Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I  for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa−/− mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa−/− macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa−/− mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.Neonatal meningitis due to  Escherichia coli K1 is the most common cause of meningitis in premature infants; the mortality rate of this disease ranges from 5% to 30%. A better understanding of the pathogenesis of E. coli K1 meningitis is needed to develop new preventative strategies. We have shown that outer membrane protein A (OmpA) of E. coli K1, independent of antibody opsonization, is critical for bacterial entrance and survival within macrophages. Using a newborn mouse model, we found that depletion of macrophages renders the animals resistant to E. coli K1 induced meningitis. OmpA binds to α-chain of Fcγ-receptor I (FcγRIa) in macrophages, but does not induce expected gamma chain association and signaling. FcγRIa knockout mice are resistant to E. coli K1 infection because their macrophages express more CR3 and are thus able to kill bacteria with greater efficiency, preventing the development of high-grade bacteremia, a pre-requisite for the onset of meningitis. These novel observations demonstrate that inhibiting OmpA binding to FcγRIa is a promising therapeutic target for treatment or prevention of neonatal meningitis. Escherichia coli K1 causes meningitis in neonates, which remains a significant problem for the last few decades with case fatality rates ranging from 5 to 40% of infected neonates E. coli K1 represent critical early steps in the pathogenesis of meningitis. During initial colonization, E. coli K1 encounters several host defense mechanisms such as complement, neutrophils, and MØ on its path to the blood-brain barrier (BBB). However, very little is known about the mechanisms by which E. coli K1 finds a niche to avoid these host defenses. Our previous studies demonstrated that E. coli K1 evades complement attack by binding to the complement pathway regulator C4bp via outer membrane protein A (OmpA), which subsequently cleaves C3b and C4b complement proteins E. coli K1 to survive in the blood 3 CFU/ml of E. coli K1 is required to resist serum bactericidal activity Professional phagocytes, including neutrophils and macrophages (MØ) express a specific set of phagocytic receptors that recognize, bind to and mediate internalization of microbial pathogens E. coli K1 with these cells is poorly defined. MØ phagocytose a broad range of pathogens by recognizing pathogen-associated molecular pattern (LPS and peptidoglycans) via pattern recognition receptors (PRR), which include TLRs, the mannose receptor and the scavenger receptor E. coli K1 enters and multiplies in both human and murine MØ, either in the presence or absence of opsonization. OmpA expression is critical for these processes E. coli OmpA interacts with any cell surface proteins of MØ for entry. Numerous studies have shown that the expression of FcγRI is increased during septicemia and meningitis caused by a variety of pathogens E. coli K1 meningitis by depleting MØ from newborn mice and utilizing a FcγRIa−/− knockout mouse model. Our studies provide evidence of a role for a novel interaction between FcγRIa and OmpA in the onset of meningitis due to E. coli K1.Despite the importance of MØ in innate and adaptive immunity, the interaction of E. coli enters and survives in human and mouse MØ E. coli K1 induced meningitis, MØ were depleted in newborn mice by the administration of carrageenan 3 CFU of E. coli K1 by intranasal instillation and examined for progression of the disease as previously described 10 CFU per ml of blood by 48 h   . Furtherbacteria . These rE. coli binds and enters MØ in vitro irrespective of opsonization status of the bacteria E. coli, although entered in lower numbers but failed to survive inside MØ. This indicates that OmpA mediated entry into MØ enables OmpA+ E. coli K1 to resist the normal antimicrobial mechanisms of MØ. Therefore, to understand the nature of the macrophage surface structures that interact with E. coli K1, biotin-labeled cell surface proteins of THP-1 cells differentiated into MØ (THP-M) and RAW 264.7 cells were incubated with OmpA+ E. coli, OmpA− E. coli or a laboratory E. coli HB101. Bound proteins were then released and analyzed by western blotting with streptavidin peroxidase. A small number of proteins bound to all the bacteria from both the cells. However, OmpA+ E. coli prominently bound to the 110 and 70 kDa proteins from both THP-M and RAW 264.7 cells, whereas OmpA− E coli bound only to the 110 kDa protein  and FcγRIa . Since OmpA+ E. coli specifically bound to the 70 kDa protein in contrast to OmpA− E. coli, the blots were reprobed with an anti-FcγRI antibody, which reacted with the 70 kDa protein, suggesting that OmpA+ E. coli binds to FcγRIa. Of note, treating the bacteria with 40% pooled human serum did not alter the binding, indicating that opsonization with complement and/or with non-specific antibody did not alter bacterial interaction with macrophage surface proteins.Our previous studies have shown that OmpA+  protein . AlthougE. coli entry into MØ. OmpA+ E. coli was incubated with Fab fragments of anti-OmpA antibody  prior to addition to MØ. In other experiments, the RAW 264.7 cells were pre-treated with antibodies to FcγRI, CR3, TLR2, TLR4 or the mannose receptor prior to addition of OmpA+ E. coli. Isotype matched antibodies or anti-S-fimbria antibodies were used as controls. Both anti-OmpA and anti-FcγRI antibodies reduced the number of bound and intracellular E. coli K1 by ∼80%, whereas other antibodies showed no significant inhibition  pre-treated with C8-deficient serum , which is known to enter MØ through the CR3 receptor E. coli entry into MØ, OmpA was purified from OmpA+ E. coli and reconstituted into liposomes as previously described E. coli K1 by approximately 50%, whereas liposomes containing outer membrane proteins from OmpA− E. coli did not show such inhibition. Increasing concentrations of OmpA liposomes showed no further increase in the inhibition, indicating that the structure of OmpA in liposomes may not be optimal to that of OmpA on E. coli K1 to bind to FcγRIa.MØ pretreated with the anti-FcγRI antibody were also infected with Group B E. coli after phagocytosis by RAW 264.7 cells was examined by immunocytochemistry after differential staining. Extracellular bacteria were stained with FITC labeled secondary antibody (green) and the intracellular bacteria were stained with a TRITC labeled secondary antibody (red) after incubation with primary anti-S-fimbria antibody. As shown in E. coli bound to RAW 264.7 cells, whereas very few OmpA− E. coli bound at 30 min post-infection. Analysis of intracellular bacteria over time revealed that OmpA+ E. coli multiplied, whereas OmpA− E. coli were degraded inside the cells. Collectively, these studies suggest that the OmpA of E. coli K1 interacts with regions of FcγRIa similar to those involved in the binding of Fc and that this interaction enables the organism to enter MØ. In addition, the data suggest that other receptors that recognize pathogen-associated molecules may not play a significant role in MØ binding and entry of E. coli K1. However, entry through other receptors in the absence of OmpA-FcγRIa interaction renders the bacteria susceptible to macrophage killing.The fate of OmpA+ E. coli entry of MØ, short hairpin RNA (shRNA) sequences for murine FcγRIa and CR3 in pGeneClip Neomycin vectors were used to transfect RAW 264.7 cells. Suppression of FcγRIa and CR3 gene transcription and expression was verified by RT-PCR and flow cytometry, respectively. The respective shRNA suppressed the transcription of FcγRIa and CR3 considerably, but had no effect on GAPDH, TLR2 or TLR4 mRNA transcript levels (E. coli phagocytosed by FcγRIa-shRNA/RAW cells compared to control or CR3-shRNA/RAW cells (p<0.001 by two-tailed t test) . To further confirm that lack of FcγRIa expression rendered bacteria susceptible to macrophage killing, FcγRIa-shRNA/RAW cells infected with E. coli K1 were examined by transmission electron microscopy. Although few numbers of FcγRIa-shRNA/RAW cells engulfed E. coli K1, several of them were either degraded or in the process of degradation by 1 h post-infection and were completely killed by 8 h post-infection  . This rerocesses . Immunocnfection . In contE. coli K1 invasion, COS-1 cells were transfected with pcDNA3 plasmids containing Myc-tagged human FcγRIa (Myc-hFcγRIa), C-terminal truncated Myc-hFcγRIa which lacks the cytoplasmic tail (CT) or Myc-hFcγRII. Expression of these proteins was verified by Western blotting using the anti-Myc antibodies . The invasion of E. coli K1 into Myc-hFcγRIa-CT/COS-1 cells was significantly reduced, although the binding of bacteria to these cells was decreased by only 30% compared to Myc-hFcγRIa+/COS-1 cells. In contrast, overexpression of FcγRII did not increase E. coli binding to, or invasion of, COS-1 cells. These data suggest that FcγRIa acts as receptor for OmpA mediated entry of E. coli K1 into COS-1 cells and that the C-terminal portion is required for this invasion.The activation of FcγRI in phagocytic cells by the binding of the Fc region of IgG requires the association of FcγRIa with the IgG γ-chain tibodies  and flowtibodies . OmpA+ Eed cells . OmpA− EE. coli. The bound proteins were released and subjected to Western blotting with antibodies to Myc or FcγRI. The purified rhFcγRIa bound to OmpA+ E. coli but not to OmpA− E. coli, whereas BSA, used as a control, did not interact with the bacteria  was purified by Myc-affinity column chromatography from COS-1 cells and incubated with OmpA+ or OmpA− bacteria . rhFcγRI control . These rE. coli K1 binds to FcγRIa at the same region as the Fc-portion of IgG in the context of whole blood. Generally, specific or even non-specific IgG in circulation binds invading bacteria and thereby presents the pathogen to FcγR receptors on MØ. Therefore, it is possible that OmpA+ E. coli may be displacing IgG for binding to FcγRI. We tested this hypothesis by performing two different competitive binding experiments. First, OmpA− E. coli were coated with anti-S-fimbria antibody and added to FcγRIa+/COS-1 cells treated with cytochalasin D to prevent internalization. The cells were washed and then various quantities of OmpA+ E. coli were added and incubated for 10 min. After washing the monolayers, the number of OmpA− E. coli that remained bound to COS-1 cells were determined by plating on agar containing tetracycline (OmpA+ E. coli is sensitive to tetracycline). As shown in E. coli bound COS-1 cells in significantly greater numbers compared to unopsonized bacteria and progressively more bacteria were released from the cells as more OmpA+ E. coli were added to the wells. In contrast, OmpA− E. coli could not displace bound OmpA− E. coli. In separate experiments, peritoneal MØ were incubated with FITC-IgG2a (1 µg) for 1 h in the presence of cytochalasin D, washed and then various quantities of OmpA+ E. coli or OmpA− E. coli were added. The cells were incubated for 10 min, washed and the amount of FITC-IgG that remained bound to the MØ was determined by flow cytometry. As shown in E. coli were added, whereas addition of OmpA− E. coli had no effect. These results indicate that the interaction of E. coli K1 with FcγRIa via OmpA can displace bound IgG2a.One important question to address in these studies is how OmpA of E. coli K1 interaction with FcγRIa avoids the association of the γ-chain. Consistent with this assumption, OmpA+ E. coli interaction with MØ in the presence or absence of IgG2a opsonization induced far less γ-chain association with FcγRIa in comparison to OmpA− E. coli, as shown by immunoprecipitation studies . The newborn animals were intranasally infected with E. coli K1 and examined for disease progression. Of note, the FcγRIa−/− animals did not develop bacteremia even at a 100 fold higher infectious dose, even though E. coli K1 entered the circulation within two hours of infection (10 CFU of E. coli K1 in blood at 72 h post-infection (−/− mice did not develop meningitis even when infected with a 100-fold greater inoculum (data not shown). These mice did not show any signs of meningitis even after 7 days of infection, whereas 90% of WT mice showed positive CSF cultures by 72 h post-infection  due to iNOS activation E. coli K1, whereas this effect was completely abrogated in CR3 siRNA transfected MØ in vitro. Other investigators have also demonstrated that CR3, TLR2 and TLR4 play important roles in the phagocytic ability of MØ E. coli K1 to survive in FcγRIa−/− mice was due to altered expression of surface receptors using flow cytometry. Peritoneal MØ isolated from infected FcγRIa−/− mice exhibited increased expression of CR3 and TLR4, but lower expression of TLR2  from FcγRIa−/− mice were significantly lower compared to WT MØ . To substantiate the role of FcγRI in E. coli K1 entry, FcγRIa−/−BMDMs were transfected with hFcγRIa, FcγRIa-CT or FcγRII and then used for binding and invasion assays. As shown in coli K1 binding to FcγRIa−/−BMDM/FcγRIa and FcγRIa−/−BMDM/FcγRIa-CT increased significantly compared to FcγRIa−/−BMDMs and FcγRIa−/−BMDM/FcγRII. However, entry was limited to binding to FcγRIa−/−BMDM/FcγRIa cells only. These results suggest that FcγRIa expression is critical for E. coli K1 binding to, and entry into, MØ and that the C-terminal domain plays a significant role for the entry. FcγRIa−/−BMDM transfected with a FcγRIa construct exhibited decreased expression of TLR4 and CR3 and increased expression of FcγRI and TLR2 in comparison with FcγRIa−/−BMDM after challenge with E. coli K1 (p<0.01)  . TransfeII cells . Taken tE. coli K1 meningitis, FcγRIa−/− mice were reconstituted with FcγRIa+/+ or FcγRIa−/− MØ and then infected with E. coli K1. FcγRIa−/− mice that received FcγRIa+/+ MØ showed higher blood bacterial numbers compared with animals replenished with FcγRIa−/− MØ  treated MØ showed enhanced expression of CR3 and TLR4 and very low levels of TLR2 and FcγRI, indicating that iNOS suppression results in decreased expression of FcγRI E. coli K1 pathogenesis, FcγRI interaction with OmpA enhances the expression of TLR2, which in turn can be utilized by the bacteria as a receptor to modulate the efficiency of phagosome formation. Alternatively, E. coli K1 interaction with FcγRIa activates non-microbicidal mechanisms for the bacterial survival in MØ. Our studies have demonstrated that E. coli K1 infected MØ also exhibit increased expression of gp96, a known chaperone for TLR2 and TLR4 E. coli K1 that eventually are responsible for the control of macrophage environment. Further studies are in progress to examine these possibilities. As cytokines are known to modulate MØ microbicidal activity, it is also possible that the surface expression of TLRs and CR3 could be controlled by the circulating cytokines in E. coli K1 infection. Of note, we have demonstrated that IL-10 administration suppressed the expression of FcγRI and enhanced the expression of TLR4 and CR3, which in turn prevented the survival of E. coli K1 in MØ E. coli K1 pathogenesis is distinct from that induced by other bacterial pathogens We recently demonstrated that iNOSE. coli K1. Similarly, Qin et al demonstrated that the CY domain induces different gene expression in murine MØ compared to MØ stably transfected with CY-deleted FcγRIa E. coli K1 meningitis.Previous studies have shown that the cytoplasmic (CY) domain of FcγRIa plays an important role in phagocytosis and antigen presentation This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal care and Use Committee (IACUC) of The Saban Research Institute of Childrens Hospital Los Angeles (Permit number: A3276-01). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.E. coli E44, a rifampin-resistant mutant of E. coli K1 strain RS 218 (serotype O18:K1:H7), has been isolated from the cerebrospinal fluid of a neonate with meningitis and invades human brain microvascular endothelial cells (HBMEC) ompA gene is disrupted (designated as OmpA− E. coli) and HB101 (a laboratory E. coli strain that expresses K-12 capsular polysaccharide) are noninvasive in HBMEC streptococcus type III strain COH-1 used in these studies was provided by Dr. Craig Rubens of Seattle Children's Hospital, Seattle 315→Stop 315) (FcγRI-CT), or with human FcγRII Murine MØ cell line RAW 264.7, human macrophage like cells, THP-1 and COS-1 cells were obtained from American Type Culture Collection . COS-1 cells were stably transfected with cDNA encoding human FcγRIa, a mutant form of FcγRIa containing a stop codon after first amino acid of the cytoplasmic domain (Lys6E. coli K1 (multiplicity of infection of 10) in experimental medium  for 60°min at 37°C, whereas COS-1 cell monolayers were infected with E. coli K1 at an MOI of 100 for 1.5 h. The monolayers were washed three times with RPMI 1640°and further incubated in experimental medium containing gentamicin (100 µg/ml) for 1 h to kill extracellular bacteria. The monolayers were washed again and lysed with 0.5% Triton X-100. The intracellular bacteria were enumerated by plating on sheep blood agar. In duplicate experiments, the total cell associated bacteria were determined as described for invasion except that the gentamicin step was omitted.Confluent MØ monolayers in 24-well plates were incubated with 1×10SureSilencing shRNA plasmids to mouse FcγRIa and CR3 (CD11b) in the pGeneClip Neomycin Vector were obtained from Super Array Inc., . RAW 264.7 cells were transfected with shRNA plasmids using Lipofectamine 2000 and later selected for G418 resistant colonies.The cell surface proteins of THP-1 cells differentiated into MØ (THP-M) and RAW 264.7 cells were biotinylated by adding to 0.1 M sodium bicarbonate buffer (pH 8.0) containing 0.5 mg/ml NHS-LC-Biotin  at a final protein concentration of 2 mg/ml in tissue culture flasks. The flasks were incubated on ice for 1 h, the cells were extensively washed with ice-cold PBS and solubilized in 5% Triton X-100 in PBS. Total membranes from the cells were isolated following extensive dialysis against PBS and then were concentrated using Centricon tubes . Biotinylated proteins (2–5 µg) were incubated with various bacteria from a 5-ml overnight culture in a volume of 0.5 ml at 37°C on a rotator for 1 h. The bacteria were then centrifuged and the pellets were washed three times with PBS containing 0.1% Triton X-100. After a final wash, the bound proteins were released with Laemmli buffer in the presence of β-mercapto-ethanol and analyzed by SDS-PAGE. The separated proteins were transferred to nitrocellulose and immunoblotted with streptavidin coupled to peroxidase. The protein bands were visualized by ECL reagent .5′-TCCTTCTGGAAAATACTGACC-3′ and RP 5′ GTTTGCTGTGGTTTGAGACC-3′; TLR2 (459 bp) FP 5′-TGAGAGTGGGAAATA TGGAC-3′; RP 5′-CCTGGCTCTATAACTCTGTC-3′; TLR4 (506 bp) FP 5′- TGGAT ACGTTTCCTTATAAG-3′ and RP 5′-GAAATGGAGGCACCCCTTC- 3′; GAPDH (479 bp), FP 5′-CACAGTCCATGCCATCACTG-3′ and RP 5′- TACTCCTTGGAG GCCATGTG -3′. Negative control assays without primers were performed in parallel for every reaction. The amplified products were separated on a 1% agarose gel and were stained with ethidium bromide.Total RNA was isolated from various transfected RAW 267.4 cells with TRIZOL-LS-reagent  and quantified using a nanodrop machine. RT-PCR was performed using the following primer sequences: FcγRIa (321 bp) FP Expression of FcγRI, CR3, TLR2 and TLR4 was detected by staining with appropriate FITC-, phycoerythrin (PE)-, PE-CY5.5-, or allophycocyanin (APC)-coupled mouse monoclonal antibodies . Cells were first pre-incubated for 20 min with IgG blocking buffer to mask non-specific binding sites and then further incubated with the indicated antibodies or an isotype control antibody for 30 min at 4°C. The cells were subsequently washed three times with PBS containing 2% FBS and fixed with BD Cytofix (BD Biosciences). Cells were then analyzed by four-color flow cytometry using FACS calibur Cell Quest Pro software . Side and forward scatter parameters for which F4/480 was used as a MØ-gating marker, which formed the collection gate and at least 5000 events within this gate were collected for analysis.E. coli. In control groups, mice were treated with equal volumes of saline.Newborn C57BL/6 mice were injected intraperitoneally with (20-mg/Kg body weight) α-carrageenan  on days 1, 2 and 3 before infecting with 3 CFU of bacteria. Control mice received pyrogen free saline through the same route. Blood was collected from the tail or facial vein at designated times post-infection and plated on LB agar containing rifampicin to assess bacteremia and level of infection. CSF samples were collected aseptically under anesthesia by cisternal puncture and directly inoculated into broth containing antibiotics. Mice were perfused intracardially with 0.9 % saline to remove blood and contaminating intravascular leukocytes. Brains were aseptically removed and homogenized in sterile PBS. Bacterial counts in all tissues were determined by plating ten-fold serial dilutions on rifampicin LB agar plates. Growth of E. coli in rifampicin containing LB broth from the CSF samples was considered positive for meningitis Three-day old mice were randomly divided into various groups and infected intranasally with 10+ and CD8+ T lymphocytes were stained with rat anti-mouse-CD4 followed by goat anti-rat-PE and anti-CD8-FITC. DCs were stained with APC conjugated anti-CD11c antibody. B lymphocytes were detected by staining with anti-CD45R (B220)-FITC. Flow cytometry was performed on a FACScan instrument  and the data were analyzed with Cell Quest Software.Determination of leukocytes in livers and spleens of untreated and carrageenan treated mice was done using flow cytometry g for 20 min at 4°C. The supernatants were collected and the protein contents determined. For immunoprecipitation studies, 300–500 µg of protein was incubated with the appropriate antibody overnight at 4°C, washed and further incubated for 1 h with protein A-agarose. The immune complexes were washed four times with cell lysate buffer and the proteins bound to agarose were eluted in SDS sample buffer for further analysis by Western blotting. Portions of the cell lysates were subjected to electrophoresis on a 10% SDS-polyacrylamide electrophoresis gel. The proteins were transferred to a nitrocellulose membrane, which was then blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 h at room temperature. The blot was then incubated with the primary antibody overnight at 4°C in 5% BSA/TBST. The blot was washed with TBST and further incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Subsequently, the blot was washed four times with TBST for 1 h, developed with SuperSignal chemiluminescence reagent, and exposed to x-ray film to visualize the proteins.Total cell lysates of RAW 264.7 cells infected with bacteria for varying time periods were centrifuged at 16,000 X E. coli K1 at an MOI of 10 for varying times, washed and then fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1. All samples were washed three times in 0.1 M cacodylate buffer for 15 minutes each. The cells were then post-fixed for 20 minutes in 1% osmium tetroxide at 4°C followed by addition of EtOH (60%). Samples were dehydrated through 70, 80, 95, and 100% EtOH , then into propylene oxide , and into a 1:1 propylene oxide/Eponate, left overnight, capped, at room temperature. The propylene oxide/Eponate mixture was decanted off and replaced with 100% Eponate mixture. The samples were polymerized at 70°C for 48 h. Thin sections (∼80 nm) were cut using a diamond knife, mounted on un-coated 300 mesh copper grids and stained with 5% uranyl acetate for 20 min. Photographs were take with a transmission electron microscope (JEOL JEM 2100 LaB6) equipped with a Gatan Ultra Scan 1000 CCD camera.RAW 264.7 cells were incubated with E. coli were incubated with anti-S-fimbria antibody for 1 h on ice, washed, and then added to the COS-1 monolayers at an MOI of 100 for 1 h. OmpA− E. coli alone infected monolayers served as controls in these experiments. The monolayers were then washed to remove unbound bacteria and incubated with OmpA+ E. coli at an MOI of 10 and 100 for 10 min, washed the monolayers, and then dissolved in 150 µl of PBS containing 0.3% Triton X-100. Serial dilutions were made and plated on agar containing tetracycline (12.5 µg/ml) in which only OmpA− E. coli grow. The number of CFU was counted and determined the percent displacement by OmpA+ E. coli. In some experiments, FITC-IgG2a (1 µg) was incubated with cytochalasin-D treated peritoneal MØ while rotating the test tube at a low speed for 30 min and washed to remove unbound IgG. Various inocula of OmpA+ E. coli or OmpA− E. coli were added to the cells and incubated for 10 min, washed and the bound FITC-IgG was determined by flow cytometry.COS-1 cells were grown in 24-well tissue culture plate to confluence and then treated with 0.5 µg/ml of cytochalasin D for 30 min prior to addition of bacteria. OmpA− E. coli K1 as described above. The monolayers were then washed with PBS and fixed in 2% paraformaldehyde for 10 min at room temperature. Subsequently, anti-S-fimbria antibody (1:1000 dilution) was added to the cells and incubated for 1 h at room temperature. The cells were then washed with PBS and incubated with secondary antibodies conjugated to FITC for 30 min at room temperature. The monolayers were washed four times with PBS and incubated with excess amounts of secondary antibody coupled to horseradish peroxidase for 1 h at RT to block the external primary antibody sites. After thorough washing of the cells, the monolayers were permeabilized with 5% normal goat serum in phosphate-buffered saline containing 1% Triton X-100 (NGS/PBST) for 30 min. The cells were again incubated with anti-S-fimbria antibody for 1 h in Triton/NGS/PBST buffer, washed and further incubated with secondary antibody coupled to Cy3 for 30 min. The cells were washed again, the chambers removed, and the slides mounted in Vectashield (Vector Laboratories) anti-fade solution containing 4′, 6-diamidino-2-phenylindole. Cells were viewed using a Leica  DMRA microscope with Plan-apochromat ×40/1.25 NA and ×63/1.40 NA oil immersion objective lenses. Image acquisition was with a SkyVision-2/VDS digital CCD  camera in unbinned or 2×2-binned models into EasyFISH software, saved as 16-bit monochrome, and merged as 24-bit RGB TIFF images . The images were assembled and labeled using Adobe PhotoShop 7.0.RAW 264.7 cells were grown in eight-well chamber slides and infected with BBB permeability was quantitatively evaluated by detection of extravasated Evans blue dye 2 to allow adherence of MØ. Non-adherent cells were removed and the flasks washed three times with Hanks' solution. The adherent cells were harvested from the flasks using a rubber policeman and were resuspended in 10% FCS-RPMI 1640 medium. MØ were then positively selected using Miltenyi biotech kit and percentage purity examined by FACS analysis using F4/80 antibody, which was >97%. Viability of MØ following interaction with bacteria was assessed using an Annexin V kit . Mouse bone marrow cells were isolated from the tibias and femurs of 6- to 10-wk-old WT and FcγRI−/− mice 2 asphyxiation, femurs were harvested and bone marrow cells aseptically flushed from the marrow cavities with ice-cold PBS. Cells were collected by centrifugation and erythrocytes were lysed by resuspending in 0.15 M NH4Cl for 3–5 min. Celle were washed with PBS and resuspended in complete DMEM medium supplemented with M-CSF (10 ng ml−1) and IL-3 (10 ng ml−1), plated and allowed to differentiate into MØ. After 5–7 days in culture, adherent MØ were washed with PBS, scraped gently into suspension and counted. The purity of the MØ was determined by flow cytometry using F4/80 antibody and found to be >95%. Fresh bone marrow derived MØ (5×106 cells) were transferred by intraperitoneal injection into mice 6 h before infecting with E. coli K1.Peritoneal MØ were isolated from mice according to the method of Mittal et al −1) and nitrate reductase followed by addition of Griess reagent. The reaction mixture was incubated at room temperature for 20 min followed by addition of TCA. Samples were centrifuged, clear supernatants were collected and optical density was recorded at 550 nm. The amounts of NO produced were determined by calibrating standard curve using sodium nitrite.NO production was determined in MØ supernatants by a modified Griess method as described earlier Half of the brain was fixed in 10% buffered formalin, routinely processed and embedded in paraffin. 4–5 µm sections were cut using a Leica microtome and stained with hematoxylin and eosin (H & E). Pictures were taken using a Zeiss Axiovert Microscope connected to a JVC 3-chip color video camera and read by the pathologist in a blinded fashion.Cytokine  levels in sera from various animals were determined using Biosource ELISA kits  according to the manufacturer's instructions.t-test were applied and p value <0.05 was considered statistically significant.For statistical analysis of the data, two tailed Fischer test, Wilcoxon signed rank test and Student's Figure S1Analysis of various cell types in MØ-depleted mice. MØ were depleted in newborn mice by the administration of carrageenan as described in (3.58 MB TIF)Click here for additional data file.Figure S2E. coli K1. WT and MØ-depleted newborn mice were infected with 103 CFU of E. coli K1 by intranasal instillation, blood samples were collected at various times, and the concentrations of IL-β (A), IL-6 (B), IFN-γ (C) and IL-12 (D) determined by ELISA as described in t test.Cytokine production in MØ-depleted mice infected with (2.54 MB TIF)Click here for additional data file.Figure S3−/−mice infected with E. coli K1. (A) WT and FcγRI−/− mice at day 3 were infected with E. coli K1, blood samples collected at various times, dilutions made and plated on blood agar containing antibiotics. The levels of IL-1β (B), IL-6 (C), IL-12 (D) in the blood samples were determined by ELISA. The data represent means ± SD of three separate experiments performed in triplicate with fifteen animals in each group.Bacteremia and cytokines levels in WT and FcγRIa(1.28 MB TIF)Click here for additional data file.Figure S4−/− mice at day 3 after birth were infected with 105 CFU of GBS intranasally. Blood was collected at 24, 48 and 72 h post-infection, dilutions were made, and plated on agar. (B) CSF samples were collected aseptically by cisternal puncture and inoculated directly into LB broth, and positive CSF cultures were considered positive for the occurrence of meningitis. (C) At 72 h post-infection brains were harvested and half of the brains were homogenized in PBS, dilutions were made and plated on agar. The data represent mean ± SD of three separate experiments with four animals each group.Bacterial load and the occurrence of meningitis in newborn mice infected with Group B streptococcus. (A) WT and FcγRIa(0.71 MB TIF)Click here for additional data file.


Establishing transcriptional regulatory networks that include protein-protein and protein-DNA interactions has become a key component to better understanding many fundamental biological processes. Although a variety of techniques are available to expose protein-protein and protein-DNA interactions, unequivocally establishing whether two proteins are targeted together to the same promoter or DNA molecule poses a very challenging endeavour. Yet, the recruitment of multiple regulatory proteins simultaneously to the same promoter provides the basis for combinatorial transcriptional regulation, central to the transcriptional regulatory network of eukaryotes. The serial ChIP (sChIP) technology was developed to fill this gap in our knowledge, and we illustrate here its application in plants.Arabidopsis RNA polymerase II (RNPII) on the promoter of the constitutively expressed actin gene (At5g09810), and the trichome-expressed GLABRA3 (GL3) gene. As anticipated, our results show a strong positive correlation between H3K9ac and RNPII and a negative correlation between H3K9me2 and RNPII on the actin gene promoter. Our findings also establish a weak positive correlation between both H3K9ac and H3K9me2 and RNPII on the GL3 gene promoter, whose expression is restricted to a discrete number of cell types. We also describe mathematical tools that allow the easy interpretation of sChIP results.Here we describe a modified sChIP protocol that provides robust and quantitative information on the co-association or exclusion of DNA-binding proteins on particular promoters. As a proof of principle, we investigated the association of histone H3 protein variants with modified tails (H3K9ac and H3K9me2) with The sChIP method described here provides a reliable tool to determine whether the tethering of two proteins to the same DNA molecule is positively or negatively correlated. With the increasing need for establishing transcriptional regulatory networks, this modified sChIP method is anticipated to provide an excellent way to explore combinatorial gene regulation in eukaryotes. In both eukaryotes and prokaryotes, regulation of gene expression is an essential process for most biological functions. Therefore, transcriptional regulation has been an active field of biological research over the past couple of decades . The proEstablishing which proteins are located on any given gene promoter at a particular time will certainly contribute to understanding transcriptional regulation and the resulting gene regulatory networks. The genome-wide identification of transcription factor-DNA-interactions (TF-DNA) by high-throughput analyses such as chromatin immunoprecipitation (ChIP) coupled with the hybridization of promoter or tiling arrays (ChIP-chip) , as wellThe serial ChIP (sChIP) technique (a.k.a. re-ChIP and double ChIP) was recently developed to study the simultaneous association between two DNA-binding proteins on the same promoter, and was applied to yeast and mammalian system -15. In bAs part of the formation of the pre-initiation complex, RNPII is recruited to a region proximal to the transcription start site (TSS) of genes. Acetylated lysine 9 on histone H3 (H3K9ac) loosens the chromatin, which in turn facilitates the recruitment of TFs resulting in transcription . In contArabidopsis RNPII and H3K9ac/H3K9me2 as examples, we show that sChIP is powerful in establishing the co-association (RNPII and H3K9ac) and exclusion (RNPII and H3K9me2) between two proteins on the same gene promoter. In addition, we show that for the GL3 gene promoter, intermediate levels of RNPII and H3K9ac/H3K9me2 association are observed, consistent with the cell-specific expression pattern (trichomes) of this gene in mature green plant tissues. Thus, this modified sChIP method provides a feasible and promising method to start uncovering the combinatorial transcriptional regulation code for plants.Although sChIP has been used in a few instances to investigate whether two proteins co-localize to a particular gene promoter in yeast and animals, the technique has not been yet applied to plants. Most of these previous studies have focused on co-localization, yet exclusion of two proteins provides equally important information. Here, we describe a robust and reliable approach for sChIP in plants. By taking The standard ChIP and sChIP procedures are often associated with significant false positive and false negative results, which require extensive validation of identified interactions. In large part, this is due to variability in cross-linking and immunoprecipitation (IP), as well as by quality of the antibody used. As a first step in increasing the reliability of sChIP results, we developed a modified sChIP procedure Fig.  based on1. "o" represents the percent of a promoter bound only by one DNA-associated protein (DBD1), "p" the percent of the same promoter bound by the second DNA-associated protein (DBD2), and "q" the percent of the promoter bound by both proteins.2. We define ChIP efficiency as the percentage of the DNA that was ChIPed with a particular antibody to the same DNA bound by a particular protein in the corresponding input fraction, which can be the original chromatin for the first round of ChIP, or the DNA ChIPed by the first antibody in the second round of ChIP.3. While it is tempting to assume that the ChIP efficiency is the same for the first and second IP using the same antibody and conditions, our experiments have shown that the ChIP efficiencies are very different (not shown). Thus, the ChIP efficiency for the DBD1 antibody at the first ChIP is "m1", and for the second ChIP is "m2". The ChIP efficiency for the DBD2 antibody is "n1", and "n2" for the second ChIP.Actin (At5g09810) provides an example of a constitutively expressed gene in Arabidopsis [4. bidopsis , and priGL3 (At5g41315) provides an example of a gene that is expressed initially in the epidermis of young leaves and, as the leaf develops, becomes restricted to trichomes [5. richomes . Primersrichomes .According to the scheme provided in Fig. actin DNA in the sample can be calculated as follows:Therefore, the ratio of final product "A or B or C or D" after sChIP to the total Then,Thus,If two proteins are always present together at the same promoter molecule  Fig. , q = o =If two proteins are never present together (exclusion) at the same promoter molecule Fig. , q = 0. actin 2/7 gene (At5g09810) was used here as the target for both histone modifications and RNPII. As a trichome-specific gene, GL3 was used as non-constitutive expressed target. Using the antibodies for H3K9ac  as examples. As a constitutive expressed gene, the P = 0.45, two-sided t test), suggesting that on the promoter of actin 2/7, H3K9ac and RNPII co-exist, in agreement with the biological evidence [After quantified by qPCR, the "A", "B", "C", "D" values were calculated as the ratio of final ChIPed target DNA after sChIP to ten times of the same DNA in the corresponding original input fraction (1/10). The C/D and A/B ratios Fig.  then werevidence . Thus, iP = 0.0002, two-sided t test), indicating that on the promoter of actin 2/7, RNPII and H3K9me2 localization are inversely correlated , in agreement with the biological evidence that H3K9me2 marks transcriptional repression or silencing [To illustrate the exclusion of two proteins on the same gene, sChIP was conducted on three biological replicates using antibodies for H3K9me2  between A/B and C/D using H3K9ac and RNPII, despite some variation between the three biological replicates. These results suggest that there is some level of exclusion between H3K9ac and RNPII on the GL3 gene promoter, which is consistent with the cell type restricted expression of GL3. Consistent with this, there is also a significant difference  between A/B and C/D using H3K9me2 and RNPII, indicating that there is some exclusion between H3K9me2 and RNPII. Interestingly, we noticed that there is negative correlation on (C/D)/(A/B) ratio between sChIP using H3K9ac/RNPII and H3K9me2/RNPII, especially between the first or third and the second biological replicate  grown under continuous light at 22°C were used for sChIP experiments. The antibodies used for ChIP were as follows: αH3K9ac from Abcam (ab12178), αH3K9me2 from Abcam (ab7312), αRNPII CTD from Abcam (ab5131).Green tissues from ten days-old wild type Buffer A: 0.4 M sucrose; 10 mM Tris pH 8.0; 1 mM EDTA; 1 mM PMSF (Freshly added from a 100 mM PMSF stock immediately before use); 1% formaldehydeLysis buffer: 50 mM HEPES pH 7.5; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; 10 mM Na-butyrate; 1 mM PMSF; 1X plant proteinase inhibitor cocktail (Sigma) (Freshly added from a 100× stock immediately before use)LNDET: 0.25 M LiCl; 1% NP40; 1% sodium deoxycholate; 1 mM EDTATE buffer: 10 mM Tris-Cl, pH 7.5; 1 mM EDTA3; 0.25 mg/ml Proteinase K (Freshly added from a 10 mg/ml stock immediately before use)Elution buffer: 1% SDS; 0.1 M NaHCOSalmon sperm/protein A-agarose: from Upstate (16–157)Arabidopsis green tissue into buffer A in a 50 ml falcon tube and keep under vacuum (15–20 psi) for 20–40 min.1. Immerse ~240 mg of 2. Add 2 M glycine to a final concentration of 0.1 M and continue vacuum for 10 min.3. Wash the tissue with excess amount of distilled water and remove as much water as possible by kimwipe paper.4. Grind tissue in liquid nitrogen and resuspend in 400 ml of lysis buffer.5. Shear DNA by sonication to a range of 100–1,000 bp (~500 bp average) in an eppendorf tube. Using a Bioruptor , the sonication conditions are as follows: 30 seconds of sonication followed by 30 seconds of break at high power with 40 minutes in total.6. Centrifuge at 10,000 × g for 10 min at 4°C.7. Check the size of the DNA on a 1.5% agarose gel.8. Pre-clear supernatant with 30 μl of salmon sperm/protein A-agarose slurry for rabbit polyclonal antibody for at least 60 min with rotation at 4°C.9. After centrifugation at 3,000 rpm for 1 minute, transfer 100 μl of supernatant into three new 1.5 ml eppendorf tubes. Keep 10 μl as the input fraction and add the antibodies into three 100 μl fractions .10. Incubate overnight with rotation at 4°C.11. Add 30 μl of salmon sperm/protein A-agarose slurry and continue incubation with rotation at 4°C for at least 2 hours.12. Centrifuge at 750 × g (3000 rpm for microcentrifuge) for 1 min at 4°C.13. Washes (at 4°C)a) Add 0.5 ml of lysis buffer, invert 6 times, centrifuge at 750 × g for 1 min and discard supernatant.b) Add 0.5 ml of lysis buffer, rotate for 5 min, centrifuge at 750 × g for 1 min and discard supernatant.c) Add 0.5 ml of LNDET, invert 6 times, centrifuge at 750 × g for 1 min and discard supernatant.d) Add 0.5 ml of LNDET, rotate for 5 min, centrifuge at 750 × g for 1 min and discard supernatant.e) Add 0.5 ml of TE, invert 6 times, centrifuge at 750 × g for 1 min and discard supernatant.f) Add 0.5 ml of TE, rotate for 5 min, centrifuge at 750 × g for 1 min and discard supernatant.14. Add 40 μl of elution buffer and incubate at 65°C for 15 min.15. Centrifuge at 750 × g for 1 min and transfer supernatant to new tube.16. Repeat eluting steps. The final elution volume is 80 μl. In parallel, add 70 μl of elution buffer into 10 μl of input fraction for the 10% input control.17. Incubate all samples overnight at 65°C.18. Extract DNA by using PCR purification kit (QIAGEN). Elute in 30 μl of EB buffer .1. The immuno-complexes from the primary ChIP were resuspended, after washing, by incubating with 10 mM DTT at 37°C for 30 minutes.2. After centrifugation at 3,000 rpm, the supernatant is diluted 1:50 in the dilution buffer .3. Using another antibody Fig. , secondaactin 2/7 (At5g09810): actin 2/7-F: 5'-CATGTACTCGTTTCGCTTTCC-3'; actin 2/7-R:5'-AGCAGCAAAATCAAGCGAAC-3'. The primers for the promoter of GL3 (At5g41315): GL3-F: 5'-AAACGGCAACTGTTTCATCA-3'; GL3-R: 5'-TTCTGTTTTGTCCGGTAGCC-3'.Using SYBR-Green, qPCR experiments were conducted with the primers targeting to TSS site Fig.  of actinactin 2/7-F and actin 2/7-R were used. The PCR program was: 95°C for 5 min; 42 cycles of 95°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min; 72°C for 10 min.For ChIP-PCR, GL3: GLABRA3; TAP-tagging: tandem affinity purification-tagging.ChIP: chromatin immunoprecipitation; DBD1: DNA-associated protein 1; DBD2: DNA-associated protein 2; H3K9ac: histone H3 lysine 9 acetylation; H3K9me2: histone H3 lysine 9 di-methylation; qPCR: quantitative real-time PCR; RNPII: RNA polymerase II; sChIP: serial ChIP; TBP: TATA-binding protein; TAFs: TBP-associated factors; TF: transcription factor; The authors declare that they have no competing interests.ZX and EG designed the protocol described. ZX performed all the experiments and analyzed the data. ZX and EG co-wrote the manuscript. Both authors have read and approved the final manuscript.


We demonstrate the preparation and characterization of a simplified packed-bed microreactor using an immobilized TEMPO catalyst shown to oxidize primary and secondary alcohols via the biphasic Anelli-Montanari protocol. Oxidations occurred in high yields with great stability over time. We observed that plugs of aqueous oxidant and organic alcohol entered the reactor as plugs but merged into an emulsion on the packed-bed. The emulsion coalesced into larger plugs upon exiting the reactor, leaving the organic product separate from the aqueous by-products. Furthermore, the microreactor oxidized a wide range of alcohols and remained active in excess of 100 trials without showing any loss of catalytic activity. Microreactors have gained attention because they can help run chemical transformations more efficiently, more selectively, and with a higher degree of safety –12. AlcoOxidations that do not require transition metal catalysts are particularly appealing since there is neither leaching nor the need for catalyst regeneration . Nitroxy® Oxirane , a commercially available resin with pendant epoxide functionalities designed for enzyme immobilization. AO is readily functionalized with a range of catalysts and works well as packing material for flow chemistry . Th. Th3, 2)pecies 5 –3437. TE) iodide –45, yiel3) and organic alcohol solutions to form plugs before reaching the packed bed , an aqueous NaOCl solution , and an aqueous KBr solution (0.5 M) were prepared. The organic phase was loaded into one syringe and a mixture of NaOCl and KBr (30 μL KBr solution per mL NaOCl solution) was added to another. The syringes were placed on separate syringe pumps and the flow rates were regulated such that 1.0 equiv alcohol min−1, 1.5 equiv NaOCl min−1, and 0.10 equiv KBr min−1 were delivered to the packed bed. Various flow rates were examined to determine the optimal flow conditions for the oxidation. For data relating flow rate to residence time, see −1  afforded quantitative conversion of benzyl alcohol to benzaldehyde, indicating that efficient mixing was occurring in the column at this flow rate. Faster flow rates (200 or 400 μL min−1) could also be used to obtain higher outputs of benzaldehyde, however these reaction conditions did not provide complete conversion of starting material.Preliminary reactions were performed using benzyl alcohol as the test substrate in order to establish the optimal flow conditions . A solutUsing these optimized flow conditions for the benzyl alcohol oxidation, a number of different substrates were examined to test the generality of the AO-TEMPO packed-bed microreactor. High conversions were achieved when using both aromatic and aliphatic alcohols  analyses were performed using an Agilent 7890A GC equipped with an Agilent 7683B autosampler, a flame ionization detector (FID), and a J&W Scientific 19091J-413 column . The temperature program for GC analysis held the temperature constant at 80 °C for 1 min, heated samples from 80 to 200 °C at 20 °C/min and held at 200 °C for 1 min. Inlet and detector temperatures were set constant at 220 and 250 °C, respectively. Cyclooctane was used as an internal standard to calculate reaction conversion and yield. Gas chromatography-mass spectrometry (GC/MS) analyses were performed using a Hewlett Packard HP 6890 Series Gas Chromatograph, a Hewlett Packard HP 5973 Mass Spectrometer Detector (MSD), and a J&W Scientific DB*-5 Column . The temperature program for the analyses held the temperature constant at 50 °C for 3 min, heated samples from 50 to 80 °C at 30 °C/min, holding at 80 °C for 2 min, then heating samples from 80 to 200 °C at 17 °C/min, and holding at 200 °C for 1.94 min. The MSD temperature was held at 300 °C for 15 min.Solvents were purified by standard procedures. All other reagents were used as received, unless otherwise noted. Sodium hypochlorite solution  was purchased from Aldrich and titrated before use. ® Oxirane  was suspended in the azide solution and the reaction mixture refluxed overnight with gentle stirring. The resin was filtered using a Buchner funnel, washed with deionized H2O (2 × 50 mL), MeOH (2 × 50 mL), Et2O (1 × 25 mL), and dried under vacuum. Elemental analysis afforded a loading of 1.0 mmol N3/g resin.Sodium azide  and ammonium chloride  were dissolved in 500 mL 90:10 v/v water in methanol. AMBERZYME4, concentrated and dried under vacuum. The product was purified using column chromatography  to yield a dark orange solid . δC : 20.619, 32.199, 44.530, 55.298, 59.198, 69.905, 74.238; MS m/z 210 (M+). To obtain NMR spectra, a few drops of phenylhydrazine were added to the NMR tube to reduce the product to the corresponding hydroxylamine.Sodium hydride  was added to DMF (10 mL) and stirred at RT. 4-hydroxy-TEMPO  in DMF (10 mL) was added drop wise to the sodium hydride suspension at 0 °C and stirred until gas evolution ceased. Propargyl bromide  in DMF (10 mL) was added at 0 °C and the reaction was allowed to warm up to RT and stirred overnight. The reaction was quenched with water and the aqueous phase was extracted with ethyl acetate (3 × 50 mL) and the combined organic extracts dried over MgSO5  and CuI  were dissolved in anhydrous THF (20 mL). 2  was added to the solution and placed under a N2 atmosphere. The suspension was shaken for 3 d. The resin was washed with THF (2 × 10 mL), MeOH (1 × 10 mL), 1 M HCl (2 × 10 mL), deionized H2O (1 × 10 mL), sat. NaHCO3 (2 × 10 mL), deionized H2O (1 × 10 mL), MeOH (1 × 10 mL) and CH2Cl2 (1 × 10 mL), and dried under vacuum to yield the white AO-TEMPO resin . The loading was calculated by mass difference.File 1Experimental methods and spectral data


MuB normally promotes integration into DNA to which it is bound, hence its removal prevents use of this DNA as target. Contrary to what might be expected from a cis-immunity mechanism, strong binding of MuB was observed throughout the Mu genome. We also show that the cis-immunity mechanism is apparently functional outside Mu ends, but that the level of protection offered by this mechanism is insufficient to explain the protection seen inside Mu. Thus, both strong binding of MuB inside and poor immunity outside Mu testify to a mechanism of immunity distinct from cis-immunity, which we call 'Mu genome immunity'. MuB has the potential to coat the Mu genome and prevent auto-integration as previously observed in vitro on synthetic A/T-only DNA, where strong MuB binding occluded the entire bound region from Mu insertions. The existence of two rival immunity mechanisms within and outside the Mu genome, both employing MuB, suggests that the replicating Mu genome must be segregated into an independent chromosomal domain. We propose a model for how formation of a 'Mu domain' may be aided by specific Mu sequences and nucleoid-associated proteins, promoting polymerization of MuB on the genome to form a barrier against self-integration.We describe a new immunity mechanism that protects actively replicating/transposing Mu from self-integration. We show that this mechanism is distinct from the established  Purified Mu phage suspended in Mu buffer  was digested by pronase (0.5 mg/ml) at 37°C overnight in the presence of 20 mM EDTA (pH 8.0). After digestion, SDS was added to final concentration of 0.5% (w/v) and the mixture was incubated at 37°C for 1 hour. Mu DNA was then purified by phenol/chloroform extraction and precipitated with an equal volume of isopropanol in the presence of 0.3 M sodium acetate (pH7.0). The DNA pellet was washed twice with 75% ethanol, vacuum-dried, and dissolved in 10 mM Tris-HCl (pH 7.5).Induction of Mu prophage and purification of phage were performed as described previously . The SJG2. Reactions were incubated at 30°C for 30 minutes. Two-step strand transfer reactions were performed as follows. In the first step, cleaved Mu complexes were assembled at 30°C for 20 minutes as described above except without MuB and ATP; in the second step, strand transfer was initiated by adding equal amounts of the reaction mixture to tubes containing 10 μg/ml of various target DNAs, 2 mM ATP and 5 μg/ml MuB, and the reactions were incubated at 30°C for 20 minutes.One-step strand transfer reactions contained 30 μg/ml of mini-Mu plasmid DNA, 20 μg/ml target, 10 μg/ml HU, 7 μg/ml MuA and 5 μg/ml MuB in 20 μl of 20 mM HEPES-KOH (pH 7.6), 2 mM ATP, 140 mM NaCl and 10 mM MgClin vitro strand transfer products were used as templates in PCR reactions with a common Mu R-end primer labeled at its 5' end with fluorescent agent 6-FAM  and a second primer annealing to different sites of the Mu or E. coli genome  and analyzed using a genetic analyzer  and interpreted using the analysis software GeneMaker .Mu phage DNA or Δfis and SJG3 Δhns strains were grown to mid-log phase (OD600 approximately 0.6) in LB at 30°C, and the temperature-sensitive Mu repressor was inactivated at 42°C for 40 minutes. Cells were cross-linked by 1% (v/v) formaldehyde (37% (v/v) solution; Fisher Scientific) at room temperature for 30 minutes, and excess formaldehyde was quenched with 125 mM glycine at room temperature for 5 minutes. The cells were collected by centrifugation, washed twice with Tris-buffered saline (pH7.5), and resuspended in lysis buffer containing 50 mM Tris-HCl, pH7.5, 1 mM EDTA, 100 mM NaCl, 4 mg/ml lysozyme (Roche) and protease inhibitors (1 tablet per 10 ml buffer) (Roche). The suspension was incubated at 37°C for 30 minutes, followed by the addition of same volume of 2× IP buffer (100 mM Tris-HCl (pH7.5), 1 mM EDTA, 200 mM NaCl and 2% (v/v) TritonX-100). The lysate was sonicated  seven times for 20 seconds each in an ice bath to shear the chromatin DNA. The size of DNA resulting from ultrasonication was 300 to 1000 bp, with an average size of 500 bp. The cell debris generated was removed by centrifugation at 21,000 g for 15 minutes at 4°C, and the supernatant was used as 'input' samples for immunoprecipitation. To immunoprecipitate c-myc-MuB-DNA or MuA-DNA, 5 μg of anti-c-myc antibody  and anti-MuA serum [®; Qiagen).SJG3, SJG3 uA serum , respect2O. The samples were heated for 2 minutes at 94°C, after which they underwent 30 cycles of 15 seconds at 94°C, 15 seconds at 55°C and 30 seconds at 68°C, followed by incubation for 7 minutes at 68°C (PTC-200 MJ Research).An aliquot (1 μl) of ChIP or mock sample was added to 5 μl of 10× Expand Long Template buffer 1 (Roche), 1.5 μl of dNTP (10 mM each), 1.5 μl of each primer (10 μM), 0.75 μl of Expand Long Template Enzyme Mix (Roche), and 38.5 μl HE. coli chromosome were measured by PCR reactions using packaged Mu DNA as template and different primers pairs, all of which had a common primer annealing within the Mu right end, with the second primer annealing within the region of interest , 0.4 μl of each primer (10 μM) and 8.2 μl of double distilled H2O. The reactions were held for 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C . Three independent biological replicates were tested, and for each biological replicate three independent technical replicates were performed.Mu insertions within the Mu genome or the ®; Qiagen) and used as templates for qPCR in the following reaction: 12.5 μl SYBR mix (Qiagen), 0.75 μl of MuR (10 μM) paired with various primers (10 μM), 1 μl of template (10-5 ng/μl) and 10 μl of ddH2O. The reactions were held for 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C . The template input amount was controlled by qPCR using the primer pair pUC19f and pUC19r. The primer efficiency of Mu1 was set as 1, and the efficiencies of the other primer pairs were normalized to that of Mu1.Primer efficiencies were determined as follows: MuR was linked to pUC18f and the various primers were linked to pUC19r; the pUC primers anneal to the pUC19 plasmid and amplify a common 180 bp fragment. The PCR products were purified . The template input amount was controlled by qPCR using primer pairs within L25. The Ct differences between Mu1 and L25 represent the copy number of Mu. When the copy number of Mu at 0 minutes was set as 1, the copy number at 40 minutes was calculated to be 18. This number is an average of three technical repeats.Mu copy number was calculated as follows. Genomic DNA was isolated from SJG3 at 0, 20 and 40 minutes after prophage induction . The purified DNA were diluted to 1 ng/μl and used as template in the following qPCR reactions: 12.5 μl SYBR mix (Qiagen), 0.75 μl of primer pairs within Mu1, 1 μl of genomic DNA and 10 μl of ddHThe authors declare that they have no competing interests.JG designed the experiments, performed the research and analyzed the data. ZL assisted with the research. RMH conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Figure S1. (a) Lysis profiles of various Mu lysogens. All cultures were grown at 30°C to an OD600 nm of 0.6, shifted to 42°C for 40 minutes for phage induction, and shifted down to 37°C until lysis. 0 indicates time of temperature shift-up. Δhns strain did not lyse naturally, so lysis was induced by addition of chloroform. Phage titers from all three strains were similar to those from the parent strain MP1999, yielding around 3 to 5 × 108 pfu/ml. ChIP samples were prepared at 40 minutes for all SJG3-derived strains. (b) Western blot of MP1999 with anti-MuB antibodies at indicated times after temperature shift-up.Click here for fileFigure S2. Moving average AT-content of Mu genome. The window for average AT-content (%) calculation is 100 bp and the moving step is 20 bp. The value of AT content at a given position represents the average AT content of 100 bp DNA starting from that position. The graph was derived from data in [Click here for fileTable S1. Sequence of primers used in this study.Click here for file


The 17HSD type 1 enzyme catalyses the interconversion of the weak oestrone (E1) to the more potent oestradiol (E2), whereas 17HSD type 2 catalyses the oxidation of E2 to E1. The aim of this study was to correlate the expression of these enzymes in the tumour with the recurrence-free survival of tamoxifen-treated breast cancer patients. We used real-time reverse transcriptase PCR to investigate the mRNA expression of 17HSD types 1 and 2 in tumour samples from 230 postmenopausal patients. For the patients with oestrogen receptor (ER)-positive breast cancer, we found a statistically significant positive correlation between recurrence-free survival and expression of 17HSD type 2 (P=0.026). We examined the ratio of 17HSD types 2 and 1, and ER-positive patients with low ratios showed a significantly higher rate of recurrence than those with higher ratios (P=0.0047). ER positive patients with high expression levels of 17HSD type 1 had a significantly higher risk for late relapse (P=0.0051). The expression of 17HSD types 1 and 2 in breast cancer differs from the expression of these enzymes in normal mammary gland, and this study indicates that the expression has prognostic significance in breast cancer.The 17 In premenopausal women, the majority of oestrogen is produced in the ovaries. In postmenopausal women, local oestrogens in breast carcinoma tissue originate through two main pathways, one involving aromatase, which converts androgens to oestrogens, and the other utilising steroid sulphatase, which converts oestrone sulphate into oestrone. 17β-Hydroxysteroid dehydrogenase type 2 uses NAD+ as a cofactor and catalyses the oxidation of testosterone and oestradiol to form androstendione and oestrone, respectively , to the biologically more potent oestradiol (E2). 17in situ regulation of oestradiol production in hormone-dependent breast carcinomas -positive breast cancer is usually treated with tamoxifen, and long-term adjuvant tamoxifen is beneficial compared with treatment of shorter duration . We hypoμg−1 DNA (or ⩾0.3 fmol μg−1 DNA with EIA) were classified as positive. The present material is a subset of the patients in the region who participated in the trial, and includes the patients for whom frozen tumour samples were available after hormone receptor analysis. The characteristics of the tumours were similar to those in the complete series as regards a positive lymph node status (71 vs 74%), tumour size larger than 20 mm (69 vs 60%) and a positive ER status (78 vs 76%). The study was approved by the regional ethics committee at Linköping University.We analysed frozen tissue from excised primary breast tumours of 230 women treated in the health-care region of southeast Sweden between 1985 and 1991. The patients were participants in a randomised multicentric trial where 2 and 5 years of adjuvant postoperative tamoxifen treatment was compared for postmenopausal patients less than 75 years of age (Swedish Breast Cancer Cooperative group). The daily dose of tamoxifen was 40 mg. All patients had primary breast cancer, stage II (UICC), without distant metastasis at the time of diagnosis. The median period of follow-up was 13.9 years. Primary surgery consisted of either modified radical mastectomy or breast-conserving surgery combined with axillary lymph node dissection. Radiotherapy (50 Gy) to the breast was offered to all patients treated with breast-conserving surgery. Lymph node-positive patients were treated with radiation directed to the breast/chest wall and regional lymph nodes. After surgery, the tumour samples were stored in a freezer (−70°C) until RNA extraction was performed. Oestrogen receptor and progesterone receptor (PgR) content was measured in clinical routine practice with isoelectric focusing before 1988 and thereafter with enzyme immunoassays (EIA) . Samples with concentrations ⩾0.1 fmol Frozen breast tumour tissue (30 mg) was homogenised in a microdismembrator , and total RNA was extracted with the SV total RNA isolation system . The purified RNA was stored at −70°C, and the RNA content was determined by spectrophotometry. We also examined the expression of 17HSD types 1 and 2 in normal mammary gland. Total RNA from a pool of human breast tissue samples from 3 women (age 46–54 years) was purchased from ADH diagnostics .μl, using Gibko BRL kit  with the following concentrations: 1 × PCR buffer, 5 mM MgCl2, 0.5 mM deoxynucleotide triphosphates, 2.5 μM random hexamers, 10 mM DTT, and 0.5 μl of Superscript reverse transcriptase  The thermal conditions used were as follows: 20°C for 10 min, 42°C for 50 min, 99°C for 5 min, and after that 5°C. The samples were stored at 4°C as the real-time PCR analysis was performed during the same day.Total RNA (500 ng) was reverse-transcribed in a final volume of 20 β-HSD types 1 and 2 cDNA sequences. We conducted Blast searches (GenBank) to confirm the specificity of nucleotide sequences chosen for the primers and probes and the absence of DNA polymorphism. To avoid detection of contaminating genomic DNA, the probe was placed in the junction between two exons. The primer and probe sequences were; 17HSD type 1: forward primer: 5′-TAT GCG AGA GTC TGG CGG TT-3′, reverse primer: 5′-TGC ACT GGG CCG CAC T-3′, probe: 5′-CGA TCA GGC TCA AGT GGA CCC CAA-3′; 17HSD type 2: forward primer: 5′-TTA CCT GTG GAT CAG AAG GCA GT-3′, reverse primer: 5′-TTG CAC AAA GCA TGG CCA-3′, probe: 5′-CCC GCA ATC ACC ACC TGT CAC CA-3′. Both primers and probes were purchased from PE Applied Biosystems, as were the primers and probes for β-actin, which was used as endogenous control gene.We used the computer software Primer Express  to design primers and probes that recognised human 17The reactions were performed in the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The design of the TaqMan probes, combined with the 5′–3′nuclease activity of AmpliTaq Gold DNA polymerase (PE Applied Biosystems), allows the direct detection of the PCR product by the release of a fluorescent reporter during the PCR.μl) was added to the reaction mixture, which had a total volume of 25 μl. With the TaqMan PCR core reagent kit (PE Applied Biosystems) the concentrations used were as follows: 1 × TaqMan buffer A, 5.0 mM MgCl2, 0.1 mM deoxynucleotide triphosphates, 0.1 μM each of forward and reversed primers, 0.1 μM probe, and 0.025 units μl−1 AmpliTaq Gold DNA polymerase. The thermal conditions used were 95°C for 10 min, 95°C for 15 s and 60°C for 1 min. Steps two and three were repeated for 40 cycles. When we used the synthesised cDNA for each tumour, the 17β-HSD types 1 and 2 and β-actin specific sequences were amplified independently in separate reaction wells in triplicate. On the same plate, we included samples for standard curves for the target genes.cDNA . The criterion for statistical significance was P<0.05.The relationships between grouped variables were analysed with the β-HSD type 1 were 0.1 and 8.2, respectively. To discriminate between low/intermediate and high expression of 17HSD type 1, the material was divided into two groups according to the mean level; lower (<2.3), and higher (>2.3). 17β-Hydroxysteroid dehydrogenase type 2 mRNA was detected in 69% of the tumours. The tenth and ninetieth percentile for 17HSD type 2 were 0.0 and 0.92, respectively. The mean expression level for 17HSD type 2 was 0.28. To categorise the patients into two groups the mean value was used as cutoff. Since 17HSD types 1 and 2 cooperate to regulate the levels of E2 and E1, we also determined the ratio between these enzymes in each tumour. The majority showed low ratios as compared to normal. Using the upper tertile as cutoff level, we divided the material into groups with higher (>0.2) or lower (<0.2) ratios. The expression of 17HSD types 1 and 2 were not significantly associated with other tumour characteristics, such as lymph node status, tumour size, ER status or PgR status , whereas no association was found among ER negative patients (P=0.34) (P=0.026) (P=0.62). The prognostic significance of type 2 hold true in multivariate analysis (P=0.042). There was no significant association between 17HSD type 1 and recurrence-free survival if the entire follow-up period was considered (P=0.0051) (Oestrogen receptor-positive patients whose tumour had a high ratio (17HSD2/17HSD1 >0.2) showed a significantly better prognosis than patients with low ratios ((P=0.34) . This st(P=0.34) . A similP=0.026) . This dinsidered . However=0.0051) .vs 2 years recurrence rate ratio, RR=0.56, 95% CI, 0.29–1.06, P=0.072). This difference could not be seen among patients with high ratios (RR=0.92 (0.28–3.0), P=0.90). The same pattern was observed for 17HSD type 2 alone, that is, ER-positive patients with low expression benefited from prolonged treatment, whereas those showing higher type 2 levels appeared to have similar recurrence-free survival with 2 and 5 years of treatment  and RR=1.01 (0.28–3.6), respectively). Oestrogen receptor-positive patients with lower levels of 17HSD type 1 showed a 40% reduced risk of recurrence with prolonged treatment (RR=0.60 (0.33–1.09)), whereas a benefit from prolonged treatment was not evident for those with increased type 1 (RR=1.33 (0.52–3.4)). However, the difference between the groups was not statistically significant.Oestrogen receptor-positive patients with low 17HSD2/17HSD1 ratios tended to have the advantage of 5 instead of 2 years tamoxifen treatment , and in the present study, this was associated with a good prognosis. In contrast, a low ratio leads to increased reductive activity (E1 → E2). Irrespective of E1 production, as a result of aromatase or steroid sulphatase activity, 17HSD types 1 and 2 are responsible for the balance of E1 and E2. It has previously been shown that in normal breast tissue the oxidative pathway (E2 → E1) dominates, whereas in malignant breast tumours the reductive pathway (E1 → E2) is dominant  at this site is a frequent and early event in breast cancer , this might be an important mechanism in the pathogenesis of ER-positive breast cancer. Previous studies have shown most interest in 17HSD type 1. This study indicates that 17HSD type 2 and the 17HSD2/17HSD1 ratio may be even more important, which motivates further investigations.


Aims. To determine the progression of body weight (BW) and body composition (BC) in patients with type 2 diabetes mellitus (T2D) on insulin therapy and the consequences on muscle strength (MS) as a reflect of free fat mass increases. Research design and methods. We analysed BC using air displacement plethysmography and MS by hand grip dynamometry in 40 T2D before and after three (M3) and six months (M6) of insulin therapy. Results. at baseline HbA1c was 9.76 ±1.6% and BW was stable with fat mass (FM) 28 ± 10.7 kg; and fat free mass (FFM) 52.4 ± 11 kg; at M6, HbA1c improved to 7.56 ± 0.8%; insulin doses tended to increase. BW gain at M6 was + 3.2 ± 4.2 kg and with an increase of only 25% by M3; it was composed of FM, whereas FFM was unchanged. MS did not increase on insulin therapy. Conclusions. In T2D, BW gain was composed exclusively of FM with no improvement in MS. Insulin is a major therapeutic tool for insulin-deficient patients with type 1 diabetes mellitus, and also for patients with type 2 diabetes mellitus (T2D), with both insulin resistance and a relative insulin deficiency  studies.Analysis of body composition (BC) changes can help determine whether insulin-induced BW gain can be considered as beneficial or not, by distinguishing the accumulation of fat (FM) from that of fat-free mass (FFM). FFM is an important nutritional parameter, linked with immune competence, functional status, and survival . PreviouThe aim of our study was to assess the effects of insulin therapy on body weight and composition, and on muscular strength, in T2D with failure to oral hypoglycaemic agents.40 type 2 diabetic patients (18 men and 22 women) who presented secondary failure to oral antidiabetic agents with HbA1c > 7% after a duration of 13.5 ± 8.7 years of diabetes mellitus were recruited during their stay in our department. The only inclusion criterion was the introduction of insulin. Exclusion criteria included a reduced autonomy, which could interfere with the plethysmographic measurement, and any condition leading to water and salt retention or any severe concurrent illness.All T2D were treated with insulin injections and their oral antidiabetic agents. All patients monitored their blood glucose and were asked to adapt the insulin doses in order to obtain HbA1c close to recommended levels . They weAll patients were fully informed of the purpose of the study and gave their informed consent to take part in the investigation. The university hospital ethics committee accepted the protocol.At baseline, all patients underwent a physical and laboratory examination to rule out any undercurrent disease. When insulin therapy was indicated, the patients were invited to participate in the study and all accepted. We carried out BC analysis and MS measurements at baseline, then 3 and 6 months later. The baseline analysis was conducted just before starting insulin.HbA1c was measured on EDTA Vacutainer samples by affinity chromatography using a Hi-AUTOA1c analyzer  initially and 3 and 6 months later.BW was measured on an electronic scale to the nearest 0.1 kg and height to the nearest 0.1 cm. Waist circumference (WC) at the umbilical midline was measured to the nearest mm with flexible tape. Air displacement plethysmographic measurements were carried out using the Bod-Pod body composition system  according to the manufacturer's recommendations . The proThree consecutive measurements of handgrip strength with the dominant hand were performed with a calibrated dynamometer , which was reset to zero before each measurement. The measurements were conducted under standardized conditions: subject seated, the shoulder adducted and neutrally rotated, with the elbow at 90° flexion and the forearm and wrist in a neutral position. Patients were encouraged using a standard phraseology (squeeze the handle as hard as possible). Mean values were recorded.P < .05 was considered significant.Data are shown as means with standard deviations. SPSS software 10.0.5 was used for the calculations . A one-way analysis of variance for repeated measures over time (ANOVA) was used to compare the values at baseline (M0), 3 months (M3), and 6 months (M6). Associations were tested by linear regression. 2, with high FM 28 ± 10.7 kg, and WC: 105.7 ± 12 cm,. FFM was 52.4 ± 11 kg; and MS was 29.1 ± 8.2 kg; Glycaemic control was poor: HbA1c 9.76 ± 1.6%.The T2D were 62 ± 12 years, their BW was stable over the previous three months: BW 85.4 ± 14.7 kg: BMI 30.9 ± 5.7 kg/mHbA1C improved during the first three months, and then stabilized. M6 HbA1c: 7.56 ± 0.8%. The doses of insulin tended to increase from M0 to M6. ( P < .012(M6/M0))whereas FFM remained unchanged, BW gain was related to changes in FM  and WC , but not to those of FFM. BW gain was related to the initial HbA1c , to the decrease in HbA1c , and to the insulin doses  in T2D.BW increased on insulin treatment to 88.5 ± 14 kg, and 75% of the increase occurred after 3 months. The composition of the BW gain: during the first three months FM and FFM were unchanged, but at six months, FM increasedr = 0.72, P < .0005) and inversely correlated with FM . MS remained stable and did not increase during the six months.MS was correlated with FFM . The course and nature of the BW gain was +3.1 ± 2.2 kg at M6 and 75% of the increase occurred after M3. All the weight gain was due to FM (+3.1 ± 2.7 kg), whereas FFM was unchanged (−0.1 ± 1.6 kg) and T2D did not gain MS on insulin therapy. The BW gain at M6 was correlated with initial HbA1c and insulin doses and with the decrease in HbA1C. One limitation of our study stems from the use of a two-compartment model for the analysis of BC by air displacement plethysmography (ADP), as hydration of FFM cannot be assumed to be constant during insulin therapy. Overall our results are in agreement with previous studies. In T2D, previous studies reported that BW gain (+1.6 ± 4.9 to +5.2 ± 2.7 kg at six months) was mainly composed of fat –20. We cOur study did not aim to explain the progression of BC during insulin treatment, but several studies investigating the role of insulin on protein metabolism in diabetes mellitus have provided useful data. In type 1 diabetes, protein turnover studies using isotopically labeled amino acid tracers have demonstrated that protein breakdown and amino acid oxidation are increased in the insulin-deprived state, and are normalized by insulin, which has an anticatabolic effect . The effInsulin therapy is clearly beneficial for glycemic control in T2D but we do not feel that the gain in FM offsets this benefit. However, the excess fat is deposited essentially in the intra-abdominal area . AlthougIn summary, BW gain of TD2 on insulin therapy was 3.1 ± 2.2 kg, it was predominantly fat with no improvement in muscular strength or FFM.


The cytosolic chaperone Hsp72 directly modulates stress sensing in response to the accumulation of unfolded proteins in the endoplasmic reticulum and promotes cell survival. Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1α, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1α or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1α. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1α in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1α enhances IRE1α/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response. The endoplasmic reticulum (ER) is responsible for production and folding of secreted proteins. When the protein folding machinery cannot keep up with demand, misfolded proteins accumulate, leading to a state of ER stress that contributes to diseases such as cancer, neurodegeneration, diabetes, and myocardial infarct. The unfolded protein response (UPR) is an intracellular signaling network activated in response to ER stress. It initially tries to restore normal ER homeostasis, but if the damage is too severe cell death pathways mediated by cytosolic and mitochondrial proteins are activated. The molecular mechanisms involved in the transition of the UPR from a protective to an apoptotic phase are unclear. IRE1α is an ER membrane protein that acts as a sensor of ER stress. A number of proteins can interact with IRE1α to regulate its function, which includes an RNase activity responsible for inducing the unconventional splicing of the transcript for a downstream signaling protein called XBP-1. Here, we report that Hsp72, a stress-inducible cytosolic molecular chaperone, can bind to and enhance the RNase activity of IRE1α, providing an important molecular link between the heat shock response and the ER stress response. Importantly, increased production of active XBP-1 was necessary for Hsp72 to exert its prosurvival effect under conditions of ER stress. Our results suggest a mechanism whereby Hsp72 overexpression helps cells adapt to long-term ER stress in vivo by enhancing the pro-survival effects of the IRE1α/XBP1 branch of the UPR. To quantify ΔΨm, TMRE, a potentiometric fluorescent dye that incorporates into mitochondria in a ΔΨm-dependent manner, was used. Cells were either left untreated or treated with 0.25 µM thapsigargin. The cells were then incubated with TMRE for 30 min and analyzed by a flow cytometer. A drop in ΔΨm was observed in Neo cells following thapsigargin treatment  and MOMP is a hallmark of apoptosis reatment . The expreatment . At 48 hreatment . Howeverreatment . To furtchondria . However72 cells . These rIPKIPK, and GRP78 was significantly enhanced in Hsp72 cells as compared to Neo cells  prior to experiment. PC12 cells were treated with 200 µM 6-OHDA for 24 h before analysis. All reagents were from Sigma-Aldrich unless otherwise stated.Rat pheochromocytoma PC12 cells (obtained from ECACC) were cultured in Dulbecco's modified Eagle's medium (DMEM) from Sigma (D6429) supplemented with 10% heat inactivated horse serum, 5% foetal bovine serum, and 1% penicillin/streptomycin (Sigma) at 37°C, 5% COThe plasmid expressing wild type Hsp72, ΔATPase-Hsp72, or ΔEEVD-Hsp72 under the CMV promoter were kind gifts from Dr. Tomomi Gotoh, Kumamoto University, Japan Viability of cells after treatment was analyzed by MTT assay. After 48 h of treatment, 1 mg/ml concentration of MTT -2, 5-diphenyl tetrazonium bromide) was added to the wells and incubated at 37°C for 3 h. The reaction was stopped with a stop mix containing 20% SDS in 40% dimethyl formamide. The color intensity is measured at 550 nm and percentage cell viability is calculated using the untreated samples as 100%.2), and incubated with annexin V-FITC or with annexin V-PE for 15 min on ice. Prior to analysis 300 µl of binding buffer containing 4 µl of PI (50 µg/ml) was added and analyzed on a FACSCalibur flow cytometer (Becton Dickinson).Externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane of apoptotic cells was assessed with annexin V-FITC as described earlier −1×mg−1 total cellular protein.Cells were harvested and pelleted by centrifugation at 350 g. After washing in PBS, cell pellets were re-suspended in 50 µl of PBS and 25 µl was transferred to duplicate wells of a microtiter plate and snap-frozen in liquid nitrogen. To initiate the reaction, 50 µM of the caspase substrate carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin  in assay buffer  was added to cell lysates. Liberated free AMC was measured by a Wallac Victor 1420 Multilabel counter (Perkin Elmer Life Sciences) using 355 nm excitation and 460 nm emission wavelengths at 37°C at 60 s intervals for 25 cycles. The data were analyzed by linear regression and enzyme activity was expressed as nM of AMC released × minMitochondrial transmembrane potential was determined by using the fluorescent probe tetramethylrhodamine ethyl ester  as previously described 5-TTACGAGAGAAAACTCATGGGC-3 and reverse primer 5-GGGTCCAACTTGTCCAGAATGC-3 specific for Rat XBP1. GAPDH  was used as an endogenous control. Real-time PCR method to determine the induction of UPR target genes has been described previouslyT method.Total RNA was isolated using RNeasy kit (Qiagen) according to the manufacturer's instructions. Reverse transcription (RT) was carried out with 2 µg RNA and Oligo dT (Invitrogen) using 20 U Superscript II Reverse Transcriptase (Invitrogen). The cDNA product was subjected to 25–35 cycles of PCR using the forward primer The cells were washed in ice-cold PBS and lysed using cell lysis and mitochondrial intact buffer (CLAMI) containing 250 mM sucrose, 70 mM KCl dissolved in 1× PBS with 0.5 mM DTT and 2.5 µg/ml Pepstatin and 0.2 g/ml digitonin. The cells were allowed to swell on ice for 5 min. The cell suspension was centrifuged at 400 g for 5 min and the pellet was removed. The supernatant was transferred to a clean eppendorf tube and the mitochondrial and microsomal fractions were separated by spinning at 20,000 g for 5 min. The cytosolic fraction was removed and prepared for Western blot by adding 5× sample buffer.2, 0.1 mM EGTA, and 1% NP-40) after stipulated time of treatments and boiled at 95°C with Laemmli's SDS-PAGE sample buffer for 5 min. Protein concentration was determined by Bradford method. Equal amounts (20 µg/lane) of protein samples were run on an SDS polyacrylamide gel. The proteins were transferred onto nitrocellulose membrane and blocked with 5% milk in PBS-0.05% Tween. The membrane was incubated with the primary antibody Hsp72 (Stressgen SPA-810), Caspase-3 , Cytochrome c , XBP1 , CHOP , phosphorylated eIF2α , total eIF2α , phosphorylated JNK , IRE1α , or β-Actin  for 2 h at room temperature or overnight at 4°C. The membrane was washed 3 times with PBS-0.05% Tween and further incubated in appropriate horseradish peroxidase-conjugated secondary antibody (Pierce) for 90 min. Signals were detected using West pico chemiluminescent substrate (Pierce).Cells were washed once in ice-cold PBS and lysed in whole cell lysis buffer . Briefly, cell lysates were incubated with HA-agarose slurry in IP column overnight. Agarose beads were washed twice with TBS containing 0.05% Tween. Protein complexes were extracted by boiling the beads with 2× lane marker buffer and analyzed by Western blotting as described above. For immunoprecipitation of Hsp72, cleared protein extracts were incubated with anti-Hsp72 polyclonal antibody (Stressgen SPA-811) overnight at 4°C, followed by 100 µl of a 12% suspension of protein A-Sepharose for 1 h at 4°C, and then washed three times with TBS-0.05% Tween. Protein complexes were eluted by boiling in 2× lane marker buffer and analyzed by Western blotting as described above.The effect of Hsp72 on activity of IREα was monitored using recombinant human IRE1αΔN-HIS produced as GST fusion protein using the Prescission Protease cleaved system. IRE1α ΔN was incubated with recombinant Hsp70 (Stressgene) in a total volume of 50 µl for 1 h at 30°C with 10 µg of total mRNA as substrate  in a buffer containing 20 mM HEPES (pH 7.3), 1 mM DTT, 10 mM magnesium acetate, 50 mM potassium acetate, and 2 mM ATP. Then, mRNA was re-extracted with 500 µl of Trizol, and the endoribonuclease activity of IRE1α was monitored by RT-PCR using the XBP1 mRNA splicing assay that employs a set of primers that closely surround the processing site. Using this method, we observed a decrease in the amount of nonspliced XBP1 mRNA due to its cleavage by IRE1αN-HIS as we previously described We generated stable subclones of PC12-Hsp72 with reduced levels of XBP1 by targeting XBP1 mRNA with shRNA using the lentiviral expression vector psiHIV-U6 (GeneCopoeia). The targeting sequences identified for rat XBP1 were XBP1 shRNA1: 5-actgcgcgagatagaaaga-3; XBP1 shRNA2: 5- gttgcctcttcagattctg-3; XBP1 shRNA3: 5-gagagccaaactaatgtgg-3; and XBP1 shRNA4: 5-ctgaggtcttcaaaggtat-3.Cells were seeded in T25 flasks 24 h prior to heat preconditioning. The flasks were sealed with parafilm and immersed in water bath set at 42°C for 1 h. The cells were left to recover for 6 h at 37°C before treating with apoptosis inducing agents. Media was changed prior to treatment.Cells were treated with 0.1 µM Tg or 0.5 µg/ml Tm or 50 µM 6-OHDA for 24 h to induce UPR. Culture media was analyzed for NGF or BDNF release using β-NGF (DY 256) or BDNF (DY 248) DuoSet ELISA development kit according to manufacturer's protocol (R&D Systems). The amount of NGF or BDNF released into the media was calculated using the standard curve generated in parallel with recombinant NGF and BDNF.t test using Graphpad (http://www.graphpad.com).All the experiments were repeated at least 2 times. Results are expressed as mean ± standard deviation. Statistical analyses of the results were done with Student's Figure S1Overexpression of Hsp72 protects PC12 cells from 6-OHDA treatment induced cell death. The control (Neo) and Hsp72 expressing (Hsp72) PC12 cells were either untreated (Un) or treated with 200 mM 6-OHDA for 24 h. Reduction in cell viability was analyzed by sub G1 peak. Average and error bars represent mean ± SD from three independent experiments performed in triplicates.(0.06 MB TIF)Click here for additional data file.Figure S2Subcellular localization of IRE1α is not affected by Hsp72. Hsp72 and Neo expressing cells were transfected with IRE1α-GFP. After 24 h of treatment with Tg, cells were fixed in 3.7% formaldehyde and the coverslips were mounted using Vectashield mounting medium with DAPI (H-1200). Cells were visualized using a Nikon microscope fitted with appropriate filters.(0.56 MB TIF)Click here for additional data file.Figure S3Overexpression of Hsp72 does not affect the half-life of IRE1. (A) Control (Neo) and Hsp72 expressing (Hsp72) PC12 cells were treated with (10 ng/ml) Tg for the indicated time. Immunoblotting of total protein was performed using antibodies against IRE1α and β-actin. (B) In the experiment described in (A), band density of IRE1α was calculated by densitometry and normalized against β-actin. Average and error bars represent mean ± SD from two independent experiments.(0.17 MB TIF)Click here for additional data file.