Aspergillus flavus remain unclear. In this study, structurally modified lentinan derivatives were prepared by carboxymethylation, sulfation, and phosphorylation to explore their inhibition activity to AF synthesis. The results demonstrated that inhibitory activity of lentinan decreased at higher or lower concentrations than 200 μg/mL. Compared with lentinan, the sulphated derivatives only performed a reduced optimal inhibition rate at a higher concentration. The phosphorylated derivatives achieved complete inhibition of AF production at 50 μg/mL, but the inhibitory activity was attenuated with an increase of concentration. The minimum concentration of carboxymethylated derivatives to completely inhibit AF synthesis was the same as that of the original lentinan, whereas their inhibition activity was not reduced at the increasing concentration. RT-PCR analyses were conducted to understand the effects of lentinan and its carboxymethylated derivatives on the transcription of certain genes associated with AF biosynthesis. The results showed that lentinan delayed the transcription of aflQ, whereas its carboxymethylated derivatives promoted the transcriptions of all the tested genes. Our results revealed that some chemical group features apart from the β-bond could play the vital role in the prevention of AF formation by polysaccharide, and highlighted the structural modifications which could promote its practicability in the control of aflatoxin contamination.The chemical properties of β-glucans leading to their inhibition on aflatoxin (AF) production by  There ad humans , and thed humans . For food humans –6. Many d humans . Among td humans .d-glucans, and specifically -β-d-glucans that are moderately branched with -β-d-glucan chains from the cell wall of Saccharomyces cerevisiae, are capable of adsorbing AFB1 through the formation of hydrogen bonds [et al. suggested that β-glucans from Lentinus edodes could inhibit AF biosynthesis and be a promising tool against AF contamination [Trametes versicolor extracts are also able to inhibit AF production in A. parasiticus [A. flavus have not been deciphered.Glucans from mushrooms, which are pharmacologically classified as biological-response modifiers and immunomodulators, have been found to attenuate aflatoxicosis . The β-den bonds . Reverbemination . Anotherasiticus . Fungal d-glucans that are moderately branched with -β-d-glucan chains -β-n chains  and haveasiticus , the dir2.2.1.2.1.1.−1 , and none of the three structurally modified lentinans resulted in any significant changes in the mycelial growth of A. flavus.As shown in Data in 2.1.3.A. flavusin response to lentinan and C-D. ApyapA orthologs are known to be part of the antioxidant response in fungi and have been found to be associated with AF biosynthesis through the modulation of the reactive oxygen species (ROS) in A. parasiticus [yapA orthologue compared to its transcription during the initial stage of AF formation in the control  in the control. However, their transcription was continuously activated in A. flavus exposed to lentinan and C-D (aflQ (a gene downstream of the AF biosynthesis pathway) compared with the control, whereas the treatment with C-D promoted aflQ transcription at a higher level.To understand the mechanism through which these β-glucans exert their inhibitory effects, we analyzed the transcription of certain genes that are known to be associated with AF production in asiticus . It was  control . The comer level . Two genynthesis . AflR isynthesis , whereasynthesis . It was ry state . Similar and C-D . It is n2.2.aflQ transcription in A. flavus exposed to lentinan and exhibited a common lag time compared with the control. This finding implicated aflQ in the lentinan-induced inhibition of AF production. Both lentinan and C-D promoted aflR transcription, which is contrary to the downregulation of aflR transcription observed in A. parasiticus exposed to the β-glucan extract from L. edodes [In this study, definite effects of lentinan on AF production were revealed. First, the valid inhibitory concentrations were restricted to a narrow range, and the inhibition was only temporary. The AF formation paralleled . edodes ,19. All in vitro and in vivo [A. flavus because this information will greatly contribute to our understanding of the relationship between β-glucans and AF formation.It has been reported that β-glucans of similar structure, molecular weight, and solution conformation exhibit vastly differing biological activities  in vivo . In this in vivo ,20. This in vivo ,22. ThesA. flavus, which might result in the inhibition of AF formation well. However, AP-1 (Yap1 othologue) is found in the promoter region of aflR and is hypothesised to attenuate the expression of aflR [aflR transcription by lentinan and C-D illustrated that the regulation of aflR transcription by Ap1 is subject to other factors. It is notable that C-D promoted the transcription of all of the tested AF biosynthesis-related genes, which was inconsistent with its inhibitory effects on AF formation. This phenomenon deserves further investigation.There is a close relationship between oxidative stress and AF production ,23. The  of aflR . The up-3.3.1.Aspergillus flavus CGMCC3.2890 was purchased from the China General Microbial Culture Collection Centre. The strain was cultured on Sabouraud Dextrose (SD) medium containing 4% (w/v) glucose, 1% peptone, and 2% agar for 3 days. Then spore suspension was made by slightly shaking the plate after the addition of 0.05% Triton X-100 and then counted by blood counting chamber under microscope. Lentinan and its derivatives were added to the SD liquid medium (without agar) at the indicated concentration  before the inoculation of the spores of A. flavus (approximately 105/mL). The inocula were incubated at 28 °C and 120 rpm for 72 h. The AFB1 in the culture was determined and compared to that in the control (without tested fractions). The fungal mycelia were also collected after the passing through two layers of cheesecloth. After washing five times with sterile water, the collected mycelia were dried in an oven at 80 °C for the determination of the dry weight.3.2.1 in A. flavus culture was extracted with chloroform in a ratio (v/v) of 1:3 (culture broth/chloroform). The extracts were dried with a nitrogen-blowing instrument , followed by dissolving in methanol, and then filtered through a 0.22 μm microporous membrane for subsequent HPLC analysis. The samples were injected through an Agilent HPLC 1100 system equipped with an ODS column  and maintained at 22 °C. The mobile phase was acetonitrile/methanol/water  at a flow rate of 1 mL/min. The column eluent passed through a variable wavelength detector operated at 365 nm and calculated against the aflatoxin B1 standard curve  [Aflatoxin B.5 μg/L) .3.3.3.3.1.et al. [Lentinan was carboxylated according to the protocol described by Chen et al. . Lentina3.3.2.According to the protocol described in a previous study , lentina3.3.3.et al. [N,N-Dimethylformamide (DMF) (20 mL) at room temperature under stirring for 30 min. Then, 3-phosphonopropionic acid (2.0 g), N,N′-Dicyclocarbodiimide (2.7 g), and 4-Dimethylaminopyridine (DMAP) (3.1 g) were added, and the mixture was allowed to react for 12 h. The mixture was dialyzed against distilled water with a dialysis tube with molecular weight cut off of 8.0 kDa for 36 h to remove DMF, DMAP, and other potential degradation products. Phosphorylated derivatives of lentinan were obtained after freeze-drying.Lentinan was phosphorylated following the procedure described by Wei et al. . Lentina3.4.−1.The derivatives of lentinan were structurally characterized with a Bruker Tensor 27 FT-IR spectrophotometer . Five milligrams of the derivatives were ground with KBr powder and then pressed into pellets for FT-IR measurement at the frequency range of 400 to 4000 cm3.5.A. flavus were sampled at 48, 72, and 96 h post-inoculation and stored in liquid nitrogen. The total RNA was extracted with an RNApure Plant Kit  according to the manufacturer’s instructions. The extracted RNA was treated with DNase I  at 37 °C for 30 min to remove the genomic DNA and then reverse-transcribed to form the first-strand cDNA. The reaction was followed by denaturation at 92 °C for 5 min and then cooling to 5 °C. The obtained cDNA was then amplified for semi-quantitative analysis. The primers of aflR were designed by Somashekar et al. [aflS, 57 °C for aflR, 49 °C for aflK, 58 °C for aflQ, 49 °C for yap, 49 °C for aflD, and 55 °C for 18S rRNA), and extension at 72 °C for 1 min. The RNA samples were tested for genomic DNA contamination with the extracted RNA directly as the PCR template prior to cDNA synthesis and under the same PCR conditions. The RT-PCR products were separated on 2% agarose gels and stained with ethidium bromide. All of the RT-PCR reactions were performed at least three times. Densitometric scanning using a computer-assisted image analysis system  was used to quantify the signal intensity of each band. The relative abundance of the transcripts were obtained by dividing the band intensity of target gene by the band density of the corresponding 18S rRNA.Mycelia of r et al. , and the3.6.p value was less than 0.05.All the data presented are the mean value ± standard errors of the means (SEM) of three determinations. One-way ANOVA was used to determine whether there are significant differences between the means in all of the experiments. The differences were considered to be significant if the 4.Of the three structurally modified lentinans, only C-D exhibited an acceptable dose-dependent activity in the prevention of aflatoxin generation. Our results revealed that some group features besides the β-bond should be considered in the activity of glucans inhibiting AF synthesis, and carboxymethylation modification could be applied in the development of glucan agents against aflatoxin contamination.


Cell survival depends on the balance between protective and apoptotic signals. When the balance of signals tips towards apoptosis, cells undergo programmed cell death. This balance has profound implications in diseases including cancer. Oncogenes and tumor suppressors are mutated to promote cell survival during tumor development, and many chemotherapeutic drugs kill tumor cells by stimulating apoptosis. BAD is a pro-apoptotic member of the Bcl-2 family of proteins, which can be phosphorylated on numerous sites to modulate binding to Bcl-2 and 14-3-3 proteins and inhibit its pro-apoptotic activities. One of the critical phosphorylation sites is the serine 112 (S112), which can be phosphorylated by several kinases including Pak1.in vitro kinase assays. We found that the primary phosphorylation site is not S112 but serine 111 (S111), a site that is sometimes found phosphorylated in vivo. In transfection assays of HEK293T cells, we showed that Pak1 required Raf-1 to stimulate phosphorylation on S112. Mutating either S111 or S112 to alanine enhanced binding to Bcl-2, but the double mutant S111/112A bound better to Bcl-2. Moreover, BAD phosphorylation at S111 was observed in several other cell lines, and treating one of them with the Pak1 inhibitor 2,2′-Dihydroxy-1,1′-dinaphthyldisulfide (IPA-3) reduced phosphorylation primarily at S112 and to a smaller extent at S111, while Raf inhibitors only reduced phosphorylation at S112.We mapped the Pak phosphorylation sites by making serine to alanine mutations in BAD and testing them as substrates in Together, these findings demonstrate that Pak1 phosphorylates BAD directly at S111, but phosphorylated S112 through Raf-1. These two sites of BAD serve as redundant regulatory sites for Bcl-2 binding. Many apoptotic stimuli disrupt the integrity of the mitochondria, causing them to release pro-apoptotic contents such as cytochrome c and apoptosis inducing factor (AIF) Raf-1 promotes cell survival through multiple mechanisms. Whereas plasma membrane targeting of Raf-1 activates the classical MEK>ERK (MAPK) cascade but does not protect cells, mitochondrial targeting of Raf-1 protects cells in vitro and in vivo. A survey of 98 signaling antibodies found that phosphorylation at S112 and S136 were pharmacodynamic markers for measuring the effectiveness of Pak inhibitors in vivoPaks (p21-activated kinases), which are the effectors for the small GTPases Rac and Cdc42, have a major role in signaling pathways to promote cell migration, cell proliferation transformation and survival in vitro mutational analysis, we showed that neither S136 nor S112 of BAD was a significant site of Pak1 phosphorylation. Instead, the vast majority of phosphorylation by Pak1 occurred at S111, a site recently identified in cells. Using transfection assays in HEK293T cells, we also confirmed that Pak1 can phosphorylate S112 through Raf-1. In addition, we found that S111 phosphorylation may precede S112 phosphorylation to facilitate S112 phosphorylation. Bad phosphorylation at S111 was observed in several other cell lines, and inhibition of Pak1 with inhibitor 2,2′-Dihydroxy-1,1′-dinaphthyldisulfide (IPA-3) reduced phosphorylation at S111 and S112.Here through Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were from Invitrogen . FuGENE 6 transfection reagent, complete protease inhibitor cocktail tablets were from Roche . Rabbit polyclonal antibodies against BAD, phospho-Ser112, phospho-Ser136, ERK, phospho-ERK, phospho-c-Raf (Raf-1) (Ser338) were from Cell Signaling Technology . Antibodies against Bcl-2 (N-19) and 14-3-3 (C-16) were purchased from Santa Cruz Biotechnology . 5-Iodo-3-[methylene]-2-indolinone (GW5074), rapamycin, and 2,2′-Dihydroxy-1,1′-dinaphthyldisulfide (IPA-3) were from CalBiochem . PD098059 and H89 were purchased from Sigma . Glutathione SepharoseTM 4B was purchased from Amersham Pharmacia Biotech .Plasmids expressing a Myc-tagged wild-type (WT), a kinase-dead (KD or K299R) and a kinase-activated (T423E) version of Pak1 cloned into the pCMV6 vector have been described elsewhere The S111-phosphorylated peptide, ETRSRH[pS]SYPAGTE, corresponding to amino acid residues 105 to 118 of mouse BAD, was synthesized , conjugated to KLH and injected into rabbits for antibody production. To affinity purify the antibody, the peptide was cross-linked to NHS (N-hydroxysuccinimide)-activated Sepharose 4 Fast Flow beads (Amersham-Pharmacia Biotech) as instructed by the manufacturer and the beads were used to purify the anti-phospho-BAD (S111) serum.2 and cultured in DMEM containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells were transfected using FuGENE 6 for 24 hr, and then starved overnight before harvesting. A human lung cancer cell line H358, and MPNST cell lines, 90-8 and STS26, were cultured in RPMI containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).HEK293T, and a malignant peripheral nerve sheath tumor (MPNST) cell line ST88-14, were grown at 37°C in 5% COc Protein Assay Kit and equal amounts of proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes . Blots were blocked for 1 hr in TBS-T  buffer supplemented with 5% nonfat milk, and incubated with primary antibodies overnight at 4°C. After washing, blots were incubated with secondary antibodies, and then detected using the enhanced chemiluminescence detection system (Amershan Pharmacia Biotech). The immunoprecipitation assay was carried out as following: whole cell lysates containing the same amount of total protein were incubated with glutathione beads for 4 hr at 4°C. Beads were collected by centrifugation and washed twice with the lysis buffer and once with 1xPBS. Proteins were eluted by boiling in 1xSDS sample buffer, and subjected to immunoblotting, and then probed with antibodies against p-Ser112 BAD, p-Ser111 BAD, Bcl-2 or 14-3-3.Cells were transfected with appropriate vectors. Cell extracts were prepared using lysis buffer  supplemented with 1 mM phenylmethlsulfonyl fluoride and the recommended concentration of Complete protease inhibitors (Roche). Protein concentration was assessed using the Bio-Rad D2, 40 mM Hepes, pH 7.4) supplemented with 5 µM ATP or where indicated 5 µCi of γ- [32P] ATP for 30 min at 30°C in a reaction volume of 25 µL. The reaction was terminated with 1XSDS sample buffer, followed by SDS-PAGE, autoradiography or Western blotting.Protein kinase assays were conducted by incubating a mixture of proteins with the indicated substrates in 1X kinase buffer  supplemented with 20 µM ATP and 1 µCi of γ- [32P] ATP for 30 min at 30°C in a reaction volume of 28 µL. The reaction was terminated with 1X Tricine SDS sample buffer (Invitrogen), run on a an SDS-PAGE tricine gel (Invitrogen) and analyzed by autoradiography.For peptide kinase assays, peptides were dissolved in Tris/EDTA buffer to make a stock concentration of 20 µg/µL. The Ninhydrin test was run with 2 µL of each sample spotted on a Flexible Thin layer Chromatography (TLC) plate to ensure the equal loading of the peptides. The peptides were incubated with Pak and Cdc42For autoradiography, the phosphorylation gel was exposed to a phosphor screen and the phosphorylated BAD bands were quantified using ImageQuant V1.2 software. The “Relative Phosphorylation” was calculated based on the level of phosphorylation in GST alone for 4HCO3, 0.1% SDS and 0.5% β-mercaptoethanol twice in a combined volume of 1.2 mL. 20 µg of carrier protein (RNase A) and TCA  were added. The mixture was kept on ice for 1 hr and then spun down at 10,000 rpm for 10 min at 4°C. After washing with 95% ethanol and dry in air, the pellet was hydrolyzed in 50 µL 6 N HCl for 1 hr at 110°C, lyophilized and dissolved in 6 µL of pH 1.9 buffer  containing 60 µg/ml unlabeled phospho-amino acids. The sample was loaded onto a TLC plate . The TLC plate was run in a Multiphor 2-D apparatus (Amersham Pharmacia Biotech). The first dimension was run at 1.5 kV in pH 1.9 buffer for 20 min. After air-drying for 30–60 min, the TLC plate was turned 90° counter-clockwise and run at 1.4 kV in pH 3.5 buffer  for 16 min. After drying, the plate was sprayed with 0.25% (V/V) ninhydrin in acetone and then incubated at 65°C for 15 min to visualize the unlabeled phosphoamino acid markers which appear as purple spots identifying to the positions of p-Ser, p-Thr and p-Tyr. The plate was then exposed to X-ray film to identify the radiolabeled amino acids.After phosphorylation and electrophoresis, the phosphorylated BAD protein band was cut out from the gel, ground up and eluted with 50 mM NH32P labeled ATP was readily incorporated into BAD when Pak1 was activated by Cdc42L61 (32P-ATP was incorporated into BAD without Cdc42L61 (in vitro kinase assay using BAD peptides (aa 105–118) showed that only mutation of S111 significantly reduced Pak1 and RacL61 phosphorylation of BAD in this region , stimulates phosphorylation of BAD at S112  lane 5–7 at S112 . These din vitro may affect the efficient S112 phosphorylation by Raf-1 and Pak1. Furthermore, these data showed that pS112 reduces phosphorylation of S111 by Raf-1 or Pak1. Together, these studies demonstrate that Pak1 phosphorylates BAD directly on S111 in vivo and indirectly on S112 through Raf-1.To determine if preventing phosphorylation at S111 would influence S112 phosphorylation, we tested if a mutation of S111 would affect phosphorylation at S112. Again, 293T cells were co-transfected with expression vectors encoding full length (FL) BAD  and Pak1 (KD or T423E). We found that mutating S111 to alanine in full length BAD reduced Pak1 stimulated phosphorylation at S112  lane 4. Phosphorylation of BAD at S112 and S136 decreases BAD/Bcl-2 complex formation and increases BAD/14-3-3 binding, which results in increased cell survival To address if S111 of BAD is phosphorylated in other cell lines besides 293T cells, we probed a panel of malignant peripheral nerve sheath tumor (MPNST) cells and one lung cancer cell line (H358) . The MPNin vitro, usually using phosphospecific antibodies SSYYWEIE and S297 of MEK, RTPGRPLSSYGMDSR. All have in common a single upstream basic amino acid, phosphorylation at the first of two serines , and a tyrosine at the +2 position. We note, however, that Pak1 substrates are difficult to predict from sequence comparisons. For example, we did not find BAD, Raf-1 or MEK listed among 1433 possible targets identified in a Scansite search of the Swiss protein database There are several reports of Pak1 stimulating BAD phosphorylation at both S136 and S112 in vivo and in vitro, S111 has only recently been found to be phosphorylated. However, both reports were large scale proteomics studies of phospho-proteins While there are numerous reports documenting phosphorylation at S112 An unexpected observation from this study is that there is a difference in the sensitivity of downstream pathways to Raf inhibitors. We found that a Raf-1 inhibitor reduced phosphorylation of BAD at S112 in 293T cells under a condition where it actually slightly increased phosphorylation of Erk1 , lane 4.The role of Raf in cell survival pathways may explain why Raf inhibitors are performing better in the clinic than MEK inhibitors Figure S1In vitro protein and peptide kinase assays. (A) Coomassie brilliant blue staining of the gel of E. coli through glutathione sephorase 4B columns except His-Pak1 which was purified from sf 9 insect cells through a Ni-NTA agarose column. Wild type (WT) and mutant GST-BAD fusion proteins contain a murine BAD fragment (aa 104-141). 2 μg of His-Pak1, 1μg of GST-Cdc42L61, 2 μg of GST and 6 μg of GST-BAD proteins were applied to 10% SDS-PAGE and stained with Coomassie Brilliant Blue. (D) Purified BAD peptides (aa 105-118) were incubated with RacL61, Pak1 or both with γ32P-ATP and subjected to autoradiography.(TIF)Click here for additional data file.Figure S232P-ATP ATP (upper panel) and subjected to autoradiography, or phosphorylated with unlabeled ATP (lower two panels) and probed with the S111 and S112 antibodies. (B) Purified BAD peptides (aa 105-118) were incubated with activated Raf-1, Pak1 or both with γ32P-ATP ATP, run on a tricine gel and subjected to autoradiography. Bottom panel of B shows phosphorimager quantification of the bands in panel D (n = 2). Student t-test was performed and * p<0.05. The arrows indicate the bands that were quantified.(A) Purified GST-BAD was incubated with or without PKA and phosphorylated with γ(TIF)Click here for additional data file.


Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers. Mammalian target of rapamycin (mTOR) is a highly conserved protein kinase . Adipose specific Raptor knockout mice phenocopied the S6K1 knockout mice with regard to adipocyte generation  were identified in ovarian cancer patients. Using biochemical assays we demonstrated that both of these mutations led to significantly reduced Sin1 phosphorylation at the T86 site, as these mutations impair the canonical AGC kinase consensus recognition motif “RxRxxpS/pT”  remain largely elusive. In this effort, we have defined a novel and independent negative feedback loop through which either S6K1 or Akt directly phosphorylates Sin1 to repress mTORC2 activation in epithelial cells or adipocytes, respectively, providing a possible molecular mechanism for mTORC1 to balance the extra- and intra- cellular signals. Moreover, systematic analyses of many signaling pathways revealed a general oscillation pattern of activation/inactivation signaling dynamics (Purvis et al., Finally, the identification of this mTORC1/S6K feedback suppression of mTORC2 expanded the critical role of mTORC1 in regulating and coordinating various growth factor signaling pathways. Compared to the previously identified negative feedback regulatory loops via IRS-1 (Harrington et al.,


Treating hazardous-infectious medical waste can be carried out on-site or off-site of health-care establishments. Nevertheless, the selection between on-site and off-site locations for treating medical waste sometimes is a controversial subject. Currently in Iran, due to policies of Health Ministry, the hospitals have selected on-site-treating method as the preferred treatment. The objectives of this study were to assess the current condition of on-site medical waste treatment facilities, compare on-site medical waste treatment facilities with off-site systems and find the best location of medical waste treatment. To assess the current on-site facilities, four provinces  were selected to participate in the survey. For comparison of on-site and off-site facilities  Analytical Hierarchy Process (AHP) was employed. The result indicated that most on-site medical waste treating systems have problems in financing, planning, determining capacity of installations, operation and maintenance. AHP synthesis (with inconsistency ratio of 0.01 < 0.1) revealed that, in total, the off-site treatment of medical waste was in much higher priority than the on-site treatment (64.1% versus 35.9%). According to the results of study it was concluded that the off-site central treatment can be considered as an alternative. An amendment could be made to Iran’s current medical waste regulations to have infectious-hazardous waste sent to a central off-site installation for treatment. To begin and test this plan and also receive the official approval, a central off-site can be put into practice, at least as a pilot in one province. Next, if it was practically successful, it could be expanded to other provinces and cities. Medical waste includes all of waste that is produced in the course of health protection, immunization, diagnosis, medical treatment of human beings or animals, scientific research and related laboratories -6. BetweBasic principles of medical waste management include preventing and/or minimizing waste production, appropriately segregating general medical waste from hazardous-infectious medical waste, sending general medical waste to the municipal waste stream for final disposal and treating hazardous-infectious medical waste carefully using special methods ,8,16-18.Currently in Iran, due to policies of Health Ministry, the hospitals  have selected on-site treating method as the preferred treatment. Because, according to Act 64 in Iran’s Medical Waste Management Regulations, all waste producers in middle-sized and large cites are responsible for treating hazardous-infectious waste and converting it into general waste in on-site facilities. Only after on-site pre-treatment of medical waste will the municipality takes the responsibility for off-site transport of waste to the final disposal site. Nevertheless, according to the same Act of the regulation, small cities and villages are allowed to use off-site  facilities for treating their hazardous-infectious waste . Based oIn the meantime, there are great concerns about operation and maintenance conditions of on-site medical waste treatment facilities in hospitals. Therefore, the primary objective of this study was to assess the current condition of on-site medical waste treatment facilities in the country, compare on-site medical waste treatment facilities with off-site systems and find the best location by employing Analytical Hierarchy Process (AHP). In addition, this study aimed to make some practical recommendations on medical waste treatment for improving the current situation.In the beginning of the study for assessing current on-site medical waste treatment facilities, 4 out of 31 provinces in Iran including East Azerbaijan, Tehran, Isfahan and Gilan were selected to participate in the survey. The selection of provinces was done in such a way to cover virtually different geographies, climates, economies, cultures. Then, 10 active hospitals with on-site medical waste treatment facilities were selected in each province  for assessing their current conditions. The selection of hospitals was carried out in such a way to cover various categories of hospitals  and sizes. Site visits  with completing checklist were conducted in all selected hospitals to gather the basic information and assess current working conditions of on-site medical waste treatment facilities. Then, to compare on-site and off-site medical waste treatment facilities, based on the experts’ perspective Analytical Hierarchy Process (AHP) was employed. Different decision-making tools have been developed for application in the environmental field such as the Matrix Method, the AHP and the Electre III method. AHP is one of the most practical multiple choice decision making techniques among the available methods widely used ,28. It sTwenty two criterion variables for on-site treatment and off-site treatment were chosen for the Analytical Hierarchy Process. Then, by brainstorming high-level experienced experts  thirteen final criterion variables (among twenty two primary criterion) for both on-site and off-site treatment was selected for calculation of treatment location . The input data for the analysis was the weighted means resulted from the primary evaluation of the panel experts, which was calculated in Microsoft Excel 2007 software. Analytical Hierarchy Process (AHP) technique was used in two steps to prioritize the objects; it prioritized the criteria in the first step and alternative locations in the second step. In the AHP procedure, as one of multiple decision-making techniques, Eigen values and eigenvectors were computed based on the input data matrix; afterwards, the priority weights were computed to rank the criteria and alternatives. In this procedure, the inconsistency ratio was calculated to investigate consistency of ranking made by the experts. This index ranged between zero (complete consistency) and one (complete inconsistency) and the values lower than 0.1 indicated a reasonable level of ranking consistency and hence confirmed the results of prioritizing the objects. The results of the analysis were presented based on absolute and normalized weights; the absolute weights ranging between 0 and 1 with higher weights showed the higher rank and higher priority of the objects. To normalize the weights, the maximum weight would be transferred to one and other weights would change correspondingly.One of the first and most important steps in making an effective decision for selection of location treatment site is to assess current experiences. Due to not using off-site medical waste treatment system in Iran, only on-site system was assessed for determining the current condition.The survey on the kind of treatment technologies which have been employed in the on-site installations of the studied hospitals indicated that most of used methods included autoclave, hydroclave, chemical treatment and incinerator successively. The result of site visiting, completing checklist and gathering the related data for operating and maintaining on-site medical waste treatment systems are presented in Table AHP presented results in several steps. First, prioritizing the criterion variables in which capital, maintenance and operation costs (CMOC = 0.093), continuous performance and monitoring the system (CPMS = 0.078) and occupational risks in treatment site (ORTS = 0.078) were prioritized as three first variables with the highest level of importance and need for skilled operators (NSO = 0.073) required space (RS = 0.073) and transportation cost of waste (TCW = 0.071) as three variables with lower level of importance Figure . But, foPractical condition assessment indicated that most of on-site medical waste treating systems had operation and maintenance problems. In additions, other problems like difficulties in preparing spare parts, insufficient financial rescores, could be considered. Analytical Hierarchy Process (AHP) technique demonstrated that off-site treatment of medical waste was in much higher priority than the on-site one. Therefore, based on the result of AHP technique, the current problems and unsuccessful experience with on-site treating facilities, off-site central treatment can be considered as an alternative. It was predicted that the advantages of off-site central systems would be more than their disadvantages. The benefits such as the following ones can be also expected: practicability of accepting and treating medical waste of minor medical producers in the city; feasibility of receiving and treating medical waste of the surrounding cities and villages; more cost-effectiveness for larger units by reducing capital, maintenance and operating costs; more economical preparation of spare capacity, easy performance of future expansion and modification; feasibility of employing private sector capacity in installing and operation of medical waste testing facilities; ensuring more efficient operation of central off-site facilities in comparison with several plants  in which skilled workers may not be readily available. Nevertheless in the planning of off-site central treatment facilities, disadvantages such as risks of waste consignment to public health and the environment or illegal recycling should be considered and minimized by strict monitoring and regulation.An amendment could be made in Iran’s current hazardous waste regulations to have infectious-hazardous waste sent to the central off-site for treatment. To begin with, this plan should be tested and official approval should be received; then, a central off-site can be put into practice as a pilot. Next, if it was practically successful, it could be expanded to other provinces and cities. The investment for off-site facilities can be carried out by privet sector or government. Each producer of medical waste should pay their own waste collection, treatment and disposal costs monthly. Meanwhile, the Health Ministry and the Environmental Protection Agency should strictly supervise medical waste collection, treating, and disposal.The authors declare that they have no competing interests.The overall implementations of this study were the results of efforts by corresponding author. All authors have made contribution into the review and finalization of this manuscript. All authors read and approved the final manuscript.


Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca2+-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane  Momordica charantia) with extracts having been used to treat diabetes-like symptoms in humans for hundreds of years. Bitter melon extracts have been reported to have hypoglycaemic, hypolipidemic, antioxidant and anti-inflammatory effects and improve skeletal muscle insulin signalling in vivoin vivo and stimulate glucose transporter 4 (GLUT4) translocation via the activation of AMP-activated protein kinase (AMPK) in both muscle and fat cells Type 2 diabetes (T2D) accounts for 90% of the world’s escalating diabetes problem, with 439 million people expected to have diabetes by 2030 AMPK is a potential target for the treatment of T2D as activation of this kinase can promote fat oxidation to counteract insulin resistance 2+/CaM binding AMPK is a heterotrimer consisting of a catalytic α subunit, a scaffolding β subunit The purpose of this study was to identify the mechanism by which BMTs increase AMPK phosphorylation. Our results in the present study rule out direct allosteric activation, as well as indirect activation of AMPK through inhibition of mitochondrial respiration. We used LKB1-deficient HeLa cells together with the CaMKKβ inhibitor and calcium chelators to confirm that BMTs activate AMPK through CaMKKβ activation without altering intracellular calcium flux. These data support BMTs as being a novel class of AMPK activators and advocates the CaMKK-AMPK pathway as a potential target for novel anti-diabetic therapeutics.32P] ATP (10 µCi/ml) was from Perkin Elmer , AMARA peptide was from Auspep ; ionomycin , bovine serum albumin (BSA), α-MEM, DMEM, foetal bovine serum (FBS) and 100×antibiotic/antimycotic and Pen/Strep/Glutamine (PSG) were from Invitrogen . EGTA-AM was from Calbiochem  and Abbott compound (A-769662) was a gift from Kei Sakamoto . The pan-AMPKβ antibody was a gift from David Carling . The 14-3-3β, CaMKI pT177 and total antibodies were from Santa Cruz Biotechnology Inc. ; all other antibodies were from Cell Signaling Technologies .Column chromatographic separations were carried out by using silica gel H60 , MCI GEL CHP20P , and Sephadex LH-20  as packing materials. HSGF254 silica gel TLC plates  were used for analytical TLC. The Analytical HPLC system was composed of Waters 2690 separations module, Waters 996 diode array detector , and All-Tech 2000 ELSD. A LiChrospher 100 RP-18e column  was used for the separation. The Preparative HPLC system composed of two PrepStar SD-1 solvent delivery modules, a ProStar UV-Vis 320 detector, and a ProStar 701 Fraction Collector . A LiChrospher 100 RP-18  column  was used for isolation. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) was obtained from Toronto Research Chemicals , STO-609 acetate was from Tocris Bioscience , EasyTide [γ-3: MeOH 100∶7∼100∶35) to yield 8 fractions. BMT-1 was purified from fraction 6 by repeated column chromatography over MCI gel (MeOH in water 40∼95%), silica gel (CHCl3: MeOH 8∶1∼8∶3), and Sephadex LH-20 (MeOH). BMT-17 was also purified from this subfraction using repeated column chromatography over silica gel (CHCl3: MeOH 12∶1∼5∶1), Sephadex LH-20 (CHCl3: MeOH 1∶1), and finally preparative HPLC . The structure of BMT-17 was determined by NMR spectroscopic data and its purity (>98%) was determined by HPLC analysis. The structures of both compounds can be seen in Purification of BMT-1 has been described previously 2 incubator. For differentiation into myotubes, L6 cells were cultured in α-MEM supplemented with 2% heat-inactivated FBS and 1% antibiotic/antimycotic for 5–7 days before interventions. Before acute treatments, cells were moved into serum-free media for 18 hrs. Cells were then treated with either vehicle (0.1% DMSO), 2 mM AICAR, or BMTs, at concentrations indicated in the 4P2O7, 2 mM Na3VO4) and 1× Complete Protease Inhibitor Cocktail . Lysates were then snap-frozen in liquid nitrogen and stored at −80°C before use. Whole cell lysates were prepared by clearing cell debris by centrifugation before quantification of protein content by BCA  or Bradford  protein assay.L6 myoblasts (up to passage 17) were cultured in α-MEM supplemented with 10% heat-inactivated FBS and 1% antibiotic/antimycotic. HeLa cells (ATCC) were cultured in DMEM (high glucose) supplemented with 10% heat-inactivated FBS, 1×PSG . Both were kept at 37°C in a humidified 5% COFor respiration experiments, L6 myotubes were cultured and differentiated in an XF24 culture plate . Differentiated L6 myotubes were then serum-starved 18 hrs in unbuffered α-MEM pH 7.4 (Invitrogen Cat No. 12000-022). Basal oxygen consumption rates (OCR) were measured in triplicate cycles of mix (3 min), wait (2 min) and measure (3 min). Repeated triplicate cycles were performed following each consecutive dose-dependent addition of vehicle, BMT-1, BMT-17 or berberine. For quality control, the OCRs were compared to those of untreated myotubes over time and total protein content of each well following the final measurement. To normalise well-well variation, the basal OCR of each well was set to 100% and the dose-dependent effects of each injection were compared to this.in vitro AMPK assays, AMPK complexes were first isolated by immunoprecipitation. Briefly, 50 µg of lysate was combined with 0.5 µl anti-pan-AMPKβ antibody , 10 µl Protein A/G Sepharose (Amersham) and 700 µl of lysis buffer in the absence of protease inhibitor cocktail and incubated rotating at 4°C for 2 h. The beads were then washed in 700 µl lysis buffer, then in 700 µl HGE buffer  and excess fluid was removed using a 30 G needle. The isolated AMPK was then incubated in assay buffer  in the absence or presence of 200 µM AMP. Assays were initiated by addition of 32P-ATP (200 cpm/pmole) at 30°C for 10–14 min. An aliquot of each sample was spotted onto P81 paper and stopped in 1% phosphoric acid. Incorporation of 32P into the AMARA peptide was assessed by β-scintillation counter.For After protein quantification, whole cell lysates were reduced in Laemmli buffer containing 15 mM DTT at 65°C for 10 mins. Lysates  were then subjected to SDS-PAGE on 9% gels and transferred onto Hybond PVDF membranes  overnight at a constant current (40 mA) in Tris/Glycine transfer buffer containing 10% methanol. Membranes were then blocked in 2% BSA in TBS containing 0.1% Tween 20 (TBST) for at least 1 hr before being incubated in rabbit polyclonal antibodies overnight (∼16 hrs) at 4°C. Membranes were then washed in TBST and then incubated in HRP-conjugated donkey anti rabbit secondary antibody  for 1–2 hrs at room temperature. Membranes were then washed in TBST and proteins were visualised by ECL  and exposed on X-ray film before scanning and densitometric analysis using ImageJ .post-hoc test. When two separate treatments were considered, individual two-way ANOVA analyses were used to assess treatment effects and interactions between treatment effects, followed by post hoc tests to compare each group to the vehicle control group. An unpaired Student’s t-test was used to assess statistical significance where one group was compared to vehicle control. For each statistical consideration, p≤0.05 was taken to indicate a significant difference.Results are presented as means ± SEM. When multiple doses were used, individual compounds were compared to vehicle control by one-way ANOVA using a Fisher’s Least Significant Difference in vitro AMPK activity, we isolated AMPK complexes from these lysates using a pan-AMPKβ antibody that recognizes the conserved C-terminus of both AMPKβ subunits essential for complex formation Initially we confirmed that the lead candidate from the original study, BMT-1 in vitro, in an allosteric manner similar to AMP or to the recently described compound A-769662 in vitro with 10 µM BMT-1 or BMT-17 or Abbott compound A-769662 in the presence or absence of 200 µM AMP for 10 min at 30°C before initiation of the in vitro AMPK assay with the addition of 32P.MgATP  only mildly increased AMPK activation may relate to the fact that L6 myotubes are derived from rat skeletal muscle which should predominantly express α2β2-containing AMPK complexes, which are more AMP- and less Abbott compound-sensitive To assess if these BMTs could directly activate AMPK 2P.MgATP . Unlike As BMTs did not activate AMPK directly, indirect mechanisms for AMPK activation were investigated. We recently showed that metformin, rosiglitazone and another natural product, berberine, could activate AMPK by altering the cellular energy charge through inhibiting Complex I of the mitochondrial respiratory chain As AMPK is phosphorylated and activated by AMPKK-dependent phosphorylation, we assessed the involvement of the AMPKKs in the BMT-dependent AMPK activation. The predominant AMPKKs are LKB1 and CaMKKβ. To evaluate the involvement of LKB1, we assessed the ability of these BMTs to increase AMPK phosphorylation and activity in LKB1-deficient HeLa cells As HeLa cells lack LKB1, we therefore investigated if inhibition of CaMKKβ could attenuate the BMT-dependent increase in AMPK phosphorylation and activation in L6 myotubes, which contain LKB1. Accordingly, L6 myotubes were pre-incubated with or without 1 µM of the CaMKKβ inhibitor STO-609 for 15 min before incubation with BMTs. As with the HeLa cells, BMTs increased AMPK phosphorylation and activation and this effect was significantly inhibited by STO-609 . Collectper se, we investigated if BMTs could also increase phosphorylation of the well-established CaMKKβ substrate, CaMKI, at Thr-177. Accordingly, L6 myotubes were incubated with increasing doses of BMT-17 for 30 min before analysis by Western blot  phosphorylate and activate AMPK. Unlike AMP and the Abbott compound A-769662, BMTs do not allosterically activate AMPK and, unlike berberine and metformin, BMTs do not alter cellular respiration. Use of the cell permeable CaMKKβ inhibitor STO-609 on both LKB1-deficient HeLa cells and L6 myotubes has demonstrated the key role of CaMKKβ in this event . We therin vitro suggesting that the BMTs do not activate AMPK in a direct manner. This finding alone is an attractive attribute in an anti-diabetic therapy, as this would allow AMPK to remain responsive to other physiological stimuli such as an exercise bout.BMTs increased AMPK activity in the absence and presence of AMP in an additive but not synergistic way suggesting the activation of AMPK by BMTs involves a mechanism distinct from that of AMP. Furthermore, unlike A-769662 and the allosteric activator AMP, neither of the BMTs could activate AMPK Metformin, rosiglitazone, berberine and resveratrol all activate AMPK at least in part by inhibiting mitochondrial respiration 2+/CaM binding 2+/CaM binding was involved in the activation of AMPK by BMTs. The inability of EGTA-AM to block the BMT-dependent AMPK activation demonstrated that the BMT-dependent AMPK activation was independent of intracellular calcium levels. Conceptually, the BMTs could be binding directly to the Ca2+/CaM binding site and thereby enhancing CaMKKβ activity. However, further experiments are needed to confirm this hypothesis. CaMKII, another Ca2+/CaM sensitive kinase can be activated by methionine oxidation or auto-phosphorylation which then maintains the protein in an active conformation 2+/CaM-independent autonomous activity 2+/CaM binding. Therefore, BMTs could be increasing CaMKKβ activity by inhibiting phosphorylation by these proteins.AMPK is also activated by phosphorylation by the major AMPKKs, LKB1 and CaMKKβ. Using LKB1-deficient HeLa cells and LKB1-proficient L6 myotubes together with the cell-permeable CaMKKβ inhibitor STO-609, we provide evidence to indicate that BMTs activate AMPK through CaMKKβ and not LKB1. As BMTs also increased phosphorylation of another CaMKKβ substrate, CaMKI, this indicates that BMTs may activate CaMKKβ itself and provides further evidence that BMTs are unlikely to activate AMPK through a direct interaction. Since binding to AMPK directs the CaMKKβ activity away from its other substrates CaMKI and CaMKIV It is also unclear whether these BMTs can enter the cell to interact with CaMKKβ. BMTs could, in fact, signal via surface receptors to increase CaMKKβ activity. Indeed, bitter melon extracts have been shown to be estrogen receptor (ER) agonists Natural products may be metabolised by specialized enzymes of living organisms to produce pharmacologically active metabolites. Due to the limited availability of BMTs, we were unable to investigate this possibility. However, our previous study showed that a number of cucurbitane-type triterpenes exert similar biological activity in stimulating GLUT4 translocation in L6 myotubes via activation of AMPK In summary, our data indicate that BMTs activate AMPK in a manner distinct from other anti-diabetic small molecules (illustrated in


These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′) Recently it has been shown that cysteines introduced into various places on monoclonal antibodies allow for the production of site-specific, homogeneous drug-antibody conjugates with many useful properties We used this paradigm to develop a combinatorial platform for producing novel multispecific and monospecific antibody variants. From a combinatorial chemistry point of view, the availability of specific, highly reactive sites at various places on the Fab domain of antibodies should allow for facile generation of Fab′2-like molecules containing the Fabs from two different antibodies (e.g. bispecific molecules) or containing two different cysteine mutations from the same parent antibody (e.g. monospecific molecules). In both cases one has exquisite control of the orientation or geometry between the two Fab arms as defined by the location of the cysteine mutations on each Fab. In addition, control over the flexibility between the Fab arms of the molecule and the distance between the antigen binding sites is possible. We reasoned that an approach that used engineering and chemical linking could provide a method for designing and developing molecules with novel activities. These are capabilities that are not available with a typical antibody format, but are more similar to domain fusions such as diabodies, where geometry and linker design are important for producing a stable, functional molecule. In this report we describe our bis-Fab technology and its application to the generation and modulation of activity of bispecific and monospecific Fab′2-like molecules directed against receptors of the epidermal growth factor family.Epidermal growth factor receptor  family members are essential regulators of cell growth, development and normal adult cellular physiology and are implicated in many abnormal physiological conditions such as cancers During our studies we created new antibody variants targeting HER2 that are identical in the complementarity-determining regions, and but vary greatly in the relative geometry of the Fab domains. These antibody variants are monospecific for HER2 yet show a spectrum of biological activities that are different from the parent antibody from which they were derived. We have used these unique HER2 selective probes as tools to detail the interaction of trastuzumab with HER2 on breast cancer cells. As an approach to antibody design, this technique shows promise for not only identifying tools to probe signal transduction pathways, but also to meet emerging engineering challenges of developing more effective and specific antibody-based therapeutics.Figure S1, panels 1–3). To this, a five-fold molar excess of crosslinker bis amine TFA (Quanta Biodesign) was added to the thio-Fab (1 mg/mL) with mixing . The completed reaction was purified by gel filtration and the dimeric peak was collected . The reaction progress during both steps was monitored by mass spectrometry and clearly showed the presence of both reactants and the formation of the bis-Fab product . The purity of the desired product after the second gel filtration was determined by mass spectrometry and SDS-PAGE. Upon reduction and SDS-PAGE analysis, irreversible crosslinking was observed by the presence of a 50 kD band representing non-reducible crosslinked chains .Thio-Fabs with an unpaired cysteine were derived from various sources as described in the following sections. After isolation and clean-up of the thio-Fabs such that the thiol from the unpaired cysteine was presented, the first thio-Fab was transferred into a buffer containing 25 mM MES, pH 5.8, 2 mM EDTA, and 300 mM NaCl  and lysed using a microfluidizer. The extract was treated with the flocculent polyethyleneimine (0.4%) adjusted to pH 9.0 for 1 hour with stirring followed by centrifugation for 45 minutes at 15,000×g. Thio-Fabs or hinge-cys-Fabs were purified by standard procedures using Protein G and cation exchange chromatography. Specifically, the supernatants were filtered through a 0.22 micron filter and then directly applied to a Hi-Trap Protein G resin . Elution of the Fab was achieved with 0.2 M acetic acid followed by capture on an SP-HP cation exchange column . Thio-Fabs or hinge-cys-Fabs were eluted with a 10 CV gradient of 0–1 M NaCl. Purified thio-Fabs were characterized by SDS-PAGE and mass spectrometry. These characterizations often showed mass increases of 275 Da and 306 Da corresponding to disulfide adducts on the unpaired cysteine. These adducts were removed by reduction and oxidation to prepare the thio-Fabs for crosslinking with bis-maleimide . Thio-Fabs were reduced for 24 hrs by the addition of 2 mM tris(2-carboxyethyl) phosphine HCl (TCEP) (Thermo Fisher Scientific) in a buffer containing 25 mM MES, pH 5.8, 300 mL NaCl, and 5 mM EDTA and re-oxidized by the addition of 5 mM dehydroascorbic acid (DHAA) (Sigma-Aldrich). The isolated thio-Fabs were analyzed by SDS-PAGE and mass spectrometry to ensure that the proteins were properly reduced and oxidized.Protein expression in Figure S2A). In one approach, cysteine substitutions were introduced into antibody constructs at various positions in the constant domains of light chains or heavy chains by site-directed mutagenesis to create thio-Mabs as described previously Several additional approaches were used to create antibody fragments with sulfhydryl groups 2 was then reduced with 1 mM TCEP in a buffer containing 25 mm MES, pH 5.8, 2 mM EDTA, and 300 mM NaCl and the Fabs were oxidized by the addition of 5 mM dehydroacorbic acid (DHAA) to reform the disulfide bond between the heavy chain and light chain. We routinely observed that under these reaction conditions, only the disulfide between the heavy chain and light chain was reformed; the two cysteine residues in the hinge region remained reduced.For the preparation of hinge-cys-Fabs from native antibodies that did not contain an engineered cysteine for use in synthesis reactions, we used the following procedure. Trastuzumab was digested with pepsin (1% w/w) by treatment in sodium acetate buffer at pH 4.5. After digestion for 1 hour, the F(ab′)Figure S2B). This reaction yielded three products: Fabs with one crosslinker and one NEM, Fabs with two NEM, and Fabs containing only one crosslinker . The Fabs containing only one crosslinker (no NEM) were found to have no free cysteine. Thus, under these reaction conditions, a single crosslinker reacted very efficiently with both cysteines resulting in a molecule where the cysteines have been cyclized by the crosslinker. The material comprising the above three reaction products was purified from the reaction mixture (to remove unwanted reaction components) by gel filtration and used in coupling to other thio-Fabs. Only hinge-cys-Fabs containing one crosslinker and one free-maleimido were able to react in the bis-Fab synthesis reactions described in the beginning section.The two thiols (cys residues) at the hinge were then reacted with 1 equivalent of N-ethylmaleimide (NEM) (Sigma Aldrich). The resultant mixture containing singly-modified, doubly modified and unmodified Fabs were then reacted with an excess of the bis-maleimido crosslinker  were calculated using a Wyatt Technology Dawn Heleos II with QELS and Optilab Rex. Samples were run over a Shodex 803-kw column run in PBS with an additional 0.15 M NaCl to isolate a mono-dispersed peak. hydR data points were chosen from 10 contiguous radii calculated at the peak maxima. Data reported in 2.Hydrodynamic radii (We generated models of how bis-Fabs may bind to the HER2 extracelluar domain (ECD) in a trimeric complex using simple manual methods and PyMol. One Herceptin Fab-HER2 ECD structure (PDB ID Code: 1N8Z) was used to generate a second structure. The two structures were maintained in the same plane in a similar manner to the cell membrane. The fourth domain of the ECD is the last domain before the transmembrane region. This was maintained as an inflexible plane. We then attempted to bring the structures together in different orientations assuming full rotational flexibility across the crosslinker. Two crosslinking sites were positioned near each other and a predetermined maximum distance was maintained. The distance between the crosslinked sites from the alpha-carbon in both of the cysteines used for crosslinking was measured using a thermodynamically minimized crosslinked structure using ChemDraw and was determined to be between 15–18 angstroms. The models show the only possible orientation that preserved the plane of the plasma membrane, maintained a crosslinking distance between 15–20 angstroms, and did not introduce steric clashes between the Fab domains for an antagonist or an agonist.BT474 cells were purchased from American Type Cell Culture and maintained in RPMI medium supplemented with 10% fetal bovine serum. Cells  were plated in 96 well plates and treated the next day with the indicated concentrations of bis-Fabs or antibody in 1% serum-containing medium. After 5 days, AlamarBlue (Invitrogen) was added to the wells and fluorescence was read using a 96-well fluorometer with excitation set at 530 nm and emission at 590 nm. The results are expressed in relative fluorescence units (RFU), or as a percentage of growth compared to control group.6 cells and grown for 24 hrs. Media was exchanged with 25 mL of fresh media containing 100 nM trastuzumab, 100 nM bis-Fab 1325, 10 nM heregulin, or no additional reagent (no treatment). Cells were harvested with 0.05% Trypsin every 12 hours for each treatment group and cells were counted with an Nexcelom Bioscience Cellometer Auto T4 cell counter. Each treatment group and time point was reported as an average from three independent plates.For the time course experiments, BT474 cells were grown in 50∶50 F12∶DMEM including 10% FBS, Glutamax (Gibco), 100 units/mL penicillin, and 100 ug/mL streptomycin. 15 cm tissue culture dishes were seeded with 1.5×105 cells/well. The following day, cells were treated with trastuzumab, bis-Fab 1325, bis-Fab 1329, and control antibody (anti-gD) at 100 nM each for 10, 30 and 120 minutes at room temperature. The cells were lysed with nondenaturing lysis buffer containing 1% Triton . Protein concentrations were determined and equal amounts of proteins were subjected to immunoblotting. The following antibodies were used for detection: pHER3 (#4791), pAKT (Ser473) (#9271), AKT (#9272), pMAPK (#9101), and MAPK (#9102) all from Cell Signaling Technology, HER3 , HER2 , pTyr  and anti-tubulin .BT474 cells were seeded into 24 well plates at 5×102SO4 was added to each well to terminate the reaction. Absorbance was measured at 490 nm with background subtracted at 650 nm. Samples were analyzed in triplicates and AKT phosphorylation levels of treated samples were compared to untreated control.The cell lysates described above were also evaluated with PathScan p-AKT1 (S473) Sandwich ELISA . Protein (10 µg) diluted in sample diluents was added to each well of the 96-well plate and the plate was incubated overnight at 4°C. After several wash steps, detection antibody was added and the plate was incubated for 1 hour at 37°C. Samples were washed 4 times, HRP-linked secondary antibody was added to the plate, and incubated for 30 minutes at 37°C. Plates were rinsed three times before the addition of substrate solution. After sufficient color development, 50 µL of 2.5 M HA HER2-specific antibody  was used to isolate HER2 from BT474 cells either before or after the different treatments. The antibody-HER2 complex was captured from cell lysates using Protein A/G resin  in the presence of phosphatase and protease inhibitors. Eluted proteins from immuno-captures were prepared for mass spectrometry by reduction in SDS-sample buffer containing 50 mM DTT (Pierce Biotechnology) at 90°C for 5 min followed by alkylation with 0.176 M n-isopropyl iodoacetamide (synthesized in house) at room temperature for 20 min. Samples were separated on a 4–20% SDS-PAGE gel (Invitrogen)  and the gel was fixed in 10% acetic acid/50% methanol (Burdick and Jackson) for 30 min. The fixed gel was stained overnight in Coomassie Brilliant Blue stain, destained and gel bands around 175 kDa (HER2 migration region) were excised, washed in 50 mM ammonium bicarbonate (Sigma) containing 5% acetonitrile   and followed by 50 mM ammonium bicarbonate in 50∶50 acetonitrile: water . Gel pieces were dehydrated with acetonitrile and digested with trypsin (Promega), in ammonium bicarbonate pH 8, 0.2 µg overnight at 37°C. Digestions of chymotrypsin or endoproteinase Glu-C (Roche) were also performed to obtain near complete coverage of the protein.via an auto-sampler onto a 75 µm×100 mm column  at a flow rate of 1 µL/min using a NanoAcquity UPLC (Waters Corp). A gradient from 98% solvent A (water +0.1% formic acid) to 80% solvent B (acetonitrile +0.08% formic acid) was applied over 40 min. Samples were analyzed on-line via nanospray ionization into a hybrid LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). Data was collected in data dependent mode with the parent ion being analyzed in the FTMS and the top 8 most abundant ions being selected for fragmentation and analysis in the LTQ. Tandem mass spectrometric data was analyzed using the Mascot search algorithm . Phosphorylation sites on HER2 were confirmed and localized by de novo interpretation. Table S1 shows the complete phosphorylation coverage of HER2.Peptides were extracted from the gel slices in 50 µl of 50∶50 v/v acetonitrile: 1% formic acid (Sigma) for 30 min followed by 50 µl of pure acetonitrile. Extractions were pooled and evaporated to near dryness and were reconstituted in 0.1% formic acid. Samples were injected 6 BT474 cells per plate were exchanged into 25 mL of fresh media (as described above) and incubated at 37°C for 2 hours. At time zero, 100 nM trastuzumab, 100 nM bis-Fab 1325, 10 nM heregulin, or media was added to the cells. Treatment groups were incubated at 37°C for 10 minutes. After treatment, the media was replaced with 1 mL/plate of ice-cold lysis buffer  and cOmplete, EDTA-free protease inhibitors (Roche)). The cells were harvested using a cell lifter. Cells were further pulsed on ice briefly with a micro-sonication probe (low power for 1 second). Anti-HER2 antibody (22 µg) that binds domain I of HER2  was added to the lysate and allowed to complex with HER2 in the presence of 100 uL Protein A/G Plus resin (Pierce Biotechnology) for 1 hour at 4°C with rotation. Lysate was removed by centrifugation and the resin was washed 3 times with ice cold PBS. Proteins were eluted from the capture beads by boiling in SDS-PAGE sample buffer and recovering the supernatant by filtration.For sample preparation for quantitative mass spectrometry of the different phosphorylation sites, 15-cm tissue culture plates with 3.0×10Table S2.Isotopically-labeled synthetic peptides containing experimentally determined HER2 phosphorylation sites were custom made by Cell Signaling Technologies. A list of the phosphorylated peptides used and their non-phosphorylated analogs are provided in Dried gel extracted tryptic peptides were reconstituted in 0.1% formic acid containing 50 fmol/uL custom HER2 synthetic peptide mixtures for phosphorylation site quantification. Samples were injected onto the LTQ-Orbitrap and analyzed by Mascot. Phosphopeptide ions were compared to their synthetic analogs using the Quan Browser in Xcalibur (Thermo Scientific) and by manual peak area assignment. Three biological replicates of each immunoprecipitation of each treatment group were utilized for quantification and the resultant digests were analyzed in triplicate.Table S1) The variance component model was used to assess the sources of variability in the relative phosphorylation levels for each peptide within each group of treated samples. Total variation was apportioned among biological and technical replicates. Due to the nesting of injections within samples, the variance component model was also used to estimate the mean relative phosphorylation level for each peptide within each treatment group . We termed these antibody-like molecules bis-Fabs to include the bis-maleimido linker in the name and to distinguish them from a F(ab′)2. By using site-specific cysteine substitutions that have been previously characterized for efficient reactivity Tumor cell growth is often driven by the activation of more than one signaling protein thus requiring multiple neutralization activities to provide effective therapies for certain cancers As a proof of concept for our screening technology we targeted signaling receptors in the HER-axis because of their critical role in cancer and the availability of well-characterized antibodies Although we suspected that different linkage positions could result in variable activity, it was remarkable that by simply changing the position of the covalent association between the Fabs we converted a potent antagonist into a potent agonist. This illustrated that a simple structural analog of an antibody was sufficient to impart radical changes in its biological activity. This observation is meaningful because, despite the importance of HER2 in driving cell proliferation, no ligand that directly binds HER2 to increase activity has been identified. Instead, HER2 activation occurs through dimerization with other ligand-binding family members Figure S2A and S2B). The molecules were combined in a matrix format to produce ten unique structural analogs . Several new agonistic molecules (EC50∼0.1 nM) were identified, including bis-Fab 1325 linked between positions LC-205 and HC-118, that showed enhanced cell proliferation activity compared to agonist bis-Fab 1321. Other bis-Fabs were either potent antagonists with activities comparable to trastuzumab (IC50∼1 nM) or produced little or no change in cell proliferation. To investigate the differences between molecules in more detail we first examined the quality of the proteins by standard techniques. Size exclusion chromatography coupled to a light scattering device indicated that the proteins were homogeneous preparations of approximately 95 kD monomeric proteins with no appreciable aggregation . Detailed Scatchard binding analyses showed that the agonistic bis-Fabs maintained the same affinity and number of binding sites as the parent F(ab′)2 2 Table 1.To determine any solution state differences we carried out dynamic light scattering analysis of bis-Fabs alone or in complexes with the HER2 ECD. The agonistic bis-Fabs showed a trend toward being more compact molecules, as judged by their hydrodynamic radii Table 1.Upon receptor dimerization allosteric interactions regulate the kinase activity in HER-family members that in turn lead to the modulation of various downstream signaling pathways Figure S4. Overall, the response of BT474 cells to bis-Fab 1325 was consistent with a HER2 pathway-activating molecule.The increase in growth rate seen with bis-Fab 1325 coincided with activation of PI3K/AKT and MAPK signaling. As shown in Despite HER2 activating properties, bis-Fab 1325 did not increase HER3 phosphorylation but rather induced a slow but noticeable decrease in levels of pHER3 (www.uniprot.org) for ERBB2_HUMAN and a comprehensive list of all sites identified are compared to annotation in other databases (Table S1).We examined whether the growth stimulatory activity of bis-Fab 1325 was, in fact, due to direct changes in the phosphorylation state of HER2. Because the basal phosphorylation level of HER2 amplified cells is high we used quantitative mass spectrometry as a more accurate measurement tool for receptor phosphorylation. This technique defined a complete signature of phosphorylation changes associated with agonistic activity at one time point. We first surveyed the basal phosphorylation state of HER2 in unstimulated BT474 cells by mass spectrometry. After immunoaffinity purification of HER2 from cell extracts and enzymatic digestion, peptides were identified by LC-MS/MS. Eighty-eight percent amino acid coverage of the intracellular domain of HER2 was obtained and seventeen phosphorylation sites in the basal state were identified. Phosphorylation was found to occur to varying levels on threonine, serine and tyrosine residues distributed throughout the entire intracellular domain. We confirmed phosphorylation on all five sites documented under the Uniprot entry (Table S3). This survey allowed a snapshot of the early phosphorylation changes induced by either agonist or antagonist treatment in BT474 cells.Quantification of the level of phosphorylation in the basal state for fifteen sites was generated by mass spectrometry by including isotopically labeled synthetic peptides in LC-MS/MS analyses of tryptic digests of isolated HER2 Figure S5). In contrast, more changes were evident with agonist treatments, for example, phosphorylation at sites T701, Y1005, S1073, and Y1139 increased above basal levels by 50 to 100%.  molecule can result in profoundly different biological activities. Recently, several reports have described disulfide-connectivity variants in antibodies found both in nature and during synthetic development 2 binding d∼1.5 nM) which is greater than the affinity of the monovalent Fab (Kd∼6 nM). In addition, the Fab domain alone has very little biological activity , we developed a site-specific crosslinking strategy to make bispecific molecules composed of two such thio-Fabs. The unpaired cysteine was incorporated into either the heavy or light chain of the Fab outside of the combining region by site-directed mutagenesis. Alternatively, the cysteines of the hinge region of the heavy chain can be used as a crosslinking site by producing a F(ab′)2 from the intact antibody by pepsin digestion followed by mild reduction. Thio-Fabs were prepared for crosslinking by removing any existing thio-adducts or dimers by reduction and reoxidation. The reduced and oxidized thio-Fab was then reacted with an excess of the bis-maleimido crosslinker and the reaction mixture was subsequently isolated by gel filtration. The monomeric species containing the thio-Fab-crosslinker complex was reacted with the second thio-Fab at a concentration greater than 5 mg/mL. The reaction was monitored by mass spectrometry and the final product (bis-Fab) composed of the crosslinked Fabs was isolated by a second gel filtration. The earlier eluting peak on the second gel filtration contained the ∼100 kDa bis-Fab irreversibly crosslinked through the two unpaired cysteines. Product purity was determined by mass spectrometry and SDS-PAGE under reducing and non-reducing conditions.(TIF)Click here for additional data file.Figure S2A matrix for combinatorial synthesis of trastuzumab bis-Fab variants. (a) Trastuzumab thio-Fabs were derived from three different sources: 1) light chain 110-Cys was obtained by expression in E. coli, 2) light chain 205-Cys and heavy chain 118-Cys were expressed as full length thio-mAbs in mammalian cells containing the cysteine mutation and subsequently obtained by digestion with lysine-endopeptidase and isolation by anion exchange chromatography, and 3) heavy chain hinge-Cys (228/230-Cys) was obtained from the native trastuzumab molecule by pepsin digestion C-terminal to the hinge-disulfide bonds followed by reduction and controlled chemistry using a thiol-reactive reagent N-ethylmaleimide (NEM) to produce a thio-Fab at the hinge region containing a single thiol. (b) Trastuzumab was digested with pepsin to release the F(ab′)2, which was subsequently purified by anion exchange chromatography. F(ab′)2 from trastuzumab was reduced with tris(2-carboxyethyl)phosphate (TCEP) to liberate the two Fab domains followed by oxidation with dehydroascorbic acid in the presence of 5 mM EDTA at pH 5.8. The reduced and oxidized thio-Fabs were reacted with 1 equivalent of NEM  as shown in the second panel. This reaction produced three products, a thio-Fab with two NEM irreversible adducts, a species with one NEM adduct and a single thiol, and an unreacted species with two thiols. The right panel shows mass spectrometric analysis of the reaction products indicating the presence of the three species. The MW of NEM is 125 Da and the unreacted thio-Fab is 48651 Da. The reaction mixture produced after addition of NEM was subsequently reacted with an excess of the crosslinker bis-Mal (XL) shown in the third panel. As determined by mass spectrometry, two new species of thio-Fab products were identified, one containing one bis-Mal crosslinker and one NEM (MW 49323) and the second containing only one bis-Mal crosslinker (+XL). The bis-Mal crosslinker reacting with the trio-Fab containing two thiols resulted in the formation of a cyclic-complex containing one bis-Mal crosslinker that had efficiently reacted with both cysteines. This species was no longer thio-active and possessed no reactive maleimide group. Only the product containing one crosslinker and one NEM reacted with a second thio-Fab in the second coupling step of the trio-Fab assembly process described in (TIF)Click here for additional data file.Figure S3Raw data for cell proliferation in response to trastuzumab bis-Fabs. Here we show the raw data that is represented in (TIF)Click here for additional data file.Figure S4Analysis of bis-Fab agonist activity in ZR75-30 and SKBR3 cells. ZR75-30 and SKBR3 cells were treated with 100 nM of trastuzumab, 100 nM of bis-Fab 1325, or 100 nM of bis-Fab 1329 for 10, 30 and 120 minutes. At times indicated, cell lysates were prepared and analyzed by immunoblotting using phospho-specific antibodies for HER3, AKT, and MAPK as well as antibodies recognizing total protein. Data are representative of three independent experiments.(TIF)Click here for additional data file.Figure S5Scatter plots showing the total level of phosphorylation of each phosphosite. These plots show levels of phosphorylation at each site in each treatment group. Each of three gel bands from the immunoisolations was derived from independent biological replicates. The gel bands containing HER2 were analyzed by mass spectrometry in triplicate. Thus, for each treatment group there are nine data points, three mass spec replicates of three biological replicates. Each phosphorylation percentage is shown as a scatter plot and the mean is indicated with a bar. Four treatment groups, No Treatment (NoTx), Trastuzumab, bis-Fab 1325 (1325), and heregulin (Hrg) are shown together for each phosphosite. The data are shown left to right in the order they occur in the sequence of HER2.(TIF)Click here for additional data file.Table S1Coverage of HER2 phosphorylation sites.(DOC)Click here for additional data file.Table S2A list of the synthetic peptides used for HER2 quantification. Isotopic label mass addition was based on the residue of incorporation (orange). Phosphorylated residues are shown in blue. A mixture of 50 fmol/µL of these 19 peptides was used for HER2 phosphoryl-quantification upon treatment.(DOC)Click here for additional data file.TableS3Quantification of HER2 phosphorylation. Blue letters indicate phosphorylated residues.(DOC)Click here for additional data file.


An annotated selection of world wide websites relevant to the topics in environmental microbiologyThis is a peer-reviewed journal article discussing the efficacy of ten bioremediation products in the cleanup of oil that had been spilled along the Alaska coast.This paper describes the effectiveness of bioremediation for treating oil spilled in Alaska by the Exxon–Valdez tanker.This site provides a large list of bioremediation products and services with links to the original websites describing the materials.This site put up by the American Society for Microbiology provides a good summary of the principles and practices of bioremediation.This site is dated but it contains specific examples of sites treated by bioremediation with links to further information.This list is provided by the Biocatalysis/Biodegradation Database and has links for each organism to its known biodegradation pathways.CandidatusAccumulibacterphosphatis removes large amounts of phosphate from waters and stores it as polyphosphate; as such this microbe is useful in phosphate bioremediation and sequestration.This webpage contains the genome sequence data for a phosphate-removing microbe used in phosphate remediation and sequestration.This site provides information on waste sites at which bioremediation is being used or considered to be used.This database defines some general terms and offers a brief list of bioremediation services provided within Hawaii.This site contains a description of a bioremediation technology for treating waters derived from unconventional oil and gas extraction, also known as hydraulic fracturing.This site contains an article that makes the case for using bioremediation as a tool for dealing with waters derived from hydraulic fracturing operations.This is an example of a technical product bulletin for a bioremediation product posted by the U.S. Environmental Protection Agency.Regenesis' products for bioremediation generally revolve around the concept of reagent biostimulation to enhance the biodegradative activity of intrinsic microorganisms.The product that is used by JRW Bioremediation is often determined by the conditions at the site and the decision of what substrate to add to support the appropriate bioremediation process.This page provides a general treatment of bioremediation and is found on the Microbe Wiki site.This web book put out by the U.S. National Academy of Sciences is a general treatise on bioremediation.This website provides describes microbial communities and methods of study that are involved in bioremediation to treat wastewater streams.This site describes the use of a microbial product to treat waste hydrocarbons from the oil and gas industries.CBI is a large company that does many types of remediation and environmental restoration, including bioremediation.This subsidiary of Haliburton deals with the microbial remediation of hydrocarbons derived from oil and gas drilling.This article describes bioremediation, largely in the context of the Deepwater Horizon spill and the ability of microbes to handle oil and derivative wastes.


Further, mechanistic analysis revealed that galanal interfered with the transcriptional function of the nuclear factor-κB and the interferon-γ signaling pathway. These effects of galanal are important for immune response. Because the inhibitory effect of galanal on IDO1 activity was stronger than that of 1-methyl tryptophan, a tryptophan analog, galanal may have great potential as the novel drug for various immune-related diseases.Indoleamine 2,3-dioxygenase (IDO) 1, that catalyzes the first and rate-limiting step in the degradation of L-tryptophan, has an important immunomodulatory function. The activity of IDO1 increases in various inflammatory diseases, including tumors, autoimmune diseases, and different kinds of inflammation. We evaluated the suppressive effect of plant extracts or phytochemicals on IDO1 induction and activity; sixteen kinds of plants extracts and fourteen kinds of phytochemicals were examined. As a result, the methanol extracts of Myoga flower buds, which are traditional Japanese foods, and labdane-type diterpene galanal derived from Myoga flowers significantly suppressed IDO1 activity. The Lineweaver-Burk plot analysis indicated that galanal is a competitive inhibitor. Galanal attenuated L-kynurenine formation with an IC Indoleamine 2,3-dioxygenase 1  is the first and rate-limiting enzyme in the tryptophan-kynurenine pathway and degrades the essential amino acid L-tryptophan (L-Trp). IDO1 is induced by interferon-γ (IFN-γ)-mediated effects of the signal transducer and activator of transcription 1α (STAT1-α), and interferon regulatory factor 1 (IRF-1) The metabolism of L-Trp via IDO1 is accompanied by the production of a series of immunoregulatory metabolites, collectively known as “kynurenines,” which can suppresses the proliferation and differentiation of effector T cells Previous researches have given direct evidence of the crucial role of natural products from plants, animals, and micro-organisms as potential sources of various modern pharmaceuticals. Currently, phytochemical research is being considered an effective approach in the discovery of novel chemical entities, with potential as drug leads. Previous reports have shown that some food compounds such as epigallocatechin gallate  and curcumin (CID 969516) inhibit the induction of IDO1Docosahexaenoic acid , CID 445580), eicosapentaenoic acid , CID 446284), epigallocatechin gallate (EGCG), L-Trp, L-kynurenine (L-Kyn) and recombinant human IFN-γ (rhIFN-γ) were purchased from WAKO Chemical . DHA and EPA were dissolved in 100% ethanol and each 20 mM solution was prepared for storing at −30°C. The purification of phytochemicals used, except EGCG from plant extracts, and the preparation of plant extracts used were conducted using the same methods as described in previous reports 2. Cells (1×106) were treated with phytochemicals (10 µM) or plant extracts (30 µg/ml), and LPS (50 ng/ml) for 24 hrs.Human acute leukemic cells, THP-1, and Human embryonic kidney, HEK293, were maintained in RPMI-1640 or DMEM medium supplemented with 10% FCS, at 37°C in a humid atmosphere of 5% COL-Kyn in each conditioned medium was measured by the method using high-performance liquid chromatography (HPLC) with a spectrophotometric detector , as described in our previous reports 2-column by affinity-binding to the N-His-tag of recombinant IDO1, as described in our previous reports The human IDO1 cDNA was expressed in E. coli, and purified by a NiIDO1 activity was determined by the methylene blue/ascorbate assay as previously described The total RNA was extracted from cell lines with ISOGEN  and the RNA concentration was determined spectrophotometrically at 260 nm. Reverse transcription-PCR was performed by using Revetra Ace Kits . Primers used in semi-quantitative analysis were as follows-GAPDH: 5′-accacagtccatgccatcac (sense) and 5′-tccaccaccctgttgctgta (antisense); IDO1: 5′-cctgacttatgagaacatggacgt (sense) and 5′-atacaccagaccgtctgatagctg (antisense). Semi-quantitative analysis of RT-PCR products was performed by using NIH ImageJ 1.34 s software and normalized to GAPDH. When the same experiment was repeated three times, the three experiments showed similar results. Therefore, the results obtained from one of the three experiments were shown.5′-GCATTTTTCAGTGTTCTTCGCATA-3′ (sense) and 5′-CATACACCAGACCGTCTGATAGCT-3′ (antisense); β-actin:5′-TGCACCACACCTTCTACAATGA-3′ (sense) and 5′-CAGCCTGGATAGCAACGTACAT-3′(antisense).To analyze the amount of IDO1 mRNA, real-time PCR was performed by the Applied biosystems 7500 real-time qPCR system using the EvaGreen PCR Master Mix . The mRNA level of IDO was normalized to that for β-actin. Primers used in real-time PCR were as follows-IDO1:Protein phosphorylation was examined with using PhosphoTracer ELISA kit . Each experiment was performed according to the manufacture protocols.Values obtained, except the kinetic study, are expressed as means ± SD. Statistically significant differences (p<0.05) were analyzed by one-way ANOVA, followed by post-hoc test with Tukey-test.The enzymatic assay of IDO1 in the presence or absence of either each plant extract or each phytochemical was performed in duplicate or triplicate; each assay was repeated at least two or three times. Based on the replicate assays of IDO1 activity in the presence or absence of galanal, the average values of the activities obtained in the replicate assays were shown in all Figures. Each value of IDO1 activity in the replicate assays varied within less than 5% of the average value of the activities.First, the effects of fourteen kinds of plants extracts and sixteen kinds of phytochemicals on IDO1 induction were examined, that is, the expression of IDO1 at its mRNA level was examined in LPS stimulated THP-1 cells treated with and without fourteen kinds of plants extracts and sixteen kinds of phytochemicals . Among tBecause quercetin, luteolin, curcumin, andrographolide and galanal clearly showed an inhibitory effect on IDO1 induction, we further examined these compounds using IDO1-overexpressed HEK293 cells, to see whether they inhibited the enzymatic activity of IDO1 or not . Among tThe inhibitory effect of galanal on IDO1 activity was examined in human IDO1-overexpressed cells and mouse IDO1-overexpressed cells in order to clarify the specificity of the compound on IDO1 species. When human IDO1-overexpressed cells and mouse IDO1-overexpressed cells were treated with various concentrations of galanal (1–5 µM), galanal inhibited both enzymatic activities of human IDO1 and mouse IDO1 in a dose-dependent manner .Results of the kinetic studies of IDO1 using two concentrations (0 and 30 µM) of galanal were plotted on the Michaelis–Menten model; galanal lowered the Vmax 0.88 folds determinations when Trp was varied, but it had pronounced effects on the apparent Michaelis constants in 50 value of galanal was 7.7 µM, proving that it inhibited IDO1 activity in a competitive manner (The mode of IDO1 inhibition by galanal was examined using the Lineweaver-Burk plot . Galanale manner .As shown in Galanal inhibited both L-Kyn production and IDO1 mRNA induction in IFN-γ-stimulated THP-1cells . The pho50 value was found to be 45 nM.Cellular IDO1 inhibition by galanal was examined in LPS-stimulated THP-1 cells. As shown in IDO1 is recognized as one of the prominent mediators of immune regulation by metabolic pathways. Therefore, the regulation of the enzymatic activity of IDO1 is useful in the treatments of tumor, inflammatory diseases, and depression. Previous data have suggested that IDO1 inhibition can modulate immune response, delay tumor growth, and enhance dendritic cell vaccines Considerable attention has been paid to the research of effective IDO1 inhibitors 50 value of inhibitor depends on the IDO1 enzyme activity in each experiment, we could not unconditionally compare the previous results. However, these results showed that galanal is an effective IDO1 inhibitor because a very low dosage of it showed inhibitory activity in the cell-based assay.Though many inhibitors for IDO1 have been found, there are few inhibitors with competitive inhibition. In the present study, galanal was found to possess more IDO1 inhibitory potency than either the most-commonly used 1-MT or other previously-published inhibitors  [14][28IDO1 is known to be induced by both the IFN-γ -dependent pathway and the IFN-γ-independent pathway, i.e., the NF-κB-dependent pathway Our results have shown that galanal is an effective compound in immunomodulation, because the compound inhibits both the certain signal transduction and enzymatic activity of IDO1 at a very low dose.The results of the present study indicate that galanal is a competitive IDO1 inhibitor. The results also indicate that a very low dose of galanal inhibits not only the enzymatic activity of IDO1 but also the expression of IDO1 mRNA induced by the NF-κB-dependent pathway. Furthermore, the results suggest that galanal could inhibit the expression of IDO1 mRNA induced by the IFN-γ -dependent pathway. Therefore, galanal and its derivatives may be useful for the treatment of inflammatory diseases.