At3g12900, encodes a scopoletin 8-hydroxylase involved in coumarin biosynthesis, and plays an important role in the iron uptake strategy of Arabidopsis.A strongly iron-responsive gene of previously unknown function,  Arabidopsis thaliana roots is induced under iron deficiency. An essential enzyme for the biosynthesis of the major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6′-Hydroxylase1 (F6′H1), which belongs to a large enzyme family of the 2-oxoglutarate and Fe2+-dependent dioxygenases. We have functionally characterized another enzyme of this family, which is a close homologue of F6′H1 and is encoded by a strongly iron-responsive gene, At3g12900. We purified At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin, via hydroxylation at the C8 position, and that it thus functions as a scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves, followed by metabolite profiling and biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various iron regimes. Our results indicate that S8H is involved in coumarin biosynthesis, as part of mechanisms used by plants to assimilate iron.Iron deficiency is a serious agricultural problem, particularly in alkaline soils. Secretion of coumarins by  In plants, chloroplasts and mitochondria have high Fe requirements . Fe defi2O3) .3+) into more soluble, and bioavailable to plants, ferrous ions (Fe2+). Fe2+ ions are then transported across the plasma membrane into the root epidermal cells by the divalent metal transporter Iron Regulated Transporter 1 (IRT1) (3+ (3+–PS complexes are then transported into the root epidermis via a high-affinity uptake system without requiring a reduction step (Higher plants have developed two different types of strategies in response to Fe limitation. The first (Strategy I), which occurs in all plants except graminaceous species, is a reduction-based strategy, where the release of protons into the rhizosphere is enhanced by Fe-deficiency-induced proton-translocating adenosine triphosphatases , such asRT1)  in many dicots . The s8h-1 (SM_3.27151) and s8h-2 (SM_3.23443) T-DNA insertional mutant lines (JXB online) were identified in the SM (Suppressor-mutator) collection . Genotyping of s8h-1 and s8h-2 was done using protocols and primers described in s8h-1 homozygous mutants were tested for S8H gene expression by quantitative real-time reverse transcription–PCR (qRT–PCR) .The qRT–PCR) . A lack 2+). Arabidopsis seeds were surface sterilized by soaking in 70% ethanol for 2 min and subsequently kept in 5% calcium hypochlorite solution for 8 min. Afterwards, seeds were rinsed three times in autoclaved deionized water. The surface-sterilized seeds were sown on tip-cut 0.65% agar-filled tubes or on microcentrifuge tube lids filled with solidified Heeg medium, which were placed into tip boxes with control hydroponic solution (40 µM Fe2+). After a few days’ stratification at 4 °C, boxes with plants were kept in a controlled environment (16 h light at 22 °C/~100 μmol m−2 s−1 and 8 h dark at 20 °C). Approximately 3 weeks later, plants were transferred either to a freshly made control solution or to Fe-deficient (10 µM Fe2+) or Fe-depleted (0 µM Fe2+) solutions. In the first set of experiments hydroponic solutions were fully changed once per week, while in the second set of experiments boxes containing old nutrient solution were replenished by the addition of fresh medium.Plants were grown in two types of modified Heeg solution  at 22 °C and 8 h dark at 20 °C].Surface-sterilized seeds were sown on Petri dishes containing different homemade Murashige and Skoog (MS) media: (i) half-strength MS medium (0.5 MS) with macroelements at half strength and microelements/vitamins at full strength, and (ii) one-quarter-strength MS medium (0.25 MS) with macroelements at one-quarter strength and microelements/vitamins at half strength. Both types of media contained 1% sucrose, 0.8% agar supplemented with 4 mg lTen-day-old seedlings from agar plates were transferred into a 250 ml wide-mouth Erlenmeyer flask containing 20 ml sterile 0.25 MS liquid medium. Plants grown in liquid cultures were incubated on rotary platform shakers at 120 rpm. Ten days after transfer, 20 ml of fresh medium was added to each flask. Plants were harvested 18 days after transfer (on the 29th day of culture); leaves and roots were frozen separately in liquid nitrogen and stored at −80 °C.N. benthamiana seeds were transplanted and cultured independently for 3 additional weeks in plant growth chambers under a photoperiod of 16 h light (120 μmol m−2 s−1) at 24 °C and 8 h dark at 22 °C with 70% humidity.Two weeks after germination, p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA) were enzymatically synthesized as described by P-coumarate and coenzyme A (CoA) were purchased from Sigma-Aldrich. Kanamycin, chloramphenicol, and isopropyl-β-d-thio-galactopyrannoside (IPTG) were purchased from Duchefa.The following acids were used: ferulic (Aldrich), coumaric (Sigma), cinnamic (Sigma), and caffeic (Fluka). Coumarins used were: coumarin (Sigma), daphnetin (Sigma), esculetin (Sigma), esculin (Sigma), fraxetin (Extrasynthèse), fraxin (Extrasynthèse), isoscopoletin (Extrasynthèse), limetin , scoparon , 6-methoxycoumarin , 7-methoxycoumarin , scopoletin , scopolin , umbelliferone (Extrasynthèse), skimmin , and 4-methylumbelliferon (Sigma). The CoA thiol esters of the cinnamates  (A. tumefaciens strain and used together with the C5851 A. tumefaciens strain containing pBIN61-P19 (N. benthamiana leaves.The amplified S8H was amplified by PCR (5ʹ primer: 5ʹ-GGATCCGGTATCAATTTCGAGGACCAAAC-3ʹ and 3ʹ primer: 5ʹ-CTCGAGCTCGGCACGTGCGAAGTCGAG-3ʹ) and cloned between the BamHI and XhoI sites of pET28a+. The recombinant plasmid was introduced into the competent E. coli Rosetta 2 (Novagen) strain by heat shock.The ORF of E. coli Rosetta 2 strain transformed with pET28a+-S8H was cultured at 37 °C overnight in 10 ml lysogeny broth (LB)  for five periods of 20 s with an interval of 30 s at 50–60% power, and subsequently centrifuged for 20 min at 10000 g at 4 °C. The purification of soluble recombinant His-tagged proteins was done using TALON Metal Affinity Resin (TAKARA) as described by the supplier. The purified proteins were eluted with 0.1 M potassium phosphate buffer and 200 mM imidazol solution (pH 8.0).The 4 (0.5 mM), α-ketoglutarate (5 mM), sodium ascorbate (5 mM) in 0.1 M potassium phosphate buffer at optimal pH (7.0), with 200 µM substrate and 2.6 µg of purified enzyme. Reaction mixtures were incubated for 10 min at an optimal temperature of 31.5 °C. Reaction mixtures with CoA esters were additionally incubated at the end of the reaction with 20 µl of 5 M NaOH for 20 min at 37 °C to hydrolyse the ester bond, and subsequently with 20 µl of acetic acid in order to close the lactone ring. In samples with acids and coumarins as substrates, the reaction was stopped by the addition of 2 µl of trifluoroacetic acid and incubation at 20 °C for 20 min. Reaction mixtures were then centrifuged for 30 min at 10000 g; the supernatant was recovered, filtered (0.22 µm), and analysed by high-performance liquid chromatography (HPLC).Enzymatic activities were assessed immediately after the purification of S8H. The protocol was adapted from –1. Separation was performed by elution with the following programme (A) H2O and 0.1% acetic acid (B) methanol and 0.1% acetic acid: 0–35 min gradient 10–70% B, 35–36 min 70–99% B, 36–39 min 99–99% B, isocratic elution and column regeneration. The wavelength was set at 338 nm to monitor the formation of fraxetin.Chromatographic separation was performed using an HPLC Shimadzu system with diode array detector/UV detector. The injected volume was 50 µl. Samples were separated on an Interchim C18 Lichrospher OD2  column with the flow speed set to 0.8 ml minin vitro metabolization of scopoletin into fraxetin, separation was conducted on an Ultimate 3000 chromatographic chain coupled with an LTQ-XL mass spectrometer , as described by Sample analysis was performed on a Nexera UHPLC system  coupled with an LCMS 2020 mass spectrometer (Shimadzu). The chromatographic column was a C18 reverse phase  150 × 2.1 mm 1.8 µm. Elution of the compounds was performed as described by A. tumefaciens  were inoculated into 40 ml LB medium containing the antibiotic corresponding to the resistance provided by the plasmids and incubated at 28 °C overnight. The cultures were centrifuged for 10 min at 4000 g and the pellet was resuspended in 20 ml sterile deionized water. This step was repeated three times in order to remove all traces of antibiotics. N. benthamiana leaves were infiltrated with a suspension of recombinant strains at an OD600 of 0.4 for pBIN61-P19 bacteria and 0.2 for pBIN-S8H or pBIN-F6ʹH2 bacteria. The infiltrated plants were stored in a growth chamber for 96 h. In parallel, for each experiment, N. benthamiana were infiltrated with LBA4404 (pBIN-GFP) as a positive control. Expression of green fluorescent protein was assessed by examination under a binocular microscope.The protocol for heterologous expression was adapted from N. benthamiana leaves were crushed in liquid nitrogen and a 100 mg sample was mixed with 800 µl 80% methanol. The solution was vigorously mixed for 30 s and then centrifuged for 30 min at 10000 g. The supernatant was transferred to a fresh tube and the pellet was submitted to a second extraction with 800 µl 80% methanol. Both supernatants were pooled and vacuum dried. The pellet was resuspended in 100 µl MeOH/H2O 80%/20% v/v and analysed by UHPLC.Freshly harvested infiltrated g. Supernatants were transferred to fresh tubes, vacuum dried, resuspended in 100 µl MeOH/H2O 80%/20% v/v, and analysed by UHPLC.Arabidopsis roots were frozen in liquid nitrogen and ground with a pestle and mortar. A 50 mg sample of plant tissue was mixed in 500 µl of 80% methanol supplemented with 2.5 µM 4-methylumbelliferone as an internal standard. Samples were sonicated for 10 min and centrifuged for 10 min at 10000 in vitro cultures were collected 18 days after the onset of Fe treatments. Phenolic compounds were retained in a BAKERBOND™ C18 column , eluted from the cartridge with 3 ml of 100% methanol, and dried in a centrifugal evaporator. Dry extracts were stored at –20 °C until further analysis.Nutrient solutions from Arabidopsis Arabidopsis roots were first lyophilized and subsequently milled before microwave (MARS)-assisted digestion in concentrated nitric acid (Aristar). Metal concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) .Chlorophyll extraction with acetone was done according to the method of t-tests, except for trace element analysis) were done using Excel 2010 (Microsoft). Statistical significance of differences observed in trace element analysis were analyzed by R version 3.3.2 (2016-10-31) (https://www.R-project.org/) with the use of the agricolae package.All treatments included at least three biological replicates. Data processing and statistical analyses . This analysis indicated that two genes involved in scopoletin biosynthesis  were clustered together, and identified At3g12900, a gene of unknown function, as sharing the highest homology with both scopoletin synthases  and the 2OG binding site (Arg 303 and Ser 305 in F6ʹH1) are highly conserved in all proteins  and encoded by yanidins  and has proteins . In contE. coli. An improved protocol was used for purification of the heterologously expressed enzyme to obtain the purified protein fraction   . This res8h-1 and s8h-2) and investigated their behaviour in various types of culture. Because the literature has reported that fraxetin accumulation is induced under conditions of Fe deficiency, we investigated the behaviour of the mutants in Fe-depleted hydroponic solutions. Plants were grown in a control hydroponic solution (40 µM Fe2+) for 3 weeks and subsequently transferred to Fe-depleted solution (0 µM Fe2+) and cultured for an additional 3 weeks. In Fe-depleted solutions both mutant lines were clearly paler in colour than Col-0 control plants (P<0.01) and scopoletin (P<0.01) concentration in roots grown in Fe-depleted conditions for both mutant lines, and a significant increase in umbelliferone accumulation in s8h-1 compared with Col-0 plants  were paler and plants grown in Fe-depleted medium (0 µM Fe2+) were chlorotic , the Fe content of both mutant lines (s8h-1 and s8h-2) was significantly lower (P<0.05) in comparison to the corresponding control (Col-0) (s8h mutants compared with control plants (P<0.05) grown in Fe-depleted medium  . The cond medium .s8h-1 phenotypes were dependent on the P to Fe ratio  in comparison to Col-0 plants under Fe-depleted (0 µM Fe2+) conditions (2+) and Fe-optimal (50 µM Fe2+) media did not show changes in trace element contents (s8h mutants grown in Fe-deficient medium (As phosphate (Pi) and Fe deficiency interact with respect to Fe-induced coumarin secretion  medium, the s8h mutants showed a strong reduction in fresh weight , mutant lines and Col-0 plants showed significantly smaller and chlorotic shoots, while roots were markedly shorter  medium were clearly larger than the WT plants; this was not the case on 0.5 MS medium with optimal Fe content  under UV light showed that both s8h mutant lines secreted a higher level of fluorescent compounds into the medium compared with WT plants (s8h plants (2+), in which the mutant plants seemed to accumulate slightly more fluorescent compounds in their roots compared with WT plants  solution, which contains 10-fold lower concentrations of microelements than the other hydroponic solution used (2+) conditions, both mutant lines were much larger than WT plants, with greatly increased fresh weight (2+) but lower amounts of other micronutrients  both s8h mutants became chlorotic   ; this coctively) . Under Fctively) ; only thIn the Arabidopsis genome there are more than 100 genes encoding enzymes sharing homologies with dioxygenases. The 2OGD family is the second largest enzyme family in plants; its members are involved in various oxygenation/hydroxylation reactions . Maintai2+- and 2OG-dependent dioxygenases, the scopoletin synthase F6ʹH1  were positively correlated with genes actively involved in the response to Fe deficiency   determined in our in vitro experiments was similar to those reported for the biosynthetic enzymes of specialized metabolism  and Fe deficiency-induced coumarin secretion  were also significantly decreased in the s8h mutant lines studied. We observed a significantly higher content of scopoletin in the root tissue in hydroponic experiments and in the root exudates in liquid cultures. Scopoletin is a substrate for the reaction catalysed by S8H. The lack of S8H in the s8h mutant background could lead to the accumulation of higher levels of scopoletin and its corresponding glycoside, scopolin.Among the numerous targeted metabolite profiling experiments conducted in this study, we repeatedly obtained results clearly showing significantly changed coumarin profiles in both s8h-1 mutant background, as well as low amounts of fraxin in some other Col-0 and s8h replicates. It cannot be excluded that these observations could be explained by the presence of another enzyme involved in fraxetin biosynthesis or an alternative metabolic pathway being induced in the s8h mutant background. The fraxetin synthesized in Col-0 plants could also be further demethylated to 6,7,8-trihydroxycoumarin, with a beneficial effect for plants under Fe-deficient conditions; alternatively, fraxetin could be directly involved in increasing Fe availability. These possibilities cannot be excluded, taking into account the catechol-type structure of fraxetin, and so they need to be further investigated. As reported by f6ʹh1 seedlings grown under low Fe availability could be reversed by exogenous application of esculetin, esculin, and scopoletin. In parallel, the result of in vitro assay showed that only esculetin was able to chelate and mobilize Fe3+ . It should also be noted that unexpectedly, in one experimental replicate, we detected relatively high amounts of fraxin and a low level of fraxetin in the The present results indicate that At3g12900 is an important candidate for having a key role in Fe acquisition by plants. Fraxetin and other root-released phenolic compounds, mainly esculetin deriving from scopoletin and scopolin, have been suggested to be Fe chelators or Fe uptake facilitators in Arabidopsis  with 0–40 µM Fe2+ content.Fig. S15. Phenotypic characterization of Col-0 and s8h plants grown in 10× Heeg hydroponic solution (fully changed once per week) with 0–40 µM Fe2+ content.Fig. S16. Phenotypic characterization of Col-0 and s8h plants grown in 1× Heeg hydroponic solution (refilled with a fresh medium) with 0–40 µM Fe2+ content.Fig. S17. Phenotypic characterization of Col-0 and s8h plants grown in 10× Heeg hydroponic solution (refilled with a fresh medium) with 0–40 µM Fe2+ content.Fig. S18. Phenotypic characterization of Col-0 and s8h mutant lines.Table S1. Genotyping of S8H transcript in the s8h mutant backgrounds.Table S2. Lack of Table S3. Modified Heeg solutions used in hydroponic cultures.S8H ORF.Table S4. PCR amplification of the in vitro substrate specificity of S8H enzyme.Table S5. Compounds used in the analysis of Table S6. Chemical analysis of soil mixes originating from different batches used in two experimental replicates.Supplementary Tables and FiguresClick here for additional data file.


Cyberlindnera fabianii 65, Pichia kudriavzevii 129, and Saccharomyces cerevisiae 131 have been isolated from the microbiota of fermented masau fruits. C. fabianii and P. kudriavzevii especially harbor promising features for biotechnology and food applications. Here, we present the draft annotated genome sequences of these isolates. Cyberlindnera fabianii 65 [previously known as Lindnera fabianii, Hansenula fabianii, and Pichia fabianii  in Zimbabwe   food products , 5–14 bu5–15–on (FDA) . C. fabiprofiles . To furtC. fabianii 65), 83.3× (P. kudriavzevii 129), and 100× (S. cerevisiae 131) based on a 12-Mb genome size. Moreover, PacBio sequencing was performed with total depths of coverage of 36.6× (C. fabianii 65), 32.1× (P. kudriavzevii 129), and 22.2× (S. cerevisiae 131). We performed hybrid assemblies using DBG2OLC with Illumina and PacBio data  and S. cerevisiae 131 (38.1%). Other assembly and annotation statistics are listed in DNA was sequenced using Illumina MiSeq paired-end 2 × 251 bp) sequencing technology, with total depths of coverage of 83.3× (Bio data . PBJellyBio data  was usedBio data . MAKER2 Bio data  was used1 bp sequAugustus  and SNAPAugustus  were trag BLASTp  against g BLASTp  analysisg BLASTp . The G+CS. cerevisiae 131. Additionally, links between phenotypes and genotypes, as well as comparative genomic studies among the three species, will reveal opportunities for industrial applications of C. fabianii 65 and P. kudriavzevii 129.The genome sequences and gene annotations can now be used to develop novel molecular tools to unravel the full metabolic repertoire of the two nonconventional yeasts compared to The annotated genome sequences are deposited at DDBJ/EMBL/Genbank under the accession numbers listed in


Peganum harmala L., exhibits broad spectrum but limited insecticidal ability against many pests. So there is an urgent need to synthesize novel derivatives with high efficiency. In the present study, a new synthetic compound, [1-(2-naphthyl)-3- β-carboline] (ZC-14), showed a strong proliferation inhibition effect against the Spodoptera frugiperda Sf9 cell line in a dose-dependent manner. Simultaneously, apoptosis induced by 7.5 μg/mL ZC-14 was confirmed with physiological and biochemical evidence, including typical apoptosis characteristics with shrinkage, apoptotic bodies, nuclear condensation/fragmentation, a clear DNA ladder, and a series of apoptotic rates. In addition, mitochondria were confirmed to be involved in apoptosis induced by ZC-14 accompanied with the loss of mitochondrial membrane potential (Δψm), the release of cytochrome c from mitochondria into the cytosol and increased expression of cleaved-caspase-3. However, harmine could not induce apoptosis at the same concentration. In summary, these data indicated that compound ZC-14 has a higher cytotoxicity than harmine against Sf9 cells. Besides, it exhibited an anti-proliferative effect in Sf9 cells via inducing apoptosis in which the mitochondrial apoptotic pathway plays a crucial role.Harmine, one of the natural β-carboline alkaloids extracted from  Peganum harmala L. which is mainly distributed in northwest China and widely used for various diseases therapies due to its remarkable pharmacological effects, including anti-tumor, anti-hypertensive and anti-inflammatory activities  was synthesized in our lab . ZC-14 wSpodoptera frugiperda Sf9 cells were cultivated in Grace’s insect medium  supplemented with 10% fetal bovine serum (FBS) , 0.33% yeast extract and 0.33% lactalbumin hydrolysate at 28 °C. Cells were changed medium every 3 days and subcultured after 80–90% confluence.4/well) and incubated at 28 °C overnight, then treated with harmine and ZC-14 by the concentrations of 5, 10, 25, 50 and 100 μg/mL. After treatment for 24 or 48 h, 50 μL of 1 mg/mL MTT solution -2,5-diphenyl) was added in each well and the plates were incubated at 28 °C in the darkness for 4 h. The supernatants were carefully discarded and 150 μL of DMSO was added to dissolve the formazan crystal. The absorbance at the wavelength of 490 nm was measured by the Multiskan FC microplate reader .Sf9 cells were seeded into 96-well plates .Cells with the density of 0.5 × 10i, Tokyo, Japan).Cells were seeded in 12-well plates and exposed to 7.5 μg/mL harmine and ZC-14 for 6, 12, 18 and 24 h. The supernatant was removed after treatments and cells were fixed with stain-fixative at room temperature for 5 min and washed with phosphate buffered saline (PBS) for two times. The fixed cells were then stained with 500 μL Hoechst 33258 solution  at 37 °C for 15 min and washed with PBS twice. The nuclear morphology was observed under a fluorescence microscope (Nikon ECLIPES 80 6/well) and incubated at 28 °C overnight. Harmine and ZC-14 were added into the medium with the final concentration of 7.5 μg/mL. After treated with compounds for 6, 12, 18 and 24 h, DNA was extracted using TIANamp Genomic DNA Kit  following manufacturer’s protocol. DNA extracted from cells which treated with 1 μg/mL camptothecin for 12 h was used as positive control. 20 μL DNA extractions were subjected to 1% agarose gel electrophoresis with 0.5 µg/mL ethidium bromide at 60 V for 1 h.Cells were seeded into a 6-well plate (0.5–1 × 106/well) and incubated overnight. Then cells were treated with 7.5 μg/mL ZC-14 for 6, 12, 18 and 24 h and stained by FITC Annexin V apoptosis Detection Kit I  according to manufacturer’s protocol. Briefly, cells with different treatments were harvested by centrifugation  and washed twice with ice-cold phosphate buffered saline (PBS). After resuspended with 100 μL 1× binding buffer, cells were stained by 5 μL Annexin V-FITC and 5 μL PI in the darkness at room temperature for 15 min. After that, another 400 μL of 1× binding buffer was added, and the samples were determined by FC500 flow cytometry . The cells with 7.5 μg/mL harmine treatment for 24 h were used as the negative control and 0.5 × 105 cells of each samples were used for analysis.Cells were seeded into a 6-well plate  and incubated in ice for 15 min. After centrifugation at 16,000× g at 4 °C for 15 min, the supernatant was transferred to a new tube and the proteins were quantified by Bradford method. For each sample, the supernatant containing 200 µg protein was mixed with 50 μL reaction buffer and 5 μL caspase-3 substrate in a 96-well plate. The plate was incubated in the darkness at 37 °C for 4 h and the absorbance at 405 nm was measured by Multiskan FC microplate reader .Sf9 cells were treated with ZC-14 at 7.5 μg/mL for 6, 12, 18 and 24 h, respectively. After incubation, cells were collected by 600× i, Tokyo, Japan).Sf9 cells were seeded on glass coverslips and incubated overnight, then treated with 7.5 μg/mL harmine and ZC-14 for 12, 18 and 24 h, respectively. The supernatant was removed after treatments and cells were stained with Rhodamine 123 (2 mg/mL)  at room temperature for 20 min. The cells on glass coverslips were washed with cold PBS twice and the fluorescence intensity was detected by fluorescence microscopy  and washed twice with PBS. Total protein was extracted by CytoBusterTM Protein Extraction Reagent  and incubated at 25 °C for 10 min, then centrifuged with 13,000× g at 4 °C for 10 min. The supernatants were used as the total proteins samples for western blot. The mitochondrial and cytosolic proteins were isolated by mitochondrial protein extraction kit  according to manufacturer’s protocol. Bradford method was used to determine the protein concentration.Sf9 cells treated with 7.5 μg/mL ZC-14 for 6, 12, 18 and 24 h were harvested by centrifugation  and transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk at room temperature for 2 h and then incubated with primary antibodies diluted 1:2000 in tris-buffered saline  overnight at 4 °C, followed by secondary antibody conjugated with horseradish peroxidase (diluted 1:4000 in TBS) for 2 h. The protein label was visualized by ECL detection system .t test. p value <0.05 was considered statistically significant.Data were presented as means ± SD (standard deviation) from three independent experiments, and the significance was evaluated by one-way analysis of variance (ANOVA) followed by Duncan’s test and student’s In summary, ZC-14, the novel compound synthesized based on the structure of harmine, has higher cytotoxicity than harmine. The effect of apoptosis induction by ZC-14 at a lower concentration of 7.5 μg/mL in Sf9 cells was confirmed by physiological and biochemical approaches. The activation of the mitochondrial apoptotic pathway by ZC-14 was reflected by the loss of mitochondrial membrane potential and cytochrome c release. Our results not only confirmed the cytotoxicity mechanism of ZC-14 in Sf9 cells, but also provide some theoretical basis for further use of ZC-14 as a pesticide.


We report adsorption behaviors of a typical fluorocarbon R22 (CHClF2) in a new series of isoreticular porous coordination frameworks [Zn4O(bpz)2(ldc)]. 2) in a new series of isoreticular porous coordination polymers [Zn4O(bpz)2(ldc)], in which the typical Zn4O clusters are connected by hydrophobic 3,3′,5,5′-tetramethyl-4,4′-bipyrazolate (bpz2–) and different linear dicarboxylates (ldc2–) to form non-interpenetrated pcu networks with variable pore sizes, shapes, and volumes. Fluorocarbon sorption measurements of these materials revealed high R22 uptakes of 0.73–0.97 g g–1 (0.62–0.65 g cm–3) at 298 K and 1 bar and working capacities of 0.41–0.72 g g–1 (0.35–0.47 g cm–3) between 273 and 313 K at about 0.13, 0.11 and 0.52 bar, respectively, as well as very large diffusion coefficients of 5.1–7.3 × 10–7 cm2 s–1. Noteworthily, the R22 sorption performance can be dramatically improved by subtle modification of the pore size and shape, demonstrating porous coordination polymer–fluorocarbon as a promising adsorbent–adsorbate pair for heat transformation applications.Adsorption heat transformation is one of the most energy-efficient technologies, which relies much on the type and performance of the adsorbent–adsorbate pair. Here, we report adsorption behaviors of a typical fluorocarbon R22 (CHClF In 1–3, the apertures along the c-axis are the same 4.3 × 6.9 Å2 since they possess the same bipyrazolate layer across the ab-plane. However, since the lengths and side groups of the dicarboxylate ligands are quite different, the pore sizes and shapes of 1–3 vary from each other  showed that 2 sorption isotherms of 1–3 measured at 77 K exhibit typical type-I characters with saturated uptakes 516, 461 and 656 cm3  g–1  g–1  and S4†,ructures , reveali2) were measured for 1–3 at ambient conditions  and 0.97 g g–1 (0.63 g cm–3) at 0.6 and 1.0 bar  and 0.85 g g–1 (0.53 g cm–3), respectively.32 This special characteristic implies that a relatively large working capacity can be easily achieved by 3 within a narrow range of pressures. At 313 K, the R22 uptakes of 3 are indeed much lower than those of 1 and 2 at a much wider range of pressures, which confirms that the working capacity for 3 in practical applications, i.e., temperature-swing isobaric adsorption–desorption,45 should be much higher than those for 1 and 2. The working capacity at a particular pressure (isobaric adsorption) depends on the difference in uptakes between the lower and higher working temperatures.49 For instance, the highest working capacities for 1–3 between 273 and 313 K can be calculated as 0.46, 0.41, and 0.72 g g–1 at about 0.13, 0.11, and 0.52 bar, respectively , 31.8(1.8), and 31.4(1.1) kJ mol–1, respectively. These enthalpies are similar to that of MIL-101 (34.6 kJ mol–1)32 and higher than those for activated carbon (22.0–28.0 kJ mol–1)46 and the standard enthalpy of vaporization for R22 (28.2 kJ mol–1), which may be associated with the more polar host of PCPs compared with activated carbon. The higher enthalpy means that the system can transfer more heat during the adsorption heat pump processes. The similar zero-coverage adsorption enthalpies indicate that the R22 molecules are initially adsorbed on very similar sites in 1–3. Actually, the cavity sizes of 1 and 2 are almost identical, while that of 3 is slightly larger, which is consistent with their enthalpy trend. On the other hand, although the aperture sizes are distinct for 1–3, their surfaces are completely lined by low-polarity C–H moieties, which are not likely the preferential adsorption sites. To explain the similar zero-coverage adsorption enthalpies and identify the primary R22 adsorption sites, the interactions between R22 molecules and 1–3 were investigated by grand canonical Monte Carlo (GCMC) simulations, which showed that the initial binding sites of R22 molecules in 1–3 are almost identical . PXRD patterns of 3 in R22 gas at 1 bar and in air were compared  at 293 K and 1.18 g g–1 (0.77 g cm–3) at 343 K, respectively. These values are similar to the highest uptakes achieved by some large-surface-area activated carbons ,46 and higher than that of MIL-101 (about 1.27 g g–1 or 0.79 g cm–3 at 298 K)  at similar conditions.46 The working capacity of 3 gradually decreases to 0.71 g g–1 or 0.46 g cm–3 at 1 bar, highlighting the good performance at higher working pressures. To ensure the regenerability of 3, R22 adsorption–desorption cycling measurements were further performed at 293 K, and there was no noticeable loss in adsorption capacity after 8 cycles , corresponding cold production can be realized at high pressure. To evaluate the performance at such conditions, high-pressure R22 adsorption isotherms for nd 343 K . The upt Table S2.32 Based Fig. S10.1–3 were analysed .50 The fast diffusion of R22 in 1–3 should be attributed to the highly uniform pore in the crystalline adsorbents. It can be seen that the larger diffusion coefficients are associated with the compound with larger pore sizes. The high diffusivity of refrigerant in adsorbent is beneficial for rapid adsorption heating and cooling processes, improving the response speed of the system. Interestingly, the pellet form of 1–3 (obtained by simple compressing at about 5 MPa) showed only slightly decreased R22 adsorption kinetics (–6.8%, –4.9% and –2.0%, Fig. S11Kinetics of R22 adsorption at ambient conditions for analysed  and S11†In summary, we demonstrated that highly porous PCPs can show large fluorocarbon adsorption capacity, high diffusivity and good regenerability, which can be candidate adsorbents in heat transformation cycles. Noteworthily, the pore size and/or porosity can impact the sorption performance, demonstrating tunable sorption performance can be obtained by the rational design of PCPs. These results may inspire future design and fabrication of novel adsorbent materials for heat transformation systems.


A family of [MIII2MII3]n+ trigonal bipyramidal cages are characterised in the solution and solid state. n+ trigonal bipyramidal cages  of formulae [Fe2Co3L6Cl6] (1), [Fe2Zn3L6Br6] (2), [Cr2Zn3L6Br6] (3), [Cr2Pd3L6(dppp)3](OTf)6 (4) and [Al2Pd3L6(dppp)3](OTf)6 (5) (where HL is 1-(4-pyridyl)butane-1,3-dione and dppp is 1,3-bis(diphenylphosphino)propane) are reported. Neutral cages 1–3 were synthesised using the tritopic [MIIIL3] metalloligand in combination with the salts CoIICl2 and ZnIIBr2, which both act as tetrahedral linkers. The assembly of the cis-protected [PdII(dppp)(OTf)2] with [MIIIL3] afforded the anionic cages 4–5 of general formula [MIII2PdII3](OTf)6. The metallic skeleton of all cages describes a trigonal bipyramid with the MIII ions occupying the two axial sites and the MII ions sitting in the three equatorial positions. Direct current (DC) magnetic susceptibility, magnetisation and heat capacity measurements on 1 reveal weak antiferromagnetic exchange between the FeIII and CoII ions. EPR spectroscopy demonstrates that the distortion imposed on the {MO6} coordination sphere of [MIIIL3] by complexation in the {MIII2MII3} supramolecules results in a small, but measurable, increase of the zero field splitting at MIII. Complete active space self-consistent field (CASSCF) calculations on the three unique CoII sites of 1 suggest DCo ≈ –14 cm–1 and E/D ≈ 0.1, consistent with the magnetothermal and spectroscopic data.A family of five [MIII2MII3] In the field of molecular magnetism, the latter is perhaps best exemplified by cyanometalate chemistry.23Molecular magnetism relies on the ability of the synthetic chemist to make an enormous breadth of structurally diverse polymetallic cages spanning the d- and f-block of the periodic table.24 the stabilisation of reactive molecules25 and photochemistry.26 Due to the difficulties associated with performing solution-based one- and two-dimensional NMR spectroscopy on paramagnetic species, where broad signals and a wide chemical shift range are commonplace,27 it is perhaps not surprising that the majority of metallosupramolecular chemistry has focused on the use of diamagnetic metal centres, albeit with some notable exceptions.28A similar synthetic approach is followed in the metallosupramolecular chemistry of diamagnetic cages and capsules where the combination of directional metal–ligand bonding and rigorously rigid ligands creates cages with permanent internal cavities capable of hosting guest molecules, constructed primarily for potential application in, for example, catalysis,29 an approach centred around the tritopic metalloligand [MIIIL3] (where HL = 1-(4-pyridyl)butane-1,3-dione), which features a tris(acac) octahedral transition metal core functionalised with three p-pyridyl donor groups butane-1,3-dione (HL) and the metalloligand   in 35 mL of dichloromethane/acetone (1 : 1 v/v) was added ZnBr2 . The solution was stirred for 30 minutes, before being evaporated to dryness. The dark-red product was re-dissolved in nitromethane, filtered and allowed to stand. Dark-red, prism-shaped X-ray quality crystals were obtained after room temperature evaporation of the mother liquor after 10 days. Yield . Elemental analysis (%) calculated for C54H48N6O12Br6Fe2Zn3: C 36.85, H 2.75, N 4.77. Found: C 36.97, H 2.87, N 4.91.To a solution of the metalloligand [FeIIIL3]  in 35 mL of dichloromethane was added ZnBr2 . After 1 hour of reaction a precipitate appeared. The dark-red solid product was isolated by filtration, re-dissolved in DMF and layered with MeOH. Dark-red, prism-shaped X-ray quality crystals were obtained after 10 days. Yield . Elemental analysis (%) calculated for C54H48N6O12Br6Cr2Zn3: C 37.01, H 2.76, N 4.80. Found: C 36.92, H 2.67, N 4.67.To a solution of the metalloligand [CrIIIL3]  in 35 mL of methanol was added [Pd(dppp)2(CF3SO3)2] propane). The solution was stirred for 30 minutes, before being filtered and allowed to stand. Orange, rod-shaped X-ray quality crystals were obtained after room temperature evaporation of the mother liquor after 5 days. Yield . Elemental analysis (%) calculated for C141H126O30N6F18P6S6Cr2Pd3: C 48.00, H 3.60, N 2.38. Found: C 47.89, H 3.47, N 2.27.To a solution of the metalloligand [CrIIIL3]  in 35 mL of acetonitrile was added [Pd(dppp)2(CF3SO3)2] . The solution was stirred for 15 hours at 50 °C, before being filtered and layered with diethyl ether. Colourless, rod-shaped X-ray quality crystals were obtained after 5 days. Yield .1H NMR : δ 8.61 , 7.79–7.67 , 7.48–7.44 , 7.42–7.39 , 7.34–7.27 , 7.26–7.22 , 7.18 , 6.13 , 3.28–3.11 , 3.10–2.92 , 2.60–2.36 , 2.15 , 1.93–1.75  ppm. 13C NMR : δ 198.79, 177.61, 151.26, 147.79, 134.91–134.82 , 133.70, 133.07–132.99 , 130.60–130.51 , 130.42–130.33 , 127.58–126.88 (m), 125.52–124.82 (m), 124.37, 122.06 , 99.58, 28.10, 22.25–21.92 (m), 18.30 ppm. 31P NMR  δ 6.97 ppm. 19F NMR  δ –79.05 ppm. Diffusion coefficient  5.99 × 10–10 m2 s–1, hydrodynamic radius 9.9 Å. ESI TOF HRMS m/z: found 1010.1238 [M – 3OTf–]3+, calculated for [C138H126Al2F9N6O21P6Pd3S3]3+ 1010.1069. Elemental analysis (%) calculated for C141H126O30N6F18P6S6Al2Pd3: C 48.69, H 3.65, N 2.42. Found: C 48.42, H 3.57, N 2.35.To a solution of the metalloligand  and compounds 1–4 at the UK National EPR Facility in Manchester.Magnetisation measurements were carried out on a Quantum Design SQUID MPMS-XL magnetometer, operating between 1.8 and 300 K for DC applied magnetic fields ranging from 0 to 5 T. Microcrystalline samples were dispersed in eicosane in order to avoid torquing of the crystallites. Heat capacity measurements were carried out for temperatures down to IIIL3] with a cis-protected square planar complex should yield a trigonal bipyramid. However, in the case of the archetypal 90° acceptor complex [(en)Pd(NO3)2],44 it had previously been shown that instead, displacement of the bidentate ethylene diamine ligand occurs to yield the [Al8Pd6]12+ cube.29 We were thus pleased to find that when we switched to the more strongly coordinating bis(diphenylphosphino)propane (dppp), we were able to isolate the [Al2Pd3]6+ trigonal bipyramidal complex, 5, in 83% yield following reaction overnight at 50 °C between [AlIIIL3] and [Pd(dppp)(OTf)2] in acetonitrile. All the spectroscopic data indicate that the structure of 5, confirmed by X-ray crystallography (see below), is preserved in solution. As well as ESI-MS, which reveals the 3+ charge state corresponding to [5 – 3OTf]3+ matching the expected isotopic distribution  octahedral complexes are also known to generate discrete metallosupramolecular cages,45 the divergent disposition of the pendant donor groups create larger closed systems, which with a dynamic system such as this will rapidly rearrange to give the entropically more favourable trigonal bipyramid. A comparison of the 1H NMR spectra of the re-dissolved crystalline sample of 5 .It is also interesting to note that the starting metalloligand  (1), [Fe2Zn3L6Br6] (2), [Cr2Zn3L6Br6] (3), [Cr2Pd3L6(dppp)3](OTf)6 (4) and [Al2Pd3L6(dppp)3](OTf)6 (5)  in acetone, methanol, acetonitrile or a mixed solvent system, with crystals isolated from slow evaporation of the mother liquor, or diffusion of Et2O or MeOH . The metallic skeletons of the cages in 1–5 describe a trigonal bipyramid with the MIII ions situated on the axial positions and the MII ions on the equatorial sites. The approximate dimensions of the [MIII2MII3]n+ metallic skeleton are MIII···MII (8.77–8.99 Å), MII···MII (11.72–12.80 Å) and MIII···MIII (10.75–11.20 Å).The heterometallic trigonal bipyramid cages [FeTf)6 (5)  and 4 weII metal ions is coordinated by two N donors from the pyridyl groups of [MIIIL3]. The N–MII–N angle of the tetrahedral CoN2Cl2/ZnN2Br2 moiety for compounds 1–3 lies in the range 90.63–103.57°; in 4–5 the equivalent N–Pd–N angle is in the range 84.40–85.39°. Each [MIIIL3] corner unit consists of a six-coordinate MIII ion with regular {MO6} octahedral geometry. For the three different [MIIIL3] metalloligands used in the synthesis the MIII–O distances and angles are: Fe–O 1.98–2.02 Å, Fe–O cis/trans angles 83.48–95.17° and 169.99–178.40°, respectively; Cr–O 1.91–1.98 Å, Cr–O cis/trans angles 86.82–94.04° and 176.35–179.85°, respectively; Al–O 1.86–1.89 Å, Al–O cis/trans angles 88.84–91.43° and 179.03–179.54°, respectively. The CoII and ZnII ions lie in distorted tetrahedral environments with bond distances in the range 2.05–2.35 Å  and bond angles around the metal centres ranging from 90.62° to 120.08°. In compounds 4 and 5, the PdII ion is 90° cis-blocked through the use of the dppp ligand (Pd–P bond distance ∼ 2.27 Å). The coordination of Pd to [MIIIL3] through the use of Pd–N bonds (ranging from 2.08–2.14 Å) creates a distorted square planar geometry around the Pd centre with cis/trans bond angles in the range 84.40–93.50° and 165.49–178.57°, respectively. While complexes 1–3 are neutral, charge balance is maintained in 4 and 5 through the presence of a total of six CF3SO3– anions, lying outside the cage.Each of the three M1–3 and 4–5. Whereas 1–3 are all homochiral racemates in which each intact capsule features two [MIIIL3] units that possess the same Λ or Δ chirality, in contrast structures 4 and 5 are both the achiral heterodiastereomer. While sorting of chiral octahedral metal motifs has been frequently observed in metallosupramolecular assembly reactions, for the vast majority homochiral assemblies are energetically preferred.47 The commonality of the [Pd(dppp)] unit in both 4 and 5 that feature different [MIIIL3] metalloligands would suggest that either the small change in angle between pyridine donors at each MII connector and/or the interactions of the dppp protecting ligand with these donors cause the change in diastereomeric preference. Solution studies with 5 would also indicate this is not simply due to selective crystallization from a complex mixture (see above). Outwith cyanometalate chemistry,34 compounds 1–4 represent the first examples of trigonal bipyramids built with paramagnetic metal ions, and join a small family of analogous compounds containing diamagnetic metal ions.52While the intrametallic distances of the five trigonal bipyramids are similar, there is nonetheless a distinct diastereomeric difference between structures χ, of a polycrystalline sample of 1 was measured in an applied magnetic field, B, of 0.1 T, over the 2–300 K temperature, T, range. The experimental results are shown in χT product, where χ = M/B, and M is the magnetisation of the sample. At room temperature, the χT product of 1 has a value of 14.4 cm3 K mol–1, in good agreement with the sum of Curie constants for a [FeIII2CoII3] unit . Note that the estimation of the g-value of the CoII ions here is an approximation and subject to error (e.g. lattice solvent lost upon sample drying will result in a variation of the samples diamagnetism), and a better measure comes from the EPR spectroscopy, which is consistent with gCo = 2.3 (vide infra). Upon cooling, the χT product of 1 remains essentially constant down to approximately 100 K, wherefrom it decreases upon further cooling to 9.5 cm3 K mol–1 at 2 K. Given that the anisotropy of FeIII is negligible, this behaviour is consistent with a relatively large single-ion magnetic anisotropy for the CoII centres and/or an antiferromagnetic exchange interaction between the FeIII and CoII centres. To better define the low-temperature magnetic properties of 1, low temperature variable-temperature-and-variable-field (VTVB) magnetisation data were measured in the temperature and magnetic field ranges T = 2–12 K and B = 0–5 T (1 is of 13.7 μB (μB is the Bohr magneton). Furthermore, when the VTVB data of 1 are plotted against the reduced quantity μBB/kT, little nesting of the VTVB data is observed. This observation indicates that the part of the energy spectrum of 1 probed under these experimental conditions does not present significant anisotropy splitting with respect to the temperature of measurement at zero magnetic field.The dc (direct current) molar magnetic susceptibility,  = 0–5 T . At the i, j run through the constitutive metal centres, gi is the g-factor of the ith centre, ŝ is a spin operator, J is the isotropic exchange interaction parameter, D is the uniaxial anisotropy parameter and S is the total spin.For the quantitative interpretation of the magnetisation data, we used spin-Hamiltonian (1)g-factors are equal to 2, SIIIFe = 5/2, SIICo = 3/2, we only consider exchange interactions between CoII and FeIII centres, and neglect the single-ion anisotropy of FeIII. Furthermore, we fix the uniaxial anisotropy of CoII to DCo = –14 cm–1, as extracted from the modelling of the EPR data and theoretical calculations, which are discussed further in the following sections. Thus, at this point our model contains only one free parameter, namely, the isotropic exchange between FeIII and CoII, JFe–Co. The χT product of 1 was fitted to spin-Hamiltonian (1) by full matrix numerical diagonalisation of the spin-Hamiltonian of the full system of dimension 2304 by 2304, through use of the Levenberg–Marquardt algorithm.53 This resulted in the best-fit parameter JFe–Co = –0.04 cm–1. In order to verify the validity of our model, JFe–Co was fixed to the determined best-fit value, JFe–Co = –0.04 cm–1, and DCo was maintained fixed at –14 cm–1. At this point our model contains no free parameters. Thereafter, the VTVB data of 1 were simulated by use of spin-Hamiltonian (1). The simulated curves are shown as solid red lines in 1 consists of four groups of densely packed states, each separated by approximately 2DCo  for zero-applied magnetic field. As is typical for molecular magnetic materials,54 lattice vibrations contribute predominantly to cp as a rapid increase above liquid-helium temperature. The lattice contribution can be described by the Debye model . Therefore, the temperature and field dependence of the cp data in B ≥ 3 T, are particularly suitable for probing the influence of crystal fields on 1, down to temperatures significantly lower than the ones obtained in the magnetisation measurements.For inset of . Such lai.e., DCo = –14 cm–1 and the here-negligible JFe–Co = –0.04 cm–1. The agreement with the experimental data is good, though not outstanding. Anticipating the discussion on the EPR spectra (vide infra), we have checked that adding a zero-field splitting (ZFS) of DFe = –0.2 cm–1 at the FeIII sites does not improve the fit. The discrepancy is most evident below ca. 1 K, where the experimental data have lower values than the calculated ones. This behaviour can be explained by a wider broadening of the low-lying energy spectrum, likely induced by higher-order anisotropy terms, which are not taken into account in Hamiltonian (1).The solid lines in 3], giving the ZFS of the CrIII, s = 3/2 ion as D = –0.55 cm–1 with a small rhombicity of |E/D| = 0.045.29 Q-Band spectra of 3 and 4 are similar to that of [CrIIIL3], and give D = –0.64 and –0.61 cm–1, respectively .55 Hence, the distortion imposed on the {CrO6} coordination sphere of [CrIIIL3] by complexation in the {CrIII2MII3} supramolecules results in a small, but measurable, increase of the ZFS at CrIII. The {CrO6} metric parameters do not appear to be very different.We previously reported EPR spectra of [CrLD is also found for the FeIII (s = 5/2) systems. X- and Q-band EPR spectra of [FeIIIL3] reveal a rather small ZFS of D = 0.08 cm–1 with |E/D| = 1/3 (D has no significance with a fully rhombic D-tensor). These values are similar to those reported for [Fe(acac)3] 56 and [Fe(dpm)3] .57 On incorporation into the {FeIII2ZnII3} complex 2, a much richer spectrum is observed (D = 0.20 cm–1 (E/D = 1/3). Angular overlap model studies on [Fe(acac)3] and [Fe(dpm)3] show D to be very sensitive to the trigonal distortion at FeIII,57 and there is a more significant structural difference in the {FeIIIO6} coordination spheres when bound in {FeIII2ZnII3}, with longer Fe–O bonds and wider O–Fe–O angles in the {py}3 face, than in the equivalent CrIII systems.Such an increase in observed  and S12,1 gives Q-band EPR spectra with very broad features at ca. 5, 9 and 12 kG that line up with the main features of the spectrum of the {FeIII2ZnII3} complex 2. Hence, the ZFS at FeIII must be similar. The very large ZFS at CoII means that only transitions within the ground Kramers doublet of this ion are observed , and there must be a significant rhombicity in order for these transitions to fall within the observed features. The spectra also show that the JFeCo exchange interaction must be very weak, resulting only in severe broadening of the peaks. Test calculations on a simple {FeIIICoII} model, with fixed ZFS at the s = 5/2 and 3/2 spins  suggest that if |JFeCo| > ca. 0.02 cm–1 then additional features would be observed in the Q-band EPR spectrum. Note that the limit for the full, five-spin system would be different.The {FeIII2CoII3} complex DFe values obtained from EPR would have a negligible effect on the calculated χT(T) and cp curves for 1, and a negligible effect on the global level structure in DFe| and |JFeCo| are ≪|DCo|. However, it would affect the detail of the states within each of the densely packed multiplets of DFe| and |JFeCo| are of similar magnitude.The II we have performed complete active space self-consistent field (CASSCF) calculations on the three unique CoII sites of 1, see the SI for details. The results suggest DCo = –14 cm–1, E/D = 0.1  octahedral transition metal core functionalised with three p-pyridyl donor groups, and a series of transition metal salts. Outwith cyanometalate chemistry, compound 1 represents the first example of such a cage containing paramagnetic metal ions. Complementary studies investigating the diamagnetic variants using 1H NMR spectroscopy reveal some interesting features about the solution self-assembly process. Firstly, the [MIIIL3] metalloligand is a highly dynamic tritopic building block as evidenced by fac configurational isomer being amplified at the expense of the mer during the course of cage formation. The self-assembly process also occurs with high and unusual stereoselectivity wherein the trigonal bipyramids are formed exclusively from twisted pyramidal components of opposite Δ/Λ-handedness. Solution stability of the cage is also confirmed via mass spectrometry. SQUID magnetometry and heat capacity measurements on 1 reveal weak antiferromagnetic exchange between the FeIII and CoII ions, with |DCo| = 14 cm–1. EPR spectroscopy reveals that the distortion imposed on the {MO6} coordination sphere of [MIIIL3] by complexation in the {MIII2MII3} supramolecules results in a small, but measurable, increase of the zero field splitting at MIII. CASSCF calculations on the three unique CoII sites of 1 suggest that the principal axes of the local ZFS tensors are oriented perpendicular to the FeIII–FeIII axis, but canted ∼120° with respect to each other.Complexes


The structure, morphology, and optical properties of the as-prepared WS2/Bi2MoO6 samples were characterized by XRD, XPS, SEM, TEM (HRTEM), and UV-vis diffuse reflectance spectra (DRS). Results confirmed the existence of an excellent nanojunction interface between layered WS2 nanoslices and Bi2MoO6 nanoflakes. Under visible light (>420 nm), the WS2/Bi2MoO6 composites exhibit significantly enhanced photocatalytic activity compared with pure Bi2MoO6 toward the decomposition of rhodamine B (RhB). Meanwhile, the active species trapping experiments indicated that holes (h+) were the main active species during the photocatalytic reaction. The enhanced photocatalytic performance can be ascribed to the effective light harvesting, fast photogenerated electron–hole pairs separation, and excellent charge carrier transport of the WS2/Bi2MoO6 heterostructures. Moreover, the prepared WS2/Bi2MoO6 composites also show good structural and activity stability in repeatability experiments.In this paper, novel WS Geneadiation . As to padiation –6. Despiadiation . It is wadiation , 9.2MoO6 has received attention in the field of visual-light-driven photocatalysis because it possesses distinct sandwiched layered structure [2MoO6 is not suitable for the utilization as an efficient visible-light photocatalyst due to the high recombination probability of photogenerated charge carrier. Therefore, some effective strategies to meet this challenge by using the architecture of proper hybrid nanostructure and especially the introduction of two dimensional (2D) nanosheets have been proved as an effectual approach to strengthen interfacial charge transfer between two components in the process of photocatalytic reaction. Obviously, it is anticipated that the heterostructure between Bi2MoO6 and 2D layered material will increase photocatalytic efficiency by visual-light irradiation [As a novel photocatalyst, Bitructure , 11. As adiation .2 have recently paid the attention of the scientific community in photocatalysis research, which ascribes the lack of interlayer coupling and the absence of inversion symmetry resulting in the photoelectric property that differ markedly from those of the bulk [2 or g-C3N4, have received significant attention to explore potential photocatalysis applications, which lead to TMD nanosheet which is often utilized as a supporter to establish the heterostructured composite photocatalysts via different energy band hybrid strategies [2/Bi2MoO6 composites exhibited an efficient performance for photocatalytic oxidation of rhodamine B under visible-light irradiation [2/Bi2MoO6 as a visual-light photocatalyst has not been reported.Layered transition metal dichalcogenides (TMDs) are widely regarded as a kind of promising loading material because of their analogous graphene reticular structure , 13. Espthe bulk , 16, 17.the bulk . In factrategies , 20. Foradiation . However2/Bi2MoO6 composite via a facile solvothermal growth method using pre-exfoliated layered WS2 nanoslices as a supporter. The WS2/Bi2MoO6 exhibits excellent photocatalytic activity towards the degradation of rhodamine B (RhB) under visible-light (λ > 420 nm) irradiation. According to the microstructure characterization analysis of XRD, XPS, SEM, and TEM, the possible photocatalytic mechanism of the few-layer WS2/Bi2MoO6 composite was also elucidated. It is believed that the formation of junctions between Bi2MoO6 and WS2 can allow the prompt migration of photogenerated charge and reduce the self-agglomeration. It is postulated that the excellent photocatalytic activity of WS2/Bi2MoO6 should be ascribed to its high migration efficiency of photo-induced carriers and the interfacial electronic interaction. These results also probably provide a valuable perspective to insight into the design of other heterostructured photocatalysts.Herein, we demonstrated a facile strategy to fabricate heterostructured WS2 was accomplished following the modified report method [2 powder  was added to 20 mL of ethanol/water with EtOH volume fractions of 40% added as dispersion solvent. The sealed flask was sonicated for 10 h, and then the dispersion was centrifuged at 3000 rpm for 20 min to remove aggregations. Finally, the supernatant was collected to obtain few-layer WS2 nanoslices. To determine the concentrations of 2D nanosheets in the supernatant, we estimated the mass remaining in the supernatant by measuring the UV-vis absorption spectrum at fixed wavelength of 630 nm. The calculation result by virtue of Lambert–Beer Law indicated that the exfoliated WS2 dispersion concentration was about 0.265 ± 0.02 mg/ml.The liquid exfoliation of layered commercial WSt method . Briefly2/Bi2MoO6 samples were synthesized using a facile solvothermal method. Typically, 2 mmol of Bi(NO3)3·5H2O was added to 10 mL of ethylene glycol solution containing dissolved Na2MoO4·2H2O with the Bi/Mo molar ratio of 2:1 under magnetic stirring. An appropriate amount of exfoliated WS2 nanoslices was dispersed into 20 mL ethanol and ultrasonicated at room temperature for 45 min. Then, it was slowly added into the above solution, followed by stirring for 10 min to form a homogeneous phase. The resulting solution was transferred into a 50-mL Teflon-lined stainless steel autoclave and kept at 160 °C for 10 h. Subsequently, the autoclave was cooled to room temperature gradually. Finally, the precipitate was centrifuged and washed with ethanol and deionized water several times and dried in a vacuum oven at 80 °C for 6 h. According to this method, WS2/Bi2MoO6 composites with different WS2 mass ratios  were synthesized. For comparison, the blank Bi2MoO6 was prepared in the absence of WS2 using the same experimental conditions.The WSλ = 1.5406 Å, USA). The ultraviolet-visible diffuse reflectance spectra (DRS) of samples were performed at room temperature in the range of 200–800 nm on a UV-vis spectrophotometer  equipped with an integrating sphere attachment. The electronic states of surface elements of the catalysts were identified using X-ray photoelectron spectroscopy .Structure and morphology of the sample was investigated by scanning electron microscopy , transmission electron microscopy , high-resolution transmission electron microscopy , and powder X-ray diffraction . The distance between the ultraviolet filter and aqueous RhB solution was about 6.5 mm. And the power density of visible light was 150 mW/cm2, which was estimated by the optical power meter . Prior to irradiation, the suspension was kept in the dark under magnetically stirred for 30 min to ensure the establishing of an adsorption/desorption equilibrium. At given time intervals, 2 mL were collected from the suspension and immediately centrifuged; the concentration of RhB after illumination was monitored at 553 nm by using UV-vis spectrophotometer . The relative concentrations (C/C0) of the RhB were determined by the absorbance (A/A0) at 553 nm. All experiments were carried out at least in duplicate. The reported values were within the experimental error range of ±2%. Combining with Lambert–Beer law, the photocatalytic degradation rate constant (k) of RhB was obtained using the following formula:In all catalytic activity of experiments, 50 mg of the samples were added to aqueous RhB solution  magnetically stirred in a Pyrex glass vessel and then radial irradiated with a 300 W Xe arc lamp  to provide visible light with C is RhB concentration at reaction time t, C0 is the adsorption/desorption equilibrium concentration of RhB at the starting reaction time, and A and A0 are the corresponding absorbance values.where +), and 2 mM p-benzoquinone , and 40 mL/min N2 (an electron quencher). The comparative trials of photocatalytic degradation were performed under the same reaction conditions as those mentioned above.In addition, to identify the active species generated during photocatalytic reactivity, various scavengers were added into the solution of RhB, including 2 mM isopropanol , 2 mM disodium ethylenediamine tetraacetic acid , (101), (103), (105), and (110) crystal planes of WS2 (JCPDS card no. 84-1398). As for the pure Bi2MoO6, the diffraction peaks of (131), (200), (151), (260), (331), and (262) planes at 2θ = 28.2°, 32.5°, 36.0°, 47.1°, 55.4°,and 58.5°, which can be indexed to orthorhombic phase of Bi2MoO6 (JCPDS card no. 76-2388). In the case of the few-layer WS2/Bi2MoO6 composite materials, the XRD pattern only displays the characteristic diffraction peaks of hexagonal phase WS2 and orthorhombic phase Bi2MoO6. Furthermore, compared with the standard data for Bi2MoO6 (no. 76-2388), the existence of few-layer WS2 did not change the diffraction peak positions of Bi2MoO6 in the composite sample, indicating Bi2MoO6 nanoflakes grown on few-layer WS2 nanoslices rather than incorporated into the WS2 lattice. There is no trace of any impurity phase under the present resolution, which suggests the high purity of the as-prepared samples.In order to confirm the composition and crystal structure of the as-prepared samples, an XRD study was carried out. As shown in Fig. 2 without sonicated treatment and exfoliated nanoslices are shown in Fig. 2 have been stripped to few-layer WS2 nanoslices. Figure 2MoO6. It can be seen that the Bi2MoO6 exhibited microsphere morphology with rough surfaces. Closer examination reveals that the microspheres consist of numerous secondary Bi2MoO6 nanoplates. Furthermore, when Bi2MoO6 was deposited onto the 2D few-layer WS2 via a facile solvothermal process  images. It is easy to observe in Fig. 2 (purple arrows) shows a clear nanosheet structure which is similar to that of graphene, proving that graphene-like tungsten disulfide is obtained. Meanwhile, Bi2MoO6 nanoplates with diameters of about 50–100 nm were observed to grow on the WS2 nanosheets. HRTEM images  planes of WS2, respectively. Therefore, the experimental results indicated that a coherent and tight heterojunction interface between few-layer WS2 and Bi2MoO6 was formed, which can benefit better charge separation and efficient electron transfer within the hybrid structure in comparison with pure Bi2MoO6.Further information about the nano-structure of the few-layer WSges Fig. , c taken2/Bi2MoO6 composites were further determined by XPS spectra. Figure 2/Bi2MoO6 (5 wt%) sample, which exhibits W, S, O, Bi, Mo, and C peaks. No peaks corresponding to other elements are observed. The peak for Bi 4f in the Bi2MoO6  toward lower binding energies as compared with the pure Bi2MoO6. Meanwhile, in the hierarchical WS2/Bi2MoO6 composite, the values of W 4f5/2 (34.2 eV) and W 4f7/2 (32.0 eV) peaks  than the pure WS2 (34.4 and 32.2 eV). Similarly, the high-resolution S 2p spectrum , the hierarchical composite display surprisingly strong absorption around 450–800 nm. These results clearly indicate that the composite photocatalyst could absorb more photons during photocatalytic reaction. Therefore, it can be revealed that the addition of WS2 nanoslices is beneficial for the visible-light absorbance of the WS2/Bi2MoO6 composite.Figure Eg) of samples were calculated by the following equation [In addition, the optical band gap energies  or indirect (n = 4)) [ahv)2 versus (hv) is converted according the UV-Vis-DRS. As shown in Fig. Eg values of pure WS2 and Bi2MoO6 have been estimated to be 1.47 and 2.72 eV, respectively.where (n = 4)) , 28. A p2MoO6 and mechanically mixed samples (5% WS2 and 95% Bi2MoO6) have also been investigated. As shown in Fig. 2MoO6 was only ~39% after 100 min of visible-light irradiation. Obviously, all the hierarchical WS2/Bi2MoO6 composites show better photocatalytic performance than the pure Bi2MoO6. ~48, ~74, ~95, and ~88% of RhB were degraded using 1% WS2/Bi2MoO6, 3% WS2/Bi2MoO6, 5% WS2/Bi2MoO6, and 7% WS2/Bi2MoO6, respectively. The results indicate that the optimal WS2 content in WS2/Bi2MoO6 composite exists when the mass ratio is 5%. Meanwhile, it was noted that the WS2/Bi2MoO6 (5 wt%) composite exhibits remarkably superior photocatalytic activity than the mechanically mixed 5% WS2 and 95% Bi2MoO6. This strongly suggests that an effective nanojunction interface contact and strong interactions between WS2 and Bi2MoO6 are extremely useful to enhance the migration, transport, and separation processes of photogenerated carriers. Furthermore, such superior photocatalytic performances could be attributed to the good crystallization and high specific surface area of composites and the small sheet thickness of the WS2 substrate.The photocatalytic activities of the as-prepared samples were measured by degrading rhodamine B (RhB) under visible-light irradiation. For comparison, photocatalytic activities of pure Bik is 0.0280 min−1 for the hierarchical WS2/Bi2MoO6 (5 wt%) composites, which is 3.8 and 7.1 times greater than those of mechanically mixed WS2 and Bi2MoO6 and pure Bi2MoO6, respectively. These results indicated that RhB could be degraded more efficiently by the hierarchical WS2/Bi2MoO6 composite photocatalyst.In addition, the pseudo-first-order kinetics model was used to fit the experimental data of the photocatalytic degradation of the RhB solution, and the results are given in Fig. 2/Bi2MoO6 (5 wt%) composite photocatalyst, which was performed to further study the photocatalytic degradation process of RhB. It can be seen that the main absorption peak of RhB gradually shifted from 552 to 537 nm, corresponding to the stepwise formation of a series of N-de-ethylated intermediates. As the visible-light irradiation process continues, the peak located at 537 nm is continued to shift and decrease, which indicates that the RhB molecules were further decomposed into smaller molecular fragments and the structure of RhB was also destroyed in the end. The two-step transition processes for photodegradation of RhB were also reported in several previous studies [Figure  studies , 30. Mea2/Bi2MoO6 composites was investigated by repeatability experiments for degradation of RhB, as shown in Fig. 2/Bi2MoO6 remains stable in the first two-cycle experiments. After four recycles, the catalysts did not show an obvious decrease in photocatalytic activity, demonstrating that WS2/Bi2MoO6 composite retained a relatively high degradation activity during the photodegradation process. Furthermore, the catalyst samples collected after four cycles was characterized by XRD measurement , 1,4-benzoquinone (BQ), and disodium ethylenediamine tetraacetic acid (EDTA) acted as the scavengers for ·OH, ·O2−, and h+, respectively. It can be observed that the addition of 2 mM IPA or BQ in the RhB solution had a little effect on the rate constant kapp, suggesting that ·OH and ·O2− are the secondary active species during the photocatalytic reaction, not the main active species during the photocatalytic reaction. On the contrary, the kapp for degradation of RhB obviously decreased after the addition of 2 mM EDTA. Therefore, it can be confirmed that h+ play a key role for degradation of RhB. Furthermore, N2 was bubbled into the RhB solution at the rate of 40 mL/min to ensure that the reaction was operated without O2 as an electron quencher. The degradation of RhB showed a slight decrease in comparison with the case of the air-equilibrated solution and further indicated that ·O2− played a minor role.Figure 2 and Bi2MoO6 potentials should be calculated. For a semiconductor, the bottom CB and top VB can be estimated by the empirical formula [ECB = X − E0 − 0.5Eg and EVB = ECB + Eg, where ECB (EVB) is the CB (VB) edge potential; X is the electronegativity of the semiconductor; E0 is the energy of free electrons of the hydrogenscale (~4.5 eV vs NHE); and Eg is the band gap energy of the semiconductor obtained from the UV-visible diffuse reflectance absorption. The X values for WS2 and Bi2MoO6 are calculated to be 5.66 and 5.55 eV, respectively [ECB and EVB values of WS2 are determined to be +0.43 and +1.9 eV and Bi2MoO6 are −0.31 and +2.41 eV, respectively.To explain the enhanced photocatalytic performance, conduction band (CB) and valence band (VB) of WS formula : ECB = ectively , 32, 33.2/Bi2MoO6 composite photocatalyst is shown in Fig. 2 and Bi2MoO6 are excited under visible-light irradiation and generate electrons and holes in their CB and VB, respectively. The electrons on CB of Bi2MoO6 will easily transfer WS2 due to the CB potential of Bi2MoO6 (−0.31 eV) is more negative than the CB potential of WS2 (0.43 eV) [2 nanoslices could act as effective electron collectors, which was favorable to the separation of electron–hole pairs in Bi2MoO6. Therefore, this fast electron and hole transfer process can decrease the recombination of charges and prolong the lifetime of holes on VB of Bi2MoO6 [2 (+0.43 eV) is more positive than E0 (O2/·O2−) (−0.046 eV) which suggests that the ·O2− radicals were not formed through electrons reducing the dissolved O2 [2MoO6 can react with dissolved O2 to yield ·O2− radicals because its potential (−0.31 eV) is more negative than E0 (O2/·O2−). Thus, the ·O2− active species played a minor role. Meanwhile, the photo-induced holes on VB of Bi2MoO6 could not also directly oxidize the adsorbed H2O molecules to ·OH radicals because its potential (+2.41 eV) was lower than E0 (·OH/H2O) (+2.68 V) [2− act as a strong oxidizing agent to oxidize the organic pollutants (RhB) to CO2 and H2O. Therefore, the hierarchical WS2/Bi2MoO6 composites exhibit improved photocatalytic activity.On the basis of the above results, a possible photocatalytic mechanism scheme of the WS0.43 eV) , 30. The Bi2MoO6 . The CB olved O2 . However+2.68 V) . Finally2/Bi2MoO6 heterostructured photocatalysts were successfully fabricated via a facile solvothermal growth method using pre-exfoliated layered WS2 nanoslices as a substrate. The hierarchical WS2/Bi2MoO6 exhibits excellent photocatalytic activity towards the degradation of rhodamine B (RhB) under visible-light irradiation. Based on the results of a series of structure and performance tests, it is believed that there formed a tight nanojunction interface between layered WS2 nanoslices and Bi2MoO6 nanoflakes, which make the photo-induced electrons be easily transferred to the WS2 substrate. As a result, the recombination of charges was decreased and the lifetime of holes was prolonged. Therefore, the hierarchical WS2/Bi2MoO6 composites exhibit much higher visible-light-driven photocatalytic activity than the pure Bi2MoO6. Furthermore, the WS2/Bi2MoO6 composites are very stable under visible-light irradiation and cycling photocatalytic tests. Thus, the as-prepared WS2/Bi2MoO6 photocatalyst has potential application for pollutant abatement.In summary, a novel WS


Bacillus thuringiensis are known to have toxicity against a variety of insects and have been exploited to control insect pests through transgenic plants and biopesticides. B. thuringiensis SBS BT-1 carrying the cry2 genes was isolated from soil samples in Pakistan. The 2-kb full length cry2Ac gene was cloned, sequenced, and submitted to the EMBL DNA database (Accession No. AM292031). For expression analysis, Escherichia coli DH5α was transformed with the fragment sub-cloned in pET22b expression vector using NdeI and HindIII restriction sites, and later confirmed by restriction endonuclease analysis. To assess the toxicity of Cry2Ac7 protein against lepidopteran and dipteran insects, BL21 (codon plus) strain of E. coli was further transformed with the recombinant plasmid. The 65-kDa protein was expressed in the form of inclusion bodies up to 180 OD units per liter of the medium. Inclusions were washed with a buffer containing 1.5% Triton-X 100 and >90% pure Cry2Ac7 was obtained. The inclusion bodies were dissolved in 50 mM K2CO3 (pH 11.5), dialyzed, and freeze-dried. This freeze-dried protein as well as inclusion bodies were used in bioassays against larvae of Helicoverpa armigera and Musca domestica. The freeze-dried protein was toxic to H. armigera larvae with an LC50 value of 131 ng/mL. However, Cry2Ac7 produced in E. coli did not show any mortality to M. domestica larvae. This is the first report of Cry2Ac protein toxic to H. armigera.The Cry  proteins from  Bacillus thuringiensis (Bt) is an aerobic, Gram-positive, endospore-forming soil bacterium. It produces parasporal protein toxins called insecticidal crystal proteins (Cry and Cyt proteins) or δ-endotoxins during the sporulation phase. These proteins are toxic to dipteran, lepidopteran, and coleopteran larvae [Bt does not have a significant history of mammalian pathogenicity [n larvae . They arn larvae . Despitegenicity .More than 250 crystal (Cry) proteins have been described ,5. The gcry2A gene encodes a protein of 65-kDa, which forms cuboidal crystals [cry2A genes viz., cry2Aa [cry2Ab [cry2Ac [cry2Aa and cry2Ac genes have a common characteristic in that these are placed at the third position in a three-gene operon. However, the crystallization of Cry2Aa protein requires the second gene, orf2, in the cry2Aa operon [orfs upstream of cry2Ac gene do not have an apparent role in the formation of Cry2Ac inclusions [cry2Ab gene is cryptic [Lymantria dispar, Heliothis virescens, and Trichoplusia ni, but not toxic to Aedes aegypti. cry2Ac gene encodes approximately 65-kDa protein, which targets lepidopteran as well as dipteran insects. In the mature toxin, the N-terminal domain (residues 1–272) is a pore-forming seven-helical bundle. The second domain (residues 273–473) is a receptor binding β prism which is a three-fold symmetric arrangement of β sheets, each with a Greek key fold. The third domain (residues 474–623) is implicated in determining both larval receptor binding and pore function, and is a lectin-like C-terminal β sandwich. Cry2A binds to different receptors on BBMV, and thus is used for resistance management. Transgenic tobacco, rice, and cotton plants expressing cry2A genes have been generated for auto-protection against insects. Bt cotton that expresses two different Bt toxins (Cry1Ac + Cry2A) is being used to manage insect resistance [Bt Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant Heliothis virescens.crystals ,12. Thre, cry2Aa ,13, cry2 [cry2Ab ,14,15, aa operon , while t cryptic ,14,17. Dsistance . Kota etsistance  demonstrBt-cotton expressing Cry1Ac. In Pakistan, Maqbool et al. [indica expressing cry2A gene, which was reported to be effective against two major rice pests in the Indian subcontinent—the yellow stem borer and the rice leaf folder. Later, Zaidi [Nicotiana tabacum with cry2A to protect it against Heliothis virescens.Besides their long-term use as a biological insecticide in the form of sprays of spore-crystal mixtures, individual Cry toxins have been expressed in transgenic plants to render crops resistant to insect pests. Since Cry1-type toxins have extensively been used in transgenic plants, there are reports that insects have developed resistance against some of these toxins. Akhurst et al.  reportedl et al.  generater, Zaidi  producedcry genes is still limited and does not cover many distinct geographic areas. There is, therefore, a need to search for novel and more potent strains with new pathogenic spectra and wider host ranges, especially in parts of the world that have not been adequately sampled. Pakistan constitutes one such area which needs to be explored for crystalliferous strains with wider host ranges in this region. The present report characterizes cry2A-type gene(s) from these strains, and evaluates the toxicity of Cry2A proteins against the target insects, Helicoverpa armigera and Musca domestica.However, information about the distribution of cry1, cry2, and cry4 genes using a universal set of primers , obtained from Cell and Molecular Biology Lab (CMBL), University of the Punjab, Lahore, revealed the presence of cry2 gene [cry1, cry1Ac, and cry4, but positive for cry2Ab and cry2Ac.All of the 50 local isolates were subjected to genotyping for  primers . Only si primers  amplifiery2 gene . SBS BT1cry2Ac gene, cloned from local isolate SBS-BT1, was sequenced from both strands and deposited to EMBL with accession number AM292031. It has now been named as Cry2Ac7 protein by B. thuringiensis Delta-Endotoxin Nomenclature Committee, becoming a new member of the Bt toxins (http://www.lifesci.susx.ac.uk/home/Neil_Crickmore/Bt/).Full length E. coli was transformed with the recombinant SBS-BT1/cry2Ac7. Ten colonies were picked up from the plate, inoculated in LB broth and induced with IPTG (1 mM) at 37 °C for 8 and 20 h. Protein profiles were analyzed with SDS-PAGE. All colonies expressed about 65 kDa Cry2Ac7 protein equally after 8 h. Furthermore, the IPTG concentration was optimized and no visual difference was noted in the expression of Cry2Ac7 when induced with 0.5, 1.0, 1.5, and 2.0 mM IPTG concentration  for freeze-dried protein and 118 ng/mL (0.118 µg/mL) for inclusion bodies, as shown in Cry2Ac7 protein, in the form of partially purified freeze-dried powder as well as crude inclusion bodies, was used in toxicity assays against M. domestica, the protein had no toxic effect on the growth and vigor of the emerging flies (data not included).In E. coli are not reflective of the true nature of the crystals, and the bioassay data reported here for H. armigera and M. domestica may not be interpretable and comparable with other Cry2A-based assays in which native Cry2A crystal/spore mixtures have been used.It may, however, be kept in mind that the inclusions produced in H. armigera) and dipteran (M. domestica) insects. PCR amplification, a sensitive method which can rapidly detect and identify target DNA sequences, had been employed by researchers to identify Bt genes [cry1, cry1Ac, cry2, and cry4 genes. The results indicated the presence of cry2 gene only, whereas cry1 and cry4 were not detected. The toxicity of the strain in the bioassays was therefore possibly because of cry2 gene(s). There are however, a number of other factors apart from Cry toxins in Bt which show insecticidal activity found within the crystals or act as enhancers for Cry or cytolysins (Cyt) toxins. Bt produces various virulence factors other than the crystal proteins, including secreted insecticidal protein toxins, α-exotoxins, β-exotoxins, hemolysins, enterotoxins, chitinases, and phospholipases [SBS-BT1 indeed showed the toxicity against lepidopteran  might be the target gene-encoding protein toxic to the tested insects. To the best of our knowledge, this is the first report of Cry2Ac-type protein toxic to American bollworm, H. armigera. Wu et al. [50 value of 4000 ng/mL, while Cry2Ac7 is 30.5-fold more toxic with an LC50 value of 131 ng/mL. Genotyping with specific primers of u et al.  reportedCry2Ac7 has 97.43% amino acid homology with Cry2Ac1. There are 16 amino acid differences between the two proteins . Most ofBacillus thuringiensis subsp. kurstaki HD-1, designated cry2Aa and cry2Ab . Their respective gene products are 87% identical but exhibit different toxicity spectra; Cry2Aa is toxic to both mosquito and tobacco hornworm larvae, whereas Cry2Ab is toxic only to the latter. They generated hybrids of the cry2Aa and cry2Ab genes as well as their resultant gene products were assayed for toxicity. A short segment of Cry2Aa corresponding to residues 307 through 382 was shown to be sufficient for altering host range specificity i.e., when this region replaced the corresponding segment of Cry2Ab, the resulting hybrid protein acquired toxicity against mosquitoes. The Cry2Aa and Cry2Ab polypeptides differ by only 18 amino acids in this region, indicating that very few amino acid changes can have a substantial effect on the toxicity spectra of these proteins.Interestingly, the difference of a few amino acids can alter the toxicity spectrum of Cry proteins. Widner and Whiteley  studied A. albopictus [cry2Ac5 encoded a toxic protein against A. albopictus, which was not in agreement with the previous reports that Cry2Ab, Cry2Ac, and Cry2Ad were only toxic to lepidopteran insects [On the other hand, the toxicity of Cry2Ac5 (Accession # DQ341379) was reported to the third instar larvae of mosquito, bopictus . Cry2Ac7 insects ,28. It mBt spores that germinated when plated on a rich agar medium after incubating 2 mL of the sample at 80 °C for 10 min. Three hundred microliters of each heat-treated sample was then spread-plated and incubated at 30 °C for 24 h on plates of a medium containing (per liter) 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate (pH 6.8), 0.005 g MnCl, and 15 g agar. Developing colonies were grown at 30 °C for 24 h on LB agar plates . Smears of these were stained with Gram’s staining. Diagnostic schemes given in Bergey’s Manual of Determinative Bacteriology [Bt subspecies.Soil samples were collected from different areas of Lahore, Pakistan. The samples were processed by the acetate selective method . One graeriology  were folFull length 16S rDNA (1692 bp) was amplified through PCR  and clonBt was prepared according to Kronstad et al. [4)2SO4 2 g, KH2PO4 6 g, sodium citrate.2H2O 1 g, MgSO4·7H2O 0.2 g, glucose 0.5%, K2HPO4·3H2O 18.3 g, tryptone 20 g, yeast extract 5 g, dissolved per liter of water) in a 2.8-L flask with shaking at 37 °C. Cells were harvested at an optical density of 0.7 at 600 nm. The cultures were harvested by centrifugation at 6000 rpm (4355 rcf) for 10 min at 4 °C in a Beckman centrifuge, and finally washed with 100 mL of a solution containing 100 mM NaCl, 10 mM Tris (pH 7.9), and 10 mM EDTA. The bacterial pellet was resuspended in 5 mL of a solution containing 150 mM NaCl and 100 mM EDTA at pH 7.9. Lysozyme was added to give a final concentration of 0.25 mg/mL, and the preparation was incubated at 37 °C for 20 min. To lyse the cells, 6.25 mL of a third solution (100 mM Tris (pH 7.9), 100 mM NaCl, 2% SDS) was added. The preparation was mixed gently by inverting the tube four or five times and incubated at 60 °C until clear . The lysate was then extracted four times with phenol-chloroform (1:1), which had been equilibrated with the above Tris-NaCl-SDS solution. The aqueous phase was extracted each time with a wide-bore pipette. After the final extraction, cold ethanol was added and the DNA was spooled out with a glass rod. The DNA was then rinsed with 70% ethanol, dissolved in TE buffer (10 mM Tris (pH 7.9), 1 mM EDTA) and stored in aliquots at −20 °C. Universal set of primers [cry1, cry2, cry4, cry1Ac, cry2Aa, cry2Ab, and cry2Ac genes in these isolates  containing Taq buffer 1x, MgCl2 1.5 mM, dNTPs 200 µM, each of the primers 50 pmol, DNA 0.2–0.8 µg, and Taq DNA polymerase 2.5–5.0 units, was run on an Applied BioSystems 2720 thermal cycler. The thermal cycle of the reaction consisted of pre-PCR heating at 94 °C for 5 min, a final extension at 72 °C for 10 min, and 30 cycles each of denaturation at 94 °C for 1 min, annealing at 50 °C for 0:45 min, and an extension at 72 °C for 2 min. For sub-cloning in expression vector pET22b, cry2Ac gene was amplified with the following primer pair using T/A cloned gene as a template:To amplify full length gene from the local pET21a-N   5′  GCTCGGTACCTCGCTGAATGCATCTAGCATATGAATAC 3′M13 R           5′-d(GAGCGGATAACAATTTCACACAGG)-3′)NdeI-HindIII digested vector and PCR fragment were used in 1:3 ratio in a 50-µL reaction containing 5 µL of 10x ligation buffer and 5 units of T4 DNA ligase. The reaction mixture was incubated at 22 °C overnight. BL21 (codon plus) strain of E. coli was transformed with the recombinant vector for expression analysis.cry2ac7 gene was amplified with N-terminal primer containing NdeI restriction site and ligated in expression vector pET22b to form a recombinant pSBS-BT1/cry2Ac7. DH5α was transformed with this recombinant DNA. Six colonies were picked up from the plate. All of them had recombinant plasmids, which was confirmed by plasmid miniprep and restriction analysis of the plasmid. +) was transformed with the recombinant plasmid pSBS-BT1/cry2Ac7. A single colony was inoculated in LB broth containing 100 μg/mL ampicillin and grown at 37 °C overnight with continuous shaking. The next day, it was seed inoculated (1%) in LB broth (ampicillin 100 μg/mL), and grown at 37 °C for about 2 h (until OD550 was 0.6–0.8) with continuous shaking. It was induced with various concentrations of IPTG  at varying temperatures  for different time periods . Expression levels of the protein were analyzed on 12% SDS-PAGE. In all experiments, BL21C+ transformed with non-recombinant pET22b plasmid was proceeded in parallel as a negative control. BL21 codon plus  50 mM, Triton X-100 1.5%, NaCl 200 mM, EDTA (pH 8.0) 10 mM, DTT 2 mM). The cells were lysed by passing through a French press under 2500 psi internal pressure and then centrifuged at 9500 rpm  for 15 min at 4 °C to harvest inclusion bodies. The pellet was washed once again with wash buffer, twice with distilled water, once with 0.5 M NaCl, and twice with water. The pellet was resuspended in 50 mM K2CO3 (pH 11.5) and incubated at 37 °C for 1.5–2 h. It was then centrifuged at 9500 rpm  for 25 min at 4 °C to harvest the insoluble inclusions. The solubilized part was dialyzed against different decreasing concentrations of K2CO3  and finally against distilled water overnight at 4 °C. The protein was then freeze-dried.The 280 (1 OD unit = 1 mg) as well as by a modified Lowry method [The freeze-dried protein was solubilized in 20 mM Tris (pH 10.6) and dialyzed against the same buffer overnight at 4 °C, centrifuged at 6000 rpm (4468 rcf) to remove any particulate matter, then filtered through 0.4-µm Millipore filter and injected in the Amersham Bioscience FPLC machine. Fractions were analyzed on SDS-PAGE and protein was quantified by taking ODy method .H. armigera and 0.217 to 8.696 µg/g for M. domestica), in two separate experiments, were thoroughly mixed in the artificial diet of H. armigera [M. domestica [Various concentrations of Cry2Ac7 protein (0.025 to 1.00 µg/g for armigera  and M. domestica . In a neH. armigera were placed over their diet surface to feed ad libitum, each in a separate vial. The vials were covered with aluminum foil and kept at 24 ± 2 °C. Mortality was recorded after 72 h. Likewise, 50 eggs of M. domestica were placed on the diet surface for the eggs to hatch within 8–16 h and the larvae were allowed to feed on the diet ad libitum at 26 ± 2 °C. The mortality was recorded after 10–13 days. All experiments were conducted in three replicas. Eight to 10 neonate larvae of The mortality was determined using Probit analysis .