Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant EPISAcagA, or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether  Helicobacter pylori, a Gram-negative bacterium, is a major cause of chronic gastritis, peptic ulcers, and gastric malignancies, including gastric non-cardia adenocarcinoma and mucosal-associated lymphoid tissue (MALT) lymphoma H. pylori infection induces both acute and chronic gastritis, which is present as superficial mucosal inflammation in the gastric mucosa. In vitro experiments demonstrate that H. pylori activates multiple intracellular pathways including mitogen-activated protein kinases (MAPK), NF-κB and activator protein-1 as well as the Wnt/β-catenin pathway, which affect various cellular functions. These include increased inflammatory cytokine production, increased apoptosis, and epithelial cell turnover cag pathogenicity island (PAI), and vacuolating cytotoxin (VacA) are responsible for these effects H. pylori VacA has also been shown to inhibit T-cell proliferation and the cell cycle, and therefore suppress the immune response H. pylori induces the transcription of thousands of host genes, while at the same time represses another set of genes Chronic infection of the human stomach by Listeria monocytogenes secretes listeriolysin O (LLO), which induces a dramatic dephosphorylation of histone H3 at serine 10 (H3 Ser10) and deacetylation of histone H4, and this correlates with changes in host gene expression during the early infection Clostridium perfringens toxin perfringolysin (PFO) and Streptococcus pneumoniae toxin pneumolysin (PLY) also induce the same dephosphorylation of histone H3 Ser10; this decreased phosphorylation of H3 Ser10 is associated with the previously reported decreased inflammatory cell responses during bacteria infection Shigella flexneri toxin OspF blocks phosphorylation of MAPK ERK2 in the nucleus; this subsequently prevents histone H3 Ser10 phosphorylaton, which is a prerequisite of NF-κB activation and downstream gene transcription, and leads to a compromised inflammation in mouse tissue Immune subversion by histone modification is an important mechanism used by multiple bacteria and viruses during infection H. pylori infection is chronic and persistently enhanced inflammation with increased inflammatory cell infiltration in the local gastric mucosa and increased inflammatory cytokine production. A small percentage of infected individuals manifest the clinical presentation of gastritis, peptic ulcer, or gastric malignancy H. pylori infection effects histone modifications has not been as thoroughly evaluated. In the present study, we investigated if H. pylori infection modulates host gastric epithelial cell histone modification. In addition, we correlated H. pylori-induced histone modification with the changes in gastric epithelial cell functions and detected bacterial genes that are responsible for these effects, and explore their impact on pathogenesis.One prominent feature of H. pylori cagPAI-dependent dephosphorylation of histone H3 Ser10 and deacetylation of H3 lysine (K) 23. The effects of H3 Ser10 dephosphorylation are independent of major H. pylori virulence factors including CagA, VacA, and flagella. In addition, this cagPAI-dependent effect is common to multiple H. pylori strains, and the histone H3 Ser10 dephosphorylation is independent of ERK and p38 pathways, and type I interferon (IFN) signaling. Additionally, H3 Ser10 dephosphorylation is associated with changes in host gene expression. These results indicate a novel mechanism of H. pylori pathogenesis through histone modifications that has potential implications on H. pylori-induced chronic and persistent infections.We demonstrate a H. pylori in gastric epithelial cells, we first monitored the histone H3 Ser10 phosphorylation status in whole cell extracts by Western blot  did not alter the dephosphorylation of H3 Ser10 in AGS cells, compared to the wild-type 60190 strain  are the cause of H. pylori-induced H3 Ser10 dephosphorylation.The major virulence factors of rylation . In addi0 strain . These rH. pylori genes products within the cagPAI are the cause of H3 Ser10 dephosphorylation in AGS cells, G27-MA (and its derivatives) did not induce H3 Ser10 dephosphorylation in MKN45 cells (data not shown), likely due to the fact that G27-MA is a MDCK cell adherent strain H. pylori Sydney strain 1 which has a handicapped cagPAI cagPAI, induced H3 Ser10 dephosphorylation (data not shown). The results suggest both pathogen and host factors may affect histone dephosphorylation.Although we showed that H. pylori can cause H3 Ser10 dephosphorylation, we next investigated if H. pylori 26695 also induced other histone modifications. We monitored several common histone protein modifications, including the acetylation of H3K9, H3K14, H3K18, H3K23, H3K9K14, H3S10P/K9Ac, H3S10P/K14Ac, dimethylation of H3K9, hyperacetylation of H4, and acetylation of H4K8 in gastric epithelial cells. The results indicated that H. pylori 26695 cagPAI induced dephosphorylation of H3S10P/K14Ac, in a similar manner to the H3 Ser10 dephosphorylation caused by H. pylori in both AGS (cagPAI-dependent (data not shown). We also detected decreased H3K23 acetylation during H. pylori infection in a cagPAI-dependent manner in AGS cells, while other modifications tested in this study revealed no difference as compared with uninfected control cells , a histone deacetylase inhibitor which non-specifically increases the chromatin acetylation status, resulted in increased multiple histone H3 acetylations (data not shown). Interestingly, this effect is associated with some commonly reported gene transcription pattern alterations in nd c-fos . We notecagA is not responsible for the H3 Ser10 dephosphorylation in gastric epithelial cells as shown above, we investigated which gene or gene product within the cagPAI was responsible for this effect. To determine this, we used a series of H. pylori cag mutant strains cag genes that are necessary for IL-8 induction  Since H. pylori or L. monocytogenes infection cxcl2, prkdc, dusp4, cox2, c-Jun, hsp70, cyclin D1, and il-8, using chromatin immunoprecipitation (ChIP). We infected AGS cells with wild-type H. pylori G27-MA, which showed dephosphorylation of H3 Ser 10, and performed RT-PCR and ChIP assays. Interestingly, we noted that c-Jun and hsp70 gene expression was correlated with the H3 Ser10 phosphorylation in the promoter region of these genes . c-Jun and hsp70 mRNA expression levels in H. pylori-stimulated AGS cells represented up- or down- regulated genes, respectively. The results are in line with previously reported gene array H. pylori selectively regulates gene expression in host cells associated with chromatin modification, which subsequently regulates cellular functions.To determine if the changes in histone modification are associated with alterations in host gene expression, we tested a group of genes that has previously reported to be changed during se genes . On the H. pylori infection status  hours post-infection  has been shown to induce H3 Ser10 phosphorylation at the IL-6 promoter and this is also associated with increased IL-6 mRNA and protein expression H. pylori has been shown to regulate p21(WAF1) expression associated with histone H4 acetylation in gastric epithelial cells c-fos, expression L. monocytogenes has been shown to regulate il-8 gene expression through histone modification, including increased H4K8 acetylation and phosphorylation of histone H3 Ser10 in endothelial cells il-8 and c-fos genes, including the up-regulation of c-fos and down-regulation of il-8 in MKN45 cells upon H. pylori infection. These results suggest that altered chromatin structure can function to “mask” or “unmask” gene transcription start sites and therefore affects their transcription. The opposite effects between il-8 and c-fos gene transcription provide additional information on the bacterial-induced gene regulation, and may suggest different roles of histone deacetylation/acetylation and transcription factors play on each of their gene promoter in gastric epithelial cells.The effects of specific histone modifications in host cells appears to be cell type and promoter specific: in the mouse macrophage, H. pylori has been shown to affect the cell cycle H. pylori infection are not clear. Interestingly, during the submission of this work, a paper by Fehri et al. H. pylori-induced cagPAI dependent H3 Ser10 dephosphorylation. H3 Ser10 dephosphorylation and cyclin B1 correlated with transient pre-mitotic arrest. Further they showed vaccinia-related kinase 1 (VRK1) activity but not Aurora B activity are responsible for this transient cell cycle delay. In addition, they pointed out that this effect was IkB kinase alpha (IKKα) dependent H. pylori-induced cell cycle arrest is independent of cagPAI H. pylori-induce a cagPAI dependent H3 Ser 10 dephosphorylation, and the current work further suggest this modification is also associated with gene transcription control. Since H3 Ser10 phosphorylation has been linked with both cell cycle and transcription control H. pylori pathogenesis remain to be established in the future.H3 Ser 10 is cell cycle mitotic marker in proliferating cells, and H. pylori-induced H3 Ser10 phosphorylation levels. SP600125, the JNK inhibitor, which has been reported to reduce global H3 Ser10 phosphorylation H. pylori-induced a fast inhibition on H3 Ser10 phosphorylation within one hour, a time we reason most likely linked to transcriptional control through chromatin remodeling. Therefore, the current results provide a signaling link between bacterial infection and H3 Ser10 dephosphorylation.MAPK pathways including ERK and p38 have been reported to mediate the increased H3 Ser 10 phosphorylation, but not decreased H3 Ser10 phosphorylation il-8, cox2, cxcl2, prkdc, dusp4, and cyclin D1 were not associated with, or were independent of, histone modification in the current infection model. However, altered c-jun and hsp70 gene expression was associated with the H3 Ser10 dephosphorylation. Increased c-Jun protein phosphorylation and mRNA and reduced hsp70 mRNA has been observed during H. pylori infection H. pylori infection is not clear, but several studies have suggested it has cytoprotective effects, by reducing stress-induced denaturation and aggregation of intracellular proteins, and protecting the mitochondria, and interfering with the stress-induced apoptotic program H. pylori infection by inhibiting the expression of iNOS from gastric epithelial cells The global gene expression profile regulated by phosphorylated histone H3 Ser10 has yet to be identified. In this work, we noted, several commonly expressed genes, including H. pylori-induced pathophysiology in human stomach related to histone modifications. The delicate interplay between the host and pathogen may potentially lead to the different outcomes of H. pylori infection. Identification of the mechanisms by which H. pylori affects chromatin structure at gene promoters will allow us a better understanding of the gene transcription control, and subsequent alteration of cellular functions.In conclusion, these observations provide novel evidence in microbial pathogenesis as well as 2 incubator.Tissue culture reagents were purchased from GIBCO . The human gastric epithelial cell line, AGS, and the mouse fibroblast cell line, L929, were purchased from American Type Culture Collection . The human gastric epithelial cell line MKN45 was purchased from JCRB Cell Bank . Cells were grown in Ham's F-12 (AGS) or RPMI 1640 (MKN45 and L929) medium supplemented with 10% fetal bovine serum (FBS) without antibiotics at 37°C in a humidified 10% COH. pylori stimulation.MAPK inhibitors, including MEK1/2 inhibitor U0126, which inhibits extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation, p38 inhibitor SB203580, and c-Jun/SAPK N-terminal kinases (JNK) inhibitor SP600125, were purchased from Calbiochem . Cells were treated with the above inhibitors 30 minutes before H. pylori infection. Controls without inhibitors were treated with medium alone and an equal concentration of DMSO.Rabbit polyclonal anti-histone antibodies including histone H3 phosphorylated at serine 10 (p-H3 Ser10); histone H3 acetylated at lysine 9 (H3K9Ac); histone H3 acetylated at lysine 18 (H3K18Ac); histone H3 acetylated at lysine 23 (H3K23Ac); histone H3 phosphorylated at serine 10 and acetylated at lysine 9 (H3S10P/K9Ac); histone H3 phosphorylated serine 10 and acetylated lysine 14 (H3S10P/K14Ac); histone H3 dimethylated at lysine 9 (dimeH3K9); total H3; and histone H4 acetylated at lysine 8 (H4K8Ac) were purchased from Cell Signaling Technology . Histone H3 acetylated at lysine 14 (H3K14Ac); histone H3 acetylated at lysine 9 and lysine 14 (H3(K9K14)Ac); and hyperacetylated H4  were purchased from Upstate Biotechnology . Specific histone deacetylase inhibitor trichostatin A (TSA) was purchased from Sigma . Stock solutions were prepared in DMSO solution at 100 mM. In some experiments, cells were treated with the above inhibitors 30 minutes before H. pylori strains were used in the current study including 26695 and its isogenic cagPAI mutant (entire cag island deletion) strain 8-1  H. pylori strain 60190 and its vacA mutant strain (60190 VacA/KO), which contains a kanamycin cassette insertion  cag+ strains   cagA, vacA, cagPAI, cagA-vacA, and the nonphosphorylateable CagA mutant EPISAcagA and EPISAvacA-cagAcagPAI genes , and strain P12, its ΔcagA and ΔcagPAI mutants 2 at 37°C, and then harvested with a sterile cotton swab and resuspended in phosphate buffered-saline (PBS) solution. The bacteria were pelleted at 1,400×g for 10 minutes and resuspended in 5 ml of culture medium and added to the cell culture media at different bacteria to cell ratios , as indicated. Infections lasted from 0.5 to 6 hours in 10% CO2 at 37°C. Under these conditions, H. pylori remained alive and motile . We also noted that wild-type 26695, P12, and G27-MA strains induced hummingbird phenotype.A total of 52 different 5) or MKN45 cells (1×106) per well in 6-well culture plates were infected with bacteria at designated doses for designated times, washed three times with PBS, and lysed with cell lysis buffer directly on the dish (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue). Prior to loading, samples were boiled at 100°C for 3 minutes, cooled down on ice, and then separated on 15% SDS polyacrylamide gels. Proteins were subsequently transferred from gels onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 hour at room temperature in Tris-buffered saline plus 0.025% Tween-20 (TBS-T) with 5% nonfat dry milk (pH 7.4). Various histone antibodies were diluted at 1∶1000 in TBS-T with 5% nonfat dry milk solution. Membranes were then incubated with antibodies at 4°C overnight and washed three times with TBS-T (pH 7.4). The secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies, were used at a 1∶1000 dilution in TBS-T with 5% nonfat dry milk and incubated at room temperature for 3 hours (pH 7.4). Bands were detected with an enhanced chemiluminescence detection kit . In some experiments, proteins were separated on 8% polyacrylamide gel, blotted on a polyvinylidene difluoride membrane, and examined for p-H3 Ser10 with protein A coupled to alkaline phosphatase. The original membrane was stripped with stripping buffer (50 mM Tris-HCl (pH 6.7), 2% SDS, and 200 mM β-mercaptoethanol) at 60°C for 30 minutes, followed by three washes with TBS-T and blocked for 1 hour with TBS-T in 5% nonfat dry milk solution (pH 7.4) and re-probed with total histone H3 antibodies to assess protein loading.AGS cells (5×10Rt–Et), where Rt is the mean threshold cycle for the reference gene HPRT and Et is the mean threshold cycle for the experimental gene. Data were expressed as arbitrary units and fold changes were adjusted to the non-stimulated control cells. Primer sequences are provided in Total RNA from AGS and MKN45 cells was purified using the RNeasy Mini Kit (Qiagen), as described previously Cells were seeded and treated essentially the same way as described for Western blot. The procedures to detect IL-8 production from AGS cell was described previously 6) per well in 150 mm dish were infected with bacteria at MOI of 150:1 for 6 hours, washed three times with PBS, and cross-linked with 1% formaldehyde (Sigma) for 10 minutes. Cells were harvested in cell lysis buffer, and sonicated with 6×10 second pulses to generate 0.4–1.2 kb size. The sonicated chromatin was then split equally into four parts, one part without any process served as input control, the other 3 parts were immunoprecipitated with: rabbit polyclonal histone H3 Ser10 antibody  H. pylori treated control. Total H3 and non-immune isogenic IgG was used as monitoring control. PCR primer for both ChIP and RT-PCR are listed in Chromatin immunoprecipitation (ChIP) assays were performed as previously described “t” tests, and differences were considered significant if P values were <0.05.All quantitative data were expressed as mean ± SEM. Data were compared by using paired or unpaired Student Figure S1cagPAI mutants on H3 Ser 10 dephosphorylation during H. pylori infection in AGS cells. AGS cells (5×105) were treated in the presence or absence of wild-type H. pylori 26695 and its various mutants strains at MOI of 150:1. The cell lysate was then subjected to immunoblot analysis with rabbit anti-phospho-histone H3 Ser10 antibodies, anti-total H3 antibodies were used to re-probe the membrane and monitor protein loading. A representative blot from each strain is presented.Effects of (0.18 MB TIF)Click here for additional data file.Figure S2cagPAI mutants on H3 Ser 10 dephosphorylation during H. pylori infection in AGS cells. Data are mean±SEM from 3–6 densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control. **P<0.05, *P<0.01 when compared with controls.Comparison of the effects of (0.25 MB TIF)Click here for additional data file.Figure S3H. pylori-induced H3 Ser10 dephosphorylation is independent of IFN α/β signaling or bacterial DNA transfer into host cells. Mouse fibroblast cell line L929 (2×105) and human gastric epithelial cell line AGS (5×105) were treated with H. pylori G27-MA (G27) or its isogenic mutant strains in antibiotic-free medium for 10 hours at an MOI of 100:1, control cells were treated with medium alone. Supernatant from L929 cells were collected and used to measure IFN α/β production (panels A and B). RNA was extracted from AGS cells with the same treatment and cDNA was made for quantitative RT-PCR assay as described in (0.21 MB TIF)Click here for additional data file.


Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac.Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac.Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.The  In nature, three main α-Gal epitopes are produced: two trisaccharides  and a pentasaccharide . These epitopes are components of glycolipids and glycoproteins displayed on the cell surface of non-primate mammals and New World monkeys via expression of an α1,3-galactosyltransferase . The α1,3-GalT was inactivated in ancestral Old World primates approximately 20-28 million years ago, resulting in the absence of α-Gal epitopes in humans, apes, and Old World monkeys today . In addiThe increasing interest in α-Gal epitopes for various medical applications necessitates an efficient and economical means of synthesizing the oligosaccharide. Traditional chemical synthesis requires numerous reaction steps, leading to low overall yields, a high cost, and a process that is not applicable for large-scale production. Enzymatic production of α-Gal epitopes can be achieved in just one step through the use of an α1,3-GalT; however, enzymatic synthesis requires provision of an expensive sugar nucleotide, UDP-galactose. To reduce cost, enzymatic synthesis schemes often employ a UDP-galactose 4'-epimerase to provide the UDP-galactose from a less expensive sugar nucleotide, UDP-glucose ,9. As UDE. coli was constructed by overexpressing five enzymes: three enzymes of the Gal operon  for UDP-galactose synthesis, a pyruvate kinase for energy production, and an α1,3-GalT. In this strategy, glucose and catalytic amounts of other cofactors  were supplied to the engineered E. coli to initiate Gal-α1,3-Lac synthesis [Pichia pastoris expressed a sucrose synthase which directly converts sucrose and UDP to UDP-glucose with fructose as co-product. With two additional enzymes , the modified P. pastoris required only sucrose, lactose, a catalytic amount of UDP, and a few essential nutrients to produce Gal-α1,3-Lac. The three recombinant enzymes in P. pastoris constituted an artificial pathway, whose operation was independent of cellular metabolism [E. coli and P. pastoris were capable of synthesizing gram-scale amounts of Gal-α1,3-Lac.Alternatively, whole cell biocatalysts can synthesize α-Gal epitopes in just one step without enzyme purification. Different hosts and engineering strategies were explored by Wang and coworkers for whole-cell Gal-α1,3-Lac synthesis. An engineered ynthesis . Alternatabolism . Both thE. coli and P. pastoris biocatalysts, permeabilization was found to be necessary [E. coli biocatalyst was engineered to synthesize the acceptor sugar in vivo. Expressing a chitin oligosaccharide synthase (NodC) and several glycosyltransferases, the recombinant E. coli produced a heptasaccharide α-Gal epitope without permeabilization of the cell membrane [in vivo substrate synthesis may hinder α-Gal epitope production.One key challenge with whole-cell catalysts is uptake of the acceptor sugar, lactose, along with the primary sugar (i.e. sucrose or glucose). In both the engineered ecessary ,13. Whilmembrane . While tAgrobacterium sp. strain ATCC 31749 is a good host for oligosaccharide production through the synthesis of β1,4-Gal disaccharides [in vivo substrate synthesis, an alternative strategy is employed to address insufficient uptake of the substrate sugar, lactose. A lactose permease (LacY) from E. coli is introduced in the Agrobacterium host to facilitate lactose transport across the cell membrane. In addition, sugar nucleotide availability is improved by eliminating curdlan production, a competing pathway for utilization of UDP-glucose  from E. coli and a truncated bovine α1,3-galactosyltransferase   Figure . InsertiAgrobacterium, ATCC 31749/pBQET, was utilized for small-scale α-Gal epitope synthesis. The synthesis process includes two phases. In the first phase, the engineered Agrobacterium is grown and induced. After recombinant protein production, the cells are transferred to a nitrogen-limited minimal media for synthesis of Gal-α1,3-Lac. Nitrogen-limited conditions are employed for α-Gal epitope synthesis as curdlan production, and hence UDP-glucose production, is activated by this environmental signal. In the α-Gal epitope synthesis reaction, the engineered strain synthesized 0.39 g/L of the desired product, Gal-α1,3-Lac  [A major challenge of whole-cell synthesis is the efficient transport of reactants across the cell membrane. This task is particularly difficult for oligosaccharide synthesis reactions such as α-Gal epitope synthesis. The synthesis reaction requires uptake of two sugars: the acceptor sugar (lactose) and a carbon source for production of the sugar nucleotide and cellular energy (sucrose) Figure . Simulta 8.5 mM) . These rlacY) from an E. coli K12 strain was expressed along with the fusion enzyme in the engineered Agrobacterium  availability, the amount of the sugar nucleotide, UDP-glucose, may also restrict α-Gal epitope synthesis by the GalE:α1,3-GalT fusion enzyme. Under the nitrogen-limited conditions of the synthesis reaction, production of the α-Gal epitope competes with curdlan synthesis for the available UDP-glucose Figure , indicatrdS [Curdlan, a β1,3-glucan polysaccharide, is not known to perform any essential or beneficial function for ATCC 31749. Therefore, by eliminating curdlan production, the amount of UDP-glucose available for α-Gal epitope synthesis will be increased without any detrimental impact on the cell. The gene responsible for the transfer of glucose from UDP-glucose to the growing curdlan polymer chain was previously determined to be curdlan synthase, crdS . CrdS haAgrobacterium sp. strain ATCC 31749; however, successful gene knockout has been reported for a related organism, Agrobacterium tumefaciens. Gene knockout in A. tumefaciens employed the standard method of insertional mutagenesis via homologous recombination of a disruption cassette [A. tumefaciens contains several tetracycline resistance genes, gentamicin was selected for the antibiotic resistance cassette in the crdS knockout plasmid. Sequences homologous to the 5' and 3' ends of crdS (500 bp) were added to each side of the gentamicin resistance cassette to allow for homologous recombination. The resulting fragment was inserted into the pBluescript II (KS-) phagemid to give the crdS knockout plasmid, pBScrdShG . The cell growth profile for ATCC 31749ΔcrdS in LB media showed no deviation from the wild-type strain, indicating that the crdS knockout did not affect cell growth. Since curdlan is not produced during cell growth, but rather, under nitrogen-limited conditions, the cell viability of ATCC 31749ΔcrdS was studied in minimal, nitrogen-free media. The cell viability of the crdS mutant under curdlan-producing conditions showed no significant difference from the wild-type strain (data not shown). Therefore, curdlan production does not appear to contribute to cell survival under nitrogen-limited conditions and should not have a detrimental effect on α-Gal epitope synthesis.This is the first reported attempt at gene knockout in cassette . To deteG Figure . After tcrdS was transformed with pBQET and pBQETY. ATCC 31749ΔcrdS/pBQETY produced 0.96 g/L of Gal-α1,3-Lac , the engineered Agrobacterium is capable of producing gram-scale quantities of the α-Gal epitope for medical research and applications.To determine the effect of the curdlan synthase knockout on α-Gal epitope synthesis, ATCC 31749ΔAgrobacterium sp. ATCC 31749. This microorganism can produce up to 93 g/L of curdlan polysaccharide [E. coli. Insufficient uptake of substrate and acceptor sugars is yet another obstacle in whole-cell oligosaccharide synthesis. To enhance sugar uptake, other strategies have used permeabilization techniques to weaken the cell membrane or have expressed additional enzymes to synthesize the acceptor sugar in vivo [Agrobacterium host to selectively increase uptake of the acceptor, lactose. This strategy increased lactose uptake without the undesirable consequences of cell permeabilization techniques, leading to a 67% improvement in Gal-α1,3-Lac synthesis. The LacY-expressing Agrobacterium may also be used to synthesize other lactose-containing oligosaccharides such as Globo-H, the major component of a vaccine for metastatic breast cancer [Agrobacterium host was exploited for α-Gal epitope synthesis, negating a need for expensive cofactors. Without the requirements of cell permeabilization or cofactors, the engineered Agrobacterium developed in this work is a suitable biocatalyst for large-scale production of the α-Gal epitope, and with only slight modification, it may be utilized for the production of other medically-relevant oligosaccharides.The whole-cell biocatalysts developed in this study provide a basis for an efficient and cost-effective means for large-scale production of the Gal-α1,3-Lac epitope. Enzymatic and whole-cell methods developed for α-Gal epitope synthesis face many obstacles including (1) low synthesis levels, (2) inefficient substrate transport across the cell membrane, and (3) expensive cofactors. This work addresses all three of these issues. Host selection is a critical factor in determining product synthesis levels. To provide an adequate host for synthesis of the oligosaccharide, Gal-α1,3-Lac, a polysaccharide-producing microorganism was selected, ccharide , demonst in vivo -14. In tt cancer . Lastly,lacY expression and crdS knockout are effective in addressing the two respective limitations, these efforts resulted in only moderate improvements in α-Gal epitope synthesis. The highest product concentration was about 1 g/L with shaker flask cultivation, far below the theoretical potential of 270 g/L (estimated from reported levels of curdlan synthesis in Agrobacterium sp.). One reason for the lower than expected improvement is that LacY expression negatively impacted expression of the fusion enzyme. As shown in Table lacY to pBQET leads to nearly a 5-fold reduction in GalE:α1,3-GalT fusion enzyme activity. In the future, chromosomal integration of lacY may be considered to improve lactose uptake while maintaining the high fusion enzyme activity of the pBQET strain. The use of shaker flask cultivation may also contribute to the low level of Gal-α1,3-Lac synthesis. Other reported methods of whole-cell α-Gal epitope synthesis utilize fermenters for either high cell density growth or both growth and the epitope synthesis reaction [Agrobacterium as high curdlan synthesis requires high levels of dissolved oxygen and pH control at pH 5.5 [crdS mutant should produce high levels of UDP-glucose for Gal-α1,3-Lac synthesis. Unfortunately, initial fermentation attempts with the engineered Agrobacterium were unsuccessful due to the low recombinant protein production. Chromosomal integration of the galE:α1,3galT fusion enzyme and expression using a natural host promoter may overcome the limited recombinant protein production to allow large-scale fermentation. The reasons for only modest improvement from crdS knockout are not clear to us at this point due to limited knowledge about the regulation of curdlan synthesis. Recently, we sequenced the genome of ATCC 31749 and a transcriptome analysis is being conducted. These efforts will lead to a better understanding of the regulation mechanism governing curdlan synthesis and its precursor, UDP-glucose. In turn, this knowledge will allow metabolic engineers to formulate better strategies for improving synthesis of α-Gal epitopes and other oligosaccharides using ATCC 31749 as host.While the results clearly show that the metabolic engineering strategies of reaction -14. Largt pH 5.5 ,27. Unde2HPO4·3H2O, and MnCl2·4H2O); Acros organics (aniline blue); V-labs, Inc. ; and Fisher .The chemicals used in this study were obtained from Sigma-Aldrich  and truncated bovine α1,3-galactosyltransferase  was constructed and inserted into the Agrobacterium expression vector, pBQ, to form pBQET for α-Gal epitope synthesis. The truncated bovine α1,3-galT was amplified from pET15b-αGalT [α1,3-galT (5'-TCGGATCCATGGAAAGCAAGCTTAAGCTATC-3') contains a BamHI site  and a start codon (underlined). The 3' primer for α1,3-galT (5'-TCGAGCTCTCAGACATTATTTCTAACCACATT-3') contains a SacI site  and a stop codon (underlined). The amplified α1,3-galT was inserted into the pGEM-T easy vector to form pT-α1,3-galT. The α1,3-galT fragment, obtained by double digestion with BamHI and SacI, was inserted into the respective restriction sites of the pT-GalE plasmid containing galE with a linker sequence [galE:α1,3-galT fusion gene for α-Gal epitope synthesis. The fusion gene fragment, obtained from BglII and SacI digestions, was fused to the BamHI and SacI sites of pBQ. The resulting plasmid, pBQET, is shown in Figure A fusion enzyme containing the 5b-αGalT . The 5' lacY) was cloned from the genomic DNA of E. coli K12 strain JM109. The 5' primer for lacY (5'-ACGAGCTCAAAGAGGAGAAAT TAACTATGTACTATTTAAAAAACACAAAC-3') contains a SacI site , a ribosome binding site (underlined), and a start codon (in bold and underlined). The 3' primer (5'-GTCTCGAGTTAAGCGACTTCATTCACCTG-3') contains an XhoI site  and stop codon (underlined). The amplified lacY fragment was inserted into the pGEM-T easy vector, yielding pT-LacY. The lacY fragment, obtained by SacI and XhoI digestions, was ligated to the SacI and SalI sites of pBQET to form pBQETY  contains a SacI site  and the start codon for crdS (underlined). The 3' primer for crdS amplification (5'-CCGGTACCTCACCCGAATGCCCGTGC-3') contains a KpnI site  and crdS stop codon (underlined). The amplified crdS gene was inserted into the pGEM-T easy vector to produce pT-crdS. Using pT-crdS as template, the pGEM-T easy vector along with 500 bp of homology at the 5' end of crdS and 500 bp of homology at the 3' end of crdS were amplified using phosphorylated primers. The 5' primer (5'-pGGCCGAATTCTCGCAATAGGTTCTTACCTC-3') contains an EcoRI site  while the 3' primer (5'-pGGCAAGCTTTTCGTGACCCTGTCTTCGGC-3') contains a HindIII site. The amplified pGEM-T easy vector with homologous regions of crdS was self-ligated to form pT-crdSh. pT-crdSh was digested using SacI and KpnI, and the resulting fragment containing crdS homology (crdSh) was inserted into the corresponding restriction sites of pBluescriptII (KS-), generating the plasmid pBScrdSh. The gentamicin resistance gene (GmR), along with its promoter and transcription terminator, was amplified from pYanni2 [GmR amplification (5'-CCGAATTCGTCTAGTGAGTAGTG GGTAC-3') contains an EcoRI site , and the 3' primer (5'-CGAAGCTT GCTTGCAAACAAAAAAACCACC-3') contains a HindIII site. The amplified fragment containing GmR was inserted into the pGEM-T easy vector, forming pT-GmR. pT-GmR was digested using EcoRI and HindIII, and the fragment containing GmR was inserted into the corresponding restriction sites in pBScrdSh to form pBScrdShG  and 8 mM of UDP-glucose  in the enzyme activity buffer described previously. The assay was conducted at 30°C for 40 min followed by 10 min at 97°C to stop the reaction. The deactivated enzyme mixture was diluted 50×, and the Gal-α1,3-Lac product was analyzed using the protocol detailed below .For the enzyme activity assay, the induced cells were collected by centrifugation at 5,000 × g and 4°C for 2 min. The cell pellet was resuspended in a buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM MnClcrdS inoculums were prepared at 30°C and 250 rpm in a culture tube with 4 mL of LB media containing 10 μg/mL of gentamicin for the crdS mutant. The inoculums were diluted 1000× and grown in 150 mL of freshly prepared LB media, supplemented with antibiotics for ATCC 31749ΔcrdS. After 12 hours of growth in LB, the cells were collected by centrifugation at 3000 × g for 10 min at 4°C. The cell pellets were washed with 10% glycerol and then resuspended in nitrogen-free media. The nitrogen-free media consisted of 70 g/L sucrose, 1 g/L K2HPO4·3H2O, 5 g/L MgSO4·7H2O, 5 g/L sodium citrate, 1 g/L MnCl2·4H2O, and 50 mM Tris-HCl (pH 7.5). The cells were cultivated in nitrogen-free media at 30°C and 250 rpm in a biological shaker for 72 hours. Samples were taken at intervals of 12 hours and diluted using sterile LB media. The diluted cultures were spread on LB/agar plates, and colony forming units (cfu's) were counted as a measure of cell viability.ATCC 31749 and ATCC 31749Δ2HPO4·3H2O, 5 g/L MgSO4·7H2O, 5 g/L sodium citrate, 1 g/L MnCl2·4H2O, and 50 mM Tris-HCl (pH 7.5). The final cell concentration was 10% wet wt/v, and the reaction vessel was a 50 mL Erlenmeyer flask. The reaction vessel was placed in a biological shaker at 30°C and 250 rpm. Samples were centrifuged for 3 min at 13,200 rpm. The supernatant was heated in boiling water for 10 min and then centrifuged again at 13,200 rpm for 3 min. The supernatant was appropriately diluted and analyzed as described in Carbohydrate analysis.The induced cells were prepared as described above . The cells were then collected by centrifugation for 10 min at 3,000 × g and 4°C. The cell pellets were washed once with sterile 10% glycerol, and then resuspended in the reaction media. The reaction media contained 50 g/L sucrose, 25 g/L lactose, 1 g/L K600 of 0.2 - 0.3 was reached, the cultures were induced with IPTG . Immediately after the addition of IPTG, the temperature was shifted to 25°C. After an induction period of 4 hours, the culture was centrifuged at 3,000 × g and 4°C for 10 min. The cell pellet was washed with 0.4 M NaCl and collected by centrifugation. A buffer containing 100 mM Tris-HCl and 100 mM NaCl (pH 8.0) with protease inhibitor cocktail (Sigma-Aldrich) was used to resuspend the cell pellet to a final concentration of 0.2 g/mL (wet weight). The cell suspension was sonicated 8 times for 10 second intervals with 1 min rest on ice. Cellular debris was removed by centrifugation at 3,000 × g and 4°C for 20 min. The GalE:α1,3-GalT fusion enzyme was purified from the supernatant using a HIS select HF nickel affinity gel (Sigma-Aldrich). A 12% Tris-HCl gel (Bio-Rad) was used for SDS-PAGE to analyze the His-tag purified and soluble protein fractions.SDS-PAGE was used to confirm the successful expression of the GalE:α1,3-GalT fusion enzyme. JM109/pBQET was inoculated in 4 mL of LB media containing 100 μg/mL of kanamycin and 100 μg/mL of ampicillin. After overnight growth at 37°C with 250 rpm, 3 mL of the inoculum was transferred to 150 mL of LB media with antibiotics and grown at 37°C with 250 rpm. When an ODDiluted samples were analyzed using a Dionex BioLC system with a CarboPac PA20 analytical column. The Dionex ED50 electrochemical detector measured carbohydrate concentrations through pulsed amperometry . Sucrose, lactose, and Gal-α1,3-Lac concentrations were determined using calibration curves prepared from standards. The mobile phase consisted of degassed 200 mM sodium hydroxide (A) and 18 MΩ-cm water (B), pressurized with inert gas (He) at a flow rate of 0.5 mL/min. The following linear gradient was used for sucrose and lactose detection: t = 0 min, 5:95 (A:B); t = 5 min, 5:95; t = 10 min, 20:80; t = 25 min, 20:80; t = 25 min, 100:0; t = 40 min, 100:0; t = 40 min, 5:95; t = 55 min, 5:95. The linear gradient for Gal-α1,3-Lac detection entails the following steps: t = 0 min, 30:70 (A:B); t = 35 min, 30:70; t = 35 min, 100:0; t = 50 min, 100:0; t = 50 min, 30:70; t = 60 min, 30:70.ATCC 31749/pBQET and ATCC 31749/pBQETY were prepared as described above . Samples (1 mL) were taken from both reaction vessels at 34 and 45 hours after the start of the synthesis reaction and centrifuged for 5 min at 5,000 × g and 4°C. The supernatant was removed and used for analysis of extracellular lactose and Gal-α1,3-Lac. The cell pellet was washed with 50 mM of Tris-HCl buffer (pH 7.5) and centrifuged a total of three times to remove any residual extracellular lactose. After washing, the cell pellet was resuspended in 600 μL of 50 mM Tris-HCl buffer (pH 7.5). The cells were disrupted using sonication . Cell debris was removed by centrifugation, and the supernatant containing the intracellular lactose was heated for 10 min in boiling water to denature any proteins. After centrifugation, the sample was diluted 50× and analyzed as described in Carbohydrate analysis to determine the amount of lactose in mmol. The intracellular volume was determined by taking another 1 mL sample and collecting the cell pellet by centrifugation. The cell pellet was washed with DI water and centrifuged three times to remove any residual media. The wet weight of the cell pellet was measured, and then, the cell pellet was dried in an oven at 80°C until a constant dry weight was obtained. The difference in weight between the wet and dry cell pellets was used to calculate the intracellular volume. The intracellular lactose concentration was then calculated using the amount of lactose determined through carbohydrate analysis and the intracellular volume.crdS was detected using an aniline blue staining method [The elimination of curdlan production in ATCC 31749Δg method . Candidaα1,3-GalT: α1,3-galactosyltransferase; ADP: adenosine diphosphate; ATP: adenosine triphosphate; Crd: curdlan; F6P: fructose-6-phosphate; Fru: fructose; G1P: glucose-1- phosphate; G6P: glucose-6-phosphate; Gal: galactose; GalE: UDP-galactose 4'-epimerase; GalK: galactokinase; GalT: galactose-1-phosphate uridylyltransferase; GalU: glucose-1-phosphate uridylyltransferase; Glc: glucose; IPTG: isopropyl β-D-1-thiogalactopyranoside; Lac: lactose; PEP: phosphoenolpyruvate; UDP: uridine diphosphate; UDPG: uridine diphosphoglucose; UTP: uridine triphosphateThe authors declare that they have no competing interests.RRC conceived of the study and participated in the analysis of data and writing of the manuscript. AMR carried out the study and data analysis and participated in the writing of the manuscript. All authors read and approved the final manuscript.


Neuraxial opioids provide excellent analgesia intraoperatively and postoperatively while allowing early ambulation of the patient by sparing sympathetic and motor nerves. A prospective, randomised double blind study was conducted involving 90 patients of ASA 1 physical status coming for elective cesarean section to evaluate the analgesic effect of neuraxial buprenorphine. They were allocated into three groups. Spinal local anaesthetic was used as the main stay of anaesthesia for surgery and spinal and epidural analgesia with opioids continued as the main stay for postoperative analgesia. All the groups were given 0.5% Bupivacaine intrathecally for the surgery. Besides this, group I was given 150 mcg Buprenorphine intrathecally and group II and III were given 150 mcg and 300 mcg Buprenorphine respectively, epidurally. In the present study, we observed that 150 mcg of Buprenorphine given intrathecally provided much longer duration of analgesia compared to 150 mcg of Buprenorphine given epidurally. Increasing the epidural dose of Buprenorphine from 150 mcg to 300 mcg proved to produce prolonged analgesia comparable to intrathecal Buprenorphine without compromising patient safety and neonatal outcome. The minor side effects were more with intrathecal Buprenorphine than epidural Buprenorphine. We concluded that 300 mcg of Buprenorphine epidurally is equianalgesic to 150 mcg Buprenorphine intrathecally. Opioids are widely used for providing postoperative analgesia and advantages of neuraxial narcotics over systemic narcotics are well established. Opioids,3There are numerous studies comparing opioid agents in varying concentrations given through either epidural route or intrathecal route separately. But there are few studies comparing the effects of the same opioid given intrathecally and epidurally and few studies on analgesic effects of neuraxial buprenorphine.The present study is undertaken to compare the effects of intrathecal and epidural buprenorphine and to find a safe equianalgesic dose of intrathecal and epidural Buprenorphine. Quality and duration of analgesia was assessed for 24 hours postoperatively. Occurrence of adverse effects like hemodynamic effects, respiratory depression, Post Dural Puncture Headache, Nausea and vomiting, drowsiness and pruritus when given through each of the routes were also assessed. Neonatal outcome was evaluated using Apgar score and neonA randomised controlled double blind prospective study was done to compare the effects of intrathecal and epidural Buprenorphine. The study was conducted after approval by the hospital Ethics Committee and an informed written consent was obtained from all the patients. A total number of 90 ASA I patients belonging to age group 20-40 years posted for elective lower segment cesarean section were divided into three groups of 30 each. Group allocation was achieved by a computer generated randomisation list. Patients with any variations from normal were excluded.The patients were kept fasting for 6 hours prior to surgery and premedicated with oral Ranitidine 150 mg at night and oral Metoclopramide 10 mg and Ranitidine 150 mg two hours prior to the surgery. NIBP, ECG, SPO2, RR were monitored up to 24 hrs postoperatively. An 18g IV canula was secured and all patients were preloaded with 750 ml of Ringer Lactate before the neuraxial block. Spinal local anaesthetic was used as the main stay of anaesthesia for surgery and spinal and epidural analgesia with buprenorphine continued as the main stay for postoperative analgesia.A single space Combined Spinal Epidural technique was chosen and the same volume of drug was injected intrathecally and epidurally in all study groups. To make the intrathecal volume of drug equal in all groups, group II and III were given 0.5% Bupivacaine 2.5 ml intrathecally while group I was given 0.5% Bupivacaine 2 ml and 0.5 ml of buprenorphine Grouping of cases was in the following manner:-The procedure was carried out in lateral decubitus position using combined spinal epidural needle. Epidural space was identified in L2-L3 region using loss of resistance technique. The anaesthetist conducting the study was blinded to the study drug which was prepared by another anaesthetist as per instructions. Epidural test dose of 3 ml of xylocaine with adrenaline was given and observed for any motor block or significant rise in heart rate. After injecting 7 ml of the epidural test drug, spinal needle was advanced into the subrachanoid space and 2.5 ml of intrathecal test drug was given. A left uterine displacement of 15° was maintained during surgery. Supplemental oxygen was given through a poly mask.Three groups were compared.Sensory block was tested by pinprick till level reached T4. The total duration of analgesia was calculated from onset of sensory block to end of analgesia i.e.; pain score of 5 or more on the Verbal Numerical Rating Scale .8]8]Verbal numerical rating scaleCriteria for hypotension was taken as a systolic BP less than 100 mm Hg and bradycardia as heart rate less than 60. Hypotension was treated with IV ephedrine and rapid infusion of fluid. Criteria for respiratory depression were a fall in oxygen saturation to <90%, a respiratory rate less than 12/min. At the end of the surgery, the overall quality of anaesthesia was judged by the surgeon and patient on a Numerical Rating Scale (NRS) from 1 (unsatisfactory) to 10 (excellent). Neonatal outcome was assessed by APGAR score at I min and 5 min and by umbilical arterial blood gas analysis. APGAR score of <7 and an umbilical arterial blood pH <7.2 was considered abnormal.Postoperatively patients were monitored in post anaesthesia care unit and complications like hypotension, respiratory depression, drowsiness, post dural puncture headache, nausea, vomiting and pruritus were assessed for 24 hours. Occurrence of urinary retention could not be assessed as the patients for LSCS are routinely catheterised for 24 hours postoperatively in our institution.P < 0.05 was considered as significant.Parametric data were analysed using Z test. P>0.05). The minimum blood pressure recorded was systolic BP of 84 mm Hg and fall in BP was transient and responded to one dose of 6mg ephedrine IV. There was no significant variation in respiratory rate and saturation in all three groups and were within acceptable limits [In all the three groups, there was no significant change in heart rate and blood pressure from the base line value, neither in intraoperative nor in the postoperative period  [The 1 minute and 5 minute Apgar score and the umbilical cord pH of babies in all the three groups were acceptable and there was no significant differences between the groups (P>.05) .In all groups the surgeon enjoyed adequate muscle relaxation for performing the surgery and patient was comfortable throughout the procedure. There was no statistically significant difference between the groups in the NRS scores .In group I , 100% of patients had analgesia till 2.5 hours and 50% had analgesia till 6 hours. At 20 hours and 24 hours 16% and 1% of patients, respectively, had analgesia.In group II  100% of patients had analgesia till 2 hours and 50% till 3 hours. Thereafter the analgesia was poor.In group III  till 3.5 hours 100% of patients had analgesia and it took 16 hours for the percentage to drop to 50%. At 20 hours and 24 hours, 20% and 4% of patients, respectively, had analgesia.Any method of postoperative analgesia must meet three basic criteria; it must be simple, safe, clinically appropriate and evidence based. The majo1012Buprenorphine is a mixed agonist – antagonist type of opioid with a long duration of action. The high lipid solubility; high affinity for opioid receptors and prolonged duration of action makes Buprenorphine a suitable choice for intrathecal and peripheral nerve site administration.4Addition of Buprenorphine 150 mcg intrathecally or epidurally and 300 mcg epidurally provided good postoperative analgesia without prolonged motor block.15 In all16One of the main concerns with Buprenorphine is respiratory depression.–20 In ouThe incidence of Nausea and Vomiting was 20% in GP I which was slightly higher than the other groups. Pruritus is one of the commonest side effects of neuraxial opiods. It is more likely to occur in obstetric patients due to the interaction of estrogen with opioid receptors. Previous studies show the incidence of pruritus after epidural administration of 50 mcg fentanyl was 47% and with 300 mcg Buprenorphine, 10%. In our oet al.,[et al.,[Incidence of PDPH, backache and drowsiness were not reported. This was comparable to the results obtained by Fuller JG et al., and Esca,[et al., The high,[et al.,Neonatal outcome was good in all the groups as assessed by 1min and 5 min Apgar and umbilical arterial blood pH. In all pOne of the drawbacks of the study was that the latency of onset of action of Buprenorphine could not be studied because it was masked by the action of subarachnoid local anaesthetic. Also the concentration and quantity of intrathecal local anaesthetic in Group I was dilute (2.0 ml of Bupivacaine and 0.5 ml of Buprenorphine). Urinary retention could not be assessed because in our institution all patients for caesarean section are catheterised for 24 hours postoperatively.Buprenorphine 150 mcg intrathecally, 150 mcg epidurally and 300 mcg epidurally provided good quality postoperative analgesia.150 mcg Buprenorphine epidurally was not equianalgesic to 150 mcg Buprenorphine given intrathecally. The duration of analgesia was much shorter with epidural 150 mcg Buprenorphine.Increasing the epidural dose of Buprenorphine from 150 to 300 mcg, prolonged postoperative analgesia up to 24hours without compromising patient safety and neonatal outcome.The minor side effects of Buprenorphine are more when administered intrathecally, compared to Buprenorphine administered epidurally.


Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis.Circulating endothelial progenitors of Down syndrome affected individuals were isolated, We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells.Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals. Down syndrome (DS) is a complex disorder caused by trisomy of the entire or a critical portion of chromosome 21 (HSA21); it represents the most frequent genetic cause of mental retardation, with a frequency of about 1/1000 new-borns, and is associated with a huge number of congenital heart defects ,4. DespiDS phenotype results from a dosage imbalance of HSA21 genes, although expression analyses have reported conflicting results ,10. The Growing interest is emerging on circulating endothelial progenitor cells (EPCs) and their pivotal role in the maintenance of endothelium integrity, repair after injury and postnatal neovascularization -17. ManyBartonella henselae, a gram-negative intracellular bacteria responsible of vasoproliferative disorders in immunocompromised individuals [Bacterial toxins may trigger pathogenic events through the over-production of cytokines and chemokines, leading to the alteration of endothelial function and capillary leakage . Particuividuals ,28, adheB. henselae model, we investigated the susceptibility of DS progenitors to this pathogen infection, also performing a detailed analysis of deregulated genes after Bartonella infection, with particular attention to angiogenesis and immune response pathways.The present study was designed to pursue the molecular mechanisms contributing to immune, vascular and haematopoietic defective DS phenotypes, by investigating the number and functions of DS EPCs compared to euploid cells, also focusing on bioinformatics analysis of differentially expressed genes. Moreover, by using the previously described DS and euploid donors were recruited at the Institute of General Pathology, Section of Clinical Pathology, Faculty of Medicine, University of Milan, and at the Second University of Naples, and an approval statement was obtained by the ethics' review boards of both Institutions. Informed consent was obtained from all persons involved in all clinical investigation of this study according to the principles expressed in the Declaration of Helsinki.All subjects recruited for EPC isolation were free of infection, and no individual was taking any medication known to affect immune system/response. DS and euploid individuals were 65% males and 35% females as gender and 28 ± 9 as mean age. The experiments, where not specified, were performed on at least six DS and age-matched euploid individuals.Plasma samples were obtained from 50 DS individuals and 30 age matched euploids subdivided into three age subgroups  as described elsewhere .EPCs were isolated from non-institutionalized individuals with DS and age-matched euploid donors.EPCs were isolated as previously described . BrieflyB. henselae strain ATCC 49882 (LGC Promochem) was grown on Columbia agar supplemented with 5% defibrinated sheep blood (Oxoid) in a humidified atmosphere at 37°C and 5% CO2. For production of bacterial stock suspensions, bacteria were harvested after 7 days of culture until they reached the mid-exponential phase of growth (109 bacteria/ml), resuspended in Tryptone Soya Broth USP (Oxoid) containing 10% glycerol, and stored at -80°C. The number of viable bacteria in the frozen stocks was determined as previously described [The escribed .For infection, Bartonella stock solutions were thawed, washed and suspended in antibiotic-free cell culture medium, and sedimented onto cultured EPCs at different multiplicity of infection (MOI) of 50, 100, 250, 500 and 1000 . The MOIUlex europaeus and counted both by fluorescence microscopy and flow cytometry as previously described [http://rsb.info.nih.gov/ij/).EPCs were dual stained with Dil-Ac-LDL and lectin from escribed ,30. ImagCommercially available SDF-1α ELISA kit  was used to determine plasma SDF-1α levels. Tests were carried out at RT on freshly thawed plasma samples of 50 DS individuals and 30 age matched euploids subdivided into three age subgroups  . ConcentB. henselae, were harvested, centrifuged and washed in PBS. After centrifugation at a speed of 400 g for 7 min, cells were fixed in 4% glutaraldehyde as described [After a short incubation with Trypsin/EDTA, DS and euploid EPCs, both infected and uninfected with escribed . Postfixescribed . SemithiOxidation-sensitive fluorescent probe, C11-BODIPY581/591 , was loaded  into the cells 30 min. before oxidative treatment. The samples were aliquoted in triplicate wells of a 24-well microplate, and fluorescence was determined with confocal laser microscopy at different times  from oxidant treatment. Between times, plates were maintained at 37°C in 5% CO2. To determine red fluorescence each microplate was excited at 543 nm (emission at 590 nm); for green fluorescence, microplates were excited at 488 nm (emission at 526 nm). Blank wells were also evaluated as well as C11-BO alone.http://www.ebi.ac.uk/arrayexpress; provisional accession number E-MTAB-312). Results were validated by qRT-PCR and semi-qRT-PCR, performed as described [Total RNA 10 μg) was isolated as previously described  [Additio0 μg was escribed , using pAnalysis of over-represented genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) ,34 and tP value < 0.05 was considered statistically significant [Additional file EPCs number, cell size differences, SDF-1α plasma levels, fluorescence intensity of C11-BO and qRT-PCRs data were reported as mean values, and results analysed by paired Student t test. P < 0.0001 vs euploid EPCs; see Figure We established that the number of EPCs isolated from peripheral blood of young and adult (mean age 28 ± 9) DS was significantly lower than age-matched euploid individuals (P = 0.02) . Thus, we first measured by ELISA its plasma levels in peripheral blood of 30 euploid and 50 DS individuals, collected in three age subgroups . Then, c) Figure .SDF-1α encoding gene, CXCL12, and of its membrane receptor CXCR4. A significant decrease in the expression of CXCL12  and CXCR4  was observed in DS endothelial progenitors compared to euploid cells . This shift was not observed in euploid hydroperoxide-treated EPCs vs untreated, as already described [To determine oxidant activities in living cells, membrane lipid peroxidation (LP) of isolated EPCs was measured by using C11-BO, a fatty acid analogue. We cultured both DS and euploid EPCs in the presence of a fixed concentration of hydroperoxide (200 nM) , and perescribed .B. henselae, known to induce - albeit at low MOI (50) - long-term endothelial cell survival and proliferation [B. henselae [To evaluate the protective effects of feration ,38, the henselae ,38.S100B, due to HSA21 trisomy, is likely to induce ROS generation, leading to increased oxidative stress in DS [SOD1 gene has been suggested to be responsible of oxidative damage to neurons [S100B and SOD1 genes in DS and euploid isolated EPCs, showing their significant over-expression in DS derived cells , were detected in this microarray analysis. Furthermore, 52  showed an evidence of differential expression in DS EPCs compared to euploid cells. In particular, 37 genes were up- and 15 down-regulated  [Additional file IFNAR1 and IFNAR2), and oxidative stress, such as SOD1, S100B and APP - recently implicated in DS neurotoxicity from elevated expression of free radicals [Database searches based on GO classification, revealed that differentially expressed genes were mostly associated to immune response (GO:0006955) and transcription regulation GO:0045449) Figure . Particuradicals  - were h5449 FiguAlthough we observed relatively small up-regulation of chromosome 21 genes, significant changes in gene expression were not limited to HSA21 genes. Indeed, we observed a global and pronounced deregulation overall the chromosomes, possibly explained by dosage imbalance of HSA21 genes encoding transcriptional factors or gene expression modulators  approach [A less frequently explored gene characteristic for microarray analysis is the chromosomal location of the genes, especially when studying diseases caused by genome alterations. First we demonstrated, by using chi-squared association tests, that differentially expressed genes are not uniformly distributed along the chromosomes. We observed that in DS EPCs the chromosomes 21 - as expected - and 19 were enriched for differentially expressed genes compared to the other chromosomes , 2913 genes (2489 of them with single probe and 424 with multiple probes) were differentially expressed, on a total of 11327 distinct genes considered. By using DAVID and PANTHER Classification System, differentially expressed genes were categorized according to their known or hypothetical biological function, and the enrichment for specific gene pathways was evaluated. The analysis revealed that most of the deregulated genes were involved in "angiogenesis", "inflammation mediated by cytokines and chemokines", "integrins" and "interleukines" signaling pathways Figure . A partiCXCL10 and other interferon-stimulated genes - were up-regulated in DS-derived progenitors, whereas, on the opposite, pro-angiogenic genes  were dramatically down-regulated  and the down-regulation of CXCR7 receptor . The reduced number of progenitors could be associated with alterations in the cell cycle; however, we did not find any significant difference between the groups [Additional file CXCL12 and CXCR4 gene transcription in their EPCs, suggest a link with the reduced number of circulating progenitors and the angiogenesis suppression observed in DS.Growing evidences indicate that chemokines and cytokines, such as SDF-1α and its receptor CXCR4, play a crucial role in the mobilization and homing of EPCs from bone marrow ,46, alsoCXCR7, IL8 and RCAN1 genes, crucial factors involved in endothelial cells' migration, homing and angiogenesis [CASP8, which has been demonstrated to have a novel apoptosis-unrelated role in proangiogenic cells [These results are strengthened by microarray analysis, indicating that DS progenitors have a pronounced perturbation, at least at transcriptional level, in the angiogenesis and cell cycle pathways. Indeed, by using this approach we demonstrated the transcriptional deregulation of ogenesis -49. Moreic cells , althougic cells .S100B and SOD1, is likely to represent a leading cause of ROS generation and increased oxidation in DS neurons [We recently demonstrated the relevance of oxidative stress on the number and function of progenitor cells . Besides neurons ,40. Oxid neurons .S100B and SOD1 genes. These findings strengthen the hypothesis of their involvement in the susceptibility to oxidation observed in DS endothelial progenitors.Here, we have assessed, by both experimental evidences and microarray analysis, that DS progenitors are more susceptible to hydroperoxide-induced LP and significantly over-express B. henselae, responsible of vasoproliferative diseases such as bacillary angiomatosis and peliosis in immunocompromised patients [B. henselae infection at low MOI on mature endothelial cells' survival [In our study we have infected DS progenitors with a human pathogen, patients ,28, prevpatients . Particusurvival ,38.In contrast, a reduced cell number in both DS and euploid groups was observed after Bartonella infection at higher MOIs; however, detrimental changes were visible only in DS EPCs, displaying a higher number of invasomes and infected cells compared to euploid cells.The molecular basis of such Bartonella-induced detrimental effect on endothelial progenitors of DS was investigated at a transcriptional level by microarray. Significant up-regulation of the Jak/STAT pathway was observed only within infected DS progenitors, whereas, on the opposite, infected euploid cells displayed a significant down-regulation. These findings strengthen the hypothesis that transcriptional analysis of EPCs is clearly of major interest in the context of this syndrome. Indeed, the activation of ISGs, involved in the immune response against infections and in tumor surveillance , also inPathologic immune and inflammatory responses are regulated by the cross-talk between interferons and TNFα , as wellPhysiological angiogenesis plays a central role in the embryogenesis and placental development; on the other hand, pathological angiogenesis represents a critical issue in the progression of many diseases, such as solid tumor growth and retinopathy.Individuals with Down syndrome, due to decreased incidence of angiogenesis-dependent diseases, have been postulated to be a systemic anti-angiogenesis model. Indeed, they exhibit a significantly increased anti-angiogenic surveillance, possibly due to increased expression of anti-angiogenic regulators on chromosome 21 .However, it has been shown a complex regulation of gene expression not only related to gene copy number, with several genes escaping the rule of "increased transcription proportional to the gene copy number" ,59. ThisOur study shows that circulating endothelial progenitors are reduced in patients with DS, possibly correlating to the low SDF-1α plasma levels, to a reduced expression of its membrane receptor in these cells, and to their higher oxidative stress and pathogen infection susceptibility compared to euploid cells. A significant perturbation in the angiogenesis and inflammation gene pathways was also observed by microarray analysis, highlighting that gene expression analysis is a crucial issue for the study of common diseases. Endothelial dysfunction, angiogenesis' suppression and infection recurrence are hallmarks of DS, and the impairment in the number and function of circulating progenitors may account for some of their pathological features. Further studies are needed to understand possible therapeutic implications of circulating EPCs in Down syndrome.The authors declare that they have no competing interests.CV, LS, ACa, BA, PS, ACi and CN designed research. CV, LS, ACa, RCo, VM, LM, CF, MR and MP carried out experimental research. VM and BA performed Transmission electron microscopy. CV and MR performed microarray analysis and quantitative RT-PCR. CV and CAn assisted with statistical analysis. BF, MMC, BS and RCa participated in the participant recruitment. CV, LS, ACa, RCo, CAn, VM, LM, BF, AG, CF, MR, MP, BA, MMC, BS, RCa, PS, ACi and CN analyzed data. CV, LS, ACa, CAn, BA, PS, ACi and CN wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/3/40/prepubSupplementary MethodsClick here for fileTable S1: Primer pairs used for quantitative and semi-quantitative RT-PCRClick here for fileFigure S1: Impaired EPC number and function. A) Representative photomicrographs of merged double-positive Dil-Ac-LDL/Lectin cells isolated from euploid (left panel) and DS (right panel) subjects (100X magnification). B) Fluorescence micrographs of EPCs labeled for 30 min with C11-BO in euploid and DS subjects. C) EPC number expressed as percentage in the different phases of cell cycle obtained by FACS. D) Curves indicate the percentage of EPC number infected with B. henselae in euploid and DS individuals. Results are representative of five different experiments in duplicate.Click here for fileTable S2: Chromosome 21 genes differentially expressed in DS vs euploidsClick here for fileFigure S2: Distribution of differentially expressed genes along the human chromosomes (DS vs euploids). A) Bar graph showing the empirical frequency distribution of differentially expressed genes along the autosomes of DS progenitors vs euploids. Asterisks indicate the significantly deregulated chromosomes. B) Representation of the robustness of our findings shown in A. The left column shows the different user-defined fold-change. For each á value used in the analysis are shown the relative p-values. C) Bar graph showing the percent of differentially expressed genes along the DS autosomes.Click here for fileFigure S3: Positional gene mapping of differentially expressed genes (DS vs euploids). Graphic representation of positional gene enrichment (PGE) approach used to map differentially expressed genes in DS vs euploids EPCs to the exact location on the chromosome.Click here for fileB. henseale-induced gene expression in DS EPCsFigure S4: . A) Bar graph showing the top-scored deregulated gene pathways after infection in DS progenitors. Ratio indicates the percent of differentially expressed genes within the related pathway. B) Semiquantitative RT-PCR of Jak/STAT genes deregulated after B. henseale infection.Click here for fileB. henselae infectionTable S3: Differentially expressed genes after Click here for file


Caenorhabditis elegans, the fly Drosophila melanogaster, and the zebrafish Danio rerio indicate that these “lower” metazoans possess unique attributes that should help in identifying, investigating, and even validating new pharmaceutical targets for these diseases. We summarize findings in these organisms that shed light on highly conserved pathways of energy homeostasis.A pandemic of metabolic diseases , unleashed by multiple social and economic factors beyond the control of most individuals, threatens to diminish human life span for the first time in the modern era. Given the redundancy and inherent complexity of processes regulating the uptake, transport, catabolism, and synthesis of nutrients, magic bullets to target these diseases will be hard to find. Recent studies using the worm  Major shifts in human populations to urban centers, engagement in sedentary employment and leisure activities, and over-abundance of calorie-dense, processed foods have created a milieu in which ancient metabolic pathways that evolved under pressures to extract enough energy from the environment to maintain optimal reproductive and immune function and to tolerate short bouts of fasting, while avoiding excessive weight gain that would hinder escape from would-be predators, are running amuck . The conThe energy-producing, storing, and transferring reactions of life are a common thermodynamic inheritance of all organisms. The large catalog of in-born errors in human metabolism includes a series of mutations ranging in phenotype from mildly hypomorphic  to completely null  in the key enzymes of cellular energetics that are shared by metazoans . AlthougC. elegans, Drosophila, and zebrafish are proving that genetically tractable lower organisms can alter our understanding of the relationship of metabolic processes underlying obesity and its related illnesses (atherosclerotic vascular disease and type 2 diabetes mellitus). Through study of these organisms, insights relating energy homeostasis to life span, reproduction, and immune function have been made [Since it is unlikely that the social and economic pressures that have generated a global pandemic of obesity will be reversed, creative tools are required to understand the signaling pathways that govern energy homeostasis and to develop new drug targets that exploit this new knowledge. In this review, we highlight metabolic research in three metazoan organisms, and argue that these studies are not mere exercises in comparative energetics . Rather,een made –7. BelowDrosophila and C. elegans. Orthologous proteins for nearly all of the intermediates in this cascade are found in vertebrates. Multiple mutations in components of the IIS pathway result in alterations in life span [The evolutionarily central insulin/insulin-like growth factor signaling pathway (IIS) has been characterized in great detail in ife span , have diife span –7. A conife span –11.C. elegans adults, triacylglycerol (TAG) is stored in gut granules  by searching for defects in the accumulation of fluorescent dyes that label acidified, mature lysosomes specifically [glo mutants have altered neutral lipid stores, altered fertility, or changes in life span as a consequence of a defect in gut granule genesis are open questions. Likewise, the involvement of these proteins in higher metazoan neutral lipid homeostasis should be investigated.Four novel genes were identified in a genetic screen for mutations causing gut granule loss  of C. elegans gut granules. [The transcription factors C/EBP and steroid regulatory element binding protein 1 (SREBP-1) are necessary and sufficient for mammalian adipogenesis in vitro. RNAi-mediated depletion of C/EBP and SREBP-1 led to lipid depletion  for the lpd phenotype [lpd-3 gene is expressed in worm intestine and mammalian adipose tissue. A short-hairpin RNA strategy to knock down LPD-3 in 3T3-L1 preadipocytes caused decreased lipid accumulation, indicating the conserved function of this protein [A secondary screen using a library of 80 RNAi vectors known to cause larval arrest  uncovered eight more proteins whose knockdown caused the henotype . LPD-3 i protein .C. elegans, but should also prioritize human and rodent investigations. For example, PGP-2 (glo-5), an ABC family transporter identified in this screen, was subsequently found to be required for gut granule acidification [glo phenotype.In a comprehensive RNAi survey, depletion of 305 proteins led to decreased intestinal lipid stores, while depletion of 112 proteins led to increased intestinal lipid stores . Most prfication –15. LikeTubby mouse has a mutation in a gene of unknown function. Mouse Tub is expressed in the central nervous system, and there is evidence that it is a transcription factor [tub-1 mutant showed a modest decrease in gut granule lipid accumulation. Five alleles of a 3-ketoacyl-coA thiolase gene, kat-1 were identified in a screen for mutations that could suppress the tub-1 mutant phenotype [tub-1 mutant phenotype was rescued through either restoring normal KAT-1 expression in muscle and intestine, or in intestine alone. Restoring KAT-1 expression in muscle alone did not affect the increased lipid mass in the intestine. Mutations causing alterations in ciliated neurogenesis recapitulated the genetic interaction of tub-1 and kat-1, suggesting that a neurohormonal mechanism regulates intestinal lipid stores [tub-1 regulation of fat stores is not regulated by the insulin receptor ortholog daf-2, nor does it require the downstream forkhead family transcription factor daf-16. Both daf-2 and daf-16 regulate life span in addition to lipid metabolism [tub-1 can selectively modulate lipid metabolism without impacting longevity [The spontaneously obese n factor ,19, an an factor , or a ren factor . A worm henotype . KAT-1 md stores . Finallytabolism ,24, indiongevity .TUB-1 interacts with a Rab GTPase activating protein RBG-3 in ciliated neurons where TUB-1 undergoes both dendritic and ciliary transport, suggesting that TUB-1′s function in the worm is to modulate chemosensory or neurohormonal functions . Given tnhr-49, which encodes a nuclear (hormone) receptor required for induction of beta oxidation, is also upregulated. Conversely, deletion of nhr-49 results in elevated fat content and shortened life span. NHR-49 regulation of life span is dependent on SCD, but NHR-49 regulation of beta oxidation was independent of SCD [Drosophila, zebrafish, mouse, and human orthologs of NHR-49 are summarized in In response to a 1-h fast of adult worms, the abundance of 18 of 97 mRNAs encoding genes involved in glucose and lipid metabolism examined by reverse transcriptase polymerase chain reaction is modulated . Inducedt of SCD . This obt of SCD . The funDrosophila fat body is a structure that stores maternally supplied lipids, and “feeds” the developing animal during its larval stages. As these lipid stores are consumed, most of the fat body cells die [The ells die , althougells die . Adult mells die . Geneticells die .Drosophila fat mass were made over four decades ago with the female sterility(2)adipose mutant , a spontaneous mutant found to have increased fat body size and female infertility [Adp mutants are also protected from starvation-induced death owing to this increased and “available” energy storage pool [adp gene encodes a WD40/tetratricopeptide-repeat-domain protein [WDTC1 is a candidate gene for obesity worth pursuing.Many of the associations among various aspects of physiology and ertility ,36. Adp age pool . The adp protein  whose fu protein . The Adp protein . In lighDrosophila fat body development and lipid accumulation [Adipocytes are mesodermal cells generated by well characterized transcriptional networks ,41. The mulation . Treatmemulation . Specifimulation . ConversDrosophila has cell types reminiscent of vertebrate metabolic organs that regulate fat body lipid stores , a neuropeptide that functions like glucagon and β-adrenergic agonists do in vertebrates in counteracting insulin action. AKH is particularly important for mobilizing energy stores for active flight muscles ,49, whenDrosophila fat body cells have shown that the proteins coating these storage structures are similar to those in mouse adipose tissue in form and function [Drosophila PAT  family lipid droplet coat protein lipid storage droplet-2 (Lsd-2) is expressed in the ovary and the fat body [lsd-2 results in defects in oogenesis [Study of lipid droplets in function . The Drofat body ,54. Mutaogenesis . These dogenesis . Dynamicogenesis  and multogenesis .AKHR), or ablation of AKH-producing CC cells leads to increased fat body TAG stores, and to an inability to release FAs from depot in response to starvation. This defect in fat body lipolysis in animals lacking AKH signaling is manifested by their earlier death when food is withdrawn [In response to AKH, fat body TAG lipase is activated, and multiple lipid droplet proteins, Lsd-2 chief among them, show increased phosphorylation. Furthermore, lipid droplet protein phosphorylation in response to AKH requires cyclic AMP and protein kinase A, reminiscent of β-adrenergic stimulation of mammalian lipolysis . Conversithdrawn .Drosophila TAG mobilization is underscored by observations involving the brummer (bmm) mutant fly and human neutral lipid storage disease: both are caused by mutations in the gene encoding adipocyte TAG lipase  [Atgl/−− mice have massive intracellular liposis in many tissues, including the heart [bmm mutant flies, increased TAG stores, frank obesity, and an increased sensitivity to starvation  are seen. This final phenotype contrasts with that seen when adp mutant flies are fasted, indicating that expanded lipid mass does not necessarily mean increased availability of calories during fasting [bmm mutant phenotype can be antagonized by introducing an lsd-2 loss of function mutation; bmm/lsd2 double mutants have normalized TAG stores [AKHR/bmm double mutants have higher fat body TAG stores than either AKHR or bmm individual mutants, and are more sensitive to starvation than either mutant [The similarity between mammalian and  PNPLA2) ,59. Humahe heart . In bmm  fasting . The bmmG stores . Mobilizr mutant .Drosophila oenocyte functions in the capacity of ketogenic hepatocyte during a fasting response [lsd-2 decreases oenocyte lipid staining during starvation. Conversely, overexpression of Brummer lipase in fat body cells promotes oenocyte lipid accumulation during fasting. Taken together, these findings suggest that oenocyte lipid stores are derived from fat body cells, much like vertebrate hepatocytes' lipids are derived from TAGs that are mobilized from adipocytes during fasting. Finally, targeted ablation of oenocytes leads to runtiness and failure to mobilize fat body lipid stores during fasting, a process that requires the lipid ω-hydroxylase Cyp4g1 [During fasting, mobilized adipose TAG stores are sent to the liver as free FAs for partial oxidation to ketone bodies, molecules that can be used by nearly all cell types as energy sources . The Droresponse . Wherease Cyp4g1 .Drosophila and C. elegans do not , and the hepatic and intestinal protein apolipoprotein B (Apob). The packaging of lipids into beta lipoprotein particles is the task of the evolutionary progenitor of this family of proteins, microsomal triglyceride transfer protein (Mtp), which is also present in the zebrafish yolk cell layer, intestine, and liver [Mtp/−− mice, with failure to transport yolk lipids and die in utero by starvation [The machinery of lipid synthesis and transport used by humans is present and active in zebrafish . Beta lind liver . Knockdoarvation .Mtp/−− mice, including death by failure to transport neutral lipids across the yolk cell layer, an intermediate phenotype of decreased, but not absent, vascular lipid staining was not seen in Mtp MO-injected zebrafish larvae. This “intermediate” phenotype is present in +/Mtp− mice and many humans with point mutations in the orthologous MTTP gene [While knockdown of zebrafish Mtp recapitulates many of the phenotypes of severe, lethal abetalipoproteinemia seen in TTP gene . This diC. elegans, which has comprehensive RNAi libraries for transient knockdown of most genes, a large pool of validated MOs is not available for systematic knockdown of zebrafish genes. One study illustrates this limitation. Following a proteomic survey to identify cotranslationally translocated (membrane and secretory) proteins in zebrafish embryos, a tractable set (150) of the corresponding genes was selected for targeted knockdown with antisense MOs [Unlike ense MOs . KnockdoAnalogous redundancy and compensation for loss of one of three Δ9 FA desaturases occurs in worms , where igonzo mutant also demonstrates the high degree of conservation of metabolic regulatory function across animal phyla: the affected gene encodes the site 1 protease responsible for processing the ER membrane-tethered transcription factor SREBP, the master regulator of de novo cholesterol and FA synthesis [gonzo mutants are incapable of responding to low cellular cholesterol by activating SREBP, and are thus genetically “starved” of lipids. This defect is manifested by an inability to mobilize the yolk lipid stores, a phenotype that is readily apparent with conventional staining methods.The ynthesis . gonzo mfat-free mutant fails to concentrate a fluorescent FA analog in its gall bladder [fat-free mutants appeared morphologically normal , they have multiple defects in intracellular transport. Since a large-scale genetic screen in medaka has found three more mutants with defective hepatobiliary processing of the same fluorescent substrate used to identify fat-free, further insights into these processes are expected from genetic studies with fish [In addition to confirming the conservation of known genes in regulating lipid metabolism, zebrafish has been useful in identifying novel proteins involved in energy homeostasis. The  bladder ; the aff bladder . While fith fish .foie gras, whose encoded protein exhibits no obvious domains and represents a completely novel pathway to inappropriate accumulation of lipids in the liver.One obesity-related disease receiving increasing attention is non-alcoholic fatty liver disease (NAFLD). The initial step in the pathogenesis of NAFLD is the accumulation of excess neutral lipid in the liver, a condition commonly seen in overweight and insulin-resistant persons . Two mutTo address the looming public health crisis of obesity and its related illnesses, increasing attention is being turned to the three work-horse organisms of modern genetics outlined in this review. Future work with these organisms must exploit their individual strengths and avoid their limitations . In thisC. elegans, integration of acute and long-term signals among the nervous system, skeletal muscles, and intestine should emerge from studies of gut granule biogenesis, ciliated neurogenesis, and feeding behavior. These studies will parallel and inform discoveries into the mechanisms by which life span and fertility are regulated.In The accelerated pace of advances in studying the molecular genetics of energy homeostasis in fruit flies should allow investigators to ask increasingly sophisticated questions about physiology. In particular, understanding the signals emanating from the fat body to the IPCs, CC cells, and oenocytes should be informative. Further genetic dissection of the regulation of Brummer lipase, particularly through interaction with PAT proteins, should tell us more about how lipid droplets are regulated. Finally, through exploration of the form and function of the proto-pancreatic and hepatic organs, we can expect new insights into the integration of energy homeostasis and other facets of life including reproduction, life span, and immune function.Future screens of energy metabolism in zebrafish must harness the optical transparency and power of transgenesis to label individual cells with fluorescent protein to monitor real-time changes in energy state . ExaminaBroader questions of central nervous system regulation of energy homeostasis could also be addressed in zebrafish. Vertebrate hypothalamic  and pituUltimately, technical advances and more detailed characterization of the known pathways regulating lower metazoan energy metabolism should aid in identifying and characterizing novel candidate genes for human diseases such as obesity, diabetes mellitus, lipodystrophy, and non-alcoholic fatty liver disease. Conversely, the function of completely novel genes implicated in obesity  and diabhttp://www.ncbi.nlm.nih.gov/sites/gquery) accession numbers for the genes, gene products, and conditions discussed in this paper are: ACAT1/acat1, OMIM: *607809; adp, FBgn0000057; AKH, FBgn0004552; Apob, OMIM: +107730; Apoc2, AL916586/OMIM: *608083; ATGL/PNPLA2, OMIM: *609059; B0412.3, WBGene00015173; B0414.8, WBGene00015176; C/EBP, OMIM: *116897; c11orf2/formerly ANG2, *601922; CG10932, FBgn0029969; CG15087, FBgn0034380; CG4841, FBgn0032622; CG8449, FBgn0038129; CG9342, FBgn0032904; chico, FBgn0024248; C05d11.7,WBGene00015484; cyp-37B1, WBGene00009226; Cyp4g1, FBgn0010019; CYP4V2, OMIM: *608614; daf-16, WBGene00000912; daf-2, WBGene00000898; desat1, FBgn0086687; F23F1.6, WBGene00017747; fat-free, NP_001036200; foie gras, ENSDARG00000004726; foxo, FBgn0038197; FOXO/Foxo OMIM: *136533; GLO-1, NP_001024837; glo-1,WBGene00011083; GLO-4, WBGene00017195; gonzo, NP_954683; gryzun, FBgn0035416; Hh, FBgn0004644; HLH106, FBgn0015234; HNF4A/Hnf4a, OMIM: *600281; InR, FBgn0013984; INSR/Insr, OMIM: *147670; kat-1, WBGene00002183; king tubby, FBgn0015721; lightoid, FBgn0002567; LOC570606, XP_699199; lpd-2, WBGene00003059; LPD-3, WBGene00003060; Lsd-2, FBgn0030608; Mdr50, FBgn0010241; Mtp, OMIM: #200100; nhr-49, WBGene00003639; PGP-2/glo-5, WBGene00003996; Rab32, OMIM: *606281; RBG-3, WBGene00007167; sbp-1/lpd-1, WBGene00004735; scd/fat-6, WBGene00001398; SCD/Scd1–4, OMIM: *604031; slbo, FBgn0005638; SLC7A3/Slc7a3, OMIM: *300443; slif, FBgn0037203; SREBP1/SREBF1, OMIM: *184756; Tub, OMIM: *601197; tub-1, WBGene00006655; and zgc:92195, NP_001002338.The National Center for Biotechnology Information (NCBI) Entrez database (


The US Servicemen's Testicular Tumor Environmental and Endocrine Determinants (STEED) case–control study of testicular germ-cell tumours (TGCTs) enrolled participants and their mothers in 2002–2005. Hours of sports or vigorous childhood physical activity per week were ascertained for three time periods; 1st–5th grades, 6th–8th grades and 9th–12th grades. Son- and mother-reports were analysed separately and included 539 control son–mother pairs and 499 case son–mother pairs. Odds ratios and 95% confidence intervals were produced. The analysis of the sons' responses found no relationship between childhood physical activity and TGCT, while the mothers' analysis found an inverse association, which was solely due to nonseminoma. Future studies should seek to validate responses further using recorded information sources such as school records. Exposurmportant . Howevermportant .Increased levels of childhood physical activity have been reported to be protective against certain malignancies . The US Details of the US STEED Study have been published elsewhere  with the hope of stimulating memory.post hoc analysis, another question was used, which asked the son and mother to name the sports in which the son competed during 1st–12th grades. For this set of responses, the son's report was set as the ‘gold standard’ against which the mother's report was assessed. The mother's score was awarded two points for corroborating a sport named by the son, deducted half a point for failing to corroborate and deducted one point for providing a sport not specified by the son. Using the median of this score, the mothers' responses were divided into low- and high-agreement groups, both of which subsequently underwent re-analysis in an attempt to assess reporting accuracy.For a Odds ratios (ORs) and 95% confidence intervals were calculated to estimate the association of childhood physical activity with risk of TGCT using conditional logistic regression. To maximise the sample size in the analyses, unconditional logistic regression was also performed. As this involved breaking the match, risk estimates derived were first minimally adjusted, taking into account only the three matching factors. Further adjustment (in the fully adjusted model) was then made for the known TGCT risk factors: history of cryptorchidism and family history of testicular cancer. The results from all the analyses did not differ and therefore, only the fully adjusted estimates derived from the unconditional model are presented. When applicable, tests for linear trend in risk according to the medians of each quartile of a given ordered categorical variable were conducted to evaluate possible dose–response relationships. In addition, stratified analyses by tumour histology were performed to assess whether risks of seminoma and nonseminoma differed.P-values for testing the equivalence of the logarithm of the ORs between the mother's and son's reports of childhood physical activity  and a family history of testicular cancer (P=0.026). Participants were mainly White and TGCT incidence peaked at ages 20–24 years. Stratified by histology, the incidence peak for seminoma was slightly later than nonseminoma. Cryptorchidism was positively associated only with nonseminoma (P<0.001), while family history of testicular cancer was positively associated only with seminoma (P=0.001).Characteristics of study participants are shown in n=767) and controls (n=928) were included in the analysis of sons' data, regardless of whether their mother had returned a completed questionnaire, the estimates remained statistically nonsignificant. Also, although height has previously been reported to be positively associated with TGCT risk in this study (P>0.05) and was, therefore, not included in the fully adjusted model.The risk estimates derived from logistic regression analyses of sons' reports are shown in P-values for linear trend. Childhood physical activity in the highest grades (9th–12th grades) was only associated with decreased risk at the highest level of exertion .The risk estimates calculated from mothers' responses  were marP-values for linear trend also lower. None of the ORs for seminoma were significant.The observed protective effect of increased childhood physical activity was solely and consistently associated with nonseminoma, all the risk estimates being lower relative to the TGCT analysis, and the χ32=14.02, P=0.003) and 6th–8th , but not for grades 9th–12th . When stratified by histologic group the χ2 estimates became statistically nonsignificant for the differences within seminoma analyses , while the nonseminoma comparisons were statistically significant for the first two time periods .The differences between sons' and mothers' responses were more pronounced at the younger ages: the ORs were statistically significant for grades 1st–5th  or not? The mothers' results would be easier to dismiss if the effect was not so consistent across time periods and, more importantly, within one histologic subgroup. The latter observation gives credence to the results; if the protective effect on TGCT was caused by bias or chance, one may expect the effect to be equal when stratified by histology. Given parental nurturing responsibilities, it is conceivable that differential misclassification could have been stronger in mothers compared with sons, but it is both unlikely and nonsensical that the bias would be associated with nonseminoma Two previous case–control studies have found physical activity to be protective against TGCT . This stFor testicular congenital abnormalities, mother's responses were used to verify or supplement self-report data . The main limitation is the lack of validation of the responses of sons and mothers.Our analysis of mothers' data suggests that increased childhood physical activity may decrease the risk of nonseminoma, although the sons' data do not support this. Studies with validated responses against other sources of information, such as school records, are needed to confirm or refute our findings.