Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.Enterotoxigenic E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.In this study, we constructed Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea. Escherichia coli (ETEC) strains are the most common bacterial cause of diarrhea. Diarrhea is the second leading cause of death to children younger than 5 years and also one of the most important diseases in young farm animals Enterotoxigenic E. coli or enteroaggregative E. coli (EAEC) strains expressing EAST1 as the only toxin were reported to cause human diarrheal outbreaks in Japan and Chile E. coli strains isolated form children or travelers with diarrhea were commonly EAST1 gene positive E. coli strains isolated from healthy or asymptomatic children and adults E. coli isolated from humans, the EAST1 gene was commonly detected among ETEC strains isolated from pigs with neonatal or post-weaning diarrhea E. coli strains isolated from pigs showing no signs of diarrhea Contradictory observation was reported regarding the role of EAST1 that could play in diarrhea. E. coli (EHEC), ETEC and enteropathogenic E. coli (EPEC) strains E. coli strains isolated from young pigs with diarrhea carried the EAST1 gene alone or together with other toxin genes Although it was initially detected among EAEC strains, EAST1 is highly prevalent among enterohemorrhagic E. coli strains to express EAST1 as the only toxin, and examined the virulence significance of EAST1 in cell lines and a pig challenge model. In addition, we examined whether EAST1 enhances LT or STa toxin of ETEC strains in stimulating intracellular cAMP and cGMP and plays a synergistic role in ETEC associated diarrhea.Significance of EAST1 in diarrhea, especially ETEC-associated diarrhea, needs to be determined. EAST1 antigen has to be included as an ETEC vaccine component if EAST1 is a virulence determinant in ETEC-associated diarrhea. In this study, we cloned the EAST1 gene and constructed recombinant astA gene encoding EAST1. Non-pathogenic porcine E. coli field isolate G58-1 in vitro and in vivo, was used to construct E. coli recombinant strains carrying different EAST1-positive plasmids. Three vectors, the single-copy pBelo-BAC, medium-copy pBR322 and high-copy pUC19, were used to clone and express the astA gene. Enteroaggregative E. coli isolate 17-2 that naturally carries the EAST1 gene was transformed to express an additional LT or STa toxin, or K88ac fimbriae. Constructed strains were cultured in LB or 4AA medium supplemented with ampicillin (100 µg/ml), chloramphenicol (20 µg/ml), or tetracycline (12 µg/ml).Bacterial strains and plasmids used in this study are listed in astA gene was cloned into different expression vectors. The astA gene that was PCR amplified with primers pUC:EAST1-F (5′-atggcctgaaaagcttccggatg-‘3) and pUC:EAST1-R (5′-tccgtgggatcctgataaatcgct-‘3) was cloned into pUC19 at the HindIII and BamHI sites, with primers pBR:EAST1-F (5′-atatcctcatcgctagcgtg-‘3) and pBR:EAST1-R (5′- gcctgctggcatgcctcttc-‘3) was cloned into pBR322 at the NheI and SphI sites, and with primers pBelo:EAST1-F (5′-atggcctgaaaagcttccggatg-‘3) and pBelo:EAST1-R (5′- gcctgctggcatgcctcttc-‘3) was cloned into pBelo-BAC11 at the HindIII and SphI sites, respectively. The amplified astA gene products were separated with gel electrophoresis and purified with a QIAGEN gel extraction kit , digested with restriction enzymes, and cloned into pUC19, pBR322, and pBelo-BAC vectors with T4 ligase , respectively. The avirulent E. coli field isolate G58-1 was transformed to express K88ac fimbriae, and the resultant E. coli (G58/K88) was used as a host strain to be further transformed with astA gene positive plasmids for isogenic strains. These isogenic strains expressed the EAST1 gene in different copy-numbers and were used for the cell line and pig challenge studies. Purification, digestion, cloning and transformation were conducted by following standard protocols astA gene in correct reading frame.To determine whether gene copy-number correlates to virulence, this taq DNA polymerase (Applied Biosystems) in a Mx3000 QPCR system  or a SmartCycler system .Total mRNA from each recombinant strain was extracted using an RNeasy Mini Kit (QIAGEN). An equal amount of mRNA extracted from each strain was used in RT-PCR with the GeneAmp RNA PCR core kit . Five microliters of each RT-PCR product were used in quantitative PCR (in triplicate) using a TaqMan probe (5′-FAM-ccgcatccagttatgcatcgtgcatatggtg-TAMRA-‘3), PCR primers astA-F (5′-atgccatcaacacagtatatc-‘3) and astA-R (5′-tcaggtcgcgagtgacgg-‘3), and gold-EAST1 competitive ELISA, which was developed by Robertson laboratory, was used to examine expression of EAST1 proteins from the constructed strains. Synthetic EAST1, with an average mass of 4109 (±0.25%) and a purity over 90%  was chemically conjugated to ovalbumin (Sigma). Ten nanogram EAST1-ovalbumin conjugates (from Crupper laboratory) were used to coat each well of a microtiter plate  and incubated at 37°C overnight. Coated plates were blocked with 2.5% casein buffer at 37°C for 2 h. Seventy-five microliters of culture supernatants, which were resulted from centrifugation of overnight grown culture (LB or 4AA) with equal amounts of cells from each strain, were mixed with 75 µl of anti-EAST1- chicken serum , and the 150 µl mixture was added to each well and further incubated at 37°C for 1 h. The plate was washed with STa ELISA washing buffer 5 T-84 cells or the IPEC-J2 cells. After 1 h incubation, intracellular cAMP and cGMP concentrations in the T-84 or IPEC-J2 cells were measured in cAMP and cGMP ELISA (acetylated version) as described previously Enterotoxicity of LT and STa of ETEC strains can be measured through stimulation of intracellular cyclic AMP or cGMP in epithelial cells. To determine whether EAST1 possesses similar biological enterotoxicity, we examined field strain 17-2 (EAST1) and EAST1-positive recombinant strains for stimulation of cAMP or cGMP levels in human cell line T-84  and porcine epithelial cells line IPEC-J2  using an EIA cAMP ELISA kit and an EIA cGMP ELISA kit . Overnight grown cultures containing equal amounts of cells, which was calculated based on culture OD values, propagated in 4AA medium from each strain were centrifuged at 3500 rpm for 30 min. One hundred and fifty microliters of resultant supernatants were added to each well containing 1–2×10Isc), which represents the algebraic sum of active ion-transport processes of the cells, were measured using a modified Ussing chamber  as described previously 3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, and 1.2 CaCl2) that was freshly prepared, maintained at 37°C, and bubbled with 5% CO2/95% O2 to maintain pH and to provide mixing. Monolayers were clamped to 0 mV using a voltage clamp apparatus . A 5 s bipolar pulse was applied every 100 s and current deflections were recorded. Data acquisition was performed at 1 Hz with an Intel based computer using Aqknowledge software  and MP100A-CE interface.Enterotoxicity of EAST1 expressed by the recombinant strains was also examined in Ussing chamber assay. Overnight culture growth of each strain was centrifuged, and 2 milliliters of each supernatant were added to chambers containing monolayers of IPEC-J2 cells or T-84 cells. Short-circuit current  and filtrated with glass wool to remove tissue debris. Brush border vesicles were collected by centrifugation, resuspended in PBS, and examined for adherence to K88ac fimbrial E. coli strains in bacterial adherence assay Pig small intestinal samples collected at necropsy were used to measure bacteria colonization using quantitative colonization assay t-test was used to compare different treatment groups. Calculated p values of <0.05 were regarded as significant when treatments were compared at two-tailed distribution and two-sample equal or unequal variance. In addition, the means procedure  was used to analyze data from multiple independent experiments to test whether EAST1 had significant synergistic effect on LT and STa toxins in stimulation of intracellular cAMP and cGMP levels.Data were analyzed by using the mixed procedure , adjusted for multiple comparison by Bonferroni. Results were expressed as means ± standard deviations. Student’s E. coli strains were constructed in this study , and strain 8722 of field isolate 17-2 to express K88ac fimbriae. These two strains were used in the piglet challenge study to examine whether an E. coli strain expressing EAST1 as the only toxin is sufficiently virulent to cause diarrhea in neonatal piglets. Strains 8724 and 8725 were derived from transformation of strain 17-2 with LT plasmid p8607 and STa plasmid p8295 to express additional LT and STa, respectively, and were used for comparative studies in EIA ELISAs to determine whether EAST1 plays a synergistic role to LT or STa toxin in stimulating intracellular cAMP and cGMP in vitro.Nine is study . The astastA gene from strains17-2 and 8722 (17-2/K88ac) were 20.6±0.01 and 20.8±0.30, with no significant differences (p = 0.64). The ΔCT values of the astA gene from strains 8321, 8322, 8323 and 8231(-) were 12.3±0.02, 14.1±0.04, 17.3±0.11 and 32.7±0.66, respectively. That showed that yields of the astA gene amplicons from strains 8321, 8322, 8323 were correlated accordingly to the astA-gene plasmid copy numbers in these strains.An equal amount of total mRNA from 17-2, 8722(17-2/K88ac), 8321(pUC:EAST1/K88ac), 8322(pBR:EAST1/K88ac), or strain 8323(pBelo:EAST1/K88ac) was used in RT-PCR and quantitative PCR. Quantitative PCR showed the ΔCT values of the amplified EAST1 protein was detected in the overnight growth supernatant of strains 17-2, 8722, 8321 and 8322, but not from strain 8323 and the negative control strain 8231 . RelativIncubation with overnight grown culture supernatant with equal amounts of 17-2, 8722, 8321 8322, or 8323 cells did not stimulate an increasing of intracellular cGMP or cAMP levels in T-84 cells . The cGMscI) changes in Ussing chamber assay. Five independent Ussing chamber assay studies, 2 using IPEC-J2 cells and 3 with T-84 cells, were carried out. Data from these studies showed that the scI changes were not significantly different in cells incubated with culture supernatants of 8322, 8321 or 8231 than that of 4AA culture medium. Among 5 studies, one study showed that incubation of culture growth supernatant of strains 8321 and 8322 caused scI changes in T-84 cells and IPEC-J2 cells  (EAST1 expressed by the strains 8321 and 8322 did not cause significant short-circuit current (J2 cells . But fin = 0.06) .9 CFUs of strain 8722 or 8321, piglets remained healthy and showed no signs of diarrhea or dehydration during 72 h post-inoculation. Analyses of blood samples collected before and after inoculation showed total proteins decreased after inoculation, suggesting no dehydration occurred among inoculated piglets. To ensure that the negative outcome results were not caused by natural resistance of challenged piglets to ETEC, i.e. the lack of diarrhea in challenged piglets was resulted from their naturally resistance to ETEC colonization, we examined expression of K88ac receptors in each piglet. Bacterial adherence assays showed the brush border vesicles extracted from ileal tissue of the challenged piglets were bound by the K88ac fimbrial E. coli strain 8231 (16–27 bacteria per brush border vesicle). In addition, data from the quantitative colonization study showed challenged piglets were well colonized. Piglets challenged with strains 8722 and 8321 had 4.4±1.3 and 8.5±4.0 (×109) CFU bacteria colonized per gram of ileal tissue at 72 h post-inoculation. Differences of colonization in piglets inoculated with these two strains were statistically not significant (p = 0.11). Colony screening indicated challenge strains had the introduced plasmids retained, as all 10 colonies (of each piglet) transferred from the LB plates grew well on ampicillin-supplemented (100 µg/ml) plates. PCR screening proved that all colonies were positive of the astA gene. In addition, microscopic images of ileal tissue section showed bacteria colonized at small intestinal epithelial cells of the challenged piglets  and low-copy (pBelo-BAC11) vectors would be used in subsequent challenge studies. After being orally challenged with 3×10 piglets .EAST1 did not act synergistically to LT or STa toxin in stimulating intracellular cAMP or cGMP in T-84 cells . SupernaExpression of LT in strains 8607 (LT) and 8724(LT/EAST1) was verified in a GM1 ELISA as described previously E. coli strain O166 that caused an outbreak of gastroenteritis in Japan in 1996 E. coli strains isolated from asymptomatic or healthy individuals E. coli strains isolated from healthy pigs 10 CFUs of strain 17-2, 2 of 6 inoculated piglets died in 18 h, another 2 became severely ill, but the other 2 remained healthy E. coli strain . In the current study, we challenged 5-day old piglets with 3×109 CFUs of inoculum. We believe that 5-day old piglets tolerated E. coli inoculation, especially at a reasonable dose, better than the newborn piglets, and could be natural in disease progression after infection.Significance of EAST1 in diarrhea has not been determined previously. EAST1 was reported as the only toxin produced by in vivo, we cultured bacteria recovered from pig small intestines on LB plates and transferred 10 colonies to grow at ampicillin-supplemented plates. Data showed all colonies grew on ampicillin plates, indicating these colonies carried the plasmids that carry ampicillin resistance gene. When examined in PCR, these colonies were positive of the EAST1 gene. Future studies to culture bacteria that are isolated from ileal segments of challenged piglets simultaneously at the LB plates and antibiotics-supplemented plates (with great sampling sizes) will help us to better assess colonization of challenge strains in vivo. When we cultured 8321 at the LB and ampicillin-supplemented plates in vitro, we observed no plasmid loss occurred through three passes . These data suggested that the EAST1 plasmids likely were stably maintained in challenge strain 8321 in vivo and in vitro. Therefore, if EAST1 were a virulence determinant in diarrhea, these challenged piglets would have developed clinic disease. Given that none of them developed any sign of diarrhea, the same as the piglets from the unchallenged negative control group, we concluded that EAST1 is unlikely a virulence determinant in porcine neonatal diarrhea. Although a positive control group was not directly included, 2 piglets from the same litter that were used in a separated project developed severe diarrhea and showed dehydration signs including backbone very prominent, skin turgor and eyes sunken in orbits, after 24 h post-inoculation with porcine ETEC strain 3030-2.In order to eliminate ambiguity caused by host genetic resistance, we used the K88ac fimbrial strains 8722  and 8321(EAST1/K88), and K88ac-receptor positive piglets in the pig challenge study. It has been demonstrated that experimental piglets would not develop disease even if being inoculated with a diarrheal ETEC strain if they lack receptors  to be attached by bacteria fimbriae or adhesins astA gene at the mRNA level and performed a competitive ELISA to determine EAST1 expression in strains 8722 and 17-2. Results showed nearly identical ΔCT values  from amplified EAST1 gene were obtained from quantitative PCR, and similar production of EAST1 (p = 0.08) was detected in EAST1 competitive ELISA. These data indicated expression of additional K88ac fimbriae did not significantly affect strain 17-2 in production of EAST1. Therefore, piglets challenged with 8722 strain would not develop diarrheal disease had they been challenged with the wild-type 17-2 strain, even were these piglets well colonized by 17-2 at the small intestines. We need to point out that we verified the cloned EAST1 gene was expressed and the EAST1 proteins were produced and secreted by the constructed strains in vitro, we were unable to verify that expression of EAST1 in the challenge strains was not affected in vivo, especially considering culture conditions and prolonged incubation inside the piglet guts; even though the same host strain and the same expression vectors were demonstrated to express LT and other heat-stable toxins including STa and STb equally effective in culture medium and pig small intestines in vitro and in vivo. Future studies to develop methods to measure expression of EAST1 inside piglet guts, or to use an EAST1 knockout mutant strain derived from a porcine diarrheagenic E. coli strain that expresses EAST1 as the only toxin, were it possible, in piglet challenge studies, will further conclusively assess the role of EAST1 in porcine diarrhea disease.To ensure expression of the additional K88ac fimbria did not negatively affect the expression of EAST1 in strain 8722 (17-2/K88), we conducted quantitative PCR to measure E. coli strains carry different copy numbers of the astA gene, and thus it was suggested the gene copy numbers may affect significance of EAST1 in disease astA gene in three different expression vectors: the single-copy pBelo-BAC11, the medium-copy pBR322, and the high-copy pUC19 and constructed strains to express astA mRNA and protein at the levels correlated to the copy numbers of the carried expression vectors. But as piglets did not develop diarrhea even after being challenged with strain 8321 that had EAST1cloned and expressed at the high-copy vector, we did not continue challenge studies using strains 8322 or 8323. We believed it is unlikely that strains 8322 and 8323, that had EAST1 cloned and expressed in a medium-copy or a single-copy vector, or perhaps EAST1-positive E. coli field isolates that carry one or a few copies of the astA gene, would cause diarrhea in pigs.It was reported that scI changes caused by strains 8321 and 8322 were not significant, even compared to those in cells incubated with the cell culture medium alone.Purified EAST1 or culture ultrafiltrate was reported previously to cause short-circuit current changes to cell lines + EAEC strains were linked to diarrhea, it is thought that not one set of virulence factors, but rather combinations of multiple virulence factors, are associated with diarrhea in vitro. Since an earlier report that indicated porcine ETEC strains trend to acquire additional toxins over time E. coli, mainly EAEC strains, cause intestinal inflammation EAST1 is often compared to STa toxin due to similarity of a peptide domain shETBA gene, as well as chloramphenicol and tetracycline resistance genes BA toxin stimulates fluid secretion and causes diarrhea, we cloned the shETBA gene into vector pBR322 and expressed it in TOP 10 E. coli cells, incubated T-84 cells with supernatants of the ShETBA recombinant strain, and measured cAMP and cGMP levels with ELISAs. We found no increasing of cAMP or cGMP levels in the T-84 cells under our conditions; inferring that ShETBA toxin may also not play a significant role in diarrhea. However, we only verified the cloned gene with DNA sequencing but not the expression or secretion of the ShETBA toxin due to lack of anti-ShETBA antibodies, neither did we conduct any animal challenge studies. Observation regarding significance of ShET toxin obtained from this study could be premature. It was also found that the astA genes carried by strains O42 and 17-2 differ at nucleotides coding the 21th amino acid. This mutation, from a more hydrophobic nonpolar alanine in strain O42 to a less hydrophobic but polar threonine in strain 17-2, was speculated to reduce EAST1 toxicity and thus virulence in strain 17-2 astA gene used in this study to construct recombinant strains is identical to that of strain O42. That suggests the substitution of this particular amino acid unlikely dictates the significance of EAST1.The EAST1-positive EAEC prototype strain O42 caused diarrhea in majority of human volunteers Data from this study suggest that EAST1 alone is not sufficient to cause diarrhea in young gnotobiotic pigs, and does not stimulate increases of intracellular cAMP or cGMP in the human colon cell line T-84 or porcine small intestinal cell line IPEC-J2. Data from this study also suggest EAST1 does not enhance LT and STa toxins in stimulating intracellular cAMP or cGMP in T-84 cells. Together, our data indicated that in the animal tested and under the test conditions used, EAST1 is not a virulence determinant in ETEC diarrhea.


X-ray crystallography with 40 mM Mg2+ in the absence of Ca2+ has shown that SERCA adopts an E1 structure with transmembrane Ca2+-binding sites I and II exposed to the cytosol, stabilized by a single Mg2+ bound to a hybrid binding site I′. This Mg2+-bound E1 intermediate state, designated E1•Mg2+, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca2+ by facilitating H+/Ca2+ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg2+ crystal structure, starting in the presence or absence of initially-bound Mg2+. Both simulations were performed for 1 µs in a solution containing 100 mM K+ and 5 mM Mg2+ in the absence of Ca2+, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg2+, SERCA site I′ maintained Mg2+ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg2+, two K+ ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg2+ and E•2K+ intermediate states of SERCA are populated in solution in the absence of Ca2+, with the more open 2K+-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K+ state acts as a functional intermediate that facilitates the E2 to E1•2Ca2+ transition through two mechanisms: by pre-organizing transport sites for Ca2+ binding, and by partially opening the cytosolic headpiece prior to Ca2+ activation of nucleotide binding.We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca This E2→E1 transition is driven by Ca2+/H+ exchange and may include steps facilitated by other cations P-type ATPases are responsible for active transport of a specific ion, such as Ca2+ binding to ATP hydrolysis are linked to structural dynamics of the cytosolic headpiece 2+-activated E1•2Ca2+ state includes an apo E1 intermediate. The negatively charged Ca2+ binding sites of SERCA probably need to be neutralized for formation of apo E1, but the occupancy of TM binding sites remains unclear for apo E1  2+ but removing Ca2+2+ binding and allosteric coupling of domain dynamics, but the time scales used in the atomistic simulations were too short for Ca2+-free E1 to populate a fully relaxed E1 intermediate state 2+-binding sites and the explicit inclusion of metal ions Experimental and computational studies have provided evidence that structural changes necessary for coupling of Ca2+ have been used recently to obtain crystal structures of SERCA in proposed apo E1 conformations, with high-affinity Ca2+ binding sites exposed to the cytosol without bound Ca2+2+, shows an apo E1 structure stabilized with a single bound Mg2+2+, shows an apo E1 structure stabilized by two bound Mg2+2+ concentration in skeletal muscle cytosol is ∼1–2 mM 2+ ion binds to the TM Ca2+ binding sites in solution 2+, designated E1•Mg2+, features a hybrid cation-binding site I′ occupied by a single Mg2+, and a semi-open cytosolic headpiece conformation that is not suitable for ATP utilization 2+ binding to the Ca2+-binding site I′ is required to stabilize the apo E1 intermediate state in the absence of Ca2+  is reported to activate SERCA + binding to TM sites has not been detected by x-ray crystallography. Thus, major questions remain regarding the role of Mg2+ and K+ in H+/Ca2+ exchange: Are the E1•Mg2+ and E1•K+ intermediate states populated in solution? If so, what mechanistic role(s) do they play in transport? To address these questions, we have performed two all-atom MD simulations of the E1•Mg2+ SERCA crystal structure, starting in the presence or absence of initially-bound Mg2+, in a solution containing physiologically appropriate concentrations of other ions .Super-physiological concentrations of Mg2+  2+ ion bound to site I′. Although the structures of recombinant E1•Mg2+ and SLN-bound E1•Mg2+ are very similar, the A domain is slightly rotated to populate an orientation between E1•Mg2+ and E1•2Ca2+2+ is small (data not shown), indicating that the crystal structure of recombinant E1•Mg2+ is an adequate starting structure to simulate the dynamics of E1. To determine the effect of metal ion binding on the structural dynamics of E1, we removed the ATP analog trinitrophenyl adenosine monophosphate (TNP-AMP) and the Mg2+ ion bound to the phosphate group of TNP-AMP. We also removed two crystallographic water molecules located in the first coordination shell of the remaining Mg2+, because water-Mg2+ interatomic distances did not converge after exhaustive energy minimization rounds. We used PROPKA to adjust the protonation states of ionizable residues, corresponding to pH 7.5 2+-binding acidic residues E771, D800, and E309 were kept unprotonated, whereas residue E908 was modeled in its protonated form. Mg2+-bound SERCA inserted in a pre-equilibrated POPC bilayer composed of 376 lipid molecules; protein-lipid systems were solvated using ∼50,000 TIP3P water molecules. K+, Mg2+, and Cl− ions were added to produce concentrations of 100 mM, 5 mM, and 110 mM, respectively. CHARMM36 force field topologies and parameters were used for the protein + and Cl−. In addition, we used a set of new CHARMM parameters for Mg2+ developed by Allnér et al. 2+ aimed at correcting the Mg2+-water exchange rate, as previous parameters do not correctly capture the water exchange kinetics between the first coordination shell and bulk water We used the crystal structure of recombinant E1•Mg2+ (PDB code: 3w5b) to construct a three-dimensional model of the E1 intermediate state in the absence of bound Mg2+. To simulate this state, we removed the Mg2+ ions located in the phosphorylation site and the TM binding site I′. In addition, the ATP analog trinitrophenyl adenosine monophosphate was removed from the crystal structure. Ca2+-binding acidic residues E771, D800 and E309 were kept unprotonated, whereas residue E908 was modeled in its protonated form. This structure of SERCA was inserted in a POPC bilayer and solvated using ∼50,000 TIP3P water molecules. K+, Mg2+, and Cl− ions were added to produce concentrations of 100 mM, 5 mM, and 110 mM, respectively. To prevent structural artifacts associated with the charge imbalance produced by Mg2+ removal from the Ca2+-binding sites, we performed a 5 ns equilibration cycle of the system with the protein heavy atoms harmonically restrained using a force constant of 2000 kcal mol−1 nm−2. This short equilibration cycle resulted in the binding of a single K+ ion to site I′ of SERCA and virtually no changes in the atomic positions of each residue of the protein. Therefore, SERCA with a single bound K+ ion was used as a starting model for the simulation of apo E1 in the absence of bound Mg2+.We used the crystal structure of E1•Mg−1 nm−2, respectively; each equilibration cycle was performed for 0.01 µs. Unrestrained production runs for E1•Mg2+ and apo E1 were performed for 1 µs.We performed MD simulations by using the program NAMD 2.9 2+-binding sites of E1•Mg2+ under solution conditions approximating the cytosol during muscle relaxation . Starting from the Mg2+-bound crystal structure, we found that the Mg2+ ion remained bound to hybrid site I′ during the entire 1 µs simulation  value of 0.04 nm. MD simulation of E1•Mg2+ demonstrated that Ca2+-binding site II remains cation-free during the entire simulation  difference ≤0.1 nm between the crystal structure and the MD trajectory (Eint) of Mg2+ in site I′ is −950 kcal mol−1, while the average Eint of Ca2+ bound to the Ca2+-binding site I is -830 kcal mol−12+ has a slow off rate from site 1′ and thus E1•Mg2+ represents an inhibitory, not activating, E1 intermediate.We investigated cation interactions with the Caajectory Table 1.2+ ion has octahedral coordination geometry and interacts with six coordinating oxygen atoms for most of the simulation time. The six coordinating ligands for Mg2+ are three carboxylic oxygen atoms from residues E771 and D800, the carbonylic oxygen from residue N796, and two water molecules  and carbonylic oxygen-Mg2+ (0.23 nm) in proteins 2+ were observed between the crystal structure and MD simulation of E1•Mg2+. First, in the crystal structure, the backbone oxygen of A305 and the side chain of N768 belong to the coordination shell of Mg2+  and K+(I′), bind in novel fashion to unique rearrangements of the two Ca2+-binding sites  and K+(I′) binding to the TM sites follow TM1 pathway + ions are guided by E55, E58, E59 and E109 toward site II before reaching sites I and I′, respectively. We did not find any evidence of another entry site to the TM domains  interacts with residues E771, T799, D800 and E908 in a location that virtually overlaps with the site occupied by Ca2+(I) in E1•2Ca2+. (Eint between K+(I) and the Ca2+-binding site I is −350 kcal mol−1, which is much weaker compared to a Eint value of −830 kcal mol−1 calculated for Ca2+ in the same site using a 0.5-µs trajectory of E1•2Ca2+ reported previously +(I′) binds to site I′ at t = 0.07 µs; it interacts weakly  with the backbone oxygen of A305, and with A305 and D800 (and+(I′) binds in a location 0.3 nm away from the site where a second high-affinity Ca2+, Ca2+(II), binds in E1•2Ca2+ (+(I′) does not engage residues E309 and N796 in metal ion-SERCA interactions, which is a requirement for metal ion occlusion in the Ca2+ -binding site II +(I′) binding does not induce the formation of the Ca2+-binding site II. We did not observe K+-Mg2+ exchange in either 1 µs MD simulation; however, it is possible that ion exchange at the Ca2+-binding sites of SERCA occurs under physiological conditions but in much longer time scales (i.e. hundreds of microseconds to milliseconds).In the absence of initially-bound Mg2+ and K+ binding on the structural dynamics of acidic residues that play a central role in Ca2+ binding δ and Cγ, respectively. The distance between E771 and E908 (E771-E908) was calculated between the protonated oxygen (Oε2) from the carboxylic group of E908 and the atom Oε1 from E771. Finally, the distance E800-E908 was calculated between atoms Oε and Cγ of E908 and D800, respectively. These distances were chosen based on the spatial arrangement between E771, D800 and E908 in the crystal structure of E1•2Ca2+. All inter-residue distances converged in both MD simulations of E1•Mg2+ and E1•2K+ , each of which contribute a carboxylate side chain. To analyze the effect of Mgd E1•2K+ Figure 32+ and E1•2K+, with a value of ∼0.65 nm  is 1.7 and 1.4 times larger than that of Mg2+ (0.09 nm) and Ca2+ (0.11 nm), respectively +(I), Mg2+ and Ca2+ between acidic residues E771 and D800, it is expected that the E771-D800 distance trend follows E1•2K+>E1•2Ca2+>E1•Mg2+, in agreement with our MD simulations  and K+(I′) in the Ca2+ sites. Coordination numbers were calculated by counting the number of nearest neighbor oxygen atoms surrounding the metal ions using a cutoff distance of 0.21 nm and 0.30 nm for Mg2+ and K+, respectively. We found that the coordination number of Mg2+ in the Ca2+ site I′ is fairly constant during the entire simulation time, with coordination numbers of either 5 or 6 (+(I) and K+(I′), with values ranging from 2 to 6  and K+(I′) have coordination numbers between 2 and 6 showed that the K+ does not have a strong preference for a specific coordination number even in a structurally restrained environment such as the Ca2+ sites  to induce local structural changes and interact with site I in a similar fashion as Ca2+ does  to evaluate the side-chain dynamics of E309 in the trajectories of E1•Mg2+ and E1•2K+. χ2 angle distributions show that in both E1•Mg2+ and E1•2K+, the side chain of E309 is in a dynamic equilibrium between two orientations (2≤−120° or +120°≤χ2≤+180°), and a side chain orientation where the carboxylic group points toward the cytosol (−119°≤χ2≤−40° or +40°≤χ2≤+119°). Calculation of percentage of time spent in each orientation showed that E309 spends 62% and 69% of the time facing the lumen in the trajectories of E1•Mg2+ and E1•2K+, respectively, indicating that the preferred geometry of E309 is the one with the carboxylic group pointing toward the lumenal face of the lipid bilayer. This finding is in agreement with crystal structures showing that E309 points toward the lumenal side of the sarcoplasmic reticulum in the presence of a bound Ca2+ ion in site II 2+-binding site II is occupied by Ca2+2+ and E1•2K+ to lock the E309 side chain in place.Residue E309 plays a central role in occluding the second Cantations Figure 52+ or K+, we calculated the backbone root-mean-square deviations (RMSD) for each functional domain of SERCA in the 1 µs MD simulation trajectories  in solution, the A and N domains undergo large conformational changes in the sub-microsecond time scale.Analysis of the time-dependent changes in the RMSD E1•2K2+ or K+ to the Ca2+-binding sites are capable of maintaining the structural integrity of the TM domain of E1 SERCA. However, we observed that binding of Mg2+ and K+ induce different structural dynamics of the cytosolic headpiece of SERCA. Structural comparison between the crystal structure ; R489-E680 (N-P domains); and T171-E680 (A-P domains). We tested two models for the distance distribution, ρ(R), of each Cα-Cα distance pair: a single Gaussian distribution and two Gaussian distributions. All distances calculated from E1•Mg2+ and E1•2K+ fit very well to an either one or two Gaussian distribution, with correlation coefficient values ≥0.97 and ≥0.99 for a one and two Gaussian distribution, respectively.Given the intrinsic flexibility of the cytosolic headpiece in solution 2+ fit to a single Gaussian distribution with means 2.7 and 0.95 nm, respectively . These values are nearly identical to those calculated in the crystal structure, indicating that the spatial arrangement of N-A and N-P interfaces in the crystal structure of E1•Mg2+ is similar to the average geometry observed in solution. Distance between residues T171-E680 (A-P domains) of E1•Mg2+ also fits well to a single Gaussian distribution; however, the mean value of the distribution is 0.5 nm larger than that calculated from the crystal structure (black line). Distances K515-T171 (N-A domains) and R489-E680 (N-P domains) calculated from the trajectory of E1•Mg2+ fit to a two Gaussian distribution . The centers of the bimodal distribution between residues T171-K515 are located at R = 3.3 nm and R = 3.8 nm, whereas the centers of the distance distribution between R489-E680 are found at R = 1.6 nm and R = 2.4 nm. These mean distances are substantially larger compared to the distances calculated from the crystal structure, indicating that K+ binding to E1 induces an increase in the spatial separation between N-P and N-A domains. The distance distribution plot of the interdomain distance between T171-E680 (A-P) of E1•Mg2+ fits a single Gaussian with a mean distance of 2.6 nm, a value very similar to the distance calculated directly from the crystal structure of E1•Mg2+ .We found that interdomain distances K515-T171 (N-A domains) and R489-E680 (N-P domains) in the trajectory of E1•Mg2+ in the absence and presence of AMPPCP. These structures are relevant to our study because they represent two opposite ends of the headpiece conformational spectrum: the crystal structure of nucleotide-free E1•2Ca2+ features a completely open and mobile headpiece conformation 2+•AMPPCP populates a compact and relatively rigid headpiece 2+ E1•2Ca2+•AMPPCP . Therefore, the E1 SERCA can be described as a broad ensemble of structural states exchanging between open and closed conformations in the µs time scale . How does SERCA selectively transport Ca2+ against other ions in a physiological environment? We found that Mg2+ or K+ stabilize E1, but fail to induce the structural arrangement of the headpiece necessary for productive ATP hydrolysis. Moreover, interdomain distance distributions revealed important structural differences between E1•Mg2+ and E1•2K+: Mg2+ prevents complete headpiece closure by increasing the distance between P and A domains  Formation of the site I. Structural comparison between E1•Mg2+ and E1•2K+ showed that only K+ binding induces the formation of a Ca2+-bound-like site I (+ binding to the Ca2+ sites is a step necessary to produce a competent site I that is capable of recognizing and binding Ca2. (ii) +-Ca2+ exchangeK. Following the formation of site I, N768 swings away from site I (+-Ca2+ exchange will also be facilitated by weak K+-SERCA interactions at sites I and I′.Moutin and Dupont have previously reported experimental evidence that supports the formation of a functional E1•2Ke site I Figure 2•Mg2+ and E•2K+ intermediate states of SERCA exist in solution in the absence of Ca2+, with the 2K+-bound state being more populated at physiological ion concentrations. Comparison between our MD simulations and published experimental data indicate that E1•Mg2+ represents an inhibited state of the pump, whereas E1•2K+ is a functional intermediate that plays a central role in the E2-to-E1•2Ca2+ transition. E1•Mg2+ and E•2K+ are structurally stable but fail to induce the structural arrangement of the headpiece necessary for productive ATP hydrolysis. E1•Mg2+ modulates the dynamics of A-P domains, whereas E1•2K+ populates an open headpiece structure by increasing the distance between N and P domains. The ability of E1 to populate different arrangements of the cytosolic headpiece in the presence of a variety of bound metal ions constitutes a checkpoint following the E2-to-E1 transition. In addition, E1•Mg2+ and E•2K+ are unable to form site II under physiological conditions. The inability of E1•Mg2+ and E1•2K+ to occlude metal ions results in the lack of charge neutralization around E309. The inability of E1•Mg2+ and E1•2K+ to neutralize the charge around E309 constitutes another checkpoint necessary to prevent unproductive ATP hydrolysis in the absence of bound Ca2+. The structural adaptability and the inability to stabilize site II effectively connects E1 dynamics with Ca2+-selectivity. We propose that E1•2K+ acts as a functional intermediate that accelerates the E2 to E1•2Ca2+ transition through two mechanisms: by pre-organizing transport sites for Ca2+ binding and by facilitating partial headpiece opening prior to Ca2+-activation of nucleotide binding. We propose that E1•2K+ is competent to act as a functional intermediate that regulates the E2 to E1•2Ca2+ transition, and that both E1•Mg2+ and E•2K+ constitute essential checkpoints for selective coupling of Ca2+ binding to ATP hydrolysis in the catalytic cycle of SERCA.Microsecond MD simulations predict that both E1


We have started inviting the relatives of our patients to remain present during our multidisciplinary team ICU ward round. The aim is to improve their understanding of the complex activity on an ICU and reduce inconsistencies in communication. In the UK it is becoming expected practice that patient satisfaction is an endpoint we should be measuring and improving . AssessiThis was a prospective study over 2 months formally inviting up to four families per day to be present for that part of the ward round involving their relative. Subsequently they were asked to complete a questionnaire anonymously on the experience.The results that reflected 31 ward round attendances were unanimous: every family agreed that their attendance had a positive impact, alleviating misconceptions about the intensive care environment and clarifying the processes involved in the care of their relative. The survey also revealed that attendance at the ward round provided an excellent opportunity to have their questions answered by consultants. All those invited wished to attend and all respondents said the experience was valuable and they would like to attend again. Comments included: 'Explanations very helpful to deal with the stress of the situation' and 'Reassuring to have information delivered professionally and compassionately'.In this single-centre survey we have demonstrated that inviting families to ICU ward rounds is feasible and we believe that this intervention could improve family satisfaction on the ICU. We are investigating the impact of this intervention with a detailed comparative survey, which we will present in the future.


A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction.Antennapedia (Antp) and wild-type, full-length p21 (Antp-p21). This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model) with differing p21 or p53 status.The fusion protein was produced from the cell-translocating peptide in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect.Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology. Uncontrolled cellular proliferation and tumour development can result from a malfunction in cell cycle control which is largely regulated by cyclins and cyclin-dependent kinases (CDKs) through phosphorylation. This is reflected by the fact that a large number of cancers involve mutations in the p53 and p21 tumour suppressor proteins ,5. SpeciReconstituting p53 in tumours has been accomplished with both retroviral and adenoviral vectors , resultiOther delivery systems, including liposomes can suffer from unstable formulations, poor pharmacokinetics, and scavenging by the immuno/reticulo-endothelial systems .drosophila homeotic transcription factor Antp, the protein product of the antennapedia gene. The mechanism of transduction relies on the presence of a cationic helix which is important for lipid interactions and for penetration of the membrane [Peptide translocation across impermeable biological membranes through a receptor-independent mechanism -21, has membrane .Antennapedia to peptides corresponding to residues 17–33 or 63–77 of p21 inhibited growth of two human ovarian cancer cell lines [in vivo[Antennapedia could only deliver truncated versions of p21. Additionally, the small size of these peptides made pharmacological development of this approach unfeasible due to rapid systemic clearance. The multi-functional role and interactions of p21 suggests its presence in a cell is required in an intact form [Others and we reasoned that anti-mitotic therapy would be less effective if administered to a tumour which is defective in the proteins needed to cause G1 arrest/apoptosis. However, if wild-type tumour-suppressor protein function is restored by protein complementation, the cells will have regained drug sensitivity. Groups have used either truncated or synthetic peptides to identify regions of p21 that affect the cell-cycle -27. Linkll lines . Similarin vitro and in vivo resulting in cell division arrest and apoptosis, leading to tumour growth inhibition and prolonged survival, an effect enhanced by anti-mitotic chemotherapy. Subcutaneous human tumour xenografts implanted into immuno-compromised mice are a well-established system for studying cancer drug behaviour allowing drug delivery in the context of living tissues and the observation of therapeutic and side effects. The significance of these results is discussed in the context of future cancer therapeutics.In the experiments presented herein, we describe a full-length Antp-p21 fusion protein in order to overcome the obstacle of cell membrane penetration. We demonstrate that the fusion protein accumulates in cells Chemicals were from Sigma, Fluka & VWR. Chromatography media and general immunochemicals from GE Healthcare, UK. Anti-p21WAF1 mouse monoclonal IgG was from Oncogene and anti-pentahistidine antibody was from Qiagen.+/p21+, p53-/p21+, p21-/p53+, p21-/p53-) was the kind gift of Prof. B. Vogelstein  [Cancer cell lines were obtained from the American Type Tissue Collection  and included SKOV3 human ovarian cancer cells that do not express p53, RKO and RKO-E6 human colon carcinoma cells which contain a stably integrated human papilloma virus (HPV) E6 oncogene which causes a decrease in normal p53 levels and functions, including the transactivation of p21. Colo-205 colorectal cancer cells express wild-type p21 and p53. A panel of HCT116 cogenic human colorectal cancer cell lines with different p53 and p21 statuses  .E. coli M15 and selected on 2TYAmp/Kan plates. The Antp and Antp-p21 proteins were expressed as insoluble inclusion bodies and refolded by denaturation and serial dialysis as described in the supplementary methods.The p21 gene was provided from pcDNA3-p21  and cloned in vector pDS56 (Novagen). This construct was used to express recombinant p21, with an N-terminal polyhistidine tag for purification (pDS56p21). The Antennapedia homeodomain (Antp) gene was subcloned from pA4C1  into vector pDS56 for bacterial expression (pDS56Antp). The fusion protein was expressed from a construct (pDS56Antp-p21) by subcloning Antp directly upstream of p21. DNA Constructs were transformed into the Proteins were identified by SDS-PAGE and immunoblotting with anti-p21WAF1 mouse monoclonal IgG or anti-pentahistidine antibody followed by peroxidase-conjugated antibodies. Samples containing pure proteins were pooled and dialysed against Tris-buffered saline  and then two changes of PBS  for 16 hours at 4°C. The dialysed proteins were concentrated by ultrafiltration to 2.5 mg/ml.Penetration of recombinant Antp and Antp-p21 was measured by incubating 100 μg/ml proteins with SKOV3 cells in serum-free media. The cells were washed with PBS containing Tween-20, fixed with 4% paraformaldehyde and stained with 1:100 anti-pentahistidine antibody and anti-mouse FITC-labelled antibody, counter-stained using hematoxylin-eosin (HE). Cells were viewed under a Zeiss fluorescent microscope for penetration and localization of recombinant proteins.3) were seeded in DMEM medium with 10% FCS in the presence of 5% CO2 in 96-well plates overnight. Recombinant proteins and/or chemotherapy were added in a concentration gradient in the absence of serum, and incubated for 48 hours. The relevant controls included untreated cells, cells treated with PBS, Antp alone, p21 alone, and cells which were totally lysed with detergent. Cell survival was determined using the Cell Tire-96 kit (Promega). Multiple experiments were performed, and cytotoxicity was expressed as mean percent survival ± SD. Differences between test and control samples were evaluated for statistical significance by t-test.Tumour cells cells (5x103) were seeded in 96-well plates, and grown for up to 36 hours. Antp-p21 fusion protein and control media alone was added and incubated for up to 3 hours. The media was removed and the cells grown for a further 3–4 hours. The cells were trypsinised, lysed and the proteins analysed on a 7% acrylamide gel and immunoblotted with anti-retinoblastoma antibodies .Cells . All procedures and observations complied with UKCCCR (UK co-ordinating committee on cancer research) guidelines.All animal groups were randomised prior to drug administration and measurements were blinded. Tumour sizes  were measured according to the formula V = length x width x width/2. Animals were purchased from Harlan UK Ltd.Female BALB/c athymic mice (6–8 weeks) were implanted with a variety of tumours. Five million cells in 200 μl complete media were subcutaneously implanted into the left flank of mice. The mice were housed in sterilized, individually-ventilated cages. Approximately 21–28 days later when the diameter of tumour was about 0.5 ± 0.2 cm mice were used. Acute mortality and mean maximum body weight loss was used as a measure of relative toxicity.® method (Pierce) and tissues analysed by gamma-counting. All microscopy slides were prepared using standard procedures, stained with anti-pentahistidine antibody and anti-mouse FITC-labelled antibody, counter-stained using HE staining and viewed under a Zeiss fluorescence microscope.For biodistribution analyses, proteins were radiolabelled with Iodine-125 using the IODO-GENOvarian and colorectal cancer models were used as described in Table Four New Zealand White Rabbits were immunized intravenously with 2.5 mg Antp-p21 without adjuvant, once per day for 5 days. Rabbits were bled once per week over a 4-month period. Control rabbits were immunized with saline. Blood samples were diluted 1:10 and 1:100 in saline, and the immune response was monitored by ELISA on native Antp-p21 (coated 50 μg/ml), detected using anti-Rabbit antibodies.In all experiments, average of all animals were expressed as mean ± SD. Mean tumour burdens in treated animals versus controls were compared at a given time point using the unpaired, two-tailed t-test. Differences between data from two animal groups were assessed by t-test and p < 0.05 was considered significant. Survival was analyzed using the method of Kaplan and Meier and plots were compared for statistical significance using the log-rank test. For tumour growth analysis, the Kruskal-Wallis non-parametric test for multiple comparison analysis was used.Expression of the recombinant proteins resulted in good yields. Antp yielded 20 mg/L of culture, p21, 10 mg/L, whilst Antp-p21 was 5 mg/L after refolding. Expression reduced upon fusing the p21 gene to the Antp gene often seen with chimaeric proteins. Antp-p21 protein precipitation was seen at concentrations higher than 2.5 mg/ml. Figure Antp and Antp-p21 were shown to comparably penetrate cancer cells at a 100 μg/ml concentration  did not alter cell survival whereas up to 40% significant inhibition was seen in p53-deficient or p21-deficient HCT116 cells. The effect was more pronounced in the presence of chemotherapy. A similar effect was seen when RKO colorectal cancer cells were compared to E6-virally inhibited p53 , however, the supplementary chemotherapy was modified into one for colorectal carcinomas . Beneficial effects were seen when this regimen was compared to the results of the previous study, suggesting a dose response effect of the total fusion protein delivered to the tumour . The growth rates of the tumours in these two animal groups demonstrated no significant difference (p = 0.773)  were more sensitive to Antp-p21 therapy compared to parental cell line controls. The effect was also seen in RKO cells. The difference was clear and significant and supports our in vivo, likely due to its small molecular weight. This may also explain the dose scheduling observations. Although detailed pharmacokinetic studies have not been carried out in mice, it is likely that the optimal administration of Antp-p21 would be in conjunction with conventional chemotherapy, possibly within a stabilizing formulation [Therapeutic efficacy was related to the frequency and intervals between agent administrations; treatment with Antp-p21 simultaneously with chemotherapy every 72 hours was significantly superior to therapy given on a weekly basis. The Antp-p21 fusion protein has a fast clearance rate mulation .As previously documented in the metastatic setting of colorectal cancer , greaterThis study proposes a new paradigm, where based on genomic selection of tumour types, the fusion protein Antp-p21 can act on its own and also enhance the efficacy of chemotherapy leading to tumour eradication in p53- or p21-mutated tumours. This work supports the concept of developing individualised therapy for cancer based on the mutation status of patients’ cancers but needs, of course, to be demonstrated in a clinical setting.Dr Kousparou and Professor Epenetos are co-founders of Trojantec who are developing the antennapedia platform. Dr Kousparou and Professor Epenetos also own equity in the form of shares in Trojantec Ltd.in vitro cell-based work, MPD and CK performed the in vivo animal model experiments, CK, MPD nad AAE analysed and interpreted the data and all four authors prepared, wrote, read and approved the manuscript.CK and AAE conceived the study ideas. CK and EY carried out the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/338/prepubFigure S1. In vitro potency of Antp-p21. Antp-p21 at 5μg/ml (black bars) and 20μg/ml (grey bars) was incubated with SKOV3 (which are p53 deficient) with various combination of chemotherapy drugs (Cisplatin (10 μg/ml), and Taxol (8.54 μg/ml). The double combination together with Antp-p21 showed an additive effect in reducing cell proliferation. Figure S2. Biodistribution of Antp-p21 in tumour-bearing mice. Radiolabelled Antp-p21 was injected into tumour-bearing mice and tissues analysed by gamma counting over 15 minutes to 18 hours. A high level of Antp-p21 was observed in the majority of the major organs which cleared after around 1 hour. No net accumulation was seen for any particular tissue. Figure S3. Immunogenicity of Antp-p21 in rabbits. Antp-p21 did not raise an immune response in immuno-competent rabbits at a dose of 12.5 mg over a week of immunizations. Figure S4. Rb-phosphorylation assay of Antp-p21 treated cells. Antp-p21 (20μg/ml) was shown to Inhibit the phosphorylation of retinoblastoma protein (Rb) in SKOV3 cells compared to a non-treated control. Figure S5. Efficacy of Antp versus Antp-p21 treatment – Dose escalation study. Mice (8/group) with s.c. SKOV-3 tumours received either Antp alone in increasing dosages (once per week for five weeks), or Antp-p21 alone in increasing dosages (once per week for five weeks), or PBS (once per week for five weeks). (A) Tumour growth during study after the first i.v. administration. (B) Kaplan-Meier survival of animals following these treatments.Click here for file


Gas hydrate model and free gas model are established, and two-phase theory (TPT) for numerical simulation of elastic wave velocity is adopted to investigate the unconsolidated deep-water sedimentary strata in Shenhu area, South China Sea. The relationships between compression wave (P wave) velocity and gas hydrate saturation, free gas saturation, and sediment porosity at site SH2 are studied, respectively, and gas hydrate saturation of research area is estimated by gas hydrate model. In depth of 50 to 245 m below seafloor (mbsf), as sediment porosity decreases, P wave velocity increases gradually; as gas hydrate saturation increases, P wave velocity increases gradually; as free gas saturation increases, P wave velocity decreases. This rule is almost consistent with the previous research result. In depth of 195 to 220 mbsf, the actual measurement of P wave velocity increases significantly relative to the P wave velocity of saturated water modeling, and this layer is determined to be rich in gas hydrate. The average value of gas hydrate saturation estimated from the TPT model is 23.2%, and the maximum saturation is 31.5%, which is basically in accordance with simplified three-phase equation (STPE), effective medium theory (EMT), resistivity log (Rt), and chloride anomaly method. Gas hydrate mainly exists in the seafloor and polar permafrost , and it Geophysical logging is an important tool for evaluating gas hydrate saturation, and valuable information can be obtained by studying the resistivity and acoustic velocity log data. According to the log data in Shenhu area, South China Sea, Chinese scholars have utilized some theoretical models and empirical formulas to estimate gas hydrate saturation –14, and Foreign scholars have carried out researches on the evaluation of the marine gas hydrate saturation earlier. A variety of theoretical models or experimental models have been proposed to estimate gas hydrate saturation, such as Wyllie et al.  time aveIn this work, based on the log data at site SH2 in Shenhu Area, we first establish gas hydrate model and free gas model by applying elastic wave velocity numerical model of the TPT method, then study the dependence of the P wave velocity on gas hydrate saturation, free gas saturation, and sediment porosity, and finally choose the gas hydrate model to estimate gas hydrate saturation at site SH2.Vp) and shear wave (S wave) velocity (Vs) is as follows:ϕeff is the effective porosity, μ is the average rigidity of the skeleton, ρm is the average density, ρf is the density of the fluid phase, β is the proportionality coefficient, k is the coupling factor, and Cb, Cf and Cm are the compressibility of the solid phase, the fluid phase, and the matrix, respectively.The TPT model , 42, whiShenhu area is considered as one of the occurrences of gas hydrate, which is located in the middle of the northern slope of the South China Sea, between the Xisha Trough and the Dongsha Islands and Baiyun Sag of Zhu II depression of the Pearl River Mouth Basin . Shenhu During April–June 2007, eight sites were drilled in Shenhu area see , among wAs previous researchers have done much work on site SH2 , 40, 50,Based on the previous research methods for the thickness of gas hydrate stability zone –53 and cSeafloor sediments containing gas hydrate are generally composed of rock grain, gas hydrate, water, and natural gas. In order to research the characteristics of gas hydrate reservoir, gas hydrate model and free gas model have been established in this section, and based on these two models, the numerical simulation method and the TPT are used to study the dependence of the elastic wave velocity on sediment porosity, gas hydrate saturation and free gas saturation.ϕs, ϕh, ϕw, and ϕg represent the volume percentage of rock grain, gas hydrate, water, and free gas in the sediments respectively, the gas hydrate model can be expressed asϕ is sediment porosity.The gas hydrate model assumes that the sediments are composed of rock grain, gas hydrate and water, and gas hydrate, is in the pore space, which is regarded as a part of the rock matrix. Supposing that Sh) and water saturation (Sw) can be written asGas hydrate saturation (Ss′) and gas hydrate in the solid phase (Sh′) can be written, respectively, asThe volume percentages of rock grain in solid phase ( (1)ϕeff) can be written as Effective porosity ( (2)ρm), density of the solid phase (ρb), and density of the fluid phase (ρf) can be written asρs is density of the rock grain, ρh is gas hydrate density and ρw is water density. Average density of sediments (  (3)Cb can be written asCs is rock grain compressibility and Ch is gas hydrate compressibility. Assume that the solid compressibility lies between the Voigt and Reuss averages . Cb can (4)Cf) isCw is water compressibility.  Compressibility of the fluid phase ( (5)Cm) indicates the compressibility of sediments without water, and it can be calculated by the following equation:Cp is pore compressibility. The algorithm [Cp is:ϕ0 is the sediment porosity at the sea bottom and Pd is differential pressure. Compressibility of the matrix β) is Proportional coefficient ( (7)μ) indicates average rigidity of the skeleton, and it can be calculated by the following equation:μh is gas hydrate rigidity and μsm is the shear modulus of solid matrix with gas hydrate [μsmKT is Kuster and Toksoz's shear modulus [μsm0 is the shear modulus of solid matrix without gas hydrate. Shear modulus ..Sh and ϕeff.According to , calculaρb, ρf, and ρm.According to  and 10)10), calcCb, Cf, and Cm.According to –14), ca, ca14), β.According to , calculaμ.According to  and 17)17), calcVp.According to , calculaConsider the following.Free gas model assumes that the sediments are composed of rock grain, water, and free gas, and it can be expressed asSw) and free gas saturation (Sg) can be expressed asWater saturation (  (1)ϕeff can be written as (2)ρm, ρb, and ρf can be written asρg is free gas density. (3)Cb can be written as (4)Cf can be written asCg is the compressibility of free gas. Assume that the fluid compressibility lies between the Voigt and Reuss averages . Cf can (5)Cm is calculated by the same equation as that used in gas hydrate model.   (6)β is calculated by the same equation as that used in gas hydrate model. (7)μ can be written asμs is rock grain rigidity. (8)k is 1–∞. Variation range of Consider the following.ϕ, or calculate it by the empirical formula.Given the Sg and Sw, calculate ϕs, ϕw, and ϕg according to (Given the rding to –21)..Sg and ϕeff.According to , calculaρb, ρf and ρm.According to  and 25)25), calcCb, Cf, and Cm.According to –14), ca, ca14), β.According to , calculaμ.According to  and 17)17), calcVp.According to , calculaConsider the following.Sediment porosity is a key parameter for the estimation of gas hydrate saturation for both gas hydrate and free gas models. Therefore, appropriate log data should be selected to estimate the sediment porosity at site SH2. The log data that can be applied to determine the sediment porosity include density, acoustic, resistivity, and neutron logging.When it comes to the determination of sediment porosity by acoustic log, the data need to be corrected by the regional core data because seafloor sediments are always loose silt and silty clay. However, the core data is always insufficient, and it is difficult to determine the compaction correction coefficient, so acoustic log is not available. When it comes to the determination of sediment porosity by resistivity log, the Archie formula should be used to calculate porosity, and the Archie constants and formation water resistivity need to be known for the Archie formula . Howeverρb). The estimation of ϕ by density log data can be expressed as [ρma is matrix density and ρf is fluid density. Considering the effect of shaly sediments, ϕ=(ρϕ=(ρρb). stratum .ρma = 2.65 g/cm3, ρf = 1.04 g/cm3, ρsh = 2.70 g/cm3 [Equations  and 30)30) are u70 g/cm3 , 35. Theh = 0, we can get the P wave velocity of water saturated sediments forward stimulated by gas hydrate model based on the TPT. From Gas hydrate model of the TPT is used to forward stimulate P wave velocity of sediment formation in different gas hydrate saturation conditions in depth of 50 to 245 mbsf at site SH2. h gradually increases, P wave velocity made by forward stimulation also increases; when Sh > 15%, P wave velocity increases significantly; when Sh = 30%, P wave velocity curve is located at the right of the actual P wave velocity logging curve. The above results indicate that the basic range of gas hydrate saturation is 0–30% in depth of 50 to 245 mbsf at site SH2.When Sh = 0) slowly increases from 1743 to 1795 m/s. But with the increase of gas hydrate saturation, the increase rate of P wave velocity is obviously accelerated, and P wave velocity  increases from 1743 to 3961 m/s. From the above analysis, the general rule between P wave velocity of forward stimulation and sediment porosity, gas hydrate saturation at site SH2 is the smaller the sediment porosity, the greater the P wave velocity; the higher the gas hydrate saturation, the greater the P wave velocity. This result is basically in accordance with the research result made by Tinivella [In order to study the dependence of P wave velocity on sediment porosity, gas hydrate saturation, it is assumed that values of gas hydrate saturation increase from 0 to 1 in the interval of 0.1. Using gas hydrate model of the TPT to model the corresponding P wave velocity of the previous gas hydrate saturations, the relation surface of previous three properties can be formed as inivella .Similarly, it is assumed that values of free gas saturation increase from 0 to 1 in the interval of 0.1 in order to study the relation between P wave velocity and free gas saturation. Using free gas model of the TPT to model the corresponding P wave velocity of the previous free gas saturations, the relation surface of P wave velocity, sediment porosity, and free gas saturation can be formed as The estimation of gas hydrate saturation for gas hydrate reservoir evaluation has an important significance. In order to estimate gas hydrate saturation in sediments, it is necessary to associate the P wave velocity of the log with the P wave velocity of gas hydrate model based on the TPT. Given an initial gas hydrate saturation, based on gas hydrate model of the TPT, the difference between P wave velocity of forward simulation and the actual P wave velocity of the log in this saturation can be acquired. If the difference is in the range of allowable error, the saturation can be treated as the actual saturation; if the difference does not satisfy the error requirement, the value of gas hydrate saturation should be modified until meeting the error precision.Using gas hydrate model of the TPT to inverse gas hydrate saturation at site SH2, the values of the main parameters are listed in It is very important to determine the porosity for the evaluation of gas hydrate saturation. In order to analyze the accuracy of porosity estimation by density log data, we use resistivity log data and combine the Archie formula  to make In order to verify the accuracy of gas hydrate saturation estimated by the TPT, we compare gas hydrate saturation in this study with that estimated by Wang et al.  in the oThe average value of gas hydrate saturations calculated by chloride anomaly method is 25%, and the peak value is 45% . The peaIn summary, the relationships between P wave velocity and gas hydrate saturation, free gas saturation, and sediment porosity at site SH2 are studie, respectively, by virtue of elastic wave velocity of numerical stimulation based on the TPT, and gas hydrate model and free gas model are established to estimate the sediment porosity in order to determine the gas hydrate saturation of the research area. Some conclusions can be drawn as follows.(1) Using the difference between P wave velocity of saturated water condition and actual P wave velocity of the log, whether the sediment contains gas hydrate or not can be identified quickly. In the interval of 195 to 220 mbsf at site SH2, the actual P wave velocity of the log increases significantly relative to P wave velocity of forward stimulation in saturated water condition, so this interval is determined to contain gas hydrate.(2) By virtue of elastic wave velocity of numerical stimulation based on the TPT, combined with log data, the dependence of P wave velocity on gas hydrate saturation, free gas saturation, and sediment porosity at site SH2 can be analyzed, respectively. In the interval of 50 to 245 mbsf, as sediment porosity decreases, P wave velocity gradually increases; as gas hydrate saturation increases, P wave velocity increases; as free gas saturation increases, P wave velocity gradually decreases.(3) The log data can be used to calculate gas hydrate saturation of the whole well, and the availability is better than the coring data. The average value of gas hydrate saturation estimated by the TPT is 23.2%, and the peak value is 31.5%, which is basically in accordance with the values estimated by the STPE model, the EMT model, the Rt model and chloride anomaly method.


This paper analyzes and compares two different video conference solutions, widely used in corporate and home environments, with a special focus on the mechanisms used for adapting the traffic to the network status. The results show how these mechanisms are able to provide a good quality in the hostile environment of the public Internet, a best effort network without delay or delivery guarantees. Both solutions are evaluated in a laboratory, where different network impairments  are set, in both the uplink and the downlink, and the reaction of the applications is measured. The tests show how these solutions modify their packet size and interpacket time, in order to increase or reduce the sent data. One of the solutions also uses a scalable video codec, able to adapt the traffic to the network status and to the end devices. The commercial success of portable electronic devices, in conjunction with the ubiquitous presence of the Internet, is changing our communications habits. In addition, user's mobility is higher, and this fact increases our need for means of communication. In this environment, new alternatives to traditional telephony are becoming more and more popular. One example is the migration from voice to data connections, which are being widely used for instant messaging or voice over IP (VoIP). Another significant example is the popularization of video conference solutions, which are no longer restricted to corporate environments but have also become popular on our daily communications.In home environments, video conference presents a lot of clear advantages with respect to a simple phone call: first, the possibility of viewing the other party provides a greater sense of closeness; second, it does not increase your phone bill at the end of the month. At the same time, in corporate environments, video conferences provide a better communication with other offices, displaced staff, or even remote clients, thus saving economic and environmental journey associated costs .As a consequence of these factors, there is a wide variety of video conference solutions, ranging from dedicated devices, developed by the leading telecom equipment vendors, to software-based solutions, whose only requirement is to install an application.best effort network which lacks delay guarantees. The public Internet presents a certain degree of unpredictability, since its network conditions may vary as a consequence of different factors   Skype [7Vidyo, the central node is called Vidyorouter, able to set up the conferences between different nodes, be it a video conference room, a desktop, a laptop, or a mobile device. In order to participate, the end user has only to download and install an application on the device. The central router is able to combine the video streams received from each device, adapting them according to the network conditions and to the characteristics of the end device. For that aim, it leverages on a scalable video codec.In client-server  architectures, there is a central node with a high processing capacity and bandwidth, which permits reducing the requirements of the clients. It should be taken into account that these systems are expected to work in portable devices as smart phones or tablets, with a limited processing capacity, bandwidth, and energy. The system management is easier due to the centralized scheme, and scalability is achieved by the addition of new components to the central server. Redundancy is recommended in order to increase the system's resilience. In the case of As said in the introduction, setting up a video conference through the Internet constitutes a challenge, due to the lack of a delay limit and the variability of the impairments that affect the traffic. It should be remarked that users are accustomed to the reliability of traditional telephony, so they are not very tolerant to drops or quality degradation during the call. Thus, this section first summarizes the mechanisms used by video conference systems in order to work properly in this environment.Joint Video Team from ITU-T VCEG and ISO/IEC MPEG.Scalable video codecs were designed with the aim of adapting the quality of a stream when bandwidth is not guaranteed. H.264/SVC is a scalability extension added to the H.264/AVC codec . These tScalable codecs rely on three kinds of scalability: temporal, which is able to tune the number of frames per second; spatial, which is able to modify the image size; and finally the image quality which can also be adapted, with different values of signal-noise relationship. A layer is defined as a combination of these characteristics. First, a “base layer” is defined, which must always be transmitted, and provides a basic quality. In addition, a number of “enhancement layers,” only transmitted when possible, are able to improve the video. They can only be decoded in junction with the base layer. Thus, a video using a scalable codec can be transmitted with different quality levels, just by sending different numbers of layers, without the need of recoding it. This is especially interesting for video conferencing, since it allows a degree of flexibility when the video has to be adapted according to the network status or due to the characteristics of the end device.Skype: in [Skype uses UDP, but it is able to switch to TCP if the firewall or NAT policies do not permit UDP traffic.In the transport layer, Skype adds redundancy to its traffic, in order to determinate the network status, sending alternatively big and small packets. After that interval, if network conditions are good enough, it stops sending big packets. As a result, bandwidth is significantly higher during the first 20 seconds of a conversation.In the first 20 seconds of a call, Different techniques are used according to the different network impairments: when packet loss is detected, some redundant information is transmitted, so packet size increases. If bandwidth gets scarce, the transmission rate is reduced, thus augmenting interpacket time.In addition to the use of robust and scalable codecs, video conference solutions use different mechanisms to react against network changes. In the literature, some studies have explored this topic, mainly focused on kype: in , 10 thisAll these mechanisms allow these services to work properly under very different conditions, and they are one of the main causes of the commercial success of these solutions in the last years.The test scenario illustrated in Vidyo, the video conference server (Vidyorouter) is located in a remote town . In the case of Skype, the communication is set between the two computers, using the Skype public network through the Internet.Two computers creating the video conference have been included in the lab in University of Zaragoza (unizar). In the case of A number of hubs, which allow us to capture the traffic, have also been included.http://media.xiph.org/video/derf/)).One of the users' computers includes a camera (resolution 800 × 450), which is pointed to a screen playing a high-movement video  (http://tldp.org/HOWTO/Traffic-Control-HOWTO/). Bandwidth is limited by the use of a token bucket Filter with a certain rate. The buffer size has been set to 104 Kbytes, and the burst parameter to 2 Kbytes. Packet loss and network delay are added by means of Linux Netem (http://www.linuxfoundation.org/collaborate/workgroups/networking/netem).An auxiliary machine has also been included, being in charge of adding different network impairments in the uplink and in the downlink. A Proxy ARP is used in order to make all the traffic pass through it. The limitations are added by means of Linux https://code.google.com/p/iperf/). The reported bandwidth is roughly 90 Mbps, which is high enough, taking into account that video conferences may require up to 2 Mbps.As a preliminary test, in order to avoid unexpected network limitations, the available bandwidth between the two networks was measured using IPERF ; the red line represents the traffic sent to the client, averaged every 5 seconds (tick = 5 s). All the video packets are UDP.The first thing that can be observed is that the application is able to adapt its traffic very fast, according to the available bandwidth. For that aim, it uses an oscillation mechanism, which alternatively increases and decreases its throughput.Vidyo server is sending a lower quality stream to the destination. The reason is that there could be other clients in the conference, so for the server it is better to get a high quality stream and to adapt it to each client, by the use of the scalable codec.It can also be observed that the grey line remains constant during the whole conference, so another conclusion can be drawn: the client that generates the video does not reduce the quality, even though the Vidyo generates 1.3 Mbps, with an average interpacket time , the traffic sent by the server is reduced. But if the bandwidth limitation becomes more severe (t = 180), a different behavior can be observed: bandwidth oscillations appear, presenting a period between 16 and 20 seconds. It can be observed that the throughput oscillates between the bandwidth limit and half the limit: from t = 180 to t = 240 it can clearly be seen that the generated bandwidth varies from 0.4 to 0.8 Mbps.When bandwidth limitation is first set , Skype reacts increasing the traffic sent from the initial 1.1 Mbps to 1.3 Mbps. This increase becomes more and more significant as the packet loss rate grows: traffic is above 2 Mbps when packet loss is 9%.In contrast with the previous behavior, Skype implements different mechanisms so as to react to network conditions. If packet loss is detected, it adds redundant data blocks to the messages. This fits the present results reporting that packet size gets increased : the bandwidth falls to 0.8 Mbps.Another change of the behavior can be observed in Skype implements different mechanisms to react against packet loss: it reduces the interpacket time, and it also adds redundancy. However, when a certain threshold is reached, a different behavior is observed in which it reduces the generated bandwidth.We can conclude this section saying that t = 60. Finally, in t = 420 the additional delay is removed.The question we will try to answer in this section is as follows: do the applications include any mechanism to compensate network delay? With that purpose, different delays (orange line) are added, from 50 to 300 ms, beginning in Vidyo, In the case of Skype also reacts to delay. It can be observed that in t = 60, when the additional delay appears for the first time, the application is able to detect this network impairment, and it then acts in a similar way to the case of packet loss; that is, it augments the generated bandwidth  when the limitation was in the downlink.t = 600) cannot be observed, so the system has in this case a less aggressive behavior. The peak in t = 680 was caused by a transient failure in the network.As a final remark, the traffic peak when limitations are removed .In this section the responsiveness of the application in both cases is compared: when limitations appear in the uplink and in the downlink. t = 360. However, if the limitations appear in the downlink, the system reacts after six seconds. This different behavior can be explained according to the different period of the bandwidth oscillations, which is shorter when the limitations are in the uplink.When the limitations appear in the uplink, the adaptation is faster: the application increases its bandwidth just after This paper has explored the mechanisms used by video conference solutions in order to react when network conditions are modified. For that aim, two different solutions, one with a client-server architecture and another one using a peer-to-peer scheme, have been selected. A test environment has been created, including an auxiliary machine able to artificially add bandwidth limitations, packet loss, and network delay.Different tests have been run, reducing and augmenting the network impairments, in order to observe the reaction of the applications, in terms of generated bandwidth, interpacket time, and packet size.The results show that both solutions dynamically react according to the network status and are able to detect the variations very fast. In some cases both applications use similar mechanisms, but in others they rely on different solutions. As an example, one of the solutions reacts against packet loss with an increase of redundancy, whereas the other one does not modify its traffic, trusting the robustness of its scalable video codec. All these tests will be useful so as to know if the mechanisms of the video conference applications are coherent with the network features that can be found in each particular case.