Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents) were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM) in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS . Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate) receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release.In corticostriatal synapses, LTD (long-term depression) and LTP (long-term potentiation) are modulated by the activation of DA (dopamine) receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A) phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5) phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) at Thr Cdk5 inhibition favors striatal LTP induction through pre- and post-synaptic mechanisms. Striatal MSN (medium spiny neurons) exhibit short-term and long-term plasticity    phosphorylates DARPP-32 at the ThrN-methyl-D-aspartate) receptors, PKA and L-type Ca  channels. It also demonstrated that plasticity induced by Roscovitine has a presynaptic and a postsynaptic component.The present study demonstrated that Roscovitine increased the percentage of experiments that exhibit LTP induction, as well as the amplitude of population spikes recorded in glutamatergic corticostriatal synapses. Furthermore, LTP induced by Roscovitine is modulated by the activation of dopamine and NMDA .All experiments were carried out with the Institutional approval in accordance with the Mexican (NOM-062-ZOO-1999), Institutional  and NHI regulations for the care and use of experimental animals  aged 4–5 weeks. They were housed in plastic cages, in groups of 5–6, in a room at 25°C with a 12/12 light-dark cycle, with free access to food and water.2+ saline solution with the following composition (in mM): 130 NaCl, 2 KCl, 5 MgCl2, 1 CaCl2, 26 NaHCO3 and 10 glucose saturated with 95% (v/v) O2 and 5% (v/v) CO2 . After 1 h of recovery, slices used for field potential recordings were transferred to a submerge chamber and superfused with ACSF  at 34°C. The ACSF composition was (in mM): 125 NaCl, 26 NaHCO3, 1 MgCl2, 3 KCl, 2 CaCl2, 10 glucose, 0.2 Thiourea, and 0.20 ascorbic acid, gassed with 95% O2/5% CO2 .Electrophysiological experiments were performed on sagittal brain slices (300 μm) obtained as follow: animals were anesthetized  in an anesthesia chamber and were then decapitated. The brains were quickly removed and brain slices containing the dorsal striatum were cut using a vibrating microtome , in an immersed cold (4°C) low CaExtracellular field recordings were performed in striatal cells by eliciting field excitatory postsynaptic synaptic spikes (population spikes) using a sharp (pencil-shaped), concentric, bipolar, tungsten-stimulating electrode  attached to an isolation unit . The stimulating electrode was placed at the corpus callosum. The recording electrode was a borosilicate glass electrode (~5 MΩ resistance), containing NaCl (3M), and placed in the dorsal striatum, ~500 μm away from the stimulating electrode. A population spikes slope of 50% of the maximum was used for baseline. After 10 min of stable baseline, three trains of 3s, 100 Hz (tetanus) with intervals of 20 s were delivered. All experiments were recorded for at least for 30 min after HFS.2, 2 CaCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 glucose saturated with 95% O2 and 5% CO2 . After 1 h of equilibration, single slices were shifted to a recording chamber that was continuously superfused with oxygenated saline (1–3 ml/min). Whole cell recordings were performed using an infrared differential interference contrast upright microscope  coupled to a CCD (charge-coupled-device) camera. After field stimulation (as described above), EPSC (excitatory postsynaptic currents) were recorded in MSN through an Axopatch 200B amplifier , digitalized with the aid of a Digidata 1322A  and acquired through pClamp 9.1 software. Series resistance was compensated (80%). Input and access resistance were continuously monitored during the experiment by evoking a transmembrane current with a voltage command. Criteria for accepting recordings were an access resistance below 30 MΩ and no changes throughout all experiments. Patch micropipettes (3–6 MΩ) were pulled  from borosilicate glass tubes  and were filled with internal saline containing (in mM): 72 KH2PO4, 36 KCl, 1.1 EGTA, 10 Hepes, 2CaCl2, 1 MgCl2, 2 ATP-Mg2, 0.3 GTP-Na and 5 QX-314 . All experiments were carried out in the presence of Bicuculline (10 μM) to block the GABAergic component. To evaluate the effects of Cdk5 inhibition, Roscovitine (20 μM) was added to the bath. Experiments designed to evaluate Cdk5 and signaling pathways were carried out in the presence of D1 and D2 receptor agonists and antagonists applied in the bath. To explore whether Cdk5 inhibition affects plasticity in both types of MSN D1-BAC mice were used, cells that did not exhibit fluorescence were assumed to be D2 expressing MSN. Finally, to identify if Cdk5 effects were mediated through an action on NMDA receptors or L-type Ca channels, some experiments were performed in the presence of AP5 (50 μM) or Nifedipine (5 μM), respectively. Data analyses were performed off-line with the aid of clampfit  and Microcal Origin (Origin Lab Corporation) software.Slices used for whole cell recordings were transferred to a saline solution containing (in mM): 130 NaCl, 3 KCl, 2 MgClFluorescent images were obtained using the upright microscope  with a 20× objective magnification (Olympus X Lum PlanFl 20X/0.95W) coupled to a Hamamatsu (Orca C4742-95) camera, and acquired with the Olympus Image acquisition system Cell M®, which sent excitation light in the range 450–490 nm through a dichroic filter (495 nm); emission was 502–538 nm.34 (1:1000), pDARPP-32 Trh75 (1:1000) and Actin (1:1000) for 12–20 h at 4°C, and with secondary antibodies (1:2000) with HRP for 2 h at room temperature. Protein detection was performed through the use of luminol with a chemiluminescence method (Millipore); images were acquired via a scan (HP Scanjet G2410) and analyzed with Image J software for the evaluation of densitometry.To evaluate the phosphorylation state of DARPP-32 in our experimental conditions, striatal slices were incubated with Roscovitine 20 μM or vehicle for 10 min in artificial brain fluid. After incubation, striatal tissue was homogenized in lysis buffer containing 26 mM Tris–HCl, 1% (v/v) Triton X-100, 1.3 M glycerol and 130 mM NaCl, with the protein phosphatase inhibitor cocktail Complete mini tab (Roche). The samples were recollected and centrifuged for 5 min at 4500 rpm; supernatants were recollected and store at −70°C. Protein quantification was made using the Bradford method and 80 μg of protein was loaded in 10% (w/v) PAGE for electrophoresis. Proteins were transferred to PVDF membranes and incubated with primary antibodies against pDARPP-32 Trh34, anti pDARPP-32 Trh 75 (Millipore) and anti Actin-(Santa Cruz); and NaCl, KCl, NaH2PO4 from JT Backer.All reagent were purchased from Sigma-Aldrich, except; antibodies, anti pDARPP-32 TrhP<0.05.Data were analyzed with the statistical software Sigma Plot  and plotted using Microcal Origin 7  and Adobe Illustrator 10 or Adobe® Creative Suite® 5 . Statistical analysis was conducted with a parametric test or a non-parametric test if data did not display normal distribution. Data were considered statistically significant if t5=6.458, P=0.000117, t test). The LTP observed in the 37.5% of the cases was also statistically significant compared to pre HFS .HFS of the corticostriatal pathway produced both striatal LTD and LTP, as previously reported A and 1B,t14=7.402, P<0.0001, t test, Comparisons of LTD with LTP obtained after HFS displayed statistical differences between  to baseline (1.794±0.166), suggesting that Cdk5 inhibition modulates glutamatergic transmission through presynaptic mechanisms.We first sought to explore whether Roscovitine had a modulatory role in corticostriatal communication. Bath application of the Roscovitine 20 μM), significantly increased the amplitude of striatal glutamatergic population spikes in comparison to baseline  protocol changed when Cdk5 was inhibited D and 2E,t4=10.15 P=0.0005, paired t test). When we compare LTP in the presence of Roscovitine against LTP produced in control conditions (without Roscovitine), we found that LTP magnitude in presence of Roscovitine significantly increased in amplitude, in comparison to control conditions , suggesting that amplitude increase after HFS in the presence of Roscovitine may have a presynaptic origin , suggesting a postsynaptic effect of DA receptor inhibition in Roscovitine LTP induction  significantly prevented LTP induction in the presence of Roscovitine ( μM signi34 was greatly increased in the presence of Roscovitine (2.12-fold compare to control)  in some experiments and L-type Ca channels were blocked with Nifedipine (10 μM) in other experiments; in both cases, population spikes was significantly reduced. AP5 significantly reduces LTP induced in the presence of Roscovitine . Comparison of PPR before and after HFS was not significant in control conditions or in the presence of Roscovitine  or after HFS, it showed a significant difference , this data illustrated that Cdk5 has to be inhibited in the presynaptic site to induce LTP. In the second series of experiments, Roscovitine was added in the recording bath to inhibit Cdk5 at the presynaptic site and the PKA inhibitor H89 was in the recording pipette to inhibit PKA pathway in the postsynaptic cell. In this conditions, HFS did not induce long-term plasticity , showing that the PKA signaling pathway is associated with to the postsynaptic component of Roscovitine-induced LTP  (direct pathway) receptors and other D2/ENK  (indirect pathway) receptors, and it has been stated that MSNs from direct pathways express LTP through PKA signaling (Reynolds and Wickens, Striatal LTD is induced by postsynaptic mechanisms; nevertheless, its expression is maintained through presynaptic mechanisms which reduce neurotransmitter release (Wang et al.,


Arabidopsisthaliana to evaluate changes in oil composition when expressed with the seed-specific Brassica napus truncated napin (FP1) promoter. Fatty acid profiles from transgenic homozygous seeds reveal that the targeted genes were silenced. The down-regulation of the AtFAD-2 gene substantially increased oleic acid from the normal levels of ∼15% to as high as 63.3 and reduced total PUFA content  from 46.8 to 4.8%. Δ12-desaturase activity was reduced to levels as low as those in the null fad-2-1 and fad-2-2 mutants. Silencing of the FAE1 gene resulted in the reduction of eicosenoic acid (20:1Δ11) to 1.9 from 15.4% and silencing of FATB resulted in the reduction of palmitic acid (16:0) to 4.4% from 8.0%. Reduction in FATB activity is comparable with a FATB knock-out mutant. These results demonstrate for the first time amiR159b constructs targeted against three endogenous seed-expressed genes are clearly able to down-regulate and generate genotypic changes that are inherited stably over three generations.Various post transcriptional gene silencing strategies have been developed and exploited to study gene function or engineer disease resistance. The recently developed artificial microRNA strategy is an alternative method of effectively silencing target genes. The Δ12-desaturase (FAD2), Fatty acid elongase (FAE1), and Fatty acyl-ACP thioesterase B (FATB) were targeted with amiR159b-based constructs in  MicroRNAs (miRNAs) are a class of 20–24-nucleotide (nt) regulatory small RNAs (sRNA) endogenous to both plants and animals which represent a recently developed miRNA-based strategy to silence endogenous genes. It has been demonstrated that the alteration of several nucleotides within the miRNA sequence does not affect its biogenesis as long as the positions of matches and mismatches within the precursor stem loop remain unaffected . Foremost among these is the ability to silence specific transcripts amongst gene families with a reduced risk of cross-silencing and, because of this, amiRNA has been described as a second-generation RNAi technology  into binary vector pORE03 , was constructed by cloning a modified version of the Agrobacterium tumefaciens strain AGL1 by electroporation and the transformed strains used to introduce the genetic construct into A. thaliana (ecotype Columbia). Plant transformation was carried out using the floral dipping method  from the transformed plants (T0 plants) were plated out on MS media  were collected at maturity and analyzed for fatty acid composition. Lines displaying an apparent high level of FAD2 silencing were again germinated on PPT media and seedlings from lines with a 3:1 surviving:susceptible seedling ratio were transferred to soil. These lines were likely to contain single locus insertions and be either heterozygous or homozygous for the transgenic insert as null lines were selected against.pJP1106, pJP1109, and pJP1110 was transformed into separate 3-HCl  at 90–100°C for 2 h in a glass test tube fitted with a Teflon-lined screw-cap. FAME were extracted into hexane:dichloromethane  and analyzed by GC. GC was performed as described by Zhou et al. (Fatty acid methyl esters (FAME) were formed by transesterification of triplicate samples of ∼200 seeds each by heating with MeOH-CHClu et al.  but usinΔ9 desaturation  to the total amount of 18:1Δ9 substrate that was available, i.e., these products of 18:1Δ9 modification plus the remaining 18:1Δ9. Arabidopsis (ecotype Columbia) typically has an ODP value of around 0.70–0.79, indicating that around 70–79% of 18:1Δ9 formed during fatty acid synthesis is subsequently converted to the polyunsaturated C18 fatty acids initially via the action of Δ12-desaturase. This parameter is useful in illustrating the effects of FAD2 gene silencing on the level of endogenous Δ12-desaturase activity.Oleic desaturation proportion represents the ratio of the total fatty acids accounting for the products of 18:1Δ9 elongation  to the total amount of 18:1Δ9 substrate that was available, i.e., the products of 18:1Δ9 modification  plus the remaining 18:1Δ9. Arabidopsis typically has an OEP value of around 0.20–0.22, indicating that around 20–22% of 18:1Δ9 formed during fatty acid synthesis is subsequently elongated via the action of FAE1. This parameter is useful in illustrating the effects of FAE1 gene silencing on the level of endogenous FAE1 activity.Similarly, oleic elongation proportion (OEP) represents the ratio of the total fatty acids accounting for the products of 18:13 and T4) were analyzed by One Way Analysis of Variance (ANOVA) using Sigmaplot (v12) by the Holm–Sidak method  to the polyunsaturated linoleic acid  and 18:1Δ9 is 12.9 ± 0.1 (Table Δ9 in T2 seed transformed with the pJP1106 construct (FAD2 target) ranged from 32.9 to 62.7% in 30 independent transgenic events compared to an average non-transgenic parental level of 12.9 ± 0.1. A highly silenced line . Similarly high levels of the 18:1Δ9 were observed in T3 seed ranging from 46.0 to 63.8% with an average of 57.3 ± 5.0% in 21 independent transgenic events. In T4 seed also similar high level of 18:1Δ9 observed from 61 to 65.8% with an average of 63.3 ± 1.0% in 23 independent transgenic events. All the means were significantly different from all others (P < 0.01). The total PUFA content  in T2 seed ranged from 6.1 to 38% but in homozygous lines the total PUFA content was further reduced and ranged from 4.3 to 5.7%. The control Arabidopsis ecotype Columbia has ODP value ranging 0.75–0.79, meaning that over 75% of oleic acid produced in the developing seed is subsequently converted to 18:2Δ9,12 and 18:3Δ9,12,15. The fad-2-1 mutant has an ODP value of 0.17, indicating about a 75% reduction in Δ12 desaturation. The ODP values ranged from 0.08 to 0.48 in T2 seed, 0.07–0.32 in T3 seed, and 0.06–0.08 in T4 seed in contrast to 0.75 in control Arabidopsis ecotype Columbia  observed in this study using amiRNA is higher than the well characterized FAD-2-2 mutant (59.4 ± 2.0%), hairpin (56.9 ± 3.6%), and hairpin-antisense approach  and FAD2 silenced line using hairpin-antisense approach  ranged from 2.2 to 15.1% in 12 transgenic events compared to an average in wild type level of 16.5% ± 0.3. A highly silenced line which has a single transgene insertion, determined by segregation ratios (3:1) of PPT forwarded to next generation (T3). Similarly, very low levels of 20:1Δ11 was observed in T3 seed ranged from 1.1 to 4.1%. The average 20:1Δ11 in T3 seed is 1.9 ± 1.0%, in contrast to 15.0% in control Columbia seed (Table Δ9 and 20:1Δ11) averages of control and transformed lines were significantly different (P < 0.01). Arabidopsis ecotype Columbia has OEP value ranging 0.20–0.22, indicating that around 20–22% of 18:1Δ9 formed during fatty acid synthesis is subsequently elongated via the action of FAE1. In FAE1 silenced lines the OEP values ranged from 0.10 to 0.20 in T2 seed and 0.01–0.04 in T3 seed in contrast to 0.20 in control Arabidopsis ecotype Columbia  ranged from 4.9 to 6.2% in 12 transgenic events compared to an average in wild type level of 8.0 ± 0.3%. A highly silenced line which has a single transgene insertion, determined by segregation ratios (3:1) of PPT was forwarded to next generation (T3). Similarly, low levels of 16:0 were observed in T3 seed ranged from 3.4 to 5.0%. The average 16:0 in T3 seed was 4.4 ± 0.5%, in contrast to 8.0 ± 0.3% in control Columbia seed . This precise silencing may be particularly useful for manipulating the seed oil composition of economically important oilseed crops.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 effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR) expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was studied in mice.in vitro were exposed to 0 µM, 0.5 µM, 2.5 µM, or 5 µM of As2O3 for 0 h, 24 h, 48 h and 72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related signal decline with As2O3 treatment. Next, mice with pharynx-implanted A431 cells received As2O3 i.p. every 48 h at 0.0, 0.5, 2.5, or 5 mg/kg/day (n = 6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.A431 OSCC In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p<0.05) with increasing As2O3 dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p<0.05) in As2O3 treated groups versus controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was reduced by As2O3 treatment in a dose responsive pattern.in vivo by NIR molecular imaging of the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As2O3 treatment.This study demonstrates for the first time that OSCC can be interrogated  Oral squamous cell carcinoma (OSCC) is one of the ten most common cancers 2O3, TRISENOX) is the most widely used and studied arsenic-based anticancer drug 2O3 is also effective on solid tumors in liver 2O3 for inhibiting growth and triggering apoptosis of cancer cells are not fully understood 2O3 was reported to inhibit epidermal growth factor receptor (EGFR) expression on the surface of OSCC cells in culture Arsenic trioxide  compared with those treated with 0.5 µM. The change in optical contrast was corroborated with the levels of EGFR expression . At 72 h post treatment, cellular EGFR expression was 57.3±3.2% (p<0.05), 29.9±2.2% (p<0.01), and 10.7±2.4% (p<0.01) in cells treated with 0.5 µM, 2.5 µM, and 5.0 µM As2O3, respectively, which were significant lower than the control group .Using flow cytometry, the EGFR expression in treatment group decreased from 0 h, 24 h, 48 h, to 72 h while in the control group, EGFR expression increased over the same time course . Before In vivo NIR fluorescent imaging of the tumor region of interest (ROI) was performed dynamically and fluorescence luminosity  was measured before treatment (day 0) and 4, 8, 12 days after titrated As2O3 treatment (2O3 treatment), the SI of the tumor ROIs among four groups  were not different: (2.20±0.54)×104 a.u., (1.87±0.53)×104 a.u., (2.10±0.66)×104 a.u. and (2.01±0.44)×104 a.u., respectively (p>0.05). From days 4 to 12, tumor SI increased progressively in the control group but gradually decreased in As2O3 groups. On day 12, the tumor SI was decreased inversely with each As2O3 dosage (1.21±0.35)×104 a.u., (0.65±0.16)×104 a.u., and (0.35±0.14)×104 a.u., respectively, versus the control group (3.18±0.63)×104 a.u.  by 17.1%, 41.3% or 56.4% with As2O3 dosages of 0.5 mg/kg/day, 2.5 mg/kg/day or 5.0 mg/kg/day, compared with control group, respectfully. In contradistinction to the effects on tumor volume, serial As2O3 treatment did not affect body weight (p>0.05), suggesting no grossly apparent toxicity.At baseline, tumor volume did not differ among the treatment groups p>0.05, . Tumor vp<0.05, p>0.05). The grade of EGFR positive stained tissue decreased as the dose of As2O3 increased, which were consistent with the in vitro and in vivo fluorescence measurements.Immunohistochemical assays were carried out to correlate the magnitude of tumor uptake  with the tumor receptor density distribution. Tumor tissue sections from the control group exhibited the highest grade of EGFR positive staining (3+) compared with high dose treatment groups  has motivated the use of As2O3 in treatment of solid tumors, even though As2O3 was initially approved as an effective chemotherapeutic drug for acute promyelocytic leukemia (APL) 2O3 activity suggest that it induces apoptosis and influences distinct signaling pathways, including mitogen-activated protein kinases (MAPK), p53, activator protein-1 or nuclear factor kappa B 2O3 also inhibits EGFR expression in cancer cells through p21 activation leading to cell death via the EGFR-Ras-Raf-ERK1/2 pathway based on in vitro or ex vivo methods in vivo therapeutic effects of As2O3 on pharyngeal OSCC tumors and the relationship of this response to EGFR expression detected noninvasively with NIR imaging has not been explored.Enthusiasm for promoting the clinical application of As2O3 concentration , agreeing with IHC detection of less EGFR expression in these treated cells  optical molecular imaging of EGFR expression, as demonstrated in this study, can noninvasively and sensitively identify pharyngeal OSCC and can be used to longitudinally monitor and guide As2O3 was determined in current experiment, the issue of potential toxicities with systematic administration of As2O3 remains. The oral superficial nature of OSCC suggests that direct local low dose therapy, applied topically  or with subcutaneous injection under image guidance would be feasible and perhaps optimal.Moreover, the accessibility of OSCC for direct topical or subcutaneous injection, may facilitate the use of EGF peptide–based contrast agents at lower the dosage requirements, with accelerate time to peak signal, and reduced residual contrast washout. In fact, one might envision very short interval between contrast administration and follow-up NIR examination that would readily be accommodated in the workflow patterns of dentists and oral maxillary surgeons. Finally, although no body weight loss due to As2O3 by quantifying EGFR expression, likely As2O3 inhibits EGFR expression through p21 activation leading to cell death via the EGFR-Ras-Raf-ERK1/2 pathway 2O3 therapy is not clear and will require further study to elucidate. Although useful for preliminary preclinical research, the use of the prototypical EGF-Cy5.5 agent in this study may require modification for translation. Specifically, the selection of an EGF receptor antagonist as homing ligand may be preferred to avoid activation of downstream EGFR signaling in vivo receptor imaging accuracy is desirable This study presented a proof-of-concept that noninvasive optical imaging could be used to evaluate the therapeutic effect of As2O3 and the response to treatment can be noninvasively assessed with EGF-Cy5.5 and NIR molecular imaging techniques. These results suggest that oral NIR molecular imaging with EGF-Cy5.5 based probes could enhance early detection as well as facilitate image based guidance for effective chemotherapeutic treatment of OSCC with As2O3.This study demonstrated that the response of EGFR expression by oral squamous carcinoma implanted within the mouse pharynx can be treated effectively with As2O (3.0 ml) in darkness at 4°C for 2 h, then the reaction was quenched by adding 3.0 ml of 5% acetic acid (HOAc). The EGF-Cy5.5 was isolated using a PD-10 disposable column , lyophilized, and resuspended in saline at a concentration of 1 mg/ml, then stored at −20°C in darkness until use.The EGFR specific targeting NIR fluorescent agent, EGF-Cy5.5, was developed by coupling EGF  to cyanine dye 5.5 (Cy5.5) molecules through a monofunctional N-hydroxysuccinimide (NHS) ester  according to our previously reported protocol 2 (19∶1) at 37°C. A431 cells were plated in flat bottomed 24-well microtiter plates on coverslips at a density of 1×105 cells/well. After 24 h, cells were washed three times with phosphate buffered saline , then treated with 0.0 µM, 0.5 µM, 2.5 µM, 5.0 µM As2O3  in calcium- 0.9% sodium chloride solution for either 0 h, 24 h, 48 h, or 72 h. After treatment, all cells were washed three times with PBS.Human epidermoid carcinoma A431 cells  that constitutively express a high levels of EGFR Cellular EGFR expression was evaluated by immunohistochemistry. Briefly, cells were fixed with 4% paraformaldehyde in 4°C PBS for 25 min then washed three times with PBS. After blocking to reduce nonspecific antibody binding for 30 min, monoclonal rabbit anti-human EGFR antibody  was incubated with the cells at 37°C for 2 h, then the unbound ligand was removed in three washes of PBS. Next, the cells were treated with a biotinylated goat anti-rabbit IgG , followed with a streptoavidin-biotin peroxidase reagent . Finally, diaminobenzidine (DAB) and 1% hydrogen peroxidase were applied as chromogen, and the cells were counterstained with hematoxylin. Imaging was performed with a Nikon E800 microscope using a Nikon DXM 1200 digital camera .The treated cells were incubated with 500 µl EGF-Cy5.5  for 30 minutes at 37°C in darkness. After incubation, all the cells were washed three times with PBS. Fluorescence microscopy (with an Olympus microscope outfitted with NIR diode sources and filters) of tumor cells was performed for visual confirmation of EGF-Cy5.5 uptake. Diamidino-phenyl-indole (DAPI) was used to stain cell nuclei. In the microscopic images EGF-Cy5.5 was pseudo-colored red (emission at 680–710 nm), while DAPI was pseudo colored blue (emission at 461 nm).After incubation with EGF-Cy5.5, A431 cells were also suspended with trypsin solution and centrifugal elutriation twice in PBS. Quantification of fluorescent intensity of EGF-Cy5.5 binding to EGFR was assessed using flow cytometry . All experiments were replicated in triplicate.All experimental protocols were pre-approved by the Experimental Animal Ethic Committee of Harbin Medical University, China . Use of animals was confirmed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .6) in 200 µl were slowly injected into the floor of the mouth of anesthetized mice with isoflurane in vivo NIR imaging.Athymic nude mice  from Vital River Laboratory Animal Technology Co. Ltd  were housed five per cage and provided with UV-sterilized pellet chow and autoclaved distilled water. Animals were maintained in a pathogen-free mouse colony at Harbin Medical University . A431 cells  which featured a 667-nm excitation pulse laser, with a 710-nm emission bandpass filter was used to image the tumor-bearing mice. The eXplore OPTX-optView software installed on the imaging system was used for data acquisition and processing.To characterize EGFR expression in A431 cells 2O3 treatment groups . As2O3 was injected intraperitoneally (i.p.) on days 0, 2, 4, 6, 8, and 10. The control group received an equal volume of saline under the identical conditions. For in vivo characterization of EGFR, mice were sedated with isoflurane and intravenously (i.v.) injected with 1 nmol/kg EGF-Cy5.5 diluted in 0.3 ml saline via the tail vein before As2O3 treatment (on day 0) and on days 4, 8, 12 thereafter (2)/2 where a and b represent the length and width of the tumor The tumor-bearing mice were divided into a single control and three Asereafter . FluoresMice were euthanized at the termination of the study. Tumors were harvested and fixed with formalin and embedded in paraffin for immunohistochemical analysis. Tissues were sectioned at 8 µm-thickness de-paraffinized, microwave pretreated, and then incubated with 0.3% hydrogen peroxide for 30 min. EGFR labeling and analysis were accomplished by the same two-step method as described above in cell IHC methods. The immunoreactivity in tumor cells were classified and scored as follows: the intensity of staining was scored as 0, no staining (<10%); 1+, weak (10–25%); 2+, moderate (26–50%); 3+, strong (51–100%), which included at least 1000cells per sample within 5 regions of interest  2O3 dose on cellular EGFR expression was statistically analyzed using a factorial design ANOVA. Fluorescence intensity was defined as total photon counts/pixel within manually inscribed region of interest (ROI) area divided by the laser pulse time (ms) and unit time 2O3 dose on fluorescence intensity, tumor size, body weights adjusted for their baseline values, respectively. Subanalysis of specific paired comparisons utilized student's t test. Nonparametric tests (Mann-Whitney) were used to compare the difference of IHC results. Analyses were performed using the SPSS statistical software package . P<0.05 was considered as statistically significant.Data were presented as mean ± standard deviation. The effect of time and AsFigure S12O3 treatment and NIR imaging.The protocol of As When the tumors reached 0.4 to 0.6 cm in diameter (1–2 weeks after inoculation), the tumor-bearing mice were divided into a single control and three As2O3 treatment groups . As2O3 was injected intraperitoneally (i.p.) on days 0, 2, 4, 6, 8, 10 (▴). The control group was injected with an equal volume of saline under the identical conditions. For in vivo NIR imaging, mice were sedated with ketamine/xylazine and intravenously (i.v.) injected with 1 nmol/kg EGF-Cy5.5 diluted in 0.3 ml saline via the tail vein before As2O3 treatment (on day 0) and on days 4, 8, 12 (▪) after As2O3 treatment. (The arrow indicated the tumor area).(TIF)Click here for additional data file.


The hydroxynaphthoquinones have been extensively investigated over the past 50 years for their anti-malarial activity. One member of this class, atovaquone, is combined with proguanil in Malarone®, an important drug for the treatment and prevention of malaria.in vitro for a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) evaluating the parasitaemia after 48 hours of incubation. Potential cytotoxicity in HEK293T cells was assessed using the MTT assay. Changes in mitochondrial membrane potential of Plasmodium were measured using the fluorescent dye Mitrotracker Red CMXROS.Anti-malarial activity was assessed 50s in the mid-micromolar range, and the most active compound, N3, had an IC50 of 443 nM. N3 disrupted mitochondrial membrane potential, and after 1 hour presented an IC50ΔΨmit of 16 μM. In an in vitro cytotoxicity assay using HEK 293T cells N3 demonstrated no cytotoxicity at concentrations up to 16 μM.Four compounds demonstrated ICPlasmodium falciparum and showed minimal cytotoxicity. N3 may serve as a starting point for the design of new hydroxynaphthoquinone anti-malarials.N3 was a potent inhibitor of mitochondrial electron transport, had nanomolar activity against cultured  Plasmodium falciparum, the main aetiological agent of human malaria, the disease is still devastating. Parasite resistance to older anti-malarials raises the need for the development of new drugs [Despite the worldwide effort to understand molecular and cellular features of ew drugs ,2. The aew drugs ,4. The lThe hydroxynaphthoquinones have been extensively investigated over the past 50 years for their anti-malarial activity . This diP. falciparum[Atovaquone is a hydroxynaphthoquinone that is used in combination with proguanil  for prophylaxis and therapy of uncomplicated malaria . Atovaqualciparum.P. falciparum. One of these compounds, N3, had an IC50 against cultured P. falciparum in the nM range, disrupted mitochondrial membrane potential, and had low toxicity against human cells suggesting potential as a lead compound for the development of new anti-malarial agents.The aim of this work was to test the activity of a new series of hydroxynaphthoquinones  against The hydroxynaphthoquinones N1-5 were recently synthesized using a new methodology .+ human erythrocytes (1-3% parasitaemia and 2% haematocrit) in RPMI-1640 media supplemented with 0.2% sodium bicarbonate, 50 mg/L hypoxanthine and 10% type A+ human serum in 92% N2, 5% CO2 and 3% O2.3D7 strain parasites were cultured and synchronized as described previously ,17. Brie2 vented tissue culture flasks at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified essential medium (Gibco BRL) supplemented with 10% (v/v) foetal bovine serum, 100 U/ml penicillin/ and 100 μg/ml streptomycin.HEK293T (human embryonic kidney) cells were cultured in 75 cm4 cells acquired on a FACSCalibur flow cytometer using CELLQUEST software (Becton Dickinson). Initial gating was carried out with unstained, uninfected erythrocytes to account for erythrocyte autofluorescence and analysis performed using Flow Jo 7.6.5 (TreeStar Inc).Infected erythrocytes at the ring stage were incubated with different concentrations of the test compounds  for 48 hours; fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours; permeabilized with 0.1% Triton X-100 and 20 μg/ml RNase; incubated for 30 minutes at 37°C; and stained with 1 nM Yoyo-1 (Molecular Probes). Parasitaemia was determined from dot plots (side scatter versus fluorescence) of 5x10m-chlorophenylhydrazone (CCCP), a protonophore that dissipate the membrane potential, was used. Results were analysed by flow cytometry as described above.Loss of parasite mitochondrial membrane potential (ΨΔmit) was determined using 5 μM Mitrotracker Red CMXROS as described previously . Culture5/well) were seeded into 48 well plates and incubated in complete medium  for 24 h. Thereafter, medium was removed and replaced with complete medium (450 μl/well); N3, atovaquone, and solvent (for controls) were added  and cultures were incubated for 48 hours. Cells were then incubated with the MTT reagent for 3 hours, and absorbance was evaluated.The toxicity of hydroxynaphthoquinone derivatives toward HEK293T cells was evaluated with the 4,5-dimethylthiazol- 2-yl-2,5- diphenyltetrazolium bromide (MTT) cell proliferation assay . Cells  was 54.6 ± 0.23 μM . Compound N3 showed an IC50ΔΨmit = 16 μM and atovaquone an IC50ΔΨmit = 4.4 μM , limited drug resistance, and high cost of production ,9,20. Woe b gene . The add complex . It was  complex . The comComparing the structures of atovaquone, N3 and BW58-C (an atovaquone precursor), these three structures are very similar in molecular volume, though N3 is much simpler to prepare and has no chiral centers Figure  and, thebc1 complex [P. falciparum, and showed minimal cytotoxicity. Optimization of N3 thus offers potential for new candidate compounds to treat and prevent malaria.Screening of a library of 2-hydroxy-naphthoquinones found compounds with alkyl side-chains that effectively inhibited the yeast  complex . In the The authors declare that they have no competing interests.in vitro assays and drafted the manuscript. SBF and DRR carried out the chemical synthesis and collaborated in the elaboration of the manuscript. LNC and MN carried out the cytotoxicity test and collaborated in the elaboration of the manuscript. MN carried out the Plasmodium culture and collaborated in the elaboration of the manuscript. CRSG and VFF conceived of the study, and participated in its design and coordination and collaborated in the elaboration of the manuscript. All authors read and approved the final manuscript.DCS carried out the P. falciparum growth. Different concentrations of compounds were incubated for 48 h with P. falciparum. Results are shown as a dose response curve for compound N1, N2, N4 and N5 incubated for 48 h. Error bars represent standard error of the mean.Structures of other hydroxynaphthoquinones and effects on Click here for file


Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5–99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55–80 °C and at a pH of 6–10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.A thermophilic lipolytic bacterium identified as  Most of the lipases used in industry are microbial enzymes, of both fungal and bacterial origin . LipasesBacillus sp. L2 using a set of consensus primers and Polymerase chain reaction (PCR) techniques, followed by expression and purification of the recombinant L2 lipase gene, and some properties of the purified enzyme including the circular dichroism (CD) spectroscopy studies.Thermostability is a desirable characteristic because it may allow the enzyme to endure conditions in industrial processes that use relatively high temperatures and/or organic solvents. Furthermore, enzymes from thermophilic bacteria have been found to be generally more resistant to denaturation than their mesophilic counterparts . In this2.2.1.Bacillus sp. L2 is an aerobic, non-motile Gram positive rod able to grow at a temperature range between 40–80 °C with an optimum growth at 70 °C and optimum pH of 7. The 16S rDNA nucleotide sequence of this strain has been deposited into GenBank data library and assigned the accession number AY 964643. BLAST results showed that Bacillus sp. L2 has highest homology with G. thermoglucosidiasus (AY 608988) with a significant value of 99% homology. From the phylogenetic tree and the G + C contents of 16S rRNA sequences, Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification  and Bacillus sp. Ak1 (L 29507). The results from BLAST by NCBI also showed that Bacillus sp. L2 has 98% homology with these two strains, and also 99% homology with G. thermoglucosidiasus (AY 608988). However, from the phylogenetic tree, it was found that this Bacillus sp. L2 did not cluster closely with G. thermoglucosidiasus (AY 608988). So far, there are no reports on lipase production from either the G. thermoglucosidiasus or the G. caldoxylosilyticus species.fication . Group 52.2.B. stearothermophilus P1 lipase gene at 99% homology. Primer P1F1 was designed based on the first 21 nucleotides of the ORF of B. stearothermophilus P1 lipase gene sequence, whereas primer P1R1 was from the last 21 nucleotides of the same sequence. The combination of these new primers (P1F1 and P1R2) was not able to amplify any PCR product. However, the combination of primer P1R1 with primer Lip F2 was successful and an 800-bp gene fragment was obtained through PCR amplification. The purified PCR product was sent for sequencing and based on the sequence, this lipase gene showed high similarities with most of the Bacillus thermostable lipase genes, especially Geobacillus sp. T1 (AY 260764) (92%).A fragment of lipase gene 300 bp in size was obtained using a pair of highly degenerate primers (Lip F2 and Lip R2). This lipase gene fragment showed the highest homology with the Geobacillus sp. T1, a new forward primer, F, was designed. The combination of primer F and Lip R2 successfully amplified a PCR product with a size of ∼700 bp. The purified DNA was sent for sequencing and the sequence obtained was later used to design another forward primer which started at the mature lipase gene sequence, which was later used to amplify a complete mature lipase gene (∼1200 bp) when combined with primer P1R1. The 1200-bp PCR product obtained was eluted from the gel and then ligated into a U-overhang vector, pQE-30. Positive transformants showed a cleared zone around the colonies on a tributyrin-LB plate with associated antibiotic agar after overnight incubation at 37 °C. The positive clones were streaked onto triolein-LB agar to screen for true lipase producers and were incubated overnight at 37 °C. The recombinant clones formed an intense blue color on the triolein-LB agar plate (data not shown).Based on the high similarities with 2.3.Bacillus sp. L2 lipase was found to be located between Ala-28 and Ala-29, based on the rules for signal peptide sequences and based on predictions from the SignalP V2.0 Web server  as host cells  cells were prepared using the protocol from .Genomic DNA of 3.3.2, 1 × PCR buffer, 0.2 mM dNTP mix, 2 U of Taq DNA Polymerase, 20 pmol of each forward and reverse primers and genomic DNA (50–100 ng). The gene was amplified with a thermocycler . The conditions for the PCR amplification were as follows: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 1 min, annealing at 58 °C for 2 min, extension at 72 °C for 1 min, final extension at 72 °C for 7 min and preservation at 4 °C.16S rDNA gene sequence was amplified via polymerase Chain Reaction (PCR) using two degenerate universal primers: 16s Forward: 5′-CCG AAT TCG TCG ACA ACA GAG TTT GAT CCT GGC TCA G-3′; and 16s Reverse: 5′-CCC GGG ATC CAA GCT TAC GGC TAC CTT GTT ACG ACT T-3′. The expected PCR product is 1500 bp. Amplification process was carried out in 100 μL of reaction mixture containing 1.5 mM MgCLE. coli, the plasmids were extracted and sent for sequencing. A DNA homology search was performed with GenBank database  cells that had recombinant plasmids with different concentrations of IPTG: 0 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1.0 mM at OD600nm ∼ 0.5 for 3 h of induction time at 37 °C. The best IPTG concentration was used for further studies.The effects of using different concentrations of inducer (IPTG) was assessed by inducing the 3.11.Time-course studies were performed by collecting the cell pellet at different post-induction times: 0, 3, 6, 9, 12, 18 and 36 h at 37 °C. The studies were also repeated with a shorter time frame (0 to 6 h). In this study, 1 mM of IPTG was used as an inducer. The best post-induction time was chosen for further studies.3.12.The cell pellet from a 200 mL culture was suspended in 20 mL of binding buffer  before sonication  and cleared by centrifugation . The clear lysate was collected and used as the crude enzyme in purification using a Ni-Sepharose affinity chromatography column. The crude L2 lipase solution was purified to homogeneity by a one-step purification protocol using immobilized metal affinity chromatography (IMAC). The purification procedure was performed using an AKTA purifier system  according to the manufacturer’s instructions. Ni-Sepharose 6 Fast Flow (5 mL) was used as the matrix in an XK 16/20  column. Equilibration was performed using binding buffer .An aliquot of 20 mL of filtered crude recombinant L2 lipase was loaded in the equilibrated column at a flow rate of 0.5 mL/min. The column was washed with five column volumes of binding buffer. The recombinant L2 lipase was eluted with 10 CV of elution buffer  using a linear gradient of imidazole ranging from 20 to 500 mM. The eluted fractions were subjected to SDS-PAGE analysis, lipase assay and Bradford assay.3.13.SDS-PAGE was carried out on 12% running gel by using the method of Laemmli (1970) . A broad3.14.2HPO3/NaOH buffer (pH 11.0–12.0). The effect of pH on lipase stability was determined by incubating aliquots of L2 lipase in buffers of different pH values for 30 min at 70 °C. Residual activity was assayed using the Kwon and Rhee method.Enzymatic activity was measured at various pH values (pH 4–12) for 30 min at 70 °C. The substrate (olive oil) was prepared in 50 mM of various buffers; acetate buffer (pH 4.0–6.0), potassium phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 8.0–9.0), glycine-NaOH buffer (pH 9.0–11.0), and Na3.15.The optimum temperature for lipase activity was determined over the range of 40–85 °C. The substrate was equilibrated at the required temperature for 5 min before the addition of enzyme. The effect of temperature on lipase stability was determined by incubating aliquots of the purified L2 lipase in Glycine-NaOH pH 9.0 at 60 °C to 80 °C in intervals of 5 °C. The residual activity was measured at the optimum temperature (70 °C).3.16.The variable temperature measurement of L2 lipase was performed by employing 10 mm cell after checking the circular dichroism (CD) value at 220 nm. The warm-up period was 50 to 95 °C, and the step was 1 degree per minute. The wavelength was set to 220 nm. The concentration was 1 mg/mL and top of the cell was completely closed using a cap. Data pitch, bandwidth, response, scanning speed, and accumulation were set to be 0.1 degree, 1 nm, 8 seconds, 1 degree per minute, 8 times, respectively.3.17.+, K+, Mg2+, Ca2+, Fe2+, Mn2+, Zn2+, and Cu2+) in 50 mM Glycine-NaOH buffer pH 9.0 at 65 °C for 30 min. The residual activity was determined at 70 °C using olive oil emulsion and expressed as a percentage of activity without the metal chlorides.The activity of the purified L2 lipase was studied following incubation with 1 mM and 10 mM concentrations of various metal chlorides (Na3.18.The influence of various effectors on purified L2 lipase activity was determined by incubating the purified L2 lipase at 65 °C for 30 min in 50 mM Glycine-NaOH buffer pH 9.0 with dithiothreitol (DTT), 2-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), ethylene diaminetetra acetic acid (EDTA), and pepstatin A at different concentrations (1 mM and 5 mM) except for pepstatin (1 mM). The residual activity was measured at 70 °C and expressed as percentage of activity without inhibitors.3.19.Purified L2 lipase in sodium phosphate buffer (pH 8.0) was analyzed with spectropolarimeter J-810  for CD spectral analysis. The warm-up periods of 50 to 95 °C and wavelength scan of 180 to 250 nm were taken into consideration.4.Bacillus sp. L2, the PCR cloning, sequencing and over-expression of a functional form of thermostable lipase L2, as well as purification and characterization studies. The full sequence of the L2 lipase gene was analyzed. A high-level expression was also successfully achieved by using the pQE-30/E. coli M15[pREP4] expression system. This system worked efficiently and overexpression of recombinant L2 was successfully carried out with a 178-fold higher activity compared to crude native L2 lipase. So far, no other lipases have been reported to successfully use this expression system. Therefore, we conclude that L2 lipase is the first lipase for which this system has been successfully used.In this study, we reported the identification of a newly isolated thermostable lipase producer, The recombinant L2 lipase was easily recovered via a single-step affinity chromatography purification method with high purity and high yield purification, leading to a low-cost method for preparation of this enzyme. The characterization of this enzyme resulted in some interesting findings, such as its thermostability profile and its wide range of pH stability, indicating that L2 lipase has significant potential for commercialization as a biocatalyst for industrial purposes, specifically, in detergent industries and food industries that use esterification process at high temperature. Therefore, in comparison with other commercially available enzymes, the use of L2 lipase for industrial applications seems promising as it is more stable at higher temperatures.


Fusariumoxysporum f.sp. tracheiphilum (Fot) in cowpea [Vignaunguiculata (L.) Walp]. In this study, we mapped loci conferring resistance to Fot race 4 in three cowpea RIL populations: IT93K-503-1 × CB46, CB27 × 24-125B-1, and CB27 × IT82E-18/Big Buff. Two independent loci which confer resistance to Fot race 4 were identified, Fot4-1 and Fot4-2. Fot4-1 was identified in the IT93K-503-1 (resistant) × CB46 (susceptible) population and was positioned on the cowpea consensus genetic map, spanning 21.57–29.40 cM on linkage group 5. The Fot4-2 locus was validated by identifying it in both the CB27 (resistant) × 24-125B-1 (susceptible) and CB27 (resistant) × IT82E-18/Big Buff (susceptible) populations. Fot4-2 was positioned on the cowpea consensus genetic map on linkage group 3; the minimum distance spanned 71.52–71.75 cM whereas the maximum distance spanned 64.44–80.23 cM. These genomic locations of Fot4-1 and Fot4-2 on the cowpea consensus genetic map, relative to Fot3-1 which was previously identified as the locus conferring resistance to Fot race 3, established that all three loci were independent. The Fot4-1 and Fot4-2 syntenic loci were examined in Glycine max, where several disease-resistance candidate genes were identified for both loci. In addition, Fot4-1 and Fot4-2 were coarsely positioned on the cowpea physical map. Fot4-1 and Fot4-2 will contribute to molecular marker development for future use in marker-assisted selection, thereby expediting introgression of Fot race 4 resistance into future cowpea cultivars.Fusarium wilt is a vascular disease caused by the fungus The online version of this article (doi:10.1007/s11032-013-9991-0) contains supplementary material, which is available to authorized users. Fusarium oxysporum f. sp. tracheiphilum (Fot) is a soil-borne fungal pathogen that causes vascular wilt disease in cowpea [Vignaunguiculata (L.) Walp]  . In addition, more than 500 diverse cowpea accessions have been SNP-genotyped and a first draft of the cowpea genome sequence has been assembled (www.harvest-blast.org). These resources will enable dissection of underlying genetic components of target agronomic traits using quantitative trait locus (QTL) analysis and association mapping. In this study, greenhouse inoculation experiments were used to identify QTLs conferring resistance against Fot race 4 in three cowpea recombinant inbred line (RIL) populations. Two loci which confer resistance to Fot race 4 were identified, Fot4-1 and Fot4-2. The target outcome of this study will be to develop molecular markers closely linked to the Fot4-1 and Fot4-2 resistance genes for application in resistance breeding.Molecular genetic tools and genomic resources have been developed for cowpea with an objective of enhancing breeding programs for the improvement of cowpea varieties for the United States, India, Brazil, and numerous countries in Africa and Asia. These genomic resources have been integrated by using a 1536-single nucleotide polymorphism (SNP) genotyping platform and include an expressed sequence tag (EST)-derived SNP cowpea consensus genetic map, known syntenic relationships between cowpea, Three cowpea RIL populations which segregate for Fot race 4 resistance were used for QTL mapping studies. The IT93K-503-1 (resistant) × CB46 (susceptible) population consisted of 113 lines advanced to the F10 generation using single seed descent. IT93K-503-1 is an advanced breeding line developed by the International Institute for Tropical Agriculture (IITA) with strong resistance to Fot race 4. CB46 was bred for resistance to Fot race 3 but is highly susceptible to Fot race 4 (Davis and Frate The CB27 (resistant) × 24-125B-1 (susceptible) population consisted of 90 lines that were advanced to the F9 generation using single seed descent. CB27 was bred for resistance to several pathogens including root-knot nematodes and Fot race 4 and also for heat tolerance  × IT82E-18/Big Buff (susceptible) population consisted of 162 RILs and was advanced to the F8 generation by single seed descent. IT82E-18 is an advanced breeding line developed at IITA which was released as cultivar Big Buff in Australia Imrie . IT82E-12 plugs were cut from frozen Fusarium-containing PDA plates and transferred aseptically to flasks containing 500 ml of potato-dextrose broth, then incubated in a shaker at 27 °C, 30 rpm under lighted conditions for 3 days. The liquid culture was strained through four layers of cheesecloth to eliminate mycelia and the spore concentration was adjusted to 1.0 × 106 microconidia per ml using a hemocytometer.Two strains of Fot race 4, which originated from infected cowpea plants in Bakersfield, California, were used for inoculation cultures. Individual isolates were developed from single spore lines. Isolates were dried and stored on sterile potato dextrose agar (PDA) plates at −80 °C. 1-cm6 spores/ml. Inoculated seedlings were transplanted into 3.8-l pots and watered daily with greenhouse day temperatures set to 28 °C and night temperatures to 16 °C.Plants were inoculated using a modified root-dip inoculation method described by Rigert and Foster . ModificPlants were evaluated 35 days post-inoculation for Fusarium disease symptoms. The wilting/stunting phenotype was evaluated by approximating the percentage of wilting or stunting to the entire plant similar to the disease severity index (DSI) utilized by the Centro International de Agricultura Tropical (CIAT)  (www.harvest-web.org).The Lucas et al.  cowpea cR2) explained by the QTL. A one-way analysis of variance using the Kruskal–Wallis (KW) package from MapQTL5.0 was used to confirm QTL loci  (www.harvest-web.org). Syntenic maps were drawn using HarvEST:Cowpea using a cut-off e-score value of −10. A minimum of five lines per linkage group was chosen to enable better viewing of syntenic relationships within the trait loci. Due to a limitation in the resolution, not all markers are presented in the screenshot images output from HarvEST:Cowpea. In order to view each individual marker, the linkage group must be magnified in the HarvEST:Cowpea database (http://harvest.ucr.edu) (www.harvest-web.org).Synteny was examined using EST-derived SNP markers from the cowpea consensus genetic map vs. 4 which were aligned to the soybean genome and functionally annotated using the most significant similarity using BLAST . Two bacterial artificial chromosome (BAC) clone libraries were developed using restriction enzymes HindIII and MboI . Contigs were assembled using the snapshot method of DNA fingerprinting by Ming Cheng Luo at the University of California, Davis .The cowpea physical map was developed using an advanced African breeding line IT93K-399-35 . However, the 7.83 cM estimated length of Fot4-1 on the cowpea consensus map is most likely the more accurate estimate due to higher recombination utilizing the 12 constituent genetic maps . Only 11 of the 26 markers in the Fot4-2 locus on the cowpea consensus map were polymorphic in the CB27 × 24-125B-1 genetic map, which also may account for the smaller QTL length on the individual map (Online Resource 7).Using the highly significant markers from the QTL study, Fot race 4 resistance was mapped using phenotyping datasets from two experiments. The QTL was identified on linkage group 1 of the individual map, spanning from 72.8 to 73.18 cM  .The QTL observed in the CB27 × IT82E-18/Big Buff population was positioned on the cowpea consensus genetic map spanning from 71.52 to 71.75 cM  on linkage group 3  were not polymorphic in the CB27 × IT82E-18/Big Buff population, and vice versa. SNP marker 1_1087 was the only marker identified as being highly significant in both populations . The maximum length of Fot4-2 was defined by the QTL identified in the CB27 × 24-125B-1 population, which spanned from 64.44 to 80.23 cM on the cowpea consensus genetic map (Online Resource 7). However, as stated previously, there was much less recombination within the Fot4-2 locus in the CB27 × 24-125B-1 population, indicated by several of the markers having the same cM position (Online Resource 7), which greatly limited the ability to narrow the QTL position. Considering that the Fot4-2 locus identified in the CB27 × IT82E-18/Big Buff population was smaller due to rapid decrease in the significance threshold of the markers outside of the 2-LOD score (Online Resource 9), the shorter length spanning from 71.52 to 71.75 cM (0.23 cM distance) on the cowpea consensus genetic map may be a more accurate estimation of Fot4-2.Subsequently, the Fot4-1 and Fot4-2 are independent of each other as observed on the cowpea consensus genetic map  × 24-125B-1 (susceptible) population spanning from 49.92 to 50.49 cM on linkage group 1 of the individual genetic map and flanked by SNP markers 1_0860 and 1_1107 . Although cowpea markers were not precisely positioned within soybean genes, three disease-resistance soybean genes were observed in the syntenic Fot4-1 locus, viz. Glyma14g17910, Glyma14g23930, and Glyma14g34880, and were considered as orthologous disease-resistance candidate genes . Two disease-resistance soybean loci were observed in the syntenic region: Glyma16g17380, which was annotated as a leucine-rich repeat protein kinase, and Glyma16g22620, which was annotated as a TIR–NBS–LRR disease-resistance gene (Table Fot4-2 locus (Table Fot4-2 (71.52–71.75 cM), all seven of the soybean genes were considered as orthologous candidate genes for the Fot4-2 locus.The Fot4-2 region of soybean chromosome 18 spanned from soybean locus Glyma18g18980 to Glyma18g38670, where five out of six soybean orthologs for cowpea SNP markers corresponded to 65.16–66.99 cM of the Fot4-2 region on cowpea linkage group 3 , which has been anchored to the cowpea consensus genetic map via SNP markers, was used to identify contigs which span the physical regions of Fot4-1 and Fot4-2.The cowpea physical map . The only significant SNP marker, 1_0030, identified in the Fot4-1 locus was identified in contig417 within BAC clones CH027H24 and CH035P21 on the cowpea physical map (Online Resource 5). SNP 1_0662, which is linked with marker 1_0030 on the cowpea consensus genetic map, was identified in BAC contig 77 within BAC clone CH095K15 (Online Resource 5). The other SNP markers within the Fot4-1 locus, 1_0557 and 1_1492, were not observed in the cowpea physical map and are probably not present in the African breeding line IT97K-499-35 which was used to create the cowpea physical map.Significant markers from the Fot4-2 locus on the cowpea consensus genetic map identified seven contigs and nine BAC clones which partially span the locus on the cowpea physical map. The significant markers for the Fot4-2 region resulting from the QTL analysis identified four contigs and five BAC clones in CB27 × 24-125B-1 and three contigs and four BACs in CB27 × IT82E-18/Big Buff (Online Resource 7). The most significant marker identified in the CB27 × IT82E-18/Big Buff population, 1_0352, was identified in contig1094, BAC clone CM052M22 (Online Resource 7). Since the Fot4-1 and Fot4-2 loci could not be narrowed further and the physical map spanning both regions was incomplete, the physical to genetic map distance was not analyzed.SNP markers from the Fot4-1 and Fot4-2, which confer resistance against F. oxysporum f.sp. tracheiphilum race 4 in cowpea. These two resistance loci were inherited from two different cowpea genotypes which differ in origin; Fot4-1 is derived from an African breeding line, IT93K-503-1, and Fot4-2 is derived from a US blackeye dry grain cultivar, CB27. In addition, Fot4-1, Fot4-2, and the previously identified Fot3-1 were positioned on the cowpea consensus genetic map, confirming that these loci which confer race-specific resistance are independent of each other. The Fot4-2 QTL was validated since it was identified in two independent populations, whose resistance locus was derived from the same CB27 resistant parent. The physical locations of Fot4-1 and Fot4-2 were roughly identified on the cowpea physical map, which will enable the generation of tightly linked markers which segregate with Fot race 4 resistance. Identification of the two independent Fot race 4 loci will enable gene pyramiding which may promote the durability of Fot race 4 resistance in future cowpea cultivars.This study has identified two independent loci, Fot4-1 and Fot4-2 loci. Previous reports of resistance to Fusarium have indicated that the resistance is a monogenic trait with dominant expression (Zink and Thomas I-2 locus for resistance to F. oxysporum f.sp. lycopersici (Fol) race 2 in tomato  in melon   family, whose proteins span the plasma membrane and respond to extracellular signals Online Resource 1 Online Resource 2 Frequency distribution of the Fot race 4 phenotypes on over 100 RIL s in the IT93K-503-1xCB46 population. The mean resistance values for the parents are indicated by the arrows. Figures 1a and 1b belong to experiment 1; Figures 2a and 2b to experiment 2 and Figures 3a and 3b to the third experiment (PPTX 591 kb)Online Resource 3 Frequency distribution of the Fot race 4 phenotypes on over 100 RIL s in the CB27x24-125B-1 population. The mean resistance values for the parents are indicated by the arrows. Figures 1a and 1b belong to the first experiment and Figures 2a and 2b belong to the second experiment (PPTX 421 kb)Online Resource 4 Frequency distribution of the Fot race 4 phenotypes on over 100 RIL s in the CB27xIT82E-18 population. The mean resistance values for the parents are indicated by the arrows. Figures 1a and 1b belong to the first experiment and Figures 2a and 2b belong to the second experiment (PPTX 451 kb)Fot4-1 in the IT93K-503-1 x CB46 population, the cowpea consensus genetic map and the cowpea physical map (PPTX 63 kb)Online Resource 5 Fot4-1 in the IT93K-503-1 x CB46 population (PPTX 56 kb)Online Resource 6 QTL analysis of Fot4-2 locus in the CB27 x 24-125B-1 and CB27 x IT82E-18/Big Buff populations, the cowpea consensus genetic map and the cowpea physical map (PPTX 63 kb)Online Resource 7 Fot4-2 in the CB27 x 24-125B-1 population (PPTX 58 kb)Online Resource 8 QTL analysis of Fot4-2 in the CB27 x IT82E-18 /Big Buff population (PPTX 54 kb)Online Resource 9 QTL analysis of Below is the link to the electronic supplementary material.


Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. Synthesis of recombinant extracellular proteins in the human 293 embryonic kidney cell (HEK293) expression system enables appropriate folding and post-translational modifications, thus generating secreted proteins that are functional and structurally similar to the native molecules Fibrillin-1 is a 350 kDa glycoprotein that multimerizes to form microfibril suprastructures in elastic and non-elastic tissues et. al, 2003 TGF-β is synthesized as a 55 kDa polypeptide that encodes the mature TGF-β at the C-terminus, and its propeptide, latency associated peptide (LAP) at the N-terminus In this study we show that recombinant fibrillin-1 fragments produced by HEK293 cells and purified by IMAC contain significant amounts of TGF-β1. TGF-β1 present in these protein preparations cannot be detected by Western blotting or mass spectrometry. However, TGF-β1 present in picomolar concentrations in these protein preparations has the capacity to trigger intracellular cell signaling through Smad2 and 3 proteins. We found that TGF-β secreted by HEK293 cells is not lost during ultrafiltration or IMAC, as it interacts with nickel ions immobilized to the column. A two-step purification procedure involving IMAC followed by gel filtration under high salt conditions is required to eliminate TGF-β1 from purified proteins. This finding has important implications for many studies using recombinant secreted proteins purified in this manner.2 atmosphere. The human fibroblast cell line, MSU 1.1, was cultured under identical conditions and was used in some cell signaling assays as indicated in the relevant figures Human skin fibroblasts (HSFs) were isolated from the foreskin of healthy individuals (2–5 years old) after a standard circumcision procedure. The local ethics committee approved this procedure (PED-06-054), and foreskin was obtained with the consent of the donor’s parents. HSFs were used between passages 4–8, and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum , 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine (PSG) at 37°C in a 5% CO2 surface areas each  in DMEM supplemented with 10% FCS, PSG and 250 µg/ml of gentamicin sulfate (Wisent) to maintain clonal selection. Cells were washed twice with 65 ml of 20 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2, pH7.4 to remove serum proteins, and cultured in serum-free DMEM (SFM). 500 ml of conditioned medium was collected every 48 h over a period of four weeks. The conditioned medium was centrifuged at 6,000×g for 15 min at 4°C in order to remove cellular debris, and was supplemented with 100 µM phenylmethanesulfonylfluoride (PMSF) to prevent protein degradation by serine proteases. The medium collections were stored at −20°C until used for protein purifications.The plasmid vectors to express the recombinant fibrillin-1 fragments  were stably transfected into HEK293 as described previously All chromatographic procedures were performed using an ÄktaPurifier 10 low pressure liquid chromatography system .2 (TBS/Ca) over a 24 h period at 4°C.Recombinant fibrillin-1 fragments were purified to homogeneity by IMAC in the following manner: 2.5 L of conditioned medium was concentrated via pressurized ultrafiltration using 30 kDa cutoff ultrafiltration membranes (Millipore) in stirred cells (Amicon) at 4°C. The concentrated conditioned medium was dialyzed twice over 24 h against 2 L of 20 mM HEPES, 500 mM NaCl, pH7.4 (running buffer) at 4°C. To eliminate any remaining debris, the concentrate was centrifuged at 10,000×g for 15 min at 4°C. The supernatant was transferred to a 50 ml superloop , and passed at 0.5 ml/min through a 5 ml Histrap HP column  preloaded with nickel ions and equilibrated in running buffer. The bound protein was eluted at 1 ml/min with a 0–200 mM linear imidazole gradient followed by 500 mM imidazole in 20 mM HEPES, 500 mM NaCl, pH7.2. Fractions containing the purified protein as assessed by Coomassie Blue staining were pooled, supplemented with 2 mM EDTA to remove traces of nickel ions and dialyzed twice against 1 L of Tris-buffered saline  containing 2 mM CaClTo test whether TGF-β1 can bind to a nickel-loaded IMAC column, 1.5 µg of active TGF-β1  was diluted in 0.12 ml of running buffer and loaded at 0.01 ml/min onto a 1 ml nickel-loaded Histrap HP column equilibrated in running buffer. Bound protein was eluted at 1 ml/min with a 0–300 mM linear imidazole gradient followed by 500 mM imidazole in 20 mM HEPES, 500 mM NaCl, pH7.2. Aliquots (0.1 ml) of eluted fractions (1 ml) were analyzed for TGF-β1 as described below. Similarly, to analyze the ability of LAP to interact with the column material, 5 µg of LAP  was diluted in 0.25 ml of running buffer and loaded onto the column under identical conditions. Aliquots (0.1 ml) of eluted fractions (1 ml) were analyzed by a direct ELISA for LAP (see below). Neither recombinant LAP nor recombinant TGF-β1 contain histidine-tags.3 staining For TGF-β1 antibody affinity chromatography, the column material was generated by coupling 0.5 mg of TGF-β1 antibody  to cyanogen bromide (CNBr) activated sepharose 4B  according to the manufacturer’s instructions. The TGF-β1 antibody column was equilibrated in TBS/Ca, pH7.4. 0.3 mg of rF20 containing TGF-β1 was loaded onto the column at a flow rate of 0.01 ml/min, and 0.25 ml fractions were collected. Antibody-bound TGF-β1 was eluted with 0.1 M glycine-HCl, pH3.0 at 0.25 ml/min and immediately neutralized with 1 M Tris. Aliquots (20 µl) of selected peak fractions were heat-denatured (95°C for 3.5 min), and separated by SDS-PAGE under reducing conditions . The protein bands were visualized by standard AgNO3 staining. Fractions containing recombinant protein were pooled and dialyzed overnight against 1 L of TBS/Ca at 4°C. The protein was stored at -20°C, and further used in cell signaling assays.For gel filtration chromatography, a Superose 12 column  was equilibrated with 50 mM Tris-HCl, 800 mM NaCl, pH7.4. 0.5 mg of protein was loaded onto the column at a flow rate of 1 ml/min, and 0.5 ml fractions were collected. Aliquots (20 µl) of the fractions were separated under reducing conditions by 7.5% SDS-PAGE, followed by AgNO2HPO4, 2 mM KH2PO4, pH7.4). Unbound antibody was washed off with an excess of PBST (0.05% Tween-20 in PBS). Non-specific binding sites on the surface were blocked for 2 h with 5% Tween-20 in PBS with 0.05% NaN3. The wells were washed with PBST and 0.1 ml of the standards and samples were incubated for 2 h. Recombinant fibrillin-1 fragments were diluted 4-fold to approximately 0.1 mg/ml in TBS/Ca and then added onto a microtiter plate. To determine total TGF-β1 (active + latent) present in fibrillin-1 protein preparations, TGF-β1 was dissociated from LAP by adding 0.02 ml of 1 M HCl  to 0.1 ml volume of protein for 10 min at 22°C. The acidification was neutralized with 0.02 ml of 0.5 M HEPES, 1 M NaOH . TGF-β1 bound to the capture antibody was detected with a biotinylated chicken anti-human TGF-β1 antibody. The detection antibody was diluted to a working concentration of 0.3 µg/ml in PBST containing 14 mg/ml delipidized bovine serum albumin . After 2 h of incubation with the detection antibody, the wells were washed and incubated with streptavidin-horse radish peroxidase (HRP) for 20 min. After washing unbound streptavidin-HRP, the color development reaction was carried out with 0.1 ml of ready-to-use peroxidase substrate containing 3,3,5,5′-tetramethylbenzidine (TMB) for 20 min , and stopped with 0.05 ml of 2N H2SO4. The absorbance was measured at 450 nm using a Beckman Coulter microplate reader (model number AD 340). The standard, recombinant human TGF-β1 was serially diluted two-fold from 2000 pg/ml to 125 pg/ml in TBS/Ca. The standard curve was generated by plotting the absorbance at 450 nm of each standard on the y-axis against the concentration (pg/ml) on the x-axis. Linear regression analysis was performed from these standard concentrations and used to determine unknown TGF-β1 concentrations present in the samples. The detection limit for the ELISA was determined at 500 pg/ml because the absorbance values obtained from TGF-β1 standards below this concentration are close to background values generated by buffer alone. This 500 pg/ml threshold value is indicated in all measurements presented.The amount of TGF-β1 present in various purified fibrillin-1 fragment preparations was determined by ELISA , and performed on 96-well microtiter plates  at 22°C. The capture antibody (mouse anti-human TGF-β1) was coated overnight at 2 µg/ml in phosphate buffered saline . Wells were washed four times with an excess of 0.05% TBST, and then incubated for 2 h with 0.5 µg/ml of LAP antibody  in 0.05% TBST containing 1 mg/ml delipidized BSA and 0.1% NaN3. Unbound antibody was removed by four washes with 0.05% TBST and then incubated for 1 h with donkey anti-goat-HRP antibody (Invitrogen) 1∶3,000 diluted in 0.05% TBST containing 1 mg/ml delipidized BSA and 0.1% NaN3. The reaction was developed with the addition of 0.1 ml TMB for 30 min, and stopped with 0.05 ml of 2N H2SO4. The standard, LAP, was serially diluted two-fold from 2000 pg/ml to 31.25 pg/ml in 0.1 ml of 20 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH7.2. After linear regression analysis of these standard concentrations unknown LAP concentrations were determined.Recombinant LAP and eluted fractions (0.1 ml) from IMAC of LAP were coated overnight at 4°C on 96-well microtiter plates . Immobilized LAP was blocked for 1.5 h at 22°C with 0.3 ml of 30 mg/ml delipidized BSA, 0.1% NaN10 µg of protein was separated on a 7.5% SDS-PAGE under reducing conditions, and stained with Coomassie Brilliant Blue G-250 colloidal protein stain . The bands were excised and subjected to a standard trypsin digestion (12 ng trypsin/µl gel volume). The peptide digest was analyzed on a Brucher HCT Ultra ion trap mass spectrometer using an electron spray ionization source. The data files generated were compared with the mammalian NCBI database (version NCBInr 20110411) using Mascot V.2.2 (Matrix Sciences).5 cells were seeded in 24-well culture plates (Corning Costar) and cultured for 3d. The cells were washed twice with 0.5 ml of SFM and incubated with 0.5 ml of SFM for 16 h to remove any growth factors remaining from the cultivation in serum-containing medium. The recombinant fibrillin-1 fragment preparations were diluted to 0.4 µM in 0.2 ml of SFM. In absolute concentrations this corresponds to 50 µg/ml of rF20, 27 µg/ml of rF18, 54.5 µg/ml of rFBN1-C, 65.6 µg/ml of rFBN1-N, and 20 µg/ml of rF23. The fragments were added onto MSU 1.1 cells or HSFs for 1 h at 37°C. This incubation period was determined by pretests in which fragments were incubated for 15 min, 60 min and 90 min (data not shown). The strongest Smad2 phosphorylation was observed at 60 min, and this incubation period was used for all subsequent cell signaling assays. As a positive control, 4 nM of recombinant TGF-β1  was diluted in 0.2 ml of SFM. In order to obtain a maximum signal, TGF-β1 was added in amounts above physiological levels. After 1 h incubation, the cells were washed twice with 0.5 ml of SFM. Cell lysis was carried out on ice with 0.1 ml radioimmunoprecipitation assay buffer  containing freshly added protease inhibitors  and phosphatase inhibitors . The lysate was centrifuged at 10,000×g for 10 min at 4°C, and the supernatant was transferred into ice-cooled microcentrifuge tubes. The protein concentrations of the lysates were determined by a bicinchoninic acid assay  in which the BSA standards were diluted in RIPA buffer.The cell signaling assay was used to test the signaling capability of TGF-β1 present in various fibrillin-1 fragment preparations in either MSU 1.1 cells or HSFs. 3×10To analyze the lysate by Western blotting, the samples were heat-denatured , and separated under reducing conditions by SDS-PAGE. To detect phospho-Smad2 (pSmad2) and Smad2/3, lysate corresponding to 12 µg of total protein were separated by 7.5% SDS-PAGE. To detect glyceraldehyde 3-phosphate dehydrogenase (GAPDH), lysates corresponding to 5 µg of total protein were separated on a 10% SDS-PAGE. The cell lysates were electrophoresed and transferred onto a 0.45 µm nitrocellulose membrane (Bio-Rad). The transfer was carried out at 0.4A in ice-cold 10 mM sodium tetraborate for 1 h. Free binding sites on the membranes were blocked with 50 mg/ml of non-fat dry milk in 0.1% TBST for 1 h at 22°C. Following 3×10 min washes with 0.2% TBST, the blots were probed with primary antibodies. Antibodies against pSmad2 (product #3101), Smad2/3 (product #3102) and GAPDH (product #2118) were purchased from Cell Signaling. The pSmad2 antibody was diluted 1∶1,000 in 50 mg/ml non-fat dry milk, 0.1% TBST. The Smad2/3  and GAPDH  antibodies were diluted in 50 mg/ml BSA in 0.1% TBST. The blots were incubated overnight at 4°C. After 3×10 min washes with 0.2% TBST, membranes were incubated with secondary antibody for 1 h in the dark. The secondary antibody, goat anti-rabbit antibody conjugated with HRP (Jackson Immunoresearch Laboratories Inc.) was used at a dilution of 1∶3,500 in the same buffer as the primary antibody. The membranes were thoroughly washed for at least 3 h with 0.2% TBST to eliminate non-specific interactions, and developed by enhanced chemiluminescence  on Hyblot CL autoradiography film.Full length fibrillins are difficult to produce and purify due to their large size and propensity to aggregate In order to test if active TGF-β1 detected in preparations of the recombinant fibrillin-1 fragments has the potential to induce Smad2 phosphorylation, equimolar amounts of the recombinant fragments rF23, rFBN1-N, rF20, rF18, and rFBN1-C were added onto fibroblasts for 1 h at 37°C. This incubation period was determined as optimal by pretests . A typical Western blot result is demonstrated in The recombinant fibrillin-1 fragments were expressed and secreted by HEK293 cells, and conditioned medium harvested was routinely purified by IMAC using nickel ions. To determine if TGF-β1 in the purified protein samples is a by-product of the expression system and purification procedure, the concentration of TGF-β1 was determined by ELISA at every step of the purification scheme of fragment rF20. The TGF-β1 levels were then compared with a control, in which non-transfected HEK293 were cultured and the conditioned medium was mock-purified under identical conditions, as detailed in Materials and Methods . The chrAnalysis of Pool 2 of the mock and rF20 purifications by SDS-PAGE demonstrated that rF20 was purified to >90% homogeneity, whereas only three faint high molecular mass proteins  were detected in Pool 2 of the mock purification . The proTGF-β1 detected in Pool 2 of the mock purification was tested for its cell signaling activity in fibroblasts for 1 h in a concentration dependent manner (3.7–22 pM) . To actiIn order to remove co-purified TGF-β1 from fibrillin-1 preparations we used three chromatographic approaches including pre-absorption on the IMAC resin, antibody affinity chromatography and gel filtration chromatography.First, we rationalized that TGF-β1 might non-specifically interact with the column resin. We pre-absorbed concentrated medium employing the IMAC resin (sepharose) in the absence of nickel ions. We tested this scheme with concentrated medium harvested from HEK293 cells expressing rFBN1-N. This recombinant protein is robustly expressed by HEK293 cells, and typically contained high amounts of total TGF-β1 see . The chr3 staining  to disrupt ionic interactions between proteins. The N-terminal half fragment rFBN1-N was subjected to gel filtration chromatography using an analytical Superose 12 column. Analysis of eluted fractions by SDS-PAGE and AgNO-10.5 ml . Based o-10.5 ml . AdditioTo confirm that gel filtration can remove TGF-β1 from preparations of fibrillin-1 fragments irrespective of their size, rF23 was subjected to gel filtration chromatography , inset. 3 staining  and LAP-TGF-β1 dimer (100 kDa) were not detected in eluted fractions when analyzed by ELISA or AgNOAs TGF-β1 does not interact with the IMAC resin , we hypoSimilarly, 5 µg of LAP (TGF-β1) was loaded onto the IMAC column, followed by quantification of LAP present in the flow-through and eluted fractions by direct ELISA . LAP eluOverall, although only 12% of active TGF-β1 and 9.8% LAP loaded onto the IMAC column were recovered, the data clearly demonstrate that TGF-β1 and LAP co-purifies with protein preparations by specifically interacting with nickel ions. Although it is not clear how exactly recombinant TGF-β1 and LAP interact with nickel, both proteins contain several clusters of histidine residues in exposed regions, which may mediate this interaction Recombinant histidine-tagged extracellular proteins produced by HEK293 cells are routinely and conveniently purified by a one-step IMAC procedure. Quality controls of the purified proteins typically include SDS-PAGE, Western blotting and mass spectrometry. Here, we demonstrate using recombinant histidine-tagged fibrillin-1 fragments, that active and latent TGF-β1 co-purifies with these fragments by IMAC. This co-purification results from the interaction of active and latent TGF-β1 with the metal-loaded IMAC resin and not from direct interactions with the various fibrillin-1 fragments. Importantly, the amounts of TGF-β1 present in the protein preparations consistently triggered the canonical TGF-β1 signaling pathway in fibroblasts through Smad2 phosphorylation, whereas conventional methods did not detect TGF-β1 in the protein preparations. TGF-β1 could be effectively eliminated from the purified proteins by gel filtration under high salt conditions.These findings are of significant importance, and their impact goes far beyond the production and purification of fibrillins by IMAC as this purification scheme is extensively used to generate secreted recombinant proteins, which are then subsequently used to examine cellular mechanisms. Based on the presented data, it is possible and even likely that a number of published experiments in the literature may be mis- or over-interpreted due to the potential and uncontrolled presence of either active or latent TGF-β1 in various secreted recombinant proteins after a one-step IMAC purification procedure. The results demonstrate the necessity of a rigorous quality control for histidine-tagged proteins purified by IMAC beyond the usual biochemical methods, if the purified protein will be used in relevant cellular analyses. In this study, we have demonstrated the presence of TGF-β1 in such protein preparations and excluded the presence of BMP-2. However, further analyses are required to determine if other potent cytokines and/or growth factors are co-purified with this chromatographic scheme.Figure S1RT-PCR of transfected HEK293 clones to analyze the mRNA expression levels of TGF-β1. The mRNA expression level of TGF-β1 among HEK293 cell clones transfected with various Fibrillin-1 fragments was analyzed by RT-PCR . The primer pairs for TGF-β1 (NM_000660.4) and GAPDH (NM_002046.4) were as follows: TGF-β1  GAPDH . The expected product sizes are 258 bp for TGF-β1, and 226 bp for GAPDH. Note the expression of TGF-β1 in the rFBN1-C transfected HEK293 cell clone is lower compared to other stably transfected cell clones.(PDF)Click here for additional data file.