Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc  and PBAld , common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like  in silico modelling, the capacity of CYP6Zs to degrade insecticide metabolites produced by esterase-mediated hydrolysis was demonstrated.The key role of mosquito cytochrome P450 monooxygenases from the CYP6Z subfamily in the resistance of mosquitoes to insecticides was revealed. By using functional expression and  Mosquitoes transmit numerous parasites and viruses responsible for severe human diseases, such as malaria or dengue. These diseases represent an important burden in tropical and subtropical regions, predominantly affecting developing countries . Indeed,In the absence of efficient treatments or vaccines, vector control often represents the most effective means for limiting disease transmission . EffectiHowever, resistance of mosquitoes to insecticides is threatening vector control programmes worldwide . ResistaCYP genes  per 0.5 g of cells. The pellet was resuspended in 10 ml of TES  and glass beads were added. Yeast cells were broken by vortex-mixing five times for 30 s at 4°C. TES (5 ml) was added to wash the beads and the supernatant was transferred into a clean tube. The beads were washed again twice, the supernatants pooled and centrifuged at 10000 g for 10 min. The supernatant was further ultracentrifuged at 27000 rev./min  for 1 h. The pelleted microsomes containing CYP6Z8 and CPR were resuspended in 500 μl of TEG  per 500 ml of yeast culture, aliquoted and stored at −80°C until use , PBAld (Rt 16 min) and two more hydrophilic metabolites, M1 and M2 (Rt 11.1 min and 12.6 min respectively). PBA accumulated gradually and was the major metabolite after 60 min incubation, followed by the M1 metabolite. PBAld never accumulated to a large extent; its proportion increased progressively over 15 min before decreasing for longer incubation times, suggesting the further conversion of this metabolite. Finally, a minor M2 metabolite gradually appeared during incubation. Kinetic parameters of PBAlc metabolism were Km=15.1±2.9 μM, Vmax=33.4±1.5 pmol/min, kcat=0.66±0.03 min−1 and kcat/Km=0.046.As pyrethroid catabolism may involve carboxylesterase-mediated hydrolysis as a first step, the conversion of intermediate metabolites, namely PBAlc and PBAld was also examined. When PBAlc was used as a substrate A, no sigWhen PBAld was used as a substrate B, a signAn. gambiae CYP6Z2, an anopheline orthologue of CYP6Z8, to metabolize PBAlc and PBAld was also investigated. These experiments performed with E. coli recombinant CYP6Z2 [http://www.biochemj.org/bj/455/bj4550075add.htm).The ability of t CYP6Z2  under idm/z respectively. PBAlc was detected 18 units below its theoretical mass (201 m/z), indicating its dehydration during LC-MS (Supplementary Table S1 at http://www.biochemj.org/bj/455/bj4550075add.htm). MS analyses did not allow the full identification of M1 and M2 metabolites. However, M1 and M2 metabolites did not appear to result from the direct or sequential hydroxylation of PBAlc or PBAld as no +16 m/z were observed. In addition, LC–MS m/z obtained for M1 and M2 did not match with any expected metabolite structure.Positive-mode electrospray LC–MS detected PBAlc, PBAld, PBA, M1 and M2 at 183, 199, 215, 200 and 228 http://www.biochemj.org/bj/455/bj4550075add.htm). The fragment shared by PBAld and PBA (171 m/z) is likely to correspond to diphenylether. This fragment could not be identified from M1 or M2, suggesting that these two metabolites rather originate from PBAlc. In addition, among all fragments, one was shared between PBAlc and M1 (155 m/z), suggesting a common backbone of these two compounds. This fragment was not shared with M2. Moreover, replacing H2O/0.1% TFA (pH 2.0) by 10 mM Tris buffer (pH 7.5) for HPLC analysis resulted in the shift of PBA and PBAld peaks (results not shown), whereas PBAlc, M1 and M2 peaks were not affected , suggesting that M1 and M2 do not possess any pH-dependent function, unlike PBAld or PBA.MS/MS analyses by positive-mode electrospray fragmentation detected various fragmentation ions associated with each compound . However metabolism appears to be prevented in CYP6Z8 by internal clashes with Tyr208, and in CYP6Z2 by residues close to the haem (Leu365 and Phe115). Steric hindrance of PBA does not appear to prevent metabolism since productive pose are predicted in CYP6Z8. However, the low binding score points to a low affinity for this molecule (Supplementary Table S3). Examination of structural models of other Ae. aegypti and An. gambiae CYP6Zs indicates they may all have the capacity to metabolize PBAlc and PBAld and produce similar metabolites . CYP6Z8 transcription was significantly induced by all compounds (2.0–3.4-fold) with a moderate dose-dependent effect.In order to assess whether Ae. aegypti CYP6Z8 and AeCPR in an heterologous system, characterizing its activity towards known P450 substrates and investigating its role in the metabolism of insecticides.The present study aimed at co-expressing CYP6Z8 sequence analysis revealed several variations in the Bora-Bora strain compared with the genome sequence . In total 49 nucleotides variations, including 13 variations leading to amino acid changes, were found, confirming the high polymorphism of mosquito CYPs. These non-synonymous variations did not occur in the conserved P450 signature motifs . Only onCYP6Z8 gene under the same promoter allowed us to produce a functional mosquito CPR–P450 membrane system. As CPR is highly conserved in mosquitoes, this new tool should allow the functional expression of any microsomal mosquito P450. This was recently confirmed by the successful expression of functional An. gambiae P450s using the W(AeR) yeast strain . To our knowledge, this is the first time a mosquito P450 has been successfully expressed in yeast, along with its redox partner and without protein sequence modification. Although E. coli is commonly used to express P450s and has higher yields, some eukaryote P450s are intractable for expression. The yeast system thereby provides a useful alternative method with the advantage of having eukaryotic cellular and redox machinery plus organelle structure appropriate for the translation of eukaryotic P450s.The yeast P450 expression system developed by Pompon et al.  was chosAe. aegypti Cyt b5 did not affect CYP6Z8 substrate specificity and activity. Whether CYP6Z8 does not strongly interact with Cyt b5 or the co-expression of AeCPR and CYP6Z8 produces such a high P450/CPR ratio making the presence of an extra electron donor, such as Cyt b5, not necessary requires further investigation [The results of the present study clearly indicate that our recombinant CYP6Z8 is functional and able to metabolize various standard P450 substrates. Kinetic data suggest that benzyloxyresorufin is preferred, followed by ethoxyresorufin. Supplementing reactions with purified tigation .An. gambiae CYP6Z2, CYP6Z8 did not metabolize permethrin and deltamethrin [Then, the ability of CYP6Z8 to metabolize various xenobiotics, including insecticides, was investigated. As for amethrin ,24. In cin vitro metabolism assays with microsomes extracted from Ae. aegypti larvae suggested that this detoxification pathway occurs in mosquitoes [An. gambiae orthologue CYP6Z2 can also metabolize PBAlc in the same manner, supporting the functional orthology of these two mosquito P450s. To our knowledge, CYP6Z8 and CYP6Z2 are the first insect P450s shown to metabolize pyrethroid metabolites generated by carboxylesterase hydrolysis.In mammals it has been demonstrated that pyrethroids can be hydrolysed by carboxylesterases leading to the production of PBAlc and PBAld and that these metabolites can be further processed by P450s into PBA –46. Recesquitoes . Becausem/z) did not correspond to the theoretical mass (201 m/z), but suggested its dehydration. Despite this, no direct additions of alcohol function (+16 m/z) were observed for M1 and M2, suggesting that these compounds are not simply hydroxylated. Previous studies have shown that the direct attack of pyrethroids by P450s often produces 4′-hydroxylation in insects [Two minor more hydrophilic metabolites (M1 and M2) were also detected. Examination of M1 and M2 metabolites by LC–MS and MS/MS did not allow the absolute identification of their chemical structure. The behaviour of PBAlc in positive-mode electrospray was not trivial as the parental ions (183  insects ,48. The Rt were not affected by pH variations as opposed to PBAld and PBA, confirming that they do not carry acid or aldehyde functions. Overall, these results support the hypothesis of M1 originating from PBAlc and M2 being a secondary metabolite of M1 (in vivo experiments revealed that PBA is the major metabolite produced and is excreted from mosquitoes without further modification. In contrast, M1 and M2 metabolites were not excreted from mosquitoes. Instead, two more polar metabolites (M1′ and M2′ in Rt were observed, suggesting that M1 and M2 are further processed before being excreted.Because PBA is not metabolized by CYP6Z8, M1 and M2 can only represent metabolites of PBAlc or PBAld. Common fragmentation ions were found between M1 and PBAlc, suggesting a similar backbone. In addition, M1 and M2 HPLC te of M1 . FinallyIn silico 3D modelling of CYP6Z–substrate interactions confirmed our experimental findings on CYP6Z8 and CYP6Z2 with good binding scores and limited clashes of pyrethroid metabolites in the active site. Comparing 3D models between various Aedes and Anopheles CYP6Zs supports the capacity of other mosquito CYP6Zs to metabolize these and produce similar metabolites (115 to π-stack with the phenyl ring, and/or Phe102 to bind with the benzyl ring [abolites . The inazyl ring ,49,50, tOverall, the results of the present study lift the veil on the pivotal role of CYP6Z8 and CYP6Z2 in pyrethroid biodegradation in mosquitoes and clarify why they have frequently been associated with resistance ,22,29,51in vitro [Genes encoding carboxylesterases have been frequently found to be overtranscribed in pyrethroid-resistant populations and their role in pyrethroid biodegradation has been established in vitro . However


Methanosarcina acetivorans was cultured in the presence of CdCl2 to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41–69% of the heavy metal from the culture and accumulated 231–539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd2+ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments. Methanogens are strict anaerobes belonging to the Archaea domain, which can be found in a broad variety of environments such as anaerobic digesters of sewage treatment plants, landfills, rice paddies, lakes and in the sea sediments, among others 2+H2, formate, CO and acetate into methane. Because heavy metal pollution may develop in some of these habitats, methanogens may be exposed to this environmental stress with the consequent perturbation of the local ecology. Heavy metal pollution of water resources is now a widespread environmental and public health problem, as a result of their elevated toxicity, which may be exacerbated by their potential bio-magnification effect and accumulation throughout the ecological food webs.Methanogenesis is the pathway by which ion (H−1 (22.6 µM) of cadmium has been found, a value far higher than permissible −1 and18, 400 µg g−1 in waters and sediments, respectively, have been determined Pollution of coastal zones by heavy metals such as Cd, Pb, Hg and Ni, is a major environmental problem in some regions of the world Methanosarcina acetivorans was used as a model to assess the effect of cadmium on methanogenesis.Most studies of microbial communities in the ocean have focused on bacterial diversity in marine sediments and the long-term impact brought about by heavy metals exposure M. thermophila, deoxyribonuclease I from bovine pancreas (DNAse I), acetyl-CoA, coenzyme A, acetyl phosphate, ATP and NADH were purchased from Sigma Chem. . Phosphoenolpyruvate, pyruvate kinase and NAD+-lactate dehydrogenase (both enzymes from rabbit muscle) were from ROCHE (Germany). Absolute methanol, acetate, and CdCl2 were of analytical grade.Acetate kinase from Methanosarcina acetivorans C2A strain, kindly provided by Prof. James G. Ferry , was cultured under anoxic conditions in the regular high salt medium (HS-medium) described elsewhere 2, 15% CO2 and 5% H2. Then, the following salts were added, in g/L: NaCl, 25.4; NaHCO3, 3.8; KCl, 1.0; MgCl2, 11; CaCl2, 0.2; NH4Cl; 1.0 and KH2PO4, 0.27 and resazurin, 0.001% (w/v) as redox indicator. Vitamin and trace mineral solutions were both added at 1% (v/v) as reported by Sowers et al 2S•9H2O (1.04 mM) to ensure complete chemical reduction of growth media. Final pH was 6.8–7.0. 50 mL of medium were poured into 100 mL serum-like bottles , sealed with a butyl rubber stopper  and secured with an aluminum crimp collar . Medium was autoclaved at 121°C for 30 min. After autoclaving a precipitate was formed in the culture media but disappeared after 24 h, approximately. Cultures were started by adding fresh cell inocula and further incubating at 37°C without shaking. Growth was determined by measuring changes in absorbance at 600 nm.−1 cm−1. Sulfide was also determined spectrophotometrically by the methylene blue formation as described by King and Morris p-phenylenediamine (DMPD) dissolved in 5 N HCl and 0.1 mL of culture medium, or different amounts of sulfide, were added by using a syringe and mixed until homogeneity. Then, 2.8 mM FeCl3 was added and incubated at room temperature for 30 min for color development (methylene blue formation). Final volume was 2.5 mL. Samples were measured at 670 nm under anoxic conditions in an anaerobic chamber. The sulfide content-absorbance relationship was linear up to 350 nmol.The concentration of the reduced cysteine and sulfide in the fresh medium was determined post column with DTNB  by HPLC as described elsewhere 2 and 1 mM EGTA (ethylene glycol tetraacetic acid) at pH 7.5. The mixture reaction assay also contained 5 mM ATP, 2 mM phosphoenolpyruvate, 0.25 mM NADH, and acetate kinase, pyruvate kinase and lactate dehydrogenase, which were added in excess  to ensure the complete phosphorylation of acetate coupled to NADH oxidation.Methane production and methanol were determined by gas chromatography (Shimadzu GC2010 apparatus), equipped with a capillary column HP-PLOT/U of 30 m length, 0.32 mm I.D. and 10 µm film  and flame ionization detector. Methane standard curve was carried out as reported by Sowers 2 concentrations  and the optical density was determined at 600 nm. Such range of cadmium concentrations has been reported to be toxic for a broad range of microorganisms from fresh To determine the effect of cadmium on growth, acetate and methanol cultures were carried out in the presence of different total CdCl2. Thereafter, 1, 10 or 100 µM total CdCl2 or other heavy metals  were added to the cultures and methane production was determined from the head space at different short times up to 12 min. For longer periods of methane synthesis (up to 60 min) in the presence of Cd2+, 20 mM acetate was further supplemented to the incubation medium.To determine the effect of cadmium on methane biosynthesis, metabolically active cell cultures in the early stationary growth phase with acetate  or methanol , respectively, were initially subjected to depletion of methane formed by gassing the culture bottles with sterile N2 and 0.02 mM ZnCl2. Then, the cell pellet was re-suspended in lysis buffer , stirred strongly for 5 min and centrifuged at 70,000× g for 30 min. The supernatant  was kept on ice and used immediately for enzyme activities determination. All activities (except for carbonic anhydrase) were determined in the direction of acetate degradation in 50 mM Na2-Hepes and 10 mM MgCl2 buffer at pH 7.0 and 27±2°C, in the presence of different CdCl2 concentrations. In all cases, the reaction assay was started by adding the enzyme (i.e. the cytosol-enriched fraction).Cell cultures of 750 mL grown on acetate were harvested under anoxic conditions in the early stationary phase by centrifuging at 3,000× g for 10 min and washed once with 4 volumes of a solution containing 50 mM Tris-HCl pH 7.5, 20 mM MgClAcetate kinase (AK) activity was determined in cytosolic enriched-fractions of 50–75 µg protein in a reaction medium that also contained 5 mM ATP, 20 mM acetate, 0.2 mM NADH, 2 mM phosphoenol pyruvate and 10 U of both, pyruvate kinase and lactate dehydrogenase. One unit of enzyme (U) is the amount of active enzyme required to transform/produce 1 µmol of substrate/product in 1 min. Phosphotransacetylase (Pta) activity was determined as follows: 3–5 µg of cytosolic protein were incubated in the Hepes-Mg buffer with 5 mM acetyl-phosphate and 160 µM CoA; aliquots were withdrawn at different times (from 5 up to 60 s), mixed with 0.1 M phosphate buffer and 1 mM DTNB and the reaction monitored at 412 nm (representative traces are shown in M. thermophilaCODH/acetylCoA synthase activity (CODH/AcCoAs) was determined anaerobically by mixing 10–25 µg protein with 80 µM acetyl-CoA and measuring the release of CoA with DTNB at 412 nm. As several different enzymes may release CoA from acetyl-CoA, this activity was also specifically determined by measuring the CO-dependent reduction of methyl viologen as reported elsewhere 2 formation, 5 µL of the head space was taken and injected at different times  in a gas chromatograph equipped with a Thermal Conductivity Detector. CO2 formed in assay reaction buffer without enzyme and with boiled enzyme was subtracted from the CO2 formed with enzyme (representative traces are shown in Carbonic anhydrase (CA) activity was determined by incubating 2.5–5 µg of cytosolic protein with 100 mM Na-bicarbonate in a sealed 10 mL bottle with rubber stopper. To detect the CO2 and 2 mM EGTA (TME buffer) at pH 7.5; the pellet was re-suspended in fresh buffer to give 5–10 mg protein/mL and frozen at −70°C until use. Aliquots of the cell suspension were digested with H2SO4+HNO3 (1∶3) for 2 h at 100°C and the intracellular cadmium content determined by atomic absorption spectrophotometry (Varian Spectra AA 640).Cells were harvested and washed as indicated above with 200 volumes of a solution containing 50 mM Tris-HCl, 2 mM MgCl2 were fixed by immersion in glutaraldehyde , after removal from the culture medium, and dehydrated in graded ethanol. Samples of 1 mm2 containing the cells were cut out in cross section with a diamond knife and embedded in 1∶1 epoxy resin. To determine cadmium and sulfur localization inside the cells, atomic-resolution high angle annular dark-field scanning-transmission electron microscopy (HAADF-STEM) was used as reported previously Methanol-grown cells with or without 100 µM CdClThe protein content was determined after cells were washed once with TME buffer by the Biuret method with bovine serum albumin as standard as described previously 2+ concentrations were estimated  for the association of the complexes were 13.4 and 20.13 for Cys-cadmium and Cys-Cd-Cys, and 6.1 for sulfide-cadmium Because cysteine and sulfide present in the culture medium bind the cadmium added with high affinity, the soluble free Cdated see  by using2  and DT values (10±2.3 h versus 11±2.7 h) were found for cells cultured in methanol without or with 100 µM total Cd2+. In contrast, in acetate cultures the GR value was significantly higher in cultures with 100 µM Cd2+ . DT did not significantly changed  for cells cultured without or with 100 µM Cd, respectively  for methanol and 4.5±0.3 (control) and 4.7±0.3 mmol methane (+100 µM CdCl2) for acetate. On the other hand, when 500 µM total CdCl2 was added to cultures with acetate or methanol as carbon source, no growth or methane synthesis were detected (data not shown) indicating that these high cadmium levels were indeed extremely toxic to the cells.The rate of methane production in acetate cultures with 100 µM total CdCl culture . It is w culture : 4.1±0.12 at 25–27°C. The concentrations of acetate and methanol remaining in the cultures were 8±3 mM  and 5±1 mM , respectively. Under these conditions, cadmium exerted a remarkably stimulating effect on the synthesis of methane in control cells not previously exposed to Cd2+; the most potent activation was reached at 10 µM total CdCl2  were incubated with CdClal CdCl2 . Moreoveal CdCl2 .2, by stationary acetate-grown cells was 18±3 and 26±4 µmol methane, and after 60 min was 24±2 and 43±8 µmol methane, respectively -containing buffer. The cell-free culture medium contained 1.4±0.1 µM total cadmium. In turn, 0.04±0.01 and 0.1±0.03 µmol total cadmium/mg cell protein were found in the supernatant after the EGTA-washing treatment in acetate- and methanol-grown cells, respectively , revealing that most of the cadmium associated with the cells was indeed intra-cellularly trapped.Under our culture conditions, in which the cysteine and sulfide concentrations were high, the added micromolar CdClpM range . It is kectively . In this protein  was dete2+ concentration, it seems likely that the complexes formed between cadmium and sulfur compounds, and not the free Cd2+, were the species that preferentially entered into cells  or presence of 0.1 (•), 1 (▴) or 10 (▾) µM total CdCl2. In the absence of protein the CoA concentration remained constant (□).(TIF)Click here for additional data file.Figure S2CODH/AcCoAs complex activity from M. acetivorans; representative traces of the activity by adding cytosolic fraction  and 80 µM AcCoA. Representative trace with: 125 (□), 62 (x) and 25 (▪) µg of cytosolic fraction without cadmium in the absence or presence of 0.01 (•), 0.1 (▴), 1 (▾) and 10 µM total CdCl2 (⧫).(TIF)Click here for additional data file.Figure S32 by CA from Formation of COM. acetivorans in the absence (chromatogram A), or presence of 1 µM CdCl2. In chromatogram C, cytosolic fraction was previously heated. The different peaks shown represent consecutive sample injections at different times. It should be noted that y-scale is higher for chromatogram B. Under these conditions, CA activity was linear for at least 1 min (st 1 min . Reverse(TIF)Click here for additional data file.Figure S4Activity of CA in the cytosolicfraction of M. acetivorans in the absence (▪) or presence of 0.01 (•), 0.1 (▴), 1 (▾) or 10 µM total CdCl2 (⧫). A representative data with heated cytosolic fraction in presence of 0.1 µM CdCl2 is also shown (□).(TIF)Click here for additional data file.Figure S5Activation of methanogenesis by cadmium. Cultures on acetate were purged by passing N2 for 5 min. Then, samples of the head space were withdrawn from the cultures at 0 and 5, 10, 20, 30 and 60 min of incubation with 0 (filled symbols) or 10 µM CdCl2 (open symbols) for GC analysis. These experiments were started with the addition of 20 mM acetate. Values are the mean ± SD of 3 independent cell preparations. P<0.05 using the Student's t-test for avs control (without cadmium).(TIF)Click here for additional data file.Figure S6Intracellular cadmium clusters in M. acetivorans. HAADF-STEM projection images of methanol-grown cells cultured in methanol in the absence (A) or in the presence of 100 µM CdCl2 for 5 days (B). The image in B revealed cadmium grains along the cell (white spots).(TIF)Click here for additional data file.Text S1Methods and Results.(DOCX)Click here for additional data file.


Thus, we treated mouse 4T1 mammary carcinoma with 1D11, a neutralizing anti-TGFβ  antibody. The treatment delayed tumor growth, but unexpectedly increased the proportion of Tregs in tumor. In vitro, 1D11 enhanced while TGFβ potently inhibited the proliferation of Tregs. To enhance the anti-tumor effects, 1D11 was administered with cyclophosphamide which was reported to eliminate intratumoral Tregs. This combination resulted in long term tumor-free survival of up to 80% of mice, and the tumor-free mice were more resistant to re-challenge with tumor. To examine the phenotype of tumor infiltrating immune cells, 4T1-tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide. This treatment markedly inhibited tumor growth, and was accompanied by massive infiltration of IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic CD11b+Gr1+ cells, and increased their expression levels of MHC II and CD80. In a spontaneous 4T1 lung metastasis model with resection of primary tumor, this combination therapy markedly increased the survival of mice, indicating it was effective in reducing lethal metastasis burden. Taken together, our data show that anti-TGFβ antibody and cyclophosphamide represents an effective chemoimmunotherapeutic combination.TGFβ is reportedly responsible for accumulation of CD4 NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the "Guide for Care and Use of Laboratory Animals" . Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of National Cancer Institute .Female wild type 8 to 12 wk old Balb/c mice were provided by the Animal Production Area of the NCI . Foxp3/gfp KI mice were kindly provided by Dr. Yasmine Belkaid at NIAID, and maintained in the NCI-Frederick. BALB/c IFNγ2.4T1 breast cancer cells were obtained from ATCC  and from Dr. Fred Miller  who firstly described this cell line Antibodies purchased from BD Biosciences  consisted of anti-CD3 (145-2C11), CD4 (GK1.5), CD16/CD32 (2.4G2), INFγ (XMG1.2). Foxp3 Staining Set (FJK-16s), anti-mouse TCRβ Ab (H57-597) and functional grade purified anti-mouse CD3e Ab (eBio500A2) were purchased from eBioscience . The anti mouse TGFβ monoclonal antibody, 1D11, which neutralizes all three isoforms of TGFβ, and an isotype-matched mouse IgG1 monoclonal antibody, 13C4, were provided by Genzyme Corp.Length × Width2)/2. “Survival” in the primary tumorigenesis studies represents the time to development of a 4 cm3 tumor or moribund, a humane endpoint that triggers euthanasia. To assess survival from metastatic burden in a spontaneous metastasis experimental format with surgical resection of the primary tumor, 40,000 4T1 cells in 40 µL of PBS were inoculated into the surgically-exposed left inguinal mammary fat pad of anesthetized mice. Primary tumors were surgically excised on day 12 as described previously 4T1 tumor cells were injected into right mammary fat pads (thoracic No. 2 mammary glands) of recipient mice in single cells suspension with 50,000 cells in 0.2 ml PBS per mouse. After indicated times, tumors were excised, minced and digested in RPMI 1640 supplemented with 1 mg/ml collagenase IV and 0.1 mg/ml DNase I. The fragments were pushed through a 70-µm pore size cell strainer to create a single-cell suspension. In some experiments, two weeks after last treatment , tumor free mice after CY+1D11 treatment were re-inoculated with 4T1 cells  into the right mammary fat pads (thoracic No. 2 mammary glands), and the same number of CT26 colon carcinoma cells were s.c. injected to the left flank. Tumor size was calculated by the formula: (Mice were treated by the following dose schedule: 1D11 or mouse IgG1 (13C4) were administered three times per week i.p at 0.1 mg in 0.2 mL PBS, starting 1 or 3 days after inoculation of 4T1 cancer cells. After 4 weeks, the three times weekly treatment was reduced to one. A single dose of CY was injected i.p. at 4 mg in 0.2 mL PBS by 3 days after cancer cell inoculation. For a reduced dose schedule, 1D11 or Mu IgG1 was i.p. administered at 0.1 mg, starting 3 days after 4T1 cancer cell inoculation. After 3 weeks, the three times weekly treatment was reduced to one. A single dose of CY was i.p. injected at 2 mg 3 days after cancer cell inoculation. For the spontaneous metastasis study with surgical resection of primary tumor, 1D11 or mouse IgG1 were administered three times per week i.p at 5 mg/kg for 2 wks starting 7 days after inoculation of 4T1 cells, followed by once a week treatment for the duration of the experiment. A single dose of 50 mg/kg CY was injected i.p. at day 14 after cancer cell inoculation.+TCRβ+(or CD3+) cells.After blocking FcR, cells were incubated with appropriately diluted antibodies. Acquisition was performed using a SLRII  and data analysis was conducted using FlowJo software . For intracellular cytokines staining, cells were re-stimulated with BD Leukocyte Activation Cocktail for 4 h. FACS analysis was gated on the live cells only by using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen Life Technologies). FACS analysis of TILs was gated on live CD45+Foxp3/gfp+ Tregs were sorted from LNs and spleens of Foxp3/gfp KI mice using Cytomation MoFlo cytometer , yielding a purity of ∼98% Tregs. T-depleted spleen cells were used as APCs and irradiated with 3,000 R. Tregs were seeded into round-bottom 96-well plate at 2×104 cells/well. The cells were stimulated with 2×105 APCs/well plus 0.5 µg/ml of soluble anti CD3 Ab, with or without murine TNF , in the presence of medium alone or increasing concentration of recombinant human TGFβ1  or 1D11 (1∼20 µg/ml). Cells were pulsed with 1 µCi [3H]thymidine  per well for the last 6 h of the 72-hour culture period.CD4t test, except for the percent tumor free data and survival data which were compared and analyzed by Logrank test, using Graphpad Prism 4.0.All data was compared and analyzed by two-tailed Student’s + cells in the tumor infiltrating CD45+ leukocytes was 28.7% in 1D11-treated mice, markedly higher than that in control IgG1- treated mice . Surprisingly, the proportion of Tregs present in tumor infiltrating CD4 cells in 1D11-treated mice was markedly increased, as compared with mice treated with control IgG1  in the control group developed measurable 4T1 and CT26 tumor by day 13 after inoculation . AlthougAlthough our original treatment regimen achieved an optimal anti-tumor effect, it did not allow us to examine Tregs and other TILs, since the majority of mice did not develop tumor at all. Therefore, we administered lower doses of both 1D11 and CY in order to allow tumor growth for analysis. The results show that 90% of mice developed tumor after treatment with reduced doses of CY and 1D11. This treatment regimen also markedly inhibited the growth of primary tumor (p<0.05∼0.001), as shown in Since tumor-free mice after CY+1D11 treatment developed partial 4T1 tumor-specific resistance, we hypothesized that T cells should be mobilized and activated. Indeed, 1D11 treatment alone, and CY treatment alone to a lesser extent, increased T cell infiltrating the tumor p<0.01, . Tumor-iTo evaluate the role of IFNγ in the anti-tumor effect of the combination treatment of CY+1D11, we examined its effect in 4T1 tumor-bearing IFNγ KO mice. As can be seen in +CD11b+ MDSCs in the spleen from mice treated with CY, CY+1D11 was markedly reduced by 43% and 54% . The cell number of Gr1+CD11b+ cells in the spleen of 4T1 tumor-bearing mice was markedly reduced by the treatment of CY or 1D11 alone or their combination . We therefore looked for alternative mechanisms. The mouse 4T1 tumor model is characterized by the accumulation of MDSCs in the spleen which causes splenomegaly  p<0.01, . The redWe further utilized the 4T1 model in a format where the primary 4T1 tumor was surgically excised and mouse survival is driven by metastatic lung disease TGFβ is a pleiotropic cytokine that plays a key role in the interplay of tumor cells and other cells in the tumor environment Thus, TGFβ has dual and biphasic effects in tumor development, and this complex nature of TGFβ in cancer biology poses the challenge for the application of TGFβ inhibitor as a sole therapeutic. Combination with other therapeutics has the potential to reinforce the beneficial anti-tumor effects, while minimizing the undesirable effects of a TGFβ inhibitor. Our study clearly shows that CY is one such chemotherapeutic. This combination treatment likely targets multiple cellular and molecular events simultaneously. Nevertheless, the activation of anti-tumor immune responses contributes substantially to the anti-tumor effect , for the reason that: 1) the combination treatment resulted in a massive infiltration of IFNγ-producing cells to the tumor; 2) anti-tumor effect of the combination therapy was reduced in IFNγ KO mice; 3) the number of splenic MDSCs was markedly reduced and the residual MDSCs showed a more mature phenotype; 4) tumor-free mice after CY+1D11 treatment developed long term anti-tumor immunity.+CD11b+ cells in the spleen  A previous report showed that the combination of CY with an anti-TGFβ receptor II antibody had an additive effect in the suppression of primary tumor growth and lung metastasis in mice bearing EMT6 mammary cancer In our studies, Mu IgG1 13C4 by itself or in combination with CY had a consistent inhibitory effect on 4T1 tumor growth . This thTaken together, our study showed a commonly used chemotherapeutic CY was able to enhance the anti-tumor effect of TGFβ inhibitor, resulting in the potent inhibition in the development of 4T1 mouse mammary carcinoma. Although multiple mechanisms may underlie the anti-tumor effect of combination therapy of CY and TGFβ inhibitor, this efficacy is in part due to improvement in the quality of anti-tumor immunity in both adaptive and innate arms. This combination regimen thus represents a successful approach to the chemoimmunotherapy of primary and metastatic cancer.


Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 , LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A  and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with  The present study characterizes integrin and DDR2 signalling networks activated by collagen. Using clustering approaches, DDR2-specific signalling components such as SHP-2 were identified. We further demonstrate that SHP-2 is phosphorylated by a subset of DDR2 lung cancer mutants. Collagen is the most abundant protein in mammals and plays a critical role in maintaining tissue structural integrity . There iDDR2 is one of two members of the DDR family. It is highly expressed in mesenchymal cells and is important for a variety of developmental processes, in particular bone and cartilage formation . DDR2 biIn the present study, we have performed an unbiased quantitative MS-based phosphoproteomic analysis of collagen receptor signalling networks upon stimulation with collagen I. Temporal analysis over seven time points identified 424 phosphorylated proteins. Using MCAM , we find that a subset of phosphorylation sites on important signalling proteins such as SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 , LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PLCL2 (phospholipase C-like 2) and PIK3C2A  strongly cluster with DDR2 phosphorylation dynamics, implicating these proteins as candidate downstream effectors of DDR2 signalling. A survey of lung SCC mutants of DDR2 using targeted proteomics show that SHP-2 is tyrosine-phosphorylated in a subset of these mutants. The present study demonstrates that SHP-2 is a key downstream component of DDR2 signalling and highlights the utility of phosphoproteomics in providing new insights into cell–matrix signalling.2 at 37°C. For expression of DDR2, HEK-293 cells were transfected with pcDNA3.1-DDR2 , immunoreactive bands were visualized by enhanced chemiluminescence  and the blots were exposed to X-ray XAR film (Kodak).HEK-293-DDR2 or mutant cells were stimulated with 20 μg/ml acetic-acid-solubilized rat tail collagen I (Sigma) at the indicated time points and lysed with RIPA lysis buffer  supplemented with protease and phosphatase inhibitors (Thermo Pierce) at 4°C. Equal amounts of protein, as determined by the bicinchroninic acid protein assay (Thermo Pierce), was applied in each lane of 4–12% or 10% Bis-Tris gels (Invitrogen). Following one-dimensional separation and transfer on to PVDF membrane, the membrane was incubated overnight at 4°C with 1:1000 dilutions of goat anti-DDR2 (R&D Systems), mouse anti-phosphotyrosine [4G10 (Millipore) or PY100 ] or rabbit anti-SHP2 (Santa Cruz Biotechnology) antibodies, a 1:500 dilution of a rabbit anti-(SHP2 p-TyrHEK-293-DDR2 cells were grown in six-well plates for 24 h. Cells were then dissociated with non-enzymatic cell dissociation solution (Sigma) and resuspended in PBS containing 1% BSA. The cells were incubated for 30 min on ice with primary mAbs  at 10 μg/ml in 100 μl of PBS/BSA. Cells were then washed three times with PBS/BSA and incubated with FITC-conjugated goat anti-mouse IgG  for 30 min on ice. After three washes as above, the cells were resuspended in 2% formaldehyde in PBS. Data were subsequently collected on a BD LSRFortessa cell analyser using BD FACSDiva software 6.0 (BD Biosciences), and further analysed on FlowJo software 7.6.4 (Tree Star). Mouse anti-(integrin α1) mAb, clone FB12, was purchased from Millipore Chemicon and mouse anti-(integrin α2) mAb, clone AK7, was from AbD Serotec. Mouse anti-(integrin β1), clone P5D2, was purified from hybridoma cells obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, U.S.A.18 Sep-Pak Plus cartridge (Waters), eluted with 25% acetonitrile and freeze-dried to dryness. Freeze-dried peptides were subjected to labelling with the iTRAQ 8-plex reagent (ABSciex) according to the manufacturer's instructions  and beads were removed by centrifugation at 5000 18) pre-column . The pre-column was attached to an analytical column  with an integrated electrospray bottleneck tip with an approximate 1 μm orifice. Peptides were eluted using a 75-min gradient with solvent A (1% acetic acid) and B : 10 min from 0 to 10% B, 45 min from 10 to 34% B, 10 min from 34 to 47% B, and 10 min from 47 to 100% B. Transitions were monitored for endogenous and heavy phosphopeptides as detailed in Supplementary Table S3 (at http://www.biochemj.org/bj/454/bj4540501add.htm). Peak areas were calculated using Analyst v1.5 software, and peak areas for endogenous peptide transitions were normalized to the corresponding transitions for the spiked heavy peptide standards.Samples were analysed using a Q-Trap 4000 instrument (ABSciex). Samples containing heavy peptide standards were loaded on to a reverse-phase (Chttp://www.biochemj.org/bj/454/bj4540501add.htm). Set pruning was determined by removing those sets whose removal improved the overall biological enrichment by 10% or greater, while not decreasing the impact of any one biological category by more than 5%. Statistical significance of biological enrichment was calculated using the PTMScout MCAM interface for GO (Gene Ontology) terms, Pfam domains, Scansite binding and kinase predictions, linear amino acid sequence motifs, and enrichment for the domain a phosphorylation site falls in. This analysis used the default PTMScout parameters of 5×10−2 for an α-corrected value, corrected by the Hochberg and Benjamini FDR procedure  and mixed with equal volumes of either (i) 200 μM ATP and 200 μM Axltide peptide substrate (KKSRGDYMTMQIG) in AB1 for loading into PAGE gels, or (ii) 200 μM ATP (with 1.5 μCi of [γ-32P]ATP) and 200 μM peptide substrate in AB1 for RFB (radiometric filter binding) assays. Each activity assay was incubated for 30 min at room temperature. RFB assay samples were stopped by spotting 20 μl on to P-81 phosphocellulose filters pre-wetted with 0.75% phosphoric acid. Filters were washed four times with 35 ml of 0.75% phosphoric acid, then immersed in 3 ml of scintillant  and counted on a Beckman LSC-1800 liquid scintillation counter.Using a TDA (template-directed assembly) approach which organizes functional complexes of membrane-associated proteins, His-tagged DDR2 and/or Src . Gels were run, stained with Coomassie Brilliant Blue and destained. Excised gel bands were diced and destained in consecutive 50% acetonitrile/50 mM TEAB (triethylammonium bicarbonate) and 50 mM TEAB washes until all of the stain was removed. The gel pieces were shrunk amd then reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The proteins were subjected to digestion with modified porcine trypsin (Promega) and incubated at 37°C overnight. After digestion, the proteolytic peptides were extracted with 50% acetonitrile/formic acid and TEAB washes, the extract solution was reduced in volume to approximately 10 μl in a speed-vac and subjected to LC-MS/MS sequencing analysis.542 levels, cells were lysed in 50 mM Hepes, 5 mM EDTA, 0.1 mM EGTA, 0.5% Nonidet P40 and 120 mM NaCl (pH 7.5). All lysis buffers were supplemented with protease and phosphatase inhibitors (Thermo Pierce). An ELISA for phospho-DDR2 was performed using the Human Phospho-DDR2 DuoSet IC ELISA kit (R&D Systems). Phospho-SHP2 was determined using the Phospho-SHP2 (Y542) DuoSet IC ELISA kit (R&D Systems). All of the remaining phosphoproteins were measured by custom sandwich ELISAs using the following capture antibodies diluted in PBS: total SHP-2 capture antibody (2 μg/ml) from the SHP-2 DuoSet IC kit (R&D Systems); anti-PLCL2 mAb clone 2D10 ; anti-LYN polyclonal antibody ; anti-SHIP2 mAb, clone T.194.8 ; and anti-NCK1 mAb, clone 714506 . ELISA plates (Costar) were coated with capture antibody overnight at room temperature and subsequently blocked with 1% BSA in PBS for 2 h at room temperature.Cell lines were serum-starved overnight before stimulation with collagen I (Sigma) or acid control at the time points indicated and lysed. For P5D2-blocking experiments, HEK-293-DDR2 cells were pre-treated with P5D2  for 30 min before stimulation with collagen for 24 h and lysis. Lysis buffer for extraction of DDR2, PLCL2, SHIP2 and SHP-2 was 1% Nonidet P40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol and 4.5 mM EDTA; for LYN lysis buffer was 1 mM EDTA, 0.5% Triton X-100 and 6 M urea in PBS (pH 7.4); and for NCK1 lysis buffer was 50 mM Hepes, 0.1 mM EDTA, 0.1 mM EGTA, 120 mM NaCl and 0.5% Nonidet P40 (pH 7.5). For determination of phospho-SHP2 Tyr2SO4. Absorbance at 450 nm was measured using an ELISA plate reader. Plates were washed five times with wash buffer [PBS (pH 7.4) containing 0.1% Tween 20] after each step.Each sample (100 μl) was loaded in duplicate and incubated for 2 h at room temperature, followed by the addition of 100 μl of HRP-conjugated phosphotyrosine mouse mAb  in diluent  (2 h at room temperature). The reaction was visualized by the addition of 100 μl of chromogenic substrate  for 30 min and the reaction was stopped with 50 μl of 1 M HCell lines were resuspended in a solution composed of 1 mg/ml rat tail collagen (BD Biosciences) in DMEM with the pH adjusted to 7.4 with 1 M NaOH. Cells (400) were seeded in each well of a 96-well plate in 100 μl of the collagen solution. After the collagen gel solidified, 100 μl of DMEM (10% FBS) was added to the top of the gel and media was changed every other day. Cells were grown for 15 days and colonies were counted.To identify the phosphorylation-mediated signalling networks downstream of DDR2 activation, HEK-293 cells were transduced to stably express DDR2 (HEK-293-DDR2 cells) which were then pooled before immunoblotting to assay for DDR2 expression. The HEK-293 cell line has been extensively used as a model system to investigate DDR signalling ,11,33. Shttp://www.biochemj.org/bj/454/bj4540501add.htm). Of the phosphopeptides that were identified, manual sequencing of mass spectra unambiguously localized the site of phosphorylation on 549 of the peptides.In order to minimize any confounding signals that may be associated with growth factors commonly found in serum in cell culture media, the HEK-293-DDR2 cells were serum-starved before stimulation with 20 μg/ml collagen I over seven time points (0–24 h) C. Cells 736 and Tyr740) are located on the activation loop of the receptor which contains three tyrosine residues (Tyr736/Tyr740/Tyr741) that are highly conserved among a number of RTKs, including the insulin receptor and c-Met  were also found in our dataset, but displayed very different profiles from the integrin study. In the integrin study, CDK1 Tyr15 showed increasing phosphorylation levels reaching a maximum at 2 h after collagen stimulation, whereas DYRK1A Tyr321 displayed high basal phosphorylation levels that steadily decreased over time. Both of these sites showed constitutive phosphorylation levels that did not vary with time in our dataset. These differences could be the result of the distinct cell lines used in the two studies, the use of soluble collagen compared with collagen-coated surfaces, or the influence of DDR2 expression on integrin activation.We have recently shown that the collagen-binding integrins are active in the HEK-293-DDR2 cells used in the present study . HoweverDDR2 gene in lung SCC by Hammerman et al. [Unlike lung adenocarcinomas, SCCs of the lung are poorly characterized and there are currently no effective targeted therapies to treat this disease. The identification of point mutations in the n et al.  suggestsn et al. ,52,53. Ln et al. . Interesn et al. ,56.In conclusion, the results of the present study highlight the use of phosphoproteomics in combination with clustering techniques to establish a high-resolution map of DDR2 signalling effectors. We further demonstrate that SHP-2 is a major downstream component of DDR2 signalling and is phosphorylated in a subset of DDR2 lung SCC mutants. These findings provide an insight into the nature of DDR2 networks and signalling modulations that occur upon acquisition of cancer-associated mutations. We have previously shown that distinct oncogenic mutations in EGFR deploy differential pathway utilization in cancer cells, which has therapeutic implications in the clinic ,27,57. T


The increased crop yields reported here has the potential to effect the world's agricultural output.This study presents a novel way of enhancing plant growth through the use of a non-petroleum based product. We report here that exposing either roots or seeds of multicellular plants to extremely low concentrations of dissolved hydrogen sulfide at any stage of life causes statistically significant increases in biomass including higher fruit yield. Individual cells in treated plants were smaller (∼13%) than those of controls. Germination success and seedling size increased in, bean, corn, wheat, and pea seeds while time to germination decreases. These findings indicated an important role of H Yet, in spite of these findings, only recently have there been analogous investigations into the potential signaling role of H2S in eukaryotes. To date, only studies on its effect on the functioning of the electron transport chain in animals, and separately on its inhibition of physiological processes enabling endothermy in birds and mammals have been conducted The biological effects of hydrogen sulfide , Pisum sativum (Pea), USU-Apogee (Space Wheat), and Zea mays L. (Corn) were exposed to variable concentrations of H2S dissolved in deionized water. Concentrations of H2S varying from 10–100 µM caused absolute stem and leaf growth (both length and mass) in seedlings to be significantly larger  for all treated plants than controls of the same species  (In addition to increasing growth rates at a finite and relatively narrow concentration of Hontrols) . Seedlinontrols) .2S, were slightly longer, however not statistically different than that of the controls (37.1±2.2 cm for 0 µM (controls), whereas 1 µM was 37.5±4.1 cm, 10 µM was 38.4±3.6 cm, 100 µM was 37.2±4.6 cm, and 500 µM was 37.2±4.7 cm). This difference between treatments is amplified when exposing the plants to H2S every seven days (36.6±2.1 cm for 0 µM (controls), whereas 1 µM was 38.6±5.2 cm, 10 µM was 39.9±2.1 cm, 100 µM was 40.3±3.6 cm, and 500 µM was 40.2±1.8 cm; P = 0.08; F = 2.6). The overall mass of the plant, roots and fruit were independently larger than the controls for all plants exposed to H2S. The mass of the entire plant was 7.0±0.3 g for 0 µM (controls), whereas 1 µM was 8.3±0.6 g, 10 µM was 9.9±0.5 g, 100 µM was 8.8±0.7 g, and 500 µM was 10.8±0.8 g, and each was statistically different from the controls . Likewise, the mass of the roots was larger in treated plants  compared to that of the controls (2.8±0.6 g for 0 µM). Beyond the macro plant, the produced fruit was larger in the treated plants: 0.95±0.1 g for 0 µM (controls), whereas 1 µM was 1.1±0.0 g, 10 µM was 1.1±0.1 g, 100 µM was 1.1±0.1 g, and 500 µM was 1.5±0.2 g . Mean change in growth was 0.17±0.02 mm for controls; 1 µM: 0.36±0.01 mm; 10 µM: 0.26±.03 mm; and 100 µM: 0.32±0.01 mm. Finally, when we compared the cells from the leaf disks it was determined that the cell size (diameter and area) was significantly smaller (∼13% and 18%) than those of the controls . Mean diameter of controls was 8.38±0.07 µm; 1 µM: 7.4±0.09 µm; 10 µM: 7.1±0.06 µm; and 100 µM: 7.3±0.05 µm.Leaf disks exposed to He.g. 29]. Additionally, high (<10 mM) but sub-lethal levels caused photosystem (PS) II to shut down, whereas PS I remained active through the experimental period through and up to plant senescence Throughout the experiments, photosynthetic activity (Qmax - a measurement of maximum photosynthetic output) of all plants was monitored using a Fluorcam. Qmax values maintained relatively comparable levels  to untreated controls until the 50 mM concentration, where significant decrease (<0.1) was observed and differences in reflected spectra were apparent [2S produces two separate kinds of increased growth rates in plants: time to seed germination; absolute mass of tissue in roots, stems, and leaves. Unlike the results in a previous study 2S, followed by a return to the slower growth rates observed in controls, unless re-exposed. This study also shows that H2S is the only chemical necessary to produce such large differences in growth rates in these species.In recent years, numerous experiments have shown that hydrogen sulfide causes an array of biological effects 2S molecules are provoking cellular division through some signaling process. The increased photosynthetic activity shown in a previous study Regardless of the physical, “macro-” changes in the plant, it is clear that the causes of these results are occurring on a cellular level. Our observation that cells increase in number rather than size suggests that the H2S molecules affecting the plants treated in this study is reasonable. We hypothesize that administered H2S could be regulating a hormonal pathway 2S is affecting the basic biology of the plants.Because hydrogen sulfide has ‘clandestic messenger’ like properties 2S load was an evolutionary response to short-term and catastrophic changes in global atmosphere during Phanerozoic mass extinction events, when a combination of higher temperature, oceanic anoxia, and release of gaseous hydrogen sulfide into the atmosphere contributed to various global mass extinctions 2S toxicity (e.g. 50 curves) decreases with plant size, even in the same species. The mechanism observed here might be that simple: at the first recognition of oncoming H2S concentrations, survivability would be affected by overall plant size. Rapid growth would be selected for. While these results are early in development, these findings may provide beneficial use to agriculture and biofuels.The origin of these effects is open to speculation. However, it may be that an increase in growth rates of some plants at the early onset of an increasing atmospheric or aqueous H2S/Sulfide Probe . The 0.5 M H2S solution was then filtered and stored in a 500 mL flask filled with nitrogen gas to maintain stability.Half-molar hydrogen sulfide was made by dissolving 78.04 grams of anhydrous sodium sulfide into 500 mL of double filtered di-water. Hydrochloric acid was then titrated into this solution in 0.01 mL increments while stirring until pH of 7.2 was reached, resulting in a solution 0.5 M±25 mM (5%) as determined by using H2O. After dilution the H2S/Sulfide Probe was used to confirm concentration.Each treatment concentration was made adding ddi-HPhaseolus vulgaris (Bean), Pisum sativum (Pea), USU-Apogee (Space Wheat), and Zea mays L. (Corn) were exposed to five levels  of liquid hydrogen sulfide dissolved in ddi-H2O. Germination rates as well as the quantity of growth was measured and compared to a control (0 µM H2S), for a seven day growth period. Thirty seeds, of each species were placed into 100 ml petri-dishes filled with 50 ml of solution. Due to the relatively short half-life of H2S when in solution −2s−1 of light daily. Temperature was ambient room temperature (∼23°C). Every day at the same hour, the number of seeds which germinated was recorded. At 72 and 168 hours, the length of the fresh seedling was recorded using digital calipersSeeds from four common crop species 2S) Seeds were wrapped with a clean paper-towel and mounted to a glass slide. The slide, containing a seed, was placed into the container and immersed in 75 ml of ddi-H2O. The containers were randomly placed on shelves serviced by grow lights which provided 12 hours of ∼100 µmol m−2s−1 of light daily. Water was replenished weekly. Temperature was ambient room temperature (∼23°C).Seedlings of the same species of pea and bean used in the germination trials were also germinated hydroponically in 250 mL containers  and relative humidity. In addition, we measured the pH of each treatment in order to rule out pH change as the reasoning for growth differences.Upon germination, seedlings were grown for fourteen days and then randomly selected for treatments. Twenty-Five seedlings were selected for each treatment and a control . Before exposure, and 24, 48 & 168 hours, photosynthetic output (Qmax of the formula Fv/Fm) was measured with a Z100 Kinetic Multispectral Fluorescence Imaging FluorCam System by P.S.I. Qmax, the maximal photochemical efficiency of PSII (Fν/Fm) was calculated according to Krause and Weis USU-Apogee, was placed in each of the four corners of a half-gallon pot filled with 400 grams of sunshine #6 soil. Each seed was submersed one cm from the surface of the soil. Six pots, each containing four seeds, were placed into each treatment. Treatments consisted of 0, 1, 10, 100 and 500 µM H2S solutions (diluted with ddi-H2O). Additionally, each treatment consisted of two sub treatments; a three day and weekly exposure regimen.One seed, of Seeded pots were watered, either weekly or every three days, with 300 milliliters of the corresponding treatment solutions . Seeded pots were haphazardly distributed in the University of Washington Botany Greenhouse. Each week the length of the shoot, number of leaves, and developmental level was measured. At the conclusion of nine weeks all plants were mature and had produced fruit. Each plant was carefully removed from the soil with root mass intact, and gently washed to remove the dirt. Whole plants were patted dry with a paper towel and weighed to the nearest 0.1grams. The roots and the fruit were independently removed from the plant and weighed to the nearest 0.1grams. Finally, the length of the plant was measured (to the nearest 0.5 cm).2S + ddi-H2O soln.) by a second individual. The first person, who took measurements, was unaware of which disks were in what treatment throughout the experiment. Initial disk size per treatment was statistically compared using ANOVA, to determine if the sample was truly random. Twenty-four and forty-eight hours after exposure, leaf disks which were determined to be random were re-measured, using digital calipers, and the growth rate was compared.Sixty-four, 6.05±0.03 millimeter diameter, leaf disks were cut from growing leaves on a bean plant, which was grown in UW Botany Greenhouse. Disks were floated in a deionized water bath. Individual disks were haphazardly selected from this water bath, measured with digital calipers and put into labeled petri-dishes. Numbers associated with the labeled petri-dishes were randomly selected using a number generator and sixteen individual leaf disks were placed into treatments  from an additional replicate of 10 disks per treatment, using the same double blind methods, were analyzed with Mitotic Image Plus 2.0 (Motic China Group Co.). Measurements consisted of: precision diameter (the largest distance across the leaf using two points); 3-point circle (3 points selected at random to form a circle of best fit); 5-point circle (5 points selected at random to form a circle of best fit); area in field of view using background exclusion. Measurements were compared to that of the calipers and differences were compared by using theoretical calculations: i.e. a theoretical area was calculated using the diameter  and treatments were compared as factors using a linear regression model with ANOVA. Both T and F statistics are listed when appropriate.


KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P21 21 21  and P21  to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling. Striga and Orobanche species Bushfires present organisms with both hardships and opportunities. For plants, fires deliver a flux of nutrients to the soil and reduce competition for light and water. Karrikins are a class of compound present in bushfire smoke that stimulate the germination of dormant seeds dwarf14 mutant of rice is insensitive to strigolactones, as are orthologous mutants in Arabidopsis and petunia KARRIKIN INSENSITIVE 2) is required for responses to karrikins, but does not appear to be essential for strigolactone responses, at least with respect to the regulation of shoot branching Discovering the molecular mechanisms of karrikin and strigolactone action has recently taken a step forward with the isolation of plant mutants. A family of α/β hydrolases has emerged as central players in mediating the responses of plants to these butenolide compounds. The The precise role that KAI2 plays in the karrikin and strigolactone signalling pathways remains unclear. Here we report the crystal structure of KAI2, which may provide valuable insight into its involvement in these pathways, especially with regard to substrate or ligand specificity. In particular, a comparison with the recently elucidated structure of DAD2 and its postulated role as both a strigolactone receptor and hydrolase is discussed.2 was prepared according to the method of Goddard-Borger et al. KARArabidopsis thaliana KAI2 coding sequence (At4g37470) was amplified by PCR using seedling-derived cDNA template and primers GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGTGTGGTAGAAGAAGC–3′5′– and GGGGACCACTTTGTACAAGAAAGCTGGGTTTCACATAGCAATGTCATTAC–3′5′– , and subsequently cloned into the pDEST17 expression vector (Life Technologies). The expression clone was introduced into the Rosetta strain . Cultures (400 ml) were grown in SOC medium at 37°C to OD600 ∼0.6. At this point, the cultures were cooled to 16°C and recombinant protein expression was induced by the addition of 0.1 mM IPTG. Growth proceeded for a further 18 hours at 16°C before harvesting by centrifugation. Wet pellets were frozen in dry ice and stored at −80°C until processing.The native http://web.expasy.org/protparam/) Cell pellets were resuspended in 50 mM sodium phosphate, 500 mM NaCl pH 8.0, at a ratio of 5 mL lysis buffer to 1 g of cell pellet and supplemented with 1 unit Benzonase nuclease  per mL lysis buffer. Cells were lysed with detergents  at room temperature (20°C) with shaking at ∼60 rpm for 20 minutes. The lysate was clarified by centrifugation at 16,000×g for 20 minutes at 4°C. To isolate His-tagged protein the clarified lysate was combined with 3 mL 50% (v/v) slurry of pre-equilibrated nickel-nitrilotriacetic acid resin (Qiagen) which was mixed end-over-end for 80 min at 4°C and 20 rpm. The bound KAI2 was washed twice with 10 mL 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole pH 8.0 by gravity flow. KAI2 protein was eluted with 5 mL 50 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole pH 8.0. Eluted protein was dialysed against 2 L 20 mM sodium phosphate 150 mM NaCl pH 8.0. Dialysed KAI2 protein was recovered at 950 μg/mL and used for optimisation of solubility. Protein concentrations were determined by spectrophotometric measurements at 280 nm using a calculated molar extinction coefficient . Cells were lysed with 4 passes through an Emulsiflex C5 high-pressure homogeniser at 10,000–15,000 kPa . Lysates were clarified by centrifugation at 16,100 rcf for 1 h at 4°C. All chromatography steps were performed at room temperature. The clarified lysate was filtered through a 0.22 μm filter and applied manually to a pre-equilibrated 5 mL HisTrap column . The loaded column was washed with four column volumes of lysis buffer at 2 mL/min. Protein was eluted using a ten column volume linear gradient from 0–100% (buffer +250 mM imidazole). KAI2 eluted at approximately 100 mM imidazole. KAI2-containing fractions were pooled, concentrated to 3.5 mL and applied to a 120 mL Superdex 200 column  for size exclusion chromatography in 100 mM Tris 150 mM NaCl pH 9.0. Pure KAI2 typically eluted as one peak, which was collected and concentrated using a 10 kDa molecular weight cut off centrifugal ultrafilter (Sartorius) to 10.2 mg/mL, yielding approximately 16 mg of highly purified KAI2 protein. In an attempt to obtain protein bound to phenylmethyl sulfonyl fluoride (PMSF), KAI2 was lysed in 20 mM Tris pH 7.0, 150 mM NaCl, 1 mM PMSF. The lysate was gently rocked at 4°C for 1 hour before 5 mL of 200 mM TRIS pH 9.0 was added and the purification was continued as previously outlined. Purified protein was further incubated with 1 mM PMSF at 4°C for 16 hours. All protein samples were divided into aliquots, flash-cooled in liquid nitrogen and stored at −80°C.2 were unsuccessful, but yielded higher resolution diffracting crystals (KAI2a). Crystals that grew in the presence of KAR2 were crystallised from a drop containing 1.8 μL protein sample (6 mg/mL), 0.6 μL KAI2 seed stock, 0.4 μL 30% glycerol and 1.2 μL reservoir solution of 1.4 M sodium/potassium phosphate pH 7.3. Prior to crystallisation, KAR2 dissolved in 100% (v/v) DMSO was added to the protein solution to a final concentration of 1 mM KAR2 and 2.5% DMSO. The N-terminal leader sequence was not removed prior to crystallisation experiments.Sparse matrix vapour diffusion crystallisation screens were performed using Hampton Index Screen (Hampton Research) with a Phenix liquid handling robot (Art Robbins) in sitting drop format in 96 well plates . Drops between 0.4 and 0.6 μL were equilibrated against 90 μL of reservoir solution. A harvestable, diffracting protein crystal grew in a condition with 0.2 μL of protein solution (10.2 mg/mL KAI2) and 0.4 μL of reservoir solution (1.4 M sodium/potassium phosphate pH 8.4). Crystals were optimised by screening buffer pH and concentration in 24 well sitting drop format where 2.5 μL of protein solution (5.0 mg/mL) was added to 2.5 μL reservoir solution and equilibrated against 500 μL of reservoir solution. A 32 μm×6 μm×6 μm multiple crystal grew in 1.5 M sodium/potassium phosphate pH 7.2 KAI2b;  and singWhere necessary, single crystals were split from the multiple crystals. Crystals mounted in a nylon loop were briefly immersed in mother liquor containing 20% glycerol then frozen in liquid nitrogen for data collection. Complete X-ray data  were collected at the Australian Synchrotron beamlines MX1 or MX2.Bacillus subtilis protein RsbQ (PDB code 1WOM) Data were integrated with XDS Arabidopsis thaliana (NCBI GI: 15235567), Ricininus communis (NCBI GI: 255567989), Populus trichocarpa (NCBI GI: 224071259), Solanum lycopersicum (NCBI GI: 225311281), Vitis vinifera (NCBI GI: 225458830), Brachypodium distachyon (PlantGDB: Brachypodium_distachyon-10841), Hordeum vulgare (NCBI GI: 326500818), Zea mays (NCBI GI: 226530032), Sorghum bicolor (NCBI GI: 242035387) and Oryza sativa cv. japonica (NCBI GI: 115453689). D14 protein sequences used in sequence alignments were from Petunia hybrida (DAD2) (NCBI GI: 404434487), Arabidopsis thaliana (NCBI GI: 18396732), Ricinus communis (NCBI GI: 255538072), Vitis vinifera (NCBI GI: 225458830), Populus trichocarpa (NCBI GI: 224129864) Solanum lycopersicum (Sol genomics network: Solyc04g077860.2.1), Hordeum vulgare (NCBI GI: 326496392), Oryza sativa cv. japonica (NCBI GI: 115451411), Sorghum bicolor (NCBI GI: 242041843) and Zea mays (NCBI GI: 226501208).Protein sequence alignments were performed using ALINE A. thaliana KAI2 was cloned into pDEST17 and expressed in E. coli resulting in protein samples of approximately 30 kDa molecular mass as determined by SDS-PAGE. An optimal buffer for solubility was identified as 0.1 M Tris pH 9.0, 150 mM NaCl. KAI2 was successfully crystallised from sodium/potassium phosphate in two crystal forms, one orthorhombic, P 21 21 21  and the other monoclinic, P 21 , each with one protein molecule in the asymmetric unit. Full details of data collected from three crystals  are presented in Bacillus subtilisNo conspicuous conformational differences were observed between KAI2 structures . KAI2 haFunctional specialisation between KAI2 and D14 proteins might be mediated by each of the proteins' respective interaction partners, so we scrutinised the conserved amino acid differences of surface exposed residues within each of the protein families. No conspicuous regions of conserved differences were detected which would indicate sites of interaction with different protein partners.3, with a classical serine hydrolase catalytic triad at its base (composed of Ser95-His246-Asp217). The KAI2 catalytic pocket is smaller than the catalytic pocket of DAD2 (448 Å3), but it is still sufficiently large to accommodate the synthetic strigolactone GR24. The two pockets are highly similar in terms of overall shape and amino acid composition (3) proximal to the catalytic residues. In DAD2, this small pocket is connected to the main cavity, helping to explain the discrepancy in size between the KAI2 and DAD2 main cavity volumes. This specific Tyr/Phe substitution is conserved within KAI2 and D14 protein families respectively  or glycerol molecule (KAI2b) nearby. As the structure of a PMSF-adduct of RsbQ had previously been reported 3) is significantly smaller than the primary pocket but is large enough to accommodate a karrikin-sized molecule. The same pocket is present within the DAD2 structure (3) and blocked from the solvent by the side chain of Phe188. The equivalent residue in KAI2 is Ser188, which permits solvent accessibility of the pocket. This Ser/Phe substitution is conserved within KAI2 and D14 protein families  can both mediate seedling responses to the synthetic strigolactone GR24


Background. It is currently recommended that diet of pregnant mothers contain 200–300 mg DHA/day. Aim. To determine whether DHA supplementation during pregnancy and lactation affects infants' immune response. Methods. 60 women in ≥3rd pregnancy studied; 30 randomly assigned to receive DHA 400 mg/day from 12th week gestation until 4 months postpartum. From breast-fed infants, blood obtained for anti-HBs antibodies, immunoglobulins, lymphocyte subset phenotyping, and intracellular cytokine production. Results. CD4+ lymphocytes did not differ between groups, but CD4CD45RA/CD4 (naïve cells) significantly higher in infants in DHA+ group. Proportion of CD4 and CD8 cells producing IFNγ significantly lower in DHA+ group, with no differences in proportion of IL4-producing cells. Immunoglobulins and anti-HBs levels did not differ between groups. Conclusions. In infants of mothers receiving DHA supplementation, a higher percentage of CD4 naïve cells and decreased CD4 and CD8 IFNγ production is compatible with attenuation of a proinflammatory response. Breast feeding is believed to confer protection against infections during infancy. Breast-fed infants have an enhanced local humoral immune response, resulting in a lower prevalence of gastrointestinal and respiratory tract infections than in formula-fed infants , 2. ExclIn infants who are breast fed, supply of these fatty acids is dependent on the maternal diet . The WesThe high demand for DHA has prompted current recommendations that during pregnancy and lactation the average dietary intake of DHA should be 200–300 mg per day , 13. Indγ and IL1) were decreased, whereas, in cord blood, levels of Th2 cytokines (IL4 and IL13) were decreased [Previous studies relating to the immune system in infants have compared infants fed human milk with infants fed either LCPUFA-supplemented or nonsupplemented formulas , 17. In ecreased . No studWe therefore questioned whether DHA supplementation during pregnancy and lactation, in a population with a high percentage of multiparous mothers with closely spaced pregnancies, may affect the humoral and cellular immune response in infants solely breast-fed up to the age of 4 months.The mothers of the babies studied belong to an ultraorthodox religious community; 60 pregnant women, age 20–35 years, in their ≥3rd pregnancy. 30 women were randomly assigned to receive DHA 400 mg/day . DHA supplementation was taken daily from the 12th week of gestation and continued until the end of the 4th postpartum month. Mothers who were not assigned to the DHA supplementation group declined intake of placebo capsules on religious grounds, as the gelatin capsules are not “kosher”, but since the DHA was regarded by the religious leaders of the community as a medication, intake of DHA capsules was permitted. In the DHA-supplementation group, 18 mothers were in their 3rd-4th pregnancy, 11 were in their 5th–7th pregnancy, and 1 was in her 8th. In the control group, 17 mothers were in their 3rd-4th pregnancy, 11 in the 5th–7th, and 2 in the ≥8th pregnancy. A nutritional intake questionnaire, with an emphasis on type of cooking oil used, amount and type of fish, and/or nuts consumed, was filled out by all mothers. Infants were exclusively breast-fed for 4 months. At age of 4 months, blood was drawn (maximum 5 mL of which 2.5 mL in EDTA and 2.5 mL in heparin) for complete blood counts (CBC), anti-HBs antibody titers, immunoglobulin levels, and lymphocyte studies.Quantitatively determined using a microparticle enzyme immunoassay 9AxSYMAVSAB, Abbot Diagnostics, Wiesbaden, Germany.Quantitative determination of immunoglobulins  was performed by means of immunonephelometry using BN ProSpec Systems, Siemens Healthcare Diagnostics, Marburg, Germany.The following monoclonal antibodies were obtained from Immunoquality Products, Groningen, the Netherlands: anti-CD8, anti-CD45RO, anti-CD45RA, and anti-CD25. Anti-CD4 and anti-CD6 mAb were purchased from eBioscience, Inc., San Diego, Calif. γ (IFNγ) was used for the detection of IFNγ.MAbs for the detection of intracytoplasmic cytokines were obtained from BD Biosciences . Fastimmune PE-labeled antihuman IL4 was used for the detection of IL4, while Fastimmune FITC-labeled antihuman interferon-3. Suspensions at a volume of 100 μL, were introduced into each test tube and stained with mAbs. The cells were incubated with either FITC- or PE-conjugated mAbs for 30 min at 4°C. The mixture of cells and antibody was incubated for 30 min at 4°C. After incubation the red blood cells were lysed with FACS lysing solution (Becton Dickinson). After two incubations of the blood with NH4CL for 10 minutes at room temperature, the suspension was centrifuged, the pellet washed twice with PBS, and cells resuspended in 1% paraformaldehyde and stored at 4°C until they were analyzed by flow cytometry [The following peripheral blood lymphocyte subsets determined by cell surface staining using a fluorescent activated cell sorter: CD4, CD4CD45RA (“naïve” T helper cells), CD4CD45RO (“memory”/activated helper cells), CD8, CD8CD45RA (“naïve” T suppressor cells), CD8CD45R0 (“memory”/activated suppressor cells), and CD16 (NK cells). Whole blood cells, in heparin, were washed twice in PBS and suspended in PBS containing 0.1% BSA and 0.1% NaNytometry .μg ionomycin. 10 μg brefeldin A (BFA) were added to each tube; BFA inhibits intracellular transport and prevents secretion of the intracellular cytokines. The suspensions were incubated at 37°C in a 5% CO2 humidified atmosphere for a 4-hour incubation.0.5 mL whole blood, in heparin, was introduced into disposable polystyrene tubes. To 0.5 mL blood, 0.5 mL RPMI-1640 medium and 2 mM L-glutamine were added. The tissue culture medium components were purchased from Biological Industries, Beit Haemek, Israel. For activation cells were exposed to mitogens, 25 ng PMA and 1.0 μL FACS permeabilizing solution, obtained from Becton Dickinson, and were left for 8–10 minutes at room temperature. The cells were washed twice in PBS containing 0.5% BSA and 0.1% NaN3. 20 μL of fluorescent conjugated mAbs were added to stain intracytoplasmic cytokines. IgG preparation of suitable isotypes was used as negative controls [For intracytoplasmic immunofluorescent staining, the cells were resuspended in 500 The study was approved by the Ethics Committee (IRB) of the Kaplan Medical Center and the Review Board of the Ministry of Health.t test for unpaired data with InStat . Results were presented as mean ± SEM.Statistical analysis for evaluation of significant differences was performed with the use of Student's Demographic characteristics of the mothers and infants studied are summarized in P = n.s).117.9 ± 34.3 IU/mL (mean ± SEM) in infants of DHA-supplemented mothers versus 111.9 ± 56.1 IU/mL in the nonsupplemented group (P = n.s). IgM levels were 99.7 ± 15.0 mg% in the DHA group versus 91.8 ± 20.8 mg% in controls (P = n.s). IgG levels were 593.6 ± 70.3 mg% in the DHA group versus 745.7 ± 162.7 mg% in controls (P = n.s).IgA levels were 33.9 ± 4.22 mg% (mean ± SEM) in the DHA group versus 54.5 ± 15.6 mg% in controls (3 (mean ± SEM) in the babies of supplemented mothers versus 6900 ± 650 /mm3 (mean ± SEM) in controls (P = n.s).Total lymphocyte count did not differ between the two groups of babies; 6598 ± 450 /mmP = n.s), but CD8+ lymphocytes were significantly lower  (P < 0.05). The percentage of memory CD4 cells, CD4+CD45RO+/CD4, did not differ between the two groups (15.79 ± 1.77 versus 12.80 ± 2.01 (P = n.s)). Of CD8+ cytotoxic suppressor cells, although the percentage of CD8 cells was lower among infants of mothers receiving DHA supplementation, the CD8+CD45RO+/CD8 fraction, representing memory cells, was higher in the DHA-supplemented group; 34.9 ± 5.9 versus 16.2 ± 5.9 (P < 0.05). The sum of the two CD8 fractions, naïve CD8+ cells (CD8+CD45RA+), and activated CD8+ cells (CD8+CD45RO+) is higher than 100%, denoting that a proportion of the cells has stained positive for both markers, and is a phenomenon observed during the transition from a naïve to an activated state. The proportion of CD16+ lymphocytes did not differ significantly between the two groups .In the babies of w3-supplemented mothers, the percentage of CD4+ lymphocytes did not differ significantly from that of the control group  . Among Cγ was significantly lower in the DHA-supplemented group (γ producing CD4+ cells was 0.99 ± 0.18% among the DHA-supplemented group versus 2.68 ± 0.69 among controls (P = 0.01). Similarly among CD8+ cells, the proportion of IFNγ producing cells was 0.62 ± 0.13 versus 1.84 ± 0.43, respectively, (P = 0.01). IL4 production in CD4+ and CD8+ cells was lower in the DHA-supplemented group, but differences did not reach statistical significance  for a 7-month period did not lead to any changes in relative proportion of CD4 and CD8 cells, in non-stimulated whole blood.γ production by peripheral blood mononuclear cells, following exposure to various stimulants/mitogens, did not differ in the LCPUFA-supplemented group [Furthermore, IFNed group . Anothered group . Notablyed group , 25–27. Most studies have assayed the level of cytokines present in supernatants of isolated peripheral blood mononuclear cells or lymphocyte cultures. In our study, utilization of a technique for the intracellular staining of cytokines enabled us to determine the selective origin of cytokines, whether from CD4 or CD8 cells. DHA supplementation did not exert an effect on the humoral immune response. Infants in both groups did not differ in levels of immunoglobulins of IgG, IgM, and IgA class.Specific antibody response as reflected by measuring the titer of antibodies to HBsAg after 2 doses of vaccine (at 2 days and at 1 month of age) was similar in both groups. All infants in our study were exclusively breast fed for the first 4 months of age. As for the diet of the mothers taking part in the study, they all ate at least 2 servings of fish per week, cooked with canola oil, which is rich in w-3 FA, and added olive oil as salad dressing. A recent consensus statement regarding dietary fat intake for pregnant and lactating women has concluded that “ the desired average intake of at least 200 mg DHA/day can be reached with the consumption of one to two portions of sea fish per week” . Thus, tOur study is unique in that it questioned whether additional DHA supplementation to breast-feeding mothers who are consuming a diet supposedly sufficient in DHA content would affect the immune response of their infants. DHA supplementation during pregnancy and lactation did not result in changes in the infants' humoral immune response, as assessed by levels of anti-HBs antibodies and total immunoglobulins, but did affect lymphocyte subset profile and cytokine production.γ production in both CD4 and CD8 cells is likely compatible with attenuation of a proinflammatory response.A lymphocyte profile with a higher percentage of CD4 naïve cells and decreased IFNγ and TNF, has been shown, in animal models, to worsen damage as incurred, for example, during ischemic strokes [γ is considered to be an important proinflammatory cytokine produced by Th1 lymphocytes and by natural killer cells, mediating both innate and adaptive immune response, recent observations indicate that IFNγ may exert suppression of the immune response and may play a role in the induction of tolerance.Local expression of proinflammatory cytokines, particularly IFN strokes . Dampeniγ has been shown to modulate T-cell activation by reducing T-cell proliferation and survival and by increasing the number of regulatory T cells (Foxp3+ Treg cells) [γ also stimulates the expression of inhibitory B7 family molecules, which in turn may abate the production of Th1 T cells [γ production in both CD4 and CD8 cells cannot be simply predicted. As for the observed higher percentage of cytotoxic activated CD8 cells, these may play a facilitatory role in encountering of viral infections. IFNg cells) , 30. IFN T cells . Thus, tThe clinical implications of the changes observed are as yet speculative, and whether supplementation of DHA to breast-feeding mothers confers an advantage to their infants remains to be elucidated.E. Granot, Principal Investigator, helped in initiating and designing of project, analysis of data, and writing of manuscript and took the responsibility for its final content. E. Jakobovich, Study Coordinator, helped in the clinical followup of mothers and infants and data collection. R. Rabinowitz and P. Levy performed lymphocyte studies and contributed to analysis of data. M. Schlesinger is director of the laboratory in which lymphocyte studies were performed and is responsible for planning of lymphocyte studies and analysis of the data. All authors read and approved the final paper.The authors have no conflict of interests to report.