Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) “head” is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines.Myosin-II thick filament formation in  A portion of the myosin tail coupled to a green fluorescent protein "head" proves a valuable model for understanding myosin self-assembly Myosin-II (hereafter referred to as myosin) is a hexameric protein composed of two heavy chains, two regulatory light chains, and two essential light chains A. The heDictyostelium discoideum assembles without the aid of a cofactor, demonstrating that the myosin molecule itself contains all the information needed to form a highly organized structure of a defined size (Dictyostelium (A bipolar thick filament (BTF) is a highly organized structure composed of individual myosin molecules. In vitro, myosin from the cellular slime mold ned size . While postelium .Dictyostelium, phosphorylation of myosin inhibits filament assembly  heavy chain of myosin is sufficient to inhibit BTF formation . These t in vivo . Mutatio in vivo . The dou in vivo .Dictyostelium myosin tail occurs every 196 a.a.  produces a chimera that is apparently indistinguishable from full-length myosin in its ability to form BTFs  and 3xAla can localize, have demonstrated the necessity of BTF assembly for proper localization of myosin . Howeverum cells . Chimerisin tail . This hasin tail .Dictyostelium BTFs has identified functional domains and repeating patterns in the coiled-coil tail. How do these properties come together to form a BTF that can be regulated by phosphorylation of only three a.a.? How do these properties contribute to regulated assembly? How do these properties enable a myosin BTF to localize to the cleavage furrow during cytokinesis? We have approached these questions by reconstituting regulated assembly of BTFs and defining the minimal part of the molecule required for regulated BTF assembly in vitro and in vivo.Characterization of We wished to test whether both the Ala 1 and the Ala 2 domains of myosin are necessary to regulate BTF assembly  or whethThick filaments and paracrystals efficiently sediment upon centrifugation, whereas unassembled molecules remain soluble. Wild-type full-length myosin efficiently assembles in buffers of intermediate ionic strength (25–100 mM NaCl), while phosphorylated full-length myosin is unassembled A Kuczma. Therefo222) (unpublished data). Therefore, both tail fragments behave as two-stranded coiled-coils in these assays.We next tested whether AD-Cterm (3xThr) and AD-Cterm (3xAsp) tail fragments form coiled-coils, similar to full-length myosin. The oligomerization state of the tail fragments was determined by sedimentation equilibrium analysis, and the secondary structure was assessed by circular dichroism (CD). Neither tail fragment is expected to assemble in the buffers used for these studies, as they contain 500 mM NaCl. Sedimentation runs were carried out as a function of protein concentration, and equilibrium traces were obtained after 20 h of centrifugation. The profiles were fit to an equation describing the sedimentation behavior of a single, non-associating, ideal species B. The diThe sedimentation equilibrium analysis demonstrates that a direct comparison of coiled-coil stability can be made by thermal denaturation at 500 mM NaCl because neither tail fragment self-assembles to form larger species in this condition. Thermal melts are reversible and at equilibrium and far-UV CD spectra show both tail fragments are α-helical at the starting temperature of the melt (4 °C) and are random coil at the ending temperature (60 °C) (unpublished data). The melting temperature for the AD-Cterm (3xThr) and AD-Cterm (3xAsp) tail fragments at both protein concentrations are identical , it forms paracrystals with a 14-nm (98-a.a.) periodicity, corresponding to the periodicity of charge in the AD-through-C-terminal region of the myosin tail  is assembled in buffer containing 50 mM NaCl with 10 mM MgClsin tail  . Both GFP-AD-Cterm (3xThr) and GFP-AD-Cterm (3xAsp) versions of this protein were made to mimic the unphosphorylated and phosphorylated states of myosin, respectively.To test whether the presence of a globular domain might induce AD-Cterm to form regulated, bipolar structures analogous to the thick filaments formed by full-length 2 and imaged them using EM. Bipolar structures first formed on a time scale of 2–5 min  apart, corresponding well to the 14.3-nm offset of full-length myosin heads in a BTF (We assembled purified GFP-AD-Cterm (3xThr) in buffer containing 50 mM NaCl with 10 mM MgCl 2–5 min B, then bhere see , it formhere see . The bipin a BTF .n = 63) at the center of the bare zone (area where heads are absent), close to the width of full-length myosin BTFs (33 ± 1 nm). For both GFP-AD-Cterm and full-length myosin, the length of the bare zone is approximately equal to the length of the coiled-coil. This length is 130–190 nm for full-length myosin and 85 ± 11 nm (n = 63) for GFP-AD-Cterm. This is an indication that GFP-AD-Cterm probably assembles in a manner similar to full-length myosin, with differences in BTF dimensions reflecting differences in myosin tail length.We compared the dimensions of GFP-AD-Cterm (3xThr) and full-length myosin BTFs . The widNotably, the solubility properties of GFP-AD-Cterm (3xThr) and GFP-AD-Cterm (3xAsp) C are simDictyostelium in vivo, we expressed our constructs in myosin-null Dictyostelium cells. However, the GFP-tail fragments were vastly overexpressed in vivo (unpublished data). Introducing the 31-a.a. regulatory light chain (RLC) binding site between the GFP and tail fragment sequences  was not (unpublished data), demonstrating that the RLC does not interfere with regulated filament formation.To test the ability of GFP-tail fragments to localize to the cleavage furrow of equences  producedDictyostelium cells . Dictyostelium cell during cytokinesis. In these cells, GFP-myosin is recruited to the cell equator early during cytokinesis and remains there until the cell divides. Thereafter, myosin remains at the back end of the daughter cell as they move away from one another. In contrast, the labeled beads of similar size show no directed motion toward the furrow  and, interestingly, this GFP-AD (1xThr) tail fragment had bulk sedimentation properties very similar to those of headless AD (1xThr), suggesting that the presence of a globular domain does not affect in vitro solubility, similar to AD-Cterm and GFP-AD-Cterm tail fragments B. The ADTo determine whether the entire 34-kDa portion of the tail C-terminal to the AD is required for regulated assembly, we generated a series of tail fragments that all start at the AD and are truncated at different positions within the C-terminal 34 kDa of the tail see C. This iTo test whether adding on a larger portion of the tail results in a greater degree of regulated assembly, we constructed tail fragments that start at the AD and end at a.a. 1,966, the end of Ala 2 (AD-Ala 2). As with other tail fragments, the aspartic acid variant of this construct is more soluble than its wild-type counterpart D. SurpriWhen the entire C-terminal domain is included in the AD-Cterm tail fragment, the inhibitory effect of Ala 2 is overcome. To test whether this stabilizing effect is specific to the C-terminal domain, we determined if Ala 1 could also stabilize assembly. A tail fragment was constructed that starts at Ala 1 and ends at Ala 2 (Ala1-Ala2 [2xThr]). The salt-dependent assembly of Ala1-Ala2 (2xThr) is more efficient than that of AD-Ala2 (2xThr) E at 5 μMThe 196-a.a. repeat is remarkably symmetric in the entire AD-Cterm region A. We cren = 118) apart, consistent with the 14-nm spacing of full-length Dictyostelium myosin. The AD-Cterm-GFP (3xThr) structures have a shorter bare zone than GFP-AD-Cterm (3xThr) structures (62 ± 8 [n = 75] versus 85 nm), but a similar width to both GFP-AD-Cterm (3xThr) and full-length myosin BTFs . While the AD-Cterm-GFP (3xThr) structures differ slightly from those formed by full-length myosin and GFP-AD-Cterm (3xThr), it is clear that the presence of the globular domain leads to a fixed length, even in C-terminally capped myosin tail fragments.To test whether AD-Cterm-GFP (3xThr) can form BTFs, we performed EM B and sedWe next tested whether AD-Cterm-GFP assembly is regulated. AD-Cterm-GFP (3xAsp) is soluble at all NaCl concentrations, whereas AD-Cterm-GFP (3xThr) sediments efficiently between 25 mM and 150 mM NaCl C, very sThe assembly properties of both AD-Cterm (3xThr) and AD-Cterm (3xAsp) closely parallel those of full-length unphosphorylated and phosphorylated myosin described by The fact that GFP-AD-Cterm (3xThr) forms BTFs whereas AD-Cterm (3xThr) does not, demonstrates that the mere presence of a globular head differentiates BTFs from paracrystals, and that neither the myosin head nor the S2 region of the myosin coiled-coil is critical for BTF formation  is less robust than full-length myosin, constitutively assembled GFP-RLC-AD-Cterm (3xAla) always localizes to the cleavage furrow in cytokinesis see . In contThe difference in localization efficiency between full-length myosin and GFP-RLC-AD-Cterm (3xThr) might be due to differences in degree of assembly. The critical concentration of full-length myosin is estimated to be less than 20 nM . One migDictyostelium cells  and AD-Cterm (3xAsp) are very similar to full-length, wild-type myosin and full-length, phosphorylated myosin, respectively, show that Ala 1 is not required for regulated assembly. This result has been confirmed in vivo by deleting Ala 1 from both full-length wild-type and 3xAsp myosin . The CD and analytical ultracentrifugation data show that AD-Cterm (3xAsp) has not simply failed to fold into a two-stranded α-helical coiled-coil. These data are consistent with rotary shadowed electron micrographs of full-length 3xAsp and phosphorylated myosin. Gross destabilization of the coiled-coil would result in a single-headed myosin, and none have been observed . Global Small numbers of assembled AD-Cterm (3xAsp) paracrystals were seen in electron micrographs of samples (unpublished data). These paracrystals, although quite rare, possess a 14-nm periodicity similar to the wild-type tail fragment. Consistent with this observation, approximately 10% of 3xAsp myosin sediments at 50 mM NaCl, demonstrating that regulation of assembly is not an all-or-none process. Together with the CD data, this result indicates that AD-Cterm (3xAsp) is structurally and functionally intact. Rather than being conformationally disrupted, the 3xAsp tail fragment fails to assemble because the critical concentration for assembly is higher than that of the wild-type tail fragment.The fact that AD-Cterm-GFP forms BTF-like structures with heads on the outside and tails in the center demonstrates the role of the 196-a.a. charge repeat in assembly. This charge repeat is symmetric in the AD-Cterm region of the tail see A. If theInterestingly, AD-Cterm-GFP (3xThr) structures are shorter than GFP-AD-Cterm (3xThr) filaments, and the bare zones are smaller as well. This may be because C-terminal sequence elements in the myosin tail may be required for proper formation of the bare zone and thereby determine the exact morphology of the BTF.The truncation analysis provides evidence of a delicate balance between forces that drive assembly and disassembly of myosin tails. This is most strikingly demonstrated by the observation that Ala 2 was actually found to inhibit the assembly of AD, while Ala 1 and the C-terminal domain help to drive assembly. We propose that that this delicate balance is related to the 196-a.a. charge repeat in the tail. The attachment of Ala 2 purple,  to the ABecause the charge repeats likely play a role in each step in the assembly pathway, a small effect, such as the introduction of negative charge at key points in the molecule, could have a large effect on overall self-assembly. The threonine phosphorylation sites are positioned near the positive clusters of charge at the end of AD and in the C-terminal domain, suggesting that regulation by phosphorylation might have its largest effect on this charge pattern.Dictyostelium expression vector (pBIG) containing either GFP (3xThr) myosin or GFP (3xAsp) myosin (The polymerase chain reaction (PCR) was used to amplify the regions of a ) myosin . An NdeIDictyostelium myosin: AD (1xThr and 1xAsp), a.a. 1,531–1,824; AD-Cterm (3xThr and 3xAsp), a.a. 1,531–2,116; extended AD (2xThr and 2xAsp), a.a. 1,531–1,840; AD-Ala 2 (2xThr and 2xAsp), a.a. 1,531–1,966; AD-2015 (2xThr), a.a. 1,531–2,015; Ala 1-Ala 2 (2xThr), a.a. 1,348–1,966; Ala 1-Cterm (3xThr), a.a. 1,348–2,116  was used to eliminate the stop codon immediately before the SacI site in the AD-Cterm tail fragments contained in pET21a. The modified GFP-UV was subcloned into this vector using SacI and NotI. All DNA sequences were verified.Dictyostelium, the myosin regulatory light chain binding site (RLCBS) sequence was ligated into plasmid pTX-GFP -RIL strain (Stratagene). LB media contained 34 μg/ml chloramphenicol and either 100 μg/ml kanamycin for pET28a or 100 μg/ml carbenicillin for pET21a. Cells were grown at 37 °C to an absorbance at 600 nm of approximately 0.6. Protein expression was induced by adding 1 mM IPTG and incubating for 1 h. Cells were harvested by centrifugation at 6,370 × g for 15 min.Harvested cells were resuspended in 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM DTT, 500 mM NaCl, 30% sucrose, and protease inhibitor (PI) cocktail . For a single protein prep, the pellet from 10 l of culture was resuspended in buffer for a final volume of 40 ml. Cells were added dropwise into liquid nitrogen and stored at –80 °C.2 were added. The cells were lysed with two passes through a French Press  at 10,000 pounds per square inch. After the first pass, a new batch of the PI cocktail was added. The lysate was centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant was boiled for 10 min, and then a new batch of the PI cocktail and 1 mM DTT was added. The boiled supernatant was centrifuged at 100,000 × g for 30 min at 4 °C and then dialyzed against DEAE Low-Salt Buffer . The protein was injected onto a column consisting of eight tandem HiTrap DEAE Sepharose Fast Flow columns . The column was washed with 5 volumes of DEAE-LSB and protein was eluted on a linear gradient from 0 to 500 mM NaCl over 10 volumes. Fractions containing protein  were identified by SDS-PAGE. Protein was precipitated by addition of 85% (NH4)2SO4, stirred at 4 °C for 30 min, and centrifuged at 100,000 × g for 30 min at 4 °C. The pellet was resuspended in a minimal volume of gel filtration buffer  and loaded on a HiLoad 26/60 Superdex 200 column (Amersham Biosciences). Fractions were analyzed by SDS-PAGE, and those containing pure tail fragments were pooled and dialyzed against Mono-Q Low Salt Buffer . Protein was injected onto a Mono Q HR 5/5 column (Amersham Biosciences) and a gradient from 0 to 1 M NaCl was run. Tail fragments typically eluted at 450 mM NaCl.Cells were thawed and 5 μg/ml RNase A , 50 μg/ml RNase-free DNase I , and 10 mM MgClProtein concentration was determined by measuring the absorbance at 280 nm in 6M guanidine hydrochloride. The extinction coefficient was calculated using the sequence of one strand of the coiled-coil as described in After harvesting, cells were resuspended in 50 mM sodium phosphate (pH 8.0) with 250 mM NaCl and containing PI cocktail. For a single protein prep, the pellet from 30 l of culture was resuspended in buffer for a final volume of 120 ml.2 was added along with the DNase I and RNase A. The supernatant was batch-bound to Ni-NTA resin  at 4 °C for 1 h. The resin was washed with 40 ml of 50 mM sodium phosphate (pH 8.0) with 250 mM NaCl (starting buffer) followed by 20 ml of starting buffer containing 12.5 mM imidazole. Protein was eluted from the column using approximately 5 ml of starting buffer with 500 mM imidazole. Fractions containing protein were loaded onto a Hi-Load 26/60 Superdex 200 prep grade gel filtration column (Amersham). Both GFP-AD-Cterm and AD-Cterm-GFP tail fragments elute between 120 ml and 150 ml. GFP-AD elutes between 152 ml and 174 ml. Protein was dialyzed against Mono-Q LSB overnight and then injected onto the Mono-Q HR 5/5 column (Amersham Biosciences) as described above. Protein elutes at about 450 mM NaCl. Protein was used immediately after purification for microscopy and sedimentation assays.Lysis and the first centrifugation step were identical to the myosin tail fragment protocol described above except that 5 mM MgCl2 concentration of 10 mM, and a final NaCl concentration of 50 mM. This solution was deposited onto glow-discharged 300-mesh carbon stabilized copper grids coated with formvar . AD-Cterm and AD proteins were assembled for 2 h, while GFP-containing proteins were assembled for 2 min. A 1% uranyl acetate solution was applied before imaging on a JEOL 1230 transmission electron microscope .Purified GFP-tail fragments were diluted to a final protein concentration of approximately 1 μM, a final MgClPurified protein was dialyzed overnight against 10 mM imidazole (pH = 7.5), 0.1 mM EDTA, and 1 mM DTT. 10 μM protein was added to an equal volume of 10 mM imidazole, 0.1 mM EDTA, 1 mM DTT, 2× mM NaCl. The final concentration of NaCl was × mM NaCl, where × = 0, 25, 50, 75, 100, 150, and 250 mM. Samples were incubated on ice for at least 30 min and then centrifuged at 132,000 × g for 15 min at 4 °C. Three replicates of each sample were made to ensure reproducibility. Samples of the supernatant and pellet were run on SDS-PAGE gels. The intensity of bands was quantified with an AlphaImager 2000 Documentation and Analysis System .222 was monitored with a 1 nm bandwidth. The temperature was increased in 1 °C increments, and the sample was equilibrated for 2 min at the new temperature before data collection. An averaging time of 30 s was used. The pmt dc voltage used was 1.0 V for 2.5 μM protein and 0.55 V for 50 μM protein. Kaleidagraph  was used for all data analysis. Plots of fraction denatured versus temperature were constructed as described in An Aviv Circular Dichroism Spectrometer model 62A DS was used . The protein was in 10 mM Tris (pH 7.4), 500 mM NaCl, 1 mM EDTA, 1 mM DTT. Wavelength scans were taken from 260 nm to 200 nm. Data were collected every 1 nm with a 1-nm bandwidth and averaging time of 10 s. The spectra presented are buffer subtracted. Thermal melts were taken from 4 °C to 60 °C. θAD-Cterm (3xThr) was assembled by diluting the NaCl concentration to 50 mM. Assembled protein was recovered by centrifugation at 100,000 × g for 15 min. Pellets were resuspended in 10 mM Tris (pH 7.4), 0.1 mM EDTA, 500 mM NaCl  and centrifuged at 100,000 × g for 15 min to remove aggregates. The AD-Cterm (3xAsp) tail fragment was centrifuged at 100,000 × g for 15 min to remove aggregates. Both tail fragments were exchanged several times into High-Salt Buffer using an Amicon Ultra-15 30,000 Dalton cut-off ultra-filtration device . The High-Salt Buffer was the blank in all analytical ultracentrifugation experiments. After buffer exchange, the protein was centrifuged at 14,000 rpm for 10 min in a microcentrifuge to remove aggregates. The protein was snap-frozen in liquid nitrogen and shipped in dry ice to the Macromolecular Interactions Facility at UNC-Chapel Hill. The protein used for all analytical ultracentrifugation experiments was centrifuged at room temperature for 15 min at 16,000 × g to remove aggregates.All sedimentation equilibrium experiments were performed at the UNC Chapel Hill Macromolecular Interactions Facility as described in Dictyostelium cells (HS1 myosin-null um cells  were traum cells  supplemeDictyostelium cells expressing the appropriate construct was centrifuged and resuspended in 10 mM imidazole (pH 7.5), 0.1 mM EDTA, and 50 mM NaCl in the presence of protease inhibitors. The cells were lysed by freezing in liquid nitrogen and thawing. Cell lysates were allowed to sit on ice for 2 h before centrifugation at 132,000 × g for 20 min. The pellets were resuspended in equal volumes of the lysis buffer. The supernatant and pellets were resolved by SDS-PAGE without boiling the samples. The GFP-fusion proteins were imaged in the gel by excitation at 532 nm on a Typhoon 8000 imager .1 ml of nearly confluent Dictyostelium genome upstream of and in-frame with the gene encoding myosin-II (mhcA) by homologous recombination. The 167 bp just upstream of mhcA through the first 500 bp of the coding region were PCR-amplified from Dictyostelium genomic DNA using primers to add a 5′ XhoI site and a 3′ BamHI site. The PCR product was cloned into pBluescript digested with XhoI and BamHI. A PstI site was added between the upstream and mhcA coding regions by Quickchange mutagenesis (Stratagene) to create plasmid pBS/upstream-mhcA. GFP was amplified from pTX-GFP , a clonal cell line, GMO8B, was created by plating FACS-sorted GFP-positive cells on Klebsiella lawns and picking spores from individual GFP-positive plaques. The correct integration of GFP upstream of mhcA was verified by PCR and imaging of GFP fluorescence in-gel after SDS-PAGE of cell lysates. The cell line was able to develop normally and grow in suspension, and GFP localization in GMO8B was very similar to GFP-myosin localization in cells expressing GFP-myosin from a plasmid  in HL5 medium. The cells were immediately scraped off of the surface with a rubber policeman and transferred to a clean glass slide. After the cells were allowed to attach for approximately 30 min, the slide was rinsed in PBS to remove excess beads not taken up by the cells. The cells were then removed from the glass slide by pipetting up and down with HL-5 medium.Glass slides were coated with 10 μg/ml polylysine overnight. Approximately 1 ml of semi-confluent GMO8B Live cells were transferred to imaging chambers  in HL-5 medium. GFP and FluoSphere fluorescence were imaged at room temperature using a Zeiss  Axiovert 200 inverted epifluorescence microscope equipped with a 63× objective (N.A. 1.3). Cells were imaged at 20-s intervals. Images were collected using Metamorph  and analyzed with ImageJ (NIH) and Photoshop .


There is genuine need to develop interventional treatment options for management of lung tumors. Radiofrequency ablation (RFA) is one such alternative being promoted to treat lung tumors recently. Larger studies should help define RFA's further development. Furthermore fluorodeoxyglucose positron emission tomography (PET) has been reported to be an accurate indicator of treatment response in variety of tumors. This study focuses on the evaluating the feasibility of RFA and usefulness of PET scan in lung tumors after RFA procedure.Between November 1999 and May 2002, 50 patients with primary or metastasis pulmonary tumors underwent RFA procedure. The electrode was guided to the target areas using computerized tomography (CT). Tumors smaller than 3.5 cm were given single RFA, while tumors larger than 3.5 cm received RFA to multiple sites. Maximum 4 lesions or 6 target areas were treated during one operating procedure. Whole body and/or lung PET images were acquired; identical site CT images and chest X-ray were taken 1 week before and after RFA.Of the 50 patients, 17 had single lesions while rest had multiple lesions. Tumors smaller than 3.5 cm were completely dissipated after RFA. In tumors larger than 3.5 cm, the part within 3.5 cm diameter dissipated. While CT showed that tumor image became larger 1 to 2 weeks after RFA procedure. PET demonstrated tumor destruction in 70% cases, compared to 38% in CT.The present study shows RFA to be safe and effective treatment option for lung tumors. PET is superior to CT in evaluation the effectiveness of RFA treatment shortly after the procedure. Lung cancer continues to be the leading cause of cancer deaths in United States . The oveRadiofrequency ablation (RFA), is an imaging-guided percutaneous ablative procedure, that has been suggested to be an effective treatment option for patients with non-small cell lung cancer (NSCLC) and metastatic disease who are not suitable candidates for surgery ,4. GuidePET has been reported to be an accurate indicator of treatment response in variety of tumors -17. HoweBetween November 1999 and May 2002, 50 patients with either primary or metastatic lung tumors were enrolled in to a prospective single group trial. Patient characteristics are detailed in Table Patients received a chest X-ray and CT for preoperative evaluation and a repeat scan after RFA procedure. A PET scan was performed one week after the treatment.The Radiofrequency ablation was carried out using RF-2000 generator and related software purchased from Radio Therapeutics Corporation, USA; PET imaging was done using an Advance 2 Scanner .Patients received general anesthesia along with local infiltration of Lidocaine. The electrodes were directed to target areas during RFA procedure using CT scan. The initial power applied was 50 W, which was subsequently increased to a maximum 90 W over several minutes. RFA continued for 5 to 15 min until roll off was achieved, which continued for 2 min to stop. Tumors smaller than 3.5 cm were given full heating energy only once, while tumors larger than 3.5 cm received multiple RFA to different areas. Maximum 4 lesions or 6 target areas were treated during one procedure.18F-FDG 296 MBq – 440 MBq (8 mCi -12 mCi) intravenously. After another period of rest lasting for 45–60 min, the whole body and/or lung images was acquired by PET scanner. PET was also acquired at 5–8 bed positions, typically from the base of skull to the mid thigh, which was identical to the CT protocol used in the present study. The complications of the treatment are detailed in table One to two weeks after the procedure and a 4 hour fast, patients were taken for PET scan. They were made to rest for 15 min, and then received After RFA procedure a number of complications were seen. Fever and/or congested pneumonia were commonest complications seen in 32% of patients; however, they were cured in a week with antibiotics treatment. Pneumothorax occurred during procedures in 18% and the patients were treated with aspiration. Five of these had small pneumothorax that did not require and treatment. One patient had hemothorax which required intercostals drainage (ICD) which was removed 2 days later. These were no life threatening events or deaths.Post procedural PET demonstrated the effectiveness of RFA on lung tumors. Tumors smaller than 3.5 cm showed complete response after RFA Figure . In tumoThe Chest X-ray and CT showed that tumor image became larger 1 to 2 weeks after RFA procedure Figure . These mSince RFA ablates lung tumors directly and locally, marginal tissues surrounding the tumor are frequently partially damaged leading to occasional pneumonia. It is difficult for regular CT and/or chest X-rays to discriminate pathological-physiological tissue damage and fibrillation from the treatment effect of RFA. PET on the other hand provides information on functional and metabolic activity anatomically, and is the only available technique which can specifically diagnose tumors or necrosis after surgery and radiotherapy effectively [28]. Our experience too proves that PET is particularly superior to CT in its ability to evaluate the effectiveness of RFA treatment early after therapy.RFA is a relatively noninvasive, well-tolerated approach. It could destruct tumor completely within the effective diameter while avoiding the surgery, side effects of radiotherapy and toxicity of high dose chemotherapy. Our observations suggest that RFA can kill lung tumors smaller than 3.5 cm after a single RFA procedure. The effect of RFA appears to be limited within 3.5 cm diameter area with the current instruments. However, this also suggests that RFA may not damage the normal tissues surrounding the small tumors. The malignant lesions dissipated in 1 to 2 weeks, while the surrounding tissue stayed intact. While at this period regular chest CT and chest X-ray may show enlarged lesion images. This is in agreement with other reports. With improvements in technology, RFA in combination with other options may further reduce the morbidity and mortality of cancer deaths . Though Despite inherent deficiency of trial design our single group study clearly demonstrates that RFA can be an effective treatment option for lung tumors. Unlike other interventional techniques, RFA provide controlled regions of coagulation necrosis with a single application to an area with 3.5 cm diameter. RFA may cure small lung tumor, reduce tumor burden in larger lesions and may be combined with external beam radiation and/or systemic chemotherapy for further improvements. PET provides functional and metabolic activity anatomically and is particularly superior to CT in evaluation the effectiveness shortly after RFA procedure. Absence of follow-up information and randomization in the current study are two major fallacies which need to be addressed in subsequent studies.SJK is the leading physician and drafted the manuscript.RL, WL, HW, XZ, YM all participated in the study, patient management, literature search and preparation of manuscript.All authors have read and approved the final version of the manuscript.None declared.


Priority setting is one of the most difficult issues facing hospitals because of funding restrictions and changing patient need. A deadly communicable disease outbreak, such as the Severe Acute Respiratory Syndrome (SARS) in Toronto in 2003, amplifies the difficulties of hospital priority setting. The purpose of this study is to describe and evaluate priority setting in a hospital in response to SARS using the ethical framework 'accountability for reasonableness'.This study was conducted at a large tertiary hospital in Toronto, Canada. There were two data sources: 1) over 200 key documents , and 2) 35 interviews with key informants. Analysis used a modified thematic technique in three phases: open coding, axial coding, and evaluation.Participants described the types of priority setting decisions, the decision making process and the reasoning used. Although the hospital leadership made an effort to meet the conditions of 'accountability for reasonableness', they acknowledged that the decision making was not ideal. We described good practices and opportunities for improvement.'Accountability for reasonableness' is a framework that can be used to guide fair priority setting in health care organizations, such as hospitals. In the midst of a crisis such as SARS where guidance is incomplete, consequences uncertain, and information constantly changing, where hour-by-hour decisions involve life and death, fairness is more important rather than less. As of August 12, 2003 there were 438 probable and suspected cases of Severe Acute Respiratory Syndrome (SARS) in Canada – the majority located in Toronto. In Toronto there were forty-four SARS related deaths and over 100 health care workers contracted SARS, placing intense pressure on Toronto's public health and hospital system [Due to both the importance of hospitals in any health system and the difficulties they face, improving priority setting  within hospitals is crucial. Priority setting is one of the most difficult issues facing hospitals because of funding restrictions and changing patient need. A deadly communicable disease outbreak, such as SARS in Toronto in 2003, amplifies the difficulties of hospital priority setting.relevance, publicity, appeals, and enforcement  over two hundred key documents , and (2) 35 interviews with key informants (senior administrators (6), physicians (10), managers (5), nurses (5), a patient (1), family members (2), and other staff (6)). Interviews were audiotaped and transcribed. Interview participants were asked to describe priority setting decisions in relation to SARS and their thoughts about it. We developed an interview guide based on previous research and improved it through pilot interviews with personnel from other hospitals. As is traditional with qualitative studies, the interview guide was modified during the study to explore emerging themes.The data were analyzed concurrent with collection using a modified thematic approach in three phases: open coding, axial coding, and evaluation. In open coding, the data were fractured by identifying chunks of data that relate to a concept or idea. In axial coding, the concepts were organized under overarching themes (i.e. the four conditions of 'accountability for reasonableness'). In evaluation, the descriptive data were compared with the conditions of 'accountability for reasonableness' – correspondence with the framework was considered 'good practice'; instances where the conditions are not met were considered 'opportunities for improvement'. Concepts were formalized and made explicit through the writing of the findings .The validity of the interpretations was enhanced in four ways . First, Approval for this project was obtained from the participating hospital's Research Ethics Board. Written informed consent was obtained from each individual before being interviewed. All data were kept confidential and viewed only by the research team. No individuals have been identified without their explicit agreement.In this section we describe one hospital's priority setting in response to SARS by focusing on the types of decisions, the decision making process, and the supportive reasoning. We then evaluate our findings using the four conditions of 'accountability for reasonableness'. We have also included verbatim quotes from participants to illustrate key points.There were two distinct phases of priority setting at the hospital during the SARS outbreak. First, during the initial days of the outbreak, decisions were made in order to contain the spread of the virus. The Ontario Ministry of Health and Long-Term Care (MOHLTC) directed Toronto acute care hospitals to establish or maintain as necessary a SARS isolation area, restrict patient visits, limit visitors and suspend selected patient transfers. Second, after the initial weeks of the outbreak, was a 'ramp up' phase during which the hospital gradually increased its level of activity. Throughout both phases, priority setting decisions can be organized according to four specific types: decisions relating to staff and patients, beds/rooms, clinical activity, and visitors.Staff were allocated for SARS patients in the SARS unit and ICU, screening at the doors, manning the site command centre, or helping out in occupational health; pregnant and immunosuppressed staff were either redeployed to low-risk activities or sent home; staff deemed non-essential were sent home with pay; students were sent home and educational rounds were cancelled. General medicine patients were transferred to other medical units to maintain the SARS isolation unit; out-patients who had been waiting for non-emergency surgery or clinic appointments were told to wait indefinitely.The SARS isolation unit required negative pressure beds. The hospital created these spaces in a specific isolation unit on a general medicine floor, and in the ICU and Emergency. Admissions to negative pressure beds were decided case-by-case and were based on the assessment of the referring physician, the hospital's infectious disease representative, and the hospital intensivist.All non-emergency surgery and ambulatory care were cancelled during the initial 7–10 days of SARS. During the ramp up phase, clinical activity volumes increased in percentages allowing for urgent cases to be seen as determined by physicians. Operating room time was allotted by division – each individual surgeon reported to their division head the cases they considered urgent. This activity was coordinated by the hospital's command center.A 'no visitors' policy was enforced during the initial stages of SARS except for compassionate grounds as determined by the nurse manager or nurse in charge of the particular unit, in consultation with the attending physician and the hospital command centre. During the ramp up phase, the hospital relaxed the no visitor policy according to changing directives from the MOHLTC and the human resources available for screening at the doors.Priority setting decisions were made across all levels of the institution. We identified four groups of key decision makers: Corporate Command, Hospital Command, Department Management/Chiefs, and Individual Clinicians. MOHLTC directives were interpreted by a team of senior administrators (corporate command) and then communicated to the hospital's command centre. The hospital's command centre implemented the recommendations from the corporate command in accordance with patient population requirements and physical logistics. Department managers and clinical staff also participated in allocating human resources and determining patient care priorities. The corporate command was in constant communication with the hospital's command centre who maintained communication with the managers and other leaders through teleconferencing and email.Throughout each stage of the SARS outbreak, safety was the primary rationale underpinning priority setting. During the early stages of SARS, decisions were made for infection control focused on protecting staff. During the ramp up phase, decisions were based more on a duty to care for patients, emphasizing the hospital's role in the community. However, in addition, there were several other reasons used in support of each decision  staff and patient safety was the primary criterion in decision making, but each decision was based on a cluster of relevant reasons – decision makers' use of clusters of relevant reasons has been identified and discussed in a previous study [opportunities for improvement: 1) patients and families did not have access to the reasons for many decisions, including the visitation policy and ramp-up of clinical activities; 2) a formal revision/appeals mechanism could help improve the quality of decision making and alleviate the unfair reliance on the 'squeaky wheel' phenomenon; 3) OR time was allocated by division, rather than by patient need, and these decisions should be discussed more fully; 4) institutional leaders should maintain two-way contact with front-line staff who are implementing priority setting decisions – this will provide support and enhance accountability for decision making by staff.We identified the following us study , 2) deci'Accountability for reasonableness' is a framework that can be used to guide legitimate and fair priority setting in health care organizations, such as hospitals. It assumes that the time and effort required for meeting the conditions of fairness is justified for two reasons: First, it is important to act ethically and be perceived to be acting ethically – in this case, fairly. Second, acting ethically can help an organization achieve 'goodwill' benefits including, but not limited to, increased trust and satisfaction and decreased complaints. However, it is clear from this scope of decisions examined in this study that time constraints imposed on a health care organization by a highly communicable and potentially fatal infectious disease creates significant priority setting difficulties. It may appear that the conditions of 'accountability for reasonableness' are too demanding to implement in the time constraints – that perhaps containment should take precedence over procedural requirements. We disagree. During the SARS outbreak the hospital's leadership developed and implemented several sophisticated processes to help with their crisis management. Tailoring those processes to meet the four conditions of 'accountability for reasonableness' is not any more difficult or demanding. Moreover, we argue, and some of the participants also argued, that in the midst of a crisis where guidance is incomplete, consequences uncertain, and information constantly changing, where hour-by-hour decisions involve life and death, fairness is more important rather than less.Our findings are limited in that they may not be generalizable to other hospitals. However, generalizability is not the goal of qualitative studies like this. We expect that other hospitals may benefit from the insights provided in this study. For example, this was the first time that 'accountability for reasonableness' has been used to evaluate priority setting in response to an infectious disease outbreak. Other hospitals in similar crises can use 'accountability for reasonableness' to help evaluate and enhance the fairness of their priority setting .The best assurance of fair priority setting in a crisis is fair priority setting everyday. A health care organization that incorporates legitimate and fair decision making everyday, where the decision making culture of the organization is permeated with the conditions of 'accountability for reasonableness', will be primed to meet the challenges of fair priority setting in a crisis. This may be the most important lesson to take from this study.SH and MB are employees of the organization studied here.JB was primarily responsible for acquisition and analysis of data. All authors made substantial contributions to conception and design of the study, data analysis and interpretation, and have been involved in drafting the article or revising it critically for important intellectual content. All authors have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:


GFP insertions within the target coding sequence are in the wrong orientation.There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all 2+ channel (IP3R).We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated CaThese new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation. GFP coding sequence lands in the correct orientation and reading frame with respect to the target coding sequence. We reasoned that it might be possible to overcome these limitations by placing two different fluorescent protein coding sequences in a single transposon, either in tandem or antiparallel. Here we present the results of our initial tests with these designs.Biosensors based on GFP-fusion proteins are powerful tools for observing real-time events within living cells. Insertion of GFP within another protein has produced biosensors capable of signaling intracellular events through intrinsic fluorescence changes ,2, fluor5 transposon [Asc I – 5' and Srf I – 3'). An identical cassette encoding cyan fluorescent protein (CFP) flanked with Asc I and Srf I sites, is positioned in the same orientation at the 3' end of the transposon. Tn5 transposition, in vitro, is only ~1% efficient [KanR) was incorporated between the two fluorescent protein cassettes. Since the YFP sequence has no start codon, it should only be translated if it inserts within another protein coding sequence in the correct orientation and reading frame. Plasmids with transposon insertions that are in-frame with respect to the target coding sequence can be rapidly identified by screening for YFP fluorescence in transiently transfected mammalian cells. Each of these clones produces a truncated fusion protein with YFP at the C-terminus. A stop codon is positioned downstream of the Srf I site to prevent translation beyond the YFP coding sequence. These truncated fusion proteins may provide additional information about which parts of the primary sequence contain trafficking signals. Full-length YFP and CFP fusion proteins are then generated in parallel from each clone by digestion with Srf I or Asc I and re-ligation. By producing identical full-length YFP and CFP fusion proteins from each in-frame insertion <EYOR> should double the efficiency with which multicolored fusion protein libraries can be generated.The mosaic ends (MEs) that define the hyperactive Tnansposon  have twofficient , so the AmpR) CMV expression plasmid, pBNJ12.5. Transposon insertions that disrupt the plasmid origin or AmpR (together ~1.5 kb) are not recovered [R and KanR were isolated by standard mini-prep procedures and transiently expressed in HEK-293 cells in a 96-well format. In a random sample of 192 transposed clones, 32 produced detectable fluorescence. Eighteen of these (~9%) were clearly localized to intracellular membranes.  [3R is a ligand gated Ca2+ channel, composed of 4 homomeric subunits, expressed within the membranes of the endoplasmic reticulum. Each IP3R subunit is over 2700 amino acids, and creating a full-length fluorescent IP3R fusion protein with such a large cDNA presents a formidable challenge for traditional molecular biological techniques. The high ratio of coding sequence to vector makes it an excellent target for transposition, however, with a predicted frequency of in-frame insertions of ~11% per fluorophore for <DBT>. At 24 hrs post-transfection, visual screening for fluorescence of 288 AmpR + KanR clones in HEK-293 cells identified 44 clones that produced green fluorescent proteins. Of these, 35 displayed a uniform cytoplasmic distribution and exclusion from the nucleus  . The IP3s Figure , 3 showes Figure . The rems Figure .GFP and DsRed to build the <DBT> transposon because their coding sequences are so dissimilar. Our concern was that if we chose two similar sequences, CFP and YFP for example, the antiparallel orientation of these coding sequences could produce extensive mRNA hybridization and secondary structure that would inhibit protein translation. It appears however, that DsRed is not well suited for insertion within other proteins. Indeed, DsRed has not been reported as a fusion protein in the middle of another protein, and even N- and C-terminal fusions with DsRed can be problematic [We chose blematic , perhapsblematic . DespiteThe transposons described here should greatly accelerate the creation of multicolored libraries of fluorescent fusion proteins. By creating identical full-length YFP and CFP fusion proteins from each in-frame insertion, the <EYOR> transposon not only facilitates the generation potential FRET pairs, it enables the direct comparison of different fluorophores in otherwise identical fusion proteins. The <DBT> design, on the other hand, has the capacity to double both the throughput of fusion protein generation by virtue of its bi-directionality as well as the total output of novel fusion proteins through the simultaneous use of multiple reading frames. Ultimately, the ability to generate large numbers of novel fusions proteins in days rather than months, should shift the limiting rate at which novel fluorescent protein biosensors are identified to functional screening rather than protein design and construction.KanR gene was obtained from pUniV5-His-TOPO (Invitrogen). The construction of the IP3 receptor expression plasmid, pCMVI-9, was previously described [PCR and standard subcloning procedures were used to create the plasmids encoding <EYOR> (pBNJ55b.1), <DBT> (pBNJ38.5) and Prestin (pBNJ12.5) . The fluorescent protein coding sequences used were Venus (YFP) , ECFP-N1escribed .5 ME (5'-CTGTCTCTTATACACATCT-3') and purified as previously described [E. coli  were transformed with 0.5 μL of the transposition reaction and plated on LB agar with Ampicillin (100 μg/mL) and Kanamycin (50 μg/mL). Parallel plating of the transformation on LB agar with Ampicillin alone was used to establish the transposition efficiency.Transposons were amplified from their host plasmids via PCR with a single primer complementary to the 19 bp Tnescribed . Purifie2O.Transposed plasmids were isolated in a 96-well format from 1.25 mL LB cultures with Eppendorf PerfectPREP-96 Vac Direct Bind miniprep kits on a PerkinElmer MultiPROBE II HT liquid handling robot and eluted in 70 μL of ddH4 cells in 100 μL of MEM-E with 10% fetal bovine serum. Transfections were performed with ~300 ng of plasmid DNA and 0.3 μL of Lipofectamine 2000™ (Gibco BRL) in a total volume of 50 μL of Opti-MEMI (Gibco BRL) per well. The cells were screened for fluorescence 24 hr after transfection with a 20× objective on a Zeiss inverted microscope with excitation and emission filter sets optimized for CFP/YFP or GFP/DsRed imaging .HEK-293 cells (American Type Culture Collection CRL-1573) were plated 24 hr prior to transfection, in 96-well glass bottom tissue culture plates  at 6 × 10YFP coding region (5'-CTGCAGGCCGTAGCC-3') or <DBT>GFP coding region (5'-TGGCCGTTTACGTCGCCGTCCA-3'). To generate full-length fusion proteins, plasmids with unique in-frame insertions were digested and re-ligated. After restriction digestion , 1 μL of the digest reaction (~20 ng DNA) was re-ligated with Fast-Link™ ligase (Epicentre Technologies) for 15 min at room temperature . After heat inactivation (70°C for 15 min.), XL-10 Gold E. coli were transformed with 0.5 μL of the ligation reaction and plated on LB agar with Ampicillin. The following day, colonies were co-inoculated in LB with Ampicillin and Ampicillin + Kanamycin to verify loss of the KanR prior to plasmid isolation.Exact insertion sites were identified for all fluorescent transposed clones by sequencing 5' out of the transposon with a primer complimentary to the <EYOR>D.S. conceived of the transposon designs and carried out their construction, performed the transposition and screening for fluorescent fusion proteins, and drafted the manuscript. T.H. participated in the study design, coordination, and analysis. All authors have read and approved the final manuscript.Supplementary Material-hughes This is a PDF file containing both plasmid maps and full sequence data for the plasmids used in this study.Click here for file


Milk, cheese, and yogurt are so ingrained in the diets of Europeans that it's easy to forget that their ancestors ever ate differently. But about 9,000 years ago, before the domestication of cows, sheep, and goats, milk was a staple only for babies. Back then—just as in most Asian and African cultures today—individuals lost their ability to digest lactose, a sugar found in milk, as they grew up.But with the domestication of animals, milk became abundant. Among herders, individuals had an advantage if they had versions of genes, also known as alleles, that allowed them to digest lactose into adulthood. They would tend to be healthier and reproduce more than those who could not digest lactose. Thus, by natural selection within herding groups, over generations those who could drink milk into adulthood became more common.Researchers have found the allele that allows adults to digest lactose, and it's one of the clearest signs of natural selection in humans. In groups with a history of herding, the vast majority of people have the allele, whereas in non-herding groups, most people lack it. Researchers can find such footprints of selection by comparing groups of people that have lived in different environments, for example, or have eaten different diets.PLoS Biology, Joshua Akey and colleagues report new-found signs of natural selection in several human genes—including a chunk of Chromosome 7 encompassing four genes, the largest footprint of selection found yet. The research group analyzed the complete sequences of 132 genes in a set of 23 European-Americans and 24 African-Americans. All these genes are involved in inflammation, blood clotting, or blood pressure regulation and were studied as part of a larger project looking for alleles that contribute to disease.In this issue of In general, solid evidence of natural selection acting on genes is hard to find. The history of selection can be obscured by a variety of processes. For one, genes can undergo “neutral changes,” in which some of its base pairs change, but without altering the sequence or function of the protein the gene codes for. Also, idiosyncrasies in the history of a population can leave marks on the gene pool. A lineage can go through a “bottleneck,” for example, if a small group splinters off from a larger population and then later multiplies. In general, the splinter group won't perfectly represent the larger population, so the frequencies of alleles for many genes will be skewed in the splinter group's lineage.TRPV5 and TRPV6, limit the rate of calcium uptake, so selection on one or both of these genes in Europeans could have originated with herding.Having first ruled out irrelevant changes in genes and population history effects, Akey and colleagues found strong signs of natural selection only in the European-Americans, suggesting this group went through significant changes in climate, diet, or culture more recently than the African-American group. This fits with the well-accepted idea that European populations came from small groups that split off from the larger African population. The researchers find evidence for such an event in the European population about 40,000 years ago. They also estimate that the region of Chromosome 7 was subjected to strong selection around 10,000 years ago, roughly when European herders began drinking milk. Interestingly, two of these genes, TRPV6 to be more active in prostate cancer cells. In addition, African-Americans suffer higher rates of prostate cancer, and Akey and colleagues found that European-Americans have alleles of TRPV6 different from those of African-Americans. Given this evidence, the researchers suggest that this gene may be involved in susceptibility to prostate cancer. This research could therefore shed light on the evolution of complex diseases such as cancer and why different populations suffer different rates of disease.Recent studies also found


In 2001 the National Cholesterol Education Program (NCEP) provided a categorical definition for metabolic syndrome (c-MetS). We studied the extent to which two ethnic groups, Blacks and Whites were affected by c-MetS. The groups were members of the Hypertension Genetic Epidemiology Network (HyperGEN), a part of the Family Blood Pressure Program, supported by the NHLBI. Although the c-MetS definition is of special interest in particular to the clinicians, the quantitative latent traits of the metabolic syndrome (MetS) are also important in order to gain further understanding of its etiology. In this study, quantitative evaluation of the MetS latent traits (q-MetS) was based on the statistical multivariate method factor analysis (FA).The prevalence of the c-MetS was 34% in Blacks and 39% in Whites. c-MetS showed predominance of obesity, hypertension, and dyslipidemia. Three and four factor domains were identified through FA, classified as "Obesity," "Blood pressure," "Lipids," and "Central obesity." They explained approximately 60% of the variance in the 11 original variables. Two factors classified as "Obesity" and "Central Obesity" overlapped when FA was performed without rotation. All four factors in FA with Varimax rotation were consistent between Blacks and Whites, between genders and also after excluding type 2 diabetes (T2D) participants. Fasting insulin (INS) associated mainly with obesity and lipids factors.MetS in the HyperGEN study has a compound phenotype with separate domains for obesity, blood pressure, and lipids. Obesity and its relationship to lipids and insulin is clearly the dominant factor in MetS. Linkage analysis on factor scores for components of MetS, in familial studies such as HyperGEN, can assist in understanding the genetic pathways for MetS and their interactions with the environment, as a first step in identifying the underlying pathophysiological causes of this syndrome. Metabolic and physiologic disorders for cardiovascular disease (CVD) and type 2 diabetes (T2D), including abdominal obesity, insulin resistance, hyperglycemia, dyslipidemia, and hypertension often cluster. This cluster is frequently identified as the "metabolic syndrome" (MetS). Reaven  related Different definitions and multivariate statistical approaches have been applied to characterize the increasing high-risk MetS premorbid state. Recently, special attention has received the categorical definition of metabolic syndrome (c-MetS) of the National Cholesterol Education Program Adult Treatment Panel III (NCEP) . The NCEa. The trait characterized as the categorical MetS (c-MetS) was studied according to the NCEP definition; b. The underlying (latent) traits or clusters of MetS (q-MetS) were evaluated by performing FA with and without Varimax rotation on 11 risk factors. All data were grouped by ethnicity and gender. Subgroups were created by excluding T2D participants, under the assumptions that T2D individuals may have a different pattern of glucose and insulin levels. Finally, our goal was to compare the expression of c-MetS and q-MetS in the Hypertension Genetic Epidemiology Network (HyperGEN) study.The objective of this study was to exemplify important facets of the MetS in the HyperGEN study. Two MetS aspects were assessed: For c-MetS the sample sizes varied from 2,025 observations for fasting triglycerides (TG) to 2,300 for high density lipoprotein (HDL) cholesterol in Blacks, and from 2,171 observations for TG to 2,471 for HDL in Whites. In the HyperGEN study, a high percentage of individuals have body waist (WAIST) and systolic blood pressure (SBP) / diastolic blood pressure (DBP) above the NCEP thresholds , fasting plasma glucose (GLUC), fasting insulin (INS), HDL, SBP, DBP, WAIST, and percent body fat (%BF), similar low density lipoprotein (LDL) cholesterol and waist to hip ratio (WHR), and lower TG. Kurtosis after adjustments varied from 0.11 for WHR to 0.85 for DBP in Blacks, and 0.17 for WHR to 1.43 for GLUC in Whites, which demonstrates normal distributions for the traits in study and also for the factors created by performing FA .Pearson correlations among the 11 adjusted and normally distributed variables are presented in Table FA with no rotation identified a factor (Factor 1) that was loaded mostly by central obesity, obesity risk factors and INS  method in S-PLUS (Insightful Corporation software), we found that the model p-values were significant, suggesting that additional factors may exist. However, although additional factors must exist to explain approximately 40 percent of the variance in the original variables, none of the remaining factors individually can explain more than about 5 percent of the variance. Therefore, we concluded that the quantitative structure of MetS can be described in terms of three to four factors when no rotation was performed, and four factors with Varimax rotation. One may even argue whether the fourth factor in the Varimax rotation is very meaningful. We chose to retain the "Central Obesity" factor particularly because it tends to reflect the well-known gender asymmetry Table . FA withWe tested the pattern of the factors between genders only for FA with Varimax rotation. This pattern was stable among ethnicities between genders Table . WHR on Another characteristic of the HyperGEN study was that at least two participants in each sibship had hypertension. A large proportion of the hypertensive participants have used anti-hypertensive and anti-cholesterol medications. Maison et al.  have comIt is a common belief that T2D participants may have a different expression of INS and GLUC, therefore it may influence also the factors pattern in the MetS analysis. In the present study, we found a consistency of the factors before and after removing type 2 diabetics. This finding is supported also by Hanson et al.  who studOther studies have provided similar results about the latent traits of MetS ,16,17. FOur study and several others have shown that FA is a useful method for studying the underlying traits of MetS. Nevertheless this methodology has not passed without been criticized. Lawlor et al.  reviewedIf c-MetS and q-MetS are explaining the same disorder in two different aspects in the HyperGEN study, can FA contribute to MetS gene finding? MetS is recognized as a precursor for cardiovascular disease and type 2 diabetes . There aThese analyses demonstrated that obesity and hypertension were the most important factors contributing to the MetS in the HyperGEN Study. Three to four distinct factor domains were identified depending on the FA rotation applied and decisions made. Results support the hypothesis that MetS is a compound phenotype, where obesity and its relationship to lipids and insulin are clearly the driving force of MetS. Insulin may play a connecting role between obesity and lipid domains.In genetic analysis, it is well known that categorical data, especially a complex trait such as MetS, encounter reduced power as compared to quantitative variables. Therefore, we suggest that genetic analysis should be performed on specific combinations of traits that belong to a factor. It is possible that some common genes may exist in the pathways for the factors identified. Linkage analysis investigating putative quantitative trait loci for MetS factor domains can be a first step which may help discover the underlying mechanisms, or generate new hypotheses, in finding the causes of MetS.The sample represents data from the HyperGEN network, part of the Family Blood Pressure Program, supported by the NHLBI as described by Williams et al.  and ProvA participant was classified as having T2D if (s)he had a fasting plasma glucose value ≥ 126 mg/dl, or is a current user of hypoglycemic medication or insulin that was documented at examination in the clinic, or if diabetes was reported in the HyperGEN questionnaire. Also, an age at diagnosis ≥ 40 years was required for T2D individuals .c-MetS according to the NCEP definition, was identified in participants by the simultaneous presence of 3 or more of the following conditions: WAIST > 102 cm in men, and > 88 cm in women; TG ≥ 150 mg/dl; high density lipoprotein (HDL) < 40 mg/dl in men, and < 50 mg/dl in women; systolic blood pressure (SBP) ≥ 130 mm Hg and/or diastolic blood pressure (DBP) ≥ 85 mm Hg or using antihypertensive medications; GLUC ≥ 110 mg/dl or on treatment for diabetes [2); WAIST measured at the level of the umbilicus in cm; WHR defined as waist circumference divided by hip circumference; GLUC in mg/dl; INS in μU/ml (where fasting time was defined as ≥ 12 hours before blood draw); LDL in mg/dl; HDL in mg/dl; TG in mg/dl; Sitting SBP in mm Hg; DBP in mm Hg ; %BF derived from the bioelectric impedance measurements based on the Lukaski formula [Factor analysis was founded on 11 variables: BMI expressed as the ratio of body weight divided by body height squared (in kg/m formula .2) and the squared transformation of %BF (%BF2) reduced the excess kurtosis considerably. The procedure transreg in SAS (version 9 for PC) was employed for finding power transformations.TG and INS had skewed distributions. A relatively skewed distribution was also present for HDL. Log transformation brought these variables distributions to approximately normal. GLUC and %BF were highly kurtotic. Using Box-Cox transformation, it was found that the inverse of the squared transformation of GLUC . Any variables with outliers beyond ± 4 standard deviations (SD) were also adjusted for heteroscedasticity of the variance. After the adjustments for each variable, outliers beyond ± 4 SD were eliminated. Each final adjusted variable was standardized to a mean 0 and variance 1.There were two field centers recruiting Blacks and four field centers recruiting Whites. Accordingly, dummy  field center variables, one for Blacks and three for Whites, were created. All 11 risk factors were adjusted within ethnicity and gender for age, agea priori information about the structure underlying the variables. FA can be used also for a confirmatory analysis, when validation (or refutation) of a postulated structure is sought. In either case, FA seeks parsimony by summarizing a large group of interrelated variables (risk factors for a complex disease such as MetS) in terms of a small number of latent factors, thereby reducing the dimensionality. Theoretical statistical descriptions of FA can be found in the literature [factanal function, in which the MLE was employed. FA evaluated latent factors underlying the 11 original variables.Prevalence of c-MetS was estimated with the FREQ procedure of SAS. The multivariate method of factor analysis was employed for reducing a group of risk factors to a subset of latent factors. The primary goal of FA is to identify the interrelationships among a set of variables. In this study FA was used for exploratory analysis, because there was no terature -26. FA wFA was performed with and without the Varimax rotation. "No rotation" achieves the simplest latent factor structure, in the extreme case loading any variable in one of the factors and almost negligible loadings in the rest of the factors. That is the reason why some studies (extracting factors with no rotation) find a concentration of the major variables' loading on the first factor. This is also the reason why some investigators named the first factor in their studies as the "Metabolic syndrome" factor ,27. ConvMetS, metabolic syndrome; c-MetS, categorical MetS; q-MetS, latent traits of MetS; FA, Factor analysis; MLE, maximum likelihood estimate; T2D, type 2 diabetes; CVD, cardiovascular disease; BMI, body mass index; INS, fasting insulin; GLUC, fasting glucose; WHR, waist to hip ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; BP, blood pressure; TG, fasting triglycerides; LDL, low density lipoprotein cholesterol; HDL, high density lipoprotein cholesterol; %BF, percent body fat.The author(s) declare that they have no competing interests.All authors were equally involved in designing the MetS study, evaluating statistics, interpreting the data, writing the manuscript, and organizing the figure and tables.Table 4. This table contains information on factor loadings result of FA with and without rotation performed on 11 risk factorsClick here for file


I would like to share my experience from nearly a decade of civil war between the Maoist rebels and the Royal Nepalese Army in Nepal in reference to the article by Zwi


Numerous malaria epidemics have occurred in western Kenya, with increasing frequency over the past 20 years. A variety of hypotheses on the etiology of these epidemics have been put forth, with different implications for surveillance and control. We investigated the ecological and socioeconomic factors promoting highland malaria vectors in the dry season after the 2002 epidemic.Investigations were conducted in Kisii District during the dry season. Aquatic habitats in were surveyed for presence of malaria vectors. Brick-making pits were further investigated for co-associations of larval densities with emergent vegetation, habitat age, and predator diversity. Indoor spray catches were completed in houses near aquatic habitats. Participatory rural appraisals (PRAs) were conducted with 147 community members.Anopheles larvae was brick-making pits. Vegetation and habitat age were positively associated with predator diversity, and negatively associated with mosquito density. Indoor spray catches found that houses close to brick-making sites had malaria vectors, whereas those next to swamps did not. PRAs revealed that brick-making has grown rapidly in highland swamps due to a variety of socioeconomic pressures in the region.The most abundant habitat type containing Brick-making, an important economic activity, also generates dry season habitats for malaria vectors in western Kenya. Specifically, functional brick making pits contain less that 50% as many predator taxa and greater than 50% more mosquito larvae when compared with nearby abandoned brick making pits. Further evaluations of these disturbed, man-made habitats in the wet season may provide information important for malaria surveillance and control. The World Health Organization estimates that 300 to 500 million people are diagnosed with malaria annually, causing 1.1 to 2.7 million deaths. Approximately 1 million of these deaths are among children in sub-Saharan Africa, where 90% of all malaria cases occur [The first recorded epidemic of malaria in the highlands of western Kenya occurred in 1918/19, with additional epidemics occurring periodically until 1950 . These iSince the 1980s, however, the incidence of highland malaria and frequency of epidemics have been increasing, with severe outbreaks in 1995, 1998/99, -9 and moFollowing the 2002 epidemic, this study was conducted to identify the ecological and socioeconomic variables affecting malaria vector densities and distributions. In this paper we connect specific man-made aquatic habitats present during the dry season with malaria vector larvae in western Kenya.The highlands of western Kenya are an ecologically diverse and densely populated region situated east of Lake Victoria. Detailed investigations have been conducted in Mosocho Division, a sub-section of Kisii Central District with a predilection for epidemics and high rates of malaria .2 and contained 491,786 people in 2001 (759 people/Km2) [2). Agriculture was the primary industry in the area, conducted on family plots which surrounded smaller industries, such as quarrying, and extended to the borders of wetlands.Kisii District covered 648 Kmple/Km2) . MosochoAnopheles mosquito larvae over the course of two weeks in the dry season (September 2002). Standing water was identified by canvassing Mosocho Division by vehicle and by foot, with the assistance of local inhabitants in the areas investigated. This technique especially relied on information from inhabitants when locating the more remote habitats, which may therefore be under-represented in the survey. Once identified, habitats were evaluated for presence of mosquito larvae using standard aquatic dippers. Dipping was performed around the perimeter of the habitat, with three dips performed at approximately one meter intervals. No effort was made to determine larval densities due to the patchiness of larval distribution within each habitat. Dipping was not performed more than one meter into the habitats, which may have led to an under representation of less abundant species.All sites with standing water within Mosocho Division were evaluated by standard dipping for presence of At each of five brick-making sites in Kisii District, three functional brick-making pits and three abandoned pits were evaluated for pit size, larval densities, percent emergent vegetation and predator taxa present in the habitat.  was perForty nine Chiefs and ninety eight brick makers were interviewed at four locations within Kisii and Gucha Districts to determine the social and economic factors promoting brick-making in the highlands, as well as the historic development of the industry. Standard Participatory Rural Appraisal methodology was used to obtain qualitative data .The means of the variables in the cross-sectional survey were compared for independence between the 15 functional and 15 abandoned brick-making pits (SPSS 11.0 t-test for independence).Anopheles in a house with distance to brick-making pits and swamp were converted into decimal degree coordinates and integrated as map layers within a Geographical Information System (ArcView 8.2). The distances from each house to the nearest brick making pit and swamp boundary were generated using the distance tool within the ArcView software. Spearman's correlation coefficient (SPSS 11.0) was calculated to correlate presence of Anopheles in a house with distance to swamp, distance to brick-making pits, and elevation.GPS coordinates taken for correlating presence of Anopheles mosquitoes. . Of the four quarries positive for Anopheles larvae, three were functional (created in the previous year) and one was abandoned (unused > two years), both with An. gambiae s.l and An. funestus. A portion of a drainage canal dug near a brick-making sites constituted an additional Anopheles-positive habitat, containing An. gambiae s.l. larvae. Of natural habitats sampled, only two contained malaria vectors. One swamp had An. funestus larvae and one tree hole in an ornamental Flamboyant tree (Delonix regia) had An. gambiae s.l. larvae; 87.5% of Anopheles-positive habitats were of direct human origin.A dry-season survey of aquatic habitats in Mosocho Division was conducted to identify potentially important larval habitats of An. gambiae s.l.  and Culex spp. . No An. funestus larvae were found in this study. This corresponded with an increase in predator biodiversity found in the abandoned sites  and increased percent emergent vegetation . , while distance from swamp was positively correlated with presence of Anopheles mosquitoes . Elevation was not correlated with presence of Anopheles in a house .Spray catches in nine houses during the dry season resulted in one According to brick-makers, making bricks is predominantly a dry-season activity due to the damage caused by heavy rains to drying bricks. Although variation in technique exists, universally used stages in the brick-making process are: Excavation, Fermentation, Moulding, Drying, and Kilning. During the Moulding stage, water is brought into the excavated clay pits and mixed with soil. During this stage, which lasts from several days to one month, water is continually supplied to the pit through irrigation systems, ground water, or is brought by bucket from a nearby water source. Once abandoned during the Kilning stage and afterwards, the pit accumulates rain water and ground water, which can be subsequently used as a water source for newly excavated pits. Over years, abandoned brick-making pits degrade until they are continuous with the surrounding swamp.Interviews with Chiefs and brick makers revealed different scales of brick-making operations, from individuals working on their own land to large-scale industries Figure  where weA primary socioeconomic factor described by brick-makers that promotes brick-making is the desire to make use of agriculturally unfavorable wetlands as human population densities increase. Nearly all brick makers work in wetlands, leading to progressive deforestation of the highland swamps. Money from brick-making was used principally for paying school fees and malaria medication. According to brick-makers, brick houses are considered to be more prestigious than traditional houses, are associated with lower long-term costs than traditional mud-thatched houses, and are less permeable to mosquitoes.Our study shows that man-made larval habitats were the predominant (87.5%) source of malaria vectors in Mosocho Division during the dry season. In particular, the continuously disturbed, functional brick-making pits contained high densities of malaria-vectoring mosquitoes. PRAs revealed that small brick-making groups have developed into large-scale industries over the past two decades, and brick-making is now dispersed throughout the highlands in unfarmable wetlands. Because brick-making occurs predominantly in the dry season, it may aid in maintaining vector populations year-round.Additionally, houses closest to brick-making pits had malaria vectors present within them. While the number of mosquitoes captured during a single transect of spray-catches was only one per house, this represents a real, if low, possibility of malaria transmission during the dry season at these locations. Thus brick-making areas may function as refugia for malaria parasites and their vectors over the dry season, facilitating spread of malaria when habitats become more plentiful in the wet season.Anopheles larval densities. More research into the ecology of small aquatic pools will help clarify the interrelationships of these variables.In this study we found emergent vegetation to be negatively associated with presence of malaria vectors in man-made habitats. This association may be a result of an association of both emergent vegetation and predator diversity with habitat age. In continually disturbed habitats , the habitats are kept at a low stage of biological succession, possessing fewer species of both plants and animals. There may also be a direct effect of vegetation on the trophic dynamics in ground pools. In structurally simple habitats, intraguild predation has been shown to suppress the diversity of important predators -23. WhetAnopheles larvae escape the high degree of predation found in the natural environment. These habitats should be specifically targeted during larval control programs. With substantial socioeconomic motivation for brick-making in the highlands, traditional source reduction (eliminating standing water) is unsustainable, and larvicides should be employed. Further research is needed on the use of these habitats by mosquito larvae during the wet season.In ground pools, predators are typically thought to regulate the density of mosquito larvae ,25. PredThe negative correlation between predator diversity and mosquito density suggests that pesticide application may exacerbate epidemics by decreasing predation pressure if used in natural habitats. Pesticide applications can be selectively harmful to larger predators such as dragonflies and large dytiscid beetles which take months to years to develop. In contThe social and economic benefits accompanying disease reduction through vector control was demonstrated in the Zambian copperbelt . As the None declaredPRA: Participatory Rural AppraisalKm: KilometerBMS: Brick making siteNGO: Non governmental organizationJCC designed and conducted evaluations of larval habitats and houses, performed statistical analysis, and wrote the initial draft of the manuscript. BDB performed GIS analysis. FXO identified BMS as sources of vector mosquitoes, designed and conducted PRAs, and aided in the production of the manuscript's final draft.The pre-publication history for this paper can be accessed here:


Involving patients in decision making on diagnostic procedures requires a basic level of statistical thinking. However, innumeracy is prevalent even among physicians. In medical teaching the 2 × 2 table is widely used as a visual help for computations whereas in psychology the frequency tree is favoured. We assumed that the 2 × 2 table is more suitable to support computations of predictive values.184 students without prior statistical training were randomised either to a step-by-step self-learning tutorial using the 2 × 2 table (n = 94) or the frequency tree (n = 90). During the training session students were instructed by two sample tasks and a total of five positive predictive values had to be computed. During a follow-up session 4 weeks later participants had to compute 5 different tasks of comparable degree of difficulty without having the tutorial instructions at their disposal. The primary outcome was the correct solution of the tasks.There were no statistically significant differences between the two groups. About 58% achieved correct solutions in 4–5 tasks following the training session and 26% in the follow-up examination.These findings do not support the hypothesis that the 2 × 2 table is more valuable to facilitate the calculation of positive predictive values than the frequency tree. Diagnostic procedures are increasingly expected by consumers to ensure their health; "certainty" has become a product . AssuminInvolving lay people in decision making on diagnostic procedures requires a basic level of statistical thinking. Help for computing Bayesian inference is needed. Statistical thinking can be enhanced by representing statistical information in terms of natural frequencies rather than probabilities ,7. This In cognitive psychology the frequency tree is used as visual help for the representation of frequencies, a variant of a tree structure often used in decision analysis to teach computing the positive predictive value the simple way Figure  4]. Thi. Thi4]. In contrast, in medical science the 2 × 2 table is the standard method to teach computing predictive values  Students attending the University of Hamburg , a vocational college  or taking part in an in-service training  were informed about the timing and content procedure of the study during their courses.The study was carried out between October 2000 and July 2001 and consisted of two supervised sessions lasting about 1 h each. The recruited 184 students were randomly assigned either to the frequency tree group (n = 94) or to the 2 × 2 table group (n = 90) using blocked randomization in blocks of 10. Concealed allocation based on computer-generated random numbers was done by an external person. In addition, the external person prepared sealed envelopes for both sessions including the tutorial with the tasks and a questionnaire for survey of age, gender, years of school, mark in mathematics and social state. The training consisted of a written step-by-step self-learning tutorial , 2, 3. TIn the follow-up examination participants were again asked to solve 5 different diagnostic problems of similar level of difficulty but without having the tutorial instructions at their disposal ,5,6. ParA solution was classified correct, when the documented positive predictive value was equivalent to the correct solution rounding up or down to the next full percentage point. If a participant used the correct computation  but made a calculation error either in the transfer of the numerical information into the visual help or within the division, we ignored calculation errors. Whenever a different computation such as rule of three – a mechanical method for solving proportions – was used or the calculation protocol was missing the rounded solutions were classified likewise as correct by congruence. If the protocol indeed showed that a correct rounded solution resulted from an incorrect computation such as positive predictive value = correct positives / false positives the answer was classified as incorrect. Tasks that had not been worked on were also classified incorrect.To evaluate the usefulness of the different visual helps, we evaluated the ability of correct transfer of the numerical information into the charts. A transfer was classified as correct, when the numerical information of the problems was inserted into the gaps provided. It was sufficient to insert the relevant values for the computation, calculation errors were ignored.The computation was classified as correct Bayesian approach when the following computation was used: positive predictive value = correct positives /  or positive predictive value = correct positives / all positives. The computation was classified as Non-Bayesian approach when the computation was used with false values. Other computations were classified as other strategies.Table Analysis is based on the intention-to-participate principle that includes all randomised participants as randomised. Drop outs were considered as having solved none of the positive predictive values correctly.Figure The groups were similar regarding demographic variables Table .Table Within the category 4–5 correct answers 27% of participants  (2 × 2 table) and 26%  (frequency tree) had correct solutions. The differences between the two study groups were not statistically significant neither in the training session (p = 0.95 {0.49 one-sided}) nor in the follow-up examination ; n = 297/450 (2 × 2 table)] were solved correctly in both groups. The amount of correct solutions decreased to 26% (n = 98/370) and 31% (n = 115/375), respectively, in the follow-up examination. Differences between groups were not statistically significant  and 76% (n = 342/450 2 × 2 table) of the tasks. In the follow-up examination in 63% (n = 234/370) and 70% (n = 264/375), respectively, the information was correctly transferred into the visual helps (Table The application of the Bayesian computation in the training session was correctly used in 65% (n = 307/470 frequency tree) and in 61% (n = 273/450 2 × 2 table). In the follow-up examination 21% (n = 76/370) and 22% (n = 83/375), respectively, used correct Bayesian computation . Mathematics literacy was stated to be poor in Germany especially in girls . A high In conclusion, our findings do not support the hypothesis that the 2 × 2 table is more valuable to facilitate the calculation of positive predictive values than the frequency tree. Regardless which visual help is used there is a need for improvement of teaching methods to approach lay people who want to participate in medical decision making.None declared.AS as the principal investigator planned and performed the study analysed the data and wrote the paper. AB contributed to planning and performance of the study. JB calculated the power of the study and carried out the statistical analysis of data. IM contributed to all parts of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Original questionnaire used in the 2 × 2 table group in the 1. session in German language.Click here for fileOriginal questionnaire used in the frequency tree group in the 1. session in German language.Click here for fileTasks used in the questionnaires of the training session in English language.Click here for fileOriginal questionnaire used in the 2 × 2 table group in the 2. session in German language.Click here for fileOriginal questionnaire used in the frequency tree group in the 2. session in German language.Click here for fileTasks used in the questionnaires of the follow-up examination in English language.Click here for file


There are no studies investigating the effect of the contrast infusion on the sensitivity and specificity of the main Doppler criteria of renal artery stenosis (RAS). Our aim was to evaluate the accuracy of these Doppler criteria prior to and following the intravenous administration of perfluorocarbon exposed sonicated albumin (PESDA) in patients suspected of having RAS. Thirty consecutive hypertensive patients  suspected of having RAS by clinical clues, were submitted to ultrasonography (US) of renal arteries before and after enhancement using continuous infusion of PESDA. All patients underwent angiography, and haemodynamically significant RAS was considered when ≥50%. At angiography, it was detected RAS ≥50% in 18 patients, 5 with bilateral stenosis. After contrast, the examination time was slightly reduced by approximately 20%. In non-enhanced US the sensitivity was better when based on resistance index (82.9%) while the specificity was better when based on renal aortic ratio (89.2%). The predictive positive value was stable for all indexes (74.0%–88.0%) while negative predictive value was low (44%–51%). The specificity and positive predictive value based on renal aortic ratio increased after PESDA injection respectively, from 89 to 97.3% and from 88 to 95%. In hypertensives suspected to have RAS the sensitivity and specificity of Duplex US is dependent of the criterion evaluated. Enhancement with continuous infusion of PESDA improves only the specificity based on renal aortic ratio but do not modify the sensitivity of any index. Renal artery stenosis is the most frequent cause of secondary hypertension  which isThe proposed criteria for the detection of renal artery stenosis by direct Doppler include an increased peak systolic velocity, an increase in renal aortic ratio and also an increased resistance index . The mea® echo-enhancing ultrasonography in hypertensives with renal artery stenosis. They demonstrated a sensitivity of 85% and a specificity of 79% without contrast and a sensitivity of 94% and a specificity of 88% with contrast, besides an important reduction in the time of procedure. The echo-enhancing agent PESDA  is a second-generation agent, containing high molecular weight gas, whose use results in higher stability and better reflections of Doppler signs . The following spectral Doppler diagnostic criterion for renal arterial stenosis were used: a) PSV > 150 cm/s [26-27]; b) RAR > 3.0 ; c) RI >We also collected clinical data, including number of drugs, serum creatinine and values of blood pressure in baseline conditions. The results of PESDA-enhanced and non-enhanced ultrasound examinations were compared with those from intraarterial angiography. A hemodynamically significant stenosis was defined as diameter reduction of 50% or more at angiography, because it has been widely used in the recent literature . The radSecondary efficacy variables included the duration of each Doppler examination, the detection of supernumerary arteries and adverse effects. The study was approved according to local legal requirements and informed consent was obtained before ultrasound examination from all patients.All patients underwent digital subtraction angiography and non-enhanced US. One patient did not receive the infusion of contrast, because the venous access was not possible. Renal arterial stenosis of 50% or greater was detected at angiography in 18 (60%) patients, 5 of whom had bilateral stenosis. Renal arterial stenosis was excluded in 12 patients. Thus, stenosis by angiography was detected in 23 arteries, while 37 arteries did not present. The clinical and demographic data of patients according to the presence of stenosis are presented in the Table Overall, all patients had the renal arteries assessable with non-enhanced US or after injection of PESDA, although in one patient with a high body mass index, the assessment was better after contrast. Despite an expectation of at least 20% accessory arteries, we did not find any in our population.As stenosis is mostly in ostium and proximal portion of the arteries, we considered for diagnosis of renal artery stenosis the indexes obtained by echo-Doppler in these arterial segments. The mean values of PSV, PDV, RAR, RI and PI in these segments are showed in the tables The sensitivity, specificity, positive predictive value and negative predictive value of each Doppler index for the detection of RAS Table  were calThe receiver operating characteristic curves for each Dopplerdiagnostic criterion showed that the area under the receiver operating characteristic curve for resistance index was greater than the area under the curve for peak systolic velocity and renal aortic ratio. For renal aortic ratio, the cutoff point that provided the best accuracy, 2.7 gave a specificity of 96% but a low sensitivity (60%). For peak systolic velocity, no precise cutoff point could be identified between arteries with stenosis and those without stenosis. For resistance index, a threshold of 0.8 led to a sensitivity of 70% and a low specificity of 56.8%. The median examination time was 35 minutes for enhanced Doppler US and 29 minutes for non-enhanced Doppler US, i.e., a small but significant reduction of 17% (p = 0.03). Only one patient presented adverse events to be potentially related to the injection of PESDA, including sensation of coldness, palpitation and dyspnea. There was no severe adverse event. Although the technique of renal arterial US scanning has been well established for years, a lot of difficulties in reliably identifying main and accessory renal arteries remain -10,25. MThe most important conclusion from this study is that both sensitivity and specificity of Doppler US of renal arteries are strongly dependent on the criterion used, and the infusion of PESDA contrast seems not to improve it significantly, although we observe a slight increase in specificity. Thus, the best sensitivity was obtained when based on resistance index <0.8 (82.9%) but at expense of a low specificity (56.8%). On the other hand, the best specificity was obtained with renal aortic ratio >3 (89.2%), but the sensitivity was low (56.2%). In addition, the sensitivity and specificity for a peak systolic velocity of 1.5 m/sec showed intermediate values, respectively, 61.9% and 64.9%. An analysis of previously published studies ,15,16,20® in hypertensives with renal artery stenosis. In a more recent multicentric study the contrast Levovist did not affect either sensitivity or specificity: sensitivity was 80.0%–83.7%, whereas specificity moderately increased from 80.8% to 83.6% or 86.2%, depending on the subgroups of comparable patients. In two single-center studies in which the value of Doppler US examination after intravenous injection of contrast agents for the diagnosis of renal arterial was also evaluated it was demonstrated an improvement in sensitivity, which increased from 83% to 95% in one study and from 75% to 100% in the other [The continuous infusion of PESDA contrast increased moderately the specificity for renal aortic ratio criteria from 89.2% to 97.3% but at the expense of a significant decrease of sensitivity from 56.2% to 33.3%. For the another criteria, peak systolic velocity and resistance index the infusion of PESDA decreased mildly or did not affect the sensitivity and specificity. MISSOURIS et al  have rephe other ,23. Howehe other  reportedThere is no consensus whether contrast agent injection potentially reduces examination duration. In our study, we found a significant reduction of mean examination time after contrast infusion (17%). In other study, it was reported that the use of Levovist dramatically reduced the mean examination time from 24.5 minutes to 13.5 minutes . This adPESDA was well tolerated and did not compromise the safety of US. This excellent patient tolerance has already been demonstrated in stress echocardiograph studies that used PESDA as contrast agent . The small number of patients impose some limitations to the present study, However, the high prevalence of renal artery stenosis in this selected group of hypertensives counterbalance this limitation. an increase in specificity based on one Doppler criterion. Also, the feasibility of US is dependent of the quality of the machine, and the infusion of contrast does not add advantages if the performance of the US machine is excellent. However, it remains unknown if the PESDA infusion can improve feasibility if the machine does not have a good imaging quality. So, there is a need for establishing a consensus opinion regarding Doppler useful criteria and thresholds for the diagnosis of renal arterial stenosis, regardless of the US equipment used or infusion of ultrasonographic contrast.In conclusion, the detection of renal artery stenosis by Doppler US depends on the criteria used and infusion of PESDA contrast seems not to improve the accuracy, despite a reduction in the examination duration and