ATM (ataxia-telangiectasia mutated) gene plays a central role in the DNA-damage response pathway. We characterized the ATM protein expression in immortalized cells from AT and AT-variant patients, and heterozygotes and correlated it with two ATM-dependent radiation responses, G1 checkpoint arrest and p53-Ser 15 phosphorylation. On Western blots, the full-length ATM protein was detected in eight of 18 AT cases, albeit at 1–32% of the normal levels, whereas a truncated ATM protein was detected in a single case, despite the prevalence among cases of truncation mutations. Of two ataxia without telangiectasia [A-(T)] cases, one expressed 20% and the other ~70% of the normal ATM levels. Noteworthy, among ten asymptomatic heterozygous carriers for AT, normal amounts of ATM protein were found in one and reduced by 40–50% in the remaining cases. The radiation-induced phosphorylation of p53 protein at serine 15, largely mediated by ATM kinase, was defective in AT, A(-T) and in 2/4 heterozygous carriers, while the G1 cell cycle checkpoint was disrupted in all AT and A(-T) cases, and in 3/10 AT heterozygotes. Altogether, our study shows that AT and A(-T) cases bearing truncation mutations of the ATM gene can produce modest amounts of full-length (and only rarely truncated) ATM protein. However, this limited expression of ATM protein provides no benefit regarding the ATM-dependent responses related to G1 arrest and p53-ser15 phosphorylation. Our study additionally shows that the majority of AT heterozygotes express almost halved levels of ATM protein, sufficient in most cases to normally regulate the ATM-dependent DNA damage-response pathway. © 2000 Cancer Research Campaign


Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1–226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005–2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in  Aedes species mosquitoes, primarily Ae. aegypti. However, the 2005–2006 CHIKV epidemic on Reunion island was unusual because the vector responsible for transmission between humans was apparently the Asian tiger mosquito, Ae. albopictus. Interestingly, the same epidemic was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). In this work we investigated the role of the E1-A226V mutation on the fitness of CHIKV in Ae. aegypti and Ae. albopictus mosquitoes. We found that E1-A226V is directly responsible for CHIKV adaptation to Ae. albopictus mosquitoes, which provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This research gives a new insight into how a simple genetic change in a human pathogen can increase its host range and therefore its geographic distribution. Ae. albopictus is abundant and widely distributed in urban areas of Europe and the United States of America, and this work suggests that these areas are now vulnerable to CHIKV establishment.Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics of arthritic disease, including one on Reunion island, where there were approximately 266,000 cases . CHIKV is transmitted by  The large-scale epidemic of the mosquito-transmitted alphavirus, Chikungunya virus (CHIKV), began in Kenya in 2004 and spread to several Indian Ocean islands including the Comoros, Mauritius, the Seychelles, Madagascar, Mayotte and Reunion. On Reunion island alone there were approximately 266,000 cases  –6. In thAedes species mosquitoes, primarily Ae. aegypti. However, the 2005–2006 CHIKV epidemic on Reunion island was unusual because the vector responsible for transmission between humans was apparently the Asian tiger mosquito, Ae. albopictus [Ae. albopictus during the epidemic, CHIKV RNA was detected . Furthermore, the species is anthropophylic, was abundant during the epidemic, and other potential vectors specifically Ae. aegypti were relatively scarce with a very limited distribution . Ae. albopictus is abundant and widely distributed in urban areas of Europe and the United States of America [Ae. albopictus transmission cycle was reported for the first time in Europe, with an estimated 254 human cases occurring in Italy [CHIKV is transmitted by bopictus ,16. This America –22. CHIK America ,23–26 ca America ,28. In Ain Italy ,30.Alphaviruses are enveloped single stranded positive sense RNA viruses. Genomic RNA, of ≈ 12,000 nt, encodes four non-structural (ns1–4) and three main structural proteins . At neutral pH, E2 and E1 exist as heterodimers in which E2 forms spikes on the virion surface that interact with cellular receptors. The E1 protein lies below E2 and mediates fusion of the viral and cellular membranes during viral entry .Ae. albopictus mosquitoes [Ae. albopictus. It is unknown if dependence on cholesterol for growth in mosquito cells is a requirement of all alphaviruses.Analysis of CHIKV genome microevolution during the 2005–2006 Indian Ocean epidemic identified an alanine to valine mutation at position 226 in the E1 envelope glycoprotein (E1-A226V) among viral isolates obtained during the outbreak . The reasquitoes . HoweverAe. albopictus and Ae. aegypti to suckling mice. We also analyzed the effect of the E1-A226V mutation on CHIKV cholesterol dependence for growth in mosquito C6/36  cells. Here we report findings that a single nucleotide change, which arose during the epidemic, significantly increases fitness of the virus for Ae. albopictus mosquitoes and was associated with CHIKV dependence on cholesterol in the mosquito cell membrane. This change likely enhanced CHIKV transmission by an atypical vector and contributed to the maintenance and scale of the epidemic.To test the hypothesis that the E1-A226V mutation might influence the fitness of CHIKV in mosquito vectors, we compared the effect of this mutation on CHIKV mosquito infectivity, the ability to disseminate into heads and salivary glands, and the relative fitness in competition assays for transmission by Ae. albopictus mosquitoes, CHIKV infectious clones derived from an epidemic Reunion island human isolate were used [To test the hypothesis that the E1-A226V mutation altered CHIKV infectivity for ere used , includiAe. albopictus mosquitoes orally exposed to serial 10-fold dilutions of CHIKV (LR-GFP-226 V or A). To determine whether infection rates correlate with blood meal titer, midguts dissected from mosquitoes at 7 days post-infection (dpi) were analyzed for foci of eGFP-expressing cells by fluorescence microscopy . To test if the infectivity phenotype was directly linked to the mutation, the complementary reverse mutation, E1-A226V, was introduced into an infectious clone of a West African CHIKV strain, 37997-GFP (37997-GFP-226A)  value for the 37997-GFP-226V virus (p<0.01) to an extent similar to that observed for LR-GFP-226V virus (50 of the 37997-GFP virus (p<0.01) in Ae. albopictus mosquitoes equivalent to that observed for the LR-GFP-226V virus  . The ReuFP-226A) C and S2D6V virus . These d6V virus .Ae. albopictus, viral competition experiments were performed. Although our CHIKV eGFP-expressing infectious clones, have similar infection properties in mosquitoes as wild-type viruses [50 values, we constructed LR-226A and LR-ApaI-226V viruses without eGFP and employed them in viral competition experiments ) was mixed with an equal amount of LR-226A virus. LR-ApaI-226V and LR-226A viruses are indistinguishable in cell culture experiments . Similarly, in three of four replicates of competition experiments, RNA from LR-ApaI-226V virus was dramatically more abundant in the heads of Ae. albopictus mosquitoes as compared to RNA from LR-226A  higher than of the LR-226A titer  of CHIKV E1-A226V mutants for Ae. albopictus means that the human viremic thresholds required for Ae. albopictus infection would likely occur earlier and be sustained for longer. Several recent studies indicate that during the course of human viremia, which last up to 6 days, CHIKV loads can reach up to 3.3x109 RNA copies per ml of the blood [10TCID50/ml [10SMICLD50/0.02 ml [10/ml based on Vero cell titration [10TCID50/ml. From these data we calculate that the maximum virus load which can be achieved in human blood is 1–2 Log10TCID50/ml higher than the Log10OID50/ml for E1-226A viruses but 3–4 Log10TCID50/ml higher than the Log10OID50/ml for E1-226V viruses. During the course of viremia there should therefore be a substantial time frame in which CHIKV blood load is high enough for E1-226V viruses to infect Ae. albopictus but below the threshold for infection with E1-226A viruses. This increased opportunity for Ae. albopictus infection, would perpetuate the selection and transmission of the mutant virus.Interestingly, our data and data from previous studies ,37 indiche blood ,39, whic/0.02 ml . Based o/0.02 ml  and a maitration , we beliAe. albopictus, although in these experiments, titers of E1-226V and E1-226A viruses were of a high enough magnitude to allow both of these viruses to efficiently infect this mosquitoes species. This indicates that there are additional mechanisms that could ensure evolutional success of the E1-A226V viruses transmitted by Ae. albopictus. It is possible that one of these mechanisms is associated with more efficient dissemination of the E1-226V as compared with E1-226A viruses. This could shorten the extrinsic incubation period (EIP)—the time from mosquito infection to transmission—and could have contributed to the evolutionary success of CHIKV during the Reunion outbreak because vectors infected with the LR-226V virus would transmit it more quickly than those infected with LR-226A viruses. Additionally, with relatively short-lived vectors such as mosquitoes [During transmission competition assays, only E1-226V virus was transmitted to suckling mice by squitoes , longer Ae. albopictus mosquitoes.Our current studies do not provide data to determine if dissemination efficiency of the E1-226V viruses into the salivary glands is a consequence of more efficient midgut infectivity or if these two phenomena are independent. In this regard, it will be of particular interest to investigate the effect of the E1-A226V mutation on CHIKV transmission by orally or intrathoracically infected Ae. albopictus, there was not a corresponding advantage in Ae. aegypti. The OID50 and midgut competition assay data indicate that E1-226V viruses were slightly less infectious for midgut cells of Ae. aegypti mosquitoes  differences associated with the E1-A226V mutation would not be sufficient to have a significant effect on the evolution of CHIKV transmitted by Ae. aegypti and would not result in accumulation of this mutation in the regions where Ae. aegypti serves as a primary vector for CHIKV. This may explain the lack of emergence of the E1-226V genotype in previous outbreaks and the predominance of E1-226A viruses during the 2006 CHIKV epidemic in India, in which Ae. aegypti is considered to be the main vector species [Ae. aegypti has never been shown to be associated with any particular mutations, therefore we believe that the same negative impact of E1-A226V would be seen in African mosquito vectors which were responsible for transmission of CHIKV strains ancestral to Reunion isolates.The E1-A226V mutation was found to have a slightly negative effect on infectivity, a negligible effect on dissemination, but a slight positive effect on transmissibility of CHIKV by  species . AdaptatAe. aegypti. Since our dissemination and transmission studies were performed using blood meal titers that were 1–2 Log10TCID50/ml higher than Log10OID50/ml values we suggest that the negative effect of decreased midgut infectivity of E1-A226V on virus transmissibility would be almost completely missed, simply because, under this condition, almost 100% of mosquitoes could become infected. In general, CHIKV requires significantly higher blood meal titers for infection of Ae. aegypti compared to Ae. albopictus [Ae. aegypti, compared to the effect of a small advantage in the ability to compete with E1-226A viruses for transmission to suckling mice. Therefore, if the E1-A226V mutation occurred in CHIKV transmitted by Ae. aegypti, it would have a weak negative effect on viral fitness and would most likely not be preferentially selected. Additional experiments are required to evaluate this hypothesis.Our data does not exclude the possibility that the E1-A226V mutation might have a negative effect on the evolution of CHIKV transmitted by bopictus ,37  and GFP-expressing full-length clone LR-GFP-226V  have been previously described [ApaI restriction site into the coding sequence of CHIK-LR ic. The resultant plasmid was designated LR-ApaI-226V. The E1-V226A mutation was introduced into CHIK-LR ic and LR-GFP-226V to generate plasmids designated as LR-226A and LR-GFP-226A, respectively. The mutation E1-A226V was also introduced into plasmids 37997–226A and 37997-GFP-226A. The resulted plasmids were designated 37997–226V and 37997-GFP-226V.The viruses and plasmids encoding full-length infectious clones of the LR2006 OPY1 strain CHIK-LR ic  following the manufacturer's instructions. The yield and integrity of synthesized RNA were analyzed by agarose gel electrophoresis in the presence of 0.25 μg/ml of ethidium bromide. RNA (10 μg) was transfected into 1x107 BHK-21 cells by electroporation as previously described [2 tissue culture flasks with 10 ml of Leibovitz L-15 (L-15) medium, and supernatants were collected at 24 and 48 h post-electroporation and stored at −80 °C. In parallel, 1x105 electroporated BHK-21 cells were serially 10-fold diluted and seeded in six-well plates for infectious centers assay as previously described [All plasmids were purified by centrifugation in CsCl gradients, linearized with escribed . Cells wAe. albopictus) were grown in the same medium at 28 °C. Ae. aegypti (white-eyed Higgs variant of the Rexville D strain) and Ae. albopictus  were reared at 27 °C and 80% relative humidity under a 16h light: 8h dark photoperiod, as previously described [BHK-21 (baby hamster kidney) cells were maintained at 37 °C in L-15 medium supplemented with 10% fetal bovine serum (FBS), 100 U penicillin, and 100 μg/ml streptomycin. C6/36 cells  for 12 h at room temperature as previously described . CHIKV g10TCID50) as previously described [Viral titers from mosquito samples and from tissue culture supernatant were determined using Vero cells and expressed as tissue culture infectious dose 50 percent endpoint titers  were soeviously , or used50), serial 10-fold dilutions of viruses were made in L-15 medium followed by mixing the samples with defibrinated sheep blood. Mosquitoes were dissected at 7 dpi and eGFP expression in infected midguts was analyzed by fluorescence microscopy. A mosquito was considered infected if at least one foci of eGFP-expressing cells was present in the midgut. The experiments were performed twice for each virus. OID50 values and confidence intervals were calculated using PriProbit (version 1.63).To estimate the Oral Infectious Dose 50% values /ml of LR-Apa-226V and 107 pfu/ml of LR-226A viruses. It had been previously found that for these two viruses the ratio of viral RNAs corresponds to the ratio of viral titers (data not shown). Midguts were collected at 7 dpi and analyzed in pools of eight to ten, and heads were collected at 12 dpi and analyzed in pools of five. RNA was extracted from the tissue pools using TRIzol reagent (Invitrogen) followed by additional purification using a Viral RNA mini kit (QIAGEN). RNAs from blood meal samples were extracted using Viral RNA Mini Kit followed by treatment with DNAse (Ambion) to destroy any residual plasmid DNA contaminant in the viral samples. RNA was reversed transcribed from random hexamer primers using Superscript III (Invitrogen) according to the manufacturer's instructions. cDNA was amplified from 41855ns-F5 (5′- ATATCTAGACATGGTGGAC) and 41855ns-R1 (5′-TATCAAAGGAGGCTATGTC) primers using Taq DNA polymerase (New England Biolabs). PCR products were purified using Zymo clean columns (Zymo Research) and were quantified by spectrophotometry. Equal amount of PCR products were digested with ApaI, separated in 2% agarose gels that were stained using ethidium bromide. Thus the LR-Apa-226V and LR-226A viruses could be distinguished by size on an agarose gel . Feeding continued until 2–3 mosquitoes per carton were fully engorged (stage ≥3+[stage ≥3+). In a pFigure S1(917 KB PDF)Click here for additional data file.Figure S210TCID50/ml.Confluent monolayers of BHK-21 and C6/36 cells in T25 tissue culture flacks were infected with LR-GFP-226V and LR-GFP-226A  or 37997-GFP-226A and 37997-GFP-226V viruses derived from electroporation at a MOI of 0.1. At the indicated times post-infection, 0.5 ml of medium was removed and stored at −80 °C for later titration on Vero cells. Viral titers are expressed as Log(372 KB PDF)Click here for additional data file.Figure S310TCID50/ml ± standard deviation of three independent experiments.Confluent monolayers of BHK-21 and C6/36 cells in T25 tissue culture flacks were infected with LR-GFP-226V and LR-GFP-226A  or 37997-GFP-226A and 37997-GFP-226V viruses derived from electroporation at a MOI of 1.0. At the indicated times post-infection, 0.5 ml of medium was removed and stored at −80 °C until titrated on Vero cells. Viral titers are expressed as Loghpi - hours post-infection.(177 KB PDF)Click here for additional data file.Figure S4Cells were infected with a 1:1 mixture of both viruses at a MOI of 0.001. 2 dpi, cell culture supernatant was collected and samples proceeded as described. The experiment was repeated three times for each of the cell types.inoc - initial ratio of CHIK-LR ic and LR-ApaI-226V in the inoculum used for infection of cells.1) and C6/36 cells (RF2), divided by the control ratio between CHIK-LR ic and LR-ApaI-226V in the inoculum.Relative fitness (RF) of CHIK-LR ic and LR-ApaI-226V was calculated as an average ratio between CHIK-LR ic and LR-ApaI-226V bands in the supernatant obtained from BHK-21 cells (RF(3.6 MB PDF)Click here for additional data file.Table S1a - amino acids at position of E1–226.in vitro transcribed RNA. 107 BHK-21 cells were transfected with 10 μg of RNA. Electroporated BHK-21 cells were 10-fold serially diluted, seeded in 6-well tissue culture plates containing 5x105 naive BHK-21 cells per well and covered with 0.5% agarose in L-15. Plaques were scored on day 2 post-transfection.b - Specific infectivity of 10TCID50/ml.c - Supernatants of electroporated BHK-21 cells were collected on days 1 and 2. Virus titers were determined by titration on Vero cells and expressed as Loghpi - hours post-infection.(34 KB DOC)Click here for additional data file.Table S2Ae. aegypti mosquitoes were orally presented with 7.24±0.4 Log10TCID50/ml of CHIKV-LR ic (summary of two experiments) and 6.52 Log10TCID50/ml of LR-ApaI-226V.Ae. albopictus mosquitoes were orally presented with 7.24±0.4 Log10TCID50/ml of CHIKV-LR ic (summary of two experiments) and 7.52 Log10TCID50/ml LR-ApaI-226V.At 7 and 14 dpi, mosquitoes were collected and triturated in 1mL of L-15 medium for titration on Vero cells.10TCID50/ml ± standard deviation.Titers are reported as Log(31 KB DOC)Click here for additional data file.


One determinant of the total K+ myometrial smooth muscle cell (MSMC) current is the large conductance, calcium- and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+, and blocking the channel by pharmacological means induces the depolarization of MSMCs and also enhances contraction strength. In MSMCs, maxi-K channels can reside in the caveolae, where they associate with the scaffolding protein caveolin-1 (cav-1). The aim of this study was to investigate the consequences of this interaction - more specifically, how disruption of the association between the maxi-K channel and cav-1 may influence the current expression and excitability of myometrial cells - with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.Myometrial biopsies were collected from women undergoing elective C-sections. From these samples, myometrial cells were isolated, cultured, infected with a virus containing either caveolin-1 (cav-1) siRNA or scrambled cav-1 siRNA, and finally subjected to patch-clamp analysis. Mutant caveolin-binding site maxi-K channel constructs were generated and transfected into mouse Ltk- fibroblasts. Channel activity, expression, association, and localization were examined by patch-clamping, Western blot, immunoprecipitation, and immunofluorescence, respectively.The caveolin-1 siRNA suppressed the total K+ current in human myometrial smooth muscle cells (hMSMC), as evident from comparison to the currents generated by both non-infected cells and cells infected with scrambled siRNA controls. The interaction between the maxi-K channel and caveolin depends on a region in the channel's C-terminal caveolin-binding site. Mutations of aromatic residues in this site  resulted in a decrease in K+ current compared to that produced by wild-type channels transfected into mouse Ltk- fibroblasts. However, mutation of all three aromatic amino acids  was necessary to disrupt the association between caveolin and the maxi-K channel, as visualized by immunofluorescence and immunoprecipitation.Our results suggest that disruption of the caveolin-binding site interferes with the cav-1/maxi-K channel interaction, and that lack of the cav-1/maxi-K channel interaction in MSMCs attenuates the total K+ channel current of the cell. One determinant of the total K+ MSMC current is the large conductance, calcium- and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+[Potassium efflux from myometrial cells results in membrane repolarization. This potassium efflux constitutes the primary ionic current responsible for maintaining resting membrane potential, and contributes significantly to uterine quiescence during pregnancy. In myometrial smooth muscle cells (MSMCs), changes in the expression or activity of Kular Ca2+, and bloular Ca2+. Also, bular Ca2+ and postular Ca2+. Adding ular Ca2+-8.+ (Kv) channel, different isoforms are normally present in distinct raft domains, with Kv1.5 present in caveolae and Kv2.1 present in non-caveolar lipid rafts [Localization to caveolae and lipid rafts has been implicated as a regulatory mechanism for a number of ion channels. For example, isoform 4 of the cyclic nucleotide-gated channel (HCN4) has been shown to localize to lipid rafts, and disruption of this association following the application of methyl-beta-cyclodextrin results in both channel redistribution within the membrane and changes in channel kinetics . In the id rafts ,11. It hid rafts .Caveolar invaginations are prevalent in human MSMCs (hMSMCs), increasing the geometric cell surface area by as much as ~ 70% . Maxi-K In spite of our knowledge of the existence of an association between the maxi-K channel and caveolin, how this interaction affects MSMC function remains unknown. In this study we investigate the consequences of this interaction--more specifically, how the association between the maxi-K channel and cav-1 influences the current expression and excitability of myometrial cells--with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.Constructs encoding mutant maxi-K channel forms were generated using the QuikChange Site-Directed Mutagenesis Kit . Primers used are as follows:a) Mutant F1012A,Forward 5'-GGTATAATATGCTTTGTGCTGGAATTTACCGGCTGAGAGATGCG-3', Reverse 5' CGCATCTCTCAGCCGGTAAATTCCAGCACAAAGCATATTATAGG-3';b) Mutant Y1007, F1012A,Forward 5'-GCA AAGCTCTGAAAACAGCTAATATGCTTTGTGCTGG-3',Reverse 5'-CCAGCACAAAGCATATTAGCTGTTTTCAGAGCTTTGC-3'; andc) Mutant Y1007A, F1015A, Y1015A,Forward 5'-CTTTGTGCTGGAATTGCCCGGCTGAGAGATGCGCACCTC-3',Reverse 5'-GAGGTGCGCATCTCTCAGCCGGGCAATTCCAGCACAAAG-3'.For both patch-clamping and immunocytochemistry experiments, mutants were cloned into the pTracer expression vector,  between restriction sites Not1 and Xba1.Human myometrial tissue from the lower uterine segment was obtained from patients who, in the absence of spontaneous or induced labor contractions, underwent elective Cesarean section in late pregnancy  while under spinal anesthesia. All patients signed written consent forms approved by the University of Iowa's Internal Review Board . Tissue was placed in Hanks' balanced salt solution or in phosphate-buffered saline (PBS) on ice, and was used to isolate hMSMCs within 1-3 h of collection.12 particles/ml of the purified siRNA adenoviral constructs and incubated in the presence of the virus for an additional 72 hours before experiments were carried out. Transduction efficiency was gauged by EGFP fluorescence every 24 hours, which was typically greater than 90% by 72 hours.Human MSMCs were isolated and cultured as previously described , and werThe mouse Ltk- fibroblasts (Ltk-)  used forLtk- cells transfected with plasmid DNA (one T-75 flask) were trypsinized  and spun down at 300 × g for 5 min. Cells were resuspended in 200 μl of Mes-buffered saline  plus 1% Triton X-100 and a Complete Protease Inhibitor tablet , incubated for 30 min on ice and dounce homogenized on ice for 1 min. A 100 μl aliquot was transferred to a microcentrifuge tube, and 300 μl of 53.33% sucrose/MBS was added and mixed in. 450 μl each of 30% and 5% sucrose/MBS were layered above this mixture, in this order, and the samples were centrifuged for 24 hours at 54,000 rpm, at 4°C. After this spin, 10 equal fractions were collected, starting at the top. We controlled for the presence of non-lipid membranes by monitoring the presence of the human transferrin receptor by Western blot, as this protein does not associate with lipid rafts.Protein G-Plus Agarose beads  were incubated in 4 ml Dulbecco's Phosphate Buffered Saline, DPBS, (Gibco-BRL) with 1% BSA, 1% Normal Donkey Serum and 1% Normal Rabbit Serum, for 2 hours at 4°C. The beads were washed 1× with DPBS and resuspended in 1 ml DPBS with 0.025% Sodium Azide. 50 μl of each floating fraction were incubated in 1 ml Solubilization Buffer  plus a Complete Protease Inhibitor Tablet (Roche), for 45 min at 4°C, and were then mixed by nutation. These samples were spun down at 3500 × g for 5 min at 4°C, and the supernatants were transferred to new tubes. 50 μl of the prepared beads were added, and the samples were agitated overnight at 4°C. One μg of a maxi-K channel antibody  was then added, and immunoprecipitates were gently mixed overnight at 4°C. The samples were spun down at 3500 × g at 4°C, and washed 3× with 1 ml Buffer A , after which the bead fractions (representing the immunoprecipitates) were resuspended in 40 μl of 2× sample loading buffer .2HPO4, 26.82 mM KCl, and 14.7 mM KH2PO4, 0.5% Tween, pH 7.2) containing 3% non-fat dry milk. Immunoreactivity was detected using HyGlo Western Blotting detection reagents .The immunoprecipitates and mini lipid-raft fractions were separated by SDS-PAGE and immunoblotted as previously described . The pri4Cl for 10 min at RT, permeabilized with 0.1% Triton X-100 for 10 min at RT, and blocked with 10% heat-inactivated FBS + 1% heat-inactivated donkey serum (blocking buffer) for 30 min at 37°C . For co-localization studies, Ltk- cells were incubated with rabbit maxi-K channel antibody  for 1 hour at 37°C, and then with a Cy3-conjugated donkey anti-rabbit  for 15 min, 37°C. They were then incubated for 15 min in blocking buffer for at 37°C, for 30 min in blocking buffer plus a mouse anti-caveolin antibody  for 30 min at 37°C, and finally for 15 min in blocking buffer plus a Cy5-conjugated donkey anti-mouse  for 15 min at 37°C. Signals were visualized using a Zeiss 510 confocal scanning microscope , and images were acquired with a Zeiss LSM 5 Image Browser (Zeiss). For controls, Ltk- cells were incubated with primary antibodies alone (30 min at 37°C) or secondary antibodies alone (15 min at 37°C). In general, Ltk- cells do not generate high background when an anti-mouse secondary antibody is used. Moreover, any background issues due to non-specific binding of the secondary antibody would be discovered by performing the experiments with the secondary antibody alone. Bleedthrough of fluorescent signals into the neighboring channels was controlled for by turning off all but one laser at a time, and recording images in all channels. Laser power, pinhole size and detector gain were then adjusted to ensure that the image appeared in its respective channel only. A nuclear counterstain was not performed since confocal microscopy of these cells showed sufficient morphological detail to identify individual cells.Ltk- cells transfected with plasmid DNA were fixed with 2% paraformaldehyde for 30 min at RT, quenched with 50 mM NH2, 10 Glucose, 2 CaCl2 and 5 Hepes. Borosilicate glass pipettes of 2-5 MΩ were filled with a pH 7.2 solution containing, in mM: 140 KCl, 0.5 MgCl2, 1 EGTA, 5 ATP and 5 Hepes. Cells expressing GFP (the reporter for the plasmid and siRNA expression vector used) were clamped, currents were measured using a holding potential of -80 mV and prepulsing to -100 mV, and currents were elicited at step potentials from -80 to 160 mV in 20-mV intervals.All patch-clamping experiments were performed at RT. Whole-cell current was measured using an Axopatch 200-B amplifier . Signals were filtered with a cutoff frequency of 5 kHz. Data acquisition was controlled using commercial pClamp 9.2 software (Molecular Devices), and data were digitized using a Digidata 1320 interface (Molecular Devices). Ltk- cells expressing wild-type and mutant channels (see Transfections), and hMSMC in which the caveolin-1 gene was inhibited (see siRNA) were placed in a pH 7.4 bath solution containing, in mM: 135 NaCl, 4.7 KCl, 1 MgClt-test for unpaired observations (siRNA and caveolin binding-site mutation experiments) and paired observations . Differences were considered significant at P < 0.05.Each experiment was performed in a minimum of X independently transfected/infected samples. Statistical significance was calculated using Student's + current, we used a siRNA to inhibit cav-1 gene expression in hMSMCs. Maxi-K currents were measured by whole-cell patch clamping and compared to those from control   Fig. . In orde15) Fig. . In ordeols Fig. . Mutant ols Fig. . Mutant ols Fig. . These fSince mutation of the caveolin-binding site had a significant effect on activation of the maxi-K channel, we next determined the role of the caveolin-binding motif in regulating localization of the maxi-K channel within lipid rafts. Taking advantage of the characteristics of lipid rafts--in particular their resistance to solubilization in nonionic detergents at low temperature and their buoyancy due to an enriched lipid content--we were able to isolate Triton X-100-insoluble complexes from mouse Ltk- cells transfected with wild-type (WT) and mutant maxi-K channels. Western blot analysis of lipid raft fractions using a maxi-K channel antibody confirmed that WT maxi-K channels were present in fractions that also harbored cav-1 Fig. . The solSince caveolae share many biochemical properties with lipid rafts, the isolation of detergent-resistant fractions is not sufficient to discriminate between these two subcellular domains. We therefore used anti-channel and anti-caveolin antibodies in immunocytochemical analysis to determine whether, in Ltk- cells expressing the WT and mutant channels, the channel proteins and caveolin localize to the same area of the membrane. As expected, WT maxi-K channels Fig. , red werTo confirm that the reduction in maxi-K current observed in cells expressing the triple mutant is due to the altered interaction between the maxi-K channel and cav-1, we immunoprecipitated the maxi-K proteins from isolated lipid rafts  relaxes to permit fetal exit. Cells isolated from the lower uterine segment of humans during late stages of pregnancy generate a current reminiscent of that typically produced by the maxi-K channel, but this channel becomes constitutively active and lacks voltage- and Caof labor -20. The + sparks ,22. ThesMyometrial smooth muscle contains an abundance of caveolae, which have been proposed to expose or shield the associated proteins to/from the extracellular milieu by opening and closing, respectively . The vas1007YNMLCFGIY1015) in its C-terminus [The maxi-K channel contains a consensus caveolin-binding motif  complex formation and 2) current modulation. Formation of the maxi-K-caveolin complex may involve redundant pathways, thus making it difficult to completely disrupt this complex. In fact, Alioua et al. have identified a second putative caveolin-binding site in the maxi-K channel protein . Thus, w another  have sho+ current [The C-terminus of the maxi-K channel harbors not only the caveolin-binding motif, but also an actin-binding domain  and is c current . FurtherOur results showing that inhibiting the cav-1 gene in hMSMCs leads to decreased maxi-K channel current demonstrate the significance of the maxi-K-caveolin interactions for the excitability of myometrial cells. These interactions appear to be regulated, in part, by the aromatic amino acids located within the caveolin-binding motif of the maxi-K channel. Because the maxi-K channel is important for regulating myometrial cell excitability and uterine contraction, the modulation of this channel by dynamic interactions with caveolin could, in part, explain novel mechanisms underlying aberrant uterine activity.The authors declare that they have no competing interests.AMB and VPK carried out all experimental work. AMB, VPK, and SKE conceived of the study and designed all experiments together. All authors participated in the writing and editing of the final manuscript and approved of the final version.


Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation.Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized.A new recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.Pentose fermenting recombinant  S. cerevisiae satisfies the last two conditions  using the XbaI restriction sites, creating plasmid YIpOB9 (Table The d YIpOB5  and inseT. reesei (H. jecorina) gene lad1 codes for a NAD+-dependent L-arabitol dehydrogenase [A. monospora ALX1 codes for a NADH-dependent L-xylulose reductase [T. reesei lad1 contains an intron of 69 base pairs [T. reesei chromosomal DNA as template. Exon N.1 was PCR amplified with primers containing the restriction site BamHI (LAD-BamHI-FW: 5'-TAGAGGATCCATGTCGCCTTCCGCAGTCGATGAC) and a 24-base region overlapping exon N.2 (LAD-Ex1-R: 5'-GGACATCTGAACCACAGATACCAGTGCTGCGGAC). Exon N.2 was PCR amplified with primers containing a 24-base region overlapping exon N.1 (LAD-Ex2-F: 5'-CTGGTATCTGTGGTTCAGATGTCCATTTCTGGCACG) and the restriction site HindIII (LAD-HindIII-RV: 5'-CAGCAAGCTTTCTAGATCAATCCAGGCTCTGAATCATG). The two PCR products were purified and pooled in a new PCR reaction, carried out with primers LAD-BamHI-FW and LAD-HindIII-RV and yielding the complete coding sequence of lad1. The PCR product was inserted into the vector p425GPD [The rogenase , while Aeductase ,45  and XhoI (ALX-Xho-RV: 5'-TTACTCGAGAGATCTTTACCAAGAAGTGAAACCACCAT). The PCR product was inserted into the vector p425GPD [ p425GPD  creatingsLAD1 and sALX1, respectively. Synthetic sequences were designed taking into account S. cerevisiae codon usage and the possible presence of secondary structure generating sequences, according to a proprietary algorithm of Genscript Corp . A fragment containing sLAD1 was excised from the vector pUCsLAD1 . The PCR product was inserted into plasmid p425GPD_sLAD1 creating plasmid p425GPD_sLAD1_sALX1 , the corresponding optimized DNA sequences were synthesized and the synthetic genes were designated  p425GPD  creating p425GPD  creatingS. cerevisiae was grown aerobically in Erlenmeyer baffled flasks filled to maximum 1/10 of the volume with medium, incubated at 30°C in a rotary shake-incubator  at 200 rpm. Cultures were inoculated at an initial O.D.620 nm of 0.20 ± 0.02 with sterile H2O-washed cells from a late-exponential YNBG pre-culture. The maximum specific growth rate, μ, was calculated from exponential fitting of growth curves from at least two biological duplicates. Anaerobic mixed-sugar batch fermentation was performed in 1.5 l working volume bioreactors , for 120 h, at 30°C, 200 rpm and at pH 5.5 automatically controlled by addition of 3 M KOH. Anaerobic conditions were established prior to inoculation by sparging the medium for at least 3 hours with nitrogen gas  at 0.2 l/min flow rate. A water-lock was placed at the fermentor gas outlet. Cells were pre-grown aerobically in shake flasks in defined mineral medium [620 nm of 0.20 ± 0.02. Fermentation experiments were performed in duplicate.l medium , harvest620 nm of 1, harvested and stored at -80°C. Proteins were extracted with Yeast Protein Extraction Reagent  according to the manufacturer's instructions. Protein concentration was determined with Coomassie Protein Assay Reagent . All activity measurements were performed on fresh extracts. Aldose reductase activity [+ and NADH were used as cofactors. Enzyme activity measurements were performed in triplicate at least on extracts from two independent cultivations. For all the activities measured, 1 U is defined as the amount of enzyme converting 1 μmol of substrate per minute in the conditions of the assay.For crude protein extract preparation, cells were grown in 100 ml YNBG medium until O.D.activity ,60 was mactivity  was measactivity , xylitolactivity  and globSamples were drawn from the fermentors after discharging the sample tubing dead-volume, cells were separated by centrifugation and the supernatant was filtered through 0.20 μm membrane filters  and stored at 4°C until further analysis.2SO4 at a flow rate of 0.6 ml/min as mobile phase. Concentration of L-arabinose, L-arabitol and xylitol was determined by HPLC  using two HPX-87P resin-based column  preceded by a Micro-Guard Carbo-P guard column . Separation was performed at 80°C, with H2O at a flow rate of 0.5 ml/min as mobile phase. All compounds were quantified by refractive index detection . For each HPLC run, a 7-point calibration curve was made for each compound to calculate concentrations. Each sample was analyzed at least in duplicate and a maximum of 10% difference between replicate analyzes was accepted.Concentration of glucose, D-xylose, acetate and glycerol and ethanol was determined by HPLC . The compounds were separated with three Aminex HPX-87H resin-based columns  connected in series and preceded by a Micro-Guard Cation-H guard column . Separation was performed at 45°C, with 5 mM HFor each fermentation experiment, cell dry weight was determined at least in three points, in triplicate for each point. The end point of the fermentation (t = 120 h) was always included. For dry weight determination, a known volume of cell culture was filtered through dry pre-weighed 0.45 μm nitrocellulose filters, which were subsequently dried in a microwave oven and weighed. Because of evaporation, ethanol concentration was calculated from the degree of reduction balance of the overall carbon stoichiometry of the fermentation . EvaporaAR: aldose reductase; ALX: L-xylulose reductase; HPLC: high performance liquid chromatography; LAD: L-arabitol dehydrogenase; NADH: nicotinamide adenine dinucleotide; NADPH: nicotinamide adenine dinucleotide phosphate; OD: optical density; PCR: polymerase chain reaction; PPP: pentose phosphate pathway; XDH: xylitol dehydrogenase; XI: D-xylose isomerase; XK: xylulokinase; XR: D-xylose reductase; YNB: yeast nitrogen base; YNBA: yeast nitrogen base/L-arabinose; YNBG: yeast nitrogen base/glucose; YNBX: yeast nitrogen base/D-xylose.The authors declare that they have no competing interests.MB participated in the design of the study, performed the experimental work and wrote the manuscript. OB performed the experimental work. BHH participated in the design of the study and commented on the manuscript. MFGG participated in the design of the study and commented on the manuscript. All the authors read and approved the final manuscript.


The purpose of this study was to identify mutations associated with bilateral retinoblastoma in a quadruplet conceived by in vitro fertilization, and to trace the parental origin of mutations in the four quadruplets and their father.Mutational screening was carried out by sequencing. Genotyping was carried out for determining quadruplet zygosity.RB1 constitutive mutation (g.2056C>G) which was not detected in her father or her unaffected sisters, and of two other mutations (g.39606 C>T and g.174351T>A) also present in two monozygotic sisters. The novel mutation probably occurred de novo while the others were of likely maternal origin. The novel mutation, affecting the Kozak consensus at the 5'UTR of RB1 and g.174351T>A were likely associated to retinoblastoma in the proband.The proband was a carrier of a novel Molecular diagnosis of retinoblastoma requires genotypic data of the family for determining hereditary transmission. In the case of children generated by IVF with oocytes from an anonymous donor which had been stored in a cell repository, this might not be successfully accomplished, making precise diagnosis impracticable for genetic counseling. RB1 gene that encodes a nuclear phosphoprotein (pRB) involved in the control cell cycle , 0.15 mRB1 promoter and exon 1 from genomic DNA. Reaction mixes were prepared containing 100 ng DNA, 0.4 pm of each primer (forward and reverse), 5% DMSO, 0.15 mM of each dNTP, 3 mM MgCl2, 1× PCR buffer  and 1 U of Platinum Taq DNA polymerase (Promega Corporation) in 50 μl reactions. PCR was performed with 40 cycles at 94°C (1 min), 50°C (40 sec) and 72°C (45 sec) in a programmable thermocycler .Forward 5'-GGTTTTTCTCAGGGGACGTT-3' and reverse 5'-AACCCAGAATCCTGTCACCA-3' primers were used to amplify the ®  using TRIZOL®  following the specifications of the manufacturer and cDNA was synthesized with SuperScript First-Strand Synthesis System for RT-PCR .RNA was extracted from blood cells of the proband isolated with Ficoll-Paque PLUSRB1, was amplified using the forward primer 5'-CTCCCCGGCGCTCCTCCACAGC-3' annealing downstream of transcription start sites -176 and -128  and [C-T-A]. Conversely, the possibility of recombination might have occurred between RB1, D13S284 and D13S317.Genotypic analysis of 13q was carried out with three intragenic The putative paternal haplotypes were [C-C-T]-213-11 and [C-C-T]-215-12, and the likely maternal haplotypes were [C-T-A]-213-9 and [C-C-T]-205-9. The mother could be a 9/9 homozygous for D13S317 or, alternatively, [C-C-T]-205-9 was a recombinant haplotype, in view that 213-9 cosegregation was more frequent than 205-9.RB1 allele, as a de novo event in germ lines or in the zygote.The father was found to be a C/C homozygote at g.2056, while genotypic data indicated that the mother was also a C/C homozygote. Thus, g.2056C>G was not constitutional in the proband's progenitors and could have occurred either in the maternal or the paternal RB1 allele because the expected paternal haplotype was [C-C-T]-213-11 and the expected maternal haplotype was [C-T-A]-213-9. A putative paternal haplotype, identical with the expected one, could be deduced in the proband, together with a putative maternal [G-T-A]-213-9 haplotype derived from the expected maternal haplotype.Alternatively, haplotypic data indicated that g.2056C>G could have occurred in the maternal Genotypic analysis indicated that both g.39606T and g.174351A alleles were of maternal origin because they were not present in the father. They were present in three quadruplets derived from two different embryos . Moreover, in the proband, in whom the paternal haplotype [C-C-T]-213-11 was deduced, these two mutations must have co-segregated with the maternal 213-9 alleles as was the case of quadruplets C and D. Moreover, the fact that child B did not carry these alleles ruled out the possibility of co-segregation with the paternal alleles 215-12 in B, C and D (Table RB1 promoter region (g.2056C>G), in position – 4 with respect to the initial translation ATG codon in an infant, born after IVF, and affected with bilateral retinoblastoma. Mutations that weaken adherence to the Kozak consensus sequence in the 5'UTR have been found to negatively affect translation initiation. In eukaryotes, sequences flanking the AUG codon modulate their ability to halt scanning of the 40S ribosomal subunit and to initiate translation  and and18] aogenitor . For estIn ART, occurrence or risk of occurrence of a genetic disorder in the family must be calculated. Medical, family and reproductive histories of female and male donors must be obtained, and appropriate genetic counseling should be offered and genetic testing implemented . These rThere are no competing interests  that may influence the authors' findings, opinions or points of view.RHB provided sequence and genotypic data of the individuals herein studied and analysed these findings. EL carried out fundoscopies and referred the patient and her family to FRV for Genetic Counseling. CRB acted as academic supervisor of RHB and contributed to the planning of this study. HNS was involved in drafting and revising the manuscript as well as giving final approval to the version to be published.The pre-publication history for this paper can be accessed here:


P < 0.001), being more rapid at higher concentrations, but reaching plateau at 10 ng ml–1 with prolonged exposure. LS174T cells showed only a small increase in apoptosis, and only at high concentrations (50–100 ng ml–1). The main effect of SN-38 in LS174T cells was prolonged cell cycle arrest, which was independent of concentration. Arrest occurred in all phases of the cell cycle, with the distribution depending on concentration (P < 0.001) and not duration (P > 0.05). With increasing concentration, LS174T cells arrested in G2/M, S and G1. Cell cycle arrest was coincident with increased p53 expression in each phase of the cell cycle. Expression in G1 increased with time and concentration , whereas in S and G2/M p53 expression increased only with time (P < 0.001). Dose-dependent p53-associated G1 arrest, in the absence of DNA synthesis indicates an additional cytotoxic mechanism for SN-38, which requires higher concentrations than the S phase mechanism, and detection of which seems to involve p53. For incubations with the same ED (exposure × duration), apoptosis in HT29 cells was significantly higher for prolonged exposure to lower concentrations, whereas in LS174T cells there was a trend towards increased apoptosis with shorter exposures to higher concentrations, indicating a schedule effect of SN-38. Although expression of wild-type p53 leads to a more rapid induction of apoptosis, SN-38 cytotoxicity was generally greater in cells with mutant p53, as wild-type cells escaped apoptosis by p53 associated prolonged cell cycle arrest. Thus, pulsed schedules with higher doses may be more effective in cells expressing wild-type p53, whereas continued exposure with protracted schedules may be more active in cells expressing mutant p53. © 1999 Cancer Research CampaignIn this study the effects of SN-38 on colon adenocarcinoma cell lines expressing wild-type p53 (LS174T) or mutant non-functional p53 (HT29) have been investigated. On exposure to SN-38, HT29 cells rapidly progressed through G1 and S and arrested in G2/M. Release and concomitant increase in apoptosis after 48 h was concentration- and time-dependent (


Salmonella pathogenicity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis.SpiC encoded within fliD and motA genes that encode for the flagella cap and motor torque proteins, respectively, were lower for a spiC mutant strain than for the wild-type Salmonella. Further, this mutant had lower expression levels of the class 2 genes including the fliA gene encoding the flagellar-specific alternative sigma factor. We also found differences in flagella assembly between the wild-type strain and the spiC mutant. Many flagella filaments were observed on the bacterial surface of the wild-type strain, whereas the spiC mutant had only few flagella. The absence of spiC led to reduced expression of the FlhD protein, which functions as the master regulator in flagella gene expression, although no significant difference at the transcription level of the flhDC operon was observed between the wild-type strain and the spiC mutant.Quantitative RT-PCR shows that the expression levels of the class 3 flhDC.The data show that SpiC is involved in flagella assembly by affecting the post-transcription expression of  Salmonella enterica serovar Typhi causes a systemic disease in human known as typhoid fever, whereas S. enterica serovar Typhimurium is responsible for gastroenteritis in humans and a systemic disease in mice similar to human typhoid fever. The ability of Salmonellae to survive within macrophages is required for systemic disease . Th. ThSalmopression . HowevermotA gene, which is necessary for motor rotation, we next investigated the motility of the respective Salmonella strains using LB semisolid plates , indicating that a decrease in class 3 genes expression in the spiC mutant is due to an FlgM-independent mechanism.To examine the mechanism by which SpiC is involved in the expression of the class 3 genes, we focused on the class 2 romoters ,34. The the cell ,36. FlgMcription -39. SpiCcription ,11, althcription ,40. TherfliA gene by fusing the transcription regulatory region of fliA to lacZ in pRL124, as described in the Materials and Methods . More research is required to clarify the molecular mechanism in how SpiC regulates the post-transcriptional expression of the flhDC.Although the molecular mechanism by which SpiC contributes to the post-transcription regulation of the on level -47,50, wechanism . CsrA issrC RNAs  where gesrC RNAs -58. One 600 of 0.7 in LB, because the spiC expression is induced at over an OD600 of 1.5 when the bacteria are grown in LB. However, the expression level of FlhD in the spiC mutant was reduced compared to the wild-type strain even in the exponential growth phase (data not shown), indicating that the FlhD expression is not strictly growth phase-dependent. We cannot explain this phenomenon until the mechanism by which SpiC regulates the post-transcriptional expression of the flhDC or the molecular mechanism of SpiC become clear.We next examined the expression of FlhD at bacterial growth phase of ODhilA and hilC genes that are the SPI-1-encoded transcription regulator genes [inv/spa and prg/org promoters on SPI-1 [hilA expression leading to SPI-1 gene expression, while RitB, a helix-turn-helix DNA binding protein, negatively regulates the expression of the flhDC [As described above, the BarA/SirA system is involved in not only the flagella gene expression but also the SPI-1 gene expression. Phosphorylated SirA directly interacts with promoters of the or genes . HilA, aon SPI-1 . In addihe flhDC . Reportshe flhDC ,61. Intehe flhDC . And thuSalmonella-infected macrophages [et al. [Salmonella leads to IL-8 expression by causing the SPI-2-dependent translocation of flagellin to a basolateral membrane domain expressing TLR5. Together with our previous results, these findings suggest the involvement of FliC in SPI-2-dependent events in the pathogenesis of Salmonella infection.Our recent studies show that the SpiC-dependent expression of FliC plays a significant role in activation of the signaling pathways leading to the induction of SOCS-3, which is involved in the inhibition of cytokine signaling, in rophages . Lyons e [et al.  also repS. enterica serovar Typhimurium. We concluded that the mechanism is due to the involvement of SpiC in the post-transcriptional expression of FlhDC. The data indicate the possibility that SPI-2 plays a role in Salmonella virulence by making use of the flagellar system.In conclusion, here we show that SpiC encoded within SPI-2 is required for flagella assembly in S. enterica serovar Typhimurium strain 14028s. The spiC::kan derivative EG10128 was described by Uchiya et al. [flhD gene was constructed using the Red recombination system [flhD or spiC gene, a kanamycin resistance gene flanked by FLP recognition target sites from plasmid pKD4 was amplified using PCR with primer regions homologous to the flhD gene (5'-TGCGGCTACGTCGCACAAAAATAAAGTTGGTTATTCTGGATGGGAGTGTAGGCTGGAGCTGCTTC-3' and 5'-CGCGAGCTTCCTGAACAATGCTTTTTTCACTCATTATCATGCCCTCATATGAATATCCTCCTTAGT-3') or the spiC gene (5'-TTGTGAGCGAATTTGATAGAAACTCCCATTTATGTCTGAGGAGGGGTGTAGGCTGGAGCTGCTTC-3' and 5'-AGATTAAACGTTTATTTACTACCATTTTATACCCCACCCGAATAACATATGAATATCCTCCTTAGT-3'). Kanamycin-resistant strains were obtained by transforming the PCR products into strain 14028s harboring λ Red recombinase on the plasmid pKD46. Disruption of the flhD or spiC gene was confirmed using PCR with flhD or spiC gene-specific primers. The kanamycin resistance gene was then removed by transforming the strain with plasmid pCP20 that expresses FLP recombinase, resulting in an in-frame deletion of the flhD or spiC gene. Plasmid pEG9127 is a derivative of pBAC108L containing the cloned spiC gene [The bacterial strains used in this study were derived from the wild-type a et al. . The deln system . To dele600 reached 0.3, 0.7, 1.1, and 1.5, the total RNA was isolated using an RNeasy kit  in accordance with the manufacturer's protocol. The RNA (50 μg) was mixed with 32P-end-labeled synthetic oligonucleotide (5'-GCAGGATGCCCATCAATAGTCATT-3'), and 50 units of SuperScript II reverse transcriptase  was added to 30-μl reaction mixtures containing 1 mM of deoxynucleoide triphosphates, 5 mM dithiothreitol, and 1 unit of RNasin/μl. The reaction was performed at 42°C for 1 h. The extension products were analyzed using electrophoresis on a 6% polyacrylamide-7 M urea gel and compared to sequence ladders initiated with the same primer.Bacteria were grown in LB. When the OD600 reached 1.6. The isolated RNA was treated with DNase I (Invitrogen) to remove contaminating DNA, and 2 μg of RNA was reverse-transcribed using SuperScript II reverse transcriptase with random primers. Real-time PCRs were performed in a 50-μl reaction mixture containing 1 μl cDNA, 0.9 μM each primer, 0.25 μM each fluorescent probe, and TaqMan Universal Master Mix . Amplification was performed in 96-well optical plates using the 7300 Real-Time PCR System (Applied Biosystems) with an initial incubation of 2 min at 50°C; followed by 10 min at 95°C; and then 40 cycles: 95°C for 15 s and 60°C for 1 min. The housekeeping gene 16S ribosomal RNA (rRNA) was used as an internal standard for quantification of the total RNA. The primer pairs and fluorescent probes were designed using Primer Express Software ver. 3.0 and were synthesized by Applied Biosystems. The specific fluorescent probes were labeled at the 5'-end with the reporter dye 6-carboxyfluorescein (FAM). The sequences of the primer-probe combinations are shown in Table Salmonella after both values were normalized to the 16S rRNA levels. Each sample was analyzed in triplicate.Bacteria were grown in LB, and the total RNA was isolated when the OD600 of 1.6 (the spiC-inducing condition). After a 1-ml sample of the culture was centrifuged at 18,500 × g for 15 min, the bacterial pellet suspended in 1 ml of cold water was mixed with trichloroacetic acid , placed on ice for 30 min, and centrifuged at 14,000 × g for 20 min. After drying, the pellets were dissolved in 100 μl of sodium dodecyl sulfate (SDS)-sample buffer and boiled for 5 min.An overnight culture in LB was inoculated into 15 ml of fresh LB at a 1:100 dilution. The cultures were grown at 37°C with mild aeration to an ODfliA or flhD promoter region to the promoterless lacZ gene using the promoter-probe vector pRL124 [fliA promoter region or a 0.73-kbp DNA fragment containing the flhD promoter region were amplified using PCR with the following primers: for fliA, 5'-ACGCGTCGACTATGCGCCTGTTAGGGCGCG-3' and 5'-CGGGGTACCCACCCAATCGCGGCTGCGTA-3'; and for flhD, 5'-ACGCGTCGACGCCACATTAATGTGAAGGAC-3' and 5'-CGGGGTACCCGGATGTATGCATTGTTCCC-3'. The PCR products digested with Sal1 and Kpn1 were ligated into the same site in pRL124, producing pRL-fliA and -flhD.To construct the transcriptional fusion of the r pRL124 , a 0.51-600 of 1.6. β-galactosidase activity was measured using the substrate o-nitrophenyl β-D-galactoside as described elsewhere [Bacteria were grown overnight in LB at 37°C and diluted to 1:100 in fresh LB and grown with aeration to an ODlsewhere . Each saBacterial cells grown in LB for 20 h at 37°C without shaking were deposited on carbon-film grids, partially dried, and stained with 2.0% uranyl acetate. The negatively stained samples were observed using a 2000EX electron microscope (JEOL) at an acceleration voltage of 100 kV.Whole-cell proteins (150 μg) from bacteria were fractionated in 16% Tricine-SDS-polyacrylamide gel, electrophoresed, and then electrotransferred onto a polyvinylidene difluoride membrane  as described previously . The banp < 0.05 was considered statistically significant.Each experiment was performed at least three times. The results are expressed as means ± the standard deviations. The data were analyzed using analysis of variance with the Dunnett's test. A value of AS performed experiments and analyses. TN helped to draft the manuscript. KU contributed to the experimental designs and drafted the manuscript. All authors read and approved the final manuscript.


CENP-C is a fundamental component of functional centromeres. The elucidation of its structure-function relationship with centromeric DNA and other kinetochore proteins is critical to the understanding of centromere assembly. CENP-C carries two regions, the central and the C-terminal domains, both of which are important for the ability of CENP-C to associate with the centromeric DNA. However, while the central region is largely divergent in CENP-C homologues, the C-terminal moiety contains two regions that are highly conserved from yeast to humans, named Mif2p homology domains (blocks II and III). The activity of these two domains in human CENP-C is not well defined. In this study we performed a functional dissection of C-terminal CENP-C region analyzing the role of single Mif2p homology domains through in vivo and in vitro assays. By immunofluorescence and Chromatin immunoprecipitation assay (ChIP) we were able to elucidate the ability of the Mif2p homology domain II to target centromere and contact alpha satellite DNA. We also investigate the interactions with other conserved inner kinetochore proteins by means of coimmunoprecipitation and bimolecular fluorescence complementation on cell nuclei. We found that the C-terminal region of CENP-C (Mif2p homology domain III) displays multiple activities ranging from the ability to form higher order structures like homo-dimers and homo-oligomers, to mediate interaction with CENP-A and histone H3. Overall, our findings support a model in which the Mif2p homology domains of CENP-C, by virtue of their ability to establish multiple contacts with DNA and centromere proteins, play a critical role in the structuring of kinethocore chromatin. Proper chromosome segregation during cell divisions depends on a specialized chromosomal site, the centromere. This nucleo-proteinaceous element performs key functions in all eukaryotes from yeast to human. The structural organization of the centromere is generally multilayered and consists of a pairing domain that maintains the cohesion between sister chromatids, a central domain that contains specific centromeric DNA, and the kinetochore, the DNA/protein complex which provides the attachment site for spindle microtubules and regulates the movement of chromosomes towards the spindle poles in vivoCENP-C is another essential kinetochore protein that localizes to the inner kinetochore plate We have previously shown that CENP-C contains two domains, the central and the C-terminal domain, that can independently target the human centromere and associate with the centromeric DNA. In vitro studies have shown that the C-terminal domain also contains a dimerization region that can induce formation of higher order structure of CENP-C First, we tested the ability of the different truncated proteins to localize at centromeres. The mutated proteins were expressed in HEK-293 cells and analyzed for centromere localization by an immunofluorescence assay. As centromeric markers, we used endogenous CENP-B signals, since it is well-known that this sequence-specific DNA-binding protein localizes to the centromeric heterochromatin beneath the kinetochore 32P-labelled alpha satellite specific probe. Hybridization signals above that of the control (pcDNA3.1HA) were interpreted as a significant enrichment of alpha satellite DNA. Signals generated by the alpha satellite probe were quantified by densitometric analysis and normalized to those obtained with an Alu sequence as previously described Second, to understand which part of the C-terminal region is involved in centromere DNA recognition, the three HA-constructs were used in a Chromatin immunoprecipitation assay. We performed a formaldehyde crosslinking technique (ChIP) that was previously employed to demonstrate direct interaction of endogenous CENP-C to the alpha-satellite DNA Then we investigated the function of the Mif2p homology domain III located within the 760/943 region. One possibility is that this domain may be required to sustain CENP-C dimerization as inferred by previous studies Taken together our findings reveal that the C-terminal end of CENP-C displays multiple functions including the ability to associate with the alpha satellite DNA and form higher order complexes.Several lines of evidence have suggested that CENP-C may interact with CENP-A. Specifically this point has been well highlighted by several papers in recent years To address this issue we looked for the formation of CENP-C/CENP-A complexes by a co-immunoprecipitation assay. A construct expressing an HA::CENP-A protein was transfected into HEK-293 cells and nuclear extracts were prepared and subjected to immunoprecipitation with anti-HA antibodies. IP-protein complexes were analyzed by Western blotting using polyclonal anti-CENP-C antibodies. As shown in Considering the high level of homology between CENP-A and histone H3, we extended our analysis to evaluate the ability of CENP-C to interact with the histone protein H3 and with the transcription factor c-Myc, used as a negative control. FLAG-tagged proteins were co-expressed with the HA-CENP-C domains and the interaction was determined by co-immunoprecipitation assay. The results of the experiments, shown in Since we could not exclude that some results of the CoIP experiments may be influenced by the presence of long centromeric chromatin in the nuclear extract, we decided to assess in vivo if the interaction between CENP-A and CENP-C takes place at human centromeres. To this purpose we adopted the Bimolecular Fluorescence Complementation (BiFC) assay. The technique is based on the observation that a fluorescent protein (in this work Enhanced-GFP) can be splitted in two fragments no longer able to emit a bright fluorescence. If the two fragments, fused to proteins that physically interact, are brought in close proximity they can functionally complement and emit bright fluorescence Drosophila and chicken can respectively, impair the proper structuring of the centromere In a previous study we have demonstrated that CENP-C contains two regions, the central one and the C-terminal one, that can both independently localize at the centromere and associate with alpha-satellite DNA S.cerevisiae Mif2p protein the two domains corresponding to human CENP-C Mif2p homology domains II and III display DNA-binding activity and dimerization ability, respectively, and both of them are required for an efficient yeast growth Second, our results indicate that the Mif2p homology domain III, corresponding to residues 890–943, is required for CENP-C dimerization and/or oligomerization. This finding is consistent with what has been proposed by Sugimoto and colleagues Third, we show that the C-terminal region of CENP-C can physically interacts with CENP-A. This is an important result since it provides a mechanistic explanation for previous correlative studies showing that the centromere localization of CENP-C is strictly dependent on the presence of CENP-A Drosophila centromeric chromatin Another important finding reported here is the interaction between the Mif2p homology domain III on the C-terminal region of CENP-C and the histone H3 protein. This result, as well as the observation of a requirement of the same CENP-C domain for the association with CENP-A are supported by two papers published while this article was under revision Overall this study assigns specific functions to the conserved Mif2p homology domains present into the human CENP-C and provides support to a structuring role of CENP-C in the organization of centromeric chromatin.AGATCTGGCCCGCGCCGCCGGAGCCG-3′5′-AAAGGG and 140R: GAATTCTCAGCCGAGTCCCTCCTCAAGG-3′5′-AAAGGG. HA-tagged full length CENP-C and carboxyl or/and N-terminal deletion mutants of CENP-C were generated by PCR as previously described GGATCCAGAAGCTCAAAGAATGAAG-3′5′-AAAGGG and 819R: GAATTCTCATCCAAGAGGTATACCTAG-3′5′-AAAGGG. The HA::760/943 mutant has been generated by PCR using the following oligonucleotides as primers: 760F: GGATTCGGAAGGCCATCAGGAGGATTCG-3′5′-AAAGGG and 943R: GAATTCTCATCTTTTTATCTGAGTAAAAAG-3′5′-AAAGGG. FLAG-tagged C- and/or N-terminal deletion mutants of CENP-C were generated by PCR; the oligonucleotide sequences used as primers to generate the different expression plasmids are listed below: i) 23/410: 23Fh: AAGCTTGCACGTGACATTAACACAGAG-3′5′-AAAGGG and 410R: GAATTCTCATGGTTTTCTGCATTCTTGG-3′5′-AAAGGG; ii) 638/943: 638Fh: AAGCTTAGAAGCTCAAAGAATGAAG-3′5′-AAAGGG and 943R iii) 638/819: 638Fh and 819R iv) 760/943: 760Fh: AAGCTTGGAAGGCCATCAGGAGGATTCG-3′5′-AAAGGG and 943R. PCR products were digested with HindIII and EcoRI and directionally cloned into the 3xFLAGpCMV10 vector  in frame with the N-terminal FLAG-tag. Full length histone H3 cDNA was amplified by RT-PCR from HeLa cells and cloned into the 3xFLAGpCMV10 vector (Sigma). Histone H3 primers: 1F3: AAGCTTGCCCGAACCAAGCAGACTGCGCGCAAG-3′5′-AAAGGG and 136R GAATTCTCAGGCCCGCTCCCCGCGGATAC-3′5′-AAAGGG. All the constructs were verified by sequencing.Full length CENP-C and CENP-A cDNAs were amplified by RT-PCR from HeLa cells and cloned into the pCDNA3.1 vector and in frame with the HA epitope tag. CENP-A primers: 1F: AAGCTTATGGTGAGCAAGGG-3′5′-GGG and NGFPCENPCH3R: AAGCTTCTTGTCGGCCATG-3′5′-GGG. The PCR products were digested with HindIII and cloned into pCDNA3.1/HA-CENPC after HindIII digestion at a HindIII target site placed 5′ with the respect to the HA epitope ATG codon and in frame with the N-terminal HA-tag. The correct orientation of the cloned N-GFP fragment has been evaluated by PCR assay using a CENP-C internal reverse primer. ii) full length C-GFP::HA::CENPC: CGFPCENPCF: 5′-ATGCAGAAGAACGGCATCAAG-3′ and CGFPCENPCR: 5′-TGTTATCTAGATCCGGTGGATCC-3′. PCR products were phosphorylated at the 5′-OH terminals with T4 polynucleotide Kinase (New England Biolabs) following manufacturer instructions and blunt-end ligated in pCDN3.1/HA::CENPC::23/943 after digestion at the Hind III site placed 5′ to the HA coding sequence and after fill-in standard procedure to make blunt HindIII protruding terminals. The correct orientation of the C-GFP fragment was evaluated on the base of BamHI digestion in the C-GFP fragment and downstream of the HA epitope coding sequence. The N-GFP::HA::CENPCΔ890/943 deletion mutant was generated by PCR starting from the described N-GFP::HA::CENPC full length construct using as primers the following oligonucleotides: NGFPCENPCPst1F: CTGCAGACCATGGTGAGCAAGGGGGG and CENPC889ER1R: GAATTCTCATATATCCTGGCCAACCCG. The PCR products were digested with PstI and EcoRI and directionally cloned into the pCDNA3.1 vector.The expression plasmids to perform the Bimolecular Fluorescence Complementation (BiFC) assay have been built as described below. To build N-GFP::HA::CENPC and C-GFP::HA::CENPC expression plasmids, the Enhanced-GFP N-terminal  and the C-Terminal  halves were generated by PCR using as template the pEGFP-C3 vector  and subcloned into the HA-tagged CENP-C full length construct previously described. The HA-epitope has been left as flexible linker between the GFP derived fragment and CENP-C. The oligonucleotide sequences used as primers and the cloning procedures to generate the different expression plasmids are described below: i) full length N-GFP::HA::CENPC: NGFPCENPCH3F: GTCGACATGGGCCCGCGCCGC-3′5′-AAA and CENPALNKSPH1R: GCATGCGCTGCTGCTGCCGCCGAGTCCCTCCTC-3′5′-AT. The PCR product was digested with SphI and SalI restriction enzymes. The N-GFP and C-GFP fragments were amplified by PCR using as template the pEGFP-C3 vector  and as primers the following oligonucleotides: i) for N-GFP amplification NGFPLNKSPH1F: GCATGCGGCAGCAGCAGCATGGTGAGCAAGGGC-3′5′-AT and NGFPER1R: GAATTCTCACTTGTCGGCCATGATATAGACGTTT; the PCR product was digested with SphI and EcoRI restriction enzymes; ii) for C-GFP amplification CGFPLNKSPH1F: GCATGCGGCAGCAGCAGCCAGAAGAACGGCATC-3′5′-AT and CAATTGCTCCTCTCAGTTATCTAGATCCGG-3′5′-CGFPMFE1R: CTCCTC. The PCR product was digested with SphI and MfeI restriction enzymes. The MfeI restriction enzyme leaves protruding ends compatible with EcoRI terminal ends. The pairs of PCR products identified by the forward and reverse primer combinations listed below where added in the same ligation reaction and directionally cloned into pCDNA3.1 after digestion with EcoR1 and SalI restriction enzymes: i) CENPASAL1F/CENPALNKSPH1R and NGFPLNKSPH1F/NGFPER1R to build the HA::CENPA::N-GFP expression plasmid; the correct cloning was verified by HindIII and EcorI digestion at internal and MCS restriction sites; ii) CENPASAL1F/CENPALNKSPH1R and CGFPLNKSPH1F/CGFPMFE1R to build the HA::CENPA::C-GFP expression plasmid; the correct cloning was verified by HindIII digestion at internal restriction sites. All the above described plasmid constructs were verified by DNA sequencing and the molecular weight of the fusion proteins produced after transfection in cells has been checked by standard SDS-PAGE/Western-Blot procedure as described below using both anti-HA and anti-CENP-C or anti-CENP-A antibodies.The HA::CENPA::N-GFP and HA::CENPA::C-GFP expression plasmids were generated as described below and notably creating an aqueous soluble and flexible 2XGly-Ser-Ser-Ser linker between the CENP-A C-terminal end and the GFP fragments N-terminal end by introducing the linker coding sequence in the oligonucleotides used as primers for the PCR reactions. HA-CENPA coding sequence has been amplified by PCR starting from the described HA-CENPA plasmid and using as primers the following oligonucleotides: CENPASAL1F: E. coli BL21 cells. GST-tagged recombinant protein was affinity purified by glutathione agarose beads and used to immunize two rabbits (387 and 386).Anti CENP-A antibodies: the DNA sequence encoding amino acids 1–40 of human CENP-A was cloned into pGEX2T vector and expressed in Anti CENP-C antibodies: the DNA sequence encoding amino-terminal half of human CENP-C (amino acids 23–410) was cloned into pFastBac HTb vector (Invitrogen). The histidine-tagged recombinant protein was expressed in SF9 cells using Bac-to-Bac baculovirus expression system (Invitrogen) and affinity purified by nickel column chromatography. The purified recombinant protein was used to immunize two rabbits (410 and 411).Anti-CENP-A and anti-CENP-C sera were produced by the Eurogentec polyclonal antibody service. The antibodies specificity has been tested by Western-Blot, ChIP and Immuno Fluorescence.5) were seeded on a collagen-coated coverslip and transfected with 3 µg of the different HA::CENP-C construct plasmid DNAs following the previously described method HEK293 cells (5×107) were cotransfected with 10 µg of the two different HA/FLAG::CENP-C construct plasmids in equal ratio by means of the lipofectamine method (Roche). After 24 hours cells were harvested and resuspended in 5× cell volumes of hypotonic buffer  allowed to swell on ice for 10 min. Cells were transferred to a glass Dounce homogenizer and homogenized through 25 up-and-down strokes using the tight pestle. Nuclei were collected by centrifugation, resuspended in 150 µl of high-salt buffer  and incubated on a rocker for 30 min at 4°C. Nuclear extracts were recovered by centrifugation at 13.000 rpm for 15 min and quantified with BCA protein assay kit (Pierce). For each immunoprecipitation 1 mg of nuclear extract was diluted 1:1 with dilution buffer  to lower the salt concentration and pre-cleared with Protein A agarose beads (Sigma) for 3 hours of incubation on a rocker; samples were recovered by centrifuging for 2 min at 2000 rpm in a chilled micro centrifuge. Precleared nuclear extracts were incubated with 50 µl of the anti-HA-affinity matrix (Sigma) and incubated overnight at 4°C on a rocker. Resin was washed 5 times with 1 ml of washing buffer . Protein complexes were separated on a pre-cast 4–12% SDS-polyacrylamide gel (Invitrogen) and transferred onto a nitrocellulose membrane. The filter was blocked with TBS  and 4% nonfat dry milk and probed with a polyclonal anti-HA (Santa Cruz Biotechnology), or a polyclonal anti-CENP-C 410 or a monoclonal anti-FLAG (Sigma) antiserum: The filter was washed three times with TBST (TBS+0.1% Tween-20) and incubated with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (Amersham). Ig/protein complexes were revealed by ECL method (Amersham).HEK-293T cells (2×10HA::CENP-C mutants were purified with the anti-HA-affinity matrix (Sigma) as described above and eluted through 2 cycles of incubation with 50 µl of 1 mg/ml HA peptide. 2 µl of the purified protein were crosslinked in a solution containing 300 mM NaCl, 20 mM HEPES pH 8.0, 1% NP40, 2 µg BSA and 0.01% glutaraldehyde for 2 min at room temperature and blocked with 100 mM Tris-Cl pH 8. Crosslinked and uncrosslinked samples were analyzed by Western blotting.ChIP was performed as described + filter (Amersham). The filter was pre-hybridized and hybridized with a solution containing 7% SDS and 0.5 M Na3PO4 pH 7.0. Probes were [α-32P]-labeled with the Megaprime kit (Amersham) and added to the hybridization solution at a specific activity of 2×109 cpm/µg. Alpha-satellite DNA probes + filter (Amersham) with a Bio-Dot Apparatus (Biorad), as previously described ***P<0,001; **P<0,01 (t-test).Total input DNA and immunoprecipitated DNA samples were transferred to a Hybond-N


Forty-two patients with clinical stage IIIA or IIIB breast cancer were treated with neoadjuvant chemotherapy followed by mastectomy and radiotherapy. The median follow-up was 32 months (range 10-72 months) and the median time to progression was 17 months (range 10-30 months). A multivariate analysis showed that a longer disease-free survival (DFS) was related to more chemotherapy cycles given (P = 0.003), a better pathological response to chemotherapy (P = 0.04) and fewer positive axillary lymph nodes (P = 0.05). A better overall survival (OS) was related to more chemotherapy cycles given (P = 0.03) and better pathological response to chemotherapy (P = 0.04). In patients with residual tumour after neoadjuvant chemotherapy, high levels of staining for Ki-67 was correlated with a worse DFS (P = 0.008). Other biological characteristics, including oestrogen receptor status, microvessel density (CD31 staining), P-glycoprotein (P-gp) staining and nuclear accumulation of p53, were not independent prognostic factors for either DFS or OS. If both P-gp and p53 were expressed, DFS and OS were worse in the uni- and multivariate analysis. The preliminary results of this phase II study suggest that coexpression of P-gp/p53 and a high level of staining for Ki-67 after chemotherapy are associated with a worse prognosis, and that prolonged neoadjuvant chemotherapy and the attainment of a pathological complete remission are important factors in determining outcome for patients with this disease.


Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures.1H-NMR profiling and two-dimensional (2D) 1H, 13C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D 1H, 13C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium, Butyrivibrio fibrisolvens. We identified trans11, cis13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by B. fibrisolvens, based on the results of 13C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects.We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds. A huge number of microorganisms are known to colonize and form complex microbial ecosystems within the human and animal gut Recently, many probiotics have emerged and their effects on human health have been partially demonstrated Escherichia coli K12 knockdown system has been reported Since bacteria grow in the gut, it is desirable to analyze their metabolic dynamics at the growth phase to elucidate their ability to produce organic acids and physiologically active substances. The multiple high-throughput metabolic analysis of To use bacteria as probiotics, it is necessary to screen their characteristics, such as pH resistance and the ability to produce bioactive substances in situ, ex vivo, and in vivo NMR real-time dependent (in vivo) events and statistical approaches have not been investigated.To understand bacterial metabolic dynamics, several analytical strategies having notable technological features have been introduced Butyrivibrio fibrisolvens, produces conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) from linoleic acid (LA) and alpha-linolenic acid (LNA), respectively B. fibrisolvens are known as health-promoting substances because of their beneficial effects on human health B. fibrisolvens, including CLA and CLNA production, is not well understood. Here we developed a novel profiling technique called real-time metabolotyping (RT-MT) to understand and evaluate metabolic dynamics when used in combination with real-time and statistical NMR analysis methods. As a result of performing 13C-labeling RT-MT experiments, we clarified the LNA hydrogenation pathway of B. fibrisolvens based on our accumulated knowledge in NMR analyses and stable isotope labeling techniques We have reported that a gastrointestinal bacterium, 1H-NMR and 1H, 13C-HSQC sequential observations during bacterial growth in an NMR tube are calculated and evaluated with several statistical methods, such as Z-score analysis, principal components analysis (PCA), and statistical total correlated spectroscopy (STOCSY). It is considered that the statistical analyses of time-dependent metabolic profiles would show meaningful biological data related to metabolic dynamics. In addition, time-dependent two-dimensional 1H, 13C-HSQC with 13C labeled substrates (2D-13C-HSQC) profiles could be used to generate three-dimensional metabolic kinetic profiles. Time-dependent 2D-13C-HSQC can provide very detailed information of specific metabolic dynamics. As anaerobic conditions can be easily created in NMR tubes, RT-MT can be applied to the analysis of anaerobic environments, such as soil, industrial plant, and animal gut. RT-MT is expected to improve our understanding of metabolic dynamics that would be necessary for the extraction of particular characteristics of metabolic changes.We have developed RT-MT to understand and evaluate the metabolic dynamics of several bacterial strains . The mosB. fibrisolvens strains revealed their metabolic dynamics, including time-varying factors (1H-NMR standard curve (data not shown), and bacterial growth rate  or linolenic acid (LNA) contributed more significantly to the difference in Z-score profiles than the difference in strain under the same conditions. Anaerobic In cultures with LA or LNA, Z-score profiles mainly around the 1 to 4 ppm region were changed compared to the cultures without LA or LNA . In partB. fibrisolvens strains could be classified into Type I (high butyrate and formate production) or Type II (high lactate production) based on their fermentation products but not their 16S rDNA sequences Different from Z-score analysis, it is possible to detect characteristic metabolites by PCA. Interestingly, the differences in PCA profiles were more remarkable among the different strains than among the different medium conditions . The PCAB. fibrisolvens MDT-10, we next analyzed the time series of STOCSY spectra In order to clarify the pathway of LNA metabolism by 1H, 13C-HSQC analysis using U-13C18 LA and U-13C18 LNA.Analyzing the metabolic response of MDT-10 to LNA, signals in the 1 to 3 ppm region were greatly changed and some of the compounds were successfully identified. For example, several organic acids produced from glucose were identified, and some signals, such as those at 1.2, 1.5, 1.8, and nearby 2.8 ppm, showed a positive correlation with glucose consumption . As thos13C-HSQC with U-13C18 LA and U-13C18 LNA. As the time-dependent 2D-13C-HSQC profiles could be used to generate three-dimensional metabolic profiles, it is possible to understand very detailed information of specific metabolic kinetics. It is well known that NMR signal intensities can be altered by such factors as the mobility of molecules, pH, and sample homogeneity 13C-labeled substrate were little affected by bacterial optical density, differences in NMR data processing, and culture pH  13C18 LA and U-13C18 LNA were largely unaltered.In the case of RT-MT with U-B. fibrisolvens hydrogenates LA and LNA to VA in order to decrease their cytotoxicity 13C-HSQC RT-MT, we found that the growth rate of MDT-10 was low at the initial stage of growth when LA concentration was high. However, the growth rate increased gradually as LA was reduced to VA through the transient accumulation of cis9, trans11-CLA , and VA. When similar analysis was performed with MDT-5 and A38, the final product of LNA hydrogenation was CLNA and t11, c15–18:2, respectively . Thus, the LNA hydrogenation pathway may be similar among B. fibrisolvens strains except A38 and MDT-5.Our past work has demonstrated that the bacterial growth inhibitory activity of unsaturated FAs increases with increasing degree of unsaturation ectively . Previout11, c15–18:2 but did not reduce t11, c15–18:2 to VA t11, c15–18:2 is isomerized once to another form, and then reduced to VA. When MDT-5, which has no CLA-R activity, was grown with t11, c15–18:2 that was produced from U-13C18 LNA by A38, only an unidentified 18:2 FA was produced . Meanwhile, MDT-10 converted the unidentified 18:2 FA produced by MDT-5 into VA. These results are consistent with the information for LNA metabolism described above.We found previously that CLA-R from MDT-10 reduces CLNA to produced , and A38t11, c15–18:2, as examined by gas chromatography-mass spectrometry  (data not shown). This FA exhibited absorbance at 233 nm in reversed-phase HPLC  (data not shown), indicating that the FA has conjugated double bonds. When we compared the structures of the intermediates produced by U-13C18 LNA hydrogenation by 2D 1H, 13C-HSQC profiling, the unidentified 18:2 FA was found to have trans and cis double bonds . TOCSY analysis revealed that CH3 and CH of the double bond of the unidentified 18:2 FA were correlated, whereas CH3 and CH of the double bond of VA were not correlated, suggesting that the double bonds of this FA are located at positions 11 and 13, respectively  purified from A38 isomerized not only LA to CLA but also LNA to CLNA B. fibrisolvens in response to unsaturated FAs can be evaluated by RT-MT. Therefore, this method is a powerful tool for the investigation and estimation of bacterial metabolic dynamics. By monitoring bacterial metabolic dynamics in real time, we would be able to understand their metabolic activities and evaluate their effectiveness as candidates for probiotics. The characteristics of the three B. fibrisolvens strains are as follows: MDT-5 has higher CLNA-producing ability than the other two strains  were described previously in vivo NMR measurement, the strains were cultured in a 5 mmф NMR tube containing 1 mL of growth medium, 10% (v/v) D2O, and 1 mM sodium 2,2- dimethyl-2- silapentane-5-sulfonate (DSS).Sources of 13C18 LA and LNA, D2O, and DSS were purchased from Shoko Tsusho Ltd. . FAs were added as a mixture with BSA, which was prepared as described previously U-1H frequency with the temperature of NMR samples maintained at 310 K. For 1D in vivo1H-NMR, spectra were observed every 5 minutes and residual water signals were suppressed by Watergate pulse sequence with 1.2 second repetitive time. 2D 13C-HSQC spectra were measured every 8 minutes according to the method of Kikuchi and Hirayama 1H inverse probe with triple axis gradient. All 2D 13C-HSQC spectra were recorded on a Bruker DRX-500 spectrometer equipped with a 1H inverse cryogenic probe with Z-axis gradient. A total of 32 complex f1 (13C) and 1024 complex f2 (1H) points were recorded with 8 scans per f1 increment. Spectral widths were 2,640 Hz and 5000 Hz for f1 and f2, respectively. To quantify signal intensities, a Lorentzian-to-Gaussian window with a Lorentzian line width of 10 Hz and a Gaussian line width of 15 Hz was applied in both dimensions, prior to Fourier transformation. A fifth-order polynomial baseline correction was subsequently applied in f1 dimension. The indirect dimension was zero-filled to 512 points in the final data matrix. NMR spectra were processed using NMRPipe software All NMR spectra were recorded on a Bruker DRX-500 spectrometer operating at 500.03 MHz 1H-NMR data were reduced by subdividing spectra into sequential 0.04 ppm designated regions between 1H chemical shifts of 0.5 to 10.0. After exclusion of water resonance, each region was integrated and normalized to the total of all resonance integral regions. 2D spectral assignments were performed using customized software .1D ijs is the intensity of the j-th bin in the i-th 1D spectrum, 〈sj〉 is the average of all the intensities of the j-th bins in all the spectra, and jσ is the standard deviation of them. Any elements that had intensity less than 3000 (arbitrary unit) in the final Z-score matrices were replaced with zero.The Z-score matrix that was used to visualize sequential changes of chemical shifts is defined as the matrix that has each element ofPCA was run on R software. Data were visualized in the form of PC score plots and loading plots. Each coordinate on the score plot represents an individual sample and each coordinate on the loading plot represents one NMR spectral data point related to metabolites. Thus, the loading plots provide information on spectral regions responsible for the positions of coordinates or clusters of samples in the corresponding score plots.i, j) is defined as a correlation coefficient between i-th and j-th bins in a set of 1D spectra (twelve spectra each acquired on an hourly basis). A positively (negatively) higher coefficient means the existence of a positive (negative) correlation between i-th and j-th peaks throughout the spectra.STOCSY was also analyzed using Excel software, as described by Cloarec et al. N H2SO4 (20∶10∶1), as reported previously 2 gas 18-AR-II .Lipids were extracted by shaking cultures with isopropanol-isooctane-6Figure S1B. fibrisolvens incubated in NMR tubes. Glucose concentration (circles) was calculated from the signal intensities observed every 5 minutes. OD (squares) was determined hourly. Mean values of triplicate experiments are shown.Glucose concentration and growth rate of three strains of (0.42 MB TIF)Click here for additional data file.Figure S2Dependence of pH on chemical shift mobility of acetate (A) and succinate (B). Mean values of triplicate experiments are shown.(0.16 MB TIF)Click here for additional data file.Figure S3B. fibrisolvens MDT-10 incubated with or without LNA. During these one-hour experiments, a total of twelve continuously acquired in vivo1H-NMR spectra were used for STOCSY analysis. Contour levels (STOCSY correlations) defined by different colors are indicated at the bottom. Blue represents high positive correlation and red represents low. Data are representative of three independent experiments.Time series of STOCSY analysis of (3.06 MB TIF)Click here for additional data file.Figure S4B. fibrisolvens MDT-10 analyzed by time-dependent 2D-13C-HSQC RT-MT. B. fibrisolvens MDT-10 was anaerobically inoculated in an NMR tube and U-13C18 LA (2.5 mM) was added to monitor LA hydrogenation by in vivo 2D-13C-HSQC. Mean values of triplicate experiments are shown. LA (circles), CLA (triangles), VA (squares), and bacterial growth (open squares) are shown.LA metabolic dynamics of (0.39 MB TIF)Click here for additional data file.Figure S513C-HSQC spectra obtained from LNA and its metabolites. Distinct signals are shown by arrows indicating Δ15 double bond (red), cis double bond (purple), and trans double bond (green). Unidentified 18:2 has trans and cis double bonds, but no Δ15 double bond.Comparison of 2D-(0.37 MB TIF)Click here for additional data file.Figure S6c9, t11,c15–18:3), B: t11,c15–18:2, C: VA (t11–18:2), D: Unidentified 18:2. Black line indicates correlation between CH3 (brown square) and CH (green square). It was shown that unidentified 18:2 exhibits correlation between CH3 and CH, meaning that one of the double bonds of this FA is located more closely to the methyl group side than Δ11.Comparison of TOCSY spectra of LNA metabolites. A: CLNA ((1.37 MB TIF)Click here for additional data file.Table S113C18 LA under various in vivo NMR conditions.Fluctuation of signal intensities of methylene group of U-(0.02 MB DOC)Click here for additional data file.Table S213C18 LA under various pH conditions.Fluctuation of signal intensities of methylene group of U-(0.02 MB DOC)Click here for additional data file.