001867 and 671 for PRGL003904) had higher expressions than GH11 xylanase-encoding genes (FPKM: 569 for PRGL000191 and 433 for PRGL003376) when xylan was used as the primary substrate [Strain PR1 has a higher xylanolytic activity than many other microorganisms . Wu and  C. japonicus Ueda107 was found to have CBM2–CBM10-GH10, CBM2–CBM35-GH10, CBM15-GH10, and CBM60-GH11 xylanases to release soluble polysaccharides and oligosaccharides [ C. mixtus J3-8. However, three CBM binding xylanases  were identified for strain PR1, and neither CBM2 nor CBM10 was detected (Table S1). It seems that the CBM15 is a specific module that is only detected in Cellvibrio strains, such as C. japonicus, Cellvibrio sp. BR, and C. mixtus [CBMs are usually considered to promote hydrolysis efficiency by binding an enzyme to the substrate. In xylanase families GH10 and GH11, the catalytic modules are appended to a range of different CBMs that target crystalline cellulose (CBM 2 and CBM10) and xylan (CBM15 and CBM35) . The C. charides . Wu and . mixtus , 10, 26. Streptomyces strains [ChiC, ChBDChiA1, etc.) and were usually found in the binding domain of GH18 chitinases [ C. japonicus Ueda107, and two CBM5-GH18 chitinases were detected in Cellvibrio sp. BR.In addition, six types of CBM  structures were detected from the binding domain of chitinases (GH18 and GH19) in strain PR1. GH18 chitinases are found in various organisms and are ordinarily composed of one catalytic domain and one or more domains that are involved in chitin binding, whereas GH19 chitinases are more likely to originate from plants and consisted of one or two domains . Many ch strains , 28. Theitinases , 29. How Cellvibrio-microalgae cocultivation model for the promotion of microalgae growth. The biomass production of Chlorella, Chlamydomonas, and Dunaliella was significantly enhanced by using this model; the comparative transcriptome analysis indicated that the xylan hydrolysates or xylose was catalyzed into some active substrates and responsible for the promotions [1 (thiamine) and vitamin B7 (biotin) syntheses . The thiamine-synthesizing genes includes thiC , thiD , thiE , thiF , thiG , thiI , thiL , thiO , thiS , and dxs . The biotin-synthesizing genes include bioA , bioB , bioC (methyltransferases), bioD , and bioF . It is known that over half of microalgal species are auxotrophic for cobalamin (vitamin B12), and 20% require thiamine, and 5% require biotin. The important role of vitamins in controlling algal growth is increasingly recognized [Strain PR1 was a syntrophic bacterium to cyanobacteria when enriching and isolating cyanobacterium under photoautotrophic condition. This strain was believed to be beneficial in cyanobacteria growth by the bacterial-algal interactions. Based on this hypothesis, we constructed aomotions . The uticognized , 31. Cellvibrio species. The genomic analysis provides some insights into the functions of strain PR1 in polysaccharide hydrolysis and monosaccharide metabolism and possible syntrophic pattern with microalgae.This study found that strain PR1 shows distinctive differences in phenotype and genome from knownTable S1: Genes encoding carbohydrate-bindingmodules (CBMs) in the genome of strain PR1. Table S2: Genes encoding carbohydrate esterases (CEs), glycosyl hydrolases (GHs), glycosyl transferases (GTs) and polysaccharide lyases (PLs) in the genome of strain PR1. Table S3: CAZyme module distributed in four species. Figure S1: Thiamine synthesis pathway. Figure S2: Biotin synthesis pathway.


Lactobacillus murinus EF-1. Whole-genome sequencing revealed a 2.30-Mb draft genome with 39.6% G+C content and 2,196 open reading frames. PHAST analysis identified three intact prophages of 26.1 kb, 25.4 kb, and 49.6 kb in size.Screening for lysogenic lactobacilli in rat fecal samples has identified  Lactobacillus murinus EF1 was isolated from a rat fecal sample and confirmed to be a lysogen by mitomycin C induction. L. murinus strains have previously been isolated and identified from rat, mice, porcine, canine, and humans  produced 18 scaffolds with a total length of 2,308,018 bp, a G+C content of 39.6%, and an N50 length of 309,081 bp; 99.46% of the sequenced mapped reads were assembled with 250× average coverage. RecA and HSP60 genes were used to confirm L. murinus identity (L. murinus open reading frames (ORFs) and rRNAs, respectively, and PHAST prophage , 2 (8.5 kb), 3 (19.7 kb), and 5 (9.8 kb). Also, one questionable CRISPR spacer was identified in scaffold 3. The identification of three intact prophages, four incomplete prophages, and a questionable CRISPR array may demonstrate a prophage preference of this strain, suggesting an environmental prophage advantage in the rat GIT for this strain. Future studies will characterize the lysogenic phages within L. murinus EF-1.To identify punitive prophages found within identity  and RNAmidentity  to confiprophage  and CRISMPSN00000000. The version described in this paper is the first version, MPSN01000000.This whole-genome shotgun project was deposited in GenBank under the accession number