We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (Kd = 0.96×10−6 M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α Normally, ROS and other oxidants are counteracted by antioxidants including the high-molecular weight enzymes superoxide dismutase (SOD), catalase, glutathione peroxidases (GPx) and the low-molecular weight non-enzymatic compounds glutathione (GSH), vitamin C and E The skin is continuously subjected to oxidative stress from both exogenous and endogenous sources. Reactive oxygen species (ROS) are generated from mitochondrial metabolism in dermal cells and enzymatic production by phagocytic and non-phagocytic NADPH-oxidases, but also from exposure of skin to ionizing radiation, UV-light irradiation, environmental toxins, chemotherapeutic compounds, etc. In conditions characterized by capillary leakage in the skin, such as during wounding, extra-vascular accumulation of red blood cells represents an oxidative threat because subsequent hemolysis leads to a massive release of free hemoglobin (Hb). Hb consists of four globin chains each carrying an iron-containing heme-group that is responsible for the oxygen-binding capacity of the molecule 1-microglobulin (A1M) in chronic leg skin-wounds Uncontrolled, excessive oxidative stress is believed to be a major factor during impaired wound repair ex vivo skin explants, keratinocyte cultures and purified collagen exposed to free heme and ROS.A1M is associated to both cells and matrix components in the epidermal and dermal layers of the skin A1M is ubiquitously localized in healthy skin as revealed by immunohistochemical staining of paraffin-embedded skin tissue sections . PericelExpression of the A1M gene in skin and in primary keratinocyte cultures obtained from skin was shown by real time PCR and radioimmunoassay (RIA) , Table 1in vitro with heme and the Fenton reaction-mix, in the presence or absence of A1M, and the response was analyzed using molecular biological and biochemical parameters  and the Fenton reaction-mix (20 µM), incubated with the explants for 20 hours, induced a significantly increased expression of the cell cycle regulatory gene p21, and heme oxygenase-1 (HO-1), a heme-degrading enzyme shown to be up-regulated by ROS and oxidants To further analyze the protection by A1M, EM was performed on skin explants , i.e. vaEM of tissue specimens, containing exogenously added A1M and incubated with gold-labeled anti-A1M, showed a localization of A1M to the plasma membrane, ER and nuclear membrane . The antThe results obtained by immunohistochemistry and EM suggest a localization of A1M to collagen fibers and a protective effect of the protein in the extracellular matrix. Therefore, the effects of heme and ROS and the protective properties of A1M on purified collagen I were investigated. Heme induced carbonyl group formation on collagen monomers immobilized to microtiter plates and this was completely inhibited by addition of A1M , left in2O2 resulted in disappearance of the collagen bands, suggesting an extensive degradation of the collagen. The degradation was inhibited by addition of 3 or 10 µM A1M but not by the lipocalin α1-acid glycoprotein (not shown). Exposure of collagen to 1 mM H2O2 also induced formation of carbonyl groups in the microtiter plate assay, and this was inhibited by addition of 5 or 10 µM A1M in a dose-dependent manner , plasma membrane rupture, destruction of the structure of the endoplasmatic reticulum and nuclear membrane, and a reduction of the keratin fibers in number and thickness. Similar effects were seen when exposing skin and keratinocytes to the Fenton reaction (not shown). The ultrastructural tissue damage seen here resemble the damage of trophoblasts described after ex vivo, i.e. addition of A1M to skin explants exposed to heme and iron reversed the disintegration of extracellular matrix . Ammonium iron (III) sulfate dodecahydrate was from Merck . A mixture of Fe3+ (10–100 µM), ascorbate (0.1–1.0 mM) and hydrogen peroxide (20–200 µM) was used to generate hydroxyl radicals by the Fenton-reaction. In this manuscript, the concentration of the Fenton reaction-mix will be referred to as the Fe3+ concentration. Collagen S  was bought from Roche . CytoTox96® Non-Radioactive Cytotoxicity Assay was from Promega . 2,4-dinitrophenylhydrazine (DNP-hydrazine) and o-phenylenediamine were from Sigma-Aldrich). Anti-DNP-keyhole limpet hemocyanin (KLH) was from Invitrogen. Swine anti-rabbit IgG-horseradish peroxidase (HRP) was purchased from Dako A/S . Recombinant human A1M, containing an N-terminal His-tag, was expressed in E. coli and purified and refolded as described Cell culture media and supplements were purchased from Invitrogen . Hemin (Ferriprotoporphyrin IX chloride) was purchased from Porphyrin Products, Inc.  and a 10 mM stock solution prepared by dissolving in dimethyl sulphoxide . “Heme” and “hemin” are sometimes used to designate free protoporphyrin IX with a bound Fe2. 20 µM heme and/or A1M (10 µM) were added to the cells as indicated in the figure legends. Culture medium was collected and cells were harvested in homogenizing buffer  after 3 and 20 hours and analyzed as described below.Human primary keratinocytes  were cultured in Keratinocyte SFM medium  containing 100 µg/mL antibiotics and 0.25 µg/mL antimycotics (amphotericin B and Fungizone®). The cells were incubated at 37°C in 5% CO3+, 0.5 mM ascorbate, 0.1 mM H2O2) and/or A1M (10 µM) were added to the skin pieces (as indicated in the figure legends), cultured for 5 hours at 37°C in 5% CO2 and analyzed using techniques described below. The skin specimens were surplus tissue from dermatological surgery, and obtained following written consent using protocols approved by the ethical committee at Lund-Malmö University Hospital (LU 762-02).Normal human skin obtained from reduction surgery was dissected into 1×10×2 mm pieces and two pieces were placed in each well of a 6-well culture dish containing Keratinocyte SFM culture medium (containing 100 µg/mL antibiotics). Heme (20 µM), Fenton reaction-mix (20 µM FeThe concentration of lactate dehydrogenase (LDH) in culture media was used to estimate the degree of cytolysis induced by heme. LDH was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay from Promega. The analysis was performed according to the instructions from the manufacturer. Briefly, at the end-point of all incubations culture medium was harvested and 50 µL was transferred to a 96-well microtiter plate. Fifty microliters reconstituted substrate mix were added to each well and the plate was incubated at room temperature (RT), protected from light. After 30 minutes 50 µL stop solution were added to each well and absorbance was read at 490 nm using a Wallac 1420 Multilabel Counter . A1M alone had no significant effect on the absorbance at 490 nm of the reagents.18 primer, 200 U reverse transcriptase and 20 U RiboLockTM Ribonuclease inhibitor in reaction buffer . Real-time PCR was then used to quantify the p21, A1M and (HO-1) mRNA. Raw data were obtained as cycle threshold values  and are shown in vice versa. The ΔΔCt-values of the controls correspond to the baseline and are not included in the Figures. Primers were designed accordingly: p21 forward primer 5′-TGGACCTGTCACTGTCTTGT-3′, reverse primer 5′-TCCTGTGGGCGGATTAG-3′; A1M forward primer 5′-CACTCGTTGGCGGAAAGG-3′, reverse primer 5′-ACTCATCATAGTTGGTGTGGAC-3′; HO-1 forward primer 5′-CAACAAAGTGCAAGA TTCTG-3′, reverse primer 5′-AAAGCCCTACAGCAACTG-3′; G3DPH forward primer 5′-TGGTATCGTGGAAGGACTC-3′, reverse primer 5′-AGTAGAGGCAGGGATGATG-3′. The expression was analyzed using iQ SYBR Green Supermix . Amplification was performed at 55°C for 40 cycles in iCycler Thermal Cycler (Bio-Rad) and data analyzed using iCycler iQ Optical System Software.Total RNA was isolated from skin and keratinocytes using the acid guanidinium phenol chloroform method supplied by QIAGEN Sciences . The OD ratio  of RNA was always greater than 1.8. Reverse transcription was performed on 3 µg total RNA at 42°C for 60 min in the presence of 0.5 µg oligo(dT)125I was done using the chloramine T method 125I-labelled A1M  and samples, or standard A1M concentrations (0.2 mL). The dilutions were done in 0.1 M sodium phosphate, pH 7.4+0.1% BSA (RIA-buffer). After incubating overnight at RT, antibody-bound antigen was precipitated by adding 0.3 mL bovine serum and 1.6 mL 15% polyethylene glycol in the RIA-buffer, centrifuged at 2500 xg for 40 min, and the 125I activity of the pellets was measured in a Wallac Wizard 1470 gamma counter (Perkin Elmer Life Sciences). Total protein in cell-homogenates was determined by Bradford protein assay. This assay was performed by adding one mL of Bradford reagent , 5% (v/v) EtOH, 6% (v/v) H3PO4 dissolved in H2O) to 100 µL of each sample, and incubating at room temperature for 5 min before determination of absorbance at 595 nm using a UV spectrophotometer . Albumin was used as a standard and plotting the absorbance at 595 nm versus protein concentration generated a standard curve.Radiolabelling of A1M with 2O2, together with A1M at different concentrations in PBS +0.1% Tween-20. Alternatively, the collagen-coated wells were first incubated with A1M at different concentrations in PBS +0.1% Tween-20, washed and then oxidized with 30 µM heme or 1 mM H2O2. Formation of protein carbonyl groups in the collagen was then quantified as described 2HPO4, H2O2) and absorbance was read at 450 nm, using a Wallac 1420 Multilabel Counter, at the onset of the reaction until a peak absorbance was obtained. Protein carbonyl groups in keratinocyte medium was determined as described Purified collagen S was coated overnight at 4°C to microtiter plates  diluted to 5 µg/ml in 20 mM Tris-HCl, 140 mM NaCl, pH 7.6, washed five times with PBS+0.1% Tween-20 and incubated with 30 µM heme or 1 mM H3+, 1.0 mM ascorbate, 200 µM H2O2), heme (20 µM) or buffer only, either with or without A1M (10 µM). Fibrils incubated without A1M during the second 24 hour-period were then incubated for a third 24 hour-period with buffer or 10 µM A1M. The samples were then adsorbed for 1 min onto carbon-coated grids, briefly washed with water, and stained with 0.75% (w/w) uranyl formate. The grids had been rendered hydrophilic by glow discharge at low pressure in air beforehand. To study binding of A1M to collagen, 5 µg/mL collagen S was allowed to form fibrils by incubation with buffer and then incubated with gold-labeled A1M (4-5 nm) for 1 hour at RT. A1M was labeled with colloidal gold as previously described (40).For negative staining collagen S (5 µg/mL) was incubated with buffer  for 24 hours at RT to allow fibrillation. Fibrils were then incubated for a second 24 hour-period at RT with a Fenton-reaction mixture . Samples were then washed with cacodylate buffer and post-fixed for 1 hour at RT in 1% osmium tetroxide in cacodylate buffer, dehydrated in a graded series of ethanol, and then embedded in Epon 812  using acetone as an intermediate solvent. Specimens were sectioned with a diamond knife into 50–70 nm-thick ultrathin sections on an LKB ultramicrotome. The ultrathin sections were stained with uranyl acetate and lead citrate. Specimens were observed in a JEOL JEM 1230 electron microscope operated at 80 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera. Keratinocytes (about 1 million cells) were pelleted by centrifugation and subsequently fixed and sectioned. Tissue samples were treated in a similar way, except that the centrifugation step was omitted. Immunolabeling of thin sections with gold-labeled anti-A1M were performed as described by Roth 2O2 (0.15–1 mM), A1M (0–10 µM), or both, in 50 mM Tris-HCl, 0.15 M NaCl, pH 7.6. After incubation for 1.5 hour at room temperature (RT), the samples were separated by SDS-PAGE as described previously Collagen, 5 µM, was incubated with H2O2 in methanol for 10 minutes. After rinsing in distilled water, followed by PBS, the slides were subsequently permeabilized with Tween 20 (0.05%) in PBS. Thereafter, sections were rinsed in PBS. After pre-incubation with normal goat serum (1%) for 30 min at RT, incubation was performed with primary antibody  diluted to 10 µg/mL in PBS  overnight at 41°C. Control sections were incubated in the same PBS solution without antibody. Antibody detection was performed with a standard avidin-biotin complex detection system after which they were developed with 3,3-diaminobenzidine tetrahydrochloride as the chromogenic substrate . Sections were mounted with Pertex  and examined and photographed (Olympus BHS photomicrographic system).Immunohistochemistry sections were deparaffinized by routine procedures and endogenous peroxidase activity was blocked with 3% H3 cpm 125I-labeled A1M and incubated overnight at 4°C. The wells were washed and the activity was determined in a Wallac Wizard 1470 gamma counter.Collagen, diluted to 5 µg/mL in 1% acetic acid +1% NaCl, 100 µL/ well, was coated on 96-well flexible assay plates  by incubation at 4°C overnight. The wells were blocked with 1% BSA in PBS +0.1% Tween 20 for 1 hour at RT. Unlabeled A1M in 50 µL PBS +0.05% Tween was added to the wells, followed by 50 µL of the same buffer containing 50×10P<0.05.Statistical analysis was performed using Origin 8 software . Students t-test was used for statistical evaluation and was considered significant when


The principal Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1) is strongly associated with nasopharyngeal carcinoma (NPC), a prevalent cancer in China. The epidermal growth factor receptor (EGFR) is important in carcinogenesis, as it is a ubiquitously expressed receptor tyrosine kinase. Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis. Our previous study demonstrated that the nuclear EGFR could bind to the cyclin D1 promoter directly in the presence of LMP1, and the correlation between EGFR and STAT3 in NPC remains to be further explored. Here, we have shown that the interaction of EGFR and STAT3 increased in the nucleus in the presence of LMP1. LMP1 promoted both EGFR and STAT3 binding to the promoter region of cyclin D1, in turn, enhancing the promoter activity of cyclin D1. Furthermore, we demonstrated that both transcriptional activity and mRNA levels of cyclin D1 were decreased by small molecule interference of EGFR and STAT3 activity. These findings may provide a novel linkage between the EGFR and STAT3 signaling pathways and the activation of cyclin D1 by LMP1 in the carcinogenesis of NPC. Epstein-Barr virus (EBV) is a ubiquitous herpes virus that is linked to multiple malignancies, including Burkitt’s lymphoma, Hodgkin’s disease, gastric cancer esophageal cancer cervical cancer and prostate cancer and nasopharyngeal carcinoma (NPC)-9. LatenElevated amounts of the epidermal growth factor receptor (EGFR) at both the protein and mRNA levels are detected in the epithelial cell carcinomas including NPC, and its expression correlates with the levels of LMP1. Our earSignal transducer and activator of transcription 3 (STAT3) is a member of the STAT family of cytoplasmic proteins that is constitutively active in many human cancers,31. UponCyclin D1 is a key regulatory protein at the G1/S checkpoint of the cell cycle. A recent census concluded that cyclin D1 gene amplification and overexpression are present in breast cancer, lung cancer, melanoma and oral squamous cell carcinomas,36,37. OIn this study, we found that LMP1 promoted the interaction of EGFR and STAT3 in the nucleus. The nuclear EGFR and STAT3 could target the cyclin D1 promoter directly, in turn, upregulating the cyclin D1 promoter activity and mRNA level. Furthermore, knockdown of EGFR and STAT3 decreased cyclin D1 promoter activity. Our results provide a novel linkage between deregulated EGFR signaling and the activation of cyclin D1 gene expression induced by LMP1 in NPC tumorigenesis.2 and 95% air at 99% humidity.CNE1 is an LMP1-negtive, poorly differentiated NPC cell line. CNE1- LMP1 is a stably transfected cell line, established by introducing LMP1 cDNA into CNE1 cells, and the cell line stably expressing LMP1,34,41-43XhoI sites of the pcDNA3.1 vector. Expression plasmid for dominant negative mutant of EGFR (EGFR-DN) had a deletion of 533 amino acids at the N terminus, which competitively inhibited the activation of EGFR, and was cloned into pcDNA3.1. The pSG5-STAT3 was obtained from whole STAT3 coding fragment cloned into XhoI sites of the pSG5 vector. Expression plasmid for dominant negative mutant of STAT3 (STAT3β) had a deletion of 55-residue in C-terminal transactivation domain of STAT3 and replaced by seven unique C-terminal residues (CT7) for 30 min on ice and centrifuged at 15,000 × g for 10 min. The supernatant was collected and stored at -70°C until used.For whole cell lysates, 107/ml cells were washed with PBS and suspended in 200 μl of lysis buffer . The cells were incubated on ice for 15 min, after which 6.5 μl of 12.5% NP-40 was added; the contents were mixed and then centrifuged for 1 min at 12,000 rpm. The supernatant was saved as cytoplasmic fraction. The pellet was resuspended in 12.5 μl of ice-cold nuclear extraction buffer  and incubated on ice for 40 min with mixing every 10 min, then they were centrifuged for 5 min at 12,000 rpm at 4°C. The supernatant was saved as nuclear fraction. The cytosolic and nuclear fractions were stored at -70°C until used.For Preparation of cytoplasmic and nuclear fractions, 10Fifty microgram (μg) of the total proteins from cell preparations were separated on 10% SDS- polyacrylamide gel electrophoresis and then electrotransfered onto the nitrocellulose membrane. The membranes were blocked with buffer containing 5% non-fat milk in PBS with 0.05% Tween-20 (PBST) for 2 hrs, and incubated with different primary antibodies (anti-EGFR or anti-STAT3) overnight at 4°C. After second wash with PBST, the membranes were incubated with anti-rabbit  or anti-mouse  horseradish peroxidase- conjugated secondary antibody for 1 hr. at room temperature and color was developed with the enhanced chemiluminescence detection kit , then, and followed by exposure to autoradiographic film. The antibodies used were as follows: EGFR , p-EGFR , STAT3 , p-STAT3 , β-actin , α-tubulin , Nucleolin , cyclin D1 .Cell extracts were prepared with harvested cells from CNE1 and CNE1-LMP1 lysed in an immunoprecipitation (IP) lysis buffer . Two milligram (mg) of protein prepared were mixed with 40 μl of protein A-Sepharose beads  in the IP assay buffer , incubated at 4°C for 2 hrs with gentle agitation and centrifuged for 10 min at 2,000 rpm for preclearing. The recovered supernatant was incubated with either 2 μg of anti-EGFR or 2 μg of anti-STAT3in the presence of 1× protease inhibitors at 4°C overnight with mild shaking. Followed by addition of 50 μl of Protein A-Sepharose beads and the incubation were continued for 2 hrs at 4°C with gentle shaking. Then, Protein A-precipitated protein complex was recovered by centrifugation for 10 sec. at 12,000 rpm and followed washed three times with IP assay buffer, the harvested beads were resuspended in 30 μl of 2× SDS PAGE sample buffer were boiled for 5 min. to release the bound protein. A 20 μg aliquot of cell lysate was used as an input control. The samples were then analyzed by Western blot. Antibodies for Western blot detection were EGFR IgG antibody and STAT3 IgG antibody.5 per well overnight and were transfected with Lipofectamine™ 2,000  as the manufacturer’s instructions. Each transfection contained 800 ng/well of pCCD1-Luc or pD1-mut-Luc firefly luciferase reporter and 80 ng/well of internal control pRL-SV40 or contained 400 ng/well of firefly luciferase reporter and 80 ng/well of internal control pRL-SV40 together with 200 ng/well of each expression plasmid or blank expression plasmid necessary to normalize the amount of DNA transfected. Twenty-four hrs. after transfection, cells were harvested at 36 hrs. after transfection and lysates were analyzed for luciferase activity using the Dual Luciferase Reporter assay  according to the manufacturer’s directions with a GloMax™ Microplate Luminometer . The luciferase reporter plasmids were co-transfected with pRL-SV40 to correct for variations in transfection efficiency. The relative luciferase activity normalized to the value of pRL-SV40 activity. Results were expressed as fold induction of pCCD1-Luc activity in CNE1 cells, which was assigned a value of 1. WHI-P131, PD98059 and AG1478 inhibited the activities of cyclin D1 induced by stable expression LMP1. CNE1-LMP1 cells were transfected with cyclin D1 promoter-reporter construct and Renilla luciferase plasmid as an internal control. The data represent the mean ± SD of the three independent experiments performed in triplicate.Cells were cultured in 24-well plates at a density of 1 × 10To observe WHI-P131, PD98059 and AG1478 inhibiting the activities of cyclin D1 induced by stable expression LMP1, 24 hrs. after transfection, cells were treated with WHI-P131 , PD98059 , AG1478  or 0.1% DMSO for 2 hr. Cells were harvested at 26 h after transfection and subjected to the luciferase assay. Empty firefly reporter vector served as the negative control.TCTTTGGCCGTCTG-3′ (wild type) and 5′-TCGCTGAGATACTCGGGCCGTCTG-3′ (mutated type). For the STAT3 binding site in cyclin D1 promoter: 5′-GTGGCGTTCTTGGAAATGCG- CCCA-3′ (wild type) and 5′-GTGGCGAGCTTGTGAATGCGCCCA-3′ (mutated type).EMSA for EGFR/STAT3 binding to cyclin D1 was performed using the LightShift™ Chemiluminesent EMSA kit  and was conducted according to the manufacturer’s protocol. Briefly, Double-stranded oligonucleotides, were labeled using the biotin 3′end labeling . Ten μg of nuclear extracts were incubated with 2 μl biotin-labeled probes in binding buffer for 20 min. at room temperature. Additionally, increasing concentrations of 200- fold of excess of a cold competitive oligonucleotide (biotin- unlabeled probe) and NF-κB biotin-unlabeled probe (as a nonspecific competitive probe) were added to confirm specificity of the interaction. The reaction mixture was then loaded onto 10% non- denaturing polyacrylamide gel containing 0.5× Tris borate (TBE) and electro- phoresed in 0.5× TBE at 4°C prior to visualization according to the manufacturer; Followed by transferred to BiodyneR B Nylon membrane, avidin-HRP to probes, and visualized and quantitated with a PhosphorImager . All the double-stranded probes were synthesized as follows: for the putative binding site of EGFR in the cyclin D1 promoter: 5′-TCGCTGAGATTo verify the involvement of EGFR, STAT3, LMP1 in the complex, DZ1, small molecular inhibitors AG1478, WHI-P 131and PD98059, was added to the mixture containing the nuclear extracts and biotin-labeled probes and incubated at room temperature or on ice for an additional 10 min.We used EGFR siRNA and STAT3 siRNA to reduce EGFR and STAT3 gene expression. The siRNA sequences for EGFR  and STAT3 , and the negative control siRNA  (silencer negative control) were purchased from Santa Cruz. Cells were plated at 30% to 40% confluency, in RPMI 1640 and 10% FCS. The indicated siRNA  was transfected in six-well plates using 10 μl Lipofect AMINE as recommended  for 6 hrs. in serum-free medium. Medium containing serum was added to bring the concentrations of serum to those indicated above.To study transcriptional activity of endogenous EGFR and STAT3, cells were transiently cotransfected with pCCD1-Luc, and 10 nM of the noncoding control siRNA as a control.Cells were transfected with the specified siRNAs and placed in RPMI 1640 with 5% FCS. Forty-eight hours later, they were harvested for RNA isolation using Trizol . RNA was reverse transcribed with random primers and SuperScript II reverse transcriptase according to Invitrogen’s protocol. The RT Real-Time SYBR/ROX PCR Master Mix was purchased from TAKARA; and PCR analysis was performed on an Applied Biosystems 7500 Real-Time PCR System, according to the instructions of the manufacturer. The RT-PCRs were performed in duplicates for four independent experiments and the results were normalized to the respective expression levels of actin. The primer sequences were for cyclin D1 (forward) 5′-CTCCACCTCACC- CCCTAAAT -3′ and (reverse) 5′-AGAGCCCAAAAGCCATCC-3′ and for actin (forward) 5′-TTCC- AGCCTTCCTTCCTGGG-3′ and (reverse) 5′-TTGCGC- TCAGGAGGAGCAAT-3′. The amplification product of cyclin D1 was 177 bp. The mean ± SD of three independent experiments is shown.5) were plated into 6-well plates and treated with the indicated siRNAs after 24 hrs. Cells were harvested after an additional 72 hrs, washed with PBS and fixed in 70% ethanol overnight at 4°C. To detect the fluorescent intensity of certain proteins, cells were counterstained in the dark with 50 μg/ml phosphatidyl inositol (PI) and 0.1% ribonuclease A (RNase A) in 400 μl of PBS at 25°C for 30 min. Stained cells were assayed and quantified using a FACSort Flow Cytometer .Flow cytometry was used to quantify cells in each phase of the cell cycle. Cells  with immunoblot analysis was performed. An anti-EGFR antibody pulled down an immunocomplex, and then Western blotting was performed to analyze the STAT3 protein in the complex. Data in Figure To confirm the interaction of EGFR with STAT3 in the nucleus under the regulation of LMP1 at the cellular sublocalization level, co-IP and Western blotting were performed from both cytosolic and nuclear fractions. Cytosolic fractions and nuclear extracts were prepared from CNE1 and CNE1-LMP1 cells, and a co-IP was performed with anti-EGFR Figure A or antihttp://www.cbrc.jp/research/db/TFSEARCH) , and both EGFR and STAT3 could target cyclin D1 promoter activity,35,46. TNuclear accumulation of EGFR and STAT3 is dependent on the activation of the related signaling pathways. CNE1-LMP1 cells were treated with the small molecule inhibitor WHI-P131, a specific inhibitor of STAT3 phosphorylation at residue tyrosine 705 and serine 727. Both the promoter activity Figure C and theNext, we addressed whether the nuclear interaction of EGFR and STAT3 associates with the cyclin D1 promoter directly using electrophoresis mobility shift assay (EMSA) in CNE1 and CNE1-LMP1 cells. The probes, which contain EGFR or STAT3 binding sites according to the previous report, were lain vitro.To address whether nuclear EGFR is involved with the cyclin D1 promoter directly, we mutated the cyclin D1 promoter sequence such that no transcription factor binds. As shown in Figure To address whether EGFR and STAT3 may be involved in cyclin D1 activity, we knocked down EGFR or STAT3 with siRNA. After we introduced EGFR siRNA or and STAT3 siRNA in CNE1-LMP1 cells Figure A, the cycyclin D1 over-expression is important in the development and progression of numerous cancers. RegulatThe mechanism by which EBV LMP1 induces EGFR and STAT3 to enhance the promoter activity and expression of cyclin D1 involves physical and functional interaction between EGFR and STAT3. This observation is in agreement with other reports that nuclear EGFR interacts with transcription factors, such as STAT3, E2F1, STAT5 and TIF2 to induce the expression of some target genes in various cancers,40,60-63Our previous studies reported EBV LMP1 induces in both expression and phosphorylation of EGFR in a dose-dependent manner,45, and It has been shown that LMP1 induces cyclin D1 expression through EGFR in NPC cells. The prein vivo without increasing toxicity[The current standard therapy for NPC is radical radiotherapy for early stage disease and concurrent chemoradiotherapy for advanced disease,68. EGFRtoxicity. It is pIn summary, we found that EBV LMP1 enhances the transcriptional activity and mRNA level of the cyclin D1 gene in CNE1 cells. This underlying mechanism for cyclin D1 regulation involves regulated binding of EGFR and STAT3 in the cyclin D1 promoter region as well as increasing the promoter activity of the cyclin D1 gene. Such a mechanism may partially contribute to the proliferation and growth of tumor cells with an LMP1-induced increase in the nuclear accumulation of EGFR and STAT3.The authors declare that they have no competing of interests.Conceived and designed the experiments: YT YC. Performed the experiments: YX, SY, QY, XL, BY and LC. Analyzed the data: YX, SY, QY, XL, BY and LC. Contributed reagents/materials/analysis tools: SY and LC. Wrote the paper: YX, YT and YC. All authors read and approved the final manuscript.LMP1 promoted the interaction of phosphorylated EGFR and phosphorylated STAT3. Two mg of protein from cell lysates were immunoprecipitated with an anti-phosphorylated EGFR antibody (p-EGFR) and analyzed by Western blotting with a phosphorylated STAT3 (p-STAT3) and p-EGFR antibodies. Negative controls included immunoprecipitation with an unrelated antibody (IgG).Click here for file


Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination.in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11−88 of 8 different HPV types (11−88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type.Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines. Persistent infection by oncogenic human papillomavirus (HPV) drives the development of cervical cancer The minor capsid protein, L2, harbors several conserved neutralizing epitopes at its amino terminus that elicits cross-protection among diverse HPV types Cost and the need for a cold chain are barriers to global implementation of HPV immunization. Unfortunately, 85% of cervical cancer cases occur in women in developing countries and even the tiered pricing for the two licensed vaccines is beyond the reach of many lower income countries Naked DNA vaccines encoding vaccine antigens have several potential advantages. Production of DNA vaccines does not require culture, inactivation of infectious pathogens, and their purification from bacteria is well standardized and comparatively inexpensive There are several alternative modes of DNA administration that can overcome inefficient delivery. The gene gun provides ballistic delivery of gold particles coated with DNA to cells in the skin including professional antigen presenting cells, termed Langerhans cells Here we show the potential of L1-expressing DNA vaccines administered with electroporation as a prophylactic vaccine. In addition, our results also demonstrate interference between L1 DNA vaccines administered at the same site with electroporation, likely reflecting co-assembly of different L1 into chimeric particles rather than immunologic competition. Finally, we find that this interference can be eliminated if L1 DNA vaccines of different HPV genotypes are spatially separated upon administration, or ameliorated if the cognate L2 proteins are co-expressed.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal studies were performed with the prior approval of the Animal Care and Use Committee of Johns Hopkins University (protocol MO08M19).Codon optimized capsid genes, L1 and L2, of HPV6, HPV16, HPV18, HPV26, and HPV51 were sub-cloned into double expression vector, pVITRO1-neo-mcs  or pcDNA , for DNA vaccination. HPV16 L2 multimer expression construct encompassing residues 11−88 of 8 HPV types (L2×8) was sub-cloned into pcDNA 3.1 for mammalian expression, and L2 α11−88×8 polypeptide was produced, purified, and dialyzed as previously described http://home.ccr.cancer.gov/Lco/pseudovirusproduction.htm). Firefly luciferase expression plasmid was employed as a reporter for PsV infection in neutralization assays and for vaginal challenge studies.Codon optimized L1 and L2 capsid genes of HPV6, 11, 16, 18, 26, 31, 45, 51, were sub-cloned into double expression vector, pVITRO1-neo-mcs . Pseudovirions (PsVs) were generated in 293TT cells following the standard PsV production protocol (v/v) glycerol, 1% (v/v) Nonidet P-40, 2 mM EDTA, and a protease inhibitor cocktail (Roche). mAb H18.F8 293TT cells grown in DMEM-10 medium were transfected with empty vector, a single expression vector for only HPV6, 16, or 18 L1, or mixture of L1 DNA of HPV6, 16, 18 in equal parts using transit 2020 , and harvested in 48 hours. Cells were lysed with non-denaturing lysis buffer containing 20 mM Tris HCl pH 8, 137 mM NaCl, 10% (http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf). Primary antibodies used were H6.C6, H16.O7, and H18.E20 Western blotting was performed with standard protocols  plasmids in pVITRO1-neo-mcs, 60 µg of mixed  plasmids in pVITRO1-neo-mcs, 60 µg of mixed  plasmids in pVITRO1-neo-mcs, or 20 µg of HPV6, 16, 18 L1 expression plasmids in pVITRO1-neo-mcs at different sites. Blood samples were collected two weeks and three months after the last vaccination. Serum was separated by centrifugation at 2,000 g for 10 min at 4°C after the blood had congealed overnight at room temperature.Groups (n = 5) of 3–4 week old female Balb/c mice were vaccinated s.c. three times at two week intervals with 25 µg of L2×8 multimer polypeptide formulated with alum (50 µg), or 50 µl of Gardasil, or i.m. with 10 µg of L2 11−88×8 multimer expression plasmid, or i.m. utilizing Mice were injected with DNA in 30 µl water i.m. into the gastrocnemius muscle, or biceps femoris muscle of hind leg. A pair of electrode needles was inserted into the muscle flanking injection site and electrical pulses were delivered utilizing an ECM830 electroporation generator . Eight pulses of 106 V each were delivered for 20 ms pulse duration at 200 ms intervals.v/v Tween 20 in PBS), HRP-sheep anti-mouse IgG diluted in 1% w/v BSA at 5000-fold was added to each well as a secondary antibody, and incubated for 1 hour at 37°C. After 3 further washes, 100 ul of ABTS solution, 2,2′ Azinobis [3-ethylbenzothiazoline-6-sulfonic acid], Roche, (Basel Switzerland) was added to each well for developing color and read by an ELISA reader, Benchmark Plus  at 405 nm.For analysis of antibody response against HPV16 L2 protein, microtiter plates were coated with L2 protein at 500 ng in 100 ul PBS/well overnight at 4°C, and blocked with PBS/1% BSA for 1 hour at 37°C. Plates were incubated with serum samples diluted 1 50 in PBS/1% BSA for 1 hour at 37°C. After 3 washes with washing buffer  for 15 min at room temperature on a rocking platform. Lysates were transferred to 96-Well black plate, and luciferase activity was measured by GloMax®-Multi Detection System  after adding 50 ul of luciferin substrate  to each well.Serum samples (4 µl) were serially diluted two-fold in culture media, and mixed with 0.03 µl of HPV PsV carrying luciferase reporter plasmid. Mixtures were incubated at 37°C for two hours, added to 3×10Mice were subcutaneously injected with 3 mg of medroxyprogesterone  four days before vaginal challenge to synchronize their estrus cycles. Viral challenge was performed by delivery of 10 µl of HPV PsV  mixed with 10 µl of 3% carboxymethyl cellulose (CMC) into the vagina before and after cytobrush treatment  while the mice were under isoflurane anesthesia. Three day after challenge, mice were anesthetized by isoflurane, and 20 µl of luciferin substrate  was delivered into the vaginal vault before imaging. Bioluminescence was acquired for 10 min with a Xenogen IVIS 100  imager, and analysis was accomplished with Living Image 2.0 software. For imaging of passively immunized mouse groups, mice were injected i.v. with 20 µl of serum one day before vaginal challenge.Exploratory statistical analyses are performed to analyze the observed titer data. Square-root data transformations were used to achieve normality in residuals for titer data. One-way ANOVA and pair wise multiple comparisons with Bonferroni adjustment were performed using SAS 9.3. When the overall significance test is not significant, the pair wise comparisons are not conducted. To claim significance, we use alpha level 0.1 for pair wise comparisons. The homogeneity of variances is examined with Levene's test.i.m. injection of patients. However, recent clinical studies suggest that in vivo electroporation of naked DNA vaccines can induce robust humoral responses in patients in vivo electroporation, or i.d. delivery via gene gun. We utilized HPV pseudovirion infection of 293TT cells to examine neutralization by serum antibody as a surrogate of wart-derived virions infecting primary human keratinocytes th of a human dose of Gardasil for which the serum titer was 2 log10 greater  shows that there is significant difference among 2 µg, 10 µg, 20 µg and 40 µg HPV16 L1 electroporation (EP) groups . With Bonferroni multiple comparison adjustment, at α level 0.1, we find a significant difference between 2 µg and 10 µg HPV 16 L1-EP groups  and between 2 µg and 40 µg HPV 16 L1-EP groups . A significant difference is also detected between 2 µg HPV16 L1-GG and 40 µg HPV 16 L1-EP groups .Vaccination of rabbits with naked DNA expressing CRPV L1 either via i.m. injection or ballistic delivery on gold beads (gene gun) protects rabbits from experimental viral challenge  greater . The resThe co-expression of the minor capsid protein L2 with L1 can enhance the efficiency of VLP assembly vice versa. However, no significant cross-neutralization was observed between these two types  and delivered i.m. by electroporation for comparison to vaccination with each type singly at doses of 2 µg or 10 µg. HPV6, HPV16 and HPV18 are each members of different papillomavirus species , whereas HPV26 and HPV51 are both members of the α5 species. The latter two types were included to determine if interference in the response to mixtures of L1 expression constructs occurred intra-species or inter-species or both Since L1-based DNA vaccine electroporation successfully induced a robust but type-restricted neutralizing antibody response, we evaluated the potential for a multivalent vaccine in which L1 DNAs of several HPV types are mixed prior to i.m. injection and in vitro neutralization titer of the serum of each mouse was tested against HPV6, HPV16 and HPV18 PsV. Vaccination with 20 µg of each L1 DNA vaccine individually induced a robust homotypic neutralizing antibody response and an equivalent titer was observed when utilizing DNA vaccines expressing L1+L2 , and these titers were higher than for 2 µg maximal dose of HPV16 L1 DNA vaccine delivered via gene gun. Although electroporation clearly enhances the efficiency of in vivo transfection of cells with the DNA vaccine, it may also trigger local inflammation, cell death and recruitment of immune cells to enhance the immune response in vivo electroporation would raise the cost and complexity of vaccination because of the need for an electroporation device and access to electricity. Delivery via a self-contained gene gun device like PD-10 is another option, but the DNA dose is much more limited than for in vivo electroporation.L1 DNA vaccines have potential as an alternative prophylactic HPV vaccine that is simple and inexpensive to produce and heat stable, properties that would facilitate widespread immunization in low resource and remote settings. However, delivery of DNA vaccines et al observed a very weak (titer of 1:50) and non-neutralizing antibody response to vaccination of mice i.m. twice with 100 µg of DNA expressing full length codon-optimized HPV16 L2 Hitzeroth Previously it was shown that HPV6 L1 and HPV16 L1 can co-assemble forming hybrid VLP in yeast The co-expression of the cognate L2 in each construct is a possible alternative to spatial separation of the L1 DNA vaccines of different genotypes during multivalent vaccination. While this approach enhanced the neutralizing antibody responses relative to a mixed multivalent L1 DNA vaccine, it did not achieve in all cases the titers obtained upon vaccinating with a single type or spatial separation with multivalent L1 DNA vaccination . NeverthBoth of the licensed HPV vaccines utilize an alum-based adjuvant and Cervarix also includes a TLR4 agonist, MPL. Naked DNA vaccines, delivered appropriately, can potentially activate TLR9 via CpG islands as well as cytoplasmic DNA sensors such as DAI and AIM2 to enhance the immune response. However, we have not examined L1 DNA vaccination with alum and utilizing electroporation. Previously, it was found that mixing a hepatitis B DNA vaccine with aluminum phosphate improved the antibody titers ∼10-fold relative to DNA alone injected i.m. without electroporation Safety issues and practical considerations surrounding the use of DNA as a vaccine need further consideration. Although it has not been an issue in clinical studies of naked DNA vaccines to date, the potential for a low frequency of integration of the vaccine DNA into the host chromosome or the induction of anti-DNA antibodies to cause disease remains a concern. Standard i.m. injection of naked DNA is very inefficient because only a small fraction of DNA is taken up by cells and expressed, but advances in delivery technologies such as ballistic delivery and electroporation are beginning to overcome this barrier. Indeed, our results suggest that vaccinating at different sites with multi-type L1 or L1+L2 DNA vaccines and using electroporation to enhance delivery shows promise as a next generation HPV vaccine candidate.


Especially,the activity of BK channel (α+β4) can be modulated by martentoxin, a 37 residues peptide, with Ca2+-dependent manner. gBK channel (glioma BK channel) and BK channel (α+β1) possessed higher Ca2+ sensitivity than other known BK channel subtypes.BK channels are usually activated by membrane depolarization and cytoplasmic Ca2+ imaging. In the presence of cytoplasmic Ca2+, martentoxin could enhance the activities of both gBK and BK channel (α+β1) subtypes in dose-dependent manner with EC50 of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (α+β1) was unrelated to the quantitive change of cytoplasmic Ca2+ concentrations though the interaction between martentoxin and BK channel (α+β1) was accelerated under higher cytoplasmic Ca2+. The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary β subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca2+, the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (α+β1) couldn't be affected by the toxin.The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca2+-hypersensitive BK channel subtypes activities in a new manner and indicate that β subunit of these BK channels plays a vital role in this enhancement by martentoxin.Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca Moreover, the interaction between martentoxin and the neuronal BK channel (α+β4) was implicated for a novel drug-docking model. Since gBK and BK channel (α+β1) possessed higher Ca2+ sensitivity than the neuronal BK channel (α+β4) in the physiological condition, it is very intriguing to investigate the modulatory characteristics of martentoxin on these two BK channel subtypes.As is known to all, neurotoxins are invaluable tools for examining structural and functional characteristics of targeting-channels. Martentoxin, a 37 residues toxin from 2, and subcultured approximately every 2∼3 days. The plasmids containing hSloα (U23767) and β1  are gifts from N.W. Davies (University of Leicester) and J.D. Lippiat (Leeds university). One day before transfection, HEK 293T cells were transferred to 24-well plates. At 90% confluence, cells were transiently transfected using Lipofectamine2000  at a ratio of 2 µL reagent with 1 µg total plasmid per well. Electrophysiological experiments were performed at 1∼2 days after transfection.All experiments were performed on the glioma cell lines U251  and HEK 293T cell lines. U251 cells and HEK 293T cells were obtained from Shanghai cell bank of Chinese Academy of Science. The cells were both cultured in Dulbecco's modified Eagle medium  supplemented with 10% heat –inactivated fetal bovine serum . Culture dishes were incubated at 37°C in a humidified atmosphere containing 5% COWhole-cell voltage-clamp experiments were performed as described previously The U251 cells were loaded with 12.5 µM fura-2 AM  in this prepared culture medium at 37°C for 1.5 h. The final concentration of 15% Pluronic acid F-127 was<0.02% (wt/vol). The HEK 293T cells were incubated with 5 µM fura-2 AM in HEPES buffered solution for 30 min at 37°C.2+ concentration by Fura-2 was performed as described previously2+ imaging system . The sampling interval of Fura-2 fluorescence measurements was 5 s.The measurement of changes in cellular Ca2 1, CaCl2 2, glucose 10, and HEPES (N-(2-hydroxyethyl) piperazine-N'- (2-ethanesulfonic acid)) acid 10(pH 7.3 with NaOH). Pipette solutions for U251 cells was composed of the following (in mM): KCl 140, MgCl2 1, EGTA 5, HEPES acid 10(pH 7.2 with KOH). The standard bath solution for HEK 293T cells was consisted of the following (in mM): NaCl 135, KCl 5, MgCl2 1.2, CdCl2 2.5, HEPES 5, glucose 10 (pH 7.4 with NaOH). Pipette solutions for HEK 293T cells was composed of the following (in mM): NaCl 10, KCl 117, MgSO4 2, HEPES 10, MgATP 2, EGTA 1 (pH 7.2 with KOH). The total Ca2+ to be added to give the desired free concentration was calculated using the program Maxchelator (http://www.stanford.edu/%7Ecpatton/maxc.html).In the patch-clamp recordings, the standard bath solution for U251 cells was consisted of the following (in mM): NaCl 150, KCl 5.6, MgCl2+ imaging, the Krebs buffered solution for U251 cells had a composition of (in mM): NaCl 130, K2HPO4 2.4, CaSO41, MgSO4 1, D-glucose 10, and HEPES 10(pH 7.4 with NaOH). The HEPES buffered solution for HEK 293T cells had a composition of (in mM): NaCl 137, KCl 5.9, MgCl2 1,CaCl2 2.5, HEPES 10, glucose 15 (pH 7.4 with NaOH).In the CaThe toxin was dissolved in the bath solution, supplemented with 1 mg/mL bovine serum albumin (BSA) in order to prevent adherence of the toxin to the vials and the perfusion apparatus. Application of 1 mg/mL BSA alone did not alter BK channel function. Unless otherwise stated, all reagents were purchased from Sigma.4 cells per well and final volume of cell culture medium in each well was 0.2 mL. The second day, medium was removed, and cells were washed once with sterile phosphate-buffered saline (PBS) and treated with serum-free media as “control”. The next day, cells were treated with various concentrations of drugs or chemicals for 48 h. IbTx was added directly to the wells at final concentrations of 100 nM, and the medium was changed daily throughout the course of the experiment. For dose-response experiments, cells were incubated in serum-free media containing 0, 20, 100, 500, and 1000 nM martentoxin. For time course experiments, cell numbers were determined daily. At the indicated time points, the cell numbers in the wells were measured by the absorbance (450 nm) of reduced WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt). The assay was conducted in five replicate wells for each sample concentration and controls. Three parallel experiments were performed. All experiments that attempted to compare growth of cells under different drug conditions were performed on serum-free culture. The cell viability of samples at each concentration of the drugs or chemicals was calculated by dividing the optical density of samples with that of control.Cell proliferation assays were performed by using Cell CountingKit-8 . Cells were plated in 96-well plates at 1×10Data were analyzed by PulseFit 8.5  and Origin 7.5 . Results of data analysis were expressed as mean±S.E.M. and n represents the number of the cells examined.f).The Statistical significance was determined using the unpaired Student's t-Test or one-way ANOVA, and an asterisk denotes P<0.05 unless otherwise stated. The degree of toxin effect was calculated by expressing the remaining current after each drug exposure as a fraction of the current magnitude of the patch prior to the first drug exposure n), where Im is maximum enhanced percentage of BK currents, and [toxin] is the concentration of martentoxin. EC50  and n denote the toxin concentration of half-maximal effect and the Hill coefficient, respectively.Dose-response curve for the percent enhancement of gBK or BK channel (α+β1) currents was drawn according to the Hill equation I =  Im/(1+([toxin]/EC1/2)/k)) +1, where V1/2 is the voltage at which half-maximal activation occurs, and k describes the slope of the fit.gBK or BK channel (α+β1) currents was elicited by the step pulses ranging from – 50 to +120 mV for 200 ms with the increments of 10 mV , respectively). For determining the voltage dependence of activation, the conductance was calculated using the formula: G(V) =  I(V)/(V–ErK), where I(V) is the currents of gBK or BK channel (α+β1) at the command voltage V, and ErK is the reversal potential. The conductance were normalized to the maximal value and the voltage dependence for activation of gBK and BK channel (α+β1) fitted to a Boltzmann equation: f(x) = –1/  or 0.14±0.01 (n = 6) of the control . The abu, n = 6) . Therefof: 0.14±0.02 compared to 0.17±0.02, p>0.05,n = 4)  . Obvious2+ concentration in the pipette solution was controlled to 150 nM, the gBK channel currents could be enhanced by martentoxin in dose-dependent manner. The EC50 of martentoxin on gBK channels was assessed to be 46.7±7.82 nM with a Hill coefficient of n = 1.40±0.26 according to the dose-response curve fitting (n = 3–8) (In the case that the free Can = 3–8) .2+ concentration in the U251 glioma cells was normally about hundreds nM level and the proliferation of human malignant glioma cells including U251 cells could be decreased by iberiotoxin, a specific gBK channel inhibitorf: 0.87±0.01 compared to 0.86±0.01, p>0.05, n = 15) (Free Ca n = 15) . This re2+-ATPase pump for 30 min at 37°C in order to deplete intracellular Ca2+ stores before performing whole cell patch recordings of gBK currents2+, the gBK channels were blocked by 100 nM martentoxin . When the free Ca2+ concentration was elevated to 150 nM or 28 µM, the activities of gBK channels were conversely enhanced by 100 nM martentoxin. But the enhancement ratios of gBK channel activities were not significantly different . . see .2+ imaging showed that the cytoplasmic Ca2+ concentration in glioma cells could not be oscillated by 100 nM or 500 nM martentoxin. Contrary to this, an obvious Ca2+ rise could be evoked by the solution with 100 mM caffeine  of activation was not significantly shifted with the application of 100 nM martentoxin  (When the free Ca p>0.05) .2+ concentration in the pipette solution remained at 700 nM, the currents of BK channel (α+β1) were increased by martentoxin in the dose-dependent manner (50 of martentoxin on BK channel (α+β1) was assessed to be 495±26.7 nM with a Hill coefficient of n = 1.39±0.09 according to the dose-response curve fitting (n = 3–5). In addition, the effects of martentoxin on steady-state activation of α+β1 subtype was studied by the same experimental voltage protocol and the same data analysis as gBK channels . A similar effect could be observed in the presence of 1 µM martentoxin. The activation curve of α+β1 BK channels and the half-maximal voltage (V1/2) of activation were also insignificantly moved  (In the case that free Cat manner . The EC5 p>0.05) .2+ was fully excluded from the pipette solution, the currents of BK channel (α+β1) evoked by +80 mV pulse could not be modulated by martentoxin even at 1 µM  were enhanced by martentoxin 1 µM  as shown in 2+ in the pipette solution. The shortest time was 185±6.4 s (n = 6) at 700 nM free Ca2+, but shortened to 79.0±6.7 s (n = 6) at 25 µM free Ca2+ (In the case that HEK293T cells transfected with BK channel (α+β1) were also pre-treated with 200 nM thapsigargin for 30 min at 37°C. When Ca at 1 µM . Howeverxin 1 µM . Similarree Ca2+ .2+ imaging showed that the cytoplasmic Ca2+ concentration in HEK 293T cells could not be oscillated by 1 µM martentoxin, suggesting that the cytoplasmic Ca2+ concentration remained almost unchanged during the recording (2+.Caecording . Therefof was 0.47±0.03 (n = 5) at 400 nM iberiotoxin. Similarly, If was 0.53±0.06 (n = 4) with the mixture of 1 µM martentoxin and 400 nM iberiotoxin (The currents of BK channel (α+β1) could be markedly inhibited by iberiotoxin, a pore blocker of BK channels, or the mixture of iberiotoxin and martentoxin. Iriotoxin .2+-hypersensitive BK channel subtypes, by martentoxin were investigated in the present study. The activities of both BK channel (α+β1) and gBK channel could be enhanced by martentoxin, depending on the presence of cytoplasmic free Ca2+.The modulatory characteristics of gBK channel and BK channel (α+β1), two Caslo) and the β regulatory subunit slo mRNA BK channels consist of two distinct subunits, the pore-forming α subunit  currents could be reduced by martentoxin in the presence of low cytoplasmic Ca2+. When Ca2+ was completely removed from the pipette solution, the activity of gBK channel was conversely inhibited, but the activity of BK channel (α+β1) was unaltered by martentoxin. It thus clearly suggested whether the presence or absence of cytoplasmic Ca2+ was also crucial to the effects of martentoxin on such BK channel subtypes.In this study, it was found that the activities of gBK and BK channel (α+β1) subtypes were both up-modulated by martentoxin only in the presence of cytoplasmic free Ca2+ binding sites of BK channels directly or indirectly. Since there is a close coupling between Ca2+ binding site and other regions especially β subunit of BK channel, martentoxin may bind with these specific regions correlated closely with Ca2+ binding sites because as a peptide, martentoxin could not reach the cytoplasm of the cell.BK channel possesses many subtypes and it still remains unknown how martentoxin modulates other BK channel subtypes. But the present results implied that the pharmacological effects of martentoxin on BK channel subtypes depended partly on the Ca− channel was not recognized by martentoxin, ii) the receptor site of gBK channel associating with martentoxin and iberiotoxin was not overlapping. As the pore region was usually regarded as the target of iberiotoxin, the docking sites of gBK channel for martentoxin might be far from the pore region. The voltage-dependence of gBK and BK channel (α+β1) was not shifted by martentoxin  activated by local CaFigure S1Sensitivity of the remaining iberiotoxin-insensitive outward currents to NPPB in U251 cells. (A) Representative whole cell current traces from U251 cells before and after the single application of iberiotoxin (IbTx) 100 nM, and simultaneous application of iberiotoxin 100 nM and NPPB 200 µM. The holding voltage was −60 mV and the currents were elicited by a pulse of +100 mV (see f) after iberiotoxin 100 nM (n = 6) and the mixture of iberiotoxin and NPPB (n = 6). P<0.001.0 mV see . (B) Sta(TIF)Click here for additional data file.Figure S22+.Effects of martentoxin on cytoplasmic Ca (A) Effects of martentoxin on cytoplasmic Ca2+ of U251 cells. After the application of martentoxin 100 nM or 500 nM, the cytoplasmic Ca2+ concentration was unchanged. (B)Effects of martentoxin on cytoplasmic Ca2+ of HEK 293T cells. After the application of martentoxin 1 µM, the cytoplasmic Ca2+ concentration was unchanged.(TIF)Click here for additional data file.Figure S3Slight effect of martentoxin on BK channels . (A) Representative current traces are shown. The channels were activated by +80 mV with a −80 mV holding potential  The time course curve confirmed the lack of sensitivity of this type  of BK channels to martentoxin.tial see . The fre(TIF)Click here for additional data file.


Also, relative HER2 expression levels in orthotopic and metastatic breast cancer models were assessed by PET imaging using the 89Zr-trastuzumab produced by this simpler method.The human epidermal growth factor receptor 2 (HER2/neu) is overexpressed in 20–30% of breast cancers and is associated with tumor growth, angiogenesis, and development of distant metastases. Trastuzumab, an anti-HER2 monoclonal antibody, is used for the treatment of HER2 positive breast cancer and clinical efficacy of this agent is dependent on HER2 expression. Targeted PET imaging of HER2 with radiolabeled trastuzumab may be used to determine HER2 expression levels and guide therapy selection. The purpose of the current study was to evaluate a facile  The human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family . HeterodTM, Genentech, South San Francisco, CA, USA) is a monoclonal antibody that targets the extracellular domain of HER2 and is widely used for the treatment of HER2 positive breast cancer .MicroPET/CT experiments were performed with the Inveon MicroPET/CT scanner . Mice were administered Tumors were harvested after completion of MicroPET/CT imaging studies and immediately fixed in 10% formalin. After allowing for the radioactivity to decay to background levels, tumors were embedded in paraffin. Five-micron sections were prepared and blocked in Dako Protein Block  for 30 min at room temperature. Antigen retrieval was performed in citrate-based buffer using a pressure cooker . The sections were incubated with rabbit polyclonal anti-HER2 primary antibody  overnight at 4 °C and visualized with Alexa Fluor 555-conjugated goat anti-rabbit IgG . Sections were mounted were mounted with SlowFade Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and cover slipped. Images were obtained with a Soft Imaging Solutions FVII cooled monochrome digital camera, Peltier cooled to −10 °C .t-test was utilized for data analysis. Differences at the 95% confidence level (P < 0.05) were considered to be statistically significant.The unpaired, two-tailed Student’s 89Zr. The radiolabeling efficiency was 78.4% ± 11.3%, the radiochemical purity was 98.7 ± 1.2%, and the specific activity was 136.9 ± 22.2 MBq/mg (3.7 ± 0.6 mCi/mg) (n = 9).Trastuzumab was successfully conjugated to Df-Bz-NCS and radiolabeled with HER2/GFP/Luc (HER2+) cells demonstrated elevated levels of HER2 expression in comparison to MDA435/LCC6Vector (HER2−) cells which demonstrated minimal expression  (89Zr-trastuzumab in HER2+ cells (The immunoreactive fraction of as 95.6% A. The up < 0.01) B. Pre-in2+ cells B.89Zr-trastuzumab were performed in athymic nude mice bearing HER2+ or HER2− tumors. Uptake of 89Zr-trastuzumab in HER2+ tumors was elevated after 24 h (26.9 ± 1.04% ID/g) and continued to remain elevated at 96 h post-injection (28.83 ± 1.33% ID/g) resulting in tumor to muscle ratios (T/M) of 8.7 ± 1.2 and 14.5 ± 2.9, respectively. In contrast, 89Zr-trastuzumab uptake in HER2− tumors was significantly lower at 24 h (8.40 ± 0.72% ID/g) and 96 h (7.91 ± 0.96% ID/g) in comparison to HER2+ tumors (P < 0.001 at 24 and 96 h), resulting in T/M of 3.0 ± 0.4 and 3.4 ± 0.6, respectively. The circulating levels of 89Zr-trastuzumab in the blood declined from 23.6 ± 2.4% ID/g at 24 h to 14.3 ± 2.1% ID/g at 96 h (P < 0.001). Bone uptake increased from 4.8 ± 0.5%ID/g at 24 h to 10.0 ± 1.4% ID/g at 96 h. The uptake for each selected organ at 24 and 96 h is shown in Biodistribution studies with 89Zr-trastuzumab in HER2+ tumors when compared to HER2− tumors at 24 h and 96 h (mean for 89Zr-trastuzumab for HER2+ and HER2− were 2.4 ± 0.3 and 1.4 ± 0.3 at 24 h (P = 0.003), and 3.1 ± 0.4 and 1.2 ± 0.1 at 96 h (P < 0.001), respectively. The level of 89Zr-trastuzumab in the blood significantly decreased from 24 to 96 h  as evidenced by visualization of the decreased radioactivity in the blood pool in the heart and the inferior vena cava. Uptake in the liver (SUVmean 0.82 ± 0.4) and kidney (SUVmean 0.58 ± 0.3) were low compared to HER2+ tumors at 96 h.MicroPET/CT imaging studies demonstrated high uptake of mean 2.6) and in the mid-thorax region (SUVmean 2.9). These lesions were readily visualized by microPET/CT demonstrating the potential use of 89Zr-trastuzumab for characterizing HER2 expression in metastatic lesions  was seen in the HER2+ tumors when compared with the HER2− tumors. DAPI (blue) was used as the counterstain for the nucleus  in subcutaneous shoulder tumors [Both an orthotopic mammary fat pad and metastatic model was utilized in the current study. t cancer ,39,40, t89Zr-trastuzumab to detect HER2 expression in distant sites of disease. The 3.3 day half-life of 89Zr is ideal for imaging full, intact antibodies (150 kDa) which require multiple days for equilibration as demonstrated in this study and previous studies [89Zr is relatively biologically inert, 89Zr-radiolabeled antibodies have low background uptake and improved stability in comparison to other radionuclides such as 64Cu, which is readily transchelated by liver superoxide dismutase resulting in elevated liver uptake, or some 124I-radiolabeled compounds which are may be dehalogenated and taken up by the thyroid. There is some residual bone uptake of 89Zr (5–10%) which may be attributed to a slow rate of intratumoral metabolism of 89Zr-trastuzumab leading to transmetalation of 89Zr4+ ions which are sequestered in the bone [89Zr-trastuzumab are underway by our group and others.The metastatic breast cancer model utilized in the current study was developed via tail vein injection of HER2+ cells. The development of tumors was initially monitored via bioluminescent imaging. This model is more of a measure of tumor invasiveness rather than metastasis as distant sites are not derived from cells migrating from a primary tumor. Despite this caveat, this model was suitable for evaluating the ability of  studies . Becausethe bone . Further89Zr-trastuzumab has significant clinical potential for the detection and characterization of lesions for HER2 status. Confirmation of HER2 status is necessary prior to initiation of trastuzumab therapy. Immunohistochemical staining and fluorescence-in-situ hybridization techniques from biopsy specimens taken at initial diagnosis are the current standards for determining HER2 expression [89Zr-trastuzumab provides a comprehensive view of the HER2 status not only within the primary tumor, but also across metastatic lesions. PET imaging with 89Zr-trastuzumab may assist in the stratification of patients by HER2 status for decision-making about trastuzumab therapy. With the increased costs of healthcare, a method to select patients that benefit from trastuzumab therapy, while avoiding the unnecessary treatment of patients with low HER2 expressing tumors could have an impact on cancer treatment.pression . Howeverpression  and HER2pression ,42. As d89Zr-trastuzumab may improve the ability to detect lesions not previously detected on routine staging studies such as CT, MRI, or bone scan. In a feasibility study involving 14 patients with metastatic breast cancer, 89Zr-trastuzumab PET imaging provided complementary information to CT, MRI, and bone scan [89Zr-trastuzumab PET imaging was able to reveal previously undetected brain metastases [89Zr-trastuzumab PET imaging and correlation with HER2 status in pathological human specimens will be challenging as it is unethical to subject patients to the discomfort of additional unnecessary biopsies.Although beyond the scope of this study, PET with one scan . Of notetastases . Determi89Zr-radiolabeled trastuzumab may also provide individualized pharmacokinetic and biodistribution information necessary to determine the optimal dose of trastuzumab for each patient. Scouting studies with 89Zr-radiolabeled trastuzumab may provide information regarding tumor uptake in comparison to critical normal organs allowing for maximal tumor targeting while minimizing normal tissue toxicity. Thus, 89Zr-trastuzumab imaging can provide this information in an efficient and safe manner, with fewer patients treated at a suboptimal dose.Quantitative pre-therapy imaging studies with p-isothiocyanatobenzyl derivative of desferrioxamine B was utilized for radiolabeling of trastuzumab. Although beyond the scope of this study, this two-step procedure may be easily applied to preparation of 89Zr-radiolabeled trastuzumab for clinical use. Biodistribution and imaging studies demonstrated comparable results to those previously reported with the succinylated deravitave of desferrioxamine used in preparation of 89Zr-trastuzumab. 89Zr-trastuzumab PET imaging is a non-invasive method for characterizing HER2 expression in primary and distant lesions. This information may be useful in selecting patients that benefit most from trastuzumab therapy.The


Since multiple factors can affect HbA2 levels, the screening can become unreliable. In 1356 healthy Arabs enrolled into a federally funded premarital BTT screening program, the effects of iron deficiency (ID), α+-thalassemia trait, gender, smoking, and tribalism on HbA2 were studied. The complete blood count and hemoglobin fractions were determined on the entire cohort; serum ferritin (<15 μg/L) in 391 subjects was used to determine ID. BTT was present in 29 (2.1%) subjects (HbA2 > 3.5%). Among 77(20.3%) subjects with ID, the mean HbA2 (2.30 ± 0.23%) was 0.2% lower than in subjects without iron deficiency . In 65 (38%)/172 subjects with phenotypic α+-thalassemia trait, the mean HbA2 (2.43 ± 0.24%) was 0.13% lower than in subjects without α+-thalassemia trait, P < 0.0001. The mean HbA2 did not differ between males and females, smokers and nonsmokers, and between the tribes. Thus, 35 (2.6%) subjects with HbA2 between 3.2 and 3.5% were at a risk of false negative diagnosis of BTT. Since iron deficiency and α+-thalassemia are both common and both lower HbA2, modifications in screening recommendations for BTT are proposed.Screening for  However, since many factors like iron deficiency, α-thalassemia, β-gene mutations, gender, and smoking may affect HbA2 levels, the screening of BTT can be compromised [β-thalassemia disease. Consanguinity is common and marriages in the native population are arranged within the same tribe, which restricts gene flow and produces a heterogeneous distribution of BTT [α-thalassemia mutations are also frequent; this is important for BTT screening since their coinheritance with β-thalassemia mutations may lower the level of HbA2. Furthermore, α-thalassemia alters MCV and MCH, which adds to the risk of a missed diagnosis of BTT [β-thalassemia mutations have been reported though their phenotype, and the prevalence of silent mutations have not been systematically investigated [2; for example, smoking is commoner among men, and the effect of smoking on HbA2 has not been studied. Women in this society are more prone to chronic nutritional disorders , which may cause a high prevalence of iron deficiency [2 and its implications for BTT screening.Screening for promised –5. The Un of BTT , 7. As as of BTT . In the stigated –13. Moreficiency . The aimn = 1356) of this study were healthy young UAE adults, who were planning to marry; as mentioned earlier, premarital screening for BTT is mandatory and government funded. Data were collected between 2007 and 2012 in the screening center in the city of Al Ain, Abu Dhabi, UAE. During two six-month long window periods, data on 965 and 391 consecutive subjects, respectively, were collected. The characteristics of some of the study subjects were previously reported [The subjects  analyzer. Hemoglobin fractions were measured using high-performance liquid chromatography  and capillary method ; the results of the two methods are comparable . All scr2 > 3.5%, MCV < 80, and there was no abnormal hemoglobin variant present. Red cell microcytosis was defined as mean corpuscular volume (MCV) <80 fL. Two definitions for iron deficiency were used: (1) ferritin < 15 μg/L (which is more specific) and (2) ferritin < 30 μg/L (which is more sensitive) [α-thalassemia trait was phenotypically defined when a subject had (1) MCV < 80 fL, (2) HbA2 ≤ 3.5%, (3) ferritin ≥ 30 μg/L, and (4) RDW ≤ 14.0. Subjects with MCV > 80 fL, HbA2 ≤ 3.5%, ferritin ≥ 30 μg/L, and RDW ≤ 14.0 were considered to have a normal genotype. In this population, α-thalassemia trait is due to α+-deletions and mutations [DNA gene tests were not performed in the study. BTT was documented when subject had the HbAnsitive) . α-thalautations . Kinship2 levels. Student-t test was used to compare means of HbA2 in both groups with other variables and between subgroups. Chi-squared test was used for no parametric variables. ANOVA test was used to test the equality of HbA2 means between major tribes. Statistical significance was defined with two-tailed P value less than or equal 0.05. Data were coded and analyzed with SPSS statistical software version 19.0 .Standard descriptive statistical methods were used. In analysis, cutoff value of >3.5% was used to separate normal from elevated HbA2 is shown in Of 1356 subjects, 50.6% were female. The mean age of males  was three years higher than that of the females . Among these 1356 consecutive cases, 29 (2.1%) had BTT. The distribution of HBA2 level was 2.61 ± 0.31%.In 1,327 subjects without BTT, the mean HbAμg/L, the mean HbA2 (2.30 ± 0.23%) was 0.2% lower than in subjects without iron deficiency . When iron deficiency was defined as ferritin <30 μg/L, iron deficient individuals (138/379) had a mean HbA2 (2.39 ± 0.25%) that was 0.11% lower than in individuals without iron deficiency . In 75/198 (38%) iron deficient females (ferritin < 15 μg/L), the mean HbA2 (2.31 ± 0.23%) was 0.17% lower than in iron sufficient females . When iron deficiency in females was defined as ferritin <30 μg/L, the mean HbA2 (2.38 ± 0.25%) was 0.1% lower than in noniron deficient females . Only 2 of 190 males had ferritin <15 μg/L.In all 77/379 (20.3%) iron deficient subjects, defined as ferritin <15 α-thalassemia trait is more reliable in males because they are rarely iron deficient. In 65 males with phenotypic α+-thalassemia trait, the mean HbA2 (2.44 ± 0.20%) was 0.13% lower than in 107 without phenotypically derived α+-thalassemia trait .The phenotypically derived diagnosis of 2 levels for females (2.68 ± 0.65%) were lower than males . However, in subjects with ferritin >30 μg/L, the mean HbA2 in females (2.52 ± 0.22%) was not different from males  implying that sex does not affect HbA2.In the entire cohort without BTT, HbA2 levels were not different between male smokers and nonsmokers (P = 0.42–0.76). Males smoked tobacco considerably more often  than females . The red cell count, mean Hb, and mean HbA2 level in subjects without BTT did not differ between 14 tribes comprising 62% of subjects (data not shown).The mean HbAμg/L), two standard deviations of HbA2 value are 0.4%. Thus, if individuals with BTT and HbA2 values between 3.6% and 3.9% are iron deficient, they could have nonthalassemic values of HbA2 that are between 3.2% and 3.5%. They are at a risk of false negative diagnosis of BTT  had lower mean HbA2 (5.24 ± 0.23%) than seven without iron deficiency . Among 12 informative cases of BTT, five with iron deficiency  than subjects who were less iron depleted (ferritin < 30 μg/L). This suggests a “dose effect” of body iron stores on the level of HbA2. In this study, two standard deviation dispersions of HbA2 value around the mean were 0.4% for all subjects and 0.46% for females with iron deficiency. Therefore, subjects with iron deficiency and HbA2 between 3.2% and 3.5% could theoretically have BTT, that is, a false negative test. Of the screened population, 2.6% were within this range of borderline HbA2 values. As it is unknown how many of them have β-thalassemia mutation , the value of routinely evaluating iron stores during BTT screening cannot be ignored. Studies have shown that iron deficiency lowers (while iron repletion increases) HbA2; thereby, the diagnosis from non-BTT carrier may change to BTT carrier and vice versa (reviewed in 1). Since 38% of females and <1% of males in our population had ferritin <15 μg/L, evaluating routinely iron stores  among females alone seems appropriate to facilitate effective screening. In addition, serum ferritin (which is not routinely measured during screening) is often requested if borderline HbA2 is encountered. In our study population, additional reasons warrant evaluation of iron stores during premarital screening. A diagnosis (and subsequent treatment) of iron deficiency in our population would benefit a large number of females and subsequently their infants since most women become pregnant after marriage. Moreover, with the high prevalence of α+-thalassemia trait, iron deficiency in females, and BTT in our population, knowledge about iron stores will help community physicians to differentiate between these disorders, all of which are common causes of microcytosis and anemia in our population [HbApulation , 15. Thuα+-thalassemia in this population, one of the highest in the world, may also affect the diagnosis of BTT [α-thalssemia trait  have lower levels of HbA2, an observation also observed by others [α+-thalssemia affects HbA2 level in subjects with BTT is less certain. In a Chinese population with BTT, the HbA2 level was unaffected by coinheritance of one α-globin gene deletion and two α-globin gene deleted in cis (α0) [α-thalassemia trait is due to two α-globin gene deleted in trans (α+) [α-thalassemia resulting in hemoglobin H disease, HbA2 was found to be normal or elevated [β-thalassemia and α-thalassemia may result in normal MCV and MCH, a type of silent BTT that could result in a false negative diagnosis of BTT [β-thalassemia mutations that result in normal level of HbA2 is unknown in our population, and such prevalence is presumed to be low. Our study was not designed to determine the frequency of such mutations. However, in some recent studies, the frequency of silent β-thalassemia mutations  is reportedly as high as 17% in some populations with endemic BTT [α-gene deletion carriers (49% in other study) in the same population increases the likelihood of their coexistence in BTT carriers [α+-thalassemia on HbA2 in BTT is unknown. Finally, recently it was found that tribalism, resulting in marriages within Kinship groups and heterogeneous distribution of BTT carriers in our population, dramatically increases odds of a BTT carrier marrying another BTT carrier [β-thalassemia mutation in all individuals with normal HbA2, who plan to marry BTT carriers.The high frequency of s of BTT , 18. We elevated . A coinhs of BTT . In our emic BTT –23. Morecarriers , 18. The2 levels were not statistically different between men and women without BTT after excluding iron deficient females from the analysis, finding which has been observed earlier [2 was also not different between males and females after the effect of iron status and type of β-thalassemia mutation were taken into account [2. Similarly, smoking in our study did not affect the level of HbA2, a finding which was also observed in one other study [In this study, we also noted that HbA earlier . In a st account . In geneer study . β-thalassemia mutation is a predictor of HbA2 level [2 level than iron deficiency [β+- and β0-thalassemia mutations resulting in the heterogeneity of HbA2 levels  is higher than appreciated. Since both iron deficiency and α+-thalassemia trait are common and both lower the level of HbA2, this decrease in HbA2 may cause some BTT carriers to be missed on screening. As iron deficiency is mostly confined to females, routinely measuring serum ferritin in women is justified. In addition, since marriage between two BTT carriers can be disastrous for the progeny, a DNA test for β-thalassemia mutation is warranted in all subjects without BTT who plan to marry BTT carriers. The benefit of this approach to BTT screening needs to be validated by larger studies.In this Arab population, the cumulative risk of all the factors that lower HbA


The aim of this study was to investigate whether perioperative volume management can influence the PaOSixteen patients were divided into two groups: one group was treated with a restrictive approach (≤8 ml/kg/hour), and the other with a liberal approach (> 8 ml/kg/hour). Patients were randomly allocated using sealed envelopes. During the thoracic part of the surgical procedure, all patients received one-lung ventilation (OLV).2/FiO2 was 288 ± 14 after intubation and 270 ± 22 before extubation. In the group treated with a liberal volume approach, fluids were replaced at 11.0 ± 2.0 ml/kg/hour. PaO2/FiO2 was 259 ± 24 after intubation and 223 ± 43 before extubation. Surgery combined with OLV was found to significantly affect the PaO2/FiO2 value . The average PaO2/FiO2 level was significantly higher in the restrictive-replacement group than in the liberal-replacement group . There was no interaction between the groups . Mean length of stay in the ICU was similar between the restrictive-replacement group (5.2 ± 2.3 days) and the liberalreplacement group (6.3 ± 1.6 days) .In the group treated with a restrictive volume approach, patients received fluids at the rate of 7.0 ± 1.0 ml/kg/hour. PaO2/FiO2 value, which restriction of perioperative volume did not significantly affect. Volume restriction also did not affect length of stay in the ICU.Results from this small sample indicate that esophageal carcinoma surgery by itself had a detrimental effect on the PaO