Based on the experimental data from current-clamp recordings, we successfully demonstrated that Kv1.3 dominated the membrane potential of T cells to manipulate the Ca2+ influx via CRAC channel. Our results revealed that the deficient expression of Kv1.3 channel would cause the less Ca2+ signal, leading to the less efficiency in secretion. This was the first successful attempt to simulate membrane potential in non-excitable cells, which laid a solid basis for quantitatively studying the regulatory mechanism and physiological role of channels in non-excitable cells.Kv1.3 channel is a delayed rectifier channel abundant in human T lymphocytes. Chronic inflammatory and autoimmune disorders lead to the over-expression of Kv1.3 in T cells. To quantitatively study the regulatory mechanism and physiological function of Kv1.3 in T cells, it is necessary to have a precise kinetic model of Kv1.3. In this study, we firstly established a kinetic model capable to precisely replicate all the kinetic features for Kv1.3 channels, and then constructed a T-cell model composed of ion channels including Ca Ca3.1) are two kinds of potassium channels in T cells. Kv1.3 channels are activated upon the depolarization of the membrane potential, while IK channels are activated by the Calcium ion CM and TEM cells + channel expression pattern with ∼300 Kv1.3 and ∼10 IK channels per cell. However, Kv1.3 channels are up regulated to ∼1500 in the activated TEM effectors and IK channels are up regulated to ∼500 in the activated TCM effectors Kv1.3 and IK  channel, intermediate K+(IK) channel, TASK channel (a two-pore domain potassium (K2P) channel), TRPM7 channel and Osmo-activated Cl− (Clswell) channel 2+ influx while depleting the endoplasmic reticulum (ER) Ca2+ store 2+ can modulate various important physiological functions such as potassium channel gene expression and secretion, the Ca2+ influx via CRAC channels may form a positive feedback to induce the larger Ca2+ signal Besides Kv1.3, five main types of ion channels have been identified at the molecular level in T cells. They are CaEM) cell contains ∼300 functional Kv1.3 channels and ∼10 functional IK channels on the surface membrane 2+ influx. A negative membrane potential enhances Ca2+ entry by optimizing the electrochemical driving force for Ca2+ movement through CRAC channels 2+ signaling remains wrapped.It is well-known that a typical resting effector memory T .2. The day before transfection, cells were transfered into a 24-well plate and transiently transfected using lipofectamine 2000 (Invitrogen) according to manufacturer's protocol. Recordings were carried out in 1–2 days after transfection.Full length cDNA for Kv1.3 was subcloned into pcDNA3.1/Zeo (Invitrogen). HEK293 cells were cultured in modified Eagle's medium  supplemented with 10% fetal bovine serum  at 37°C incubator with 5% CO+ antibody and maintained in RPMI 1640 medium supplemented with 1 mM L-glutamine and 10% fetal bovine serum (Gibco) in a humidified, 5% CO2 incubator at 37°C. Recordings were carried out in 24 hours after T lymphocyte cells preparation.Peripheral venous blood was obtained from healthy volunteers. Mononuclear cells were isolated by Ficoll-Hypaque density gradient. T cells were separated from PBMCs by using FACS with PE conjugated CD32, 10 HEPES, 1 EGTA, 2 K2ATP (pH 7.4) titrated with KOH, bath solution contained the following (in mM): 145 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES (pH 7.4) titrated with NaOH. All the chemicals were attained from Sigma.For whole-cell patch recordings, the pipette solution contained the following (in mM): 130 KCl, 2.5 MgClPatch pipettes pulled from borosilicate glass capillaries with resistance of 2–3megohms in transfected HEK293 cell experiments and 6–8megohmsin T lymphocyte cell experiments when filled with pipette solution. All experiments were performed using an EPC-9 patch-clamp amplifier and corresponding software . Currents were typically digitized at 20 kHz and filtered at 2.9 kHz (Bessel). The 85% of series resistances were electronically compensated. During recording, the corresponding solution was puffed onto cells via a puffer pipette containing seven solution channels. All experiments were performed at room temperature (20–24°C).2, 1.8 mM CaCl2, 10 mM Hepes (pH 7.4), 10 mM glucose, 2 µM fura-2/AM (Invitrogen), 2 mM Probenecid, 0.05% Pluronic F-127 and 0.1% bovine serum albumin at 37°C for 30 min. After the incubation, cells were placed in the platform for optical imaging. The imaging of intracellular Ca2+ was performed on Olympus-IX70 microscope system with a polychromatic light source . The excitation wavelengths of fura-2 fluorescence were 340 nm and 380 nm, coming from the bottom of plate at 1 Hz. All of the experiments were performed at room temperature.T lymphocyte cells were incubated in 100 µl of extracellular solution (ECS) containing 140 mM NaCl, 5 mM KCl, 1 mM MgClpeak/(V-Ekv1.3)I, where peakI is the peak current, kv1.3E is the reversal potential of Kv1.3. The conductance-voltage (G-V) curves of activation were fitted by the Boltzmann equation:maxG is the maximum conductance, 50V is the half maximal conductance voltage and κ is a slope factor. The steady-state inactivation curve was fitted to the Boltzmann equation:maxI is the maximal peak current, 50V is the half maximal availability voltage and the κ is a slope factor.Recording data were analyzed with IGOR , Clampfit  and Sigmaplot software . Unless stated otherwise, the data are presented as mean ± S.D. The conductance of Kv1.3 channels was calculated from G = V is the voltage in mV, mC is the capacity of the cell membrane in pF, t is the time in ms, injI is the injection current in pA, TASKI is the leak current in pA, CRACI is the CRAC current in pA, Kv1.3I is the Kv1.3 current in pA and IKI is the IK current in pA. In this study, mC = 1.5 pF and the resting potential Rest = −55 mVVT lymphocyte model cell has been described as the following equations injI can be written as follows:TASKG = 0.13 nS TASKE = −100 mV CRACG = 0.09 nS CRACE = +80 mV Kv1.3G = Variable, n is the number of the v1.3K channel, the a single-channel conductance Kv1.3g = 15 pS Kv1.3E = −73 mV oP(V) comes from the v1.3K model.IKg = 11 pS in normal Ringer saline n is the number of the IK channel, the total conductance IKG = 0.11 nS, assuming that 10 channels per TEM cell IKE = −75 mV oP(Ca) comes from the oP(Ca)-[2+Ca]i curve reported by Grissmer et al. All currents except IK channel is triggered by a global Ca2+ concentration in nM, which usually is tremendously smaller than the local Ca2+ in µM 2+ concentration based on an assumption proportional to the local Ca2+ with a average intracellular Ca2+ (about 500 nM) after CRAC channel opening, which could be measured in T cells Considering that the 2+ concentration, depending strictly on net influx and local diffusion of Ca2+ near the CRAC channel in T cells. Assuming that secretory proteins, i.e. complexes of synaptobrevin, syntaxin and SNAP-25 termed SNARE complexes are colocalized to individual Ca2+ sources such as CRAC channel in T cells 2+ influx to a local Ca2+ concentration:2+Ca]i is the intracellular Ca2+ concentration, etrans is an arbitrary transfer coefficient that scales calcium influx to the calcium concentration, and ediff is the calcium diffusion coefficient trans = 0.006 µM*pA−1*ms−1 and ediff = 0.003 ms−12+ concentration high enough (≥10 µM) to trigger secretion d and quantity of various buffers and the calcium efflux releasing from stores. All of those factors can be simply solved by changing the diffusion and transfer coefficients to reveal Ca2+ properties in T cells.To trigger the secretion in T cells, we need to calculate the local CaThe differential equations for the kinetic modeling were solved numerically, using a Q-Matrix or Five-order Runge-Kutta integration method. The fitting procedure is based on a PSO-GSS algorithm for direct estimation of rate constants from macroscopic currents 2+ signaling and thereby to determine Kv1.3 channel physiological role in T cells.To understand the role Kv1.3 channels play in T cells, it is extremely necessary to establish a kinetic model capable to precisely describe its detailed kinetic characteristics under physiological conditions. After that, we can use this Kv1.3 channel model and other four channel models to construct a model T-cell for mimicking the behaviors of membrane potentials and the intracellular Cai) to the open (O) to the inactivated (I) states, the deactivation process depends on the backward rates β and B from O to Ci and the inactivation process depends on the rates η and φ from O to I. All the rates can be determined by automatically fitting the model to the experimental data with the software CeL Several non-sequential kinetic models of Kv1.3 had been reported for simulating the kinetics of Kv1.3 currents, but they focused on the individual activation, inactivation or recovery characteristics Many studies have confirmed that the heterologously expressed Kv1.3 channels possess biophysical and pharmacological signature closely resemble that of native Kv1.3 channels in T lymphocytes cells To complete the kinetic model of Kv1.3, experiments were performed on Kv1.3-transfected HEK293 cells to acquire the activation , steady-50 = −28.4±1.6 mV, and the normalized steady-state inactivation (availability) curve tested at 50 mV had a V50 = −43.5±3.7 mV (a) (i) (d)  curves (black) of Kv1.3 had an averaged value of V5±3.7 mV  left, co7 mV (a)  bottom,  (a) (i)  top, and (i) (d)  were alsBased on the perfect fitness to the activation, inactivation, deactivation and steady-state inactivation data, all the parameters in Kv1.3 model were basically determined. Then we further examined whether this model matched the data of recovery from inactivation by subtly readjusting the parameters in the Kv1.3 model. Since the time course of recovery from inactivation is mono-exponential, the sequential model with only one inactivation state is good enough for the Kv1.3 kinetics. This model conferred by and large a rough fit to both the currents recovering at −45 and −90 mV. As we reported previously, the engineered scorpion toxin ADWX-1 is a potent specific inhibitor of Kv1.3. It has the highest affinity in ∼pM to Kv1.3 channels Based on the sequential kinetic model of Kv1.3 channels, their inhibition features were further investigated in T cells. We first examined the voltage-activated currents in T cells in the absence and presence of 30 and 300 pM ADWX-1. Our results demonstrated that the slow inactivated currents were sensitive to ADWX-1 . And aft2+-release activated calcium (CRAC) channel, intermediate K+ (IK or KCa3.1) channel and leak K+(TASK) channel o of each channels . For more precise, we calculated the time- and voltage-dependent total conductance of Kv1.3 via the open probability Po of Kv1.3 kinetic model rather than via the formulized term m3 h in classic Hodgkin-Huxley model (See Kv1.3 ∼300) for control, 2.0 nS (NKv1.3 ∼133) for 30 nM ADWX-1 and 0.4 nS (NKv1.3 ∼27) for 300 pM ADWX-1  channel by swelling T cell, leading depolarization of T cell 2+ concentration showed a Ca2+ peak at beginning, following by the higher Ca2+ plateau, comparable with that as predicted by the model T cell  in applying with the lower osmolarity solution The CRAC channel was triggered by IP3-induced depletion of the ER Cal T cell . ObviousEM cell, composed of Kv1.3highKCa3.1low has been confirmed for several human autoimmune diseases, such as multiple sclerosis (MS), type 1 diabetes mellitus (T1DM) and rheumatoid arthritis (RA) EM cells that are key mediators of autoimmune diseases, a thoroughly studying the physiological role of Kv1.3 channel plays in T cell not only helps to understand the working mechanism of T cell, but also provide a crucial clue for treatment of diseases.The effector memory T2+ signaling along with changes of membrane potentials.In this study, we systematically studied the basic characteristics of Kv1.3 channel and constructed a sequential kinetic model capable to precisely replicate all the kinetics of this channel. Moreover, we examined the Kv1.3 currents in T lymphocyte cell by a specific inhibitor ADWX-1 toxin, consistent to that of expressed in HEK293. To explore the physiological role of Kv1.3 channel, we further built a T model cell, which was composed of four basic channels prominently in T lymphocyte cell. Finally, we demonstrated that the Kv1.3 channels could dominate the membrane potentials of T lymphocyte cell, and that the CRAC channels manipulated the intracellular Ca− (Clswell), and TRPC3/6 were excluded from this T-cell model. The TRPM7 (or a Mg2+-inhibitor-Ca2+ (MIC) permeable channel) is a stretch- and swelling-activated cation channel, which can be activated by PIP2 and inhibited by Mg2+swell channel conducts an outwardly rectifying chloride current (or volume-regulated anion current), leading an anion and osmolyte efflux that is activated by cell swelling 2+. Besides CRAC channel, TRPC3/6 channels provide another Ca2+ entry pathway, but it seems that these two channels are little expressed in T cells, the amplitude of TRPC3 inward current is only around 2 pA/pF (or 1–3 pA/cell) at −50 mV Using the model T-cell composed of four channels, we have examined the mechanism of Kv1.3 channel regulating T lymphocyte cells. Actually, a couple of channels, i.e., TRPM7, Osmo-activated Cl2+ source in T cells, which provides a continuous calcium influx to elevate the intracellular Ca2+ level for T-cell exocytosis. With TASK channels together, Kv1.3 channels also play a role in maintaining the rest potential.During the whole depolarization process of membrane potentials, CRAC contributes much less current than that of Kv1.3, due to the small total conductance of 0.09 nS compared with ∼4.5 nS of Kv1.3. And the membrane potential was much less sensitive to the CRAC channel conductance in simulations, suggesting that Kv1.3 played the key role in dominating membrane potentials in T cells. While CRAC channel was the major Ca2+ via CRAC channels, and finally to start the IL-2 (interleukin 2) promoter production program In this study, we identified the physiological role of Kv1.3 channels, which further provided us an opportunity to better understand the activation mechanism of T cells. How can a CRAC channel in a T cell be activated following antigen stimulation? In CM, B and sperm cells.In this study, we provided a precise model of Kv1.3 and a novel non-excitable model cell for quantitatively investigating the regulatory mechanism of T cell, which laid a solid basis for further studying other channel such as IK, TASK roles in T cell and the events of membrane potentials, calcium signaling and secretion in other non-excitable cells such as T


Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.Most Y. pestis by flow cytometry and whole-cell ELISA.Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding  Yersinia pestis is a gram-negative, non-spore-forming bacterium belonging to the family Enterobacteriaceae that is known to have evolved from the enteric pathogen Yersinia pseudotuberculosis approximately 20,000 years ago Yersinia species three are pathogenic to humans; Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolytica, while all others are harmless Y. pestis is the causative agent of the plague; an illness that manifests itself in bubonic, pneumonic or septicaemic forms that has resulted in the death of an estimated 200 million people throughout history Y. pestis is listed as a National Institute of Allergy and Infectious Disease, Biodefense Category A Priority pathogen (http://www3.niaid.nih.gov/topics/BiodefenseRelated/Biodefense/research/CatA.htm), and is viewed as a high-priority agent that poses a risk to national security because it is relatively easy to acquire from the environment, and can be effectively dried and converted into an aerosol form. Therefore the development of methods for Y. pestis detection is relevant to public health and biosurveillance.Currently, Specific detection of a microorganism is based on recognition of a genotypical or phenotypical feature unique to that microorganism for which nucleic acid-based or immunological detection technologies are adopted respectively. Immunological detection is inherently more rapid and therefore whenever possible preferable to nucleic acid-based detection since minimal sample preparation is required. Generally the development of immunoassays uses polyclonal or monoclonal full-length antibodies (mAb). The procedure to generate mAbs is time consuming (2–3 months), labor intensive and requires immunization. Furthermore, all procedures to derive mAbs with specific recognition characteristics  are carried out at the screening stage. Because of the limitations of the number of clones which can be grown up this limits the number of mAbs that can be assessed and the specificities that can be obtained.in vitro methods for antibody fragments discovery has been extremely successful, however the use of scFv as reagents in research, diagnosis or detection has been limited by handling issues: low production levels, aggregation and poor stability in long-term storage. Where it is worth the additional effort, this problem has been overcome by the transformation of these antibody fragments into full-length antibodies. Unfortunately, this conversion is resource intensive and cannot be practically carried out on all selected antibody fragments.In recent years, antibody phage display has become a very popular method to isolate specific antibodies, bypassing hybridoma technology and even the need for immunization. In general, libraries are made up of either single chain Fv (scFv) or Fab fragments, and comprise billions of different clones, from which specific binders can be isolated by recursive selection cycles. One of the advantages of using phage antibody libraries is that several antibody fragments, binding to different epitopes, are usually selected, providing a greater likelihood that useful binders will be obtained. Traditional screening methods involve ELISA, a time and cost ineffective way to analyze a large number of clones for binding specificity. We have recently reported a multiplex flow cytometry screening method Y. pestisF1 antigen is a capsular protein unique to most Phage display technology was used to select multiple scFv clones that specifically bound to recombinant F1 antigen. Following library screening, the DNA pool representing the selected scFv population was recovered, and full-length scFv open reading frames were cloned into the pEP-AP vector . This exBinding of the 8 selected αF1 scFv antibodies, to recombinant F1 antigen, was confirmed by one-step ELISA as well as multiplex flow cytometry . One-ste+13 and 8×10+11 as shown in Phage antibody libraries are a source of antibody fragments with exquisite binding specificities Reactivity of the αF1 phage-displayed scFv (CT1 through 8) with recombinant F1 was carried out by multiplexed flow cytometry using a microsphere triplex bound to biotin, biotinylated Lysozyme or biotinylated F1, with the first two bead sets acting as negative controls for assessing specificity of F1 binding . Upon inY. pestis cells by flow cytometry  Nairobi, Y. pseudotuberculosis 0104 (YPT 0104) and Y. enterocolytica (YE) as F1-negative controls. Phage binding events were reported using horseradish peroxidase-conjugated anti M13 antibody (αM13-HRP). Background noise coming from either the buffers or the cells adhering to the wells was evaluated by including wells with no added phage . Purified F1 protein was biotinylated using EZ-Link® NHS-LC-LC Biotin  according to the manufacturer's instructions. The level of biotinylation was quantified as 3–5 molecules of biotin per mole of protein, using the EZ™ Biotin Quantification Kit .+12 colony forming units (cfu) of scFv phage library were blocked using 2% BSA PBS + 0.01% Tween 20 (PBS-LT), at room temperature (RT) for 1 h in a final volume of 134 µL. In the first selection round, 1.8 µg of biotinylated F1 antigen was incubated with the blocked scFv library at room temperature (RT) for 1 h, in a final volume of 190 µL. Following incubation, the scFv phage-F1 complexes were captured onto 2×10+7 streptavidin magnetic beads  by incubation at RT for 15 min. The bead complex was then washed 3 times in 190 µL of PBS-LT and 3 times with 190 µL of PBS + 0.1% Tween 20 (PBS-T). The final scFv phage binding population was eluted via incubation at RT for 3 min and 30 s using 180 µL of 0.1 M glycine pH 2.2, and neutralized to pH 7.5 by the addition of 50 µL 1 M Tris-HCl (pH 8.8). The eluted scFv phage population was recovered by infecting 100 µL into 1 mL of Escherichia coli DH5αF' cells at OD600  =  0.5, by static incubation at 37°C for 45 minutes. The phage-infected bacterial cells were plated onto 2XYT agar containing 100 µg/mL carbenicillin and 3% Glucose (2XYT/Carb/Glu) and incubated overnight (O/N) at 30°C. The phage-infected bacterial cells were recovered in 2 mL of 2XYT/Carb/Glu broth, 10 µL of the bacterial suspension was inoculated into 10 mL of 2XYT/Carb/Glu broth and incubated with shaking (260 rpm) at 37°C until the OD600 was 0.5. The cells were subsequently co-infected with 1.3×10+13 cfu of M13K07 helper phage (Amersham Pharmacia) by static incubation for 30 min at 37°C. Following infection, the cells were recovered by centrifugation at 3000 rpm for 30 min and re-suspended in 10 mL of 2XYT broth containing 100 µg/mL carbenicillin, 25 µg/mL kanamycin and incubated O/N at 30°C with shaking (260 rpm). The amplified scFv phage were recovered within the media supernatant by centrifugation at 3000 rpm for 30 min. 171 µL of the supernatant was used directly in the subsequent selection round. The amplified phage particles were precipitated from the remainder of the media supernatant by two rounds of PEG/NaCl precipitation and stored at 4°C. The stringency of the selection conditions was increased through 3 subsequent rounds of panning. The biotinylated F1 antigen concentration was decreased 10 fold (from 600 nM to 60 nM) from the first to second, and third round and wash times were increased  with each subsequent round.The scFv phage library has been previously described BssHII and NheI, purified using QIAquick gel extraction kit  and ligated into BssHII and NheI digested pEP-AP vector . The DNA, encoding full-length scFv antibodies, was recovered by digestion with NEB restriction enzymes P vector . 1 µL ofp-Nitrophenyl Phosphate Disodium Salt . In a second Maxisorp 96 well plate, individual scFv expression levels were determined by addition of 10 µL of scFv antibody periplasmic extract to 80 µL of AP substrate. Immediately following addition of AP substrate, the absorbance at 405 nm was measured using the SPECTRA fluor Plus spectrofluorometer. The time zero value was deducted from subsequent absorbance values generated as the AP signal increased over time.The expressed αF1-AP scFv fusion proteins were analyzed by one-step ELISA for binding to recombinant F1 antigen, or cross reactivity to chicken Lysozyme. The ELISA was automated using the liquid handling Genesis 2000 workstation (Tecan). 192 wells of a 384 Maxisorp plate (NUNC #464718) were coated with 0.1 µg of F1 antigen and all remaining wells were pre-coated with 0.1 µg of chicken Lysozyme , allowing duplicate analysis of 96 different scFv clones against both antigens. The plate was incubated O/N at 4°C and the following day, unbound antigen was removed, the plate washed twice with PBS-LT and blocked with 100 µL of 1% w/v BSA , by incubation for 1 h at RT. The block buffer was removed and the plate washed twice with PBS-LT. 50 µL of scFv antibody periplasmic extract was added, in duplicate, to wells containing either Lysozyme or F1 antigen and incubated at RT for 1 h. The plate was washed 3 times with PBS-T and 3 times with PBS-LT. Binding events were detected following addition of 80 µL of Alkaline Phosphatase (AP) substrate TAATACGACTCACTATA 3′) and reverse primer APRev (5′ CAGGTTTATCGCTAAGAGAAT 3′). The resulting DNA sequence was analyzed using Vector NTI Advance 10 ContigExpress (Invitrogen) and translated, and aligned, using Vector NTI Advance 10 Alignx (Invitrogen) to identify different scFv groups.62 clones, identified as positively binding to F1 antigen by ELISA, were sequenced (Ohio State University Plant-Microbe Genomics Facility) using forward primer T7 Promoter (5′ BssHII and NheI as described previously. Clones were sequenced with both T7 Promoter (5′ TAATACGACTCACTATA 3′) and APRev (5′ CAGGTTTATCGCTAAGAGAAT 3′). The eight different αF1 scFv antibodies were expressed in BL21 Gold DE3 , as described earlier, and analyzed by multiplex flow cytometry as reported in The DNA encoding 8 different scFv antibodies was sub-cloned from the pEP-AP vector into pEP-AP-Ecoil vector  by digesBssHII and NheI digested DNA fragments encoding each αF1 scFv antibody (described earlier) were ligated into BssHII and NheI digested pDAN5 E. coli DH5aF'. Transformants were plated on 2XYT/Carb/Glu agar. Two colonies were picked for each αF1 scFv, inoculated onto 2XYT/Carb/Glu broth and incubated O/N at 37°C. The DNA from each clone was recovered using the QIAprep spin miniprep kit (Qiagen) according to the manufacturer's instructions. Plasmids were used as template for amplification of scFv genes with primers PDPH3′ (5′ TAACGTCTGGAAAGACGACAA 3′) and PDPH5′ (5′GCAGCCGCTGGATTGTTATTA 3′). A PCR master mix was prepared containing 395 µL Milli-Q autoclaved water, 50 µL Taq thermo buffer (NEB), 20 µL dNTP , 10 µL of 10 µM PDPH3′ primer, 10 µL of 10 µM PDPH5′ primer and 5 µL (25 units) of Taq Polymerase (NEB). 19.6 µL master mix was mixed with 0.4 µL plasmid miniprep and amplification occurred under the following conditions: 94°C for 5 min,  ×40 cycles, 72°C for 7 min. The identity of the clones was confirmed by size, DNA fingerprinting . Following O/N incubation at 30°C with shaking (260 rpm) the cultures were centrifuged at 4,000 rpm for 30 min. The supernatant containing phage was recovered and the phage was PEG/NaCl precipitated at 4°C O/N. Phage precipitates were recovered by centrifugation at 10,000 rpm for 30 min. The resulting phage pellet was re-suspended in 15 mL PBS and the suspension was cleared by centrifugation at 7,000 rpm for 15 min. The phage sample was PEG/NaCl precipitated on ice for 1 h. Precipitated phage was recovered following centrifugation at 10,000 rpm for 30 min, phage pellets were resuspended in 1 mL PBS, cleared by centrifugation and stored at 4°C. Phage was amplified from the phage stocks according to the following method. M13cp-CT helper cells overnight cultures were diluted 1∶50 into 10 mL of 2XYT-Cap broth and incubated at 37°C to OD600  =  0.5. Each phage stock was diluted in 2XYT medium to a final concentration of 1.8×10+9 cfu/mL. 1.8E+9 cfu of each phage was added to 10 mL of M13cp-CT cell (1∶1 multiplicity of infection). Infection occurred at 37°C for 30 min, cells were harvested by centrifugation and resuspended in 50 mL 2XYT-Carb. Additional steps are as for previously described phage production protocol. Phage concentration was determined by either titration  or densitometry. For the latter method 10-fold diluted phage solutions were analyzed by SDS-PAGE together with serial dilution of wild type phage (M13K07) of known titer. The p8 band intensity of scFv-phage was translated into concentrations using the equation defining the standard curve obtained by plotting known M13K07 concentrations (determined by titration) versus corresponding p8 band intensities.DNA plasmids, encoding each selected αF1 and αLysozyme (negative control) scFv were transformed into M13cp-CT helper cells 494/71000) using unlabelled phage as blank. The degree of labeling was determined by dividing FITC concentration by phage concentration (both expressed as particles/volume), resulting in a level of labeling ranging between 30 and 150 fluorophore molecules per phagemid particle.CT1-8, CTD1.3 and wt phage was labeled according to a slight modification of a previously described procedure +12 phage/mL determined by the densitometry-based method described above) at RT for 1 h with mixing. Upon washing the beads were re-suspend in 100 µl of 10-fold diluted wonderblock  in PBS and 1∶500 diluted mouse αM13  following incubation at RT for 1 h with shaking. After washing, phage-bound beads were re-suspended in 100 µl of 10-fold diluted WB in PBS and 1∶1000 diluted goat anti-mouse-PE . After more washing the beads were re-suspended in 200 µl of PBS and analyzed by flow cytometry as described above.The multiplex flow cytometry assay was performed according to a slight modification of a previously described method Y. pestis strains are described in pestis A1122, Kim, India, India 195, CO92 are F1-positive strains while Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 are F1-negative strains. 1 µl of cell glycerol stock was inoculated into 5 ml TSB (tryptone soya broth) medium in a 50 mL falcon tube. The culture was incubated at the 28°C with moderate shaking (240 rpm) for 15–16 hours. Live cells were directly re-suspended in PBS and used in the experimental assay. Whereas those cells to be fixed were washed 3 times with PBS and re-suspended in 4% paraformaldehyde. Cell death was confirmed by culturing paraformaldehyde treated cells on TSB agar plates for 2 days at 37°C.All 600 of 0.1 and incubated either at 4°C O/N or at RT for 1 h. Cells were harvested by centrifugation  and re-suspended in a volume of PBS 10-fold lower than the volume of blocking solution. 1 µL of blocked cells (about 1×10+6 cells) were incubated with 8 µL of FITC-labeled CT1-8 (6×10+11 cfu/mL) or 8 µL negative control phage (wt or CTD1.3 4×10+12 cfu/mL) in 5-fold diluted WB. Cells were incubated at RT for 2.5 h, washed twice with PBS and analyzed by flow cytometry. The fluorescence intensity of each cell type was measured on a FACSCalibur  flow cytometer with laser excitation at 488 nm and fluorescence emission collection through a 530/30 nm filter. Untreated cells were also analyzed to assess auto fluorescence, which was negligible. Resultant data were analyzed by CellQuest software.Fixed cells were blocked by re-suspension in WB to a final ODYersinia cells (in PBS OD600  =  0.2) were transferred into each well of 96-well flat bottom Nunc Maxisorp plate . The plate was incubated O/N at 4°C. Unattached cells were aspirated and plate was washed once with PBS + 0.05% Tween 20 (wash buffer). All the washes were performed by aspiration to eliminate the creation of aerosols. The plate was blocked by addition of 250 µl WB for 1 h at 37°C. scFv-phage (CT1-8 or CTD1.3 as a negative control) was diluted to 4.5×10+9 cfu/mL (determined by densitometry) in WB. 60 µL of each phage solution was added to each well and the plate was incubated at RT with gentle agitation for 2 h and then washed four times with wash buffer and additional four times with PBS + 0.005% Tween 20. aM13-HRP antibody conjugate  was diluted 1∶1000 into 10-fold diluted WB and 60 µL was added to each well of the plate. Following incubation at RT for 1 h, with shaking, the plate was washed as described previously. 100 µL of TMB substrate  was added to each well and the reaction was stopped by the addition of 50 µL 1 M H2SO4. The absorbance was read at 450 nm using victor3 plate reader (PerkinElmer).The ELISA was performed in triplicate under Containment Level 3 Biosafety conditions. 100 µL live or fixed 3 . Phage clones were stored in various conditions including 4°C for 6 months; 4°C for 9 months followed by room temperature for 1 month; 4°C for 9 months followed by room temperature for 2 months. These phage samples and their freshly prepared counterpart were tested by phage ELISA using live Y. pestis A1122 as described above, in triplicate, using a fixed concentration of phage (4.5×10+9 cfu/mL).CT4 CT6 and CT5 (negative control) were solubilized in WB, protease inhibitor cocktail  and NaNFigure S1Genetic maps of pEP-AP (A) and pEP-APEcoil (B) vectors.(TIFF)Click here for additional data file.Table S1A typical set of phage concentrations: Concentration of phage displaying αF1 (CT1-8) or αLysozyme (CTD1.3) scFv were obtained by standard titration and by densitometry. Each value corresponds to the average of two experiments.(DOCX)Click here for additional data file.Table S2A typical set of phage labeling efficiencies: Upon labeling, concentration of phage displaying αF1 or αLysozyme scFv (CT1-8 and CTD1.3 respectively) were determined by densitometry and concentration of FITC was determined by absorbance at 494 nm. Ratio of FITC to phage concentration allowed determination of labeling efficiency. Each value corresponds to the average of two experiments.(DOCX)Click here for additional data file.


We developed a xenograft model of human Chronic Eosinophilic Leukemia (CEL) to study disease progression and remission-induction under therapy with tyrosine kinase inhibitors using imatinib and nilotinib as examples. The FIP1L1/PDGFRA+ human CEL cell lineEOL-1 was injected intravenously into scid mice, and MR imaging and FACS analysis of mouse blood samples were performed to monitor disease development and the effects of imatinib and nilotinib. Organ infiltration was analyzed in detail by immunohistochemistry after sacrifice. All animals developed CEL and within one week of therapy, complete remissions were seen with both imatinib and nilotinib, resulting in reduced total tumor volumes by MR-imaging and almost complete disappearance of EOL-1 cells in the peripheral blood and in tissues. The new model system is feasible for the evaluation of new tyrosine kinase inhibitors and our data suggest that nilotinib may be a valuable additional targeted drug active in patients with FIP1L1/PDGFRA+ CEL. EOL-1 cells from cell culture clearly displayed HLA-DR expression on their surface as analyzed by flow cytometry . EOL-1 cThe percentage of EOL-1 of the non-erythrocyte cells in the mouse blood was analyzed by flow cytometry. On day 28 after injection, EOL-1 cells ranging from below 0.1 to 4% of the non-erythrocytes could be detected in all animals. For part B of the experiment, this percentage dropped to zero in the blood of the imatinib-treated mice already on day 31 (4 days after therapy start) and EOL-1 cells were still undetectable on day 35 (therapy day 8) in the therapy group . In the 3) decreased from a mean of 428.9±149.1 on day 27  to 156.0±67.4 on day 30  and further to 11.9±5.2 on day 34 , massive tumor masses could be detected in all mice by MR imaging. Under imatinib therapy the total tumor volume (given in mm3): From 153.6±49.1 on day 27 (range 45.1 to 343.9 mm3) to a mean of 446.8±65.7 on day 30 (range 252.6 to 599.1 mm3) and to a mean of 1233±172.4 on day 34  all animals were sacrificed. Staining for human HLA-DR revealed EOL-1 cells in sections of paraffin-embedded tissue samples. Chloromas (granulocytic sarcomas) constituted large proportions of the total tumor volume in most animals, however, in the imatinib-treated mice most of the chloromas had gone into complete remission after seven days of therapy. In chloromas from mice of the placebo group, most of the cells were EOL-1  were detectable in the placebo group. Total tumor volume as determined by MR images was drastically reduced after 7 days of therapy in all nilotinib treated mice. Development of total tumor volume for each animal are shown 3 the mean total tumor volumes for the nilotinib treated group from days 27, 31 and 34  were 163.8±50.4, 26.2±10.7 and 3.4±1.4 and 63.93±14.89, 108.3±24.2 and 229.3±47.6 for the placebo group, respectively. Both on day 30 (therapy day 4) and 34 (therapy day 7) the differences between the therapy and the placebo group were highly significant  New in our model is the monitoring of the therapy in the mouse blood using FACS analysis, which was not performed in previous models using tyrosine kinase inhibitors or aurora inhibitors, respectively in vitro data . Long term remissions with molecular remissions can be achieved in more than 90% 3H-thymidine from GE Healthcare , and an antibody against activate caspase 3 (C92-605) from Becton Dickinson Biosciences .The tyrosine kinase inhibitors imatinib (Gleevec®) and nilotinib (Tasigna®) were kindly provided by Novartis . RPMI 1640 medium and fetal calf serum (FCS) were purchased from PAA laboratories , −1 penicillin and 100 µgml−1 streptomycin  at 37°C in a humidified atmosphere of 5% CO2. To investigate drug effects, EOL- 1 cells were incubated with control medium or in various concentrations of imatinib (0.1–100 nM) or nilotinib (0.1–100 nM) at 37°C for 48 hours. Thereafter, the number of apoptotic cells and 3H-thymidine uptake were measured. All experiments were performed at least three times. In select experiments, expression of active caspase 3 in EOL-1 cells was determined by flow cytometry after drug exposure.EOL-1 cells  were cultured in RPMI 1640 medium, supplemented with 10% FCS, 2 mM L-glutamine, 100 UmlAll animals used were pathogen-free Balb/c severe combined immunodeficient scid mice aged 9–14 weeks with a weight of 25–30 g at the beginning of the experiments. The animals were housed in filter-top cages and provided food and water ad libitum.The methodology for carrying out the animal experiments was consistent with the UKCCR guidelines for the welfare of animals in experimental neoplasia part A) was first carried out with 10 mice  and repeated (part B) with 12 mice . The mice were designated with the letter t (for therapy group) or p (placebo group) and a running number. In the following, each individual animal will be referred to as for example “tB3” . For injection, EOL-1 cells were washed and resuspended in PBS at 2×107 cells per ml. All mice received an intravenous injection of 2×106 EOL-1 cells (in 100 µl PBS) and were either treated with intraperitoneal injections of imatinib (100 mg/kg body weight in 100 µl PBS) or PBS as placebo control. The animals each received one injection per day for 10 days.The experiment  and additionally on days 31 and 35 (part B only). All mice were sacrificed on day 35 after 7 days of treatment.The animal experiment using nilotinib for EOL-treatment was performed as described for imatinib above, except as follows.Nilotinib was applied orally via gavage once per day for 7 days (100 mg/kg body weight in 100 µl sterile water), placebo control was sterile water.−1 and a slew rate of 50 mT−1 s−1. For signal reception a dedicated four-element mouse coil  with an inner diameter of 2.5 cm was used. The MR sequence protocol consisted of a T1 weighted localizer sequence in three orthogonal planes, an additional T2 weighted localizer in sagittal orientation followed by a high-resolution fat saturated T1 weighted 2D turbo spin-echo sequence (TSE) in coronar orientation, a fat saturated T2 weighted 2D TSE in coronar and sagittal orientation. Image parameters of the whole body coronar T1 TSE sequence were as follows: time of repetition (TR)/time of echo (TE)/flip angle (FA) = 1275 ms/33 ms/90°; field-of-view (FOV) = 100×35 mm2; matrix = 464×464; slice thickness (ST) = 1 mm, number of slices (NS) = 14; number of acquisitions (NA) = 3; spatial resolution = 0,22×0,22×1 mm; acquisition time = 5 minutes 18 seconds. Image parameters of the fat saturated whole body coronar and sagittal T2 TSE sequence were as follows: TR/TE/FA = 2361 ms/90 ms/90°; FOV = 100×35 mm2; matrix = 448×448; ST = 1 mm, NS = 14; NA = 3; spatial resolution = 0,22×0,22×1 mm; acquisition time = 3 minutes 37 seconds. The tumor size was determined by measuring the largest dimensions of the tumors height, length and width in the coronar and sagittal plane.The animals were anesthesized for approximately 45 minutes by intraperitoneal injection of a weight-adapted dose (1 µl/g body-weight) of a mixture of 1.2 ml Ketamin , 0.8 ml Rompun  und 8 ml physiological saline solution (Invitrogen). MRI was performed on a clinical 3.0T MRI scanner . The scanner was equipped with a conventional body transmit coil and gradient system allowing a maximal amplitude of 30 mT mEach blood sample for FACS-analysis was prepared from 200 µl of fresh EDTA-blood as follows: Erythrolysis was performed according to the manufacturer's instructions using the Mouse Erythrolysis Kit . Cells were stained for 30 min on ice with phycoerythrin-labelled mouse anti-human HLA-DR , diluted 1∶50 in 100 µl FACS buffer  or the corresponding isotype control . After washing, cells were subjected to fluorescence assisted flow cytometry on a FACSCalibur (BD). Files were analyzed using Win MDI 2.9 software.Immunohistochemistry using sections of paraffin-imbedded tumours was carried out as previously described Statistical Analysis was done using GraphPad Prism 5 . Tests used are given for each value of P in the


In the stomach of rodents clusters of brush cells are arranged at the “gastric groove,” immediately at the transition zone from the non-glandular reservoir compartment to the glandular digestive compartment. Based on their taste cell-like molecular phenotype it has been speculated that the cells may be capable to sense constituents of the ingested food, however, searches for nutrient receptors have not been successful. In this study, it was hypothesized that the cells may express receptors for short-chain fatty acids, metabolites generated by microorganisms during the storage of ingested food in the murine forestomach, which lacks the acidic milieu of more posterior regions of the stomach and is colonized with numerous microbiota. Experimental approaches, including RT-PCR analysis and immunohistochemical studies, revealed that the majority of these brush cells express the G-protein coupled receptor types GPR41 (FFAR3) and GPR43 (FFAR2), which are activated by short-chain fatty acids. Both, the GPR41 receptor proteins as well as an appropriate G-protein, α-gustducin, were found to be segregated at the apical brush border of the cells, indicating a direct contact with the luminal content of this gastric region. The exposure of microvillar processes with appropriate receptors and signaling elements to the gastric lumen suggests that the brush cells may in fact be capable to sense the short-chain fatty acids which originate from fermentation processes during the retention of ingested food in the anterior part of the stomach. The stomach of rodents comprises two well-defined areas, a thin-walled, non-glandular compartment, the forestomach (fundus), which serves as a reservoir for food and the thick-walled, glandular section, the corpus, where secretory glands produce digestive enzymes and where the actual digestive processes take place . Animals were fed with standard laboratory chow Total RNA was isolated from dissected tissue preparations of the stomach compartments with a Nucleo Spin RNA kit  according to the manufacturer's protocol. To ensure the complete removal of DNA, a DNase digestion  step was included. Subsequently, 1.0 μ g total RNA was reverse transcribed using oligo (dT) primers and SuperScript III Reverse Transcriptase . RNA integrity of each sample was confirmed by the amplification of the housekeeping gene for the ribosomal protein L8 (RpL8) with intron spanning primers to verify the DNA removal.RT-PCR amplification was conducted by using normalized cDNA from different tissues of the stomach compartments. PCR amplifications were performed with the following primer combinations:GPR41 forward, 5′- CAG AGT GCC AGT TGT CCA ATA-3′; GPR41 reverse, 5′-ATG CCA GGA ACC AAC AGA CT-3′; GPR43 forward, 5′-CAA ACT CGG GAT GCT TCA G-3′; GPR43 reverse, 5′-AGC AGC AAC AGG AGC AAG TC-3′.RT-PCR was carried out using High Fidelity PCR Enzyme Mix  and a Peltier PTC-200 thermo cycler (MJ Research). For amplification the following PCR cycling profiles were used with annealing temperatures adjusted to the used primer combinations and optimized numbers of amplification cycles, as specified in the following.For GPR41:One cycle: 4 min at 94°C; 20 cycles: 30s at 94°C, 30 s at 65°C with −0.5°C per cycle, 40 s at 72°C; 25 cycles: 30 s at 94°C, 30 s at 55°C, 40 s at 72°C; and one cycle: 3 min at 72°C.For GPR43:One cycle: 4min at 94°C; 8 cycles: 30 s at 94°C, 30 s at 68°C with −0.5°C per cycle, 40 s at 72°C; 25 cycles: 30 s at 94°C, 30 s at 64°C, 40 s at 72°C; and one cycle: 3 min at 72°C.PCR products were run on 1.5% agarose gels containing EtdBr. Amplification of a 205 bp fragment from mouse housekeeping control gene ribosomal protein l8 (rpl8) was used as control to confirm equal quality and quantity of the cDNA preparations.For immunohistochemistry, stomachs of adult mice were dissected in 1 × PBS and fixed as described below.2 and perfused via the left heart ventricle with 1 × PBS followed by 4% ice-cold paraformaldehyde with 0.1% glutardialdehyde . After perfusion the tissue was fixed in 1:1 4% PFA:1 × PBS for 24 h.For immunoreactivity to GPR41, CK18, TRPM5, and gustducin antibodies mice were gassed with COFor double-labeling experiments with GPR43 and TRPM5 antibodies, the stomach was fixed in 4% paraformaldehyde  for 7 h at 4°C.After fixation the tissue was cryoprotected by incubation in 25% sucrose overnight at 4°C. Finally, the tissue was embedded in Tissue Freezing Medium and quickly frozen on liquid nitrogen. Cryosections (6 μm) were generated using a CM3050S cryostat  and adhered to Superfrost Plus microscope slides .Cryosections were air-dried, rinsed in 1 × PBS for 10 min at room temperature and blocked in 0.3% Triton X-100 in 1 × PBS containing either 10% normal goat serum  or 10% normal donkey serum  for 30 min at room temperature. For immunostaining with CK18-antibody and in part for GPR41-antibody cryosections underwent citrate-antigen-retrieval. Therefore, frozen sections were incubated in sodium citrate buffer  for 45 min at 4°C. Afterwards sections were immersed in the same sodium citrate buffer for 1–5 min at 100°C. After three rinses for 5 min in 1× PBS, cryosections were blocked in 0.3% Triton X-100 in 1 × PBS containing either 10% normal goat serum (NGS) or 10% normal donkey serum (NDS) for 30 min at room temperature. For single- and double-labeling experiments, primary antibodies were diluted in 0.3% Triton X-100 in 1 × PBS containing either 10% NGS or 10% NDS. Antibodies were used in the following dilutions: rabbit-anti-GPR41  1:30; goat-anti-GPR41  1:30; goat-anti-GPR43  mouse anti-cytokeratin18  1:80; rabbit anti-TRPM5 serum73 [purified antibody (AB-321) described in Kaske et al. ] 1:1000;Blocked sections were incubated with the diluted primary antibodies overnight at 4°C. After washing in 1 × PBS, the bound primary antibodies were visualized using appropriate secondary antibodies conjugated to Alexa 488 or Alexa 568  diluted in 1 × PBS with 0.3% Triton X-100 containing either 10% NGS or 10% NDS for 2 h at room temperature. After three rinses for 5 min in 1 × PBS, the sections were counterstained with 4,6-diamidino-2-phenylindole  for 3 min at room temperature, rinsed with bidest and finally mounted in MOWIOL. No immunoreactivity could be observed when the primary antibodies were omitted.Immunohistochemical staining was documented by using a Zeiss Axiophot microscope . Images were captured using a “Sensi-Cam” CCD-camera . Images were adjusted for contrast in AxioVision LE Rel. 4.3  and arranged in PowerPoint (Microsoft).boxed area) while the cytoplasm exhibited fainter fluorescence. By contrast, CK18 immunofluorescence . This appearance is very reminiscent of the staining pattern obtained for GPR41. For technical reason (antibodies from the same species) double labeling experiments for α-gustducin and GPR41 were not possible. Therefore, thin consecutive tissue sections (6 μm) were used for immunohistochemical studies with antibodies for α-gustducin  and the activation of intracellular Ca2+ stores. Interestingly, brush cells at the “gastric groove” express phospholipase C beta2 (PLCβ2) which mediates the formation of IP3 (Eberle et al., In the stomach of rodents, clusters of brush cells are arranged in a band-like manner between the proximal keratinized reservoir compartment and the more distal glandular part. Due to the expression of molecular elements typical of gustatory sensory cells, it has been suggested that these brush cells may have chemosensory capacity (Höfer et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The adolescent years are among the most stressful times in a person’s life  and if tPatients fulfilling that criteria were included this study 8-17 years of age, diagnosed with AIS, at least two months of experience wearing the brace. The BSSQ consists of 8 Likert-scale items. A maximum score of 24 indicates the lowest level of stress.38 patients  who had came Formed Physical Therapy and Rehabilitation Clinic between March 2008 and November 2010 were included in this study and fulfilled the questionnaire. They were using Cheneau brace for 11.65±11.37 months (range 2-30) 7 were male and 31 were female. Mean Cobb angle was 34.45° ± 10.89 (range 18-57) and mean rotation angle was 6.13° ± 4.19 (range 0-22). The average stress value was 11.57 ± 5.25 (range 22-3). There was a negative correlation between brace using time and stress ; and there was no correlation between age and stress .Previous studies suggested that the BSS questionnaire is reliable for deformity related stress. We used the BSSQ and our study showed that braces in AIS treatment seem to produce stress.


P<0.05). The ratio of ε and βH1 to β2m in EBs was higher than both BMP-4 containing groups (P<0.05) and βmajor was not expressed in EB cells. These findings showed in serum free condition the effects of BMP-4 on the erythroid differentiation was prominent than serum supplemented group.This study was done to compare the effects of bone morphogenetic protein-4 (BMP-4) on mouse embryonic stem cells (ESC) differentiation to erythroid lineage in serum free and serum supplemented media. The embryoid bodies (EBs) cells were seeded in semisolid serum free and serum supplemented media in the presence of different concentrations of BMP-4. The erythroid colonies were assessed morphologically, ultrastructurally and by benzidine staining. The expression of the epsilon (ε), βH1 and βmajor globins, Runx1 and β2m genes was evaluated by Real time PCR. The colony size and the percent of benzidine-positive colonies increased in both BMP-4 supplementd groups but the number of colonies were lower in these groups than control. Erythropoiesis related genes were expressed in both serum free and serum supplemented groups. There were not significant differences between the ratios of genes expression to β2m in these groups except the ratio of Runx1 was significantly higher in serum free group ( Therein vitro –9.Differentiation of ESC into hematopoietic cell types depends upon the addition of some cytokines and growth factors to semisolid culture media which allowed the development of embryoid bodies derived cells in either defined serum free media or serum supplemented media –14. Thesin vitro differentiation systems  supplemented media. Also we tried to determine the erythropoiesis pattern (primitive and definitive) in differentiated cells by analysis of the specific erythropoiesis related gene expression using real time PCR technique.2, and 95% humidity  with 20% fetal bovine serum , 0.1 mM non-essential amino acids , 0.1 mM β-mercaptoethanol  and 1000 U/ml leukemia inhibitory factor . Undifferentiated ESCs were passaged every two days by trypsin /EDTA  dissociation and cultured at 37°C, 5% COhumidity , 24.−4 mol/L; Sigma, USA), ascorbic acid , and l-glutamine . After reaching confluence, the cells were dissociated with trypsin/EDTA and plated on plastic culture dishes for 5 days in the absence of LIF to promote EBs formation (5 × 103 cells/ml)  with FBS (15%) and monothioglycerol (MTG) (1.5 × 10ells/ml) ,25.After dissociation of EBs cells with trypsin/EDTA, their erythroid differentiation was compared in the following experimental and control groups: A; in semisolid serum free media containning different concentrations of BMP-4  and B; in semisolid media supplemented with FBS and different concentrations of BMP-4 . The media without BMP-4 were considered as their controls. Then the expression of the epsilon (ε), βH1 and βmajor globins, Runx1 and β2m genes was evaluated by Real time PCR in the group with high perent of erythroid like colonies.4 cells in IMDM, knock out serum replacement , MTG , ascorbic acid (12.5 ng/ml), l-glutamine , penicillin , streptomycin , and different concentration of BMP-4 .One milliliter of semisolid serum free medium  contained 5 × 104 cells in IMDM, FBS , MTG , ascorbic acid (12.5 ng/ml), l-glutamine , penicillin , streptomycin , and different concentration of BMP-4 .One milliliter of semisolid medium  contained 5 × 10Colony assays were carried out after 14 days for all experimental and control groups. The size and total number of colonies were evaluated using an inverted microscope. The number and percent of erythroid colonies determined morphologically and using benzidine staining.2O2, while benzidine-negative colonies were light yellow  staining to identify hemoglobin-containing cells was carried out according to the Palis method , with slt yellow .The group with high percent of erythroid colonies were selected for transmission electron microscopy (TEM) evaluation therefore, according to the statistical analysis the colonies were collected from semisolid serum free media group containing 20ng/ml of BMP-4 and fixed in 2.5% glutaraldehyde in PBS (pH 7.4) for 2 h and post-fixed with 1% osmium tetroxide in the same buffer for 2 h. After dehydration in a graded ethanol series, they were placed in propylene oxide and embedded in Epon 812. Semi-thin sections (0.5 μm) were stained with toluidine blue for light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy (Zeiss EM 900).After collection of the data and statistical analysis of all experimental and control groups at the first step of study, the colonies formed in 20 ng/ml of BMP-4 in serum free and serum supplemented media were pooled on day 5 of culturing and evaluated for epsilon (ε), beta H1 (βH1) and βmajor globin, Runx1 and β2m genes expression. Also the undifferentiated embryoid bodies were considered as control.Total RNA was extracted using Qiagen kit (USA) according to the manufacturer's instructions and the extracted RNA integrity was confirmed using gel electrophoresis and concentration of each sample was determined by ultraviolet spectrophotometry. After RNA extraction, DNase treatment was done immediately to eliminate genomic DNA contamination according to Fermentas RNase free-DNase kit. Total RNA was stored at −80°C and triplicates were prepared for each colony sample.After extraction of total RNA, one step real time PCR was performed on Rotor-gene 3000 (Corbett) real time thermal Cycler according to QuantiTect SYBR Green RT-PCR kit .For target sequence amplifications, 500ng of RNA was used per 25 μL reaction volume. After completing the PCR run, melt curve analysis was used to confirm the amplified product. The real time PCR products were further verified by agarose gel electrophoresis. For each sample, the reference gene (β2M) and the target genes  were amplified in the same run. Standard curves were obtained by using the logarithmic dilution series of total RNA. Then relative quantitation of target genes was determined by Pfaffl method. The primers sequences for amplification of erythropoiesis related genes are listed in the Table P<0.05).Statistical analysis was done with SPSS software. Data are shown as mean ± SD or as indicated. Colony assays were repeated eight times and the results were compared by Tukey and Pearson Chi-Square tests. The Real time PCR experiments were repeated three times and the results were compared by LSD test (Cultured CCE mouse embryonic stem cells (CCE) formed colonies with irregular borders in undifferentiated state Fig. . Day fivThe colonies with morphology similar to erythroid colonies (BFU-e) were illustrated in Fig. P<0.05).The colonies size in semisolid serum free media containing different concentrations of BMP-4  were 170.24 ± 70, 197.12 ± 73.92, 266.56 ± 58.80, 330 ± 96 and 258 ± 56.56 µm, respectively and there were significant differences between the control and BMP-4-containing groups. The largest colonies were observed in the group containing 20 ng/ml BMP-4 (P<0.05).The colonies size in group with FBS and 0, 5, 10, 20 and 40 ng/ml BMP-4 were 97.6 ± 11.52, 140.76 ± 47.73, 172.80 ± 46.25, 155.90 ± 46.78 and 149 ± 43.58 µm respectively and satatistically differences were seen in the control and BMP-4-containing groups (P<0.05).The number of colonies which formed in serum free media with 0, 5, 10, 20 and 40 ng/ml BMP-4 were 115.13 ± 14.89, 88.75 ± 11.90, 81.5 ± 9.48, 87.75 ± 11.32 and 72.75 ± 11.48 respectively . These rates were statistically different in serum supplemented media with 40ng/ml of BMP-4 and other concentration of BMP-4 and its control groups (P<0.05).The number and percent of benzidine-positive colonies in serum free condition and supplementation with 0, 5, 10, 20 and 40 ng/ml BMP-4 were 7.38 ± 3.37 (6.41%), 14.50 ± 6.90 (16.33%), 29.25 ± 11.63 (35.88%), 30.75 ± 11.53 (35.04%) and 16.75 ± 8.27 (23.02%)  and housekeeping gene (β2m) were expressed in the colonies in serum free and serum supplemented media but in the EBs cells which were not treated with BMP-4 (the control group) the βmajor globin gene was not expressed.t) for each gene, relative quantitation of one step real time PCR were done using Pfaffl method and the ratio of target genes to β2m were calculated and shown in Fig. After obtaining the standard curve and determination of Cycle Thresholds (CP<0.05).A high ratio of ε-globin to β2m gene expression were observed in embryoid bodies cells (0.191 ± 0.045) and there were significant differences between control (EBs cells) and the serum free (0.110 ± 0.032) and serum supplemented media .As were shown in Fig. P<0.05). However this ratio was higher in serum free group than serum supplemented group (P<0.05).The ratio of Runx1 to β2m gene expression was significantly increased in both BMP-4 treated groups: in serum free (0.747 ± 0.142) and serum supplemented groups (0.548 ± 0.079) in comparison to embryoid body's cells  and serum supplemented groups (0.321 ± 0.029) but there was no expression in embryoid body's cells. No significant differences were observed between the ratio of this gene expression in serum free and serum supplemented group.The potential of human, nonhuman primate and murine embryonic stem cells to differentiate to hematopoietic cells were widely investigated using different culture conditions , 15, 20.et al.  groups and this technique provided us additional evidence in this study.P<0.05) and the other genes expression ratios did not show any significant difference between these two groups. Runx1 is an important regulator of hematopoietic development and plays a critical role in the development of both primitive and definitive hematopoietic cells.Our results showed that, all erythropoiesis related genes including epsilon and β H1 globin, Runx1, βmajor globin genes were expressed in the colonies of both systems. Since the ratio of Runx1 to β2m gene expression was higher in serum free group than serum supplemented group  were seen in serum free and serum supplemented conditions on the other hands these results suggested that the cells in both systems exhibit similar gene expression patterns. By contrast Kaufman et al.  showed t  showed that in serum free condition the effect of BMP-4 on the erythroid differentiation was prominent in comparison with serum supplemented group.


The median DOP < 0.000 (Figure P < 0.000 (Figure 2I in the postoperative period. Patients who achieved a DO2I of 600 ml/minute/m2 were less likely to suffer postoperative complications and have a significantly reduced length of hospital stay.Postoperative GDT was able to increase DO


Robinia pseudoacacia L.) is adaptable to salt stress. Here, we compared morphological, physiological, ultrastructural, and proteomic traits of leaves in tetraploid black locust and its diploid relatives under salt stress. The results showed that diploid (2×) plants suffered from greater negative effects than those of tetraploid (4×) plants. After salt treatment, plant growth was inhibited, photosynthesis was reduced, reactive oxygen species, malondialdehyde content, and relative electrolyte leakage increased, and defense-related enzyme activities decreased in 2× compared to those in 4×. In addition, salt stress resulted in distorted chloroplasts, swollen thylakoid membranes, accumulation of plastoglobules, and increased starch grains in 2× compared to those in 4×. However, 4× developed diverse responses under salt stress. A comparative proteomic analysis revealed that 41 and 37 proteins were differentially expressed in 2× and 4×, respectively. These proteins were mainly involved in photosynthesis, stress and defense, energy, metabolism, transcription/translation, and transportation. Distinct patterns of protein changes between 2× and 4× were analyzed. Collectively, our results suggest that the plants showed significantly different responses to salt stress based on ploidy level of the plant. The 4× possessed a better salt protection mechanism than that of 2×, suggesting salt tolerance in the polyploid plant.Tetraploid black locust ( In geneal level . Proteomal level ,7. EarliPolyploidy arises from the doubling of chromosomes of a single species (autopolyploidy) or the hybrids between two species . Polyploidy usually changes anatomical and morphological characteristics such as an increase in leaf thickness and pubescence ,9. MoreoRobinia pseudoacacia L.), which is native to Korea, is a preferred tree species in the timber forest due to its rapid growth and good wood texture. Moreover, it can be used as fine feed for domestic fowl and livestock because its fleshy leaves are rich in vitamins and minerals. Importantly, tetraploid black locust is a pioneer tree species due to it wide ranging adaptability to adverse environments such as salt, drought, cold, and pests. Accordingly, tetraploid black locust has high ecological and economic value.Tetraploid (4×) black locust  to determine growth and physiological traits under salt stress in 2× and 4× black locust (2) to improve the understanding of the molecular mechanism of differential salt tolerance in different ploidy species, and (3) to aid in the rational engineering of plants with enhanced salt tolerance.2.2.1.Diploid plants began to wilt after 10 days and some plants died after 15 days under salt stress, as shown in 2− and H2O2 contents in both 2× and 4× plants  content and relative electrolyte leakage (REL) increased in 2× and 4× after salt treatment, and 2× showed much higher levels than that of 4×  and stomatal conductance (Gs) were higher in 4× than those in 2×  of 2× and 4× did not decrease significantly under salt treatment  activity decreased in 2× and 4× after salt treatment, but 4× showed higher SOD activity than that of 2× . The actPn and Gs for 4× were not sensitive in response to salt stress after RWC decreases are detectable. It may be a reason that the salt stress caused less pronounced inhibition of photosynthesis in 4× than in 2×. In other words, 4× plants have higher water use efficiency under salt stress. Thus, 4× can keep photosynthesis stability under low RWC to adapt salt stress. At the same time, RWC is not only factor influencing Pn. The decrease in growth and stomatal conductance may result from reduced leaf water content. Gs plays an important role in the change in Pn. Little change in Ci was observed compared with that of Gs  in 4× plants increased under salt stress. All antioxidant enzymes play an important role adjusting the cellular redox state [Salt stress induces the accumulation of reactive oxygen species (ROS) such as Hproteins . H2O2 anox state . In contox state . However2.2.Cs)/thylakoid (Th) of the mesophyll cells of 2× and 4× plants after one day of salt treatment  changes under the salt treatments were submitted for protein identification. All 41 protein spots from 2× gels and 37 protein spots from 4× gels were detected as differentially expressed spots  and increased growth rate. This result substantiated that 4× can tolerate high salt stress.Salt stress is a major environmental factor that limits the efficiency of photosynthesis. In our study, nearly half the proteins (27 in 2× and 23 in 4×) were photosynthesis-related proteins. Among them, a number of proteins (20 in 2× and 17 in 4×) were assigned to the Rubisco large subunit from different plant species but are visualized at positions with very different pIs and molecular weights  and 2. T2 fixation. Down-regulation of RuBPCase leads to a reduction in photosynthetic capacity. But, changes in these enzymes were not identified in 4× under salt stress. A possible reason is that 2× and 4× used different strategies to combat salt stress. Particularly, ATP synthase CF1 alpha subunit showed increased levels in 4× but decreased levels in 2× at 10 days of salt treatment. The increase in ATP synthase CF1 alpha subunit increases photosynthesis rate and down-regulates the Calvin cycle enzyme phosphoglycerate kinase [Photosystem II (PSII), as the core complex in photosynthesis, mediates oxygen evolution activity. PSII proteins (spot 578) in 2× were down-regulated under salt stress, suggesting that PSII activity was inhibited. These results were similar to previous observations . In addie kinase . Clearly2.3.2.2O2 to H2O and plays an important role resisting salt stress in plants [Salt stress can lead to ion imbalance, hyperosmotic stress, and oxidative damage. Thus, plants induce various stress response proteins, many of which are crucial components of the plants self-defense network. Five and four proteins had altered translation levels in response to salinity stress in 2× and 4×, respectively. Here, they were only visible in the treated 2× and 4× plants. Agglutinin I polypeptide B and phenylalanine ammonia lyase were significantly up-regulated after salt treatment in 2×, whereas heat shock protein 70 and plastidic aldolase decreased under salt stress . In 4×, n plants . These rLectins are carbohydrate-binding proteins expressed in plants upon exposure to biotic or abiotic stressors such as drought, salinity, heat, hormone treatment, pathogen attack, or insect herbivory. In the present study, the abundance of agglutinin I polypeptide B in 2× and chain A, a legume lectin of the bark in 4×, increased after salt treatment. Lectins reduce the detrimental effects of salinity-induced oxidative stress in wheat seedlings, which is consistent with other findings . Clearly2.3.3.When plants are exposed to high levels of salt, extra energy may be consumed to prevent damage. Phosphoglycerate kinase (PGK) and mitochondrial F1-ATPase beta subunits increased in 4× under salinity stress . Some rein vivo and in vitro.The same PGK (spot 494) was down-regulated in 2× under salt stress. However, another PGK was up-regulated. This was inconsistent with what was found in 4×. This different response to salt stress between the two plants is interesting. However, the PGK protein was found in two spots, which is presumably due to post-transcriptional modification or proteolytic degradation of proteins 2.3.4.Aconitate hydratase and pyruvate kinase are essential respiratory metabolism enzymes in plants. The abundance of aconitate hydratase and pyruvate kinase suggests that the tricarboxylic acid (TCA) and glycolysis increase under salinity stress. Enhancement of TCA and glycolysis would produce more ATP. Increases in aconitate hydratase and pyruvate kinase involved in respiratory metabolism showed the capacity of 2× plants to recover from salt stress. In contrast, no proteins associated with TCA or glycolysis changed significantly under salt stress in 4× plants. Besides, we found that the levels of chalcone synthase (CHS) and enolase changed remarkably in 4× under salt stress. A large number of investigations have shown that CHS and enolase are induced by various environmental stressors. Therefore, it was considered that 4× plants used another pathway to resist salt stress, or 4× as a halophyte had a high capacity to prevent damage resulting from oxidative stress.In this study, to keep genetic stability, we propagated plants by cutting. Generally, cutting propagation can produce genetically identical progeny compared with hybrids propagation. Thus, cutting reproduction strongly influences genetic variation. Now, other methods such as flow cytometry and chromosome counting have been used for ploidy estimation (11).3.3.1.v/v) mixture of soil and sand. The experiments were carried out at Harbin Experimental Forest Farm of Northeast Forestry University in June 2011. Potted plants were grown in the greenhouse  for 1 month. Previous research showed that 4× tolerate high salt stress (500 mM NaCl) for a short time. Thus, in this study, we treated plants with 500 mM NaCl for 15 days. After 1, 5, 10, and 15 days of treatment, the leaves were harvested, immediately frozen in liquid nitrogen, and stored at −80 °C prior to physiological and proteomic experiments. At least three independent biological experiments for each treatment were replicated.All materials are introduced directly from South Korea to China by Beijing Forestry University. The diploid and tetraploid plants are from a germplasm. Thus, they have the same genetic origin. Thirty uniform plants (2-years-old) from 2× and 4× black locust were collected from Beijing Forestry University in Beijing, China and planted in plastic pots (18 cm in diameter and 18 cm in depth) filled with 2:1 (3.2.h′2 − h′1)/(h2 − h1), where h′1 is the height of salt-stressed plant at the beginning, h′2 is the height of salt-stressed plants at the end, h1 is the height of control plants at the beginning, h2 is the height of control plant at the end. RGR = (r′2 − r′1)/(r2 − r1), where r′1 is the range of stressed plants at the beginning, r′2 is the range of stressed plants at the end, r1 is the range of control plants at the beginning, r2 is the range of control plants at the end. BGR = (b′2 − b′1)/(b2 − b1), where b′1 is the stem basal diameter of stressed plants at the beginning, b′2 is the stem basal diameter of stressed plants at the end, b1 is the stem basal diameter of control plants at the beginning, b2 is the stem basal diameter of control plants at the end. At 9:30 AM, the fully expanded leaves were collected to measure relative water content (RWC) of leaves using the following formula: RWC (%) = (FW − DW)/(TW − DW) × 100, where FW is fresh weight, TW is turgid weight after rehydrating the samples for 24 h, and DW is dry weight after oven-drying the samples at 85 °C for 24 h.The morphological traits of all plants were observed and photographed at 1, 5, 10, and 15 days of treatment. At the beginning and end of the salt treatments, height, range, and stem basal diameter of plants and leaf water content were recorded. The HGR, RGR, and the relative growth rate of stem basal diameter (BGR) were calculated using the following formulas: HGR = (3.3.3 (v/v)) using the method of Wolf (1982) to measure Na+ and K+[+ and K+ were analyzed by flame emission using atomic absorption spectrophotometry . Cl− was quantified by a modified silver titration method described by Chen et al. (2001) [Leaves were dried at 80 °C and digested in nitric acid (1% HNOa+ and K+. Na+ and. (2001) .3.4.w/v) PVP, 0.1 mM PMSF, and 0.2% (v/v) Triton X-100. The mixture was centrifuged at 12,000× g for 15 min at 4 °C. One millilitre the supernatant was mixed with a mixture of 1 mL hydroxylamine hydrochloride for 1 h, 1 mL β-aminobenzene sulfonic acid, and 1 mL α-naphthylamine, and then the solution was incubated at 25 °C for 20 min. The concentration of superoxide radical was determined by measuring the absorbance of the mixture at 530 nm using a NaNO2 standard curve. Hydrogen peroxide (H2O2) content was determined by measuring the absorbance of the H2O2 titanium complex at 410 nm using known concentrations of H2O2 as the standard curve. Lipid peroxidation was estimated as MDA content using a modified method. Leaves (0.2 g) were homogenized in 5 mL of 5% of trichloroacetic acid (TCA) and centrifuged at 12,000× g for 15 min. Two ml of supernatant was added to a test tube containing the mixture of 2 mL of 20% TCA, 0.01% butylated hydroxytoluene, and 0.6% thiobarbituric acid. The mixture was heated in boiling water for 30 min, and then quickly cooled on ice. After centrifugation at 12,000× g for 10 min, the absorbance of the supernatant was determined at 450, 532 and 600 nm.Leaves (0.5 g) were ground at 4 °C and then homogenized in 5 mL 50 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 1% (2) from the third to fifth fully expanded leaves were vacuum-infiltrated in 10 mL deionized water for 30 min and maintained in water for 6 h. Using a portable conductivity detector , the conductivity of the bathing solution (C1) was determined. Then, the leaf discs and the bathing solution were boiled for 10 min and thoroughly cooled to room temperature, and the conductivity of the resulting solution (C2) was determined. The REL was calculated (REL (%) = (C1/C2) × 100).Twenty leaf discs (1.0 cm3.5.Pn), Gs, and intercellular CO2 concentration (Ci) were measured at 0, 5, and 10 days of salt treatment from 9:00 to 11:30 in the morning with a portable photosynthesis measuring system LI-COR 6400 .Net photosynthetic rate  activity was assayed using the method of Nordhoff et al. (1993) [2O2 at a final concentration of 0.1 mM.SOD (EC1.15.1.1) activity was measured following the method of Roth and Gilbert (1984) . The rea. (1993) . GR acti3.7.v/v) at 4 °C for 2 h, washed twice in 0.1 M PBS  at 4 °C. Then, they were postfixed in 2% osmium tetraoxide (OsO4) for 2 h, sequentially dehydrated in 50%, 70%, 90%, and 100% acetone, and embedded in Epon 812 for 2 h. Ultra-thin sections (70 nm) were sliced, stained with uranyl acetate and lead citrate, and then mounted on copper grids for viewing on the H-600 IV TEM  at an accelerating voltage of 60 kV.Fresh leaf segments (about 1.5 cm in length and 0.5 cm in width) were fixed in 2.5% glutaral pentanedial , and the supernatant was collected by centrifugation at 15,000× g for 50 min at 4 °C. The protein content was assayed using bovine serum albumin as the standard according to the method of Bradford (1976) [Total leaf protein of plants at different treatment times  was extracted using the method of Hurkman and Tanaka (1986) . Fresh ld (1976) .3.9.et al. (1982). A mixture of 300 μg protein sample in 350 μL of rehydration buffer containing 7 M urea, 2 M thiourea, 2% w/v CHAPS, 40 mM DTT, and 0.5% v/v IPG buffers, pH 4–7 , and 0.01% bromophenol blue was prepared. The mixture was loaded onto IPG strips . After overnight rehydration of the IPG strips at 20 °C, isoelectric focusing was carried out on an Ettan IPGphorII  at 20 °C. Isoelectric focusing was performed using the following procedure: 30 V for 2 h, 100 V for 1 h, 500 V for 1 h, 1000 V for 1 h, gradient 8000 V for 0.5 h, and 8,000 V rapid focus for 6 h. After focusing, the strips were equilibrated by reduction with DTT and carboxymethylation with iodoacetamide. The second dimension was performed on 12.5% (w/v) polyacrylamide gels at 15 mA per gel for 30 min followed by 30 mA until the bromophenol blue ran off the bottom of the gel. Gels were stained with Coomassie Brilliant Blue R-250 and destained the next day.2-DE was carried out according to the method of Bjellqvist p < 0.05 and a change of more than two-fold in abundance, were considered to be differentially accumulated proteins, and they had to be consistently present in three replications.Gel images were scanned using an ImageScanner III . Images were analyzed with ImageMaster 2D Platinum 7.0 software . The average volume percent values were calculated from three technical replicates to represent the final volume percent values of each biological replicate. The experimental molecular mass and PI of the protein spots were determined by 2-DE standards and interpolation of missing values on the IPG strips. Spots were quantified based on total density of the gels by the percentage volume. Significantly different spots, which were determined as 3.10.v/v) acetonitrile in 0.1 M NH4HCO3, and dried at room temperature. Proteins were reduced with 1 mM DTT and 2 mM NH4HCO3 at 55 °C for 1 h and alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 in the dark at room temperature for 45 min. The gel pieces were thoroughly washed with 25 mM NH4HCO3, 50% ACN, 100% ACN, and dried. The proteins were digested in 10 mL modified trypsin  solution (1 ng mL−1 in 25 mM ammonium bicarbonate) during an overnight incubation at 37 °C. Digests were immediately spotted onto 600 mm Anchorchips . Spotting was achieved by pipetting 1 mL of analyte onto the MALDI target plate in duplicate and then adding 0.05 mL of 20 mg mL−1 α-CHCA in 0.1% TFA/33% (v/v) ACN, which contained 2 mM ammonium phosphate. All samples were analyzed in the positive-ion reflectron mode on a TOF Ultraflex II mass spectrometer . Each acquired mass spectra  was processed using FlexAnalysis v2.4 software . Proteins were identified with Mascot software (http://www.matrixscience.com) based on the mass signals to search for proteins in the SwissProt, NCBInr, and MSDB databases.Selected protein spots were excised, washed with 50% . All parameters are presented as mean ± standard error and were obtained from at least three replicates and analyzed using Duncan’s multiple range test or Student’s 4.Tetraploid black locust has high ecological and economic value. Until now, little was known about the implications of ploidy level in black locust on the physiological and proteomic responses under salt stress. In this study, the physiological and proteomic responses of 2× and 4× black locust were detected under salt stress during different times. Our results demonstrated that 2× plants suffered from greater negative effects than those of 4× plants according to their morphological and physiological characteristics under salt stress. In addition, 2-DE was used to analyze ploidy differences of the black locust leaf proteome under salt stress. Therefore, 4× plants have higher levels of several ROS scavenging enzymes and accumulate photosynthesis-related enzymes, defense protein and energy proteins to cope with salt stress compared with 2×. These results contribute to selection of some specific proteins during ploidy process. In addition, the results suggested that 4× had a greater capability to defend against salt stress by accumulating more related proteins. Our results provide more information to further understand the molecular and physiological basis of stress tolerance in polyploid plants.


The S2 values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The Rex values of Rho residues R49 and D51 were 0.91 s−1 and 1.42 s−1; however, no Rex was found for those of the P48A mutant. The τe values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5β1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5β1.Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4–11.5 times more actively inhibited integrin α5β1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The values of the  The tripeptide sequence Arg-Gly-Asp (RGD) is the consensus sequence of many adhesive proteins, such as fibronectin, fibrinogen, vitronectin, and von Willebrand factor W sequence have a higher affinity for binding with the integrin αIIbβ3, whereas disintegrins with an ARGDN sequence preferentially bind with integrins αvβ3 and α5β1 ARGDMP sequence, diminishes its binding to integrin α5β1 Many studies have shown that the residues flanking the RGD motif of RGD-containing proteins affect their binding specificities and affinities on integrins Calloselasma rhodostoma venom and belongs to the family of disintegrins Pichia pastoris (P. pastoris) has the same function and structure as native protein In this study, we used Rho as the model protein to investigate the effect of the N-terminal proline residue adjacent to the RGD motif on the dynamics of disintegrin and the structure-activity relationships of RGD-containing proteins. Rho is obtained from P. pastoris was accomplished by following protocols previously described Pichia strain, X33, using a Pichia EasyComp kit from Invitrogen.The expression of Rho and eleven mutants  in 15N-labelled Rho and its mutants were produced by following protocols previously described −5% biotin). Methanol (1% w/v) was added once every 12 h to induce protein expression for 2 days. The 15N-labelled proteins were produced as follows: 100 µL of cell stock grew at 30°C in 100 mL of 15N minimal medium  in 100 mM of potassium phosphate buffer with 100 µg/mL of Zeocin for 48 h. The cells were then transferred into 900 mL of 15N minimal medium. After another 24 h, the cells were collected by centrifugation and grown in 1 L of 15N minimal medium in 100 mM of potassium phosphate buffer with 4×10−5% biotin. The methanol in the medium was maintained at 1% (w/v) in order to induce protein expression for 48 h.Unlabelled and 2O and once against 5 L of binding buffer (50 mM Tris-HCl buffer at pH 8.0). The dialyzed solution was loaded into a Ni2+-chelating column and proteins were eluted using elution buffer containing 200 mM of imidazole. Proteins were then purified using C18 reversed-phase HPLC with a gradient of 20–30% acetonitrile. The recombinant proteins were more than 95% pure, as determined using tricine-SDS-PAGE.The supernatant was collected by centrifugation and dialyzed twice against 10 L of H3, and 1 mM benzamidine. The resin was washed with 1 M NaCl and 1 mM benzamidine at pH 7, and fibronectin then was eluted using 1 M urea and 1 mM benzamidine at pH 7. The fractions were dialyzed three times against 4 L of PBS buffer at pH 7.4 and concentrated using Amicon with a 10-kDa cutoff membrane. The yields of fibronectin were 15–20 mg, and the purification of human fibronectin was greater than 95% as determined using SDS-PAGE. Purified fibronectin was stored at −70°C until it was used.Fibronectin was purified from citrated human plasma using gelatin-Sepharose 4B affinity chromatography as previously described The molecular weights of Rho mutant proteins were confirmed using an API 365 triple quadrupole mass spectrometer equipped with a TurboIonSpray source . Protein solutions (1–10 µM in 50–90% methanol or acetonitrile with 0.1% formic acid) were infused into the mass spectrometer using a syringe pump  at a flow rate of 12–20 µL/min to acquire full-scan mass spectra. The electrospray voltage at the spraying needle was optimized at 5000–5300 V. The molecular weights of proteins were calculated using computer software provided with the API 365 mass spectrometer.g for 10 min to obtain platelet-rich plasma (PRP) and allowed to stand for 5 min. Then, PRP was collected. Platelet-poor plasma (PPP) was prepared from the remaining blood by centrifuging it at 800× g for 10 min. The PPP platelet count was measured on a haematology analyzer, and the platelets were diluted to 250,000 platelets/µL. A solution of PRP (190 µL) and either Rho or PBS buffer (10 µL) was incubated for 5 min in a Hema Tracer 601 aggregometer at 37°C. Ten microlitres of 200 mM ADP was added to monitor the response of platelet aggregation by light transmission.Venous blood (9 parts) from healthy donors who had not received any medication for at least two weeks were collected in 3.8% sodium citrate (1 part). Blood was centrifuged at 70× A cell adhesion assay was done using protocols previously described 4, 2 mM CaCl2, and 500 µM MnCl2. CHO and human erythroleukemia K562 cells were diluted to 3 and 2.5×105 cells/mL, respectively, and 100 µL of the cells were used for the assay. Rho and its mutants (0.001–500 µM), which were used as inhibitors, were added to the cells and incubated at 37°C in a 5% CO2 atmosphere for 15 min. The treated cells were then added to the coated plate and reacted at 37°C (5% CO2) for 1 h. The reacting solution was then discarded and non-adhered cells were removed by washing them twice with 200 µL of PBS. After the non-adhered cells had been removed by rinsing the wells with the same buffer, adhered cells were quantified using a crystal violet assay. The well was fixed with 100 µL of 10% formalin for 10 min and then dried. A solution of 50 µL of 0.05% crystal violet was added to the well at room temperature for 20 min. Each well was then washed four times with 200 µL of distilled water and dried. Colorization was done by adding 150 µL of colorizing solution . The resulting absorbance was read at 600 nm and the readings were correlated with the number of adhering cells. Inhibition was defined as50 values are the average of at least three separate experiments.Chinese hamster ovary (CHO) cells that expressed the integrins αvβ3 (CHO-αvβ3) and αIIbβ3 (CHO-αIIbβ3) were kindly provided by Dr. Y. Takada (Scripps Research Institute) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) medium 2O/90% H2O or 100% D2O at a concentration of 3 mM; pH was adjusted with 100 mM KOD to 6.0. The data were processed with Topspin Version 1.3 software and analyzed with Aurelia software. 2D NOESY, TOCSY, and DQF-COSY NMR spectra were recorded in the phase-sensitive absorption mode with quadrature detection in both F1 and F2 dimensions. A concentration of 2 mM 15N labeled Rho and its P48A mutant was used for the 2D 1H-15N HSQC, 3D 1H-15N edited-TOCSY-, and NOESY-HSQC experiments. Mixing times of 30–90 ms and 60–150 ms were used for TOCSY and NOESY experiments, respectively. The centre frequencies of double resonance experiments were 4.75 ppm (1H) and 118 ppm (15N). The observed 1H chemical shifts were referenced with respect to the H2O or HOD signal, which was 4.754 ppm downfield from external sodium 3-trimethylsilylpropionate-2,2,3,3-d4 (TSP) in D2O (0.0 ppm) at 300°K. The nitrogen chemical shift was referenced to external 15NH4Cl (3 mM in 1 M HCl) at 300°K, which is 24.93 ppm downfield from liquid NH3.NMR experiments were done at 27°C on a Bruker Avance 600- and 700-MHz spectrometer equipped with pulse field gradients and xyz-gradient triple-resonance probes. In these experiments, samples were dissolved in 10% D3JNHα coupling constants. For 3JNHα values less than 5 Hz, ϕ values were restricted from −30° to −90°, and for 3JNHα values greater than 10 Hz, ϕ values were restricted from −100° to −170°. Two restraints were used for each NH-CO backbone hydrogen bond with dN-O restricted to 2.4–3.3 Å and d H-O to 1.7–2.3 Å. A family of 100 structures was generated using NOE distance, dihedral angle, and hydrogen bond restraints. The S-S covalent bonds were deleted and reintroduced as pseudo-NOE distances with the S-S distances constrained to the upper limit of 2.1 Å. During the first phase of dynamics at 2000°K, the value of the force constant of the NOE term was kept constant at 50 kcal/mol−1 Å−2. The repulsion term was gradually increased from 0.03 to 4.0 kcal/mol−1 Å−2, and the torsion angle term from 5 to 200 kcal/mol−1rad−2. The simulated annealing refinement consisted of a 9-ps cooling dynamic followed by 200 cycles of Powell minimization. The twenty lowest-energy structures were accepted based on violations of distance restraints less than 0.5 Å, dihedral angle restraints less than 5°, a van der Waals energy cutoff value of 35 kcal/mol, and an NOE energy cutoff value of 55 kcal/mol. The structure figures were prepared using the MOLMOL or the PyMOL program Structures were calculated using the program X-PLOR with the hybrid distance geometry-dynamical simulated annealing method 15N- spin-lattice (1R) and spin-spin (2R) relaxation rate constants and steady-state 1H-15N NOEs were measured from 1H-detected 1H-15N correlation spectra recorded with sensitivity-enhanced pulse sequences. A recycle delay of 6 s was used, and 128 complex T1 increments of 32 scans were acquired. A series of 10 experiments with relaxation delays of 30, 100, 150, 300, 450, 600, 800, 1000, 1500, and 3000 ms were done to measure T1. A series of 10 experiments with relaxation delays of 18, 36, 48, 72, 90, 100, 120, 150, 300 and 500 ms were done to measure T2. The longitudinal and transverse relaxation rate constants, 1R and 2R, were obtained from exponential fits of the peak height data using least-squares fit software SigmaPlot (Jandel Scientific). The reported iR values are the mean values of two independent data sets. In the NOE experiment, two spectra—one with the NOE and one without—were collected. The NOE was calculated as the ratio of peak heights in spectra collected with and without NOE. The reported NOE value was the average value of three pairs of NOE experiments.Backbone dynamics of Rho and its P48A mutant were studied by two-dimensional proton-detected heteronuclear NMR spectroscopy. The 15N relaxation rate constants, 1R and 2R, and the 1H-15N steady state NOE values were analyzed using the FastModelFree program m is the overall rotational correlation time of the molecule, τe is the effective correlation time for the motions on the slower of the two time scale, S2 is the square of the generalized order parameter, and S2s and S2f are the squares of order parameters for the motion on the slow and fast time scale, respectively The heteronuclear 2+ of the MIDAS  of β1. Additional 0.5 Å distance was added to the upper and lower limits in the direct interaction restraints. Using these restraints, the standard HADDOCK protocol for protein docking was done with minor modifications. This protocol combines three stages of molecular dynamics calculations, including heating and cooling with a progressive increase of the flexibility at the binding interface. In the first stage, 500 conformations were calculated using a rigid-body docking protocol. The best 100 structures in terms of their inter-molecular energies were refined by semi-flexible simulated annealing in the second stage. Both the side-chains and the backbone atoms of the residues 46–54 were defined as flexible and allowed the residues to move in a semi-rigid-body docking protocol to search for conformational rearrangements. The resulting 100 structures with the lowest intermolecular energy values were refined with explicit water molecules in the last stage. The structures were classified by clustering based on the pairwise RMSD differences. The structures were found by fitting them over the RGD residues with an average RMSD value <1.5 Å for the backbone atoms of all the amino acids in 100 integrin complexes.The dockings of Rho and its P48A mutant to integrin α5β1 were done using the docking program HADDOCK 2.1 with hydrogen bond and distance restraints The coordinates of 20 calculated structures of P48A mutant were deposited in the Protein Data Bank under accession number 2PJI.The ethics approval for our study was approved by an independent ethics committee of National Cheng Kung University. The collection of human serum was followed the guidelines and regulation. We have obtained written informed consent from all participants involved in this study.P. pastoris X33 strain. Rho and its P48A, M52W, P48A/M52W, M52N, P48A/M52N, M52G/P53W, P48A/M52G/P53W, M52D/P53L, P48A/M52D/P53L, M52D/P53M, and P48A/M52D/P53M mutants produced in P. pastoris were purified to homogeneity, according to SDS-polyacrylamide gel electrophoresis (data not shown), using Ni2+-chelating chromatography and C18 reversed-phase HPLC. The yields of Rho and its mutants were 10–25 mg/L. Mass spectrometry showed that the experimental molecular weights deviated less than 1 Da when compared with the theoretical values, which had been calculated by assuming that all cysteines form disulfide bonds, and would result in the formation of six disulfide bonds in Rho and its mutants  (G/A)RGD but not PRGD sequences for binding.Proline residue is the only common imino acid in proteins with a bulky pyrrolidine ring that restricts the conformational range of its adjacent residues. The lack of a proton on the imino nitrogen of proline blocks the hydrogen-bond formation required for α-helix and β-sheet secondary structures, and thus disrupts the propagation of neighbouring secondary structures. Therefore, it is commonly found in the turn and loop structures of proteins 2R values of the R and D residues, which resulted in a decrease of the rigidity, the disappearance of conformational exchange, and the increase of fast motion on the ps-ns timescale of the RGD motif. The RGD motif, the integrin-binding site, of P48A mutant is clearly more flexible on the ps-ns timescale than on that of Rho, as indicated by both the dramatically lower-order parameters and much larger effective internal correlation times. This effect may facilitate the P48A mutants to interact with integrin α5β1. This is consistent with the dynamic features of the RGD motif in fibronectin, which preferentially binds to integrin α5β1 The synergy between structure and dynamics is essential to the function of biological complexes. We found that the effect of the N-terminal proline residue of the RGD motif in Rho on reducing its binding affinity to integrin α5β1 may be due to its effect on the dynamic properties of the RGD motif. The proline effect is commonly attributed to the limitation of its backbone entropy. The cis-trans isomerisation of the proline peptide bond is also responsible for the activities of many proteins and peptides In conclusion, we found that the N-terminal proline residue adjacent to the RGD motif affects the function and dynamic properties of the RGD motif, which shows that the dynamic properties of the RGD motif in RGD-containing proteins may be important for integrin recognition. Our functional analysis also showed that the integrin α5β1 ligands prefer to have N-terminal residue that contains either glycine or alanine amino acid. These results provide important dynamic information for designing potent RGD mimetics and serve as the basis for exploring the structure and the functional relationships of RGD-binding integrins and their ligands.Figure S1Mass spectra of recombinant Rho variants.(TIF)Click here for additional data file.Figure S2Map of the NOE connectivities of detected in Rho (A) and its P48A mutant (B). NOEs involving the sidechain resonances are plotted below the diagonal, and those involving only mainchain resonances are plotted above the diagonal.(TIF)Click here for additional data file.Figure S3Amide strips from I47 to M52 of Rho (A) and its P48A mutant (B) at pH 6.0. The dNN  and dαN  NOE connectivities are shown.(TIF)Click here for additional data file.Figure S4Comparison of the relaxation parameters of Rho (▪) and its P48A mutant (□).15N 1R with error (A). 15N 2R with error (B).1H-15N steady-state NOE with error (C) These experiments were acquired using 700 MHz NMR.(TIF)Click here for additional data file.Figure S5Comparison of model-free parameters of Rho (▪) and its P48A mutant (Δ). Generalized order parameters S2, τe, and Rex . Gaps indicate the proline residues, and the β-sheet secondary structure is shown. Comparison of internal timescale parameters, τe, of Rho and its P48A Mutant (B). Only some fitting models resulted in a τe term. Comparison of the conformational exchange terms, Rex, for Rho and its P48A mutant (C). Only some fitting models resulted in an Rex term.(TIF)Click here for additional data file.Table S1Molecular weights of recombinant Rho variants.(DOC)Click here for additional data file.Table S2Summary of the interactions between protein and integrin.(DOC)Click here for additional data file.Table S3Relaxation data and dynamic parameters of Rho and P48A mutant (700 MHz).(DOC)Click here for additional data file.