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The accession number GSE15920 is registered with the Gene Expression Omnibus (GEO)database and available at |
Na3V2(PO4)3 belongs to the family of NASICON-related structures and is built up from isolated [VO6] octahedra (3. symmetry) and [PO4] tetrahedra (.2 symmetry) interlinked via corners to establish the framework anion [V2(PO4)3]3−. The two independent Na+ cations are partially occupied [site-occupancy factors = 0.805 (18) and 0.731 (7)] and are located in channels with two different oxygen environments, viz sixfold coordination for the first tris(orthophosphate), were grown from a self-flux in the system Na DOI: 10.1107/S1600536810002801/wm2293Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: |
Prolonged release drug delivery system of pilocarpine nitrate was made by optimizing thin layer film hydration method. Egg phosphatidylcholine and cholesterol were used to make multilamellar vesicles. Effects of charges over the vesicles were studied by incorporating dicetylphosphate and stearylamine. Various factors, which may affect the size, shape, encapsulation efficiency and release rate, were studied. Liposomes in the size range 0.2 to 1 µm were obtained by optimizing the process. Encapsulation efficiency of neutral, positive and negatively charged liposomes were found to be 32.5, 35.4 and 34.2 percent, respectively. In vitro drug release rate was studied on specially designed model. Biological response in terms of reduction in intraocular pressure was observed on rabbit eyes. Pilocarpine nitrate liposomes were lyophilized and stability studies were conducted. The importance of liposomes as drug delivery vehicle is now becoming well established. This applies particularly to the ability of liposomes to buffer the toxicity of entrapped drugs while maintaining efficacy, some ar8in vivo[The behavior of liposomes in vivo is stronin vivo. The ratThe circulation time is also sensitive to lipid dose; higher doses lead to longer circulation times. These effects are presumably related to saturation of RES at higher dose. Other fIn vitro release rate studies were conducted on specially designed In vitro model and in vivo studies of liposomes were performed on albino rabbit eyes.Multilamellar vesicles (MLVs) were prepared using thin lipid film hydration method. Various factors such as phosphatidylcholine and cholesterol ratio, lipid and drug ratio, incorporation of charged species and pH etc. were studied which may affect the size, shape and incorporation efficiency of liposomes. Liposomes were evaluated by optical microscope as well as transmission electron microscope. Analysis of drug content was carried on UV spectrophotometer. Pilocarpine nitrate (PN) was a gift sample from JT Baker Chemicals Co., (Philipsburg NJ), and analyzed in our laboratory. Phosphatidylcholine, cholesterol, dicetylphosphate and stearylamine were purchased from Sigma chemicals U.S. All other chemicals and reagents used were of analytical grade.Stock solution containing phosphatidylcholine and cholesterol in 10:4 molar ratios were prepared in chloroform. Appropriate volume of these solution and 10 g glass beads were transferred to a 250 ml round bottom flask and attached to the rotary vacuum evaporator. The flask was kept immersed in a thermostat water bath, with the temperature set at 30° and rotated at about 100 rpm. Process was allowed to continue till all the liquid had evaporated from the solution and a dry lipid film had deposited on the wall of the flask. Flask was rotated under vacuum for another 15 min and then flushed with nitrogen to remove the last traces of solvent. Aqueous phase (5 ml containing drug) was added to the flask and the flask was rotated with the same speed as before for 30 min or until all the lipid had been removed from the wall of the flask. The suspension was allowed to stand for an optimized period of 2 h at room temperature in order to complete the hydration.The inclusion of negatively charged lipid such as dicetylphosphate or positively charged surfactant such as stearyl amine tend to increase the interlamellar repeat distances between successive bilayers in the MLV, swelling the structure with the greatest proportion of the aqueous phase. These effects lead to a greater overall entrapped volume. Hence two batches of liposomes were prepared containing phosphatidylcholine, cholesterol, stearylamine and phosphatidylcholine, cholesterol and dicetylphosphate in the ratio 10:4:1 and percent drug incorporation was calculated. Antioxidant such as 1 mol % α-tocopherol was usedVibronics-250W probe type ultrasonicator was used for size reduction of MLV dispersion21. SamplSample (4 ml) was placed in ultracentrifuge tubes at 20,000 rpm. Ice cold water was added to enhance centrifugation and mixture was centrifuged for 20 min. Solid particles left in the tube was collected and suitable volume of aqueous medium was added. Nitrogen gas was flushed to avoid peroxidation of lipids.After optimization of process variables, following batches were prepared. SL-1 (phosphatidyl choline:cholesterol) 10:4, SL-2 (phophatidyl choline:cholesterol:dicetylphosphate) 10:4:1, SL-3 (phophatidyl choline:cholesterol:stearyl amine) 10:4:1.Every sample prepared was examined under optical microscope using oil immersion lens. Samples showing fragments of non-dispersed lipid film or PN precipitates were discarded. Electron microscopic studies were carried out on transmission electron microscope . Grids precoated with colophony resins were used. Liposome sample was applied to the grid and kept for settling for 5 min. The grid was then coated with 1.5% phosphotugustate and dried at room temperature. The same was examined under transmission electron microscope and photographs were taken .To aliquots of liposome sample (0.5 ml), 5 ml of 10% sodium laurylsulphate (SLS) was added and the volume was made up to 50 ml. The sample was warmed on water bath at 70° for 30 min; similarly a blank solution in 50 ml was prepared. This procedure was employed for the analysis of PN in various batches. Drug concentration was estimated by measuring the absorbance at 215 nm using spectrophotometer with reference to the blank solution prepared .Drug release was determined with the help of modified USP XXI dissolution rate model. The model comprises of a beaker (250 ml) and a plastic tube of diameter 17.5 mm opened from both the ends. The tube was tied at one end with treated cellophane membrane and dipped into the beaker containing dissolution medium. The beaker was filled with 90 ml phosphate buffer (pH 7.4) and temperature was maintained at 37±1°. Liposomic preparation (10 ml) was added in the tube and a paddle type stirrer was attached in the center of the beaker. The speed was maintained at 100 rpm dissolution sample (5 ml) were withdrawn at 0.5, 1, 2, 4, 6 and 8 h and analyzed spectrophotometrically. Results were tabulated and graph was plotted as percentage drug release versus time for all three formulations .rt = I0 - It / I0 were I0 is the initial IOP and It = IOP at time t were instilled in lower cul-de-sac of one eye and the same amount of the normal saline in the other. Intraocular pressure (IOP) was measured periodically and observations were recorded in Tables t time t . Marketet time t .Lyophilization–24 was cThe aim of the use of the drug loaded liposome in ocular formulation is to increase the availability of the encapsulated drug to the eye in terms of both uptake and residence time. To achieve this goal, liposomes with positive and negative surface charges were prepared using negatively and positively charged components and evaluated. Thin lipid film-hydration method was used to prepare liposomes.Process was optimized for solvent selection, lipid:lipid ratio, lipid:drug ratio, buffer system, pH, charged species added. In selection of solvent system, chloroform alone and mixture of chloroform and methanol (in ratio 2:1 and 1:1) were tried for lipid dissolution and film formation. Inclusion of methanol resulted in precipitation of some part of phospholipid before drying and/ or non-uniform film; hence chloroform alone was used. In the absence of glass beads, the lipid film formed on the wall of the flask was thick. It took longer time for hydration and fragments of non-dispersed films were formed. Hence glass beads (10g) were added to increase the surface area available for deposition of the film. Phosphatidylcholine and cholesterol in the molar ratio 10:2, 10:4 and 10:8 were tried to form liposomes. All the three batches were seen under the microscope and drug entrapment was calculated. 10:2 and 10:8 molar ratio showed some fragments of nondispersed lipids. At 10:4 ratio liposomes formed were uniform and entrapment efficiency was more . Hence tPhospholipid:drug molar ratio was selected on the basis that lipid bilayer can intercalate drug to a maximum extent of 10% by molar ratio. On weight basis it becomes 15:1 phospholipids to drug ratio. Thus dispersion containing different molar proportions of drug was prepared . Pilocarpine nitrate added in concentration higher than 15:1 invariably precipitated out in the system. Therefore, in all the studies, concentration of lipid and PN was kept 15:1. Drug was dispersed in buffers of different pH viz. 6.0, 6.6 7.0, 7.4 and 8.0 phosphate buffer. Buffer of pH 6.0 and 6.7 showed poor swelling of lipids with fragments of nondispersed film whereas normal saline containing sodium bicarbonate (pH 7.4) resulted in well shaped liposomes. This swelling process depends on the pH of aqueous medium. This particular lipid composition showed better swelling properties in pH range above 7. Aqueous phase of pH 7.4 was selected for the study at which drug exists in both ionized and unionized form.Size reduction of MLV system was attempted by ultrasonication. Ice was used to lower the temperature during sonication. For determination of total drug content, the entrapped drug should be made free from liposomes. This was achieved by the addition of alcohol or detergent. Addition of SLS at higher concentration (10%) yielded a clear solution on warming at (60-70°) and did not absorb at 215 nm in UV spectrophotometer.Optical microscopy was found to be the fast and useful method for preliminary experiment. Transmission electron microscopy enabled the determination of particle size of liposomes formed. The method for the preparation of the grid was standardized. The successes of the preparation of the grid depended on setting of the liposome particle on the coated grid surface. Settling time was kept high and a very small droplet was applied to the grid. Scanning of the grids prepared in such a manner showed a heterogeneous dispersion . Photomicrographs of liposomes were taken at 10 000 X .in vivo and In vitro. In general charged liposomes were more stable against aggregation and fusion than uncharged liposomes. However physically stable neutral liposomes have been described. The inclusion of negatively charged lipid such as stearylamine tends to increase the entrapped volume. (in vivo studies of liposome formulation. Positively charged liposome showed greater duration of action (DR 468 min) and AUC (23.5) compared to negatively charged liposomes (DR 396 min) and AUC (20.8) . The resThe characteristics of lyophilized and non-lyophilized liposomes were examined. Lyophilization of liposomes had no substantial effect on size and shape. They could be easily dispersed to give a suspension of aggregate free liposomes. The drug content of freshly prepared liposomes and lyophilized liposome were found to be the same. No difference was found in the drug release rate. Both lyophilized and freshly prepared liposomes were stored in the refrigerator at 4°. Studies revealed that non-lyophilised liposomes were stable for 6 weeks only whereas lyophilized liposomes were stable for 6 months and above. Mannitol was used in the ratio 2:1 mannitol to lipid for lyophilization, which helped in reducing the time required for the process as well as acted as cryoprotectant.In conclusion, delivery of liposome encapsulated drugs as eye drops improved the extent of uptake and residence time compared to the free drug solution. Positively charged liposomes interact more with the corneal surface thus prolong the residence time. |
Tumors and cancer cell lines were surveyed with end-sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes.in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor.In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets. Cancer is driven by selection for certain somatic mutations, including both point mutations and large-scale rearrangements of the genome; thus, the genomes of most human solid tumors are substantially diverged from the host genome. Many copy number aberrations have been shown to be recurrent across multiple cancer samples. These recurrent copy number aberrations frequently contain oncogenes and tumor suppressor genes, and are associated with tumor progression, clinical course, or response to therapy . MoreoveStructural rearrangements, particularly translocations, are frequently observed in solid and hematopoietic tumors. In hematopoietic malignancies the importance of translocations is well established, but their biologic and clinical significance in solid tumors remains largely enigmatic because of technical difficulties and complex karyotypes that defy interpretation. Recently, a bioinformatics approach identified recurrent translocations in about 50% of prostate tumors . This diEnd sequencing profiling (ESP) is a technique that maps and clones all types of rearrangements while generating reagents for functional studies -7. To pein situ hybridization (FISH) confirmed the presence of translocations predicted by ESP in BT474 and SKBR3 cells. Sequencing of 41 BAC clones from cell lines and primary tumors validated a total 90 rearrangement breakpoints. Mapping these breakpoints in multiple breakpoint spanning clones provided evidence of numerous genomic rearrangements that share similar but not identical breakpoints, a phenomenon analogous to the inter-patient variability of breakpoint locations in many fusion genes identified in haematopoietic cancers. Comparison of rearrangements shared across multiple tumors and/or cell lines suggests recurrent rearrangements, some of which confirm or suggest new germline structural variants, whereas others may be recurrent somatic variants. Analysis of single nucleotide polymorphisms (SNPs) in BAC end sequences revealed putative somatic mutations and suggests a higher mutation rate in the ovarian tumor.We performed ESP on the following: one sample each of primary tumors of brain, breast, and ovary; one metastatic prostate tumor; and two breast cancer cell lines, namely BT474 and SKBR3. Hundreds of rearrangements were identified in each sample, some of which may encode fusion genes. Fluorescence ESP complements other strategies for tumor genome analysis including array comparative genomic hybridization (aCGH) and exon resequencing by providing structural information that is otherwise not available. New sequencing technologies promise ZNF217 locus on 20q13 and very high amplifications at chromosome 17. Table BAC libraries were constructed from frozen samples from two breast tumors and single tumors from the brain, ovary, and prostate, demonstrating that there is no tumor-specific bias for BAC library construction. Approximately 50 mg to 200 mg of fresh frozen tumor specimen was used in the construction of each library. All tumors were dissected to minimize contamination with normal tissue. BAC libraries from the breast cancer cell lines BT474 and SKBR3 were also constructed. Breast cancer cell lines were included in this study because their genomes and transcriptomes are similar to those identified in primary breast ,11 and aEnd sequences of 4,198 BAC clones from the brain tumor library, 5,013 clones from the metastatic prostate library, 5,570 clones from ovary tumor library, 9,401 and 7,623 clones each from primary breast libraries, 9,580 clones from the BT474, and 9,267 clones from the SKBR3 breast cancer cell lines were generated. The end sequences were mapped to the reference human genome sequence, and the results are summarized in Table Each clone with uniquely mapped ends gives a BAC end sequence (BES) pair. A BES pair is a valid pair if distance between ends mapped on the normal human genome sequence and the orientation of these ends and are consistent with those for a BAC clone insert; otherwise, the BES pair is invalid Figure . An invain silico BAC libraries . This apparent reduction in coverage is probably a result of differing amounts of aneuploidy and genomic heterogeneity in the samples.For each library, we formed BES clusters grouping invalid pairs with close locations and identical orientations that are consistent with the same genome rearrangement . Each BEWe performed low coverage sequencing of 37 BAC clones corresponding to invalid BES pairs and combined these data with ten previously sequenced MCF7 BACs ) are consistent with NHEJ repair and either span microhomology regions ranging in size from 1 to 33 base pairs (45/51) or lack any homology (6/51) between the two regions involved in a particular rearrangement. We find insertions at the junction site ranging from 1 to 31 base pairs in 7 out of 51 NHEJ events. Twenty of the 106 breakpoint sites deduced from the nonredundant junction analyses are located within regions of known structural variation.EFCAB3, METTL2A, TLK2, MRC2, and RNF190). An additional seven breakpoints are located within genes and may result in intragenic rearrangements . The remaining eight breakpoints are either rearrangements involving intergenic regions or microrearrangements within introns.Of the 90 breakpoints, 72 are predicted to alter gene structure, resulting in either gene fusions or fusions of gene fragments to intergenic regions. This high proportion reflects a nonrandom selection of clones for sequencing, with priority given to clones that are likely to encode fusion genes . Of the BCR-ABL gene in leukemia with chrom1 = chrom2, loc1 < loc2, strand1 = '+', and strand2 = '-') were identified. The distribution of distances between mapped ends (loc2 to loc1) was used to define the length distribution of the BAC libraries. BES pairs with ends on the same chromosome and having convergent orientations on opposite strands and distances in the 99.5% quantile of this distribution were classified as valid. Other BES pairs were classified as invalid and thus candidate rearrangements in the tumor. Note that the distance criterion was very permissive and might misclassify clones harboring small indels as valid. Overlapping valid pairs were combined into 'contigs', whereas invalid pairs were clustered into sets according to whether their locations were close enough to be explained by a single rearrangement event -7. InvalCustom software was used to visualize the mapping results, as described by Volik and cowokers . A plot Locations of previously reported structural variants were downloaded from the Database of Genomic Variants ,57. ClusBAC DNA was purified from 250 ml overnight culture using the Qiagen columns . Approximately 2 μg of BAC DNA was mechanically sheared using the HydroShear , end-repaired with the Klenow enzyme and T4 DNA polymerase, size selected for 3 ± 0.5 kb fragments on agarose gels, and cloned into a pUC19 vector. Individually picked subclones were grown on 96-well plates overnight in LB plus 200 μg/ml ampicilin and 10% glycerol. Plasmid DNA was prepared from the arrayed cells using the TempliPhi kit , in accordance with the manufacturer's protocol. Three-kilobase subclones were end sequenced using BigDye terminators and capillary sequencers. The quality of the sequence reads were determined by Phred score , and onlBreakpoint junction sequences were aligned to the Human Genome Assembly using BLAT , and the2 test. Enrichment of Gene Ontology terms for the genes containing candidate SNPs was computed with the DAVID tool [P values for enrichment correcting for the size of the gene sets in each term. We used the LiftOver tool from the UCSC Genome Browser [Out of the approximately 70,000 clones sequenced for this and previous studies, we selected the 97,860 BESs that mapped to unique loci on the hg17 reference genome with a minimum BLAT identity score of 97%. The mean phred score of theseVID tool , which c Browser to identRT-PCR experiments were carried out as described by Zardo and coworkers . Primer Cells lines were shipped to Dr. Padilla-Nash. When cell lines reached 70% confluence, cells were treated with colcemid for 1 hour to arrest the cells in mitosis. Metaphase chromosome suspensions were prepared first by treating cells with a hypotonic solution (0.075 mol/l KCl); next, the cells were fixed using methanol:acetic acid and dropped onto slides in a humidity controlled chamber. The slides were aged at 37°C for approximately 1 week. Chromosome preparations were hybridized with either FISH probes or spectral karyotyping (SKY) probes for 72 hours. The protocols for preparation of FISH/SKY probes, slide pre-treatment, slide denaturation, detection, and imaging have been described previously and are available on the internet . Ten to in situ hybridization; kb, kilobase; Mb, megabases; NCBI, National Center for Biotechnology Information; NHEJ, nonhomologous end joining; PCR, polymerase chain reaction; RT, reverse transcription; SKY, spectral karyotyping; SNP, single nucleotide polymorphism; UCSF, University of California, San Francisco.aCGH, array comparative genomic hybridization; BAC, bacterial artificial chromosome; BES, BAC end sequence; BLAT, BLAST-like alignment tool; DAVID, Database for Annotation, Visualization, and Integrated Discovery; DSB, double strand break; ESP, end sequencing profiling; EST, expressed sequence tag; FISH, fluorescence BJR analyzed the data including identification and analysis of sequence variants, clustering of the identified breakpoints, and comparison of ESP and aCGH data. SV and CC developed the ESP methodology and BES mapping algorithms, analyzed the data and coordinated the clinical samples, sequencing, and experimental validation of ESP results. PY and CW integrated ESP and public EST data, and identified fusion transcripts. EL and BT performed analysis of sequenced breakpoints and contributed to paper writing. FW selected and managed the breast clinical specimens and developed the FISH methods of breakpoint validation. JC, KJP, and GBM managed and selected the brain, prostate, ovary tumor samples, respectively. PP, KB, YK, G-QH, and SS performed experimental validation. AB, RB, and SJA performed analysis of fusion genes and sequence variants. JWG and J-FC sequenced BAC clones. QT, PdJ, and MN constructed BAC libraries. HP-N and TR performed FISH validation and experimental validation of ESP breakpoints. BJR, SV, and CC wrote the paper. All authors read and approved the final manuscript.The following additional data are available with the online version of this paper. Additional data file Supplemental text and tables, including a description of all supplemental tables.Click here for fileThree supplemental figures.Click here for fileSupplemental tables.Click here for file |
PSA: P = 6.9×10−11). However, rs2395029 encoding the G2V polymorphism within the class I gene HCP5 (combined P = 2.13×10−26 in U.S. cases) yielded the highest ORs with both PS and PSA (4.1 and 3.2 respectively). This variant is associated with low viral set point following HIV infection and its effect is independent of rs10484554. We replicated the previously reported association with interleukin 23 receptor and interleukin 12B (IL12B) polymorphisms in PS and PSA cohorts and detected an independent association in the IL23R region with a SNP 4 kb upstream from IL12RB2 (P = 0.001). Novel associations replicated in the U.S. PS cohort included the region harboring lipoma HMGIC fusion partner (LHFP) and conserved oligomeric golgi complex component 6 (COG6) genes on chromosome 13q13 , the late cornified envelope gene cluster (LCE) from the Epidermal Differentiation Complex (PSORS4) and a region of LD at 15q21 . This region is of interest because it harbors ubiquitin-specific protease-8 whose processed pseudogene lies upstream from HLA-C. This region of 15q21 also harbors the gene for SPPL2A which activates tumor necrosis factor alpha by cleavage, triggering the expression of IL12 in human dendritic cells. We also identified a novel PSA locus on chromosome 4q27. This region harbors the interleukin 2 (IL2) and interleukin 21 (IL21) genes and was recently shown to be associated with four autoimmune diseases .A genome-wide association study was performed to identify genetic factors involved in susceptibility to psoriasis (PS) and psoriatic arthritis (PSA), inflammatory diseases of the skin and joints in humans. 223 PS cases (including 91 with PSA) were genotyped with 311,398 single nucleotide polymorphisms (SNPs), and results were compared with those from 519 Northern European controls. Replications were performed with an independent cohort of 577 PS cases and 737 controls from the U.S., and 576 PSA patients and 480 controls from the U.K.. Strongest associations were with the class I region of the major histocompatibility complex (MHC). The most highly associated SNP was rs10484554, which lies 34.7 kb upstream from HLA-C (P = 7.8×10 Psoriasis (PS) and psoriatic arthritis (PSA) are common inflammatory diseases of humans affecting the skin and joints. Approximately 2% of Europeans are affected with PS, and ∼10–30% of patients develop PSA. Genetic variation in the MHC (multiple histocompatibility locus antigen cluster) increases risk of developing PS. However, only ∼10% of individuals with this risk factor develop PS, indicating that other genetic effects and environmental triggers are important. Recent approaches using a case/control approach and genome wide association studies with DNA markers known as SNPs (single nucleotide polymorphisms) have been fruitful in identifying genetic factors for common diseases. This study describes the first large scale genome wide scan for additional PS and PSA susceptibility genes using 233 cases and 519 controls. It revealed that the MHC is truly the most important risk factor for PS and that it plays a very major role in PSA, confirmed recently identified associations with interleukin 23 receptor and interleukin 12B in both PS and PSA, and identified new associations. These include a region on chromosome 4q27 that contains genes for interleukin 2 and interleukin 21 that has been recently implicated in other autoimmune diseases, and seven additional regions that include chromosome 13q13 and 15q21. S) of PSA is high, and estimates of 27–47 have been proposed S of PS which is estimated to be between 4 and 11Psoriasis (PS) is a chronic inflammatory disease of the skin affecting 2–3% of the population PS and PSA are interrelated disorders, and the prevalence of PS is 19 times higher among first degree relatives of probands with PSA compared with the general population A number of regions in the genome have been reported to be associated with PS In order to systematically search for other susceptibility loci, we undertook a genome wide association (GWA) scan to identify genetic factors predisposing to PS and PSA. Besides detecting strong association with the HLA class I region in the combined and PSA cohort, and replicating the recently reported associations with IL23R and IL12B, we identified a number of novel associations. These include a region on chromosome 13q13 harboring LHFP and COG6, a region on chromosome 15q21 harboring USP8-SPPL2A-TNFAIP8L3, association with the LCE cluster of genes on chromosome 1q21 from the PSORS4 locus, and a region of chromosome 4q27 recently reported to be associated with several other autoimmune diseases and associated with PSA and potentially PS.−5, we had 70% power of detecting a locus with a genotype relative risk (GRR) of 2.0, and over 99% power to detect a locus with a GRR of 3.0 such as the MHC (see below). However, many replicated associations have small GRRs For our “discovery” phase, 223 PS cases (132 cases with PS without arthritis and 91 PS cases with arthritis (PSA) were typed on the Illumina HumanHap300 arrays. We compared case data to publicly available genotype data of 519 European controls from the New York Cancer ProjectFollowing the genotyping of samples, stringent quality control measures were implemented. We required that all samples used for the discovery phase passed a 93% genotyping call rate threshold, and that all SNPs passed a 95% call rate threshold. Justification for this threshold is based on the evaluation of empirical distributions and S2. For the discovery phase, a total of 311,398 SNPs were pruned to 305,983 SNPs after filtering for low call rate, minor allele frequency <0.01 and deviation from Hardy-Weinberg equilibrium (P<0.001). Quality control also led to the removal of 29 samples, leaving 218 cases for further analysis . The aveGCGC = 1 indicating no inflation). We also performed analysis on the same set of AIM SNPs with STRUCTURE softwareGC = 1.101 before correction . Following adjustment of P values with the genomic control method, λ = 1˜ The discovery P values adjusted by the genomic control method as implemented in PLINKTo investigate other biases−5; a level that we would informally expect to observe by chance roughly 15 times in this scan given the number of tests performed if all SNPs were independent. A subset of SNPs from 120 regions were investigated further. Criteria for selection included the strength of the discovery P value, particularly when several SNPs from a single region showed evidence for association, a possible biological role of a gene harboring a SNP with some evidence for association, or localization of SNPs with moderate evidence for association to a known psoriasis susceptibility locus (e.g. PSORS4). We also included the previously reported associated SNPs in IL23R and IL12BTo detect associations, we first performed a preliminary analysis with a Cochran-Armitage trend test An independent cohort of 577 PS cases from the U.S. and 737 U.S. controls were used for the replication stage; 94 of these cases had also been diagnosed with PSA. To examine the potential role of variants upon PSA susceptibility specifically, 576 PSA cases from the UK and 480 controls from the UK were also employed. An alternative genotyping technology was used for the replication phase. The platforms used for the discovery and replication phases gave very similar results: Concordance rates on the basis of 116 samples and 301 SNPs typed with both platforms was 98.74%.Our 100 top ranked SNPs with any cohort are listed in −5 , Table 1−11) , althoug−26 in the U.S. cohort and 1.86×10−10 in the U.K. PSA cohort (2 = 0.33 in European CEPH HapMap samples and r2 = 0.23 in our U.S. case/control cohort). Conditioning upon rs10484554, the P value for rs2395029 was still significant (P = 7×10−10), hence effects from this SNP are likely to be independent.A second SNP from the HLA class I region lying between MICA and MICB (rs2395029) was highly associated with PS and PSA. This SNP results in the G2V polymorphism of the class I gene HCP5 (HLA complex P5) which encodes an endogenous retroviral element. For this SNP, PS was associated with a combined P = 2.13×10A cohort , Table 2http://smd-www.stanford.edu/), consistent with a potential role in autoimmunity. This allele was recently shown to explain 9.6% of the total variation in viral set point following HIV-1 infectionHCP5 is expressed primarily in cells of the immune system such as spleen, blood and thymus 2 = 0.031 in HapMap CEPH European samples; 0.009 in cases; 0.026 in controls). Conditioning upon rs11209026, the P value for rs11209026 was 0.013. Hence, effects from this SNP may be independent of rs11209026 and its association with PS should be investigated in other cohorts.In our discovery cohort, the most significant association in the IL23R interval was obtained with a different SNP from that described previously as being associated with PS (rs11209026). This SNP, where P = 0.0039 in the discovery cohort , has noth1 cells and plays a role in Th1 differentiationSNP rs12131065 lies downstream from IL23R (63 kb from rs11209026) and 4.041 kb upstream from the gene for interleukin 12 receptor B2 (IL12RB2) . IL12RB2−4 and 0.001 respectively (−4), with the rs11209026*T allele being found at frequencies of ∼0.04 in cases and ∼0.08 in controls (Association with the previously reported IL23R associated SNP rs11209026 in the discovery cohort was not significant (adjusted P = 0.081). Genotyping of rs11209026 and rs12131065 in the U.S. replication cohort yielded combined P values of 1.4×10ectively consistecontrols .−5 and replicated previously reported associations . These were: rs1186468, rs4514547, rs4569133 and rs7993214 . Rs3812888, was the only SNP where replication results would remain significant following the stringent Bonferroni correction for multiple tests (P = 0.048). The OR of the rs3812888*C allele was 1.38 (95% CI: 1.15–1.66). The rs3812888*C allele was found at frequencies of 0.43 in cases and 0.35 in controls.In the discovery cohort, there were four SNPs from 13q13 where P<5×10s7993214 . These Ss7993214 . ResultsC. elegans, a COG complex is required to glycosylate an ADAM protease http://smd-www.stanford.edu/) indicates that highest levels are found in the ear and spinal cord.COG6 is a component of the conserved oligomeric golgi (COG) complex and is involved in intracellular transport and glycoprotein modification−5 was analyzed separately, four SNPs from a region on chromosome 15q21 between ubiquitin specific protease 8 (USP8) and tumor necrosis factor, alpha-induced protein 8-like 3 (TNFAIP8L3) were associated with P<5×10−5 . In the This region is also of interest however, because a processed pseudogene for one of the genes in this region (USP8) is found upstream from HLA-C−5 (OR 1.45) , which harbors a previously established psoriasis locus (PSORS4)OR 1.45) . This SN−5 combined). For this SNP, the G allele was found at a frequency of ∼0.47 in cases and ∼0.39 in controls . GNLY is of considerable interest with respect to psoriasis. It is present in cytotoxic granules of cytotoxic T lymphocytes and natural killer cells, and is released upon antigen stimulationM. tuberculosis and other organisms. Priming of granulysin in CD4 is dysregulated in the CD4+ T cells of HIV-infected patientsThere were two other regions selected for follow-up where P values were <0.05 in the PS replication cohort, and where evidence for association increased in the combined cohort . One wasOther genes that should be evaluated in additional PS cohorts on the basis of replication P values <0.05, and increased significance in combined cohorts are calp−5 (adjusted P<10−4). These were rs13151961, rs6822844 and rs6840978 are also present in psoriasis lesions. IL-2 may influence how a common precursor T-cell differentiates into either a Treg or a Th17 T-cell, since addition of IL-2 has been shown to suppress the differentiation of Th17 T-cells in miceAs reported elsewhere the 4q27 locus that contains these associated SNPs corresponds to two closely correlated ∼439 kb and ∼40 kb haplotype blocks h17 polarization). It then acts in an autocrine or paracrine fashion on T-cells to up-regulate expression of the IL23 receptor which has already been implicated in psoriasis pathogenesis. IL23R sensitizes cells to IL-23 which stimulates IL17 synthesis and/or prolongs the survival of Th17 cells h17” mediated disease. Therefore IL21 may play a role in Th17 formation in this and other autoimmune diseases where these cells are pathogenic.The epidermal staining for IL-21 is much lower than for IL-2 and appeExtensive resequencing of IL2 coding and flanking regions has already been performed in T1D samples and no coding or obvious regulatory/splice variants were identified The observed associations in the current study are of interest for several reasons. It is noteworthy that the strongest association is with the MHC. Even in PSA, where associations are reportedly less than with PS (without PSA), associations with the class I region appear to be more significant than with any other region. We were also able to replicate previously reported associations with IL12B and IL23R and detected a potentially novel association upstream from IL12RB2. Novel associations within COG6 and the region on chromosome 15q21 harboring USP8 and SPPL2A are of interest. These and other regions reported here are worthy of follow up in other cohorts. Moreover, the association with chromosome 4q27 provides further evidence that this region is a common locus for multiple forms of autoimmune disease.A recent study reported the IL13/IL4 region from chromosome 5q31 as being associated with PSThe ability to identify low risk variants for common diseases such as PS and PSA will be limited by the cohort size, and larger numbers of cases and controls will be necessary to identify the majority of genetic factors for these diseases. Moreover, some of the SNPs with borderline discovery P values are also likely to be genetic risk factors for disease. It is worth noting that our discovery P value for the associated IL23R R381Q SNP did not reach significance although allele frequencies in the discovery dataset revealed a M.A.F. of 0.041 in cases and 0.065 in controls, which is similar to what has been reported in other studiesThe cohorts for the discovery and replication phases of this study are all of European descent and are described in http://intragen.c2b2.columbia.edu/. These were random controls and there was no specific information about autoimmune/inflammatory disease. Recent large genome-wide association studies using controls of this type have been shown to be successful, leading to only a modest effect on power unless the event of misclassification bias is substantialGenotypes of 519 European controls obtained following hybridization to the Illumina HumanHap300 array were from the New York Cancer Project (NYCP) Blood samples were obtained by venipuncture for all subjects, and genomic DNA was isolated from whole blood by standard procedures.Replication cohorts were from both the U.K. and the U.S. The U.K. cohort consisted of 576 PSA patients from the UK and are described elsewhere The replication cohort from the U.S. for cases consisted of 577 patients with PS , ascertained at the University of California, San Francisco, CA or at Texas Dermatology, Dallas, TX The replication cohort for controls consisted of 479 unrelated Caucasian individuals from the University of California, San Francisco, ascertained as a set of healthy controls, for cardiovascular studies. A separate cohort of 258 controls was ascertained in Texas. The latter controls were all >40 years of age and were ascertained on the basis of not having PS, PSA, or any other inflammatory or autoimmune disease.DNA was normalized to a concentration of 100 ng/µl (diluted in 10 mM Tris/1 mM EDTA). Samples were quantitated with a Nanodrop Spectrophotometer (ND-1000). For the discovery phase, approximately 1 µg of genomic DNA was used to genotype each sample on the Illumina HumanHap300v2A Genotyping BeadChip. This was performed at the Robert S. Boas Center for Genomics and Human Genetics at The Feinstein Institute for Medical Research, Manhasset, NY. This assay relies on allele specific primer extension and the use of a single fluorochrome. Samples were processed according to the standard Illumina Infinium II automated protocol. This involved whole genome amplification, fragmentation, precipitation, resuspension in hybridization buffer and hybridization to the Illumina Bead Chips for a minimum of 16 h at 48°C. After hybridization the BeadChips were processed for the single base extension reaction, followed by staining and imaging on an Illumina Bead Array Reader. Normalized bead intensity data were loaded into the Illumina Beadstudio 2.0 software which converted fluorescence intensities into SNP genotypes.http://hg.wustl.edu/info/Sequenom_description.html.Genotyping for all the replication studies was performed with the Sequenom MassArray system (iPlex assay). This involves primer extension chemistry and mass spectrometric analysis described at our web site Before analysis, we performed quality filtering of both samples and SNPs to ensure robust association tests. Based on previous criteria In the case of the replication studies, 57 individuals from the total of 2370 individuals in the replication study were removed because of low genotyping . SNPs with <75% call rates were also excluded from analysis to obtain an average genotyping rate of 0.902. Genotypes were also evaluated for departure from HWE in the controls and SNPs with P<0.001 were removed from further analysis. After pruning, 244 SNPs remained.A total of 463 ancestry informative SNPs (AIM) present on the Illumina HumanHap300v2A Genotype BeadChip were used to check for possible confounding population substructure in the discovery sample with STRUCTURE software [To investigate other biaseshttp://pngu.mgh.harvard.edu/purcell/plink). However, several SNPs in the current study that exhibited significant differences in cases/controls, were also different when allele frequencies in controls were compared with those from European CEPH typed for SNPs in the HapMap project. NYCP participants are quite diverse with respect to European origin, and many SNPs are reported to show differences among European subgroupsThe Cochran-Armitage Test for trend2, and allele frequencies were based on precomputed scores from the International HapMap website or were computed locally from HapMap genotypes or from our own case and control genotypes with Haploview 3.2 (http://www.broad.mit.edu/mpg/haploview/). Power calculations for association were calculated at: http://pngu.mgh.harvard.edu/purcell/gpc/. Association localization plots (http://cbdb.nimh.nih.gov/kristin/snp.plotter.html) and Regional Association Plot (http://www.broad.mit.edu/diabetes/scandinavs/figures.html).Measures of linkage disequilibrium, D' and ron plots ,6,S4–S5 Family based association tests on 271 nuclear families were performed with the Pedigree Transmission Disequibrium Test Tissue sections were fixed with acetone and stained with 10 ug/mL purified mouse anti-human monoclonal antibodies to IL-2 , IL-21 , COG6 and SPPL2A . Biotin labeled horse anti-mouse antibodies (Vector Laboratories) were amplified with avidin-biotin complex (Vector Laboratories) and developed with chromogen 3-amino-9-ethylcarbazole (Sigma Aldrich).Table S1Summary of cases and controls used in discovery and replication stages.(0.03 MB DOC)Click here for additional data file.Table S2−5 in discovery cohorts .Top ranking SNPs where P<5×10(0.25 MB PDF)Click here for additional data file.Figure S1Heterozygosity of sample versus genotyping call rate.(0.95 MB TIF)Click here for additional data file.Figure S2Distribution of SNP success rate in the discovery study.(1.36 MB TIF)Click here for additional data file.Figure S3GC = 1.101) and red dots indicate the negative logarithm of adjusted trend P values (λGC = 1).Q-Q plot of GWA analyses in unrelated individuals used in the discovery study obtained with PLINK. Black dots indicate the negative log of unadjusted trend P values (λ(6.03 MB TIF)Click here for additional data file.Figure S4Association localization plot for replicated region at chromosome 1q21. Results for SNPs used in the discovery phase (adjusted for GC) are presented as diamonds. Negative LOG P values are provided on the Y axis. The X axis corresponds to the locations of SNPs. The P value for all samples are shown as circles. The P value obtained with the most highly associated SNP is shown as a red circle. The SNPs shown as orange diamonds are in r2>0.8 (European HapMap CEPH (CEU) samples) with the most significant SNP identified in our study. The recombination rate based on the CEU HapMap is shown in light blue along the x axis . The green arrows indicate the locations of select genes. The LD relationship of Illumina discovery SNPs derived from CEU HapMap genotypes are shown below the graph. The most highly associated SNP, rs6701216 is indicated with an asterisk above the LD plot.(9.70 MB TIF)Click here for additional data file.Figure S5Association localization plots for autoimmune locus on chromosome 4q27 showing P values obtained in discovery sample. Symbols are the same as those used in Figure S4. The asterisk above the LD plot corresponds to rs6840978.(9.59 MB TIF)Click here for additional data file. |
Atrial fibrillation (AFib) is one of the prominent causes of stroke, and its risk increases with age. We need to detect AFib correctly as early as possible to avoid medical disaster because it is likely to proceed into a more serious form in short time. If we can make a portable AFib monitoring system, it will be helpful to many old people because we cannot predict when a patient will have a spasm of AFib.We analyzed heart beat variability from inter-beat intervals obtained by a wavelet-based detector. We made a Poincare plot using the inter-beat intervals. By analyzing the plot, we extracted three feature measures characterizing AFib and non-AFib: the number of clusters, mean stepping increment of inter-beat intervals, and dispersion of the points around a diagonal line in the plot. We divided distribution of the number of clusters into two and calculated mean value of the lower part by k-means clustering method. We classified data whose number of clusters is more than one and less than this mean value as non-AFib data. In the other case, we tried to discriminate AFib from non-AFib using support vector machine with the other feature measures: the mean stepping increment and dispersion of the points in the Poincare plot.We found that Poincare plot from non-AFib data showed some pattern, while the plot from AFib data showed irregularly irregular shape. In case of non-AFib data, the definite pattern in the plot manifested itself with some limited number of clusters or closely packed one cluster. In case of AFib data, the number of clusters in the plot was one or too many. We evaluated the accuracy using leave-one-out cross-validation. Mean sensitivity and mean specificity were 91.4% and 92.9% respectively.Because pulse beats of ventricles are less likely to be influenced by baseline wandering and noise, we used the inter-beat intervals to diagnose AFib. We visually displayed regularity of the inter-beat intervals by way of Poincare plot. We tried to design an automated algorithm which did not require any human intervention and any specific threshold, and could be installed in a portable AFib monitoring system. There is a growing tendency that atrial fibrillation (AFib) related disease affects quality of life. Its risk increases with age ; in factAFib can be classified into three grades: paroxysmal, persistent and permanent AFib. The paroxysmal AFib can be a preceding omen of the persistent AFib. Takahashi, Seki and Imatak observed that their patients with the paroxysmal AFib were highly affected with the more serious form of AFib; 25.3% of paroxysmal AFib patients developed into the more serious form of AFib in one year .Electrical remodeling is one of the features of AFib and it is related to decreased conduction velocity of electricity signals . When thThere are several studies about screening AFib by palpating an electrocardiogram (ECG) manually. Sudlow et al. tried to screen AFib by two methods: digoxin prescriptions and pulse palpation of ECG. Sensitivity and specificity using digoxin prescriptions were somewhat low. Sensitivity and specificity using pulse palpation were 93%, 71%) in case of women elder than 75, 100%, 86%) in case of 65-74 aged women, in case of men elder than 75, in case of 65-74 aged men, respectively [1% in cas6% in casThere are lots of studies about detecting AFib. Xu et al. chose five feature parameters which were input regularity, input atrial rate, energy distribution, time interval corresponding to zero amplitude signal, and number of points reaching baseline. They used Bayesian discriminator to classify the input data as one of sinus rhythm, AFib or atrial flutter . PetruccIf the paroxysm of AFib occurs, variability of the inter-beat intervals increases from the onset to the end of AFib ; hence, We used two databases, Computers in Cardiology challenge 2001 and 2004 of physionet ,18. The We obtained inter-beat intervals from input ECG data by using the wavelet method . We presAN is an approximation coefficient vector, and Di, is a detail coefficient vector. We chose one detail coefficient vector Di by a criterion [Di-1, Di-2,⋯, D1. Figure where riterion , and asswhere 0 means a zero vector. Figure Next we tried to find time position of each QRS complex which is protruded substantially above the baseline. The QRS complexes designate the heart beats. We calculated the approximation and detail coefficient vectors e vector . We assiWe determined the most adequate wavelet scale by comparing the Pearson correlation coefficients . Figure I1, I2, I3, I4, I5,⋯, In like Figure I1, I2), , , ,⋯, . We connected the consecutive points with lines to observe dynamics of the inter-beat intervals.If we represent the inter-beat intervals as a sequence x-coordinate of a point in Poincaré plot is x1, y-coordinate of the point is mathematically related to x axis of phase plane is x, the y axis corresponds to The Poincaré plot applicable to discrete data is closely related to a conventional phase plane of continuous data. If an lated to . If the I1, I2, I3, I4, I5, I6. Figure I1, I2), , , , , and we drew the lines between the consecutive points to observe the dynamics more easily. The points revolve clockwise and make a wedge-shaped diagram. This is because the inter-beat intervals changed around the PVC.Figure O means the QRS complex detector found the time position corresponding to the ventricular activity. Figure The Poincaré plots from non-AFib data show several typical patterns. Figure Figure I1, I2, I3, I4, I5,⋯, In. The points in Poincaré plot will be , ,⋯, in order. If we designate two consecutive points as and , the distance between two points in the Poincaré plot will be Let us assume that we were given inter-beat intervals, I1, I2, I3, I4, I5,⋯, In, the points of the Poincaré plot consist of , , ,⋯, . We tried to find a central point minimizing sum of distance squares from this point to all the other points in the Poincaré plot. If we designate this sum as E(x), this will be represented as follows.Let us calculate coordinates of a central point on the diagonal line in Poincaré plot. If the inter-beat intervals are x. From a, a) as follows.To find the point minimizing this sum, we calculated a derivative with respect to the variable Ij, Ij+1) to the diagonal line y = x is represented as The distance from a point , , , where I1, I2, I4 and I5 are almost same but less than I3.Poincaré plot helped us to catch a fault of our QRS complex detector. If the QRS complex detector misses one QRS complex like Figure Ij, Ij+1) and . We calculated the coordinates of the central point on diagonal line in Poincaré plot in the above section. We can think of the central point as a new origin; then the coordinates of two consecutive points will be and with regard to this new origin. We identified a mistake done by the QRS complex detector when the distances We tried to correct this by identifying the triangle in the Poincaré plot as follows. Let us designate two consecutive points as , Mean specificity = Total True Negative/. Mean sensitivity and mean specificity were 91.4% and 92.9% respectively.We tried to propose an automated detection method which did not require any human intervention. Usually an ECG data is recorded for some period of time; then, an expert reads the data to find an abnormality in it. However, this process is tedious and inconvenient for patients since they should be in some place equipped with an electrocardiograph. Because pulse beats of ventricles are less likely to be influenced by baseline wandering and noise, we used the inter-beat intervals to diagnose AFib. It will be useful to make a real time portable monitoring electrocardiograph because we cannot predict when the paroxysm of AFib will come about. Our algorithm requires only one lead of ECG to acquire inter-beat intervals. We tried to design our algorithm not using any specific threshold.Heart rate variability is closely related to homeostasis of the autonomous nervous system. The dynamics of inter-beat intervals come to change after the onset of AFib. Therefore we used the Poincaré plot because it was a useful tool in studying dynamics of ECG data. We found that people without any AFib showed some patterns in the Poincaré plots and these patterns were regular. The plots of AFib patients, however, were very irregular and changed too much in the course of time.We described feature selection process and classifier using clustering and support vector machine method. We captured inter-beat intervals from an input ECG data, and drew Poincaré plot using them. By analyzing the Poincaré plot, we obtained three feature measures: the number of clusters, mean stepping increment of inter-beat intervals, and dispersion of the points in the plot. These three feature measures were dimensionless quantities.There are some limitations in this paper. First, one feature, the dispersion of points in Poincaré plot, was not powerful to discriminate AFib from non-AFib as you can see in the Figure The authors declare that they have no competing interests.JP has worked on the wavelet analysis, extracted three features by investigating the Poincaré plots, and written the whole manuscript. SL has worked on classification using the support vector machine. MJ has designed the detection strategy of AFib, and corrected the manuscript. All authors have read and approved the final manuscript. |
Extravagant ornaments evolved to advertise their bearers' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules . Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties. Testosterone plays a pivotal role in regulating the expression of many animal ornaments . A main Lagopus lagopus scoticus). This bird displays supra-orbital red combs, the coloration of which is carotenoid based , (iii) testosterone implants, no parasite challenge , and (iv) testosterone implants, challenge with T. tenuis . We sampled males before treatments (S1) and again after 10 days (S2) and 17 days ornamentation (comb area), (iv) total antioxidant status and (v) plasma concentrations of malondialdehyde (MDA), a measure of oxidative damage. TAS measures the capacity of the plasma to quench a free radical cation and the pooled effect of all extracellular, non-enzymatic antioxidants in plasma . MDA is We predicted that (i) increased testosterone levels would enhance ornamentation but increase oxidative damage, measured in terms of MDA, (ii) a developing parasite infection would reduce circulating antioxidants, cause oxidative damage and reduced ornamentation, (iii) the cost of each treatment (oxidative damage) would depend on initial ornament size, with males displaying smaller combs showing a greater increase in oxidative damage (MDA) relative to larger combed males, and (iv) the treatment effects on oxidative damage and the ability of males to increase antioxidant activity (TAS) to reduce this damage would explain changes in male ornamentation.T. tenuis nematodes , 10 days later (S2) and 17 days later (S3). Details on the timing and data sampling for the experiment are given in We worked on Edinglassie and Catterick moors (UK) in 2006. In September, we caught 20 male red grouse on each site, by dazzling and netting them at night . Upon fiematodes . We starWe measured comb area (maximum length×width of flattened comb) as an index of ornament size .Plasma testosterone concentration was measured using a commercially available testosterone enzyme immunoassay , an assay that has been developed and validated for determining testosterone levels in small volume (20 μl) avian plasma samples phase was collected and transferred into an HPLC vial for analysis. Samples (10 μl) were injected into a Dionex HPLC system fitted with a 5 μm Octadecyl-silica (ODS) guard column and a Hewlett-Packard Hypersil 5 μm ODS 100×4.6 mm column maintained at 37°C in a thermostatted column compartment . The mobile phase was methanol buffer , the buffer being a 50 mM anhydrous solution of potassium monobasic phosphate at pH 6.8 (adjusted using 5 M potassium hydroxide solution), running isocratically over 3.5 min at a flow rate of 1 ml min−1. The data were collected using a fluorescence detector set at 515 nm (excitation) and 553 nm (emission). For calibration, a standard curve was prepared using a TEP stock solution (3 μM in 40% ethanol) serially diluted using 40 per cent ethanol. TEP standards assayed in duplicate showed high repeatability .Plasma concentrations of MDA were calculated by HPLC using fluorescence detection . All che2O2) was then added and the sample was incubated for 195 s. Hydrogen peroxide (H2O2) addition induces the production of the radical cation ABTS, which generates a blue-green colour. Colour is measured at 600 nm before and after H2O2 addition, thus determining the change in colour. Antioxidants in the plasma sample cause suppression of this colour change to a degree that is proportional to their concentration. Results are given as mmol l−1 of plasma. Repeatability was determined on a subsample measured twice .TAS concentration of plasma was assessed by means of commercial kits adapted to an automated spectrophotometer . Plasma samples were incubated for 15 s with a chromogen composed of metmyoglobin and ABTS . Hydrogen peroxide or direct worm counts . Caecal egg counts provide reliable estimates of worm burdens and were used to calculate T. tenuis abundance as the difference between the final and initial values, corrected for the initial value . We tested whether these changes over time differed according to testosterone treatment (TTreat), parasite treatment (PTreat) and their interaction using general linear models. All models also included ‘site’ as a fixed effect to control for possible differences between sites. Experimental results did not differ between sites.We used SAS v. 8.01 . Counts T. tenuis eggs in their faeces , indicating that the initial parasite purging had been effective. Testosterone levels increased more in T+ than in T− males between S1 and S2 (a) and remained higher afterwards (S2 until S3). Parasite challenges had no effect on testosterone levels (a), but increased T. tenuis abundance. When challenged (S1), males had no detectable T. tenuis worms. However, by S3, average T. tenuis abundance was higher in P+ (mean 147 worms) than in P− males (15 worms), irrespective of the testosterone treatment .At the start of the experiment (S1), males that had been dosed previously with anthelminthic (at S0) had no 1 and S2 a and reme levels a, but inc). MDA concentration increased more in T+ than in T− males (d).Consistent with prediction 1, testosterone implants increased comb area and the concentration of MDA. Comb area increased more in T+ than in T− males between S1 and S2 and remained larger afterwards c. MDA coT− males d.d). Parasite challenge reduced ornamentation, but only in T− males and with a time lag .Consistent with prediction 2, parasite challenge caused oxidative damage . In T− males, changes in MDA were explained by parasite treatment, irrespective of initial comb area .Consistent with prediction 3, changes in MDA were dependent on initial comb area, but in T+ males only. In T+ males, changes in MDA were not explained by parasite treatment, but by initial comb area . In T− males, parasite treatment reduced TAS .TAS increased more in T+ than in T− males, while parasite treatment reduced TAS, but depending on testosterone treatment . In T+ mF1,30=2.41, p=0.13; slope ±s.d.: +0.499±0.323). Changes in MDA between S1 and S2 were not significantly related to changes in TAS . However, changes in TAS and MDA explained the impact of treatments on changes in comb area after taking into account treatment group level effects , while no such effect was found in T− males .Before testosterone and parasite treatments (at S1), MDA was not significantly related to TAS . In T− males, a reduction in TAS was associated with a decrease in ornament size (c). In T+ males, a greater increase in TAS was associated with a continued increase in ornamentation (d).Consistent with prediction 4, changes in TAS explained lagged changes in ornamentation, between S2 and S3 c,d. In Tent size c. In T+ entation d.T. tenuis infection levels . There was no short-term effect of testosterone on the effectiveness of parasite challenges, although previous work showed that elevated testosterone can indirectly increase T. tenuis abundance 1 year after challenge , but were still within the natural range (Trichostrongylus tenuis larvae impact most on metabolism 12–16 days after infection (Testosterone implants enhanced ornamentation (prediction 1), while parasite challenges reduced ornamentation (prediction 2), but only in T− males, and with a time lag. Testosterone implants increased oxidative damage, as indexed by MDA concentrations (prediction 1). This might be because testosterone increased metabolic rates e.g. .in vitro, suggesting that the antioxidant defences were upregulated in vivo. However, this was not sufficient to prevent oxidative damage. Parasite challenges also increased oxidative damage (prediction 2) but reduced TAS when these were not increased by testosterone . Overall, parasite challenge caused more oxidative damage than experimental testosterone increase, possibly because of the contrasted effects of these manipulations on circulating antioxidant defences.Interestingly, elevated testosterone resulted in an increase in antioxidant defences as measured by the TAS assay T. tenuis parasites were shown by experiment to reduce circulating carotenoids: in vivo has been both supported and questioned, and remains controversial but at the cost of investing these same resources to sexual signal expression. Such a trade-off was supported for carotenoid-based coloured traits controlled by testosterone: elevated testosterone may increase or decrease signal coloration in a direct trade-off with circulating plasma carotenoids explained short- and medium-term treatment effects on male ornamentation, showing for the first time that the ability to express a testosterone-dependent ornament is tightly related to an individual's oxidative balance and susceptibility to oxidative stress. The extent to which testosterone and parasites cause oxidative damage depends on an individual's ability to increase circulating antioxidant defences (by acquiring more or mobilizing stored antioxidants) and to resist parasites (the ability of its activated immune system to raise an appropriate immune response that finds the right target and at the same time avoid immunopathological damage; Ornaments have evolved to facilitate the assessment of individual quality , such as |
In the current discussion of surgical treatment of arthroses in the ankle joint, arthrodesis is in competition with artificial joint replacement. Up until now, no valid biomechanical findings have existed on the changes in intraarticular loads following arthrodesis. One argument against tibiotalar arthrodesis is the frequently associated, long-term degeneration of the talonavicular joint, which can be attributed to changes in biomechanical stresses.We used a dynamic model to determine the changes in intraarticular forces and peak-pressure in the talonavicular joint and in the calcaneocuboid joint on 8 cadaver feet under stress in a simulated stance phase following tibiotalar arthrodesis.The change seen after arthrodesis was a tendency of relocation of average force and maximum pressure from the lateral onto the medial column of the foot. The average force increased from native 92 N to 100 N upon arthrodesis in the talonavicular joint and decreased in the calcaneocuboid joint from 54 N to 48 N. The peak pressure increased from native 3.9 MPa to 4.4 MPa in the talonavicular joint and in the calcaneocuboid joint from 3.3 MPa to 3.4 MPa. The increase of force and peak pressure on the talonavicular joint and decrease of force on the calcaneocuboid joint is statistically significant.The increase in imparted force and peak pressure on the medial column of the foot following tibiotalar arthrodesis, as was demonstrated in a dynamic model, biomechanically explains the clinically observed phenomenon of cartilage degeneration on the medial dorsum of the foot in the long term. As a clinical conclusion from the measurements, it would be desirable to reduce the force imparted on the medial column with displacement onto the lateral forefoot, say by suitable shoe adjustment, in order to achieve a more favourable long-term clinical result. The surgical treatment of ankle joint arthrodeses is currently a subject of hot debate, where many authors view arthrodesis as the standard treatment -3. Provi2) upon which the foot „walked“ was mounted on a hydraulically activated translation platform. The platform was elevated by means of a force-controlled hydraulic cylinder which was programmed and calibrated to simulate the vertical component of the ground reaction force. The translation stage was further allowed to move freely in the medial-lateral, and anterior-posterior directions. The platform was tilted by a second angle-controlled hydraulic cylinder in order to simulate tibial inclination .Eight macroscopically and roentgenographically normal foot specimens were tested comparing tibiotalar arthodesis vs. nativ situation on a dynamic gait simulator. The stance-phase of walking was simulated from heel-contact to toe-off. Ground reaction forces were simulated by a tilting angle- and force-controlled translation stage upon which a pressure measuring platform was mounted . Force were applied to the tendons of the foot flexor and extensor muscle groups by cables attached to an additional set of six force-controlled hydraulic cylinders . Tibial rotation was produced by an electrical servo motor . The foot simulator functions inversely and was described previously . The preLower limb specimens were obtained by transection at approximately mid-tibial length. Soft tissues were removed to roughly 4 cm above the ankle joint. The tendons of the lower-leg muscles were blunt dissected free of the leg to allow attachment via clamps to cable pulls by means of which force was applied. Nine tendons of the foot were simulated with six hydraulic cylinders: the triceps surae, tibialis posterior, flexor hallucis longus and flexor digitorum longus combined, tibialis anterior, peroneus longus and brevis combined, and combined extensor digitorum longus and extensor hallucis longus.The proximal ends of the specimen tibia and fibula were prepared clear to the surface of the bone and potted in their relative anatomical positions in a cold-curing methylmethacrylate resin . The potted tibia and fibula were mounted concentrically and vertically oriented in an aluminium tibia mounting cylinder using centring screws. Neutral rotation of the tibia was defined prior to testing by turning the mounting cylinder to orient the foot in 12° of external rotation with respect to the long axis of the pressure measuring platform.Physiological gait was simulated from heel strike (0%) to toe-off 100%) over a standardised time of 60 s . The sensor sheets with a thickness of 0.3 mm consists of 264 different sensors arranged in an 11 by 24 matrix with a spatial resolution of 1,8 mm and a maximum pressure range of 2000 PSI . The pressure values were recorded with the manufactures software (Iscan) with a recording rate of 2 Hz for a time span of 60 seconds to achieve 120 pressure frames. Raw data were then exported as ASCII-Files and further analyzed in MatLab .Separate sheets were used for the talonavicular and the calcaneocuboid joints, with measurements taken simultaneously during the experiment. Therefore the initial sheet was cut along appropriate conducting path to get sensor areas of 9 by 24 mm and 11 by 24 mm respectively that fitted to the anatomic conditions of the joints. The cutted sheets were sealed carefully to avoid wetting and fixed inside the joint with sutures to the capsular tissue. During the experiments, the position was closely followed by the investigators in order to exclude displacement of the sensors during foot motion. Each experiment was performed with five repetitions. The applied load of the joint was characterized by the time course of the force acted upon the sensor area for each pressure frame and the maximum peak pressure value (mpp) within each frame. Mean values for the time course of the 5 repetitions and the 95%-confidence interval were calculated to visualize possible differences in the time course of force and mpp graphically.Tibiotalar arthrodesis was performed with a stabile fixation using external fixator without removing the cartilage. One pin was placed in the distal tibia and another two pins were placed in the talus body. The pins were connected under maximal compression in a triangle construction medial and lateral. Each specimen was checked for correct placement of the pins using x-ray images and checked for stability under load application prior to start the nativ measuring cycles before fixing the foot in the model, free movement of the neighbouring joints was controlled as well. An additional pin was placed in the tuber calcanei for later study of subtalar arthrodesis. Special concern was taken not to affect tendon-gliding by the Steinmann pinsMeasurements were taken first for nativ load for five cycles with the fixateur pins still in situ. After completing the nativ measuring the pins were connected to simulate tibiotalar arthrodesis without changing the simulating conditions to get same conditions for the five arthrodesis runs. Tibial rotation, muscleforce simulation and ground reacting forces were not changed.The data were analyzed using the statistical software JMP IN 5.1 . For each of the 8 feet and each of the 5 replications, the measurements consisted of the values for force and peak pressure for 120 points in time. As dependent variables, we took the mean of the 120 values of the force and the maximum of the 120 peak pressures to get overall values for the whole stance phase. Since these were not normally distributed, we used the Box-Cox transformation of the data to obtain normally distributed data with equal variances. An analysis of variance was performed with the state (native vs. arthrodesis), the joint, their interaction and the length of the foot as fixed factors and the foot number as random factor. Furthermore, an analysis of variance was carried out for each joint separately, now with the state and the length of the foot as fixed factors and the foot number as random factor. For obtaining the median and 95% confidence intervals (95% CI), the means and 95% CIs of the Box-Cox transformations were transformed back.We present a mean normalised time course for the force values in the talonavicular joint in figure The values for median force during stance phase of both parts of Chopart's joint for 8 foot specimen is given in table We observed a varied increase in intraarticular force and peak pressure in the talonavicular joint following arthrodesis. The median force changed from a native value of 92 N (95% CI: 64 N–145 N) to 100 N (95% CI: 69 N–164 N) upon arthrodesis. Over all feet, the increase in force on the talonavicular joint was statistically significant (p = 0.027), while we also saw a significant increase in the peak pressure (p = 0.0005) from native 3.9 MPa (95% CI: 3.1 MPa–5.7 MPa) to 4.4 MPa (95% CI: 3.5 MPa–6.2 MPa).In the calcaneocuboid joint, we recorded lower values for force upon arthrodeses, the effect was statistically significant (p = 0.029). We observed a median decrease from the native load, from 54 N (95% CI: 28 N–91 N) to 48 N (95% CI: 24 N–82 N) upon arthrodesis; regarding intraarticular peak pressure, we saw a change from native 3.3 MPa (95% CI: 2.7 MPa–4.4 MPa) to 3.4 MPa (95% CI: 2.6 MPa–4.2 MPa). This effect was not significant (p = 0.098).Our measurements indicate a tendency of relocation of the force onto the talonavicular joint after arthrodesis with a reduction of the force on the calcaneocuboid joint. Overall, this relocation of force is statistically not significant . The effect of the relocation of maximum peak pressure onto the talonavicular joint is also not detectable with statistical significance (p = 0.11).In our experiment, there is a trend correlating the size of the foot preparations with the imparted force, where larger preparations in the model produced higher values than smaller preparations regarding the sum of the two parts of the Chopart joint (p = 0.0043). There is no statistically significant correlation of peak pressure with foot size (p = 0.07).The transfer of force from the tibia to the ground when walking is imparted through an extension/flexion movement of the foot against the lower leg and also an eversion/inversion movement in the hindfoot and an abduction/adduction movement in the forefoot. Previous examinations show here that about 4/5 of the extension/flexion movement is imparted through the ankle joint and 1/5 through the Chopart joint . The latThe complex anatomy of the Chopart joint, with a ball-and-socket articulation at the medial column of the foot and a saddle articulation at the lateral column, is an expedient biomechanical construction for accomplishing the complex tasks of the foot, namely optimally adapting to uneven surfaces upon first heel contact and forcefully pushing the foot off the ground at the end of the walk cycle. The locking mechanism of the Chopart joint upon calcaneal inversion was described by Elftman 1960 . This loSince it is not possible to make in-vivo measurements of intraarticular pressures or analyse the imparted forces in people, the biomechanical joint stresses must be simulated in a model. In this regard, computer simulations and cadaver experiments are possible approaches. Cadaver experiments are thus, even in the present study, only a model in the simulation of physiological processes and thus limitations of their meaning must be taken into account. An essential advantage of our experiment is the ability of the test apparatus to simulate even extrinsic tendon pull on the foot. As such, our simulation is a test setup that corresponds more closely to reality than previously published experiments. The inability of earlier cadaver experiments to simulate this has been described as significantly disadvantageous .We observed much interindividual variance among our preparations. This can be explained by the fact that the preparations were of different sizes, and a fluctuation of the results in the range of 100% can be reasonably explained by different individual anatomic conditions, such as body weight. We were able to make a statistical correlation between imparted force and foot size. Since we activated the same muscles in all preparations, we assume relatively too high measurements for the smaller feet and too low measurements for the larger feet. After averaging the results, we obtained a realistic average value.One specific possibility for error in our experiments was the possibility that forces were being imparted off to the side of the pressure films, which cannot be ruled out with absolute certainty in the narrow joints, even after careful preparation and placement of the films.Determining force and pressure conditions in the articulations of the hindfoot is technically difficult. In a previous study the loads on the subtalar joint and the talonavicular joint following tibiotalar arthrodesis were determined in a servohydraulic test machine . The FujAfter tibiotalar arthrodesis, we measured inconsistent interindividual changes in the Chopart joint. The imparted force and, consequently, the peak pressure both rise on the medial column of the foot in the talonavicular joint and the force fall on the lateral column in the calcaneocuboid joint. When seen in connection with the clinically observed and scientifically described adaptation processes – with an increase in the degree of movement in the talonavicular joint after ankle joint arthrodesis, increased tibial rotation and increased calcaneal eversion – this biomechanical fact impressively explains the appearance of cartilage degeneration on the medial dorsum of the foot. The increase in peak pressure after tibiotalar arthrodesis at the point of highest loading during the forceful push-off phase of the foot must be particularly problematic. As a conclusion of our experiment, it would be desirable to reduce the force imparted on the medial column that results from arthrodesis, with lateral relocation of the imparted force. Further clinical investigations are required to test such an effect, say, by simple shoe adjustment with support of the lateral forefoot or a restriction of hindfoot eversion after tibiotalar arthrodesis, the objective being to improve the long-term results.The author(s) declare that they have no competing interests.AS: planning, writing and leading the study; TH: statistical analyses and support in mathematical preparation of the complex interpretation of data; OM: Preparation of the data writing MatLab programms to interpret raw data and creates figures; NW: organized financial support and constructed the testing apparatusThe pre-publication history for this paper can be accessed here: |
Approximately 28% of patients with cancer pain die with severe untreated pain despite effective multidisciplinary techniques that should treat these patients effectively. Despite Interventional pain management procedures are most often used in concert with standard analgesic regimens to reduce opioid side effects or gain better analgesic efficacy. The interventional analgesic technique may be used at any time during the course of cancer treatment but is often employed during the more aggressive phases of disease. IntervenBefore jumping into interventional technique, patients should receive appropriate trials of opioid and non-opioid analgesics. A complete pain evaluation with pain history, appropriate physical examination (including a neurologic examination), and a tentative etiology for the pain is necessary prior to any invasive procedures. A neurologic examination identifies any preexisting neurologic deficits as well as areas of minor motor or sensory reduction, Informed consent is a prerequisite, especially with the neurolytic blocks that may result in permanent motor/sensory deficits. A local anesthetic block is usually performed prior to any “permanent” neurolytic procedure to evaluate the likelihood of success and identify neurologic deficits that may be intolerable.Virtually all studies, to date, recommend the proper selection of patients so that less invasive analgesic techniques may be utilized as first-line therapy.The wide variety of interventional pain management techniques available in the present situation should encourage physicians to consider “something further” in almost all cases of cancer pain. The appropriate use of anesthesiologic interventions to help manage cancer pain will improve the quality of life for all patients.There is a need for research to determine clear indication and benefit from interventional pain management techniques in cancer pain patients. |
Models were stratified by active versus retired work status and by years employed before the baseline survey (< 5 and ≥ 5 years).This prospective cohort study followed 447 cotton textile workers from 1981 to 2006.at approximately 5-year intervals. We used a generalized estimating equations approach to model FEV1 level among retired cotton workers. Among all cotton workers, past exposure was more strongly associated with reduced FEV1 for those hired < 5 years before baseline than for those who were hired ≥ 5 years after baseline. Recent endotoxin exposure was significantly associated with byssinosis, chronic bronchitis, and chronic cough.Past exposure to endotoxin was associated with reduced FEV Occupational exposure to cotton dust can cause acute respiratory responses such as chest tightness and bronchoconstriction and resp1). In fact, a doubling of exposure was associated with a 19-mL increase in the average annual decline of FEV1 cotton workers and 243 (51.5%) silk workers participated in all six surveys. The institutional review boards (IRBs) of the Harvard School of Public Health, the Putuo District People’s Hospital, and the Human Resources Administration of China approved the study. We complied with all applicable requirements of the United States and international regulations ; the participants gave written informed consent before the study.Stationary measurements of airborne cotton dust were performed with a vertical elutriator in the two cotton textile mills, in six work areas where yarn was prepared . Samplin3). Cumulative exposure to endotoxin (EU/m3-years) estimated for each participant was derived from work area samples and from detailed work histories. A limited number of full-shift samples were also taken in the silk mill, and measurements for endotoxin were nondetectable (below the limit of detection); thus, silk workers were considered unexposed to endotoxin and to cotton dust.To measure airborne endotoxin concentrations, endotoxin assays were performed on the cotton dust sample filters using the Limulus amoebocyte lysate assay, chromogenic method . Endotox1. Acceptable FEV1 tracings varied by no more than 5% or 100 mL, whichever was greater, and all values were corrected for body temperature and pressure saturated with water vapor . The highest FEV1 values from technically acceptable tests were used in the analyses. At the 2006 survey, 30 cotton workers and 44 silk workers did not perform the spirometry testing because of poor health.Tests of pulmonary function were conducted according to American Thoracic Society (ATS) criteria and were supervised and recorded by trained technicians at the cotton mills , 2001. FWe used a modified ATS standardized respiratory symptom questionnaire , transla1 level (at each survey over 25 years) and a logit link function when the outcome was chronic bronchitis, chronic cough, dyspnea, or byssinosis.We used generalized estimating equations (GEEs) to accour = 0.92) among the cotton workers. All models for FEV1 level and symptoms were adjusted for age (year), height (centimeters), sex , smoking status (current or former smoker with never smoker as reference), work status (active vs. retired) and years since cessation of exposure. All covariates were treated as time-dependent factors except sex and height (defined as the average of the first three surveys). Years since cessation of exposure was defined as zero during active employment and as the time (years) between the date last worked in an endotoxin-exposed job in the cotton mill and the date of the current survey. Models for FEV1 level were stratified on employment status (active workers vs. retirees) and by years hired prior to the baseline (1981) survey (< 5 and ≥ 5 years). A p-value < 0.05 was selected to indicate statistical significance.Cumulative exposure to endotoxins that was estimated at each survey was divided into two time windows: past endotoxin exposure, from date of hire up to the start of each survey interval, and recent endotoxin exposure within each current interval . Cumulat1 was also modeled as a smoothed function of past endotoxin exposure, recent exposure, and years since cessation of exposure, using penalized splines in generalized additive mixed models . By contrast, evidence from penalized splines suggested associations with past exposure were nonlinear. Thus we treated past exposure as a discrete variable with high-, medium-, and low-level categories that were derived from tertile cutoffs of past cumulative endotoxin exposure at each survey.We modeled both recent exposure and years since cessation of exposure as continuous terms because results from penalized spline models (data not shown) suggested that associations with FEV1 relative to low level of past exposure, but only the association with the medium level was statistically significant among retired cotton workers, whereas the association between recent endotoxin exposure and FEV1 level was inverse among active workers and positive among retired workers. Among all cotton workers, interactions between high and medium levels of past endotoxin exposure and retired status and between recent endotoxin exposure and retired status (p = 0.55) were not statistically significant.When cotton workers were examined as a whole, high and medium levels of past endotoxin exposure were associated with higher FEVnificant . A posit1 level and past endotoxin exposure for cotton workers who were employed < 5 years before the baseline evaluation than for workers who had worked for a longer period of time were observed between < 5-year work tenure and high and medium levels of past endotoxin exposure.Cotton workers worked for 16.4 years, on average, before the baseline survey, and only 14.8% had worked < 5 years. We observed a stronger inverse association between FEV of time . SignifiOdds ratios for self-reported byssinosis and dyspnea were significantly increased for continuous recent endotoxin exposure . Years s1 level for retired workers with higher past cumulative exposure to endotoxin. Among all exposed workers, the inverse effect of past cumulative exposure on FEV1 level was largest for those with shorter work histories before the baseline survey. In contrast, recent exposure to endotoxin, rather than past exposure, was associated with byssinosis and chronic bronchitis.In the most recent follow-up of a 25-year prospective cohort study of cotton textile workers, we observed decrements in FEV1 level after retirement. A lag period between exposure and disease onset has been observed for occupational exposure-related diseases such as cancer and pneumoconiosis compared with participants who completed spirometry testing at the last survey (31 mL/year). This may represent another potential source for downward bias on the association between past cumulative endotoxin exposure and decline in FEV1 level.The present study had several limitations. Because of the inherent limitation of the study design, surveys were conducted at approximately 5-year intervals over 25 years of follow-up. More frequent follow-up with exposure assessment and FEV3) between two laboratories performing an analysis of duplicate samples of airborne cotton dust. In general, background levels of endotoxin in the environment are < 10 EU/m3. However, mean endotoxin concentrations in several occupational settings with airborne exposure to organic dusts or decaying organic matter have ranged from several EU/m3 estimated in a meta-analysis of airborne endotoxin concentration in the cotton textile industry , rather than past cumulative exposure, was associated with chronic respiratory symptoms in retirees. The data suggest that timing of occupational endotoxin exposure may influence the rate of FEV1 decline as well as the incidence of respiratory symptoms among textile workers.In summary, past rather than recent cumulative exposure to endotoxins before baseline FEV |
This study assesses the validity and reliability of the Spanish version of DN4 questionnaire as a tool for differential diagnosis of pain syndromes associated to a neuropathic (NP) or somatic component .A study was conducted consisting of two phases: cultural adaptation into the Spanish language by means of conceptual equivalence, including forward and backward translations in duplicate and cognitive debriefing, and testing of psychometric properties in patients with NP and NNP. The analysis of psychometric properties included reliability and validity .A sample of 164 subjects , 94 (57.3%) with NP and 70 with NNP was enrolled. The questionnaire was reliable and valid for a cut-off value ≥ 4 points, which was the best value to discriminate between NP and NNP subjects.This study, representing the first validation of the DN4 questionnaire into another language different than the original, not only supported its high discriminatory value for identification of neuropathic pain, but also provided supplemental psychometric validation and showed its validity in mixed pain syndromes. Appropriate therapeutic management of pain requires an accurate diagnosis, distinguishing its cause and origin. According to the International Association for the Study of Pain (IASP), neuropathic pain (NP) is defined as a pain initiated or caused by a primary lesion (or dysfunction) of the nervous system, and comprises a very large group of neurological conditions including diabetic and other sensory polyneuropathies, trigeminal neuralgia, post-herpetic neuralgia, stroke, spinal cord injury, and multiple sclerosis, as well as common conditions such as lumbar or cervical radiculopathies, traumatic or postsurgical nerve injuries, etc -4. VirtuDue to the lack of validated or consensual diagnostic criteria, diagnosis of NP has traditionally been based on identification of the neurological lesion through the medical history, neurological examination, and appropriate electrophysiological or imaging investigations ,12. A seIn order to test this hypothesis, this study analyzed the psychometric properties of the DN4 questionnaire translated into Spanish. The DN4 questionnaire was originally developed and validated in French . It is aThus, the aim of this study was not only the translation and cultural adaptation of the DN4 questionnaire into Spanish, but to provide also a supplemental psychometric validation of the questionnaire. In particular, in addition to the general diagnostic properties of our translated questionnaire (i.e. sensitivity and specificity), its test-retest reliability was verified, and the influence of various factors, such as pain intensity and educational level, on the DN4 results was analyzed. The DN4 was also administered to a group of patients with a combination of neuropathic and non-neuropathic pain to support its validity in mixed pain syndromes.The DN4 questionnaire consistsThe adaptation into Spanish of the DN4 questionnaire and the subsequent assessment of its psychometric properties have been performed following the traditional recommendations for adaptation and validity of health questionnaires and diagnostic tests -25. In tPatients of both sexes with chronic pain for more than 3 months aged 18 years or over, with an adequate cultural level to understand health questionnaires administered in Spanish were included in this study. Chronic pain could be of a neuropathic, non-neuropathic, or mixed (i.e. both neuropathic and non-neuropathic components) origin. Pain was classified based on the medical history, physical examination, and any procedures considered appropriate by the clinicians to establish the diagnosis of the type of pain. All patients gave their written informed consent before entering the study. Tables In addition to the DN4 questionnaire, average daily pain intensity was measured using a 100 mm visual analogue scale (VAS), and the short-form McGill Pain Questionnaire (SF-MPQ) was usedAll investigators were pain experts. At each participating center, the main investigator collected the sociodemographic and clinical data required for patient characterization and was required to classify patients into two broad diagnostic categories corresponding to NP and NNP, and to further subcategorize NP into peripheral, central or mixed pain. The diagnosis of the principal investigator was considered as the reference (gold standard) diagnosis. Patients were then separately seen within 2 days by two other investigators (raters A and B) blinded to the diagnosis proposed by the principal investigator, who administered the DN4 and SF-McGill questionnaires.This study was conducted according with usual standard of care in each participant centre, and it did not involve any drug or therapeutic management. Nevertheless, the study design was approved by the IRB of the Universidad Autónoma de Madrid and was conducted in compliance with the Helsinki Declaration for research in humans.The internal consistency of the Spanish version of the DN4 questionnaire was separately established for raters A and B by calculating Cronbach's α coefficient that assesses the contribution of each item to the precision of measurements by the instrument. Cronbach's α coefficient was assessed in the complete questionnaire and after removing each item from it to assess the independent contribution of each item to the measurement error in the instrument.Inter-rater reliability was assessed by the agreement of the results obtained by raters A and B for each item and the total score of the DN4 questionnaire. Agreement was determined by calculating the Cohen's kappa coefficient. Inter-rater agreement was also determined by calculating the intra-class correlation coefficient of the total scores assigned by each pair of raters to the same subject, and the answers to each individual item, using a two-factor model and mixed effects.In a subject sub-sample (n = 68), the test-retest reliability of the questionnaire was assessed by a third administration, after at least 48 hours, of the DN4 scale by rater B. Stability of the questionnaire was analyzed by measuring the intra-class correlation coefficient between the scores of clinicians completing test and retest and using Cohen's kappa coefficient of agreement for the total retest sample and in subgroups by type of pain.A ROC (receiver operating characteristic) curve analysis was performed to determine the cut-off value of the questionnaire score providing the best values of sensitivity and specificity for NP diagnosis and determination of Youden's index for the total patient sample included in the study. This was repeated in two sub-samples excluding either patients with mixed pain only or patients with mixed and subjects with central NP . For each cut-off value of the scale score, in addition to sensitivity and specificity and Youden's index, the positive (PPV) and negative predictive value (NPV), the kappa coefficient of agreement with the diagnosis according to the standard criterion or reference diagnosis, and the area under the curve (AUC) calculated by the trapezoid method and its significance level were estimated. The 95% confidence intervals (95% CI) of the estimators of the cut-off point selected as optimum were calculated. Validity indicators were recalculated individually for the presence of each descriptor symptom of the DN4 questionnaire. The Youden's index was calculated by the equation: sensitivity + specificity - 1 .Descriptive statistics were prepared for the variables tested in this study to assess central positition, dispersion, and distribution of variables tested by the Kolmogorov- Smirnov test. A Student's t test for independent groups or a Mann-Whitney's U test was used in the case of a non-normal distribution to compare continuous or ordinal variables respectively between patients with NP and NNP. All statistical tests were two-tailed, and an α error of < 0.05 was accepted as statistically significant. Data were analyzed using SPSS version 12.0 statistical software.The study was conducted from June to November 2005. Table Table Inter-rater reliability, which is essential for an instrument that contains a semi-structured interview subject to interviewer interpretation, was good to very good. The total scores in each diagnostic group did not show statistically significant differences, and the intra-class correlation coefficient values ranged from 0.84 and 0.93 were removed, and worsening when such patients were included between both studies. In particular, our analysis of the properties of the questionnaire according to pain intensity revealed significantly lower sensitivity and specificity in patients with mild pain intensity. The influence of pain intensity on the psychometric properties of neuropathic pain screening tools had not been previously tested with the DN4 or with other questionnaires. The data reported here suggest that these tools might be much less discriminant and should therefore be used with caution in patients with reduced pain intensity. However, this would have to be confirmed in further studies with the DN4 or other diagnostic questionnaires, because the subgroup of patients with mild pain intensity was relatively small in this study. By contrast, our results suggest that the discriminant value of the DN4 questionnaire did not depend on the educational level, confirming that these simple symptom-based questionnaires are easily understood and that their administration should not be restricted to highly educated patients. A supplemental validation investigated in this study concerned the stability over time (i.e. test-retest reliability) of the DN4 questionnaire, which was excellent in our patients with or without NP.The results of the DN4 were similar in patients with "pure" neuropathic pain or mixed pain syndromes, (i.e. the combination of neuropathic and non neuropathic pain in the same patient). Thus, the present data suggest that the neuropathic component of the "mixed pain" syndrome has clinical characteristics similar to those of "pure" neuropathic pain. Interestingly, this is consistent with the results recently reported by Freynhagen et al , showingThe results of this study support the transcultural validity of the DN4 questionnaire. More generally, our data tend to confirm the interest of symptom-based diagnostic tools for identification of the neuropathic pain component. These simple clinical tools may be used not only in daily practice, but also in the clinical research setting . Future studies directly comparing the performance of the different available tools are warranted.DN4: Douleur Neuropathique 4 questions;NP: Neuropathic pain;NNP: Non Neuropathic pain;IASP: International Association for Study of Pain;VAS: Visual Analogue Scale;SF-MPQ: Short-form McGill Pain Questionnaire;ROC: Receiver Operating Characteristic;PPV: Positive Predictive Value;NPV: Negative Predictive Value;AUC: Area under the curve;PCP: Primary Care Physicians.Javier Rejas is employed at Pfizer Spain. The author(s) declare that they have no competing interests.CP and JR designed the study and were responsible for the idea of the study. CP, RG, SH and JI participated in patient enrrollment and manuscript elaboration. DB wrotte the manuscript in part and was responsible for consultation and scientific support of the project. SD was responsible for monitoring, logistic and evaluation of data. All authors read and approved the final manuscript.Cuestionario DN4. This appendix includes the Spanish version of DN4 questionnaire.Click here for file |
While access and utilization form core components in assessing the effectiveness of a health service, the concept of coverage is often neglected. In this study we propose to develop a GIS-based methodological framework for the measurement of district-based geographic coverage to examine the service effectiveness of methadone treatment programme (MTP) in Hong Kong on a regular basis.To overcome the incompatibility of spatial units, population data and data of heroin addiction of the year 2001 are interpolated by population-weighted and area-weighted algorithms. Standard overlay and proximity analytical functions are used to delineate altogether 20 accessible zones around each methadone clinic at a fixed 1.5 km Euclidean distance. Geographic coverage here is defined as the percentage of heroin addicts covered by a methadone clinic within the accessible zone by district.A total of 6413 out of 11000 reported heroin addicts are found geographically covered. The average geographic coverage in Hong Kong is 44.6%, with the figure varying from 0% to 96% by district. One district having no clinic results in 0% coverage whereas another without a clinic yields 15.3% coverage from the clinic in adjacent district. Maps illustrating district-based geographic coverage are generated.As continuous data collection is required for a monitoring system, the simplified approach facilitates the handling of large volume data and relevant data analysis. It is concluded that the number of methadone clinics is as important as their locations. Geographic coverage could become an important consideration for monitoring harm reduction. Methadone maintenance is one of the well-known harm reduction strategies for public health intervention in heroin addiction. The significance of methadone treatment in preventing needle sharing, which in turn reduces the risk of HIV and HCV transmission among injectors, has been well cited. -4 MethadTo achieve maximum effectiveness, methadone clinic should be situated at where it satisfies the needs of local community of heroin users. The identification of the service zone of a health service and an examination of whether it is reachable are crucial. In this connection, the concept of coverage is useful. This is particularly meaningful for evaluating the provision of methadone maintenance programme, a service that delivers public health good. While large proportion of studies to date have focused on access and utilization of health care systems -10, few In this study, Hong Kong is taken as an example to pilot the measurement of the district-based geographic coverage of methadone clinics in terms of their availability within defined accessible catchment areas. The study aims at developing a simplified methodological framework to measure the geographic coverage of methadone clinics regularly, which enables a monitoring system to be developed for evaluating harm reduction strategies.Hong Kong is a metropolitan city located in Southeast Asia, bordering the South China Sea and mainland China . The totThe MTP has been operated by the Department of Health for 30 years. It adopts an out-patient and low threshold approach. Services are provided promptly to all heroin addicts who come forward for methadone treatment. For those with life-threatening medical condition, referral for treatment is made before being started on methadone. Besides prescribing methadone, other related services including patient assessment, counselling services and referral services are provided in methadone clinics . CurrentEssential data for this study included population data and data of heroin addiction. All data obtained were of the year 2001, when consistent data types from different services could be assured. As a study to demonstrate the methodological framework of coverage measurement, the use of the 2001 data, instead of more updated but inconsistent datasets, would be more important to enable reliable calculations to be made. District-based and TPU-based population data were collected from the Census and Statistics Department, while district-based data of heroin addiction was acquired from the Central Registry of Drug Abuse (CRDA), Narcotics Division of Security Bureau. CRDA has been established for 35 years to capture drug abuse data, which are provided by the reporting agencies on a voluntary basis with their full cooperation. The statistics is used to monitor the trend of drug abuse and to facilitate the planning of drug rehabilitation programmes in Hong Kong.Digital base maps including the boundary map of districts, boundary map of TPUs, map of highlands and water bodies were obtained from the Survey and Mapping Office (SMO), Lands Department of Hong Kong Government. Locations of methadone clinics were stored as X, Y projected coordinates in the GeoCommunity Database table (Version 2.1.2), which was also acquired from the SMO. These data, after synchronization, were input into a GIS software, ArcGIS 9.1 , for datThe landscape in Hong Kong is mountainous. About one-fifth of land area is over 200 metres in altitude, which is defined as uninhabitable highland in this study. Furthermore, the result will be highly unreliable if heroin addicts are assumed to distribute over the entire district, considering that only part of the district is populated. District-based heroin addicts' data were apportioned into TPUs by areal interpolation before measuring district-based geographic coverage. From the interactions with current and former heroin users, three assumptions for this study were made. First, walking is a preferred means of going to methadone clinics. Second, heroin addiction is assumed to be rather localized. Heroin addicts seldom go to the methadone clinic that is not located in their residence district. Third, heroin addicts are willing to walk at most for 30 minutes to their local methadone clinics.In preparing the Hong Kong base maps for spatial analysis, all water bodies, including reservoir and rivers, and land elevated over 200 metres were eliminated. is proportional to its population, sharing the same ratio of heroin addicts by district over population by district. Figure Thus theNumber of heroin addicts by district / population by district * population by a specific TPUEuclidean distance was defined as accessible zone for each clinic, assuming that addicts will cover this distance in 30 minutes. statements were used in the GIS to calculate both the area of entire intersected TPUs and the area of intersected portion of TPU. Percentage of area covered by the accessible zones in each intersected TPU was calculated and TPU-based heroin addicts covered were estimated by using the percentage. The following equation was used:Area of a TPU within the accessible zone / Area of the entire corresponding TPU* Interpolated number of heroin addicts in that TPUNumber of addicts from all TPUs in a district covered within accessible zones was aggregated into their corresponding district. However, the number of methadone clinics in each district varied considerably. The number of district-based addicts covered was adjusted and the mean number of heroin addicts covered by one methadone clinic by district was calculated. District-based geographic coverage was expressed as the percentage of heroin addicts covered within those accessible zones in each district among all heroin addicts in that district, using the following formula:Number of heroin addicts covered within a accessible zone in a district / Total number of heroin addicts in that corresponding district* 100%In the year 2001, there were about 11000 reported heroin addicts, giving a prevalence of 166 heroin addicts per 100,000 population in Hong Kong. The prevalence in eight out of 18 districts was higher than the average, with Yau Tsim Mong yielding the highest rate of 403. The distribution of methadone clinic was uneven. While one district was having three methadone clinics, two districts including Kwai Tsing and Sai Kung did not have any. Most of the districts (13 out of 18) had one methadone clinic, with two districts having two. After the elimination of uninhabitable areas, all TPUs (N = 197) remained populated and were therefore valid for conducting areal interpolation. The population-weighted areal interpolation produced interpolated values of heroin addicts by TPUs ranging from 1 to 369, with one-forth of TPUs (50/197) having less than 10 heroin addicts. Though all TPUs could be subjected to population-weighted areal interpolation, only 125 of them gave an intersection with the defined accessible zones for a clinic. In fact, an average of 66% of TPUs in a district was covered by the accessible zones. Accessible zones in four districts even covered all TPUs in that particular district.Using the definition developed in this study, a total of 6413 of 11000 heroin addicts were geographically covered by all methadone clinics in 2001. Since there were more than one methadone clinic in some districts, adjusted number of heroin addicts covered in each district was calculated. Only one district, Sai Kung, showed no coverage by any methadone clinic. Geographic coverage varied from the lowest 15.3% in Kwai Tsing to the highest figure 96.2% in Yau Tsim Mong. In the remaining districts, the average geographic coverage was 44.6% in 2001, though eight out of 18 districts demonstrated over 50% geographic coverage. declare that they have no competing interests.SSL was involved in the conceptualization and research design of the study. PTTP conducted the cartographic design and GIS analyses. This manuscript was drafted by PTTP and edited by SSL. Both authors read and approved the final manuscript. |
The effect of renal failure on melphalan pharmacology and toxicity has been poorly understood. Such information is of interest because melphalan is the most commonly used anticancer drug in the treatment of multiple myeloma, which is frequently associated with renal failure. We have studied the disposition and marrow toxicity of parenteral melphalan in dogs before and after induction of renal failure with subtotal nephrectomy. The surgical procedure decreased the creatinine clearance by an average of 62% (P = 0.001). The lowest neutrophil counts following i.v. melphalan (1 mg/kg) averaged 4.9 x 10(3)/mm3 pre-nephrectomy and 0.9 x 10(3)/mm3 post-nephrectomy, respectively (P = 0.002). The mean lowest recorded platelet counts after melphalan (1 mg/kg) were 115 x 10(3)/mm3 in the pre-nephrectomized dogs, and 9.7 x 10(3/mm3 in those who had been nephrectomized (P = 0.002). Following nephrectomy, i.v. melphalan's terminal-phase plasma half-life and renal clearance were both raised (P = 0.02) to 75% over pre-nephrectomy values. These studies show that i.v. melphalan-induced myelosuppression is markedly increased and its plasma elimination and renal clearance significantly decreased in the presence of renal dysfunction in dogs. These data suggest that parenteral melphalan's starting dose be decreased by at least 50% when used in myeloma patients with renal failure. |
Development of acute kidney injury (AKI) during the perioperative period is associated with increases in morbidity and mortality. Our aim was to evaluate the incidence and determinants of postoperative AKI after major noncardiac surgery in patients with previously normal renal function.This retrospective cohort study was carried out in the multidisciplinary Post-Anaesthesia Care Unit (PACU) with five intensive care beds. The study population consisted of 1166 patients with no previous renal insufficiency who were admitted to these intensive care unit (ICU) beds over 2 years. After admission patients were followed for the development of AKI, defined as proposed by The Acute Kidney Injury Network . Patient preoperative characteristics, intraoperative management and outcome were evaluated for associations with acute kidney injury using an univariate and multiple logistic regression model.P = 0.005).A total of 1597 patients were admitted to the PACU and of these, 1166 met the inclusion criteria. Eighty-seven patients (7.5%) met AKI criteria. Univariate analysis identified age, American Society of Anesthesiologists (ASA) physical status, emergency surgery, high risk surgery, ischemic heart disease, congestive heart disease and Revised Cardiac Risk Index (RCRI) score as independent preoperative determinants for AKI in the postoperative period. Multivariate analysis identified ASA physical status, RCRI score, high risk surgery and congestive heart disease as preoperative determinants for AKI in the postoperative period. Patients that developed AKI had higher Simplified Acute Physiology Score (SAPS) II and Acute Physiology and Chronic Health Evaluation (APACHE) II, higher PACU length of stay (LOS), higher PACU mortality, higher hospital mortality and higher mortality at 6 months follow-up. AKI was an independent risk factor for hospital mortality (OR 3.12, 95% CI 1.41 to 6.93, This study shows that age, emergency and high risk surgery, ischemic heart disease, congestive heart disease, ASA physical status and RCRI score were considered risk factors for the development of AKI, in patients needing intensive care after surgery. AKI has serious impact on PACU length of stay and mortality. AKI was an independent risk factor for hospital mortality. Acute kidney injury (AKI) is commonly seen in the perioperative period and in the intensive care unit (ICU). It is associated with a prolonged hospital stay and high morbidity and mortality -6.Acute renal failure is a complex disorder that occurs in a variety of settings with clinical manifestations ranging from a minimal elevation in serum creatinine to anuric renal failure.acute renal failure, renal insufficiency, kidney injury, and renal impairment, and various definitions have been used in previous publications. Furthermore the term acute kidney injury has been put forth as the preferred nomenclature to replace acute renal failure with the understanding that the spectrum of AKI is broad and includes different degrees of severity.To date, there is no universally accepted definition for acute kidney dysfunction. Varying terms, including We used the definition of AKI proposed by the Acute Kidney Injury Network (AKIN) that was formed to facilitate the development and execution of initiatives to ensure the best outcomes for patients with AKI . As an iAKI occurs in approximately 1 to 5% of all hospitalized patients and is increasingly prevalent . It is dAlthough predictors of postoperative acute renal failure after noncardiac surgery in patients with previously normal renal function had been studied previously , little The purpose of our study was to evaluate the incidence and determinants of the development of AKI in the immediate postoperative period in patients with previous normal renal function.The Institutional Review Board of the Hospital de São João approved the study and waived the requirement for informed consent for the retrospective review of medical records. This retrospective cohort study was carried out at the Hospital São João, a 1124-bed community teaching hospital in Porto, Portugal, in the multidisciplinary post-anesthesia care unit (PACU). The PACU includes a surgical ICU with five beds in which surgical critically ill patients are admitted and closely monitored and treated.All postoperative patients admitted to the surgical ICU area of the PACU, aged 18 years or more, who underwent scheduled or emergency noncardiac surgery between 1 March 2006 and 1 March 2008 with an overnight admission and more than 12 hours of PACU stay were eligible for the study. Patients were included only if a preoperative Scr within 30 days of the operative data was available. Patients readmitted during the study period were enrolled in relation to the time of their first admission. The PACU admits all surgical patients, with the exception of cardiothoracic patients.Exclusion criteria were pre-existing renal dysfunction requiring renal replacement therapy or a preoperative Scr higher than 1.6 mg/dL for men and 1.4 mg/dL for women. Preoperative Scr values were defined as the most recent Scr (mg/dL) measured within 30 days of the surgery.The primary outcome was development of AKI during PACU stay.Patients were classified as having AKI if they had an increment of Scr of 0.3 mg/dL or higher or 50% or more increase within any 48-hour interval and/or an episode of less than 0.5 mL/kg/hr urine output for more than six hours despite fluid challenge of 500 mL or more normal saline, when appropriate.The following variables were recorded on admission to the PACU: age, gender, body mass index (BMI), American Society of Anesthesiologists physical status (ASA-PS), preadmission comorbilities , duration of anesthesia, type of anesthesia, core temperature, and troponin I blood levels.Intraoperative data recorded for each case included administration of crystalloids, colloids, erythrocytes, and fresh frozen plasma.Intraoperative and PACU data were collected as well as hospital length of stay (LOS). Mortality was recorded for all patients. The Acute Physiology and Chronic Health Evaluation (APACHE) II and the Adapting a classification scheme developed by Lee and colleagues , we calcPatients' demographics, and intraoperative and postoperative data were collected.Physiologic data were recorded using customized data entry forms. Included was Scr that was recorded for each day and for PACU admission. These data were also recorded 24 hours before meeting criteria for AKI, at the time of AKI, 24 hours after AKI, and 48 hours or longer after AKI.PACU and hospital LOS were also recorded. For mortality we have registered PACU mortality, hospital mortality, and mortality at six months after PACU discharge.Descriptive analyses of variables were used to summarize data and the Mann-Whitney U test was used to compare continuous variables; Chi-squared or Fisher's exact test were used to compare proportions between two groups of subjects.P ≤ 0.002.To evaluate the determinants of postoperative AKI and to identify independent predictors of hospital mortality univariate analysis were performed using simple binary logistic regression with an odds ratio (OR) and 95% confidence interval (CI) with the following independent variables: age, gender, BMI, ASA-PS, type of surgery, comorbidities, RCRI score, type of anaesthesia, intraoperative fluid administration, troponin I blood levels at admission, length of anaesthesia, and temperature at admission . To reduce the risk of a type II error we have controlled the significance level for multiple comparisons applying the Bonferroni's correction for multiple comparisons (test-wise significance level divided by the number of tests performed). All variables were deemed to be significant if P ≤ 0.002 in the respective univariate analysis were entered (applying the Bonferroni's correction for multiple comparisons).Multiple regression binary logistic with forward conditional elimination was used to examine covariate effects of each factor on AKI and to identify independent predictors of hospital mortality. In these models covariates with a univariate Data were analyzed using SPSS for Windows version 16.0 .A total of 1597 patients were admitted to the PACU during the study period and 1166 patients met the inclusion criteria and were followed for the development of AKI after PACU admission. One-hundred and twenty-one patients were excluded because they had abnormal renal function preoperatively (defined as Scr higher than 1.6 mg/dL for men and 1.4 mg/dL for women); 196 were excluded because they stayed less than 12 hours and did not had an overnight admission in the PACU; 52 were excluded because they were less than 18 years of age; 44 were admitted more than once to the PACU and therefore excluded; 12 were excluded because they were not surgical patients; and 6 had no preoperative Scr measurement and were excluded.P = 0.002), had lower BMI , were more likely to have been submitted for general anesthesia , had higher troponin I at admission , were more likely to have been submitted to emergency surgery or high risk surgery , had more frequently ischemic heart disease and congestive heart disease , were more likely to be ASA-PS IV/V and have RCRI scores of more than 2 , and had higher volume of intraoperative fluids administered .The remaining 1166 were followed for the development of AKI after PACU admission. Eighty-seven (7.5%) developed AKI. The characteristics of patients with and without AKI are summarized in Table P < 0.001 and median APACHE II 12 versus 7, P < 0.001), and stayed longer in the PACU . For the six months follow-up we obtained the postoperative vital status of all patients. The unadjusted mortality rate at six months follow-up of patients with AKI was 36%, nearly four times the mortality rate of those without AKI . The increased mortality observed among patients with AKI was even greater for hospital mortality , and PACU mortality .Table P-values are summarized in Table P ≤ 0.002): age , emergency surgery , ASA-PS , high-risk surgery , ischemic heart disease , congestive heart disease , and RCRI score .Univariate analysis for determinants of AKI and their relevant P < 0.001 and OR 1.18, 95% CI 1.14 to 1.23, P < 0.001 respectively). The perioperative onset of AKI in patients with previously normal renal function was associated with significantly increased in PACU LOS , higher PACU mortality , higher hospital mortality and higher mortality at six months follow-up .Univariate analysis for severity of disease, LOS, and mortality are summarized in table P < 0.001, for ASA-PS IV/V patients), RCRI , high-risk surgery , and congestive heart disease .Multiple regression logistic analysis was used to examine covariate effects of each factor on AKI Table . This reP = 0.005) after adjustment for age, ASA-PS, high-risk surgery, congestive heart failure, emergency surgery, SAPS II, APACHE II, PACU LOS, RCRI, and AKI. Other independent predictors of hospital mortality were ASA-PS , high-risk surgery , congestive heart disease , and SAPS II .Multiple logistic regression analyses was used to examine covariate effects of each factor on hospital mortality Table . The regWe report the incidence of postoperative AKI among patients with normal preoperative renal function to be 7.5%. This incidence is consistent with that reported among general hospitalized patients, ranging from 1 to 5% . Acute rUnfortunately, acute renal failure is often unrecognized as definitions for the disease range from quantitative and qualitative alterations in Scr to alterations in urine output and dialysis requirement. The lack of a universally recognized definition of ARF has posed a significant limitation contributing to the lack of clinical success. Recognizing the need for uniform standards, the Acute Dialysis Quality Initiative group in 2002 proposed consensus recommendations of the Risk, Injury, Failure, Loss and End-stage kidney disease (RIFLE) criteria for the definition and staging of acute renal failure . The widPostoperative AKI remains a leading cause of morbidity, mortality, prolonged hospital stay, and increased hospital cost. In this study, we have attempted to correlate and predict factors predisposing to AKI. By doing so, one might be able to predict those at high risk and interventional measures might be planned in advance to improve outcome after such a complication, achieving a better outcome and a more efficient use of hospital and intensive care resources.We intentionally excluded patients with documented preoperatively raised Scr in order to predict AKI among patients with preoperatively normal renal function, as dictated by a Scr less than 1.6 mg/dL for men and 1.4 mg/dL for women.There have been a variety of predictive models developed to stratify risk in patients undergoing cardiac surgery ,28 and tIn his study with similar objectives but with different methodology Kheterpal and colleagues found seThe ASA-PS score, a preoperative evaluation used routinely for every patient, was never intended to be a perioperative risk score, but all large-scale studies have suggested that a high ASA-PS score is one of the best predictors of postoperative morbidity ,30. ThisThe RCRI is a prediction tool for major cardiac complications after noncardiac surgery ,31. It wIn fact, to assume that both cardiac and renal complications are based on ischemic injury to sensitive organs may be a plausible explanation for RCRI as an index. Even some of the individualized risk factors that compose it appears as a risk factor for postoperative renal failure.We noted congestive heart failure to be an independent predictor for hospital-acquired AKI as stated by Drawz and colleagues and thatAcute renal injury without the need for renal replacement therapy is associated with increased mortality in critically ill patients and in postoperative cardiac and noncardiac surgery ,13,33,34Mortality of patients with acute renal failure is high. In the study by Barrantes and colleagues , the autIn our study patients with AKI had significantly higher hospital mortality than patients without AKI. Even when controlled for other variables with a multiple variable regression analysis, AKI remains an independent risk factor for hospital mortality. This is consistent with previous data ,6,35.The study by Chertow and colleagues first deAs in the previous focused study by Barrantes and colleagues , patientWe should note some limitations to our analysis. First, our study was retrospective in nature; we were unable to identify and analyse all potential confounding factors. The data were collected as part of the delivered clinical cares. As a result, the data reflect the electronic medical record, and no additional detail is available. Documentation of urine output, the use of vasoactive substances and hemodynamic stability during surgery was less reliable. In our analysis we could only quantify volume of intraoperative fluid administration and we were unable to address the complete role of intravenous hydration in AKI because several data elements involved in the quantification of resuscitation volume were not accurately collected in the medical records. This might be an important aspect of pathophysiology for AKI development and could not be included in our analysis.In the study we do not analyse time points for AKI development and we did not make exclusion criteria to prevent the admission of patients with late development of AKI. With this approach we could have considered AKI that could have been due to complications other than surgery. We did not assess other potential consequential effects of AKI and the studied outcomes may not necessarily capture all relevant consequences of AKI. The mortality data are based on all-cause mortality and no additional detail regarding the cause of death are available for review. This may limit the ability to interpret our data on mortality.Using the interim consensus definition of AKI we could predict meaningful clinical outcomes and were able to identify risk factors for the development of AKI in patients needing intensive care after surgery.• AKI is commonly seen in the postoperative period after major surgery. In our study 7.5% of patients developed AKI after surgery.• Age, emergency surgery, ASA-PS, high-risk surgery, ischemic heart disease, congestive heart disease, and total RCRI score were considered independent predictors for the development of AKI.• Patients that developed AKI stayed longer in the PACU.• The perioperative onset of AKI was associated with significantly increased PACU LOS and higher mortality rates at the hospital and at six months follow-up.• AKI was an independent risk factor for hospital mortality.AKI: acute kidney injury; AKIN: Acute Kidney Injury Network; APACHE II: Acute Physiology and Chronic Health Evaluation; ASA-PS: American Society of Anesthesiologists physical status; BMI: body mass index; CI: confidence interval; ICU: intensive care unit; LOS: length of stay; OR: odds ratio; PACU: post anesthesia care unit; RCRI: Revised Cardiac Risk Index; RIFLE: Risk, Injury, Failure, Loss and End-stage kidney disease; SAPS: Simplified Acute Physiology Score; Scr: serum creatinine.The authors declare that they have no competing interests.FA participated in conception, design, acquisition of the data, analysis of the data, statistical analysis, critical revision of the manuscript, and supervision. MB and VF participated in conception, design, acquisition of the data, analysis of the data, and critical revision of the manuscript. HB was involved in drafting the manuscript, analysis of the data, and revising it critically for important content. All authors read and approved the final manuscript. |
Irregular uterine bleeding is the major side effect of, and cause for, discontinuation of long-term progestin-only contraceptives (LTPOCs). The endometria of LTPOC-treated women display abnormally enlarged, fragile blood vessels (BV), decreased endometrial blood flow and oxidative stress. However, obtaining sufficient, good quality tissues have precluded elucidation of the mechanisms underlying these morphological and functional vascular changes.The current study assessed the suitability of the guinea pig (GP) as a model for evaluating the uterine effects of LTPOC administration. Thus GPs were treated with a transdermal pellet for 21 days and examined for endometrial histology, angiogenic markers as well as markers of oxidative stress and apoptosis.We now demonstrate that GP uteri were enlarged by both estradiol (E2) and medroxyprogesterone acetate (MPA) (p < 0.001). Effects of MPA on uterine weight differed significantly depending on E2 levels (p < 0.001), where MPA opposed the E2 effect in combined treatments. Angiogenesis parameters were similarly impacted upon: MPA alone increased BV density (p = 0.036) and BV average area (p = 0.002). The presence of E2 significantly decreased these parameters. These changes were associated with highly elevated of the lipid peroxidation product, 8-isoprostane (8-isoP) content in E2+MPA-treated and by nuclear 8-OH-deoxyguanosine (8oxoG) staining compared to all other groups (p < 0.001). Abnormalities in the E2+MPA group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased apoptosis as observed by a marked increase in TUNEL labeling.in vivo change observed in the human uterus following LTPOC administration. Hence, the GP is an excellent model for the study of LTPOC effects on the uterus and will be extremely useful in determining the mechanistic pathways involved in this process which cannot be conducted on humans.LTPOC exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. These findings are remarkably similar to Because of their safety and efficacy, long-term progestin-only contraceptives (LTPOCs) are well-suited for women with restricted access to health care or in whom estrogen containing contraceptives are contraindicated. Unfortunately, administration of LTPOCs leads to irregular uterine bleeding in the majority of users ,2. Such Endometria from LTPOC-treated patients display dilated, thin walled, fragile vessels that are irregularly distributed across the endometrial surface -5. PreviWhile past studies produced descriptive information regarding the possible causes of abnormal uterine bleeding following LTPOC treatment, considerations of difficulty in attaining good quality tissues from humans preclude functional studies of the mechanistic pathways involved in this process. Poor understanding of the mechanisms underlying bleeding has limited the development of effective therapies for abnormal bleeding with LTPOC. To further understand these mechanisms, we determined whether the guinea pig (GP) was a relevant model to study the uterine effects of LTPOC administration. The GP was chosen because its endometria display functional estrogen and progesterone receptors as well Eighteen nulliparous female GPs, aged 2-6 months, were subjected to bilateral oopherectomy and then given subcutaneous implants of 50 mg medroxyprogesterone acetate (MPA)-cholesterol-based 21 day time-release pellets or 5 mg estradiol (E2) cholesterol based 21 day time-release pellets or both. Thus, animals received the treatment as follows: MPA (n = 6), E2 (n = 6), E2+MPA (n = 3) or placebo (n = 3). After three weeks, hysterectomy was performed and the right uterine horn was formalin fixed whereas the left horn was snap frozen for subsequent studies. These studies were approved by both Charles River and Yale University IACUC offices.The specimens were weighed and then formalin fixed, and paraffin embedded. Five micron sections were cut and stained with Hematoxylin-Eosin or Trichrome Mason by conventional histological procedures as described or used Sections were stained for von Willebrand factor (vWF) with the AB6994 primary antibody at 1-10,000 dilution or 8-oxoG with the x 24326 primary antibody at 1-100 . For negative controls, normal IgG isotypes which were derived from the animals from which the antibodies were prepared and used at the same concentrations as the primary antibody. The sections were washed and the appropriate secondary biotinylated antibody was added per the manufacturer's instructions.The antigen-antibody complex was detected with 3,3'-diaminobenzidine with or without nickel sulfate as the chromogen solution (Vector Laboratories). When nickel sulfate was used, no hematoxylin counterstaining was conducted. For each condition, 3 different slides were assessed and at least three independent areas of each slide photographed. For vWF, 6 randomly selected fields were digitally captured at 200× magnification using an Olympus microscope with digital camera. Vessel size, density and heterogeneity were measured in each field by computerized selection of the stained vessels with the public domain image analysis software Image J as previously described by others ,22. All The isoprostanes are a family of eicosanoids of non-enzymatic origin produced by the random oxidation of tissue phospholipids by oxygen radicals . The levin situ. To assess differences in apoptotic levels, photographs were taken from 3 representative areas of each slide at x200 magnitude under identical camera settings. The slides were analyzed with Image J by setting the minimum threshold that allowed for the visualization of the stained nuclei only. This threshold was maintained throughout the analysis of all subsequent slides. All values were subjected to analysis with Sigma Stat utilizing the recommended ANOVA provided by the program based on sample distribution. The mean particle stained average +/- standard error of the mean were then represented by bar graphs.Assessment of apoptosis was conducted on formalin-fixed, paraffin-embedded tissues with ApoTag peroxidase labeling kit as per the manufacturer's instructions. The procedure is based on detection of free 3'OH DNA termini Statistical analysis was conducted by ANOVA using the Sigma Stat Program .Figure Histological analysis of the samples was conducted after fixation and tissues were cut at 5 μ. Samples were stained with H&E or Mason Trichrome Figure . Sub-endFigure Thus, angiogenic parameters were impacted upon as follows: MPA alone increased BV density (p = 0.036) and BV average area (p = 0.002). The presence of E2 significantly decreased these parameters .Levels of 8-isoprostane (8-IsoP) production were evaluated in endometrial extracts obtained from uteri treated with the various steroids. Figure Expression of 8-oxoG was significantly higher in E2+MPA treated groups compared to E2 alone. A significant, though diminished effect was observed with MPA alone. However, no statistical differences were observed between E2 and the placebo control.In humans, LTPOC treatment results in enhanced apoptosis of the endometrial glands and stroma . Figure Endometria from LTPOC-treated patients display dilated vessels that are irregularly distributed across the endometrial surface -5. TheseThese studies demonstrate that treatment of guinea pigs with progestin and in particular with E2+MPA resulted in changes in endometrial vascular morphology, as well as increased markers of apoptosis and oxidative stress similar to that observed in human LTPOC-treated endometria. In humans, LTPOC treatment occurs in the setting of continuous low-level ovarian-derived estrogen production. Since the GPs used in the current study are ovariectomized, the E2+MPA treated animals are likely to most closely resemble LTPOC-treated humans.In prior studies, we have demonstrated that LTPOC results in both reduced endometrial blood flow and increased oxidative stress ,6. TheseThe authors declare that they have no competing interests.GK and CL conceived and designed the experiments and wrote the manuscript. IB, FS and LB carried out the ELISAs and immunohistochemical procedures and statistical analysis. MH contributed in the critical analysis of the paper. |
Light and dark patterns are the major synchronizer of circadian rhythms to the 24-hour solar day. Disruption of circadian rhythms has been associated with a variety of maladies. Ecological studies of human exposures to light are virtually nonexistent, however, making it difficult to determine if, in fact, light-induced circadian disruption directly affects human health.A newly developed field measurement device recorded circadian light exposures and activity from day-shift and rotating-shift nurses. Circadian disruption defined in terms of behavioral entrainment was quantified for these two groups using phasor analyses of the circular cross-correlations between light exposure and activity. Circadian disruption also was determined for rats subjected to a consistent 12-hour light/12-hour dark pattern (12L:12D) and ones subjected to a "jet-lagged" schedule.Day-shift nurses and rats exposed to the consistent light-dark pattern exhibited pronounced similarities in their circular cross-correlation functions and 24-hour phasor representations except for an approximate 12-hour phase difference between species. The phase difference reflects the diurnal versus nocturnal behavior of humans versus rodents. Phase differences within species likely reflect chronotype differences among individuals. Rotating-shift nurses and rats subjected to the "jet-lagged" schedule exhibited significant reductions in phasor magnitudes compared to the day-shift nurses and the 12L:12D rats. The reductions in the 24-hour phasor magnitudes indicate a loss of behavioral entrainment compared to the nurses and the rats with regular light-dark exposure patterns.This paper provides a quantitative foundation for systematically studying the impact of light-induced circadian disruption in humans and in animal models. Ecological light and activity data are needed to develop the essential insights into circadian entrainment/disruption actually experienced by modern people. These data can now be obtained and analyzed to reveal the interrelationship between actual light exposures and markers of circadian rhythm such as rest-activity patterns, core body temperature, and melatonin synthesis. Moreover, it should now be possible to bridge ecological studies of circadian disruption in humans to parametric studies of the relationships between circadian disruption and health outcomes using animal models. As the earth rotates, all species on the surface of the planet are exposed to 24-hour patterns of light and darkness. In response to these regular, daily oscillations to the natural light-dark cycle, these species have evolved endogenous circadian rhythms that repeat approximately every 24 hours ,2. ExampMaintaining the phase-relation ordering of the various circadian rhythms from molecular to behavioral levels appears to be crucial for coordinated functions throughout the human body. Lack of synchrony between the master clock and the peripheral clocks can lead to asynchronies within cells and between organ systems . This breakdown in synchrony, as demonstrated most profoundly with jet lag, disrupts sleep , digestiactually experienced by people . Tw. Tw20]. in vivo, and thus it is impossible to measure entrainment in the purest sense in living and active humans; the term "behavioral entrainment" is used in this paper to describe the observed levels of synchrony between light-dark exposures and activity-rest responses as measured by the Daysimeter.It should be emphasized that activity as measured by the Daysimeter is not a direct measure of the endogenous clock in the SCN. Like every downstream measure of circadian function, behavior can only yield partial insight into circadian entrainment. It is presently impossible to directly measure SCN activity The Daysimeter was sent to nurses throughout the United States to measure their actual CS exposures and activity for seven consecutive days. Forty-three pre-menopausal female nurses, both day-shift (n = 32) and rotating-shift nurses (n = 11), participated in the study. They wore the Daysimeter for seven consecutive days and were scheduled to work at least two and no more than three consecutive days during that period. The Daysimeter was worn while nurses were awake. The nurses were instructed to place the Daysimeter next to them when they slept or bathed. After the seven-day recording session, they returned the device for data analyses. In addition to wearing the Daysimeter, participating nurses provided urine samples, obtained every four hours, for subsequent melatonin assay and filled out a chronotype questionnaire [Horne-Östberg Morningness-Eveningness Questionnaire (MEQ)] and a lighting survey. The nurses were also asked to keep a sleep log, writing down the times they went to bed and any other information about their sleep schedules. These sleep logs were used to match the exact time nurses started wearing the device. Presented here are only the Daysimeter data.Rattus norvegicus) were housed in individual cages illuminated by a lighting system previously developed by Bullough et al. [murine). Based upon the mouse phase response curve (PRC) obtained in that study, a spectral power distribution and irradiance (approximately 5 μW/cm2 on the cage floor) were selected to provide the light stimulus to the Sprague-Dawley rats. This particular light stimulus for nocturnal rodents was estimated to be above threshold and below saturation for stimulation of the rat circadian system. The light stimulus for the rats was precisely controlled using a light-emitting diode (LED) light-delivery system fabricated and installed in every cage. The light-delivery system provided better controlled and more biologically meaningful circadian light stimulation to the rats than the fluorescent ceiling lighting traditionally used to provide bright, ambient illumination throughout an animal colony [Forty albino female Sprague-Dawley rats followed by 12 hours of darkness (12D), and another 20 rats (the "jet-lagged" group) were exposed to a 12L:12D pattern where the phase of the light-dark cycle was reversed every 48 hours (as if this group of rats instantly travelled back and forth from Asia to the Americas every other day). Animals were housed individually and allowed to eat and drink Wheel running was measured continuously throughout the experimental session and used as the measure of activity-rest in these animals. The accumulated number of wheel revolutions was recorded at 10-minute time intervals. At the start of the experiment, the photoperiods for both groups were in phase with each other, and the animals exhibited typical nocturnal behavior . To allow for acclimation to the cages and to the lighting by the rats, wheel-running data were not collected until the third day of the study, by which time the photoperiod for the "jet-lagged" group had reversed. Most of the activity in the "jet-lagged" group on that day occurred during the light phase. As shown below, the animals in this group were unable to entrain to the regularly reversing photoperiod and exhibited behavior similar to free-running, with similar amounts of activity in the light and in the dark throughout the eight-day observation period.Figure Figures Examination of Figure Although many analyses of the activity and of the transformed CS data are possible, the data in Figure Figure The wheel running data from the 12L:12D . Conversely, greater amounts of activity near the offset of circadian light exposure than near the onset of circadian light exposure produces a phasor extending above the zero-phase line. Researchers have useThe rats exposed to the consistent 12L:12D light-dark cycle produced average (n = 8) phasors with magnitudes similar to the day-shift nurses, but with directions to the left, clustered around a 12-hour phase shift between light and activity, as would be expected for an entrained nocturnal animal Figure . The "jeboth human and animal models.Figure Considering only the degree of behavioral entrainment, Figure zeitgebers, or time givers [zeitgeber.The interdaily stability (IS) and the intradaily variability (IV) statistics have beee givers -32. Unlizeitgeber for entrainment, that is, light.It is possible, however, to compare phasor magnitudes Figure and IS vThe IV statistic was also calculated from the activity data from nurses and rats, but the values showed no significant difference between the two groups of nurses nor between the two groups of rats; the mean IV values for day-shift and rotating-shift nurses were 0.50 and 0.54 respectively with standard deviations of 0.20 and 0.16 respectively, and the mean values for the entrained and "jet-lagged" rats were 1.10 and 1.21 with standard deviations of 0.28 and 0.27 respectively. This lack of separation in IV values for the two groups of nurses and for the two groups of rats suggests that consolidation of activity patterns is not systematically related to the degree of circadian behavioral entrainment as measured either with IS values or with phasor magnitudes.This paper provides a new framework for the study of the effects of circadian entrainment/disruption on human health, emphasizing three important links in the logical chain relating circadian disruption to maladies such as breast cancer, obesity, and sleep disorders .First, circadian light (and dark) for humans and for animal models can now be quantitatively defined to such a degree that meaningful studies of light as a stimulus for circadian disruption can be undertaken, not only in humans but in nocturnal rodents as well. Without quantitative definitions of the light stimuli, it would simply be impossible to understand the results of any ecological study of circadian disruption on human health or how laboratory studies using animal models relate to the human condition. Second, with an understanding of circadian light, it is now possible to measure the synchrony between light-dark and activity-rest patterns in actual human living environments using tools like the Daysimeter . These eIt should be emphasized, too, that the methods presented here are not limited to the study of behavioral entrainment. Rather, this analysis provides the basis for assessing entrainment of other outcome measures from the circadian system, such as core body temperature or melatonin synthesis, to light-dark patterns. From these envisioned studies, modern maladies like diabetes, obesity, and poor sleep, as well as breast cancer and cardiovascular disease, can be meaningfully and systematically investigated. More important perhaps, forging the links identified in this paper will significantly accelerate a deeper understanding of the role of circadian disruption on human health and therThe authors declare that they have no competing interests.MSR conceived the study, lead the team in its execution and drafted major sections of the paper, AB formulated the analyses and drafted portions of the Results and Discussion sections, MGF was instrumental in acquiring the nurse data, drafted sections of the paper and provided expertise while preparing the manuscript, JDB was instrumental in acquiring the rat data and provided expertise while preparing the manuscript. All authors participated equally in discussions and the exchange of ideas during the study, and all reviewed and approved the final manuscript. |
The animals were subjected to intraarticular injection of anti-bovine sertLm albumin (BSA) IgG antibodies followed by i.v. injection of BSA. Histopathological analysis of the synovial membranes disclosed infiltration of polymorphonuclear (PMN) cells and vascular congestion. Slight increase in vascular permeability, measured by Evans blue dye extravasation into the joints, was detected after 3 h of arthritis. Cellular influx into the articular cavities was most evident at the sixth hour of arthritis with predominance of PMN. Pretreatment with either indomethacin, a cyclooxygenase inhibitor, or L-660,711, a peptido-leukotriene antagonist, did not inhibit cell infiltration, whereas pretreatment with either L-663,536, a 5-lipoxygenase inhibitor, or L-655,240, a thromboxane antagonist, significantly inhibited the phenomenon. Pretreatment with WEB 2170, a platelet activating factor (PAF) antagonist, also significantly inhibited cell influx. These results suggest that thromboxane, LTB |
The X-ray structure showed that this product represents a trans isomer with respect to the amino and hydroxy substituents in the cyclohexyl ring; the dihedral angle between the aminopyrimidine plane and the mirror plane of the substituted cyclohexyl fragment is 33.6 (3)°. Only two of the four potentially ‘active’ H atoms participate in intermolecular N—H⋯O and O—H⋯N hydrogen bonds, linking the molecules into layers parallel to the (10The title compound, C N-pyrimidine derivative of aminocyclohexane, see Melguizo et al. Å b = 7.1879 (11) Å c = 19.566 (4) Å β = 91.053 (3)°V = 1399.3 (4) Å3 Z = 4 Kα radiationMo −1 μ = 2.93 mmT = 198 K 0.10 × 0.10 × 0.08 mm Siemens P4 with APEX CCD area-detector diffractometerSADABS; Bruker, 2001T min = 0.758, T max = 0.799Absorption correction: multi-scan (8853 measured reflections3251 independent reflectionsI > 2σ(I)2500 reflections with R int = 0.030 R[F 2 > 2σ(F 2)] = 0.041 wR(F 2) = 0.114 S = 1.05 3251 reflections166 parametersH-atom parameters constrainedmax = 0.83 e Å−3 Δρmin = −0.77 e Å−3 Δρ SMART used to solve structure: SIR2004 (Burla et al., 2005SHELXL97 (Sheldrick, 2008ORTEP-32 (Farrugia, 1997WinGX (Farrugia, 1999Data collection: 10.1107/S1600536809035533/bg2293sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809035533/bg2293Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: |
HCP5, HLA-C and ZNRD1 confer restriction against HIV-1 viral replication or disease progression. Here, we also find that these alleles are associated with different aspects of HIV disease, albeit mainly in European Americans. Additionally, we offer that because the GWAS cohort was a subset of HIV-positive individuals, selected based in part on having a low viral load, the observed associations for viral load are magnified compared with those we detect in a large well-characterized prospective natural history cohort of HIV-1-infected persons. We also find that because of linkage disequilibrium (LD) patterns, the dominant viral load- and disease-influencing associations for the ZNRD1 or HLA-C and HCP5 alleles are apparent mainly when these alleles are present in HLA-A10- or HLA-B*57-containing haplotypes, respectively. ZNRD1 alleles lacking HLA-A10 did not confer disease protection whereas ZNRD1-A10 haplotypes did. When examined in isolation, the HCP5-G allele associates with a slow disease course and lower viral loads. However, in multivariate models, after partitioning out the protective effects of B*57, the HCP5-G allele associates with disease-acceleration and enhanced viral replication; these associations for HCP5-G are otherwise obscured because of the very strong LD between this allele and a subset of protective B*57 alleles. Furthermore, HCP5 and HLA-C alleles stratify B*57-containing genotypes into those that associate with either striking disease retardation or progressive disease, providing one explanation for the long-standing conundrum of why some HLA-B*57-carrying individuals are long-term non-progressors, whereas others exhibit progressive disease. Collectively, these data generally underscore the strong dependence of genotype-phenotype relationships upon cohort design, phenotype selection, LD patterns and populations studied. They specifically demonstrate that the influence of ZNRD1 alleles on disease progression rates are attributable to HLA-A10, help clarify the relationship between the HCP5, HLA-C and HLA-B*57 alleles, and reaffirm a critical role of HLA-B*57 alleles in HIV disease. Furthermore, as the protective B*57-containing genotypes convey striking salutary effects independent of their strong impact on viral control, it is conceivable that T cell-based therapeutic vaccine strategies aimed at reducing viral loads may be inadequate for limiting AIDS progression, raising the potential need for complementary strategies that target viral load-independent determinants of pathogenesis.A recent genome-wide association study (GWAS) suggested that polymorphisms in or around the genes However, this is proving to be an immensely complex task because it is difficult to predict which immunological response(s) in candidate vaccine-challenged animals or HLA) genes, which reside in the Major Histocompatibility Complex (MHC) locus, may be especially informative HLA alleles are critical determinants of HIV-AIDS pathogenesis HLA-B*57 is among the most intensively studied alleles in the HIV-AIDS field as it is among the most protective HLA class I alleles. In multiple cohorts of HIV-positive individuals possession of a B*57 allele is associated with a slower rate of disease progression and strong virologic control HLA class I alleles might have benefit in terms of reducing population-level viral load, and consequently, as modeling studies suggest, the pace of the epidemic In this quest to identify the correlates of protection that might have relevance for vaccine development, the immunological features linked to alleles of class I Human Leukocyte Antigen because nearly 50% of the top 50 single nucleotide polymorphisms (SNPs) that associated with virologic control were in or around the MHC locus at 6p21.3 + individual of European descent achieving a steady-state viral load or having a stable low viral load , without knowledge of the date of seroconversion HLA-B*57: the first was a SNP in an endogenous retroviral element designated as HLA complex P5 (HCP5), and the second SNP was in the 5′-region of the HLA-C gene RNF39) and zinc ribbon domain–containing 1 (ZNRD1), which are in close proximity to the HLA-A locus The importance of HLA-C and ZNRD1 are key independent host determinants associated with control of HIV replication and disease progression, respectively, and (ii) HCP5 may contribute positively to the well-described protective effects attributed to HLA-B*57, as this allele was found to be in nearly 100% linkage disequilibrium (LD) with the HLA-B*5701 allele. This GWAS clearly represents an advance for the HIV-AIDS field, and the results served as a fulcrum to probe further the importance of the MHC locus in HIV-AIDS pathogenesis. To understand further the role of HLA alleles in AIDS pathogenesis we reflected on the following three points.Thus, the main inferences of this GWAS were: (i) HCP5 and HLA-C accounted for ∼9.6% and ∼6.5% of the variability in steady-state viral load, respectively HLA-C allele and viral load exceeded the threshold for statistical significance imposed by a GWAS, but the association of this allele for disease progression was not statistically significant HCP5 was not identified in the GWAS screen for disease progression, despite being in nearly 100% LD with the HLA-B*5701 allele, which in multiple cohorts has been associated with better virologic control, as well as a slower rate of disease progression HLA-C and HCP5 for viral load, but not for disease progression) suggested that perhaps epidemiological considerations, such as the combined analyses of subjects who achieve steady-state viral load and HIV controllers First, the SNPs in HLA alleles HCP5, HLA-C and ZNRD1) were due, in part, to their LD with HLA class I alleles that affect viral load and/or disease course. Third, we and others have shown that the host genetic determinants of HIV-AIDS pathogenesis may be population-specific Second, there is a high degree of LD between SNPs in the MHC locus and HCP5, HLA-C and ZNRD1 before and after accounting for patterns of LD with HLA alleles that were in close proximity to these SNPs in the entire cohort, and separately in subjects of European and African descent.For these three reasons, we sought to achieve greater clarity regarding the phenotypic effects associated with the SNPs in the three genes identified in the GWAS on steady-state viral load and disease progression rates. We conducted these analyses in a large, well-characterized U.S.-based natural history cohort of HIV-1-positive individuals, whose clinical characteristics have been described before + and HIV− subjects in the Wilford Hall Medical Center (WHMC) cohort for the SNP in HCP5 , the 5′-region of the HLA-C gene , as well as for the seven SNPs in or near the ZNRD1 and RNF39 genes . Hence, the results reported here are for a single SNP near ZNRD1 with rs2301751 by the HapMap project in Utah residents of European descent. Accordingly, we found a very strong LD between the ZNRD1 SNP with not only HLA-A*26, but also with A*25, which along with HLA-A*34 and HLA-A*66 make up the serogroup HLA-A10, bottom). Among the WHMC HIV+ subjects, the HLA-A10 group was composed mainly of HLA-A*25 and HLA-A*26 alleles in European Americans (EAs), whereas HLA-A*34 and HLA-A*66 alleles were more common in African Americans . This difference in the distribution of alleles that belong to the HLA-A10 serogroup across the two major ethnic groups in the WHMC HIV+ cohort was statistically significant .The spatial proximity between the HCP5 SNP and B*5701 . Moreover, there was a high degree of LD between the HCP5-G, HLA-C5′-C and HLA-B*57 alleles, and the HLA-C5′-C/HLA-B*57/HCP5-G-containing haplotype was found almost exclusively in European Americans .Consistent with the results of the GWAS ZNRD1 or HCP5 and HLA-C were independent of HLA-A10 and HLA-B*57, respectively.Because of the observed LD patterns, in the analyses described below, we examined whether the influence on disease course of the SNPs in + subjects, heterozygosity but not homozygosity for the ZNRD1-C allele was associated with disease-retardation . These results are consistent with the observations by Fellay et al ZNRD1-C allele is associated with a slower disease course in subjects of European descent. However, we also found that this allele does not influence HIV disease course in HIV-positive African Americans in the WHMC cohort.In the entire cohort of WHMC HIVZNRD1-C allele were due to those associated with alleles categorized to the HLA-A10 serogroup for three reasons. First, the ZNRD1-C allele was in high LD with HLA-A10 . Notably, the strength of the disease-retarding effects associated with HLA-A10 were similar before (RH = 0.62) and after (RH = 0.44) adjustment for explanatory covariates that in previous studies we had found influenced disease progression in this cohort = 0.028–0.93; P = 0.028 for comparison of those possessing and lacking HLA-A10; HLA-A10 and ZNRD1-C each had independent effects on disease, then the protective effects of the ZNRD1-C allele that are not because of its LD with HLA-A10 should be evident in those 85 individuals who possessed at least one ZNRD1-C allele but lacked alleles corresponding to A10 serogroup. For these reasons, we stratified HIV+ EAs into six genotypic groups based on their ZNRD1-C allele and/or HLA-A10 status . This suggested that in itself the ZNRD1-C allele was not associated with disease-retarding effects in the HIV+ EAs we examined had a slower rate of disease progression (P = 0.004) or group 2 (P = 0.057)). Additionally, although the number of subjects was small, those who were HLA-A10+ but lacked the ZNRD1-C allele (group 4) also had a slower disease course that was comparable to those in group 5 who were A10+ but possessed a ZNRD1-C allele than those who had one allele each of ZNRD1-C and HLA-A10 .Subjects possessing one ZNRD1-C suggested that it might be associated with a rapid rate of disease progression (P = 0.013). Although there were few subjects who were homozygous for the ZNRD1-C allele with (group 6) or without HLA-A10 (group 3), it was interesting to note that subjects assigned to either of these two groups had a progressive disease course and HLA-A10 associated with a reduced rate of disease progression but not the ZNRD1-C allele was associated with a reduced rate of disease progression . However, we found that subjects who had one ZNRD1-C allele but lacked an HLA-A10 allele (group 2) were similar to those who lacked both ZNRD1-C and HLA-A10 alleles (group 1) with respect to their baseline, cumulative and nadir CD4+ T cell counts, as well as steady-state viral load and delayed type hypersensitivity skin test reactions (a measure of cell mediated immunity HLA-A10/ZNRD1-C-containing haplotype) were also similar to those in group 1 with respect to most of these laboratory and immunologic characteristics, except those in group 5 had lower cumulative CD4+ T cell counts, a parameter that reflects the amount of CD4+ T cell loss over disease course and is a parameter that is highly correlated with development of AIDS as a phenotypic endpoint. Therefore, it was plausible that although the ZNRD1-C allele and steady-state viral load, we determined the influence of ZNRD1-C and HLA-A10 on viral load using univariate and multivariate linear regression analyses. In these analyses, the alleles were considered as independent variables and the steady-state viral load was considered as the dependent variable. These analyses revealed that the impact of the ZNRD1 allele on viral load was minimal and HLA-A10 had no influence on viral load the previously reported dominant protective role of B*57 alleles in virologic control and disease progression, and (iii) the high degree of LD between the HLA-C5′-C, HLA-B*57 and HCP5-G alleles their proximity to alleles , top, we alleles Figure 3B*57 allele were each associated with significant protective effects on the rate of disease progression to AIDS. The impact of the HLA-C5′-C allele on disease progression was greater in EAs than AAs .In univariate analyses, possession of a HLA-B*57 allele, including the B*5701 allele . However, the statistical significance of the protective effects associated with HLA-C5′-C in multivariate models were slightly lower than those observed in univariate models . This was most prominent for homozygosity for HLA-C5′-C as the hazard ratios for the rate of progression to AIDS were 0.66 (P = 0.025) and 0.72 (P = 0.067) in univariate and multivariate analyses, respectively , suggesting that this allele might convey weak disease-retarding effects , whereas when examined in the context of a multivariate model with HCP5-G, the hazard ratio was lower (RH = 0.25) and the strength of the association increased . When HLA-C5′-C heterozygosity and homozygosity were also added to the multivariate model .In univariate analysis, the hazard ratio for the rate of disease progression to AIDS associated with the HCP5-G allele in the univariate and multivariate analyses, and the accentuation of the protective effects of the B*57 in the multivariate models, convey the following: when examined at the haplotype level, those haplotypes which contain both B*57 and HCP5-G alleles are associated with a faster rate of disease progression compared to those B*57-containing haplotypes that bear the HCP5-T allele. Consequently, in multivariate models when the overall beneficial effects of B*57 are partitioned out or accounted for, the HCP5-G allele, which is in nearly 100% LD with a subset of HLA-B*57 alleles, associates with disease-acceleration. Conversely, in multivariate models when the effects of B*57/HCP5-G haplotypes are partitioned out, the overall beneficial effects associated with B*57 are accentuated. Concordant results (and inferences) were obtained when we studied the association of the HLA-C5′–HLA-B–HCP5 haplotypes shown in We inferred that the opposing direction of the disease-influencing effects associated with the HLA-B*57, HCP5 and HLA-C5′ alleles on rates of disease progression are similar to those observed when the steady-state viral load is used as a phenotypic endpoint. The hierarchal order of the extent to which the B*57, HLA-C and HCP5 alleles explained the variability in the steady-state viral load in the WHMC HIV+ cohort >HLA-C >HCP5 . These findings indicated that at least within the context of a natural history cohort, the SNPs in HLA-C and HCP5 explained only one-third to one-tenth of the variability in steady-state viral load than the B*57 allele, and several fold less than the reported estimates in the GWAS We next examined whether the direction and the relative strength of the effects observed for the HLA-B*57 allele, (ii) one (heterozygosity) or two (homozygosity) copies of the HLA-C5′-C allele, and (iii) a HCP5-G allele each associated with a lower steady-state viral load . However, the viral load-mitigating effects observed for possession of the HCP5-G allele in the univariate analysis was not evident when this allele was examined in a multivariate context with B*57 alone , or with the B*57 allele and HLA-C5′ genotypes together . Thus, the opposing direction of the viral load-influencing effects of the HCP5-G allele observed in the univariate , but also a higher viral load a HLA-B*57, HLA-C5′ and HCP5 alleles together explained 4.66% of the variability in the steady-state viral load in the WHMC HIV+ cohort .Analyses of the final multivariate regression model also revealed that the B*57 and conferred differential rates of disease progression or homozygous (group 6) for the HLA-C5′-C allele had comparable rates of disease progression . Thus, the genotype-phenotype relationship for group 2A may explain why when examined in a multivariate model, possession of the HCP5-G allele was associated with both disease acceleration and higher viral loads but not two (group 3) HLA-C5′-C alleles .These survival curves also underscore two other points: First, a B*57 have a progressive disease course HLA-B*57-containing genotypes classify into two categories.There is clinical, virologic and immunologic data indicating a conundrum: some subjects despite possessing a ‘protective’ , B and C), and collectively these genotypes are designated here as B*57-NPD genotypes in B*57-NPD genotypes conferred a nearly 83% slower rate of disease progression compared to genotypes that lack a B*57 allele course or when the analyses were restricted to subjects who did not receive HAART .The second group are those subjects categorized to group 2A and, compared to those with + T cell profiles and DTH skin test reactivity) and virologic profiles would reveal a step-wise worsening in subjects categorized to B*57-NPD, B*57-PD and those lacking a B*57 allele. In general agreement with this, as a general rule, there was a step-wise decrease in the baseline, cumulative and nadir CD4+ T cell counts as well as DTH responses in subjects assigned to B*57-NPD, B*57-PD and those lacking B*57 had a progressive disease course whereas most of those who bear two copies of the HLA-C5′-C allele have a nonprogressive disease course. Fourth, after partitioning out the protective effects of B*57, the HCP5-G allele was associated with disease-acceleration and enhanced viral replication, and these associations for HCP5-G were otherwise masked because of its LD with the protective B*57 alleles. Fifth, ZNRD1 alleles influenced disease course without impacting on the viral load. Sixth, the influence of the HLA-C on the steady-state viral load was much lower than that of HLA-B*57 alleles, and the extent of variability in steady-state viral load that can be explained by HLA-C alleles independent of HLA-B*57 was significantly lower than that reported previously B*57-containing genotypes could be stratified into two categories, one of which associated with striking disease retardation, the other with a disease course that is similar to that observed in individuals who lack a B*57 allele. Finally, we found that B*57-containing genotypes that associate with a slower disease course influenced progression rates by impacting both the viral load as well as parameters that are independent of the viral load. Below, we discuss the seven major implications of these findings within the context of how they apply to AIDS pathogenesis, vaccine development, and genotype-phenotype association studies.The salient genetic-epidemiologic findings of this study conducted in a natural history cohort of HIV-1 subjects are as follows. First, because of their LD patterns, the associations for ZNRD1 in HIV pathogenesis. Using several different analytical approaches we find that in the European American component of the HIV+ cohort we studied, associations of the alleles in or around ZNRD1 or RNF39 with disease progression are not attributable to an independent effect of these alleles on HIV disease course, but rather to their strong LD with HLA-A10 alleles. In the cohort studied, there was a higher proportion of subjects who had ZNRD1-containing haplotypes that lacked HLA-A10 than individuals with ZNRD1-C/A10-containing haplotypes. Despite this, ZNRD1-C/A10-lacking haplotypes were not associated with a slower rate of disease progression whereas the ZNRD1-C/A10-containing haplotypes were. Additionally, HLA-A10-containing genotypes that lacked ZNRD1 alleles were also associated with a slower disease course. Collectively, these findings suggest that HLA-A10 may be an important determinant of the rate of HIV disease progression in European Americans. Previous association studies also suggest that serogroup HLA-A10 may convey a protective effect HLA-A*25 (a subtype of A10) is known to present gag epitopes HLA-A*26 (a subtype of A10) restricted gag responses have been also reported HLA-A10 carriers elicit protective immune responses against key HIV-1-derived peptides, and consideration of this possibility might have value for understanding the host factors that influence AIDS pathogenesis and for vaccine development. Nevertheless, future studies are clearly warranted to clarify the role of ZNRD1 in AIDS pathogenesis as it was among the more than 250 candidate genes identified by a large-scale siRNA screen used to identify host factors required by HIV-1 First, our results provide a basis to clarify the role of HCP5 and HLA-C. (i) In multivariate models, after accounting for the protective effects of HLA-B*57 alleles, we find that the HCP5-G appears to have detrimental effects on both viral load and disease progression. Thus, we surmise that when examined in isolation the true phenotypic effects associated with the HCP5-G allele might be obscured because of its strong LD with protective B*57 alleles. We propose that the protective associations detected previously for the HCP5-G allele B*57-containing genotypes, i.e., those that are also homozygous for the HLA-C5′-C allele We suggest that the differences in the extent to which the HLA-C5′ and HCP5 alleles explained the variability in the steady-state viral load detected in a natural history cohort versus that observed in the GWAS might relate to epidemiological considerations. The combined analysis of subjects who achieved a steady-state viral load and those who had virologic characteristics similar to those of spontaneous HIV controllers HLA-C5′ and HCP5 alleles, yet these alleles were not among those detected when using disease progression as a phenotypic endpoint HLA-C5′ and HCP5 alleles and restriction of viral replication Second, our results may provide a basis to clarify the disease- or viral load-influencing effects of the SNPs in HLA haplotypes that have relevance to HIV-AIDS have been identified HLA alleles might provide a basis for why a large proportion of the SNPs identified in the GWAS were in the MHC locus, including additional HCP5 and HLA-C SNPs HLA-B*57 and other HLA alleles play pathogenic roles. For example, a recent GWAS implicated the HCP5-G allele as a determinant of psoriasis Third, the LD patterns detected herein are not completely unanticipated because the MHC locus is known to have small recombination rates and a high degree of LD, hence, increasing the number of extended haplotypic blocks in this genomic area B*57-carrying individuals have a progressive disease course whereas others exhibit a strikingly slow clinical course B*57 allele confers strong protection against early viral replication and disease progression. However, the studies especially of Migueles et al + subjects who possess a B*57 allele, including those possessing B*5701 can have a progressive disease course. A very striking example of this conundrum was reported recently by Bailey et al who studied a HIV-1 transmission pair HLA-B*57 positive, the transmitter progressed to AIDS, whereas the recipient was an elite controller. The contrasting clinical phenotypes of nonprogressive vs progressive disease course associated with carriage of a HLA-B*57 allele has been attributed to differences in cytotoxic T lymphocyte (CTL) escape mutations and CTL activity against epitopes in Gag as well as to differential viral replication capacities HLA-C5′/HLA-B*57/HCP5 containing genotypes may represent multiplex ‘genetic signatures’ that have differential effects on HIV disease course. These genetic findings have broad biologic and public health significance as the immunologic features linked to protective vs non-protective B*57-containing genotypes may have practical value in achieving a detailed understanding of the immunologic correlates of virologic control and nonprogressive disease.Fourth, we provide a genetic basis for the long-standing but highly underappreciated conundrum of why some HLA-A10 affects disease course without influencing the viral load, and (ii) HLA-B*57-containing genotypes that convey a nonprogressive disease course do so partly by restricting viral replication and also by impacting on parameters that are independent of the viral load. These observations are reminiscent of our previous results where we found that CCL3L1-CCR5 genotypes also influence disease progression, in part, by impacting on parameters that are independent of the viral load Fifth, our findings place a spotlight on that aspect of pathogenesis that is influenced by parameters whose effects are independent of the viral burden. This point is illustrated by two observations (i) HLA-B*57 genotypes in subjects who remain AIDS free for a prolonged duration. This gradual shift emphasizes that the relative contribution of a genetic factor that confers the phenotype of nonprogressive disease within the context of a natural history cohort may not be identical to the contribution of the same genetic factor in subjects who are selected for analyses based on specific clinical characteristics such as those with nonprogessive disease , or virologic characteristics such as untreated subjects who maintain very low viral loads during some period of the disease course HLA-B*5701 allele is dramatically overrepresented in long-term nonprogressors. When studied within the context of a natural HIV history cohort, we find that 6.6% of EAs possess a B*5701 allele and of these 64% have progressive disease. Thus, the very high frequency of the HLA-B*5701 allele among those selected because of nonprogressive disease or a low viral load may represent an estimate specific to this selected subset of HIV-positive subjects, because HIV-positive individuals who have progressive disease are unlikely to be well represented in such a study group. Hence, association studies in such selected patients are more likely to identify genetic factors that are skewed towards having a significant impact on restriction of viral replication or nonprogressive disease, and may not necessarily identify the full realm of host factors that influence pathogenesis in the context of the natural clinical course of HIV disease, especially because a proportion of AIDS pathogenesis is independent of the viral load Sixth, within the context of a natural history cohort of HIV-positive individuals we find that there is a gradual enrichment of protective A10/ZNRD1, HLA-C and HCP5 alleles on disease course were evident mainly in subjects of European descent. Some of these race-specific effects are not related to the differential distribution in the frequency of these polymorphisms in European and African Americans. The exact basis for these race/ethnicity-specific effects on HIV disease is unclear. Given the differing evolutionary histories of the populations examined, one possibility is that the race/ethnicity-specific effects observed might relate to the interplay between polymorphisms in different gene systems that played an ancestral role in the contrasting host-microbe interplay found in subjects of European and African descent. For example, we found recently that the disease-accelerating effects of the CCL5 -471A/A genotype in African Americans is evident only in those who do not bear the African-specific -46C/C genotype of Duffy Antigen Receptor for Chemokines (DARC), a genotype which is thought to have arisen due to the selective pressure of specific malaria-causing species Finally, the results of the present study support our previous thesis that the host determinants of HIV pathogenesis are likely to be highly population-specific HIV-positive subjects were from the Department of Defense (DoD) HIV Natural History Study (NHS) cohort followed at Wilford Hall Medical Center (WHMC) and more recently at the Brooke Army Medical Center (BAMC), San Antonio, TX. The studied population is the local component of a prospective multisite observational cohort from the United States Military's Tri-Service AIDS Clinical Consortium (TACC) HIV Natural History Study. Unidentified cast-off blood from subjects participating in training at Lackland AFB, TX was used for the HIV-negative control population. The demographic and clinical characteristics of the HIV-positive WHMC cohort have been described extensively HLA-B*57 and HLA-B*5701 subtypes was undertaken in 1,143 HIV+ subjects. Polymorphisms in the HCP5 (rs2395029), HLA-C5′ (rs9264942) and seven SNPs in and around ZNRD1-RNF39 genes Click here for additional data file.Figure S1Disease-influencing effects associated with HLA-A10 status in HIV-positive EA subjects from the WHMC cohort who had not received HAART.(0.26 MB TIF)Click here for additional data file.Figure S2Association between HLA-A10-ZNRD1 haplotypes and rates of HIV disease progression in the EA component of the WHMC cohort.(0.58 MB TIF)Click here for additional data file.Figure S3Association between HLA-C5′-HLA-B-HCP5 haplotypes and rates of HIV disease progression in the WHMC cohort.(1.06 MB TIF)Click here for additional data file.Figure S4Disease-influencing effects associated with HLA-B*57-NPD and HLA-B*57-PD genotypes in subjects who had not received HAART.(0.33 MB TIF)Click here for additional data file. |
Sir,Lancefield group A beta-hemolytic streptococcus (GABHS) is known to cause a wide range of infections. However shoulder pain due to isolated subpectoral abscess caused by group A beta haemolytic streptococcus is extremely rare with only two previously reported cases in the literature.2 The onsOn physical examination he was a well-built and well-nourished boy. Inspection revealed erythema and fullness of the right supraclavicular fossa and pectoral region. There was no bruise, scars or other lesions on the overlying skin. He had an oral temperature of 38.6°C and a pulse rate of 90/min. He had tenderness around right axilla with no palpable lymphadenopathy. External rotation and active abduction beyond 90° were painful in the right shoulder. The remainder of shoulder movements were normal.3 with 85% neutrophils, 10% lymphocytes, 4% monocytes, 0.6% eosinophils, 0.1% basophils with erythrocyte sedimentation rate of 84 mm/hr, and C-reactive protein level of 224 mg/dl. Blood and urine cultures were obtained.Preliminary blood tests revealed that white blood cell count was 24300/mmChest and right shoulder radiographs revealed no significant abnormality.Ultrasound scan of the right shoulder and the pectoral region showed a loculated collection deep to the pectoral muscles. Magnetic resonance imaging of the right shoulder confirmed the loculated abscess of size 3 × 3 × 9 cm lying deep to the pectoral muscles and extending into the right axilla. There was no involvement of the shoulder joint or proximal humerus .Incision and drainage of the abscess was performed the following day and approximately 25 ml of pus was drained. Gram stain of the purulent material showed Gram positive cocci in chains and it was sent for aerobic, anaerobic, mycobacterial, and fungal cultures. Group A beta-hemolytic streptococci (GABHS) were isolated from microbiological culture of the abscess fluid with sensitivity to penicillin, erythromycin, and tetracycline. Blood and urine cultures remained sterile on routine and extended culture and sensitivity tests.The patient was commenced on intravenous benzyl penicillin for a period of 48 h followed by an oral course of Penicillin V for further 2 weeks. At follow-up visit 2 weeks after discharge his operative wounds had healed well. At subsequent 3 month review, he had made good recovery from the procedure with full range of movements in the right shoulder and complete resolution of the pectoral swelling.Lancefield group A beta-haemolytic streptococcus (GABHS) is known to cause wide range of infections with varied severity and clinical course. Commonly reported forms of infection include pharyngitis, rheumatic fever, pneumonia, streptococcal toxic shock syndrome (STSS), necrotizing fasciitis, and osteFactors predisposing to GABHS infection include skin lesions, wounds, diabetes, nonsteroidal anti-inflammatory drug use, malignancy, and primary varicella infection. Skin lesPrevious authors suggested that the suppurative lymphadenitis of the deltoideopectoral lymph nodes which drain the lymphatics from the thumb and lateral half of the index finger resulted in the formation of subpectoral abscess. In patie3Subpectoral abscess due to GABHS causing shoulder symptoms is a rare presentation. Given the paucity of the literature in such cases diagnosis hinges on clinical suspicion and appropriate diagnostic studies need to be obtained early. Due to the size, loculated nature, and the extent of the abscess our patient had an incision and drainage. Once the diagnosis of subpectoral abscess was established in our patient, the functional results following treatment were remarkable.Subpectoral abscess due to GABHS has been previously reported by Rathore MH in a 22-The clinical course of GABHS infections is often precipitous with high mortality.5 However |
Many adult cigarette smokers initiated the habit as adolescents. Adolescent tobacco use may be a marker of other unhealthy behaviours. There are limited data on the prevalence and correlates of cigarette smoking among in-school adolescents in Iraq. We aimed to estimate the prevalence of, and assess the socio-demographic correlates of current cigarette smoking among in-school adolescents in Kurdistan region of Iraq.Secondary data analysis of the Global Youth Tobacco Survey, conducted in the region of Kurdistan, Iraq in 2006. Logistic regression analysis was conducted to assess the association between current cigarette smoking and explanatory variables.One thousand nine hundred eighty-nine adolescents participated in the Kurdistan-Iraq Global Youth Tobacco Survey. Of these, 58.1% and 41.9% were boys and girls respectively. The overall prevalence of current cigarette smoking was 15.3%; 25.1% and 2.7% in boys and girls respectively. The factors associated with adolescent smoking were: parents' smoking, smoking in closest friends, male gender, having pocket money and perceptions that boys or girls who smoked were attractive.We suggest that public health interventions aimed to curb adolescent cigarette smoking should be designed, implemented and evaluated with due recognition to the factors that are associated with the habit. Tobacco use is a leading cause of morbidity and mortality from non-communicable diseases globally ,2. BarzaOver the past 10 years, there has been growing research interest to estimate the prevalence of tobacco use among adolescents. This effort has largely been driven by the Global Youth Tobacco Survey Collaborating Group as part of the Global Tobacco Surveillance System initiated by the World Health Organization (WHO), CDC, and the Canadian Public Health Association.Adolescent cigarette smoking is of public health significance. Many adult smokers had initiated the habit as adolescents. Also smoking among adolescents has short to medium term health effects in the smokers as well as peers who may be exposed to environmental smoke. Smoking in adolescents may also be a marker of other harmful lifestyles such as engagement in illicit drug use, alcohol use, psychiatric illnesses and sexual intercourse -6.The Centers for Disease Control and Prevention (CDC) has reported on the prevalence of tobacco use including cigarette smoking among in-school adolescents in Kurdistan, Iraq . This stThe CDC's paper reported on prevalence of tobacco use and other social characteristics among in-school. This paper restricted data analysis to 13 to 15 year olds and did not assess the socio-demographic correlates of being a current cigarette smoker. While estimates of prevalence of tobacco use will certainly quantify the burden of the problem, it is not possible to identify factors that are associated with cigarette smoking without a deliberate assessment of the potential correlated. We therefore carried out a secondary analysis of the Kurdistan-Iraq Global Youth Tobacco Survey to identify the factors that are associated with current cigarette smoking among in-school adolescents in this region of Iraq.The Kurdistan-Iraq Global Youth Tobacco Survey was conducted in the region which covers the governorates of Irbil, as-Sulaymaniyah, and Dahuk. The Kurdistan region borders Iran to the east, Turkey to the north and Syria to the west. Its capital is the city of Erbil.The GYTS is a cross-sectional survey which aims to collect data from 13 to 15 year old in-school adolescents. Although the GYTS aims to collect data from 13 to 15 year olds, all students in the selected classes are invited to participate. In the Kurdistan region of Iraq, this age range is covered by the first through fourth years of secondary education. The GYTS uses a cross sectional two-stage sampling design. In the first stage of sampling, the sampling frame included all schools containing secondary school grades 1 to 4. The probability of a school being selected was proportional to the enrolment size of the school in the selected grades. In the second stage of sampling, classes within the selected schools were selected randomly. All students present in the classes that had been selected were eligible to participate in the survey. There was no age restriction once a class had been selected. A total of 1,989 students completed the GYTS . The school response rate was 100% (25 schools), the student and overall response rates was 95.6%.The data from the Kurdistan-Iraq GYTS (2006) were collected anonymously from the eligible students who were present on the day of the survey at their school. A self-completed, computer scannable questionnaire was used. Students were asked questions about their smoking practices, socio-demographic information and exposure to tobacco-related media.For the purpose of the current analysis, we were interested in the prevalence and factors associated with current cigarette smoking. Current cigarette smoking (the main outcome) was defined as self-report of having smoked a cigarette, even a single puff, within the last 30 days. No biomarkers were used to verify the self reports. We also assessed the distribution of social characteristics among the study population. These characteristics included: gender, age, amount of pocket money usually received each month, parental smoking, smoking among closest friends, exposure to tobacco-related media and perceptions towards smoking.A weighting factor was used in the analysis to obtain prevalence estimates to reflect the likelihood of sampling each student and to reduce bias by compensating for differing patterns of non response.We also conducted weighted backward logistic regression analysis using SPSS software version 14.0 to assess what relationship existed between a selected list of explanatory variables and the main outcome. The explanatory variables were identified from the literature as having been associated with adolescent smoking in other settings. These variables included: gender, age, parental smoking, smoking in friends, exposure to tobacco-related media, perceptions towards tobacco and the amount of pocket money at the disposal of the adolescent -12.One thousand nine hundred eighty-nine adolescents participated in the Kurdistan-Iraq Global Youth Tobacco Survey. Of these, 58.1% and 41.9% were boys and girls respectively. The overall prevalence of current cigarette smoking was 15.3%; 25.1% and 2.7% in boys and girls respectively. Further description of the study participants is shown in Table Table Also as shown in Table We also assessed whether exposure to various sorts of media was associated with being a current smoker. As shown in Table The prevalence of current cigarette smoking among the study participants in the Kurdistan-Iraq Global Youth Tobacco Survey was 15.3%, higher among boys compared to girls (25.1% versus 2.1%). This prevalence is much higher than the 10.4% reported by Kyrlesi among Greek adolescents 13 to 15 years because Current cigarette smoking among Ethiopian in-school adolescents was 4.5% among boys and 1.0% among females in 2003 . Our estMaziak et al reported on prevalence of cigarette smoking among university students in Syria . In thisOur study found that current smoking prevalence was higher among boys compared to girls (25.1% versus 2.1%). The male predominance in cigarette smoking has been reported elsewhere ,13-17. HWhile male predominance in smoking has been reported in many settings, this finding is not universal. Steele et al had reported that girls were more likely to be current and former smokers in Minnesota, United States .We also found that adolescents who had friends or parents who were smokers were also more likely to be smokers themselves. Similar findings have been reported elsewhere ,11,12,22In the case of having friends who smoke as a factor associated with smoking, Simmons-Morton and HoffWhen we assessed whether exposure to tobacco related advertisements was associated with being a current smoker, we found inconsistent results; some analysis showed exposure positively associated while others negatively associated with smoking , strict adherence to these regulations has not been possible.The findings in this report are subject to some limitations. Firstly, the sample that we participated in the survey was limited to adolescents enrolled in school and present on the day of the survey. As such our findings may not be representative to all adolescents in the Kurdistan region of Iraq. We however believe that the findings are likely to be representative of the school-going adolescents as overall response rate was high i.e. 95.6%.Secondly, the data were based on self-reports of students, who might have underreported or over-reported their behavior or attitudes. Brener et al has repoWe also examined the variables as operating at the individual level. However authors like Nichter and WilcFurthermore, our study was based on a secondary analysis of already existing data. We therefore did not have control of which variables to collect from the study participants. The literature on adolescent smoking has reported that other variables such sibling smoking status ,32, alcoWhile we have assessed the relationship between the amount of pocket money and being a smoker, we also realize that like in many developing and emerging markets countries, smuggling of tobacco products, including cigarettes, may be an important source of tobacco among adolescents -41. TherWe are also unaware how the overall current conflict situation in Iraq may or may not have influence on the prevalence and associated factors of smoking in Kurdistan region of Iraq. We do recognize though that unlike the other areas of the country, this region has to a large extent been administered as an autonomous region of Iraq and has not been active geographic area of the conflict.We have estimated adolescent smoking prevalence as 15.3% with males having higher prevalence than females. Evidence-based public health intervention should make use of our knowledge of the factors that are associated with smoking in Iraqi-Kurdistan. We suggest that research be conducted to assess the effectiveness of the interventions that are being provided in the region to prevent adolescent smoking. Also future studies should consider measuring other variables such as alcohol use, sibling smoking status and religiosity to enable assessment of how these factors may be associated with smoking in Iraq.CDC: Centers for Disease Control and PreventionGYTS: Global Youth Tobacco SurveyWHO: World Health OrganizationThe author(s) declare that they have no competing interests.SS: conducted the data analysis, participated in the drafting of manuscript.ASM: participated in the interpretation of the results and drafting of the manuscript.ER: participated in the interpretation of results and drafting of manuscript. |
After the publication of this work , we disc |
Osx knock-out mice lack bone completely and cartilage is normal. This opens a new window to the whole research field of bone formation. Osx inhibits Wnt pathway signaling, a possible mechanism for Osx to inhibit osteoblast proliferation. These reports demonstrate that Osx is the master gene that controls osteoblast lineage commitment and the subsequent osteoblast proliferation and differentiation. This review is to highlight recent progress in understanding the molecular mechanisms of transcriptional regulation of bone formation by Osx.Bone formation is a complex developmental process involving the differentiation of mesenchymal stem cells to osteoblasts. Osteoblast differentiation occurs through a multi-step molecular pathway regulated by different transcription factors and signaling proteins. Osx is the only osteoblast-specific transcriptional factor identified so far which is required for osteoblast differentiation and bone formation. Runx2-null mutants, no endochondral and no membranous bones form [Runx2 is required for the differentiation of mesenchymal cells into preosteoblasts. As a downstream gene of Runx2, Osx is required for the differentiation of preosteoblasts into mature osteoblasts. Osx is specifically expressed in all osteoblasts. In Osx-null embryos, cartilage is formed normally, but the embryos completely lack bone formation [β-catenin in either skeletal progenitor cells or at a later stage of osteoblast development in mouse embryos blocks osteoblast differentiation [Bone formation takes place through two distinct processes: endochondral ossification involving a cartilage model and intramembranous ossification by which bones form directly from condensations of mesenchymal cells without a cartilage intermediate. Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Osteoblasts produce a characteristic extracellular collagenous matrix that subsequently becomes mineralized after hydroxyapatite crystals deposition. Much progress has been made in understanding the factors that control the gene expression program through the osteoblast induction, proliferation, differentiation, and maturation. Osteoblast differentiation occurs through a multistep molecular pathway regulated by different transcription factors and signaling proteins Table . Indian ormation . Wnt signtiation -7. Otherntiation -12. ThisOsx was discovered as a bone morphogenic protein-2 (BMP2) induced gene in mouse pluripotent mesenchymal cells, encoding a transcription factor that is highly specific to osteoblasts [Osx gene is located in chromosome 15 in mouse and in chromosome 12 in human. There are only two exons in the Osx gene. Exon 1 sequence encodes the seven N-terminal amino acids of Osx, and exon 2 contains the remaining open reading frame (ORF) and 3-prime UTR. The mouse Osx protein is a 428 amino acid polypeptide with a molecular mass of about 46 kDa as shown in Figureeoblasts . Osx is Osx transcripts are not detected before embryonic stage E13 [Osx first appears in differentiating chondrocytes, the surrounding perichondrium, and mesenchymal condensations of future membranous bones of E13.5 embryos. After E15.5, Osx is strongly expressed in cells that are associated with all bone trabeculae and bone collar formation. Weak expression of Osx is observed in the prehypertrophic zone. Osx is highly expressed in bone trabeculae and in secondary ossification centers after birth. Osx is only expressed in cells in the bone matrix and the inner (endosteum) and outer (periosteum) bone surfaces.During mouse embryogenesis, Osx gene was inactivated in the mouse embryonic stem (ES) cells using homologous recombination to understand Osx function. Most of the exon2 coding sequence was deleted. As a result, the Osx gene was inactivated. Heterozygous Osx mutant mice were normal and fertile. Homozygous Osx mutant mice were lethal and these mice had difficulty in breathing, rapidly became cyanotic, and died within 15 min of birth. Newborn homozygous mutant mice showed severe inward bending of forelimbs and hindlimbs [Osx-null embryos have normal cartilage development, they completely lack bone formation, so neither endochondral nor intramembranous bone formation occurs. The mesenchymal cells in Osx-null mice do not deposit bone matrix, and cells in the periosteum and the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. In the endochondral skeletal elements of Osx-null mutants, a dense mesenchyme emerges from the perichondrium/periosteum and invades the zone of hypertrophic chondrocytes along with blood vessels. However, cells in this mesenchyme are arrested during differentiation. A similar, dense mesenchyme is also found in the membranous skeletal elements. Bone trabeculae are completely absent in all skeletal elements. Although mineralization does not occur in membranous skeletal elements, it does in the endochondral skeleton because of the physiological mineralization of the zone of hypertrophic chondrocytes. No mineralization occurs in the periosteum, suggesting that bone collars do not form.It has been demonstrated that Osx is necessary for bone formation and mineralization in vivo . The Osxindlimbs . AlthougOsx-null mutant embryos, expression of type I collagen (Col1a1) in the condensed mesenchyme of the membranous skeleton and the periosteum and mesenchyme of the endochondral skeleton is severely reduced. Expressions of the osteoblast-specific markers such as osteonectin, osteopontin and bone sialoprotein (BSP) cannot be detected in these mesenchymes. In E18.5 Osx-null embryos, osteocalcin, a late, highly specific osteoblast marker, is not expressed in endochondral and membranous skeletal elements. Despite a lack of osteoblast markers expression, Runx2 expression in Osx-null mutants remains comparable to that of wild-type osteoblasts in the dense mesenchyme of both membranous and endochondral skeletal elements. Thus, osteoblast differentiation is completely arrested in Osx-null embryos, even though similar expression of Runx2 remains compared to wild-type embryos. On other hand, over-expressed Osx in vitro has been shown to induce expression of osteocalcin and collagen type 1a1.In Osx-null embryos the number of TRAP-positive cells appear to be reduced compared to wild-type embryos. In long bones of Osx-null embryos cells from the periosteum invade the zone of the hypertrophy chondrocyte as a wedge-shaped expansion of the periosteum in which osteoblast precursors are arrested in their differentiation. These observations are supported by the evidence that expressions of both OPG and RANKL are downregulated, but the ratio of OPG/RANKL increases in E18.5 Osx-null calvarial cells [In the skeletal elements of E18.5 al cells . ExpressLrp5-null mice, which phenocopy the osteoporosis-pseudoglioma syndrome in humans [Lrp5 lead to high bone mass syndrome in patients [Dkk1 mutant mice increase with an increased number of osteoblasts [Dkk1 in osteoblasts causes severe osteopenia with decreased osteoblast numbers [It has been demonstrated that canonical Wnt signaling is required for normal osteoblast proliferation. A marked increase in osteoblast proliferation occurs when β-catenin is stabilized in osteoblasts during mouse embryonic development . Moreoven humans , developn humans . In contpatients and in mpatients . The Wnteoblasts . In cont numbers . These dOsx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. It has been demonstrated that Osx binds to and activates the Dkk1 promoter. Osx is shown to inhibit β-catenin-induced Topflash reporter activity and also inhibit β-catenin-induced secondary axis formation in Xenopus embryos. Moreover, this study showed that in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, β-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo [Recent studies in our research group have provided evidences showing that the osteoblast-specific transcription factor Osx is able to inhibit Wnt pathway activity during osteoblast differentiation . In calv in vivo . Osx canThe PRR region of Osx is responsible for disruption of Tcf1 binding to DNA, and for inhibition of β-catenin transcriptional activity. These findings indicate that Osx negatively controls the activity of β-catenin in two different mechanisms shown in FigureOsx-null calvaria showed greater BrdU incorporation than wild-type calvaria, and primary calvarial cells from Osx-null E18.5 embryos also grew faster than wild-type cells. On the other hand, Osx over-expression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, it is speculated that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/β-catenin activity. These results add a new layer of control to Wnt signaling in bone formation.We have shown that Osx decreases osteoblast proliferation . E18.5 OALP), bone sialoprotein (BSP) and type I collagen, as they commence to produce and mature osteogenic extracellular matrix. Finally, they express genes involved in mineralization of the extracellular matrix such as osteocalcin (OC), osteopontin. This highly regulated program of gene expression and cellular differentiation is governed by the expression and activity of different transcription factors. These factors do not act alone but interact with each other to integrate diverse signals and fine-tune gene expression.Osx is necessary for the osteoblast lineage ,13. FollOsx-null mutant phenotype and recent studies, the following brief model for osteoblast differentiation is proposed as shown in FigureRunx2-expressing biopotential progenitors can differentiate into either osteoblast or chondrocyte depending on cell signaling. Then cells differentiate into preosteoblasts, a process in which Runx2 play an essential role. At this stage, preosteoblasts express early osteoblast marker genes like ALP. Next step, preosteoblasts differentiate into mature osteoblast, a process in which Osx plays a critical role. Mature functioning osteoblasts strongly express characteristic later osteoblast marker genes such as OC and BSP. In the membranous and endochondral skeletons, Osx-null preosteoblasts are blocked from differentiating into osteoblasts, so there is no mature osteoblast without Osx. In Osx-null embryos, osteoblast differentiation markers, such as OC, BSP and osteonectin, are not expressed. Because the promoter regions of several osteoblast marker genes contain binding sites for Runx2 that are functional in DNA transfection experiments [in vivo and produce a bone-specific matrix.Based on the characterization of the eriments ,21, it iWnt/β-catenin signaling has an essential role in osteoblast differentiation during embryonic development and has a major role in stimulating osteoblast proliferation during both embryonic and postnatal development. Osx is an osteoblast-specific transcription factor, required for osteoblast differentiation. The inhibition of Wnt/β-catenin signaling activity by Osx, also constitutes a possible mechanism for the inhibition of osteoblast proliferation by Osx. Recent observations that Osx inhibits Wnt signaling pathway in vitro and in vivo provide novel concept of feedback control mechanisms involved in bone formation .Osx-null mice, while no Osx transcripts are detected in skeletal elements in Runx2-knockout mice [Osx gene promoter [Osx is believed to be downstream of Runx2 in the pathway of osteoblast differentiation because Runx2 expression is normal in out mice . This ispromoter . It is n2 vitamin D3, which have positive roles in osteoblast function, have also been shown to up-regulate Osx expression [The mechanism underlying the regulation of Osx expression in osteoblasts is still unclear. Several studies have reported that some factors can modulate Osx expression. Both BMP-2 and insulin-like growth factor-1 (IGF-1) can induce Osx expression in undifferentiated mesenchymal stem cells . IGF-I-mpression ,26. It wIn vivo studies provide genetic evidence that p53 tumor suppressor blocks osteoblast differentiation and bone development [Osx promoter.Some studies indicate that negative regulators of osteoblastogenesis can inhibit Osx expression. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life ,28. Inhielopment ,28. Prolelopment . PTH inhelopment . Recentlelopment . ATF4-reDespite these interesting findings, the details concerning the regulation and function of Osx are incompletely understood.Osteoporosis is characterized by reduced bone mass, alterations in the microarchitecture of bone tissue, reduced bone strength, and an increased risk of fracture . OsteopoOsx are associated with bone mineral density (BMD) in children and adults probably through primary effects on growth [Osx (SP7) locus. A meta-analysis of these existing studies revealed strong association between SNPs in the Osx region and adult lumbar spine BMD. In light of these findings, this research group genotyped a further 3692 individuals from ALSPAC who had whole body BMD and confirmed the association in children as well.Recent study has indicated that genetic variants in the chromosomal region of n growth . A genomAlthough Osx has been identified to be associated with osteoporosis-related phenotypes, further investigation needs to be done to determine whether Osx will represent a useful diagnostic index of osteoporosis or molecular target for therapeutic manipulation.Runx2 , which is an upstream of Osx, have been shown to be the cause of the human genetic disease cleidocranial dysplasia [Osx mutation leads to any clinical human disease.Osx is indispensable for the commitment of the osteoblast lineage and the expression of the osteoblast-specific matrix proteins, including type I collagen, bone sialoprotein, osteonectin, and osteocalcin. No pharmacological approach to target Osx in osteoblasts has been reported. Heterozygous mutations in ysplasia . There iThe extensive studies by many laboratories to explore how to control the Wnt signaling pathway in osteoblasts stems from the realization that this pathway has an essential role in bone mass determination in the adult skeleton. There is also an expectation that efforts to pharmacologically target this pathway should yield promising agents to treat bone diseases such as osteoporosis. Results in our group showing that Osx inhibits Wnt/β-catenin signaling add an important new layer of control to the complex regulation of the Wnt pathway in osteoblasts .Osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Using an in vitro migration assay, Osx suppressed the migration of tumor cells to lung extracts. These results suggest that Osx expression may play a role in osteosarcoma tumor growth and metastasis, and that osteolytic activity of tumor cells may be regulated by Osx via down-regulation of interleukin-1 gene transcription [It was observed that the Osx expression was decreased in two mouse osteosarcoma cell lines and in three human osteosarcoma cell lines . Transfecription . It is rcription .Bone formation is essential for maintenance and healing of the skeleton following injury and operative interventions, such as osteotomies and limb lengthening. In numerous orthopedic conditions, such as congenital pseudoarthrosis of tibia, femoral head osteonecrosis, and large bone lengthening, bone healing and regeneration remain challenging goal to achieve. Most therapy for skeletal diseases with less bone such as osteoporosis and osteonecrosis is aimed at inhibiting bone resorption, but to cure these diseases, it is also critically important to stimulate new bone formation. Therefore, there is currently great interest in understanding the regulation of osteoblast differentiation and activity to guide the development of anabolic therapies. Although no pharmacological approach to target Osx in osteoblasts has identified yet, an interesting future research direction is to look for upstream genes or molecules which can selectively target Osx expression and activity. We speculate that Osx could become a therapeutic target in efforts to stimulate the anabolic pathway of bone synthesis.Bone formation is a complex process regulated by multiple factors and pathways; it is clearly shown that Osx is required for the final commitment of osteoblast lineage. Although recent molecular and genetic studies using gene targeting in mice have established Osx as a master regulator of osteoblast differentiation during bone formation, the mechanisms of Osx regulation of osteoblast differentiation and function are still under investigation. Future studies to decipher the Osx direct upstream or downstream molecular targets, Osx expression regulation and Osx functional partners are required to clarify the detailed mechanism of the temporal and spatial regulation of Osx for bone formation and homeostatic regulation of skeletal system. The need to develop novel drugs that stimulate bone formation and thereby elevate bone mass (anabolic agents) has opened new research areas for therapeutic intervention in the treatment of bone-related diseases.Col1a1: type I collagen; OC: osteocalcin; BSP: bone sialoprotein; ALP: alkaline phosphatase; PRR: proline-rich region; BMP2: bone morphogenic protein-2; BMD: bone mineral density.Osx: Osterix; OB: osteoblast; E18.5: embryonic day 18.5; Ihh: Indian hedgehog; The authors declare that they have no competing interests.The author contributed to the article. The author has read and approved the final manuscript. |
Mood and anxiety disorders are highly prevalent and have a large impact on the lives of the affected individuals. Therefore, optimal treatment of these disorders is highly important. In this study we will examine the effectiveness of a stepped care program for primary care patients with mood and anxiety disorders. A stepped care program is characterized by different treatment steps that are arranged in order of increasing intensity.This study is a randomised controlled trial with two conditions: stepped care and care as usual, whereby the latter forms the control group. The stepped care program consists of four evidence based interventions: (1) Watchful waiting, (2) Guided self-help, (3) Problem Solving Treatment and (4) Medication and/or specialized mental health care. The study population consists of primary care attendees aged 18–65 years. Screeners are sent to all patients of the participating general practitioners. Individuals with a Diagnostic and Statistical Manual of mental disorders (DSM) diagnosis of major depression, dysthymia, panic disorder (with or without agoraphobia), generalized anxiety disorder, or social phobia are included as well as individuals with minor depression and anxiety disorders. Primary focus is the reduction of depressive and anxiety symptoms. Both conditions are monitored at 8, 16 and 24 weeks.This study evaluates the effectiveness of a stepped care program for patients with depressive and anxiety disorder. If effective, a stepped care program can form a worthwhile alternative for care as usual. Strengths and limitations of this study are discussed.Current Controlled Trails: ISRCTN17831610. Depressive and anxiety disorders are highly prevalent . CurrentThere are ongoing discussions regarding the effectiveness of the management of common mental health problems in general practice settings. Both anxiety disorders and depression can be treated effectively by pharmacotherapy as well as psychotherapy -15. AlthThrough its objective of initiating interventions at the right time and as adequately as possible, the stepped care model could provide a solution for the problem of applying effective, evidence based care for depression and anxiety. Care is offered not earlier or more intense than necessary and not later or less intense than needed ,28. In aAlthough the stepped care model seems to be a promising care model -30 to inThis study is a randomised controlled trial. We recruit patients by screening all patients of the participating GPs during an inclusion period of 1,5 years. All patients with a positive screen for depression and/or anxiety are screened again after 4 to 6 weeks. Those with a second positive screening (baseline) are contacted by telephone for a diagnostic interview. During this interview all in- and exclusion criteria are checked. Those who are eligible for participation are being asked for informed consent to participate in the study. All patients who give informed consent are subsequently randomised to either (1) stepped care or (2) care as usual. All patients are asked to fill out questionnaires again after 8, 16 and 24 weeks. The study protocol, information brochure and informed consent are approved by the Medical Ethics Committee of the VU University Medical Center (registration number 2006/248).We include adults aged 18 – 65 years with one or more of the following DSM-IV diagnoses: major depression (single episode or recurrent), dysthymia, panic disorder (with or without agoraphobia), social phobia or generalized anxiety disorder, including comorbid diagnoses. We also include patients with a minor depression or a minor anxiety disorder. We use the DSM-IV research criteria to define minor depression. The main difference between the criteria of minor and major depression is that for minor depression only two to four out of nine symptoms have to be present, of which at least one has to be a core symptom . As there are no DSM criteria, we define a minor anxiety disorder as a score of 12 or more on the Hospital Anxiety and Depression Scale and dysfIn the present study we collaborate with two mental health centers in Amsterdam (GGZ inGeest and Mentrum). Both of these mental health centers employ psychiatric nurses and psychologists, who work for a few hours per week in a GP practice. Usually, GPs refer patients to these psychiatric nurses/psychologists for short-term treatments. First we want to approach psychiatric nurses and psychologists and secondly we want to invite the corresponding GPs to collaborate in this study.and return their informed consent are randomised.Patients are recruited by screening all patients of the participating GPs during the inclusion period of 1,5 year. All patients with a positive screen for depression and/or anxiety are assigned to a watchful waiting period of 4 weeks. After 4 weeks every patient is screened again to exclude the patients who recover spontaneously. This second screener is the baseline questionnaire (T0) and is sent to each patient together with general information about the project and an informed consent form. Two weeks later the patients are approached for a diagnostic interview by telepRandomisation takes place at an individual level. Patients are randomised into two groups, stratifying by care manager and using blocks of 4 to prevent overburdening the care managers. An independent researcher not involved in the current project uses computer generated block randomisation to produce sealed envelops. After every inclusion the researcher opens a sealed envelop.(1) Watchful waiting. Patients with mood and anxiety disorders often recover spontaneously over time [Guided self-help. Guided self-help starts with one 30 minute session with a care manager. This session enables the care manager to check exclusion criteria , to give psychoeducation and to explain the self-help interventions. In this study we have two different self-help interventions available. The first is a generic intervention based on problem solving treatment, which aims at patients with symptoms of mood and/or anxiety disorders. The Dutch version of self-examination therapy is called "Alles onder controle" and is available as a book and through the Internet. The patient can choose to get feedback by email or by telephone. In this study feedback is provided by coaches (members from the research group) from the VU University. The focus of the feedback is to guide the patient through the intervention by motivating and activating them. When patients send their assignments to their coach, they receive feedback within 3 working days. The second self-help intervention is specifically aimed at patients with phobias and is based on exposure therapy. In this course patients first have to make a list of all the situations that provoke anxiety and put them in order of intensity. Next they have to make a plan to practice exposure to these situations based on this anxiety hierarchy. This course takes six weeks to complete and is only available as a book. Feedback is therefore provided by telephone. During the first session, the care manager and the patient together decide which self-help course is most suitable. Patients who do not recover using self-help treatment start with (3) Problem Solving Treatment (PST). PST is a short psychological intervention, 5 sessions of 45 minutes each, provided by the care manager [Problem-solving treatment for anxiety and depression: A practical Guide' by Laurence Mynors-Wallis [Pharmacotherapy and/or specialized mental health care. In case patients do not recover from PST they have one more session with the care manager to discuss the next step: either pharmacotherapy or more specialized mental health care. In case a patient chooses pharmacotherapy, the care manager sets up an appointment for the patient with the GP. In case the patient chooses specialized mental health care, the care manager sets up an appointment with a mental health care specialist.The stepped care intervention consists of four steps: ver time . In the manager . The tres-Wallis . Recent s-Wallis ,37-42. TEven though there is no clear evidence that patients with more severe symptoms of anxiety or depression do not benefit from low intensity (self-help) interventions, we decided that patients with more severe disorders should be referred to more specialized mental health care and/or pharmacotherapy directly and skip the preceding steps. Severity of the disorders is based on questions about daily functioning on the Work and Social Adjustment Scale (WSAS) . If the Patients randomised to the 'care as usual' will be informed about this outcome. They are informed that they can choose to find health care and are allowed to discuss this with their GP.After each step in the stepped care intervention, patients are monitored: after 8 weeks (T1), 16 weeks (T2) and 24 weeks (T3). During each assessment we measure depressive symptoms, symptoms of anxiety and daily functioning. Recovery is defined as a score below the cut-off score on all three of the following questionnaires: having a score of less than 14 on the Inventory of Depressive Symptomatology (IDS) , a score). The extended version contains five extra items for anxiety. The anxiety items can only be answered "yes" or "no". Compared to the K10, the extended K10 has a higher sensitivity and specificity for detecting both depressive and anxiety disorders [The Kessler Psychological Distress Scale (K10) is used isorders .Respondents screen positive when they score higher than the cut-off score of 20 and/or reported at least one "yes" on the anxiety items. Respondents with a negative screener were excluded from the study.th edition [The CIDI (version 2.1), a structured interview developed by the World Health Organisation , enables edition . The ass edition . In thisWe will use the Inventory of Depressive Symptomatology (IDS) to measure depressive symptoms. The IDS consists of 26 items and the total score varies between 0 and 79. Scores below 14 indicate absence of depressive symptoms. We use this cut-off score as an indication of recovery from depressive symptoms ,46. InteFor identifying anxiety disorders we will use the Hospital Anxiety and Depression Scale (HADS) , which iWe will measure daily functioning of the patient via four questions of the Work and Social Adjustment Scale (WSAS) ,56. The Quality of life is measured through the MOS Short-Form general health survey (SF-20) which identifies the health related quality of life . This seThe Four Dimensional Symptom Questionnaire (4DSQ) is developed for GPs to distinguish depression, anxiety and somatisation from more general distress . The disThe Alcohol Use Disorder Identification Test (AUDIT) is a lisThe Neuroticism, Extraversion, Openness to New Experience-Five Factor Inventory (NEO-FFI) is a questionnaire that contains 60 items on the five personality domains; neuroticism, extraversion, openness to experience, agreeableness and conscientiousness. The item responses are given on a 0 – 5 scale . A studyWe use the Trimbos and iMTA questionnaire on Costs associated with Psychiatric illness (TiC-P) to colleFor the measurement of health related absenteeism, we used the earlier mentioned TiC-P.We will examine the societal costs which are based on the health care utilisation and health related absenteeism.This is measured via the QUOTE, which stands for Quality Of care Through the patient's Eyes. This questionnaire gives insight in the patient's opinions and believes about the quality of the received care . There aWe assess the amount of perceived control through the Pearlin Mastery Scale . This scA meta-analysis on the effects of psychological treatment on patients with sub-clinical depression shows an effect size of 0.40. Based on a power of 0.80 in a two-tailed test and an alpha of 0.05, we need 100 patients in each condition. Therefore, the total sample size is set at 200.d [All analyses will be conducted according to the intention-to-treat principle. All respondents who have been randomised will be included in the analyses examining the effects of the intervention. Missing data will be imputed using regression imputation. The effectiveness of the stepped care program is measured via the primary outcomes: the HADS (anxiety) and IDS (depression). It is useful to know not only wether the effectiveness is statistically significant, We will use Cohens' d to measud . AdditioThis paper describes the design of a study to investigate the effectiveness of stepped care for patients with minor and/or major depressive and anxiety disorders in comparison with care as usual in general practice. The following four steps are included in the stepped care model: watchful waiting, guided self-help, PST and pharmacotherapy and/or referral to mental health care. All these interventions are evidence based but there is a lack of studies regarding the effectiveness of stepped care as a whole. Results of this study could offer encouragement for the implementation of an effective stepped care model.A major strength of this study is that it is a pragmatic randomised trial. In a pragmatic trial, patients and therapists are the same as those seen in daily practice. This means that the sample of patients may be quite heterogeneous (may have mild to severe depression/anxiety with or without psychiatric or somatic co-morbidity) and that the therapists (psychiatric nurses or psychologists) have average qualifications (instead of top level therapists from an academic centre). This enhances external validity which means that the results of this study will reflect the 'real' effects of daily practice. If an intervention is shown to have a significant beneficial effect in a pragmatic trial then it has been shown not only that it can work, but also does work in real life .This advantage of the study has also some risks. First of all, it is difficult to maintain treatment integrity when conducting a study in day-to-day clinical practice and using personell not specifically hired for research. We hope to minimize this limitation by organizing strict supervision of the care managers.Secondly, it is possible that there is a bias in our GP recruitment. To conduct this study, we need GPs who work together with a psychiatric nurse. This could mean that these particular GPs are aimed at mental health problems more than their colleagues who work without psychiatric nurse in their practice. If the GPs in our study pay more attention to mental health problems then GPs without psychiatric nurses, then our care as usual might be more adequate then care as usual in general.Another limitation of our study could be the fact that we choose to recruit people with a diagnosis who do not receive any form of care and/or medication. There is a possibility that these patients have visited the GP before, but were not recognized as mental health patients. These patients might differ in a number of aspects from those who were recognized as mental health patients by their GP. It is possible that this leads to selection bias. To examine this we will conduct a non-response research.It is widely acknowledged that depression recognition and management in primary care can be improved. As a consequence many collaborative care models, or disease management strategies, have been developed in recent years. The core elements of these care models are (1) an enhanced case management role for nonmedical specialists such as practice nurses and (2) integrated working relationships between primary care and specialist/secondary services . In geneThe authors declare that they have no competing interests.AvS and PC obtained funding for the study. WS coordinates the recruitment and data collection during the study. AB and PC are responsible for the overall supervision. HvM, AvS and PC provided the setting of the project. All authors provided comments, read and approved the final manuscript.The pre-publication history for this paper can be accessed here: |
XRCC1 gene single nucleotide polymorphisms (SNPs) and NAC response.Platinum-based neoadjuvant chemotherapy (NAC) is new therapeutic strategy for locally advanced cervical carcinoma, but the variables used to predict NAC response are still infrequently reported. The aim of our study was to investigate the association between XRCC1 (at codon 194 and 399) and XRCC1 protein expression were detected. The association of XRCC1 gene SNPs and protein expression with NAC response were analyzed.Seventy patients with locally advanced cervical carcinoma who underwent NAC were collected. SNPs of XRCC1 at codon 194, while responses were significantly different in genotypes Arg/Arg, Arg/Gln, Gln/Gln of XRCC1 at codon 399 . The risk of failure to chemotherapy in the patients with a Gln allele(Arg/Gln+Gln/Gln) was significantly greater than that with Arg/Arg. The expression level of XRCC1 protein was significantly associated with response to NAC. Moreover, the genotype with the Gln allele(Arg/Gln+Gln/Gln) at codon 399, but not codon at 194, presented a significantly higher level of XRCC1 protein expression than that with Arg/Arg genotype .Response to NAC was not statistically significant in three genotypes, Arg/Arg, Arg/Trp, Trp/Trp of XRCC1 gene at codon 399 influences the response of cervical carcinoma to platinum-based NAC. This is probably due to changes in expression of XRCC1 protein, affecting response to chemotherapy.SNP of Cervical carcinoma is the second most common malignancy, and continues to be a leading cause of cancer death in women. It is generally accepted that radical surgery or radiotherapy can be curative for the majority of patients with early-stage cervical carcinoma. However, the prognosis of locally advanced or bulky disease remains very poor, and the optimal management for those patients is still a matter of debate, new therapeutic strategies, such as neoadjuvant chemotherapy (NAC) and concurrent chemoradiation, have been adopted to improve the prognosis for those patients .Many clinical studies have revealed that NAC is highly effective for patients with locally advanced cervical carcinoma, the use of NAC followed by radical surgery and/or radiation for the treatment of cervical carcinoma has been investigated extensively in the past decade, it has been reported that NAC with cisplatinum-based chemotherapeutic regimens have high response rates (ranging from 53% to 94%) ,2. HowevCisplatin is considered to be the most effective drug for the treatment of cervical carcinoma, and usually is an essential element in the NAC regimen, but the mechanisms dictating variable response to chemotherapy among individuals are still unknown. Because platinum compounds produce adducts and breaks in the DNA double helix, individual variability of DNA repair may be relevant in modulating the efficacy of such cytotoxic agents. In resent years, some studies have shown that the molecular condition of DNA repair genes can predict the response of chemotherapy in some human cancers . The preXRCC1) is one of the most important DNA repair genes. The XRCC1 protein physically interacts with ligase III and poly(ADP-robose) polymerase, acting as a scaffold in the removal of adducts through both single-strand break repair and base excision repair (BER), and in the repair of other types of cisplatin-induced damage, including double-strand breaks, through a nonhomologous end-joining pathway were 90.0%, 0% (0/2), and 83.33%, respectively . Logistic regression analysis showed a significantly increased rate of failure of NAC in patients with at least one Gln allele [Arg/Gln(GA)+Gln/Gln(AA)] versus the Arg/Arg (GG) genotype .t = 13.64, P = 0.008).The level of XRCC1 protein expression was significantly higher in patients with poor response to NAC (SD+PD) than it was in those with good response (CR+PR) ; however, there was a statistically significant association between codon 399 polymorphism and XRCC1 protein expression .The association of the variant genotypes at codon 194 and 399 with expression of the XRCC1 protein in locally advanced cervical carcinoma tissues were further evaluated, as shown in Table P = 0.009).In addition, the level of expression of XRCC1 protein in patients with at least one Gln allele [Arg/Gln (GA) + Gln/Gln (AA)] was significantly higher than that in the patients with the Arg/Arg (GG) genotype [XRCC1 contributed to susceptibility to cancer or to sensitivity to chemotherapy. These inconsistent results may be related to the different types of cancers studied in different ethnic populations [Some studies have assessed the association between g cancer . Studies. 20.3%) . In pati. 20.3%) . On the ulations ,16.Only one study assessed the association between XRCC1 gene polymorphisms at codon 194 and NAC response in cervical cancer, recently, Kim and his colleagues reported 66 patients with cervical cancer undergoing platinum-based NAC, the results showed that the genotypes of XRCC1 Arg194Trp was associated with the response . But OurXRCC1 at codon 399 may influence the outcome of cisplatinum-based chemotherapy in some human carcinomas, but the results are also variable. Wang and his colleagues reported that in patients with non-small cell lung cancer who received the platinum-based chemotherapy, the response rate was significantly higher in patients with the Arg/Arg genotype than that in those with at least one Gln allele (41.5% vs. 21.2%). In contrast, other studies of patients with neck cancer revealed that sensitivity to chemotherapy was higher in patients with a Gln allele than in those with other genotypes [XRCC1 at codon 399 and the response to chemotherapy in non-small cell lung cancer [It has been suggested that the SNPs of enotypes ,17. Moreenotypes . While ig cancer .XRCC1 gene at codon 399 influences the response of cervical carcinoma to platinum-based neoadjuvant chemotherapy, and that the genotype carrying at least one Gln allele may be considered to be a candidate molecular marker to predict poor response to NAC in locally advanced cervical carcinoma.Our study showed that the response to chemotherapy in locally advanced cervical carcinoma was significantly higher in patients with the Arg/Arg genotype at codon 399 than in those with the Arg/Gln or Gln/Gln genotype (90.0% vs. 76.92%). The risk of failure of NAC therapy was 3.254 fold higher in patients carrying at least one Gln allele compared with those carrying no Gln allele. Our findings suggest that SNPs of the XRCC1 at codon 399 influences response to NAC in locally advanced cervical carcinoma affirms previous results reported by the studies of other carcinomas, but the exact mechanism remains unknown [The fact that SNPs of unknown ,17,19. S unknown . Dabholk unknown .XRCC1 genotype variation at codon 399, the protein expression was significantly higher in the patients with a Gln allele (Arg/Gln or Gln/Gln) than that with the Arg/Arg genotype in locally advanced cervical carcinoma. Our findings suggest that the genotype with at least one Gln allele probably increases the expression of XRCC1 protein, and consequently, results in poor response to platinum-based chemotherapy in patients with locally advanced cervical carcinoma. To our knowledge, this is the first investigation of XRCC1 gene SNPs, protein expression, and their association with response to chemotherapy. Further study is needed to clarify the mechanism behind this phenomenon.Similarly, our results also showed that the level of XRCC1 protein expression was significantly higher in patients with poor response than in those with good response to NAC in locally advanced cervical carcinoma. In addition, we found that this altered expression of the XRCC1 protein was associated with XRCC1 gene at codon 399 influence the response of patients with locally advanced cervical carcinoma to platinum-based NAC. Patients with a genotype carrying at least one Gln allele have an increased risk of failure to respond to chemotherapy compared with those carrying no Gln allele. This reduced response to chemotherapy is probably due to elevated expression of XRCC1 protein in those patients who have at least one Gln allele.We have demonstrated that SNPs of the The authors declare that they have no competing interests.XDC have made substantial contributions to conception, and drafting the manuscript. WGL have made substantial contributions to patients sample collection. FY carried out the molecular genetic studies. XYW carried out the protein expression detection and performed the statistical analysis. XX conceived of the study, and participated in its design, and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. |
Tumour lysis syndrome (TLS) is a rare side effect of chemotherapy for solid tumours. Itdescribes the metabolic derangements following rapid and extensive tumour cell death following a good response to chemotherapy. Symptoms are those of metabolic derangement and renal failure. Treatment involves rehydration and correction of metabolic abnormalities. TLS should be considered in high risk groups. We report a case of TLS in a patient with metastatic gastrointestinal stromal tumour treated with imatinib mesylate. To our knowledge, this is the first reported case. Gastrointestinal stromal tumour (GIST) is derived from mesodermal tissue but its pathogenesis remains unclear . It predMost GISTs have a gain of function mutation in the c-kit proto-oncogene resulting in ligand independent activation of the KIT receptor tyrosine kinase and unopposedstimulus for cell growth. Glivec selectively binds to and inhibits the activityof a small number of tyrosine kinases including ABL, BCR ABL, platelet derivedgrowth factor receptor, and KIT , 4. ResuTumour lysis syndrome (TLS) is commonly seen following the treatment of exquisitely chemosensitive tumours, such as haematological malignancies, and has been reported in various solid tumours , but to An 81-year-old manpresented in 2002 with a two-day history of melaena and dizziness. He gave no history of peptic ulcer disease, NSAID use, and denied significant alcohol intake. His past medical history included systemic hypertension, noninsulin dependent diabetes mellitus, ischaemic heart disease and gout which were all well controlled on medication includingallopurinol. He was also under regular review for chronic lymphocytic leukaemia.This was stable and he was not receiving active treatment.On examination he was haemodynamically stable and his abdomen was soft and nontender, with no palpable masses. Per rectum, digitalexamination revealed only tarry black stools consistent with melaena. Haemoglobin on admission was 13 g/dL, urea 28 mmol/L, creatinine 142 mmol/L, liver function tests and clotting werenormal. Endoscopy showed a large focallyulcerative gastric mass on the greater curve with prominent underlyingvasculature and there was an isolated rise in alkaline phosphatase (456 IU/L). Computerised tomography (CT) scandemonstrated a large abdominal mass involving the mesentery and abdominal wallmusculature measuring 20 × 11 × 25 cm . Although the perforation may have contributedto morbidity, it is our opinion, due to its limited extent, that it was not theprimary cause of death. Histologically, the vast majority of themain mass was hypocellular consisting of scattered tumour cells stainingpositive with CD117 with pyknotic nuclei and condensed cytoplasm (resemblingmast cells) in an abundant myxoid stroma .% [% of cases, severe toxicity grade 3 to 4 adverse events occur including gastrointestinal or intratumourhaemorrhage due to massive tumour necrosis, which rarely can be fatal [Primary surgical excision with good margins is the mainstay of treatment of GIST with an overall five-year survival rate for localised disease of 54% . The res% –5. The r% , 8. Imat% –5. In ge% , 4. In aImatinib is alsoan effective treatment for chronic myeloid leukaemia and a case report has beenpublished of TLS occurring in one patient .The most reliableparameters for TLS are laboratory indices and clinical signs and symptoms,which typically occur 12–72 hours aftercommencement of cytotoxic therapy .Although nonhaematologicalmalignancies are considered low risk for developing TLS due to longer doublingtime, low growth fraction, and slow response to treatment compared withlymphoproliferative malignancies, there are numerous case reports in patientswith different tumours including small cell lung cancer, medulloblastoma butnone in patients with GIST. Such solidtumours tend to be very chemosensitive, although TLS can occur in less sensitivetumours if bulky metastatic disease is present.Risk factors for TLS include bulky disease, elevated pretreatment LDH, compromised renal function, potentially nephrotoxic drugs, and raised uric acid levels. The patient discussed here had several riskfactors: dehydration secondary to diarrhoea, compromised renal function, bulkytumour, and use of nephrotoxic drugs .% in hyperuricamic patients) [Much of theliterature on TLS stresses the importance of prevention by having a high indexof suspicion for patients with large chemosenstitive tumours. This can be achieved by prophylactic treatment with allopurinol, pretreatment hydration, and alkalinization of urine . There aatients) and the atients) . An alteatients) . This dr>50 × 109/L and/or high LDH levels), elevated uric acid levels,intensive cytoreductive therapy, and poor hydration [We thus advise caution when treating advanced GIST with imatinib and suggest a full assessment of risk factors for TLS which include high tumour burden (high white cell count |
ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (ΔN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-γ-S Cytoplasmic coat proteins govern the transport of proteins between membrane-bound compartments of the secretory and endocytic pathways by shaping the membrane of the donor organelle into vesicular or tubular transport carriers and selecting appropriate cargo into them –3. The win vitroGLO3, named ret4-1arf1-16 and arf1-17 mutants, which display strong defects in retrograde trafficking for several cargo proteins in vitro. Our data strongly suggest that ARFGAP1, ARFGAP2 and ARFGAP3 co-operate to perform essential, overlapping functions in COP-I-mediated trafficking in mammalian cells.Schizosaccharomyces pombe, we were able to identify a small conserved signature motif, named the Glo3 motif, found in 19 ARFGAP proteins from 14 different species. Using this information, we found two distinct human orthologues of yeast Glo3p, ARFGAP2 and ARFGAP3. In summary, searching the increasing amount of data available from systematic sequencing projects, we identified two distinct human sequence orthologues of the yeast ARFGAP Glo3p.We wished to identify a human orthologue for the yeast ARFGAP Glo3p, which in yeast has been demonstrated to have a prominent role in coatomer-mediated trafficking. Among over 16 human proteins with an ARFGAP domain, we found human Gcs1p, which is named ARFGAP1, and four novel proteins with a closely related ARFGAP domain. In order to identify a true orthologue of yeast Glo3p, we searched for proteins with sequence similarity beyond the ARFGAP domain. Making use of a Glo3 protein from S.pombe, as well as rat and human ARFGAP1, and the human ARFGAP2 and ARFGAP3. Note the presence of the four conserved cysteine residues characteristic for the zinc finger domain of ARFGAPs highlighted in yellow.Both Glo3p and Gcs1p share high sequence homology in their amino terminal catalytic zinc finger domain, which is known to engage ARF. Plasmodium yoelii and the fruit fly Drosophila melanogaster. In these organisms, only a single Ile-Ser-Ile tripeptide is present in the second repeat, whereas only the first repeat is well conserved in the nematode Caenorhabditis elegans. During preparation of this manuscript, Nakano’s group also noted the presence of the Glo3 motif and could demonstrate that it is important for Glo3p function in yeast The conserved Glo3 motif, which is characteristic for Glo3 proteins from a wide variety of eukaryotic organisms including plants, is about 45 amino acid residues in length and is situated in the carboxy terminal part of the Glo3 proteins see . It consin vitro had been noted and determined their intracellular localization. By IF studies on methanol–acetone-fixed NRK cells, we find the majority of ARFGAP2 as well as ARFGAP3 associated with a juxtanuclear densely packed structure typical for the mammalian Golgi complex and endosomal compartments Again, this is not the case for ARFGAP2 and ARFGAP3 when compared with the well-characterized early Golgi marker GM130 in vitro. For this we employed the ‘classic’ budding assay developed by the Rothman/Wieland labs and expressed at low level in Vero cells. This CFP fusion protein localized to the Golgi complex [as identified using the Golgi enzyme marker galactosyl transferase tagged with yellow fluorescent protein ] and also localized to punctate structures scattered through the cytoplasm .in vivo, we employed a model cargo protein previously described by us . At elevated expression levels (12 h after transfection), a strong cytoplasmic pattern was seen in addition to the Golgi pattern (To test whether ARFGAP2 may be involved in COP-I-dependent transport At even longer time-points after transfection (16 h), cells expressing ΔN-ARFGAP2–CFP displayed a strikingly higher frequency of having two or even three nuclei . No significant cell death was observed in single knock-downs or in any pairwise knock-down, compared with cells transfected with a control oligo. However, a triple knock-down of ARFGAP1, ARFGAP2 and ARFGAP3 in HeLa cells resulted in strongly significant cell death. More than 75% cell death was routinely observed after three sets of siRNA transfections at the 72-h time-point after the last transfection. Very similar results were observed in NRK cells (data not shown). We conclude that at least one of the three ARFGAPs is required to maintain cell viability.In order to further investigate the role of ARFGAP2 and ARFGAP3 in COP-I-mediated transport, we analysed NRK and HeLa cells upon knock-down of individual ARFGAP transcripts. Protein levels were monitored by immunoblots of total cell extracts from NRK cells 72 h after transfection with appropriate small interfering RNA oligonucleotides (siRNAs). As shown in in vitro in the presence of the non-hydrolysable GTP analogue, GTP-γ-S.We wish to understand the role of ARFGAPs in the regulation of COP-I-dependent trafficking. In yeast, the two ARFGAPs Glo3p and Gcs1p together provide an overlapping, essential function in COP-I-dependent membrane traffic. In mammalian cells, only the Gcs1p orthologue, ARFGAP1 has been characterized so far. In this study, we provide an initial cell biological characterization of two recently identified human ARFGAPs, ARFGAP2 and ARFGAP3. We unambiguously demonstrate by sequence analysis that they are the human orthologues of yeast Glo3p, through the presence of a conserved ‘signature’ motif at the carboxy terminus, the Glo3 motif. Using novel antibodies, we demonstrate that they are localized to the Golgi complex and peripheral punctate structures scattered throughout the cytoplasm in NRK cells. Both ARFGAP2 and ARFGAP3 significantly colocalize with COP I subunits in both locations, strongly suggesting that they are associated with COP I on both ERGIC/VTC structures and at the Golgi complex. Here we further show that both ARFGAP2 and ARFGAP3 are associated with COP I vesicles produced The Golgi and VTC localization of ARFGAP2 and ARFGAP3 appears to be dynamic, as treatment with brefeldin A (BFA) rapidly renders their localization completely diffuse-cytoplasmic within 2–5 min of adding the drug, with kinetics very similar to those of β-COP and ARFGAP1 (unpublished data). Similarly, re-recruitment of ARFGAP2 and ARFGAP3 to Golgi membranes occurs rapidly upon washout of the drug. Future experiments will need to address the dynamics of ARFGAP2/3 using live cell imaging approaches, similar to detailed analysis that has been reported for ARFGAP1 arf1-16 and arf1-17, suggesting that the motif is important for Glo3p function The Nakano group has recently reported that Glo3p but not Gcs1p can suppress arf-Ts mutants with a severe defect in Golgi-to-ER retrieval in an allele-specific manner in vitro interactions with mammalian coatomer. The same phenomenon was observed for their yeast counterpart Glo3p in binding experiments using yeast cytosol. It has recently been shown that ARFGAP1 can also directly interact, albeit more weakly, with coatomer. However, this interaction involves two distinct binding sites on ARFGAP1, one in its catalytic domain and the other in the non-catalytic region . Multiple alignments were performed with the program MegAlign using the clustal V algorithm.image consortium. A hypothetical intron–exon structure established for the ARFGAP2 locus on chromosome II allowed to explain a number of splice variants and artefacts found in the expressed sequence tag (EST) database. Splice variants most closely related to yeast Glo3p were chosen for further experiments: image clone 650′690 for ARFGAP3 and a combination of image clone 2′961′498 (ATG to NcoI site) and clone 2′986′241 (NcoI to TGA) for ARFGAP2. The coding region of several image clones with full-length inserts were sequenced.Full-length clones for both human Glo3 proteins were obtained from the To obtain His6-tagged recombinant proteins as antigen and for binding experiments, DNAs encoding full-length ARFGAP2 and ARFGAP3 were cloned into pET21d (Novagen) and expressed in λDE3 lysogens of strain BL21 and purified by Ni-NTA chromatography as described m glutamine and antibiotics penicillin and streptomycin . In some experiments, BFA was added to the cell culture medium at a concentration of 5 μg/mL. A stock solution of BFA was stored at −20°C.HeLa cells, NRK and Vero (green monkey kidney fibroblast) cells were maintained in DMEM medium + 10% bovine calf serum, 2 mFor colocalization analysis by IF, we used monoclonal antibodies against GM130 , p115 , γ-adaptin and β-COP .NRK or HeLa cells grown on glass coverslips were fixed in methanol at −20°C for 4 min followed by fixation for 30 seconds with −20°C acetone. Cells were mounted in 50% glycerol on glass slides and epifluorescence microscopy was performed on a Zeiss Axioplan microscope with a 63×, 1.4 oil immersion objective, with images taken using a CoolSnap CCD camera.His6- or GST-tagged proteins were used to pull down coatomer from yeast or rat liver cytosol, under conditions described by us previously In vitro budding of COP-I Golgi-derived vesicles from purified rat liver Golgi membranes was performed in the presence of pig brain cytosol as a source of coatomer, ATP and the nonhydrolysable GTP analogue, GTP-γ-S as described by the Rothman lab was a kind gift from J. Lippincott-Schwartz (NIH). We constructed CFP-tagged full-length or ΔN96-ARFGAP2 expression clones, with the CFP moiety at the carboxy terminus of the proteins, using standard cloning procedures in the vector pECFP-C1 (ClonTech).−1 cm−1. From this presence of approximately 1.5–2 dye molecules per A-subunit were determined.As described by us http://www.Dharmacon.com/). A green fluorescent protein duplex was used as a control oligo for all experiments .Sequences were designed following the parameters set out by Dharmacon by analysing the human genomic sequence for each protein. The following sequences were selected: ARFGAP1, AAG GUG GUC GCU CUG GCC GAA G (siACE-RNAi OPTION C DUPLEX); ARFGAP2, AAG CUA UGG GGU GUU UCU CUG (siACE-RNAi OPTION C DUPLEX); and ARFGAP3, AAC CUA UGG AGU GUU CCU UUG (siACE-RNA OPTION C DUPLEX). All oligos were custom synthesized by Dharmacon and streptomycin (100 μg/mL). siRNAs were used at a final concentration of 20 nm in all transfection experiments. Triple knock-down of all three ARFGAPs and combinations of these with controls were performed in a staggered 10-day protocol, using oligofectamine. For double transfections, paired siRNA oligos were added to 15 nm final concentrations. Cells were seeded on day 0 and transfected on day 1 with either control, single ARFGAP or ARFGAP2 and 3 oligos. On day 2, cells were split into new wells, allowed to adhere to the dish for 6 h, then transfected with either control or ARFGAP1 oligo. On day 3, the media was changed to remove transfection reagents. On day 5, cells were split, allowed to adhere for 6 h and transfected as on day 1. On day 6, the media were refreshed, and on day 7, cells were transfected as on day 2. On day 8, cells were split (some cells were reseeded onto coverslips for 4′-6′-diamidino-2-phenylindole (DAPI) staining. On day 9, the cells rested, and on day 10, cells were collected for analysis. For immunoblot samples, cells washed with PBS, then lysed directly in the plate by addition of 3× Laemmli sample buffer, pipetted out and immediately boiled at 95°C for 5 min. DNA was sheared by passing the samples through a narrow gauge needle and vortexing at maximum speed. Samples were then loaded onto SDS–PAGE gels. Cells growing on coverslips for DAPI staining were washed three times in PBS, then methanol–acetone fixed and mounted on to glass slides in DAPI-containing mounting medium . A similar protocol was used for NRK cells, yielding almost identical results.HeLa M cells were maintained in DMEM medium supplemented with 10% foetal bovine serum, 2 m |
The changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches.2+ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca2+ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization.A rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and CaIschemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation. The fibers were typically <250 µm in diameter and <1.5 mm in length. Prepared fibers were permeabilized for 2 h at 4°C with pCa8.0 relaxing solution containing 1% Triton X-100. To test the impact of cAMP-dependent protein kinase (PKA) activation on perfused and ischemic anterolateral papillary muscle fibers, the cell permeable analog 8-Br-cAMP was included during the permeabilization step.This model was described in detail previously 2+, 10 mM total EGTA, 25 mM total BES, pH 7, and when appropriate, 25 mM creatine phosphate and 15 units/mL creatine kinase. Ionic strength was maintained at 200 mM using potassium methanesulphonate. Activating solutions of varying free Ca2+ were prepared using an iterative program The workstation to test permeabilized fiber contraction has been described previously 2+ relationship, the fibers were activated with solutions of varying free [Ca2+] between pCa8.0 and 4.0. For the measurement of the influence of ADP or Pi on the force-Ca2+ relationship, a separate batch of free [Ca2+] solutions were mixed including either 1 mM added ADP or 5 mM added Pi. All forces were normalized to cross-sectional area with the assumption that fibers were elliptical in cross-section. Data were fit using the Hill equation to determine the concentration of free Ca2+ required for half-maximal force (EC50). Fiber stiffness was measured using four consecutive fiber length releases of 0.05, 0.1, 0.15 and 0.2% F) was measured by a single exponential fit of the force recovery profile following a 5% decrease in fiber length within 3 ms t-test using a Bonferroni adjustment when multiple comparisons were necessary between three surgery groups.To test fiber mechanics, an initial control contraction was recorded from each fiber by changing the bathing solution from pCa8.0 to pCa4.0. To measure the force-CaAct) was determined for perfused, ischemic and ischemia-reperfused fibers. The fiber was placed in a pCa8.0 relaxing solution that was prepared as before except for the substitution of 20 mM PIPES for BES and inclusion of 10 mM dithiothreitol 2+-rigor (kATP) was measured by flash photolysis of NPE-ATP. A fiber in pCa8.0 relaxing solution was transferred to pCa8.0 rigor solution (0 mM ATP) including 1 unit/mL apyrase Act and kATP, were determined from the force transients by single exponential fits.The rate constant of force development upon flash photolysis of NP-EGTA -1 and -2 phosphoforms essentially as described To measure the extent of Ser23/24 phosphorylation of TnI, a multiplex Western blotting strategy was used. Samples resolved by SDS-PAGE were transferred to PVDF membrane and incubated simultaneously with a mouse anti-cardiac muscle TnI monoclonal antibody and a phospho-Ser23/24 TnI rabbit polyclonal antibody . Following washing, the blots were incubated simultaneously with Cy3 labeled anti-mouse IgG and Cy5 labeled anti-rabbit IgG (GE Lifesciences). Blots were scanned on a Typhoon 9410 imager at the appropriate channels, and the scanned images were analyzed using ImageQuant TL software. The relative extent of Ser23/24 phosphorylation of TnI was determined to be the Ser23/24 phosphospecific antibody signal divided by the total TnI mAb signal for each sample. To determine the extent of Ser23/24 phosphorylation of TnI in perfused versus ischemic hearts, homogenates of the anterolateral papillary muscle from five perfused hearts and five ischemic hearts were resolved on a single gel and blotted. The extent of Ser23/24 phosphorylation in the ischemic samples was reported relative to the perfused samples. To determine the effect of 1 mM 8-Br-cAMP treatment on Ser23/24 phosphorylation of TnI in perfused and ischemic rat hearts, ten fibers from the anterolateral papillary muscle were dispersed into two groups, with one group receiving 8-Br-cAMP treatment during permeabilization and the other group remaining untreated. All ten samples from each individual rat were resolved on a single gel and Western blotted to measure the extent of Ser23/24 phosphorylation of TnI. The extent of phosphorylation in the five treated samples was reported relative to the untreated samples. The experiments were repeated with an independent pair of rats, resulting in measurements from two perfused rat hearts and two ischemic rat hearts.The overall phosphorylation state of the thick filament associated protein myosin binding protein-C (MYBP-C) was examined by sequential Pro-Q Diamond phosphoprotein and Deep Purple total protein staining as previously described in vivo rat model was used to determine the effect of myocardial ischemia-reperfusion on the maximum force per cross-sectional area (Fmax), maximum fiber stiffness or the force – Ca2+ relationship of permeabilized small fiber bundles isolated from the anterolateral papillary muscle. Following thirty minutes of ischemia, Fmax fell from 50.1±2.5 mN/mm2 (n = 12) to 36.1±2.4 mN/mm2 , which increased significantly in ischemic fibers to 3.61±0.11 µM (n = 8) compared to 3.32±0.13 µM in perfused fibers (n = 7). This loss of force during ischemia was partially recovered by reperfusion to 41.3±5.2 mN/mm2 (n = 5) whereas the EC50 of reperfused fibers was 3.57±0.12 µM (n = 5). The Hill coefficients were not different for any of the conditions tested (data not shown). To determine if the change in Fmax correlated with a change in crossbridge attachment, we measured maximum stiffness per cross-sectional area based on the assumption that measured changes in stiffness would be proportional to the number of attached crossbridges 2/mm (n = 7) to 350.2±40.5 mN/mm2/mm (n = 8) with ischemia (P<0.016), and there was not full recovery with reperfusion .An n = 12; . A subse2+ relationship of perfused, ischemic and ischemia - reperfused fibers in the presence of 1 mM added ADP. In agreement with previous studies, the addition of 1 mM ADP decreased the EC50 of fibers from the three surgery groups We probed the ADP-dependent transition of the actomyosin crossbridge cycle by steady state measurements of the force - Ca2 to 35.9±3.0 mN/mm22.The phosphate-bound transitions of the crossbridge cycle were examined at steady state by similar experiments. As expected, the addition of 5 mM Pi decreased the Fmax of perfused fibers from 50.1±2.5 mN/mmP<0.016). These data were complemented by measurements of the rate constant of force redevelopment, kF, following a length release F was significantly reduced in ischemic (19.3±0.8 s−1) and ischemia – reperfused fibers (20.7±0.6 s−1) as compared to perfused fibers (25.9±1.1 s−1). For all fibers, increasing [Pi] increased the rate of force redevelopment, but ischemic fibers had significantly reduced rates at all Pi concentrations tested, whereas ischemia – reperfused fibers were intermediate (F = k1+(k−1 [Pi])/(K2+[Pi]) according to Scheme 1 1/k−1) whereas K2 is the equilibrium constant for the Pi release step:We measured the effect of Pi on the Fmax of perfused, ischemic and ischemia-reperfused fibers to further explore the steady state response to exogenous Pi. We hypothesized that if the relative insensitivity of ischemic fibers to 5 mM Pi is reflective of a change in the rate of the transitions between Pi-bound states, then the effect should manifest at other Pi concentrations. Fibers from a new cohort of rats were activated at pCa4.5 and then moved to pCa4.5 solutions containing 20 and 30 mM Pi. When compared to zero added Pi, relative force declined to 61.3±1.7% (n = 14) of maximum in ischemic fibers as compared to 54.2±0.9% (n = 17) and 50.0±1.9% (n = 12) in perfused and reperfused fibers. This relationship was similar at 30 mM Pi, wherein the relative force maintained was 51.3±1.7% (n = 14) in ischemic fibers as compared to 41.9±0.9% (n = 17) and 40.0±1.5% (n = 12) in perfused and reperfused fibers, respectively. Under all cases, the maintained relative force in ischemic fibers was significantly higher than for perfused and reperfused fibers (rmediate . We fit 1 and k−1 were reduced (18.9±0.8 s−1 and 30.7±1.7 s−1 respectively) versus perfused fibers . Values were intermediate for ischemia – reperfused fibers where k1 was 20.4±0.6 s−1 and k−1 was 37.4±2.9 s−1. The value of K2 determined from the fits was 39.4±4.6 mM for perfused fibers, which was significantly higher than in ischemic fibers but not different from ischemia-reperfused fibers (29.4±3.3 mM).In this scheme, the necessary transition that activates crossbridges from non-cycling to cycling states 2+ release following flash photolysis of NP-EGTA resulted in a rapid rise in force, with a maximum value of 58.0±4.2 mN/mm2 and a rate constant of 22.0±1.2 s−1 (n = 11). Both the force produced and kAct were significantly reduced in ischemic fibers to 33.6±2.9 mN/mm2 and 15.4±1.1 s−1, respectively . For reperfused fibers, the force produced and rate constant of activation were intermediate at 42.1±1.6 mN/mm2 and 19.3±1.6 s−1 (n = 9). As the major difference in rate and force was observed between perfused and ischemic fibers, the NP-EGTA activation data were complemented with NPE-ATP activation data for perfused and ischemic fibers in Ca2+-rigor. Force produced by transferring perfused and ischemic fibers from relaxing to Ca2+-rigor solution was 18.4±2.2 mN/mm2 and 11.9±1.5 mN/mm2, respectively (P<0.05). Following photolysis of NPE-ATP, there was a small, brief drop in force prior to force activation . Total force produced (Ca2+-rigor + active) in these experiments was 55.6±5.8 mN/mm2 (n = 13) for perfused fibers and 35.3±3.5 mN/mm2 (n = 12) for ischemic fibers. However the rate constant of force activation from Ca2+-rigor, kATP, was not different between perfused and ischemic fibers, measuring 58.5±3.0 s−1 (n = 13) and 56.3±2.9 s−1 (n = 12) respectively and the percentage of diphosphorylated TnT decreased to 60.3±2.9% (The levels of phosphorylation of MLC-1 and MLC-2 were determined in perfused and ischemic fibers. Both MLC-1 and MLC-2 were present largely as nonphosphorylated and singly phosphorylated forms 0.3±2.9% .P<0.05). These data were complemented by an examination of the phosphorylation state of MYBP-C as determined by phosphoprotein and total protein staining (P<0.05). As both TnI and MYBP-C are downstream targets of β-adrenergic signaling P<0.05). However, 8-Br-cAMP treatment of anterolateral papillary muscle fibers from ischemic rat hearts did not result in an increase in Ser23/24 phosphorylation of TnI. In untreated ischemic fibers, the relative level of phosphorylation was 100±5.9% (n = 10 fibers from two rats) whereas treatment with 8-Br-cAMP resulted in a relative level of 95.2±5.5% whereas k−1 was significantly increased from 36.7±3.0 s−1 to 49.7±3.9 s−1 (P<0.05). In contrast, neither k1 nor k−1 (23.3±1.5 s−1 vs 25.7±2.5 s−1) was significantly influenced by 8-Br-cAMP treatment of ischemic fibers. Compared to untreated perfused fibers, 8-Br-cAMP treated perfused fibers trended towards higher Fmax when activated by pCa4.5 in the presence of 0, 5, 10, 20 and 30 mM Pi. In fibers from ischemic rat, the Fmax at all [Pi] increased significantly following 8-Br-cAMP treatment (P<0.05).To determine the impact of 8-Br-cAMP treatment on the Fmax and force redevelopment in the presence of Pi, 8-Br-cAMP treated and untreated fibers from perfused and ischemic rat hearts were examined . Treatme±3.9 s−1 ; P<0.05.2+ availability 2+] relationships were determined in the presence and absence of exogenous Pi and ADP. These products of ATP hydrolysis influenced the force – [Ca2+] relationship of fibers generally as expected 2+-sensitization of muscle fibers from all surgery groups, although the measured effects were not significantly different between groups. By contrast, Pi decreased the Ca2+-sensitivity of force production in all surgery groups. However, ischemic fibers better retained Ca2+ sensitivity and force in the presence of 5, 20 and 30 mM Pi when compared to perfused and reperfused fibers. This distinct response suggested that the cellular events associated with ischemia impacted the Pi-bound states of the actomyosin crossbridge cycle. In the presence of Pi, the higher Ca2+-sensitivity of ischemic fibers may signal retention of attached crossbridges through near-neighbor thin filament regulatory unit interactions that better preserve the level of activation Deciphering the mechanisms contributing to the decline in contractility of the heart is fundamental to developing new treatment modalities. We have previously demonstrated that permeabilized fibers from ischemic hearts produce submaximal force regardless of maximum ATP or Ca2+ and ATP. Both the maximum force and the rate constant of force activation with Ca2+, kAct, were lower for ischemic fibers as compared to perfused fibers , crossbridges readily cycle between force bearing and non-force bearing states with a higher apparent rate for k1 (Scheme 1), allowing kATP to be faster than both kF and kAct. Therefore, at high Ca2+, thin filaments freely permit re-attachment of detached crossbridges, allowing them to readily transition between force bearing and non-force bearing states 2+ release is best described as a change in the rate of crossbridge activation into cycling states, which itself is a function of the molecular events that facilitate thin filament activation through an inter-related mechanism mediated by Ca2+ and strong crossbridge attachment Act of ischemic fibers is slowed as a result of their reduced Ca2+ sensitivity, which may alter the cooperative recruitment of crossbridges into activated, cycling states.These fiber mechanics measurements therefore allow us to speculate about the steps of muscle activation that limit the magnitude and rate of force production during ischemia. Since koduction . In the han kAct but unaleriments is due tvivoin vivo sites of cardiac muscle TnT phosphorylation. A recent study suggested an in vivo site of phosphorylation within the first 29 amino terminal residues in vitro, with Thr206 phosphorylation having a dominant, depressive effect on fiber contraction in vitro work using in vivo phosphorylated or dephosphorylated TnT will be required to address this mechanism.To begin to decipher the underlying molecular basis for reduced contractility during ischemia, we concentrated on the regulatory troponin complex of the thin filament. Both TnT and TnI of the troponin complex are phosphorylated in 2+ sensitivity under β-adrenergic stimulation 2+ sensitivity with reduced Ser23/24 phosphorylation in this and a previous model −1 without a change in k1 (To examine the possible impact of TnI phosphorylation, we focused on the adjacent residues Ser23 and 24, two sites that are especially relevant for β-adrenergic signaling. This signaling pathway may be altered in heart failure through a reduction in the content of PKA regulatory subunits, reduction in β-adrenergic receptor density, or desensitization of β-adrenergic receptors ge in k1 , which wge in k1 . Nonethege in k1 , 2.et al−1 and force following 8-Br-cAMP treatment of perfused fibers (32P into MYBP-C was significantly less in magnitude than other prominent targets such as TnI and phospholamban 32P incorporation over baseline At this point, it is difficult to determine how the change in MYBP-C phosphorylation contributed to the accumulated results. Previous studies have reported reduced MYBP-C phosphorylation in models of altered myocardial blood flow, suggesting that this is a conserved target d fibers occurredd fibers . Althoug2+ but not ATP is reflective of altered thin filament regulatory function and crossbridge recruitment during ischemia that modulates the apparent rates of activation of the actomyosin crossbridge cycle without impacting the intrinsic activity of the myosin enzyme. The in vivo model demonstrated reduced phosphorylation of TnT and TnI, suggesting that the integrated effect of altered troponin phosphorylation may be involved in the observed contractile response. Future studies will benefit from a focus on the thin filament and the post-translational state of regulatory proteins during ischemia in order to understand their individual and combined contributions to pathophysiological states marked by reduced contractility.In conclusion, the present study demonstrates that the actomyosin crossbridge cycle during ischemia is unique for a reduction in the forward rate constant of the isomerization of the Pi-bound actomyosin transition, which may be closely linked to the transition of non-cycling crossbridges to activated, cycling crossbridges. Additionally, the decrease in the rate of force activation with Ca |
Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material.A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30.The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections. As early-stage tumor sample size is usually small, therefore it is only possible to obtain small amounts of material, for example, fine needle aspiration Multiplexed immunoassays based on protein microarray platforms have been broadly employed in the discovery and validation of disease-associated biomarkers as well as in clinical diagnostics research Multiplexed immunoassay can be performed in a capillary requiring only a minute amount of sample material. Phillips et al. (2007) analyzed the expression of twelve cytokines in dissected tissue lysates. The cytokines were captured by a mixture of immobilized capture antibodies, and subsequently labeled with a flurophore. Captured and labeled cytokines were separated by electrophoresis and the quantification of individual cytokine peaks was performed The schematic set up for µFBI is shown in The relevant parameters of the µFBI were optimized using an artificial capture assay consisting of an immobilized anti-biotin antibody and biotinylated PE as the analyte. The optimal number of antibody coupled beads was defined in a first experiment. 250, 500, 1000, or 2000 beads were pumped into the capillary and incubated for 5 min with Biotin-PE (100 ng/ml). The median fluorescent intensity (MFI) and the number of recovered beads were analyzed . The MFINext, the influence of the coating surface of capillary was investigated. Capillaries with four different physical surface properties were tested for their performance of the µFBI assay . The higThirdly, the optimal assay volume was determined using the Streptavidin - Biotin-PE assay. 1000 anti-biotin antibody coated beads were incubated with Biotin-PE (100 ng/ml) for 5 min by changing the flow rate from 0.1, 0.2, 0.4, 0.8, 1.6 up to 3.2 µl/min. The generated MFI and the number of recovered beads are given in The optimal incubation time was determined using the Streptavidin - Biotin-PE assay. The different incubation periods of the anti-biotin antibody coated beads with 1 µL Biotin-PE (100 ng/ml) were achieved by adjusting the flow rate from 1 µl/min, down to 0.008 µl/min. The results revealed that the MFI value increased with longer incubation times whereas the number of recovered beads decreased below 50 when the incubation time was extended above 60 min . These dFinally, the performance of µFBI assay was evaluated by the quantitative analysis of different biotin-PE concentrations. Anti-biotin antibody coated beads were incubated with 1 µl of Biotin-PE using concentrations ranging from 0 to 5000 ng/ml for 5 min, respectively. As a negative control BSA coated beads of a differently color-coded bead type were used in the same assay. The data are shown in These sets of experiments allowed to define the number of beads (1000), the surface coating (phenyl-silicon), the analyte volume (1 µL), incubation time (60 min) and quantitation ability of the µFBI assay. In a next step, the µFBI was applied to the analysis of Receptor Tyrosine Kinases in tissue lysates.A commercially available bead-based sandwich immunoassay - WideScreenTM Receptor Tyrosine Kinase Assay (RTK) Kit - was selected to evaluate the performance of the µFBI. RTKs are critical regulators of numerous cell signaling pathways. Ligand binding to the extracellular domain of transmembrane RTKs triggers receptor dimerization and autophosphorylation of an intracellular kinase domain. This event ultimately triggers activation of downstream pathway proteins via phosphotyrosine-SH2 domain interactions. RTKs have been shown to be not only key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer Considering the relationship between the assay sensitivity and incubation time as well as influence of flow rate , the capMultiplexing of beads in the µFBI is not limited due to the very low sample volume of only 1 µL. In such a volume 100 different bead types can be incubated simultaneously and subsequently analyzed in a Luminex instrument. A set of 100 different bead types were incubated into the capillary for 1 h mimicking real incubation conditions. After this incubation time beads were pumped out of the capillary and counted in a Luminex 100 instrument. For each bead type a sufficient number of beads above 35 were counted which are sufficient to fulfill statistical requirements, when measuring MFIs .Several aspects have to be considered to perform a successful µFBI experiment. Firstly, bubbles in the capillary have to be avoided, as bubbles severely influence the accuracy of bead numbers or sample volume during the assay. According to our experience, this problem could be significantly minimized by tightly sealing the connection between the microfilter and capillary and a carefully operation. In addition the dead volume of beads or sample has to be controlled. In our experiment, the capillaries were washed thoroughly with washing buffer in between consecutive assay runs. After extensive washing no residual beads could be observed, which allowed us to reuse the setup. In the here presented experimental setup two capillaries were used allowing to perform two assays in parallel. To increase sample throughput the number of capillaries can be increased as described recently by Bainer et al. 6 carboxylated colour coded beads with an diameter of 5.6 µm were obtained from Luminex, Inc. , and were coupled with 25 µg (100 µg/ml) Bovine serum albumin ) or 25 µg (100 µg/ml) anti-biotin antibody according to the manufacturer's protocol. The coupled beads were stored at 4°C in Roche buffer with 0.05% sodium azide. Coupling controls were used to assess the immobilization efficiency by staining 1500 of each type of microspheres with 0, 0.1, 0.3, 1 and 3 ug/ml R-phycoerythrin conjugated goat anti-mouse IgG .2.5×10The µFBI set up consists of a syringe pump, incubation zone and two capillaries . The KDSThe WideScreen™ Receptor Tyrosine Kinase Assay Kit was obtained from EMD Chemicals Inc. . This kit enables the quantification of a set of key RTKs, including EGFR, IGFR, HGFR, PDGFRb, HER-2, VEGFR2, and Tie-2. Breast cancer tissue and normal tissue were kindly provided by Georg Sauer and Helmut Deissler, University hospital Ulm, Ulm Germany Before starting the assay, the capillaries and pumps were rinsed thoroughly to remove any remaining sample or beads with 20 µL 70% isopropanol . 1 µL diluted antibody-coupled beads, 1,000 for each analyte, were pumped into each capillary at a flow rate of 0.2 µl/min. The prepared tissue lysates (200 µg/ml) were then pumped into the capillary and incubated with the mixed antibody-coupled beads for 1 h at a flow rate of 0.016 µL/min. Subsequently, the mixed biotinylated detection antibodies and Strepavidin-Phycoerythrin were pumped into the capillary and incubated with the analytes captured on the beads for 1 h and 45 min, respectively. After the assay was finished, the beads were pumped out of the capillary in a volume of 5 µL and diluted into 100 µL with assay diluent (1X). Finally, the diluted beads were transferred into a 96-well ELISA microplate . Followed by incubation for 5 min on a thermomixer at 650 rpm to mix the beads in solution, the resulting beads were submitted to a Luminex 100 IS system for fluorescent signal readout . The MFI value was extracted to perform data analysis.The assay was carried out according to the instructions given by the manufacturer for the WideScreen™ Receptor Tyrosine Kinase Assay Kit with minor modifications. In short, the MultiScreen HTS™ 96-well filter plate was pre-wetted with assay diluent for 5 min, 100 µL/well. Then, 50 µl diluted (1X) capture beads were transferred to each well. After removing the excess solution by filtration, 100 µL of the same tissue lysates (200 µg/ml) were added to each well to incubate with the beads at 23°C for 1 h on a thermomixer (650 rpm). Subsequently, the beads were washed 3 times with 100 µL washing buffer in each well. Then, 30 µL mixed detection antibodies (1X), 60 µL assay diluent, 30 µL streptavidin-phycoerythrin (1X), 60 µL assay diluent were transferred to each well and incubated at 23°C for 1 h and 45 min on a thermomixer at 650 rpm, respectively. Followed by 3 washing steps, 120 µL assay diluent (1X) were transferred to each well and incubated for 5 min on a thermomixer (650 rpm). After these procedures, the beads were submitted to a Luminex 100 IS system for fluorescent signal readout .The graphs were drawn with Excel 2003 and origin 7.0 . The data of |
Dear Editor,et al.,[We thank Chalisgoankar et al., for haviet al., Our poinWe agree that number of cases and controls was rather less. One of the reasons was that the study was conducted over a short period (21 months) during which we could select only 23 cases and 12 age and sex-matched controls who fulfilled all the requisite inclusion criteria. Patients receiving corticosteroids in any form within last one month or with other conditions which could independently alter endogenous cortisol levels were excluded.Though it appears reasonable to assume that ″endogenous cortisol may not act pathologically unless it is outside normal range″, our study has documented an interesting finding that pathological changes of idiopathic central serous chorioretinopathy (ICSC) occurred even when cortisol levels were within normal range. It, however, clearly showed that mean serum cortisol levels were significantly higher in patients with ICSC as compared to controls. Whereas 12 (52.17%) cases had levels > 500 nmol/L, all controls had lower levels. Moreover, to the best of our knowledge, ours is the only study where serum cortisol levels were tabulated for each and every case.Morning cortisol levels were estimated since cortisol levels follow a relatively predictable circadian rhythm with an early morning peak after awakening, a rapid decrease over the next few hours, followed by a more gradual decline over the course of the day to very low levels by bedtime. A singleThe learned authors have mentioned some interesting observations of their own study. However, since we are not aware of their exact methodological details as well as criteria for exclusion, it would not be appropriate to either compare the two studies or comment on the findings of their study. It may be worthwhile to mention that many a times patients first seek treatment from local practitioners who invariably prescribe steroids in some form. Exogenous steroid intake in any form leads to suppression of the hypothalamo–pituitary–adrenal axis resulting in lowering of endogenous serum cortisol for variable periods of time.Even as the precise mechanism of steroids in the etiopathogenesis of ICSC has not been established, some of their effects on choriocapillaries and retinal pigment epithelium (RPE) have been mentioned in various studies. Glucocorticoids are known to affect choroidal circulation, inhibit We do strongly feel that more, preferably larger studies on endocrinological association of ICSC are needed in order to have a better understanding of this important ophthalmological problem. |
In the indole ring system, the benzene and pyrrole rings are nearly coplanar, forming a dihedral angle of 1.57 (15)°. The cyclohexenone and tetrahydrofuran rings have envelope conformations, while the dithiolane ring adopts a twist conformation. In the crystal structure, pairs of weak intermolecular N—H⋯S hydrogen bonds link the molecules into centrosymmetric dimers with R 2 2(16) ring motifs. Weak C—H⋯π interactions may further stabilize the structure.The title compound, C Å b = 8.4992 (2) Å c = 15.2115 (3) Å V = 2813.47 (11) Å3 Z = 8 Kα radiationMo −1 μ = 0.37 mmT = 294 K 0.35 × 0.20 × 0.15 mm Enraf–Nonius TurboCAD-4 diffractometeret al., 1968T min = 0.913, T max = 0.944Absorption correction: ψ scan (North 8196 measured reflections2289 independent reflectionsI > 2σ(I)1105 reflections with R int = 0.149 3 standard reflections frequency: 120 min intensity decay: 1% R[F 2 > 2σ(F 2)] = 0.041 wR(F 2) = 0.108 S = 0.98 2289 reflections185 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.24 e Å−3 Δρmin = −0.23 e Å−3 Δρ CAD-4 EXPRESS (Enraf–Nonius, 1994CAD-4 EXPRESS; data reduction: XCAD4 (Harms & Wocadlo, 1995SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997WinGX (Farrugia, 1999PLATON (Spek, 2009Data collection: 10.1107/S1600536809006035/xu2478sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809006035/xu2478Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: |
P values in the article, "Employer Adoption of Evidence-Based Chronic Disease Prevention Practices: A Pilot Study," were expressed incorrectly. The P values are located in Table 3 of the manuscript and should have been expressed as "P > .99," not "P < .99." Corrections were made to our website on August 7, 2008, and appear online at www.cdc.gov/pcd/issues/2008/jul/07_0070.htm. We regret any confusion or inconvenience this error may have caused.Because of an editing error, 2 |
In the November 2007 issue of Emerging Infectious Diseases, Gayer et al. . Symbols indicate outbreaks of emerging or reemerging infectious diseases during this period . Circles indicate diseases of viral origin, stars indicate diseases of bacterial origin, and triangles indicate diseases of parasitic origin. CCHF, Crimean-Congo hemorrhagic fever; SARS-CoV, severe acute respiratory syndrome coronavirusGeographic distribution of recent emerging or reemerging infectious disease outbreaks and countries affected by conflict, 1990–2006. Countries in yellow were affected by conflict during this period (source: Office for the Coordination of Humanitarian Affairs, World Health Organization, We agree with Kelly-Hope on the propensity for cholera outbreaks to occur in conflict-affected countries and the need to monitor and respond more effectively to such events. In 2006, cholera was reported from 33 countries in Africa, and 88% of all reported cases were from conflict-affected countries (As highlighted in our November 2007 article on conflict and emerging infectious diseases (Vibriocholerae persists in the environment, little is known about the exact conditions that trigger a cholera outbreak at a particular time. Further elucidation is needed about the factors that influence the duration of an outbreak, disease severity, and duration of individual protective immunity after an episode of cholera.More precise research on cholera and conflict is indeed necessary. However, despite cholera being a disease that has been around for a long time and that causes frequent outbreaks to this day, much information about this disease, beyond its relationship with conflict, remains unknown. For example, although Cholera, which is closely linked to a country’s social and economic development ( |
Introduction. Acute adrenal crisis in relation to nasal steroid overuse has been reported very scantly in English medical literature and remains an underdiagnosed condition. Case presentation. A 55 year-old male presented with altered mental status, retrograde amnesia, fluid refractory hypotension, abdominal pain, fever, and chest pain. Physical examination revealed amnesia, bradypsychia, tachycardia, decreased muscle tone and hyporeflexia. Overuse of nasal steroid was suspected by history. Random early morning cortisol level was < 0.2 mcg/dL. The patient was started on hydrocortisone and within 24 hours he had a full recovery. Conclusion. This one-of-a-kind case describes acute adrenal crisis secondary to withdrawal from inhaled nasal corticosteroids overuse in a patient with particular risk factors. Prevention and early recognition of this disorder can significantly reduce its morbidity and mortality. Every day, emergency departments face patients presenting with peripheral vascular collapse, shock, hypotension refractory to fluid replacement, change in mental status, and/or fever. The diagnosis of acute adrenal crisis is not typically considered, even when the patient is known to have adrenal insufficiency (AI). Without appropriate therapy, it is a potential life-threatening disorder, which usually progresses to coma and death.A 55-year-old Caucasian male presented to the emergency department with altered mental status, generalized weakness, diarrhea, and vomiting. He had been in his usual state of health the previous day, but when his wife returned from an overnight trip, she found the patient in his current state with signs of fecal and urinary incontinence. On arrival, the patient had no signs of trauma, was lethargic with decreased responsiveness, and was unable to recall recent events.The patient has a longstanding history of seizure disorder and takes phenytoin and phenobarbital. Additional history includes hypothyroidism (diagnosed seven years ago), hypercholesterolemia, osteoarthritis, and chronic knee pain. He had craniotomy and brain tumor removal as a child (presumably a meningioma), a recent arthrocentesis, and a right hip replacement. The patient is listed as disabled due to the chronic knee pain, does not smoke or use illicit drugs, and occasionally consumes alcohol. He is known to suffer from seasonal allergies and uses a nasal spray as needed. Two weeks prior, the patient was diagnosed with rhinitis medicamentosa and was prescribed fluticasone propionate (Fp) nasal spray one puff BID.3 cells/mm3, Hemoglobin and Hematocrit of 11.4 g/dL and 32.6%, respectively, and platelet count of 132 × 103 cells/mm3. His liver function test showed AST of 71 U/L, alkaline phosphatase of 371 U/L, and GGTP of 275 U/L; his cardiac enzymes revealed an elevated CPK of 768 U/L and CK-MB of 18.9 U/L EKG was normal. Basic metabolic panel revealed sodium level of 133 mEq/L potassium of 3.4 meq/L; his BUN and Creatinine were 29 mg/dL and 2.3 mg/dL, respectively, and his Glucose level was 89 mg/dL. The urinalysis was positive for blood but no cells were seen, and the toxicology screen was positive for barbiturates. The patient's TSH level was 0.162 mU/L. At this point, a diagnosis of altered mental status, gastroenteritis, acute renal failure secondary to volume depletion, mild rhabdomyolysis from prolonged recumbent position, and a possible postictal state versus phenytoin intoxication was made in this patient. On physical examination, the patient was found to be lethargic and having anterograde and retrograde amnesia, but he was arousable. Heart rate was 100 beats/minute; his blood pressure was 90/60 mmHg; and his respiratory rate was 17 breaths/minute with room-air oxygen saturation of 98%. He was disoriented and was found to have bradypsychia; generalized decreased muscular tone and strength and deep tendon reflexes were noted. The rest of his physical examination was unremarkable. Laboratory studies on admission showed a WBC count of 4.3 × 10After IV hydration, he continued to be hypotensive, lethargic, and disoriented but showed no evident ictal activity. Head CT reported craniectomy with encephalomalacia in the right frontal lobe, but no acute pathology. Brain MRI/MRA and EEG were ordered. Further laboratory results revealed low free T4, a CPK level that had spiked to 1506 U/L, BUN 13 mg/dL, and creatinine 1.2 mg/dL. His calcium was 6.5 mg/dL, and albumin was 2.3 g/dL. These electrolyte abnormalities were corrected. Due to abdominal pain and distention, an abdominal X-ray was ordered revealing a nonspecific bowel gas pattern, moderate left hip degenerative changes, osteopenic bone changes, and right hip arthroplasty .As the first day continued, the patient developed new onset atrial fibrillation with RVR. He was started on digoxin and heparin protocol. Blood cultures were negative and his phenytoin level was relatively low at 7.1 mg/L. Dopamine infusion was required to maintain hemodynamic stability. After reinterviewing the patient's wife, it was learned that the patient had used his prescribed 2 bottles (approx. 16 g each) of nasal spray several times a day until he ran out of medication 3 days prior to admission. It was at this point that AI was suspected. A random early morning cortisol level was ordered and the patient was started on 50 mg of Hydrocortisone intravenously every 8 hours .3 cells/mm3, Hemoglobin and Hematocrit of 10.6 g/dL and 29.3%, respectively, and a platelet count of 117 × 103 cells/mm3. Fecal occult blood test, C. difficile, and fecal leukocyte studies were negative, and his diarrhea stopped. A 2D echocardiogram showed normal ejection fraction. A nonenhanced MRI revealed a large area of encephalomalacia in the right frontal lobe related to previous craniotomy, with no acute underlying pathology is caused by insufficient cortisol secretion from the adrenal gland , 2. AcutThe widespread and habitual use of ICSs for the treatment of asthma and allergic rhinitis may lead clinicians to overlook and underdiagnosis of this condition. An abrupt withdrawal of corticosteroids such as fluticasone may promote an adrenal crisis , 2. Whiln = 24), budesonide (n = 2), and beclometasone dipropionate (n = 5) [There are few literature reports regarding this condition. Todd et al. described AI in a 33-year-old male with difficult-to-control asthma who had been taking 1,000–2,000 mcg/day of Fp for 3 years . In 2005 (n = 5) . Indeed,On the other hand, fluticasone is known to have greater dose-related systemic bioactivity compared with other available inhaled corticosteroids, particularly at doses above 800 micg inhaled via pressurized metered-dose inhaler . Some reAI related to inhaled nasal corticosteroid overuse in adults is an overlooked phenomenon scantly described in the English medical literature. This entity should be suspected in the appropriate clinical scenario whenever patients experience fluid administration refractory shock, especially if we have a history of abrupt withdrawal from glucocorticoid therapy, either oral, systemic, or inhaled. This case also stresses the importance of adequate patient education on medication administration in order to prevent unexpected adverse events. |
Virtual Muscle 3D (VMus3D).Myogenesis is an ordered process whereby mononucleated muscle precursor cells (myoblasts) fuse into multinucleated myotubes that eventually differentiate into myofibres, involving substantial changes in gene expression and the organisation of structural components of the cells. To gain further insight into the orchestration of these structural changes we have overlaid the spatial organisation of the protein components of a muscle cell with their gene expression changes during differentiation using a new 3D visualisation tool: the in vitro, as well as a mouse tissue survey dataset. Visualisation of the expression data in VMus3D yielded novel observations with significant relationships between the spatial location and the temporal expression profiles of the structural protein products of these genes. A muscle specificity index was calculated based on muscle expression relative to the median expression in all tissues and, as expected, genes with the highest muscle specificity were also expressed most dynamically during differentiation. Interestingly, most genes encoding costamere as well as some Z-disk proteins appeared to be broadly expressed across most tissues and showed little change in expression during muscle differentiation, in line with the broader cellular role described for some of these proteins.Sets of generic striated muscle costamere, Z-disk and filament proteins were constructed from the literature and protein-interaction databases. Expression profiles of the genes encoding these proteins were obtained from mouse C2C12 cells undergoing myogenesis By studying gene expression patterns from a structural perspective we have demonstrated that not all genes encoding proteins that are part of muscle specific structures are simply up-regulated during muscle cell differentiation. Indeed, a group of genes whose expression program appears to be minimally affected by the differentiation process, code for proteins participating in vital skeletal muscle structures. Expression alone is a poor metric of gene behaviour. Instead, the "connectivity model of muscle development" is proposed as a mechanism for muscle development: whereby the closer to the myofibril core of muscle cells, the greater the gene expression changes during muscle differentiation and the greater the muscle specificity. Skeletal muscle exhibits organisation and uniformity in anatomical structure between vertebrate species whilst displaying characteristic differences between muscle types as a functional requirement. This structural organisation is due to the sarcomere, the basic contractile unit and core structural component responsible for the striated appearance of skeletal and cardiac muscle fibres ,2. SarcoMyogenesis is an ordered process whereby mono-nucleated muscle precursor cells (myoblasts) fuse into multi-nucleated myotubes that mature into the different classes of myofibre ,7. SucceWe have developed the virtual muscle (VMus3D), an animated 3D computer graphics model to visualise the relative locations of the products of muscle gene expression which characterises striated muscle structure at molecular resolution. To test the novelty of the VMus3D in terms of its ability to contribute to the understanding of myogenesis, we have used it to analyse a number of publicly available muscle gene expression datasets.) and HPRD . The paralogs were included to enable the analysis and address the issue of paralog swapping. Gene expression data for this set of genes was obtained from a mouse C2C12 in vitro muscle cell line before and after differentiation from the NCBI GEO (Gene Expression Omnibus) database [The set of generic mammalian genes encoding the costamere, Z-disk and filament system proteins and their paralogs was built using the published literature and protein-protein interaction databases including BIND of the expression of each of the genes was ascertained by interrogating the SymAtlas mouse GNF1M dataset which contains a tissue survey of mouse gene expression promoting the development of organised sarcomeres, which later integrate muscle-specific proteins to structurally support expanding and contracting myofibres ,22,23. OThere are four major mechanisms for the modification of the structures of multi-subunit complexes: (1) recruitment of entirely new proteins; (2) paralog exchange; (3) alternative splice variants; and (4) modification of existing proteins (such as phosphorylation). Construction of the costamere and Z-disk clearly involves the first process, and in some cases the second, although this is most strongly associated with the filament proteins. The relationship of paralog exchange with location also occurs in developed muscle, whereby the filament system also uses paralog exchange as a fundamental mechanism for altering muscle characteristics and hence function; for example via differential incorporation of myosin heavy/light chains and other proteins to drive fibre type changes. In some circumstances, these paralogs maybe co-expressed within an adult cell during fibre type transitions (due to the hybrid nature of myosin heavy/light chains) but the repertoire of paralogs is reduced compared to those expressed and selected for or against during the transition of cell state during early development, as observed herein . In addiIn general, the construction of the costamere and to a lesser extent the Z-disk, during muscle differentiation involves the recruitment of broadly expressed proteins. However a small number of genes encoding costamere proteins and more Z-disks proteins are induced during differentiation and these proteins may serve to nucleate the construction of muscle specific structures. Our approach using VMus3D and combining gene expression datasets provides a spatial method of gene expression analysis at molecular resolution in the context of the core structural components of a skeletal muscle cell and these findings highlight the utility of this approach.The mouse C2C12 muscle cell line , and in vitro differentiation have been used in a number of studies ,13,32. T[In addition, a mouse GNF1M (MAS5 compressed) dataset was downloaded from the GNF SymAtlas Download portal . This daTo eliminate unnecessary relational querying, these two datasets were combined into a single dataset on the basis of matching gene symbols. The criterion chosen (due to multiple probes possibly representing the same gene) was: for every gene symbol from the C2C12 differentiation dataset to include every matching gene symbol and its relative tissue expression level from the processed SymAtlas dataset. Therefore, all possible tissue expression profiles for any of the probes representing a gene were included. Multiple gene expression profiles for a gene were reduced to a single profile for each gene symbol by selecting only the highest normalised mean gene expression value from C2C12 differentiation dataset and tabulated tissue specificity index.AC3D version 6 was used to build de-convoluted 3D models of the structural proteins and their arrangements within skeletal muscle . The datThese foundation models formed the basis of an internet accessible browser , whereby the models were displayed using X3D (extensible 3D) and web access interaction occurred via its SAI (Scene Authoring Interface) which was supported by Media Machines Flux Player plug-in. Combination of JavaScript, PHP and MySQL databases permitted the querying and compilation of results for real-time injection of data into the X3D scenes, a process known as Ajax (Asynchronous JavaScript and XML) and more recently, Ajax3D when combined with X3D. This injection provided the nodes with the required information to connect with additional internal databases (e.g. to view raw data/different probes/other datasets etc.) and also for connecting to external repositories that relate to the gene expression data chosen.i, two parameters were explored as follows: (1) MMi: its differential expression computed from the log ratio of the normalised gene expression values from myoblast to myotube and; (2) SPi: its skeletal muscle specificity computed from the difference between the normalised mean expression in skeletal muscle and the median expression in the remaining tissues : 2 dimensions belonging to time (myoblast and myotube: GDS2412) and another dimension belonging to the difference between the normalised mean expression of a gene in skeletal muscle and its median gene expression across the remaining tissues sampled (GNF SymAtlas). Available paralogs were automatically extracted from the datasets via a script that retrieved all numerical variants of a gene and selected alphabetical subtypes. The retrieved genes and their paralogs were categorised and colour coded into three groups corresponding to the costamere (blue), Z-disk (red) and filament system (green).MMi) and muscle specificity (SPi) and for each gene in i, and see which genes have relative expression and specificity levels far away from the majority [MMi and SPi were assumed to be independent observations from a two-component mixture model with probability density function:A two-component mixture model analysis was performed on 150 genes to determine if two groups of genes could be classified as distinct clusters based on their differential gene expression from myoblast to myotube and skeletal muscle specificity index observed in the VMus3D and a 2 × 2 covariance matrix V0 (not necessarily identity) and corresponding to non-differentially expressed and non-muscle specific genes;denotes the empirical null bi-variate normal density with 2-dimensional mean vector μ0 and π1 are constrained to be non-negative and sum to unity. In the present study, parameters of the mixture model were estimated using the EMMIX-GENE software [i-th gene is differentially expressed and/or muscle specific is given bydenotes the bi-variate normal density function corresponding to genes that are differentially expressed and/or muscle-specific; finally, the mixing proportions πsoftware . Once thA simulation was also performed by generating 100,000 elements drawn from the estimated two-component mixture model. Genes lacking either skeletal muscle specificity or myogenesis differential expression data were appropriated a probability of belonging to cluster 1 or 2 from their average p-values of the components from the 100,000 simulation that fell into a range of +/-0.1 from the log ratios . Costamere: green; Filament: blue; Z-disk: red.Click here for fileCluster analysis of myoblast to myotube including skeletal muscle specificity index. Initial two clusters calculated. Cluster 1: black dots; Cluster 2; yellow dots.Click here for file |
Chromosomal regions 3p21 and 5q11–q13 were found hemizygously deleted in 46% and 23% of patients respectively. Half of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlapping (SRO) deletions were delimited by markers D3S1298 and D3S1266 at 3p21 and by D5S647 and D5S418 at 5q11–q13. Currently, the genes hMLH1 (3p21) and hMSH3 (5q11–q13) are the only known candidates located within these regions. The consequence of these allelic losses is still unclear because none of the breast cancers examined displayed microsatellite instability, a hallmark of mismatch-repair defect during replication error correction. We suggest that hMLH1 and hMSH3 could be involved in breast tumorigenesis through cellular functions other than replication error correction. © 1999 Cancer Research CampaignTo study the involvement of DNA mismatch-repair genes in sporadic breast cancer, matched normal and tumoral DNA samples of 22 patients were analysed for genetic instability and loss of heterozygosity (LOH) with 42 microsatellites at or linked to |
Psychology is also a popular and important program of undergraduate study for students who then pursue advanced education and training in a wide range of fields, including Medicine. Undergraduate Psychology courses that are popular with premedical students include Biological Psychology and Abnormal Psychology.Psychology became an integral and popular part of the undergraduate curriculum in the United States by the 1920s. It continued to grow in popularity throughout the century and now, in the 21th century, Psychology was not considered a necessary part of medical education and psychologists were considered adjunctive in the practice of Medicine. However, by the last third of the 20th century, the situation had begun to change. In this article, I describe efforts from early in the 20th century to make Psychology a more formal part of medical education and suggest some reasons for why they failed to gain a purchase in the medical curriculum. I then briefly describe some factors that led to a greater acceptance of Psychology in medical training and settings and suggest some reasons for this acceptance. At the beginning of the 21st century, lessons can be drawn from these examples to help articulate a viable role for psychologists in an integrated health-care system, both in India and in the United States.Even with the growth and popularity of Psychology and its increasing relevance for health care, there has been resistance to incorporating it in the training of physicians. For most of the 20th century, there were many opportunities for interaction between psychologists and the medical profession in the United States.[In the first half of the 20d States. In a fewd States. The questh century were a period of social change in American history. The social, political and professional order was transformed; new scientific disciplines were formed and professions were reformed. For scientific psychologists at the beginning of the 20th century, one point of potential application for their discipline was through an alliance with the medical profession.The years around the turn of the 20th century. This resulted in greater success in the treatment of some diseases and a higher status for medicine in American society. The medical sciences became increasingly centered on the laboratory as the locus of their work.[The practice of medicine was placed on a more scientific basis in the last quarter of the 19eir work.5 Medicaleir work. The reoreir work. The ideaeir work. In this What did physicians think about psychology as part of medical training? Physicians who wrote about this issue wanted a psychology that was suited to their needs, practical and applicable to patient care. Perhaps a typical example of this came from George Dearborn of Tufts Medical School in the Boston, Massachusetts, area. What the medical curriculum needed from psychology, in Dearborn's view, was two things: 1) an explication of normal mental processes so that a physician could recognize a deviation from the normal and 2) an explanation of the psychological factors in the relationship of doctor and patient, especially as this affects disease and recovery from disease. Dearborn believed that a psychology able to make these contributions would be welcomed into the reformed medical curriculum.10What did psychologists think about psychology in the training of physicians? Psychologists who addressed this issue typically wanted to introduce some version of standard experimental psychology into the training of medical students, while also contending that psychology could benefit both physicians and their patients by helping them understand the psychological aspects of daily life.After the Flexner report was published in 1910, there were several years of intense debate and struggle over the medical curriculum. It was in this period that psychologists made their strongest case for inclusion of psychology in the curriculum. Despite their efforts, psychology did not become a basic course of instruction at this time.The medical and psychological literature of the time had numerous articles pro and con in the debate over inclusion. For example, a special session on the issue was held at the 1911 meeting of the American Psychological Association (APA), with psychologists, psychiatrists and neurologists involved. All agreThe issue was important enough that surveys were conducted to ascertain what was being done in medical education vis-à-vis psychology. In 1913, E. Stanley Abbot surveyed 85 medical schools. Only four schools required a course in normal psychology, while thirty ignored it altogether. Two schools required a course in abnormal psychology and two offered such a course as an elective. Eleven schools offered instruction in psychology in other courses, while seven planned to do so. Abbot found that the total number of schools requiring, advising or teaching psychology was 26 out of the 58 that replied. Of course, as Abbot noted, these numbers did not indicate the type, the quality or the quantity of psychological instruction. They did indicate that psychology was not ignored in the competition for curricular space. What was clear from this survey was that psychology was not considered basic to medical training.[The APA formed a Committee on Psychology and Medical Education that sent out a survey in 1913 to medical schools in order to discover interest in psychology, the amount of instruction currently given to psychological topics, what amount, if any, of such instruction was planned for the near future and who did the instruction (psychologists or physicians). The Committee received replies from 71 out of 114 medical schools. Overall, the results were discouraging for psychologists. Few schools offered instruction in psychology, although ten planned to do so in the near future. Additionally, there appeared to be great confusion on the part of those replying as to what was meant by psychology, which reflected the confusion within the discipline itself. The one encouraging note came from the data from the highest-ranked schools. Several of these schools already offered instruction in some form in psychology, while several more planned to do so.[Psychologist Fred Wells argued that the onus was on psychology to provide medical students with “something they can use.” Medicine, he pointed out, deals with problems of human suffering, while psychology is concerned with scientific investigation of psychological phenomena not necessarily related to medical problems. Unlike most other psychologists who wrote about this issue, Wells argued that it was useless for psychologists to expect medical schools to include a psychology that was not oriented to the practical needs of medicine.When seen within the larger context of the reform of medical education and the contested content of the medical curriculum, the suggestions of psychologists and psychiatrists can be understood as united in their plea for a place for psychology in the medical curriculum. These participants all were invested in the importance of psychological instruction in the medical school.Why then did they not succeed? Two reasons stand out. First, psychologists and their partners in psychiatry and neurology were marginal to the leaders of medical education. At the time, psychiatrists and neurologists were not as well respected as they later became. Psychologists and their allies were bargaining from a position of weakness in the debate over the extension of the medical school curriculum to include psychology. The medical reform leaders, like Abraham Flexner and William Henry Welch, did not see psychology as crucial to medical science or practice. When theth century.[The role of psychology in medical training in the USA did become a reality much later in the 20 century.17 What fAs psychologists developed sound clinical skills in the years after World War II, they were able to gradually add clinical contributions to their research work in health and disease. In settiAfter the early failure to find a place in formal medical training, psychologists took advantage of the opportunities that emerged from their past experiences to move in the new directions that emerged as the social and intellectual ecosystem of which they were a part shifted and changed. In the current flux over the provision of health care, new opportunities exist for psychologists as a vital part of an integrated health-care system. If psychologists are able to learn from their own history how to forge effective alliances and how to engage other health-care professions and health policy makers from a position of demonstrated excellence, then the future is bright for psychology in health care, whether in the United States or in India. |
Subsurface fluids from deep-sea hydrocarbon seeps undergo methane- and sulfur-cycling microbial transformations near the sediment surface. Hydrocarbon seep habitats are naturally patchy, with a mosaic of active seep sediments and non-seep sediments. Microbial community shifts and changing activity patterns on small spatial scales from seep to non-seep sediment remain to be examined in a comprehensive habitat study.Beggiatoa spp. mat at Mississippi Canyon 118 (MC118) in the Gulf of Mexico. High process rates within the mat (∼400 cm and ∼10 cm from the mat's edge) contrasted with sharply diminished activity at ∼50 cm outside the mat, as shown by sulfate and methane concentration profiles, radiotracer rates of sulfate reduction and methane oxidation, and stable carbon isotopes. Likewise, 16S ribosomal rRNA, dsrAB (dissimilatory sulfite reductase) and mcrA (methyl coenzyme M reductase) mRNA transcripts of sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae) and methane-cycling archaea (ANME-1 and ANME-2) were prevalent at the sediment surface under the mat and at its edge. Outside the mat at the surface, 16S rRNA sequences indicated mostly aerobes commonly found in seawater. The seep-related communities persisted at 12–20 cm depth inside and outside the mat. 16S rRNA transcripts and V6-tags reveal that bacterial and archaeal diversity underneath the mat are similar to each other, in contrast to oxic or microoxic habitats that have higher bacterial diversity.We conducted a transect of biogeochemical measurements and gene expression related to methane- and sulfur-cycling at different sediment depths across a broad The visual patchiness of microbial mats reflects sharp discontinuities in microbial community structure and activity over sub-meter spatial scales; these discontinuities have to be taken into account in geochemical and microbiological inventories of seep environments. In contrast, 12–20 cm deep in the sediments microbial communities performing methane-cycling and sulfate reduction persist at lower metabolic rates regardless of mat cover, and may increase activity rapidly when subsurface flow changes. Beggiatoa spp. These large, filamentous bacteria can be white, yellow, or orange and form extensive microbial mats with diameters of up to several meters, which cover the seafloor at methane seeps and hydrate sites in complex, patchy patterns In deep-sea hydrocarbon seeps, fluids that originate from thermal maturation of deeply buried fossil organic carbon seep into the upper sediment column, where they often solidify into methane-rich hydrates and may contribute to global climate forcing in episodic releases Beggiatoa spp. mats are often used as visual locators of active hydrocarbon seeps and seep-related microbial communities Beggiatoa spp. mat at a hydrocarbon seep in the Gulf of Mexico (Mississippi Canyon 118) (yon 118) . This hadsrAB) for sulfate reduction mcrA) for methanogenesis and possibly also anaerobic methane oxidation 14C-fixation rates mcrA is closely linked to metabolism in both Methanococcus vanielii, where mcrA has a maximum half-life of 15 minutes Methanosarcina acetivorans, where mutants can nonetheless arise that are capable of constitutive expression dsrAB genes is also coupled to sulfate-reducing activity in sediments Desulfobacterium autotrophicumEstablishing microbial activity with analysis of nucleic acids in the environment is difficult since DNA from inactive cells may be stable in cold anoxic sediments dsrAB and mcrA mRNA, as well as bacterial and archaeal 16S rRNA, were analyzed in conjunction with DNA-based V6-tag sequencing, porewater concentrations of methane and sulfate, radiotracer measurements of sulfate reduction and methane oxidation rates, and stable carbon isotopic values of methane to describe the spatial structure and activity patterns of sediment microorganisms with respect to Beggiatoa spp. mat location and hydrocarbon seep geochemistry at MC118.Sequences of reverse-transcribed in situ pressure.Steep sulfate and methane gradients were observed directly under the mat as well as at its edge . Sulfate13C-depletion trends in the Mat-A and Edge cores also indicate methanogenesis . Mat-A wbacteria . Some ofbacteria , Fig. S3bacteria , Fig. S3Similarly to the RT-PCR clone libraries, the V6-tag sequences (available from 12–15 cmbsf inside and outside the mat) were dominated by members of the Deltaproteobacteria . Deep sadsrAB transcripts were recovered from all depths and the majority of them were related to the uncultivated Cluster B group dsrAB transcripts are found only in the surface under the mat and include uncultured Group IV Desulfobacterium anilini. No dsrAB transcripts of the Desulfobulbaceae were detected, even though they were present in the 16S rRNA transcript libraries. Primer bias most likely explains this result; one of the internal dsrAB primers used in this study had between three and four mismatches to cultured members of the Desulfobulbaceae . Outside the mat bacterial community richness was higher than that of archaea; it peaked in surface sediments outside the mat and decreased with depth outside the mat. All samples from under the mat had lower bacterial diversity than those from outside the mat. Archaeal community richness peaked at 12–15 cmbsf outside the mat. No consistent trends were seen for archaea relative to depth or presence of overlying mat.dsrAB transcripts , ANME populations could switch to methanogenesis, as has been suggested previously Deeper in the sediments underneath the mat, the tight spatial coupling between active microbial community and measured geochemical processes is absent . The traUnlike surficial sediments, no large-scale changes were observed in the active microbial communities at 12–15 cmbsf between mat, edge, and outside cores . This deBeggiatoa spp. mat show decreased diversity, possibly a consequence of strongly reducing conditions in sulfate-reducing and methane-cycling sediments. The archaeal diversity trends do not show this strong contrast between sediments underneath the mat and outside the mat. Archaeal diversity peaked at 13.5 cmbsf outside the mat, suggesting an additive effect of overlapping surface and seep archaeal communities; 16S rRNA clone libraries show that pelagic Marine Group I archaea and anaerobic ANME archaea were both present. Under the mat, the active archaeal and bacterial communities showed similar diversity (Chao1 indices of 9 to 63 for bacteria vs. 8 to 42 for archaea), in contrast to the oxic sediments outside the mat that strongly favored bacterial diversity. In fact, three out of the five clone libraries under the mat and at its edge had higher archaeal than bacterial diversity. To our knowledge, this is the first documentation of higher archaeal than bacterial diversity in marine sediments. Bacterial diversity has generally been found to be much higher than archaeal diversity in a given environment Among the many possible controls that affect bacterial and archaeal diversity, we consider the roles of electron acceptor and carbon substrate availability in determining relative bacterial and archaeal diversity. Bacterial diversity was highest at 1.5 cmbsf outside the mat, suggesting the combined effects of pelagically-derived organic matter and energetically advantageous electron acceptors such as oxygen and nitrate. By comparison, all bacterial samples under the Our molecular analysis of bacterial and archaeal community structure and stratification has focused on the level of gene expression via RNA, not gene presence via DNA. Environmental microbial communities are most often studied by extracting, amplifying, and sequencing bulk DNA dsrAB mRNA, as detectable by our primers. Unless these particular SRB have special adaptations for post-translational control of DSR protein, it is likely that they were not actively reducing sulfate. Given that the intracellular lifetime of mcrA mRNA molecules is on the scale of minutes and that of dsrAB is on the scale of days in laboratory cultures Indeed, we found that mRNA was more sensitive to environmental conditions than rRNA. In surface sediments outside the mat a few 16S rRNA transcripts from SRB were present, but these were not accompanied by Apparent changes in diversity might be impacted by detection issues. For example, rare groups that are still detected in sediments with high biomass may fall below detection level in sediments that have a lower biomass, resulting in erroneously low diversity indices Even if we assume equal detection sensitivity for different bacterial and archaeal groups, clone library representation can be read only as a relative, not as an absolute measure of their abundance. For example, what appears to be an increased contribution of sulfate reducing bacteria and ANME archaea at 12–15 cmbsf compared to the surface layer outside the mat, may instead reflect decreased contributions of surface-layer bacteria and archaea at depth, leaving SRB and ANMEs to comprise a larger percentage of the 12–15 cmbsf community. Finally, the 16S rRNA primers for bacteria and archaea are in principle subject to primer bias and mismatch problems Beggiatoa spp. mats are occasionally observed and recorded on JSL 2006 dive tapes, but an extensive video survey suggests that they cover only a small proportion of the seafloor, mostly in the northwestern crater of MC118, and to a lesser extent in the southeastern area One of the most interesting implications of this mat study is the extremely uneven spatial distribution of mat-associated microbial processes in surficial sediments at seep sites. Microbial mats cover only a small fraction of the total sediment surface area at methane and hydrocarbon seep sites. At MC118, −2 d−1; they drop to 2.1±0.8 outside the mat (Beggiatoa spp. mats with no tubeworms (26.9±25.9 mmol m−2 d−1) −2 d−1) and methanogenesis rates as well for this site, as no others of this size have been documented. A photomosaic survey of a limited area near the sampling site indicate the mat . These v as well . Thus, mBeggiatoa spp. mat suggest that the boundaries of rising methane-and hydrocarbon-rich fluids are delineated by overlying mat cover. Rates at the center and edge of the mat are nearly identical, and then drop sharply less than a meter outside the mat. These clear geochemical boundaries are reflected in the compositions of the active surface microbial community, with consistent community compositions of active sulfate reducers and methane-cycling microorganisms in the center and edge of the mat, but a large drop in their RNA expression levels immediately outside the mat.High radiotracer rates of sulfate reduction, and methane oxidation, as well as steep methane and sulfate gradients in the center and edge of the Beggiatoa spp. mats. As a result, microbial mat formation and the establishment of a sulfur-and methane-cycling, mat-associated microbial community in surficial sediments would be rapid and accessible to continuous in-situ observation over days and weeks The deeper microbial communities outside the mat, however, look more similar to those under the mat. Therefore visually undistinguished sediments without conspicuous mat cover (and no porewater evidence for hydrocarbon seepage) can still harbor anaerobic methane- and sulfur-cycling communities that express genes for metabolic activities, but remain below detection limit in the geochemical measurements. High levels of sulfate reduction and methane oxidation in these sediments could resume quickly at the onset or reintroduction of active seeping, resulting in sulfide production and the rapid development of These results validate that the often-observed patchiness and small-scale spatial architecture of microbial mats and methane seeps correspond to a profound reorganization of microbial community composition, activity patterns and geochemical imprint on spatial scales of tens of centimeters both vertically and horizontally in the sediments. Microbial mats play an important role as indicators of subsurface microbial heterogeneity and activity, a role proposed previously for seafloor fauna Mississippi Canyon Block 118 (MC118) in the Gulf of Mexico is characterized by seafloor-breaching methane hydrate deposits and thermogenic hydrocarbon-rich fluids pushed upwards through fractures in the sediments by salt domes The mosaic was generated from a self-contained digital still stereo camera package developed at the Australian Centre for Field Robotics. The camera system was mounted on the Johnson-Sea-Link II and used to acquire 12 bit, 1.4 Mpixel imagery at 1 Hz from an altitude of 3 to 4 m from the seafloor. The speed of the submersible was such that high overlap (over 75%) was typical. The imagery was assembled into a composite view using the approach described in 13C (‰) = [R(sample)/R(PDB standard)–1]*1000, where R is the ratio of the heavy to light isotope relative to the Pee Dee Belemnite standard 13C of methane with depth.For sulfate measurements, plastic 15 ml tubes filled completely with sediment were centrifuged and the resulting porewater was filtered at 0.2 µm, acidified with 10% HCl, and measured shipboard using a 2010i Dionex ion chromatograph , as previously described O is molecular diffusivity, CPW is the concentration of the solute in sediment porewater, x is the depth interval in the sediment, ω is the sedimentation rate, α is the bioirrigation coefficient, CPW is the concentration of the solute in the overlying water, and RPW is the reaction rate of the porewater constituent. The first term in the equation accounts for molecular diffusion, the second for sedimentation and compaction, the third for bioirrigation, and the fourth for reaction rate.A 1-D, inverse, reaction-transport model was used to compare concentration profiles to radiotracer rate measurements based on the following equation Total RNA was extracted following previously described methods dsrAB transcript cDNAs were amplified with DSR1f-DSR4r mcrA transcript cDNA were amplified with ME1-ME2 Bacterial 16S rRNA cDNAs were amplified with B8f-B1492r Each 25 µl RT-PCR reaction contained 1 µl RNA template, 0.15 µl each primer solution (100 pmol/µl), 1 µl bovine serum albumin , as well as the following products from the Takara OneStep RT-PCR kit Version 3.0: 12.5 µl buffer, 0.5 µl RNase inhibitor, 0.5 µl HotStar Taq, and 0.5 µl reverse transcriptase. Each 25 µl nested PCR reaction contained 1 µl cDNA template, 0.15 µl each primer solution (100 pmol/µl), 1 µl bovine serum albumin (10 mg/ml), 4 µl deoxynucleotide triphosphate , 2.5 µl 10× FastBuffer I (Takara), and 0.125 µl SpeedStar Taq (Takara).dsrAB mRNA cDNA and mcrA mRNA cDNA, each consisting of 5 s denaturation at 95°C, 15 s at primer annealing temperature (see above), and 20 s elongation at 72°C, plus a final elongation at 72°C for 10 min. Nested PCR for dsrAB required the following protocol: 94°C polymerase activation for 2 min, followed by 40 cycles of 98°C denaturation for 10 s, 48°C annealing for 15 s, and 72°C extension for 20 s, plus a final elongation at 72°C for 10 min. All PCR and RT-PCR products were purified using either a MoBio PCR Clean-up kit or purification in a 1% agarose gel and MoBio UltraSpin for gel purification. Purified products were cloned using the TOPO TA PCR cloning Kit, and transformed into E. coli by electroporation following the manufacturer's protocols . Sequences were obtained at the Josephine Bay Paul Center at the Marine Biological Laboratory , using an ABI 3730 sequencer, or at Genewiz on an ABI Prism 3730xl sequencer. Vector and primer sequences were removed from sequences and forward and reverse reads were assembled into contigs using Sequencher 4.7. Ribosomal sequences were aligned against the 2007 Silva release with ARB (www.arb-home.de). Sequences were deposited in NCBI Genbank with accession numbers GU190968-GU191015 for archaeal 16S, GU302419-GU302497 for bacterial 16S, GU302498-GU302509 for mcrA, and GU302510-GU302521 for dsrAB.Conditions for RT-PCR in a Bio-Rad iCycler were as follows: reverse transcription at 42°C for 15 min, reverse transcriptase inactivation and polymerase activation at 95°C for 2 min, followed by 25 cycles for bacterial 16S rRNA cDNA and archaeal 16S rRNA cDNA and 40 cycles for www.vamps.mbl.edu; see also 17). Quality control included removing sequences with ambiguous base calls, or ones that did not match the primers perfectly http://vamps.mbl.edu as the following datasets: GMS_0003_2006_09_14 , GMS_0004_2006_09_14 , GMS_0005_2006_09_14 and GMS_0006_2006_09_14.DNA was extracted from 0.5 g of sediment using the MoBio DNA Power Soil Kit . Using the methods of the International Census of Marine Microbes (ICoMM), the variable 6 (V6) region of the 16S rRNA gene was amplified and subjected to 454 pyrosequencing on a Roche GS20. All PCR methods, primers and analysis tools are detailed on the ICoMM website these PCR products were gel purified alongside concentrated RT-PCR products used to guide the cutting of the invisible bands, and 2) two of the three No RT clone libraries were made with www.arb-home.de), and phylogenetic groups were determined. Only full-length reads were included in the phylogenetic trees, which were made in PAUP. Chao1 diversity estimates were calculated using the methods of DOTUR Chao1) were calculated with a bias correction for the presence of singletons as SChao1 = Sobs+n1 (n1−1)/(2*(n2+1)), where Sobs is the observed number of species, n1 is the number of OTUs with only one sequence, and n2 is the nmber of OTUs with only two sequences. Chao1 is a method for predicting actual diversity, assuming that only a subset of the total population has been sampled; and works well at a low average sample capture probability Operational taxonomic units (OTUs) were determined by aligning 500–600bp of each forward read in ClustalX, and grouping into 99% similar OTUs using a distance matrix generated in PAUP4.b10 Table S1Comparison of depth-integrated sulfate reduction and methane oxidation rates (mmol m-2 d-1) to concentrations fluxes of sulfate and methane (mmol m-2 d-1), respectively.(0.04 MB DOC)Click here for additional data file.Table S2Primer sequences used in the study, their annealing temperatures, target groups, and known mismatches.(0.07 MB DOC)Click here for additional data file.Figure S1Model fit to sulfate concentration data (red line), or sulfate reduction rate data (blue line) for Edge core. Yellow markers are the data from (9.28 MB TIF)Click here for additional data file.Figure S2Neighbor-joining tree of cDNA of full-length Deltaproteobacterial 16S rRNA sequences for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. OTUs are based on 98% similarity. Sequences from dive 3570 are in colors corresponding to those of (10.24 MB TIF)Click here for additional data file.Figure S3Neighbor-joining tree of cDNA of full-length non-Deltaproteobacterial 16S rRNA sequences for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. OTUs are based on 98% similarity. Sequences from dive 3570 are in colors corresponding to those of (0.66 MB TIF)Click here for additional data file.Figure S4Comparison of Blast hits for sequence tags from V6 tag pyrosequencing and 16S rRNA sequences from RT-PCR clone libraries for 2 samples . Shown are 100% bar charts for A) bacteria and B) archaea.(9.72 MB TIF)Click here for additional data file.Figure S5Neighbor-joining tree of amino acid translations of dsrAB transcripts for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. Sequences from dive 3570 are in colors corresponding to those of (5.47 MB TIF)Click here for additional data file.Figure S6Neighbor-joining tree of cDNA of full-length archaeal 16S rRNA sequences for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. OTUs are based on 98% similarity. Sequences from dive 3570 are in colors corresponding to those of (5.38 MB TIF)Click here for additional data file.Figure S7Neighbor-joining tree of amino acid translations of mcrA transcripts for all samples. The nodes are labeled with parsimony-based boostrap values (1000 repetitions) that were over 60%. Sequences from dive 3570 are in colors corresponding to those of (7.09 MB TIF)Click here for additional data file. |
We therefore addressed the involvement of mGluR1 and mGluR5 in long-term potentiation (LTP) and long-term depression (LTD) in the hippocampal CA1 region of adult male rats in vitro. The mGluR1 antagonist (S)-(+)-α-amino-4-carboxy-2-methylbenzene-acetic acid (LY367385) impaired both induction and late phases of both LTP and LTD, when applied before high-frequency tetanization or low-frequency stimulation , respectively. Application after either HFT or LFS had no effect. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), when given before HFT, inhibited both the induction and late phases of LTP. When given after HFT, late LTP was inhibited. MPEP, given prior to LFS, impaired LTD induction, although stable LTD was still expressed. Application after LFS significantly impaired late phases of LTD. Activation of protein synthesis may comprise a key mechanism underlying the group I mGluR contribution to synaptic plasticity. The mGluR5 agonist -2-chloro-5-hydroxyphenylglycine (CHPG) converted short-term depression into LTD. Effects were prevented by application of the protein synthesis inhibitor anisomycin, suggesting that protein synthesis is triggered by group I mGluR activation to enable persistency of synaptic plasticity. Taken together, these data support the notion that both mGluR1 and mGluR5 are critically involved in bidirectional synaptic plasticity in the CA1 region and may enable functional differences in information encoding through LTP and LTD.The group I metabotropic glutamate receptors, mGluR1 and mGluR5, exhibit differences in their regulation of synaptic plasticity, suggesting that these receptors may subserve separate functional roles in information storage. In addition, although effects Group Iin vivo may be mediated by alterations in intracellular calcium release or disruption of other mGluR1-mediated processes such as depolarization of CA1 pyramidal neurons, depression of the slow afterhyperpolarization (In previous work we have demonstrated that selective antagonism of either mGluR1 or mGluR5 results in a significant impairment of both induction and maintenance of long-term potentiation (LTP) in freely moving adult rats, an impairment that is associated with disruption of spatial memory . The imprization . Transgein vivo are strikingly consistent in hippocampal slice preparation. As group I mGluRs can influence NMDA receptor currents with an oxygenated Ringer's solution at 35 °C. Following 30 min equilibration, the slices were submerged by filling the chamber to a volume of 3 mL with warmed (35 °C) O2/CO2 Ringer's solution. The flow rate was then adjusted to 0.8 mL/min.Immediately after preparation, slices (400 µm) were placed on a nylon net in a 2-mL circulation chamber at the interface between the incubation medium and a humidified atmosphere of 95% OMonopolar platinum-tipped silver chloride electrodes were positioned in the stratum radiatum of the CA1 region for stimulation and in the CA1 dendritic area for recording . For each time point, five evoked responses were averaged. The slope of the field excitatory postsynaptic potential (fEPSP) was measured as the maximum slope through the five steepest points obtained on the first positive deflection of the potential. By means of input–output curve determination the maximum fEPSP slope was found for each individual animal, and all potentials employed as baseline criteria were evoked at a stimulus intensity which produced 40% of this maximum.LTP was induced from one stimulation input only; the other input was used to generate test-pulse responses. LTP was induced with high-frequency tetanization comprising three stimulus trains, at 5-min intervals, of 100 pulses. Short-term potentiation (STP) was induced with HFT (100 Hz) comprising one stimulus train of 100 pulses. Persistent LTD was induced with LFS at 2 Hz (1200 pulses) whereas short-term depression (STD) was induced with LFS at 1 Hz (900 pulses).The metabotropic glutamate receptor antagonists -2-chloro-5-hydroxyphenylglycine (CHPG), LY367385 and 2-methyl-6-(phenylethynyl)pyridine (MPEP) were obtained from Tocris Cookson, Bristol, UK. The protein synthesis inhibitor anisomycin was obtained from Sigma-Aldrich, Germany.anova with repeated measures, followed by post hoc Student's t-tests. Within-group and between-group analysis was conducted. The probability level interpreted as statistically significant was P < 0.05.The baseline fEPSP data were obtained by averaging the response to stimulating the Schaffer collaterals, to obtain five sweeps at 0.025 Hz, every 5 or 15 min as described above. The data were then expressed as mean percentage preinjection baseline reading ± SEM. Statistical significance was estimated using 50, 8.8 µm). It fails to interact with other mGluR subtypes up to 100 µm (LY367385 is a highly selective antagonist of mGluR1 receptors. This compound antagonizes mGluR1α receptors in recombinant cells in the low micromolar range (ICm) for 20 min immediately prior to HFT resulted in a significant inhibition of LTP in hippocampal slices (n = 5) compared to controls . A significant effect on the induction phase was evident (P < 0.05). In addition, the expression phase of LTP was markedly impaired compared to controls (Application of LY367385 (100 µcontrols . LTP in n = 8), no significant effect on the profile of LTP was seen . LY367385 (100 µm) had no effect on basal synaptic transmission compared to controls . These data suggest that mGluR1 contributes to LTP processes by modulating the induction phase.When LY367385 was applied after the tetanus (controls . (anova:50 of 36 nm at mGluR5 with no activity at any other mGluR subtype (MPEP is a highly selective antagonist at mGluR5. This compound exhibits an ICm MPEP (n = 9) for 20 min prior to HFT resulted in a significant impairment of both induction and expression of LTP . Taking into account that activation of mGluR5 can modulate NMDAR-mediated currents (m (n = 8) also caused a significant impairment of LTP . This effect did not derive from effects on basal synaptic transmission, which remained stable over the 4-h monitoring period and did not differ after MPEP treatment when compared with control slices prior to LFS resulted in a significant impairment of both LTD induction and expression . When LY367385 was applied after LFS (n = 7) no significant effect on LTD was evident . These data suggest that antagonism of mGluR1 interferes with both the induction and late phases of LTD, and that mGluR1 must be active during the induction of LTD in order for persistent plasticity to occur.Application of LY367385 (100 µpression comparedm MPEP (n = 8) prior to LFS resulted in a significant impairment of the LTD induction phase (P < 0.05). However, persistent LTD still occurred and it was not significantly different from controls . This suggests that although antagonism of mGluR5 may result in a reduction in NMDA receptor currents was applied immediately after LFS (n = 13) a significant impairment of LTD occurred . These data suggest that antagonism of mGluR5 interferes with the late phases of LTD.When MPEP was evoked by LFS at 1 Hz (900 pulses). Treatment with CHPG (100 µm) prior to application of weak LFS resulted in persistent LTD .STD prior to application of CHPG (100 µm) and weak LFS. Under these circumstances CHPG treatment did not convert STD into LTD . This suggests that mGluR5 may trigger protein synthesis to enable persistency of synaptic plasticity.Chemical LTD, induced by activation of group I mGluRs, is protein synthesis-dependent in the CA1 region via Gq proteins and are typically expected to mediate signalling processes through stimulation of diacylglycerol and inositol trisphosphate which trigger, respectively, stimulation of protein kinase C and calcium release from intracellular stores. However, they also mediate an increase in neuronal excitability that occurs independently of activation of PLC and inositol trisphosphate .In the CA1 region mGluR1 also alters excitability via mechanisms that are distinct from those used by mGluR5. For example, mGluR1 mediates an increased frequency of spontaneous inhibitory postsynaptic potentials and a direct neuronal depolarization , and sug+ currents (in vivo (MGluR1 activation mediates NMDA receptor cycling ((in vivo .IAHP) and potentiation of NMDA receptor currents (MGluR5 antagonism affected LTP regardless of whether the antagonist was given before or after the tetanus. When MPEP was applied before the tetanus a marked impairment of LTP induction was evident. Activation of mGlu5 results in suppression of the calcium-activated potassium current (de novo protein synthesis to enable long-lasting LTP (The inhibition of late phases of LTP that occurred when MPEP was given after the tetanus suggests that mGluR5 facilitates persistent LTP by additional mechanisms. A time window has been reported for the post-tetanic facilitation, by the group I agonist DHPG, of STP into LTP . MetabotCuriously, application of MPEP prior to LFS impaired the induction of LTD but did not prevent persistent LTD from being expressed. In contrast, application of the antagonist after LFS prevented late LTD. These effects may derive from the temporal dynamics of mGluR5 facilitation of LTD. It is possible that mGluR5 must be active immediately after LFS in order for persistent LTD to occur. This could relate to a lingering spillover of glutamate after conclusion of LFS that activates perisynaptically localized mGluR5 (The bidirectional modulation of synaptic plasticity by mGluR1 has interesting implications for information storage in the CA1 region. LTP and LTD appear to engage in the encoding of different functional aspects of spatial memory (The results of this study demonstrate that activation of both mGluR1 and mGluR5 is critically required for persistent LTP and LTD in the hippocampal CA1 region. Whereas activation of mGluR1 during the plasticity-inducing protocol is essential for persistent bidirectional plasticity, the time window for activation of mGluR5 to enable LTP or LTD extends to periods after cessation of the plasticity-inducing protocol. Furthermore, the regulation by mGluR5 of late phases of plasticity appears to be mediated by stimulation of protein synthesis. These data emphasize an important functional role for group I mGluRs in the regulation of hippocampal synaptic plasticity and highlight the importance of this receptor for information storage in the CA1 region. |
AGT gene has been related to an increased risk of hypertension. This finding may also suggest an increased risk of coronary heart disease (CHD).The M235T polymorphism in the P = 0.28). This result was not changed by adjustment nor by using dominant, recessive and pairwise genetic models. Analyses for AMI risk under the additive genetic model also did not show any statistically significant association . To evaluate the association, a comprehensive systematic review and meta-analysis were undertaken of all studies published up to February 2007 . The meta-analysis (38 studies with 13284 cases and 18722 controls) showed a per-allele odds ratio (OR) of 1.08 . Moderate to large levels of heterogeneity were identified between studies. Hardy-Weinberg equilibrium (HWE) violation and the mean age of cases were statistically significant sources of the observed variation. In a stratum of non-HWE violation studies, there was no effect. An asymmetric funnel plot, the Egger's test (P = 0.066), and the Begg-Mazumdar test (P = 0.074) were all suggestive of the presence of publication bias.A case-cohort study was conducted in 1,732 unrelated middle-age women from a prospective cohort of 15,236 initially healthy Dutch women. We applied a Cox proportional hazards model to study the association of the polymorphism with acute myocardial infarction (AMI) (n = 71) and CHD. In the case-cohort study, no increased risk for CHD was found under the additive genetic model (hazard ratio [HR] = 1.20; 95% confidence interval [CI], 0.86 to 1.68; AGT gene. However, the relevance of this weakly positive overall association remains uncertain because it may be due to various residual biases, including HWE-violation and publication biases.The pooled OR of the present meta-analysis, including our own data, presented evidence that there is an increase in the risk of CHD conferred by the M235T variant of the AGT gene, encoding a threonine instead of a methionine at residue 235 of the mature protein, has been associated with a higher plasma AGT level and higher BP in patients homozygous for the T allele and occurs among various ethnic populations Angiotensinogen (AGT) is a liver protein that interacts with renin to produce angiotensin I, the pro-hormone of angiotensin II. Angiotensin II is the major effector molecule of the renin-angiotensin-aldosterone system (RAAS) and plays a key role in the regulation of blood pressure (BP) by increasing vascular tone and promoting sodium retention. Genetic variants in the angiotensinogen gene modify the plasma concentration of angiotensinogen, which has been directly related to arterial blood pressure AGT variant with increased CHD risk; however, this relationship was not confirmed in several other studies AGT gene with acute myocardial infarction (AMI) and CHD in a large population-based cohort of middle-aged Dutch women and conducted an updated meta-analysis of the available studies to clarify the role of the M235T polymorphism in CHD risk.Given the importance of hypertension in the occurrence of coronary heart disease Study design, general questionnaire, anthropometric and laboratory measurements have been described in detail elsewhere st 2000 were selected as cases. These were 211 CHD cases, including 71 AMIs. For all case subjects, follow-up ended at the date of diagnosis or at the date of death due to cardiovascular disease.We applied the case-cohort design introduced by Prentice Genetic analysis was performed at the Cardiovascular Genotyping (CAGT) laboratory of the Department of Internal Medicine of the University Hospital Maastricht. Genomic DNA was extracted from buffy coats using the QIAamp® Blood Kit . Genotyping of the polymorphisms was performed using a multilocus genotyping assay for candidate markers of cardiovascular disease risk 2 test among the controls. Allele frequencies were estimated by gene counting. We used the ANOVA F test to estimate relationships among the M235T genotypes and continuous variables, while we tested the significance of any difference in proportions by applying the χ2 statistic. A p-value <0.05 (2-sided) was considered statistically significant.Hardy-Weinberg equilibrium (HWE) was tested with the χAGT gene with the outcome, we used a Cox proportional hazards model with an estimation procedure adapted for case-cohort designs. We used the unweighted method by Prentice http://lib.stat.cmu.edu/general/robphreg. A previous meta-analysis AGT M235T variant on its intermediate phenotype (plasma angiotensinogen level) follows an additive model according to the number of T alleles [5% (95% CI: 2 to 8%) increase for the MT and 11% (95% CI: 7 to 15%) increase for the TT genotype versus the MM genotype]. Therefore, our priori hypothesis was that the association between the M235T polymorphism in the AGT gene and CHD follows an additive model according to the number of T alleles. However, other genetic models were evaluated as well. We considered different modes of inheritance as follows: the additive “per-allele” model, the T allele was compared between cases and controls by assigning scores of 0, 1, and 2 to homozygotes for the M allele, heterozygotes, and homozygotes for the T allele, respectively; the recessive model, the TT genotype versus the MT and MM combined genotypes; and the dominant model, the MT and TT genotypes combined versus the MM genotype. We also performed separate pairwise comparisons of the MT and TT genotypes versus the MM genotype.To assess the relationship of the M235T polymorphism in the AGT gene and CHD. Terms used for the search contained both medical subject heading terms and text words: (Met235Thr OR M235T OR T704C) AND (angiotensinogen OR AGT) AND (polymorphism OR mutation OR genetic OR genotype) AND . We also retrieved additional studies by hand searching the bibliographies of original research reports and review articles and through the MEDLINE option “related articles”. Search results were limited to articles published in English and studies on human subjects.We searched PubMed/MEDLINE, Web of Science, and EMBASE up to February 2007 for observational studies evaluating an association between the M235T polymorphism in the AGT M235T genotype frequencies were provided by case-control status (studies without controls were excluded); (ii) risk of CHD or MI was evaluated (studies on recurrent coronary events were excluded); (iii) relevant data were presented to calculate the effect size and its 95% CI; (iv) non-overlapping data were contained. For duplicate publications, the study with the smaller data set was excluded.All studies were considered potentially eligible if they aimed to investigate the relationship between the M235T genotypes and risk of CHD or MI. Any observational study, regardless of sample size, which fulfilled the following criteria, was included: (i) AGT gene genotypes based on different genetic models . We again considered a dominant, a recessive, an additive “per-allele” model and pairwise comparisons. Data were extracted independently and entered into separate databases by two authors (performed by MHZ and MLB). Results were compared, and disagreements were resolved by a consensus.The following information was extracted from each study that we included: the first author's name; country; year of publication; the population evaluated; study design; mean age or age range for case-patients and controls; definition and number of cases and controls; allele frequencies and genotype distribution in case-patients and controls ; consistency of genotype frequencies with HWE ; gender in the evaluated population and male percentage, matching variables, use of blinding of genotyping staff, performing regenotyping of a random sample, and crude ORs and 95% CIs for development of CHD or MI related to the priori hypothesis was the additive model. In each study, we tested for HWE by using the χ2 test or an exact test among the controls by using the genhwi command in Stata 9.2 The method of Mantel-Haenszel was used to calculate the odds ratio for the pooled data in a fixed-effects model, and, if there was evidence for heterogeneity, the DerSimonian-Laird method was used for the pooled odds ratio in a random-effects model, under pairwise comparisons of the different genotypes and dominant, recessive, and additive inheritance models. For all the models used, the T allele was considered the risk allele. The genetic model to be considered as the 2 – based Q statistic for between-study heterogeneity, which is considered to be significant for P<0.10, as well as the 2I statistic for estimation of inconsistency in meta-analyses 2I represents the percentage of the observed between-study variability due to heterogeneity rather than to chance. It ranges between 0% and 100%, where a value of 0% indicates no observed heterogeneity, and larger values indicate an increasing degree of heterogeneity In addition, we used Cochran's χP<0.10. Meta-analysis was carried out using STATA 9.2. We used random effect meta-regression models with restricted maximum likelihood estimation to evaluate the extent to which different variables explained heterogeneity among the individual ORs. The pre-specified characteristics for assessment of sources of inter-study heterogeneity were: study size under different genetic models, odds ratios, and variances were corrected by using the HWE-predicted genotype counts in the control instead of the observed counts as previously suggested 2 = 0.020; P = 0.89). General and clinical characteristics of CHD cases and controls are shown in The general characteristics of the randomly sampled participants of the cohort (N = 1522) are given in P = 0.28), which did not alter after adjustment . The same was true for other comparisons . AGT T235M polymorphism with CHD risk under different genetic contrasts. When a recessive model was evaluated, a significant association was found between individuals homozygous for the T allele (T235T genotype) and CHD risk, when compared to carriers of the M allele . Under the dominant model, the association was not significant. Under pairwise comparisons, there was a significant modest association between the T235T genotype and CHD risk, as compared with the M235M genotype . There was evidence for moderate to large between-study heterogeneity under all models . Next, single covariates were added in a series of univariate models. We performed the regression analysis for ten pre-defined potential sources of heterogeneity, including ethnicity, sex, mean age of cases, study size, case definition, source of controls, HWE-violation, blinding in genotyping, performing a sub-sample regenotyping, and matching (we hypothesized that studies that used matching might produce more conservative estimates of association). Univariate regression analyses showed that violation of HWE (β coefficient = 0.27 (0.06 to 0.48); HetP = 0.015, τ2 = 0.019), the mean age of cases (β = −0.01 (−0.02 to 0.0008); HetP = 0.066, τ2 = 0.024), and the method of case definition, clinically diagnosed CHD versus WHO criteria adjusted for other definitions (β = 0.26 (0.02 to 0.50); HetP = 0.038, τ2 = 0.020), were significant sources of heterogeneity among studies. The study size , the ethnicity , the male percentage in the study , blinded genotyping , sub-sample regenotyping , the source of controls , and matching were not significant sources of heterogeneity among studies. Violation of HWE in multivariable regression analysis remained a statistically significant source of heterogeneity after adjustment for the effect of study size . Adding the mean age of cases and method of case definition to the model with violation of HWE decreased the τ2 value to 0.017 . It also showed that the effect of method of case definition on the variation among the studies was through the effect of the mean age on the heterogeneity and not as an independent factor. A model that included only violation of HWE and the mean age of cases reduced the τ2 value to 0.018 .First, an empty regression was run with only the log of the effect estimate of pooled studies under the additive model to determine the baseline value for τFirst, the influence of deviation from the HWE on effect estimates was examined by using HWE-deviated adjusted ORs. P-values equal to 0.066 and 0.074, respectively) suggested the presence of a publication bias. By using the trim and fill method, we showed that, if the publication bias was the only source of the funnel plot asymmetry, it needed seven more studies to be symmetrical the absence of a biological effect, (ii) the presence of real genetic heterogeneity according to ethnic background, or (iii) failure to detect a small effect because the epidemiologic risk for an individual genetic variant is likely to be small and a large sample size is needed for adequate statistical power. It has been commonly proposed that, as well as a need for much larger and more rigorous studies those that are currently used, there is a greater need for international collaborations, particularly for a complex disease like CHD In this prospective study of healthy women aged 49 to 70 years, we investigated the relationship between the M235T polymorphism in the AGT genotypes in Hardy-Weinberg equilibrium. The limitations of this study were the relatively short period of follow-up and the small number of cases. Moreover, because this cohort was exclusively composed of Dutch women, these results cannot be generalized to men or other ethnic groups, for whom the rates of the events or the allele frequency are known to differ.In our study, the data collection was prospective, before the diagnosis of AMI or CHD and equal for all participants. This ensures that the cases and the randomly selected controls are comparable AGT gene in CHD risk. Although a pooled per-allele OR was suggestive of a modest increase in the risk of CHD of 1.08 , the robustness of this summary estimate is uncertain. First, in the pre-specified sub-groups analyses in the meta-analysis, larger studies, those with validated genotyping quality controls, and studies that used standardized criteria for case definition did not provide strong evidence for a positive statistically significant association between the M235T variant of the AGT gene and CHD risk. Second, the meta-regression analysis revealed that the HWE violation was a significant source of the moderate to large heterogeneity in the meta-analysis. Taking violation of HWE into account in the meta-analysis decreased the overall effect or bias AGT gene and CHD was found, the relevance of this weakly positive overall association remains uncertain because it may be due to various residual biases. Moderate to large heterogeneity was identified between studies, and violation of HWE and the mean age of cases were statistically significant sources of the observed variation.In conclusion, the present meta-analysis, including our own data, indicated that, although a weak association between the M235T variant in the |
Coronary artery bypass graft (CABG) surgery represents the standard treatment of advanced coronary artery disease. Two major types of anastomosis exist to connect the graft to the coronary artery, i.e., by using an end-to-side or a side-to-side anastomosis. There is still controversy because of the differences in the patency rates of the two types of anastomosis. The purpose of this paper is to non-invasively quantify hemodynamic parameters, such as mass flow and wall shear stress (WSS), in end-to-side and side-to-side anastomoses of patients with CABG using computational fluid dynamics (CFD).One patient with saphenous CABG and end-to-side anastomosis and one patient with saphenous CABG and side-to-side anastomosis underwent 16-detector row computed tomography (CT). Geometric models of coronary arteries and bypasses were reconstructed for CFD analysis. Blood flow was considered pulsatile, laminar, incompressible and Newtonian. Peri-anastomotic mass flow and WSS were quantified and flow patterns visualized.CFD analysis based on in-vivo CT coronary angiography data was feasible in both patients. For both types of CABG, flow patterns were characterized by a retrograde flow into the native coronary artery. WSS variations were found in both anastomoses types, with highest WSS values at the heel and lowest WSS values at the floor of the end-to-side anastomosis. In contrast, the highest WSS values of the side-to-side anastomosis configuration were found in stenotic vessel segments and not in the close vicinity of the anastomosis. Flow stagnation zones were found in end-to-side but not in side-to-side anastomosis, the latter also demonstrating a smoother stream division throughout the cardiac cycle.CFD analysis of venous CABG based on in-vivo CT datasets in patients was feasible producing qualitative and quantitative information on mass flow and WSS. Differences were found between the two types of anastomosis warranting further systematic application of the presented methodology on multiple patient datasets. Coronary artery bypass graft (CABG) surgery represents the standard treatment of advanced coronary artery disease (CAD). Since the pioneering work of Favaloro , variousNeointimal hyperplasia, defined as the accumulation of smooth muscle cells and extracellular matrix in the intimal compartment, is the major disease process in venous CABG in the first year after surgery and sets the foundations for later development of graft atheroma . In suppIn attempts to prevent acute and late graft occlusion, much effort has been invested in identifying the etiology of anastomotic neointimal hyperplasia and the plausibility of its prevention. Hypotheses related to this subject include the concept of compliance mismatch between graft and host artery , high frHemodynamic patterns at distal CABG anastomoses are thought to exhibit flow separation, recirculation and moving stagnation zones. Such flow features correspond typically to low time averaged WSS, long residence times, shear oscillation and increased spatial WSS gradients. These are the main hemodynamic features that have been connected with atherogenesis and intimal hyperplasia ,18. In aAn alternative to invasive or non-invasive flow measurements is the simulation of blood flow by using computational fluid dynamics (CFD) and/or ex-vivo experimental flow setups. Numerical studies on physiological coronary flow as well as on CABG flow have been published extensively during the last 15 years. Steinman presenteResearch on the hemodynamics of anastomoses has largely focused on determining the influence of the geometric characteristics of end-to-side configurations on WSS distributions. Fei presenteIt is worth noticing that most of the published CFD studies on anastomosis hemodynamics have been evaluated versus equivalent flow experiments by utilizing a variety of flow visualization, velocity and WSS measurement techniques such as in -40. CombAdvances in the CFD software and its corresponding hardware along with medical imaging are leading to the generation of increasingly reliable computational models. We can retrace a similar development into the fluid dynamics investigations of physiological coronary flow. Intracoronary flow can now be addressed in anatomically accurate configurations ranging from stiff multi-branched models to movinTwo male patients, 64 and 55-years old respectively, underwent cardiac CT. Patient 1 suffered from CAD with recurrent episodes of angina pectoris and a recent myocardial infarction. Invasive coronary angiography revealed a significant stenosis of the proximal right coronary artery (RCA) and non-significant stenoses of the middle left anterior descending (LAD) and distal left circumflex artery (LCX). Subsequently, the patient underwent saphenous CABG surgery with an end-to-side anastomosis onto the distal RCA. Patient 2 suffered from dyspnoea and instable angina pectoris. Invasive coronary angiography showed serial significant stenoses of the proximal LAD and a significant stenosis of the distal LCX. Saphenous CABG surgery was performed with a sequential side-to-side anastomosis onto the middle LAD and an end-to-side anastomosis onto the distal LCX.CT was performed 10 and 15 days after surgery, respectively, on a 16-detector row scanner using the following parameters: detector collimation 16 × 0.75 mm, gantry rotation time 0.37 sec, pitch 0.38, tube potential 120 kV, tube current time product 400 mAs. A bolus of 150 ml iodinated contrast material followed by 30 ml saline solution was continuously injected into a right antecubital vein via a 18-gauge catheter at a flow rate of 5 ml/sec. Bolus tracking was performed with a region of interest in the ascending aorta and image acquisition was automatically started 5 sec after signal attenuation reached a threshold of 140 HU. Synchronized to the electrocardiogram (ECG), CT data sets were retrospectively reconstructed throughout the cardiac cycle in 5% steps of the R-R interval with a slice thickness of 1 mm and an increment of 0.5 mm using a medium soft-tissue convolution kernel (B30f). The adaptive cardio volume approach was used for image reconstruction and ECG-pulsing was applied to reduce radiation exposure. The reconstruction phase providing best image quality with the lowest degree of motion artifacts was determined by two readers in consensus and was used for further post-processing. The local ethics committee approved the study protocol and written informed consent was obtained from both patients.Axial CT images were digitally processed to extract geometrical contours representing the coronary arteries and the CABGs. The lumen of all coronary arteries and grafts of the two patients were semi-automatically segmented using a commercially available software package . In regions of reduced arterial opacification, segmentation was manually complemented. The outflows and inflows of the vessels were separately marked to allow the imposition of boundary conditions. As a next step, an unstructured surface mesh of triangles was generated covering the segmented volume using the marching cube algorithm. Manual smoothing and low-pass spatial filtering was then applied to further reduce fine-scale surface irregularities. The final model depicted the real three dimensional (3D) geometry of the coronary arteries and bypass grafts was based on standard data [The flow for the simulation was considered transient, 3D, incompressible, and laminar. Corresponding to standard values from the literature, blood was assumed Newtonian with a viscosity of 0.0037 Pa· sec and a density of 1060 kg/mard data ,46 refleThe finite element software FIDAP was used to perform the CFD simulations by solving the Navier-Stokes equations with linear basis functions. Calculated flow variables were flow velocities and pressure following a segregated solution approach. A convergence criterion of four orders of magnitude was adopted for the residuals in velocity and pressure. The instantaneous flow field was acquired at 100 steps per cardiac cycle with a constant time step using backward Euler implicit time integration. All the results presented herein belong to the third computational cardiac cycle to allow ample time for the attenuation of the effects of the initial conditions. The sensitivity of the numerical results on the underlying grid was examined under steady flow conditions. The time averaged values of the inflow velocities, shown in Figure Mean heart rate during CT scanning was 47 ± 9 bpm for patient 1 and 51 ± 6 bpm for patient 2. The percent phase providing best image quality was found between 50 and 60% of the RR-interval for the RCA and between 60 and 70% for the left main artery (LMA), LAD, and LCX.The mean volumetric flow through the right coronary artery was 3.07 ml/sec. The mean volumetric flow through the CABG was 1.81 ml/sec Figure , leadingThe WSS characterizes the tangential fluid forces that act on the vessel wall. The changes of WSS throughout the cardiac cycle showed a correlation with flow velocities, with WSS forces being high when blood flow was fast. Near the end-to-side anastomosis the maximum WSS spatial variation was approximately 1.5 Pa Figures and 5. TThe mean volumetric flow through the left coronary artery was 3.35 ml/sec. The mean volumetric flow through the CABG was 1.51 ml/sec. Quantifications of volumetric flow through the proximal and distal segment of the LAD and the proximal and distal bypass close to the side-to-side anastomosis are demonstrated in Figure Comparing side-to-side to end-to-side anastomosis, the mean WSS values were lower in the side-to-side anastomosis with a time-averaged value of 0.29 Pa in the perianastomotic region Figure and 8. TThe results from our study indicate that considerable variations of blood flow exist in the distal perianastomotic region of venous CABG that may coincide with areas of neointimal hyperplasia. We found WSS variations in both types of anastomoses, with highest WSS values at the heel and lowest WSS values at the floor of the end-to-side anastomosis case. In contrast, high WSS in the side-to-side anastomosis configuration was only found in stenotic vessel segments but not in the close vicinity of the anastomosis. Across both types of anastomoses, elevated WSS values did coincide with vessel stenosis, being either caused by atherosclerotic wall changes or by a surgical clip.CFD provided detailed information on instantaneous mass flow. In the case of side-to-side anastomosis, this information was important to predict the mass flow reaching the next anastomosis. In addition, the amount of blood running retrograde in the coronary artery and eventually reaching a more proximal branch can be quantitatively measured. The numerical results in our patients revealed considerable retrograde flow into the host coronary artery in both types of anastomoses, a finding that has been previously described in experimental models . On the When investigating hemodynamic features of blood flow, numerical simulation has become an important tool. This study presents realistic, patient-specific models based on CT angiography datasets of coronary and CABG anatomy. This approach circumvents the need for idealization of the main geometrical features. Most of the CFD studies that were mentioned in the introduction had to rely on some sort of geometrical regularization by introducing pre-specified parameters such as the CABG angle, the host to graft diameter ratio and the planarity or non-planarity of the entire configuration. There are merits in the latter methodology. There is the ability to parametrically study the hemodynamic effects of these strictly defined morphological parameters. Furthermore the generation of the underlying surface and volumetric grids is much easier and can lead to numerical results of greater accuracy. On the other hand, a patient specific model is much more difficult to construct and to ensure sufficient numerical accuracy. It is a closer match to reality but at the same time it is harder to parameterize the results and extract generalized conclusions.CT angiography currently provides the most accurate representation of real anatomy. In contrast to most computational studies, our geometric models did not only include a small part of a coronary artery or CABG bypass but entailed the coronary tree and bypass grafts from their origin to the distal anastomoses. This became possible with the improvement of multi-detector row CT scanner technology with fast gantry rotation times enabling accurate and reliable depiction of coronary and bypass graft anatomy in a short imaging time ,48. WithThe following study limitations have to be acknowledged. Firstly, 16-detector row CT does not offer currently the best imaging for coronary arteries and CABG as compared to newer scanner technology. In addition, geometric data for CFD was obtained from a single reconstruction time-point in early to mid-diastole and vessel contours and diameters might be different at other reconstruction time-points. Secondly, the assumptions concerning inflow and outflow may not be valid under pathologic conditions and thus leading to exaggerated mass flow in the bypass grafts. The localization of the bypasses and native coronary inlets is extracted directly from the CT scans. It is expected that a realistic inflow velocity profile would not exhibit developed flow characteristics, as in Poisseuille or Womersley theories. The main discrepancy lies on the significant presence of in plane secondary flow patterns due to tMoreover, both the arterial and bypass walls were simplified as being stiff. Heart movement and changing pressure at the outer wall of the vessels due to the myocardium contraction were not included in the present calculations. The importance of the large scale coronary motion induced by the beating heart is hard to quantify in CABG configurations. Although the coronary arteries move considerably throughout the cardiac cycle, it has been shown that pulsatility is the main characterizing factor of WSS distributions . AdditioFinally, the assumption that blood behaves as a Newtonian liquid must be examined further. Although it is assumed that blood exhibits shear thinning behavior in vessels of smaller diameter than the coronary arteries, there is a multitude of patient dependent parameters like the hematocrit and the intake of drugs that can alter blood viscosity. Numerical simulations of non-Newtonian flow in a two dimensional end-to-side anastomosis under pulsatile flow conditions have shown only minor effects on WSS distributions . HoweverOur study has investigated the influence of patient specific geometry of end-to-side and side-to-side anastomosis on perianastomotic hemodynamics to identify geometrically driven flow features that might increase the propensity of venous graft failure. CFD analysis allowed us to differentiate hemodynamics in the two types of anastomoses and indicated significant spatial WSS variations especially in the end-to-side anastomosis configuration and an absence of stagnation areas in side-to-side anastomosis. The CFD simulations were performed in only two patients, a fact that limits the significance of our findings, which might differ in patients with different geometry. The numerical method applied herein may provide the basis for future prospective investigations correlating hemodynamic features with neointimal hyperplasia after CABG surgery.The author(s) declare that they have no competing interests.Guarantor of integrity of entire study: TFStudy concepts: TF, BMStudy design: TF, EBLiterature research: TF, EB, HAClinical studies: TS, LH, SLComputational studies: TF, EB, DPData acquisition: LH, SLData analysis/interpretation: TF, EB, HAStatistical analysis: TF, EBManuscript preparation: TF, EBManuscript editing: TF, EB HA, DP, BMManuscript revision/review: TF, EB, HA, DP, BMAll authors read and approved the final version of the manuscript. |
Dear Editor,et al.[We read with interest the article by Bhende et al. We wouldThe title of the article mentions pars plana vitrectomy. We feel that in these young eyes the pars plana is still not developed and the sclerotomies are actually through the pars plicata.Not all cases of Stage 4A retinopathy of prematurity (ROP) require surgery. Some of these remain stable and some get better spontaneously. Only those eyes which are progressing in spite of good laser or unlasered late referrals with vascular activity should be operated upon.It is commendable that in spite of having iatrogenic breaks in three cases, two had a favorable anatomical and visual outcome. In our experience, all the With the advent of 23 and 25-gauge systems, lens-sparing vitrectomy (LSV) has become more popular. The small instruments allow the surgeon easy access to anterior membranes in peripheral detachments in these small eyes.[Triamcinolone acetonide-assisted vitrectomy has been useful in adults. It has also been used in Stage 5 ROP. We are o |
Carbon nanotubes represent a class of nanomaterials having broad application potentials and documented cellular uptake and ecotoxicological effects that raise the possibility that they may bioaccumulate in living organisms.Lumbriculus variegatus.Radioactively labeled nanotubes were synthesized using a novel methane chemical vapor deposition procedure. Single-walled carbon nanotubes (SWNTs), multiwalled carbon nanotubes (MWNTs), and pyrene were spiked to sediment samples, and the respective uptake and depuration of these nanotubes and pyrene were assessed by the oligochaete, 14C-labeled carbon nanotubes were developed for these experiments to overcome significant previous limitations for quantifying nanotube materials in environmental and biological media. Biota-sediment accumulation factors for SWNTs and MWNTs were observed to be almost an order of magnitude lower than those for pyrene, a four-ringed polycyclic aromatic hydrocarbon (PAH). The depuration behaviors of the oligochaete suggested that the nanotubes detected in these organisms were associated with sediments remaining in the organism guts and not absorbed into cellular tissues as was the pyrene. The results suggest that, unlike PAHs, purified carbon nanotubes do not readily absorb into organism tissues. Single-walled nanotubes (SWNTs) and multiwalled nanotubes (MWNTs) make up the two principal classes of carbon nanotubes. SWNTs are one-layer graphitic cylinders having diameters on the order of a few nanometers, whereas MWNTs are composed of numerous concentric cylinders having much larger diameters.Carbon nanotubes have been the subject of extensive research over the past decade because of potential breakthroughs in a broad range of applications. Discovered by Sumio Iijima in 1991 , nanotubDaphnia magna using modified chemical vapor deposition procedures. These nanotubes were spiked to sediments and assessed with respect to their uptake by Lumbriculus variegatus, a sediment-burrowing oligochaete. Oligochaetes have been used extensively as bioindicators of pollution as the freshwater organism of choice for assessing bioaccumulation . Helium gas (99.95%), argon gas (99.998%), and methane gas (99.97%) were from Cryogenic Gases .Nickel nitrate hexahydrate (99%), magnesium nitrate hexahydrate (reagent grade), ferric nitrate (reagent grade), and citric acid were purchased from Fisher Scientific . Alkaline magnesium carbonate was obtained from Sigma Aldrich . 14C-labeled methane and regular methane as feedstock gases .SWNTs were similarly synthesized using a methane chemical vapor deposition method with a catalyst composed of iron on a magnesium oxide support matrix . AlkalinCarbon nanotubes were analyzed microscopically by transmission electron microscopy (TEM). Samples were prepared by adding dispersed nanotubes onto holey carbon film grids and viewing the grids using a 3011 TEM operating at 300 kV. Thermal gravimetric analysis (TGA) was also used to analyze the nanotube samples with respect to the presence of amorphous carbon impurities and residual catalyst materials. Because of the less stable chemical structure of amorphous carbon impurities, their presence can be assayed by analyzing derivatives of mass change with respect to temperature; in addition to the principal peak representative of the carbon nanotubes, a peak at a lower temperature represents the oxidation of carbon impurities. The percentage of original mass remaining after oxidation represents the fraction of residual catalyst in the sample. Raman spectra were obtained using a Renishaw inVia Raman microscope equipped with a Leica microscope, RenCam CCD detector, 785 nm diode laser, 1,200 lines/mm grating, and 50-μm slit. The radioactivity of the nanotube samples was assessed using biological oxidation . Determination of the nanotube radioactivity by their direct addition to scintillation cocktail underestimated the nanotube radioactivity compared with combustion in a biological oxidizer, perhaps as a result of the absorption of beta emissions in nanotube bundles.L. variegatus obtained from Carolina Biological Supply Co. were used to assess carbon nanotube availability to biological uptake and accumulation. The organisms were cultured in aquaria containing artificial freshwater [oC under photo-period (light:dark) ratios of 16:8 hr. We changed the overlying water and fed aquatic worms at least 2 times per week.eshwater and unblCarbon nanotubes and pyrene were added separately to either mixtures of 90% sediment with 10% Michigan (MI) Peat (by mass) or to un-amended sediment. The addition of 10% MI Peat allowed for a larger number of worms to be used for the bioavailability experiments, with a 50:1 ratio of sediment organic carbon to dry weight of the aquatic worms . Sedimen14C-SWNTs (0.03 or 0.003 mg/g dry sediment) and MWNTs (0.37 or 0.037 mg/g dry sediment) were dispersed by sonication in water prior to addition to the sediment. 14C-labeled pyrene in methanol and nonradioactive pyrene were dissolved in acetone and added to sediment to give a final mass ratio of 0.054 mg/g dry sediment. The samples were thoroughly tumbled, and the acetone from the pyrene samples allowed to volatilize, then the samples were refrigerated. Sediment samples were freeze dried, combusted using the biological oxidizer, and radioactivity was measured by scintillation counting to determine initial concentrations of materials in the sediments and the homogeneities of their distributions. Elevated nanotube concentrations were detected occasionally, likely as a result of carbon nanotube aggregates not being fully dispersed during sonication. Samples for which sediment radioactivities were greater than 2 times mean values were excluded from calculations of mean sediment concentration. Sediment samples spiked with nonradioactive carbon nanotubes or pyrene and unspiked sediment samples were prepared as controls.Uptake experiments were conducted according to a modified U.S. EPA method . 14C-SWNSix days after spiking with carbon nanotubes or pyrene, a 50-g (dry weight) quantity of amended or unamended sediment sample was added to 300-mL lipless beakers, and twice-daily artificial freshwater renewal was initiated . Aquaticd-mannitol to aid combustion. After drying overnight, the worms were combusted in the biological oxidizer and radioactivity was measured by liquid scintillation counting.We sieved aquatic worms from the sediments after predetermined intervals to determine the uptake of desired entities. The worms were collected from the sediment and placed in beakers with 500 mL of new artificial freshwater for 6 hr, a period shown to allow these organisms to purge > 98% of their gut content but also minimize tissue depuration of non-polar hydrophobic chemicals . The wor14C-labeled compounds. Lipid content was measured using a spectrophotometric method for the aquatic worms from nanotube-and pyrene-spiked sediments was rapidly reached because of lack of absorption in the lipids of the organisms.Differences between the uptake and depuration behaviors of carbon nanotubes and pyrene might be attributable to several factors. A. tenuiremis (14C nanotubes developed here are ideally suited for such investigations.The sizes of carbon nanotubes may be another factor in the lack of absorption by organisms. Size-dependent toxicities of SWNTs have been shown previously for the copepod nuiremis , and thenuiremis . The lownuiremis , but theBeyond the risks posed by nanotubes themselves, it is entirely possible that such materials may influence the bioaccumulation and fate of other pollutants in environmental systems. Carbon nanotubes possess strong sorptive capacities for such metals as lead, cadmium, and copper and various hydrophobic organic chemicals . Hypothe |
Skin is an organ that has a primary function in tactile receptivity and reacts directly upon emotional stimuli. Dermatological practice involves a psychosomatic dimension. A relationship between psychological factors and skin diseases has long been hypothesized. Psychodermatology addresses the interaction between mind and skin. It is divided into three categories according to the relationship between skin diseases and mental disorders. This article reviews different dermatological conditions under each of the three categories namely psychosomatic disorders, dermatological conditions due to primary and secondary psychiatric disorders. Dermatological conditions resulting from psychiatric conditions like stress/depression and those caused by psychiatric disorders are discussed. This review intends to present the relationship between the ‘skin’ and the ‘mind’ specifically from the dermatology point of view. The effects on the quality of life as a result of psychodermatological conditions are highlighted. A multidisciplinary approach for treatment from both dermatologic and psychiatric viewpoints are suggested. Skin has a special place in psychiatry with its responsiveness to emotional stimuli and ability to express emotions such as anger, fear, shame and frustration, and by providing self-esteem, the skin plays an important role in the socialization process, which continues from childhood to adulthood. The rela612Although there is no single universally accepted classification system of psychocutaneous disorders and many of the conditions are overlapped into different categories, the most widely accepted system is that devised by Koo and Lee.Psychodermatology is divided into three categories according to the relationship between skin diseases and mental disorders : 1) PsycHere psychiatric factors are instrumental in the etiology and course of skin conditions. The skin disease is not caused by stress but appears to be precipitated or exacerbated by stress.Psoriasis is a relatively common, chronic and inflammatory and hyperproliferative skin disease that occasionally requires systemic therapy. Stress hThe onset or exacerbation of atopic dermatitis often follows stressful life events. Symptom 3133Psychological stress may be an acquired factor affecting the expression of atopic dermatitis. Atopic iSevere emotional stress may exacerbate preexisting urticaria. Increase3840The psychosocial effect of acne was first recognized in 1948, when Sulzberger and Zaidens wrote, ’There is no single disease which causes more psychic trauma and more maladjustment between parents and children, more general insecurity and feelings of inferiority, and greater sums of psychic assessment than does acne vulgaris’. Acne hasPrimary psychiatric disorders are encountered less often than psychophysiologic disorders. These disorders have received little emphasis in the psychiatry or dermatology literature, even though they may be associated with suicide and unnecessary surgical procedures. Most of these disorders occur in the context of somatoform disorder, anxiety disorder, factitious disorder, impulse-control disorder or eating disorder.Psychogenic excoriation occurs in 2% of dermatology patients mostly in women. It is an uncommon psychodermatological condition, which responds well to serotonin reuptake inhibitors and behavioral therapy. It is characterized by excessive scratching or picking of the skin. The lesions are usually found on face, upper limbs and upper back. It is a The most common form of monosymptomatic hypochondriacal psychosis encountered among patients with skin problems is called delusions of parasitosis. DelusionTrichotillomania, according to the dermatologic use of the word, is a condition in which a person pulls out his or her own hair. The psychiatric definition of trichotillomania requires the presence of ‘impulsivity’. The mostPatients usually present to dermatologists because of skin lesions resulting from scratching, picking, and other self-injurious behaviors. They typically have an increased level of psychiatric symptomatology compared with age and sex matched controls taken from the general population of dermatology patients, and many patients experience negative stigmatization in their daily life.–62 CommoThis condition is also called body dysmorphic disorder or dermatological non-disease. Patients68This is an artifactual skin disease caused entirely by the actions of the fully aware patient on the skin, hair, nails or mucosa, with no rational motive for this behavior. The condition is more common in women than in men (3 : 1 to 20 : 1). The lesi71In this disorder, there are cycles of stress leading to pruritus as well as of the pruritus contributing to stress. Psychologic stress and comorbid psychiatric conditions may lower the itch threshold or aggravate itch sensitivity. Stress lThis category includes patients who have emotional problems as a result of having skin disease. The skin disease in these patients may be more severe than the psychiatric symptoms, and, even if not life-threatening, it may be considered ‘life-ruining’. SymptomsThe role of psychological factors in the pathogenesis of alopecia areata (AA) has long been the subject of debate. The inflVitiligo is a specific type of leukoderma characterized by depigmentation of the epidermis. In some studies, patients with vitiligo have been found to have significantly more stressful life events compared with controls, suggesting that psychologic distress may contribute to onset. Links be86Psychodermatologic disorders are conditions involving interaction between the mind and the skin. They fall into three categories; psychosomatic, primary psychiatric disorders and secondary psychiatric disorders. Atopic dermatitis, eczema, urticaria, psoriasis, herpes simplex, alopecia areata, rosacea, etc are regarded among dermatological psychosomatic disorders with psychogenic manifestation/exacerbation. It is suggested to use a biopsychological model, which takes into account the psychological and social factors, in addition to the primary dermatologic factors, in the management of the disease. The treatment of psychodermatological disorders should be carried out through the liaison therapy, which enables multidisciplinary approach, including family physician, dermatologist, psychiatrist and psychologist. It is very important to educate dermatologists in the diagnostic procedures and therapy of psychiatric disorders, which sometimes coexist with the skin disease. Majority of psychodermatological disorders can be treated with cognitive-behavioral psychotherapy, psychotherapeutic stress-and-anxiety-management techniques and psychotropic drugs. Psychopharmacologic treatment includes anxiolytics, antidepressants, anti-psychotics and mood stabilizer. The cooperation of the dermatologist and a psychiatrist in order to increase the life quality of the patients is of utmost importance. A dermatologist’s lack of knowledge on the psychiatric morbidity rates in dermatological diseases may delay the diagnosis of psychiatric condition and hinder the treatment, and hence establishment of separate psychodermatology units and multicenter research about the relationship of skin and psyche is necessary in the form of prospective case-controlled studies, and multisite therapeutic trials can provide more insight into this interesting and exciting field of medicine. The management of psychodermatologic disorders requires evaluation of the skin manifestation and the social, familial and occupational issues underlying the problem. Once the disorder has been diagnosed, management requires a dual approach, addressing both dermatologic and psychologic aspects. A mutual, respectful collaboration between dermatologists and mental health professionals might be of help for many psychiatric patients. Therefore, understanding of biopsychosocial approaches and liaison approach involving general practice, psychiatrist, dermatologist and psychologist treatment in this field is essential. |
Tobacco use is the single most preventable cause of morbidity and mortality in humans. Limited data exist regarding the extent of the problem among Cyprus youth. We use the Global Youth Tobacco Survey to assess the prevalence of cigarette smoking among middle and high school students as well as the social environment in which this is taking place.The survey was conducted by the Cyprus International Institute for the Environment and Public Health in association with Harvard School of Public Health. A two-stage cluster sample design was used to select a representative sample of students from middle and high schools registered with the Republic of Cyprus in 2005–2006. The study questionnaire consisted of 99 questions and participation in the survey was voluntary. Statistical analyses were performed taking into consideration the specific design of the study and the sample weights associated with each completed questionnaire.The prevalence of current smoking, defined as having smoked cigarettes on one or more days of the past 30 days, is 13% among boys and 7% among girls in middle schools, and 36% among boys and 23% among girls in high schools. Furthermore, 16% of middle school students and more than 24% of high school students that had never smoked indicated that they are likely to initiate smoking within the next year. Exposure to environmental tobacco smoke is also very high with 91% of students reporting being exposed to smoke in places outside home. In addition, more than 95% of current smokers reported that they had bought cigarettes in a store during the past month and were not refused cigarettes because of their age.Smoking prevalence among Cyprus middle and high school students is high and there are indications of an increase in the prevalence of smoking among girls over the last few years. Susceptibility rates, exposure to second-hand smoke, and access to and availability of cigarettes to youth are also high and concerning. The present survey indicates that the problem of cigarette smoking among youth in Cyprus is significant and requires collective action immediately. Tobacco smoking is a major public health problem worldwide and it is the single most preventable cause of morbidity and mortality in humans -3. ApproAdult smokers almost always initiate tobacco use before the age of 18 when theThe World Health Organization Framework Convention on Tobacco Control (WHO FCTC) reinforces the need for data on adolescent tobacco use by calling on countries to establish surveillance programs of "the magnitude, patterns, determinants, and consequences of tobacco consumption and exposure to tobacco smoke" . Cyprus In Cyprus limited data exist regarding smoking among youth. A survey conducted by the Ministry of Health in 1997 revealed that 6.5% of all youth 15–19 years old were daily smokers whereas The present study uses data from the 2006 Cyprus GYTS to assess cigarette smoking prevalence (the dominant form of tobacco use in Cyprus) among middle school students (with ages ranging from 12 to 15) and among high school students (with ages ranging from 15 to 18) and study the psychosocial, environmental, and behavioral factors associated with smoking among youth in these ages. The goal of the study is to provide scientific information and evidence in order to help create effective smoking prevention and cessation programs and reduce the smoking rate among youth in Cyprus. Results are reported on the prevalence and factors that influence youth smoking; exposure to second-hand smoke (SHS); access to and availability of tobacco products; exposure to media and advertising; cessation programs; and smoking-related education in Cyprus.The GYTS was conducted in 2006 by the CII, in collaboration with the Harvard School of Public Health, the WHO, the CDC, and the Ministries of Health and Education and Culture of the Republic of Cyprus.st, 2nd, and 3rd grades and high schools with 4th, 5th, and 6th grades were sampled with the probability of selection being equal to the school's enrollment size; in the second stage, classes within chosen schools were selected using a systematic equal-probability sampling with a random start. All students in the selected classes were eligible to participate in the survey.All middle schools and high schools registered in the academic year 2005–2006 with the Ministry of Education and Culture in Cyprus with a school size of 40 or more students were included in the sampling frame for the GYTS. A two-stage cluster sample design was implemented by the CDC with the goal of selecting a representative sample of students from the Republic of Cyprus drawn from the country's five government-controlled regions, namely Lefkosia, Lemesos, Paphos, Larnaka, and Ammochostos. In the first stage, all middle schools with 1The study questionnaire was developed based on the Greek translation of the GYTS core questionnaire used in Greece in 2005 [Standardized methodology was followed for the preparation of the questionnaires, the selection of the sample, the field work, and the processing of the data, details of which are described elsewhere ,17-19. PAnalyses were performed using the Statistical Analysis Software (SAS) version 9.1. The analyses took into account the specific sampling frame and the specific weight associated with each one of the questionnaires. This weight is the product of the inverse of the probability of selecting the school, the inverse of the probability of selecting a classroom within the school, the school-level non-response adjustment factor calculated by school size category , the class adjustment factor calculated by school, and the student-level non-response adjustment factor calculated by class. Weights are used to reflect the likelihood of sampling of each student and to reduce bias by compensating for differing patterns of non-response. Current smoking is defined as having smoked cigarettes on one or more days of the last 30 days. Qualitative variables are described as percentages together with their corresponding 95% confidence intervals and quantitative variables are described as mean (SD). All statistical tests reported are two-sided, and a p-value < 0.05 is considered to be statistically significant.A total of 13,246 students from 90 schools (51 middle schools and 39 high schools) participated in the survey. Out of these, there were 3,651 middle school boys, 3,579 middle school girls, 2,619 high school boys, and 3,312 high school girls that provided gender information significantly more likely to have done so than girls 51.2%) ; about an equal proportion (16.1%) reported that they had ever been offered free cigarettes by a tobacco company representative want to stop smoking; 60.9% reported that they tried unsuccessfully to stop smoking during the past year and 70.7% reported that they received help to stop smoking at some point that smoked daily in the last month reported to be 38.1% and the corresponding rate for boys 15–19 years old reported to be 22.6% [Second, and even more concerning, are the smoking rates among high school girls; the prevalence of smoking is 23.2%, with 19.0% of all girls 17–18 years old and 11.8% of all 15-year old or older girls reporting being daily smokers. In 2003, among all adult females 15 years old or older 10.5% were daily smokers, with only 5.7% of girls ages 15–19 being daily smokers ,22.Third, the susceptibility rates reported are high. Girls in both middle and high school that have never smoked and boys in middle school that have never smoked indicated that they are likely to initiate smoking within the next year at rates higher than the existing prevalence of current smokers. If that holds true then an increase in prevalence rates can be expected, especially among teenage girls in high school whose susceptibility rates are significantly higher than those of high school boys.When compared with other countries that administered the GYTS, Cyprus has higher current cigarette smoking rates for both boys and girls aged 13–15 than the average of the Eastern Mediterranean region countries sampled (though it has lower rates for current use of other tobacco products) . On the It is clear that smoking is an important problem in Cyprus. Conservative estimates suggest that between 600 and 700 people die every year in Cyprus because of smoking . In lighSeveral tobacco control measures exist in Cyprus including regulations regarding the manufacture and sale of tobacco products , advertising bans and restrictions , mandatory package warning labels (e.g. 'Smoking Kills' warning covering 32% of the area of one of the two largest surfaces of the outside of the packet), and policies on secondhand smoke . However, despite laws that prohibit the sale of tobacco to minors and ban smoking in public places, the present research suggests that these laws are not being observed. Exposure to SHS is extremely high both in the homes of students, where 84.5% report being exposed to smoke, and in other public spaces outside the home, where 91.2% report exposure. SHS is a serious danger even at low levels and is classified by the International Agency for Research on Cancer (IARC) as a known cause of cancer . MeasureThe present study is limited by the subjectivity of self-administered questionnaires, introducing the possibility of information bias, as well as some non-response . Nevertheless, because of the high response rate, the anonymous administration of questionnaires, the good test-retest reliability of similar data, and the openness in Cyprus about the subject of smoking, biases introduced are likely to be minimal ,29. The Another limitation of the study is the fact that many students in technical schools were not surveyed. However, most of the students in Cyprus attend non-technical schools so we believe that the sample is representative of the whole population .Our recommendations include for the Cyprus government to ensure that the current laws regarding the sale of tobacco to minors and smoking in public spaces are enforced and to further strengthen the existing legislation. In addition, youth programs and anti-tobacco advertising campaigns need to be implemented, and increased professional help for cessation should be made available to persons who want to quit. Public awareness of the dangers of smoking should be promoted through public education campaigns and policy efforts need to be coordinated to address the problem. Further research is also needed in order to understand the complexity of the tobacco problem in Cyprus, the reasons for the high prevalence rates and the increase among females, as well as the specific measures that would effectively reduce tobacco use and its hazardous health effects. The Republic of Cyprus and the Ministry of Health have already adopted the 'Strategic Plan for Tobacco Control in Cyprus' prepared by the Tobacco Research Program of the CII . This plThough further research is needed in Cyprus in relation to tobacco use and more scientific and updated data should be periodically collected, the GYTS indicates that the problem of cigarette smoking among youth in Cyprus is significant and that collective action needs to be taken immediately.The authors declare that they have no competing interests.PD, GNC, CWW, and NRJ conceived the study, participated in the design, and helped in the development of the methodology. OK supervised and helped in the collection and compilation of the data. CAC performed the statistical analysis, developed the first draft of the manuscript, and revised the manuscript before final submission. HRA and CWW critically revised the manuscript. All authors contributed towards the revision of the manuscript to its final version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: |
A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 μm diameter and 150 μm depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber ; and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated. Clean the wafer using a razor blade if needed. Clean with Acetone, Methanol, and IPA. Perform plasma treatment at 50 Watts for 30 s.Spin SU8-100 @ 2000 rpm. .[NOTE: do not hold the wafer with tweezers after spinning SU8]Soft bake the wafer at 65 °C for 20 min and at 95 °C for 50 min. 2.The wafer is exposed to UV light through a desired mask. Dose for 150 μm height is 650 mJ/cmPost exposure bake the wafer at 65 °C for 1 min and at 95 °C for 12 min. Develop the wafer in SU8 developer for 15 min.Clean the wafer using a razor blade if needed. Clean with Acetone, Methanol, and IPA. Perform plasma treatment at 50 Watts for 30 s.Spin SU8-2150 @ 1000 rpm. .Soft bake the wafer at 65 °C for 15 min and at 95 °C for 2 hrs and 30 min. 2.The wafer is exposed to UV light through a desired mask. Exposure dose for 650 μm height is 685 mJ/cmPost exposure bake the wafer at 65 °C for 5 min and at 95 °C for 35 min. Develop the wafer in SU8 developer for 20-30 min.Polydimethylsiloxane (PDMS) solution is prepared by thoroughly mixing 10 parts of silicone elastomer with 1 part of curing agent of a standard Sylgard 184 kit.The bubbles generated in the PDMS solution during the mixing process are removed using a vacuum desiccator.The bubble-free PDMS solution is slowly dispensed onto the SU8 masters and an empty Petri dish for the third layer. The temperature of the hot plate is set to 75 °C and the PDMS is cured at this temperature for 2 hrsInlets, outlets and exchange wells are punched out using the appropriate size hole puncher.The layers are bonded together on a glass slide (size 0.1 mm) using a handheld plasma device.Flow 70 % ethanol through the micro-device to sterilize. Flow DI water to wash out the ethanol. Perfuse 50 ml of 0.5 %BSA through the device to prevent non specific adsorption of insulin to the microchannel walls.The glucose ramp gradient and other related gradients generated by LabView software that communicates with the syringe pumps are tested to make sure the gradients are stable.25-30 mice islets were incubated with 5 μM Fura-2/AM and 2.5 μM Rhodamine 123 for 30 min at 37 °C in Krebs-Ringer buffer (KRB) containing 2 mM glucose The mice islets are carefully pipetted into microfluidic perifusion device through the inlet port. The microfluidic network is then setup by connecting the inlet to the syringe pumps using Tygon tubing and a Y-connector and the outlet to fraction collector. The device sits on a heating stage (37 °C) on the microscope and the inlet tubing is heated on a hotplate to keep the temperature of the chamber and solution at 37 °C. Immediately after setup, the mice islets are perifused with KRB containing 2 mM glucose for 10 min and then a glucose ramp (2mM 25mM) for 25 min. Time-lapse images are collected and analyzed every 15 s by SimplePCI software. The perifusate is also collected every minute using a fraction collector to analyze the insulin secretion using ELISA kit.Mouse islets were perifused with a linear gradient of 2-25 mM glucose. As shown in Figure 1, calcium influx and insulin secretion are triggered after about 13 minutes of perifusion, corresponding to 6 mM glucose. Changes in Mitochondrial potentials are seen earlier as expected, at about 11 minutes. This data demonstrates the advantage of using this microfluidic network to characterize islet physiology. Figure 1. Physiological responses to islet cell stimulation. Traditional islet perifusion systems have some limitations including complex of setup and design, high technical requirements, and difficulty to create user-prescribed chemical gradients in the system. The microfluidic perifusion system described here overcomes these limitations with simple geometry of design and fabrication. More important, this system can be integrated with fluorescence imaging approach that provide as a unique tool to study islet physiology. The system demonstrated with high signal-noise ratio and spatial-temporal resolution of these fluorescence signals. The current prototype device also can hold multiple perifusion setups in one chip and provide different types of microenvironments for widespread application purposes. |
This study aimed to examine the changes in cognition associated with long-term antipsychotic treatment and to evaluate the effect of the type of antipsychotic or novel antipsychotics (n = 26) at baseline and at two years after.In this naturalistic study, we used a comprehensive neuropsychological battery of tests to assess a sample of schizophrenia patients taking either conventional (Continuous antipsychotic treatment regardless of class was associated with improvement on verbal fluency, executive functions, and visual and verbal memory. Patients taking atypical antipsychotics did not show greater cognitive enhancement over two years than patients taking conventional antipsychotics.Although long-term antipsychotic treatment slightly improved cognitive function, the switch from conventional to atypical antipsychotic treatment should not be based exclusively on the presence of these cognitive deficits. Cognitive disturbances are a core feature of schizophrenia and have been extensively studied in recent years . CognitiThe positive action of conventional antipsychotics drugs (APDs) on cognition is considered mild or moderate and is lRegarding novel antipsychotics, this supposed cognitive enhancement would be mediated by their capability to raise the level of dopamine and acetylcholine in prefrontal regions . HoweverStudies that attempted to demonstrate the association of a greater cognitive enhancement with novel versus conventional APDs are susceptible to biases and face several difficulties, which include confounding effects of clinical symptoms, previous and adjunctive medications , and practice effects, especially when intervals between assessments are short ,20.Many studies have compared the procognitive properties of conventional and atypical APDs. Results that stemmed from these various reports were compiled by Woodward et al. in a metDespite all these data, the benefits of atypical APDs over conventional APDs for the full range of cognitive disturbances in schizophrenia remain controversial, especially for the most severe impairments, i.e., serial learning, executive functioning, vigilance, motor speed, and verbal fluency . SeveralMost of the studies comparing typical versus atypical antipsychotics on cognitive performance are randomized blind trials. Since many clinicians consider changing from conventional to novel antipsychotics in order to enhance cognition, we think that naturalistic studies may provide a closer look to real clinical practice.In the present naturalistic, retrospective study, we used a comprehensive neuropsychological battery of tests to assess the cognitive outcome in two groups of schizophrenic patients: the first group was treated with one or more conventional APDs over two years, while the second group was treated with one atypical APD. The treatments were not discontinued at any time and there were no switches in the type of APD administered. Patients' performance on tasks of executive functions, verbal working memory, short-term memory, verbal memory, visual memory, speed of processing, verbal fluency, and motor speed were assessed twice, two years apart.The first objective of this study was to examine changes in cognitive impairment associated with long-term APD treatment. The second objective was to assess the effect of the type of antipsychotic treatment on change of cognition over two years and describe the potential differences between the groups.n = 13, 9 men, 4 women) was composed of patients being treated with conventional antipsychotics and the second group comprised patients who were taking an atypical antipsychotic .Subjects who participated in this observational and naturalistic study were enrolled in the Valencia Follow-Up Study of Schizophrenia and Bipolar I Disorder -26. FiftAll participants were assessed at baseline (T1) and two years later (T2). Most patients in this study were treated in settings that did not offer psychosocial rehabilitation programs. At each time point, all patients were medicated by their psychiatrists in a naturalistic manner. The average daily dose of APD was converted into chlorpromazine equivalent (CPZ) units, for statistical purposes ,30. The Written informed consent was obtained from all participants after an explanation of the study procedures. The Ethics Committee of the University Clinic Hospital of Valencia approved the research protocol.The clinical evaluation of each patient was rated according to the Positive and Negative Symptom Scale (PANSS) ,32 and tAll patients completed a battery of tests, which were described in three previous publications ,7,37. Th1) Executive Functions/Reasoning and Problem Solving .2) Short-term Memory (Digit Span Forward Test)3) Working Memory .4) Verbal Memory 5) Visual Memory . Immediate and differed (30 minutes) recall.6) Visual-Motor Processing/Speed of Processing .7) Semantic Verbal Fluency .8) Motor Speed .The variable years of education was used as a measure of premorbid intelligence.t test to compare the means (T1 vs. T2) for all patients, as well as for each treatment group at T1. In each group, changes in cognitive and clinical scores were analyzed using an analysis of variance (ANOVA) with repeated measures. Cognitive performance at T1 and T2 were the dependent variables and the type of APD was the independent variable. As CPZ units and PANSS positive scores at T1 were the only variables differing between that reached statistical significance, they were entered as covariates in these analyses.Data analyses were carried out using the SPSS software (version 15.0 for Windows). An alpha level of 0.05 was used for all statistical tests. Data were analyzed using Student's paired Pearson correlations were calculated among the clinical, treatment, and outcome variables at T1 and for the difference in neurocognitive scores (T2-T1) for every cognitive variable. This new variable was calculated to better assess the evolution of cognitive performance. Two sets of correlation analyses were carried out by splitting the cohort of patients according to the type of antipsychotic medication taken.P ≤ 0.05) in any of the two correlations were entered in the regression models as independent variables. The dependent variable was the T2-T1 difference in performance on each neuropsychological test.Linear regression analyses with a forward stepwise procedure were performed to assess the relative contributions of the variables cited above. In this model, the type of antipsychotic medication and the clinical, outcome, and treatment variables at baseline that significantly correlated with neurocognitive measures years, the mean length of education was 10.1 (SD = 3.01) years, the mean age at onset was 24.94 (SD = 7.05) years, and the mean number of prior episodes was 2.25 (SD = 1.59). The mean dose of APDs was 773.97 (SD = 514.63) CPZ units and the mean dose of anticholinergic medication (biperiden) was 0.53 mg (SD = 1.33). The mean dose of benzodiazepines was 3.55 (SD = 8.94) diazepam equivalent units. At baseline, nine out of the 39 patients were taking benzodiazepines. At endpoint eight were taking benzodiazepines . Eight patients were on biperiden at baseline (4 from each group) and four at endpoint (2 each).Patients' cognitive performance on the different tests ranged around 2-3 standard deviations under normative data for age and education-matched healthy Spanish population, with exception of the digit span test. [Mean whole sample of patients for TMA test = 65.20 vs. 24.40 (SD = 8.71) for normal population; Mean TMB test = 162.71 vs. 50.68 (SD = 12.36); Rey Figure = 13.44 vs. 21.48 (SD = 5.54); FAS = 25.05 vs. 38.75 (SD = 4.80); Mean for Stroop test (word/color interference) = 82.0 vs. 49 (SD = 15.5). See Ardila et al [t = 2.69; P = 0.01) and committed more total errors in the WCST . Sex distribution was equal in both groups.The baseline between-group comparison is summarized in Table Significant differences between T1 and T2 were observed for the following cognitive measures.t = -4.14; P < 0.000; d =.664) and FAS Test .A) Semantic verbal fluency, as assessed using the CIG test and for perseverative errors , the Color/Word interference part of the Stroop Test , and the Trail Making Test part B .B) Executive functions, as assessed using the WCST for total errors .C) Auditory verbal memory, as assessed using the Digit Span Forward Test and delayed visual recall.D) Visual memory, as assessed using the Rey-Osterrieth Complex Figure Test for immediate on any of the cognitive tests used Table ; howeverSignificant correlations between outcome, clinical, and treatment variables at T1 and T2-T1 and neuropsychological change scores for the two patient groups are shown in supplemental material. Age, age at onset, length of illness, and number of prior episodes and hospitalizations did not correlate with any of the neurocognitive variables.2 = 0.132; P = 0.02); WCST errors ; and Finger Tapping Test, right motor performance . Diazepam equivalent units significantly predicted the number of total errors in the WCST test .The type of antipsychotic medication did not predict the performance in any neuropsychological test. Dosage of biperiden significantly predicted the performance in the Stroop Test .Concerning clinical variables, the PANSS General Psychopathology subscale significantly predicted the performance in the Category Instant Generation Test , where size effects were all over 0.6. In the opposite, after two years of treatment no cognitive improvements were observed in visual/motor processing, motor speed and working memory tasks.As patients did not participate in psychotherapeutic interventions or programs aimed at improving cognitive function and considering the absence of significant differences between T1 and T2 in the PANSS scores, we suggest that these cognitive gains may be in part the result of continuous exposure to antipsychotic medication. Moreover, the characteristics of the sample in the present study correspond to clinically stable, chronic patients, previously stabilized by the ongoing antipsychotic medication, so the effects psychopathological changes on cognition, although not measured, are probably limited.The relatively long time interval between the baseline and the endpoint would rule out, at least in part, learning effects associated with repeated testing ,20.The cognitive improvement observed in our cohort supports the results obtained in the neurocognitive component of the CATIE trial and in aOur results do not support the hypothesis of a better cognitive outcome in patients treated with atypical APD. The variable "type of APD" did not predict the improvement of performance in any of the neuropsychological tests, as assessed using regression analyses; however, intake of anticholinergic drugs seems to predict cognitive changes significantly. These results support other studies that concluded that concurrent use of anticholinergic drugs, especially their acute administration , is anotIn contrast to the similar effect on cognitive evolution observed for both types of APDs, a significant improvement in the PANSS positive subscale score was detected in the group treated with conventional APDs. Interestingly, another significant effect, which translated into an improvement in the PANSS General Subscale score, was observed in the group treated with atypical APDs, which suggests a positive effect of these new drugs on the nonpsychotic symptoms of schizophrenia.The limitations of the present study should be taken into account. Firstly, the relatively small sample size and the consequent lack of statistical power may have masked possible differences between the groups. In addition, these factors did not allow us to perform head-to-head comparisons of specific APDs. Secondly, as this was not a blind trial and patients were not randomly assigned to medication groups, the differences detected among groups may simply reflect a prescription bias. According to a recent meta-analysis , the difData from the literature showing that cognitive measures can predict functional outcomes emphasizBearing in mind that this pilot study lacks a representative sample of patients, these preliminary finding are consistent with the recent literature in which atypical antipsychotics have not demonstrated cognitive benefits over typical or conventional antipsychotics. Our results did not show a clear advantage of atypical over conventional APDs on cognitive performance, however, the general improved tolerability profile of second generation antipsychotics regarding neurological side effects may facilitate treatment adherence, which in turn may result in cognitive improvement. Anyway, in the absence of other reasons to change ongoing treatment, which include negative or affective symptoms or lack of compliance with the regimen, the switch from typical APD at low or moderate doses to atypical APDs is not justified if based solely on the expectation of a more favorable cognitive outcome. Nevertheless, other psychopharmacological and psychosocial strategies should be implemented to enhance cognitive outcome in schizophrenic patients.Role of funding sourceFunding for this study was provided by CIBERSAM, which had no further role in study design, the collection, analysis, and interpretation of data, the writing of the report, or in the decision to submit the paper for publication.Dr. Vieta has received grants from or acted as a consultant for the following companies: AstraZeneca, Bristol Myers-Squibb, Forest Research Institute, Glaxo SmithKline, Janssen, Jazz Pharmaceuticals, Eli-Lilly, Lundbeck, MSD, Novartis, Organon, Otsuka, Pfizer, Sanofi-Aventis, Servier, Schering-Plough, Solvay, Takeda, United Biosource Corporation, and Wyeth.Dr. Tabarés-Seisdedos has received grants from or acted as a consultant for the following companies: AstraZeneca, Janssen, Eli-Lilly, Lundbeck, Novartis, Pfizer, Sanofi-Aventis, and Wyeth that were deposited into research accounts at the University of Valencia.Dr. Balanzá-Martínez has received grants from or acted as a consultant for the following companies: AstraZeneca, Boehringer Ingelheim, Bristol-Myers-Squibb/Otsuka, Janssen-Cilag, Pfizer, and Wyeth.All listed authors have contributed significantly to the manuscript and consent to their names on the manuscript. GSV, RTS, VBM and JSF conceived of the study, participated in its design and coordination and helped to draft the manuscript. PC collected data and carried out the neuropsychological assessments. JSM, AMA and EV revised the article critically for important intellectual content. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/10/47/prepub |
The IGF receptor type 1 (IGF-1R) pathway is frequently deregulated in human tumors and has become a target of interest for anti-cancer therapy.IGF-1R as assessed by independent SNP array analysis. Addition of erlotinib or paclitaxel to R1507 led to further growth inhibition in sensitive but not resistant lines. In one EGFR mutant lung adenocarcinoma cell line (11–18), R1507 and erlotinib co-treatment induced apoptosis, whereas treatment with either drug alone induced only cell cycle arrest. Apoptosis was mediated, in part, by the survival-related AKT pathway. Additionally, immunohistochemical (IHC) staining of total IGF-1R with an anti-total IGF-1R Ab was performed on tissue microarrays (TMAs) containing 270 independent NSCLC tumor samples. Staining intensity was scored on a scale of 0 to 3+. 39.3% of tumors showed medium to high IGF-1R IHC staining , while 16.7% had scores of 3+.We used a panel of 22 non-small cell lung cancer (NSCLC) cell lines to investigate predictive biomarkers of response to R1507, a fully-humanized anti-IGF-1R monoclonal antibody . 5 lines were moderately sensitive (25–50% growth inhibition) to R1507 alone. While levels of phospho-IGF-1R did not correlate with drug sensitivity, 4 out of 5 sensitive lines displayed high levels of total IGF-1R versus 1 out of 17 resistant lines . Sensitive lines also harbored higher copy numbers of In NSCLC cell lines, high levels of total IGF-1R are associated with moderate sensitivity to R1507. These results suggest a possible enrichment strategy for clinical trials with anti-IGF-1R therapy. Lung cancer is the leading cause of cancer-related death in the United States and worldwide One emerging approach involves targeting of the type I insulin-like growth factor receptor (IGF-1R) pathway The IGF-1R pathway appears to play important roles in tumorigenesis, metastasis, and resistance to existing forms of anti-cancer therapy Here, we sought to identify molecular biomarkers in NSCLC that may predict for benefit from anti-IGF-1R directed therapy. Specifically, we analyzed parameters that may be associated with sensitivity of 22 NSCLC lines to R1507 , a fully humanized IgG1 monoclonal antibody directed against the extracellular portion of IGF-1R. It binds with high selectivity to the extracellular domain of IGF-1R (and not to IR), leading to displacement of IGF-1 binding and loss of protein at the cell surface due to receptor internalization and degradation. After identifying one potential biomarker, we analyzed signaling properties affected by anti-IGF-1R treatment in various cell lines and evaluated expression status of the biomarker in 270 NSCLC patient samples.2 at 37°C. SK-MES-1 cells were grown in Eagle's Minimum Essential medium (ATCC) supplemented with 10% fetal bovine serum and Pen-Strep Solution. SW900 cells were cultured in Leibovitz's L-15 Medium (ATCC) supplemented with 10% fetal bovine serum and Pen-Strep Solution .The human lung adenocarcinoma cell lines H3255, PC-9 and H1975 were described previously Anti-IGF-1R antibody R1507 was kindly provided by Hoffmann-La Roche Inc . Erlotinib was synthesized by the Organic Synthesis Core Facility at Memorial Sloan-Kettering Cancer Center. Paclitaxel was purchased from EMD Biosciences . The Vybrant® Apoptosis Assay Kit #2 was from Invitrogen . The phospho-receptor tyrosine kinase (RTK) array kit, human IGF-1R ELISA kit, human phospho-IGF-1R ELISA kit, and recombinant human IGF-1 were from R&D Systems . Recombinant human EGF was from Cell Signaling Technology .Anti- pERK(Thr202/Tyr204), -ERK, -pAKT (Ser473), -AKT, -pEGFR (Tyr1092), -pIGF-1Rβ (Tyr 1135/1136) and -IGF-1Rβ antibodies, and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology . Anti-total EGFR antibodies were from Santa Cruz Biotechnology .Cells were seeded in 96-well plates at a density of 5,000 cells in triplicate and treated with different concentrations of drugs on the following day. The growth status of drug-treated cells was measured at 72 hours post treatment using CellTiter Blue Reagent . Annexin V/PI apoptosis assays (Invitrogen) were performed according to manufacturer's instructions.Cells were scraped from 10 cm petri dishes, washed twice with PBS, and then incubated in RIPA lysis buffer containing protease inhibitor cocktail , 40 mM sodium fluoride and 1 mM sodium orthovanadate for 30 min. The supernatants were subjected to SDS-polyacrylamide gel electrophoresis (Invitrogen) followed by blotting with indicated antibodies. Signals were detected by Supersignal® West Pico Luminol/Enhancer Solution .Phospho- and total IGF-1R ELISAs were performed according to manufacturer's instructions (R&D Systems). For phospho-IGF-1R ELISAs, 100 µg of total protein was used for each cell line. For total IGF-1R ELISAs, 25 µg of total protein was used for most of the cell lines. For cells lines with high IGF-1R expression , only 5 µg of total protein was used.Phospho-RTK array was performed according to manufacturer's instructions (R&D Systems). 300 µg of total protein was used for each membrane.5 cells/well. 24 hour later, cells were transfected with siRNAs against GFP , human IGF-1R or human GAPDH using DharmaFECT 1 transfection reagent as per manufacturer's instruction. Cells were harvested 48 hours after transfection to analyze the level of protein expression by immunoblotting analysis.11–18 cells were seed into 6-well plates at a density of 1.25×10Tissue microarrays (TMAs) were constructed using a fully automated Beecher Instrument, ATA-27, with triplicate cores for each case. Use of human tissues was approved with an institutional waiver and by the human bioutilization committee. The study cohort was comprised of NSCLCs consecutively ascertained at the Memorial Sloan-Kettering Cancer Center (MSKCC) between 1999 and 2006. All biopsies were evaluated at MSKCC, and the histologic diagnosis was based on hematoxylin-eosin staining. TMAs were stained as per manufacturer's instructions on the Ventana Benchmark XT with the anti-IGF-1R rabbit monoclonal antibody directed against the C-terminus of the beta chain. Images were obtained with the Olympus DP20 Camera and taken with the 40X/0.75 objective. Image acquisition and processing software was performed using Adobe Photoshop 7.0 and DP20 software. Cores were scored as follows: 0, no staining; 1, weak focal staining; 2, moderate staining; 3, strong staining with at least 10% of the core showing complete membranous staining. Cores were assigned 1 score and read by 2 pathologists (JTF and MA).For IHC of cell lines, 30 million cells per line were fixed in 4% paraformaldehyde for 10 minutes, washed with 70% ethanol, and spun down into pellets. The pellets were kept in 70% ethanol in 4°C until they were paraffin-embedded and then processed as above.www.broad.mit.edu/cancer/software/genepattern/) and R (http://www.r-project.org/). All data is MIAME compliant and the raw data has been deposited in a MIAME compliant database GEO (accession number GSE17247).As part of a larger effort to characterize the genomes of NSCLC, some authors (MP and RKT) analyzed 84 NSCLC cell lines for chromosomal gene copy number alterations, gene mutations, transcriptional changes and drug response. The detailed description of this collection was published elsewhere IGF-1R obtained from R1507 sensitive lines and select resistant lines (HCC95 and H1975) were sequenced via direct Sanger-based sequencing of PCR fragments amplified with M13 tagged primer pairs .The full-length cDNAs of .R1507 (RO4858696) is a fully human IgG1 monoclonal antibody directed against the extracellular portion of the human IGF-1R. To confirm that R1507 binds selectively to human IGF-1R, cell lysates from two cell lines (H3255 and 11–18) were immunoprecipitated with R1507 and then immunoblotted with a commercial antibody that recognizes the beta chain subunit of IGF-1R. Compared to whole cell extracts, immunoprecipitation with R1507 readily enriched detection of IGF-1R in lysates from both lines EGFR/KRAS/NRAS/HRAS/PI3K mutations . Sensitivity was assessed using a growth inhibition assay that measures a colorimetric signal produced by conversion of resazurin to resorufin, which is directly proportional to the numbers of viable cells. Lines were grown in 10% serum for 72 h in different concentrations (0.1–25 µg/ml) of R1507. None of the lines displayed high sensitivity . Thus, we could not calculate the concentration of drug needed to inhibit growth by 50% (GI50) for each line. Instead, we compared the maximum levels of growth inhibition observed across all cell lines that were achieved using an R1507 concentration of 25 µg/ml . Among the 22 cell lines, 5 displayed 25–50% growth inhibition, whereas the remaining 17 cell lines showed less than 20% inhibition. We chose to define the former lines as “sensitive”, while the latter lines were deemed “resistant”. There was no obvious correlation between R1507 sensitivity and lung cancer histology or mutation status.We next established the sensitivity to R1507 of 22 NSCLC cell lines. These included 12 adenocarcinomas, 9 squamous cell carcinomas and 1 large cell carcinoma, all examined for known utations . SensitiTo assess whether specific activated receptor tyrosine kinases might correlate with R1507 sensitivity, we incubated lysates from sensitive and resistant lines with a human phospho-RTK array containing antibodies that capture 42 different phosphorylated RTKs. Cell lines were grown in the absence or presence of serum. Lysates from 11–18 cells .We next asked whether expression levels of total IGF-1R correlated with R1507 sensitivity in NSCLC cells. Protein levels were measured quantitatively by ELISA using an antibody specific for both unphosphorylated and phosphorylated forms of the protein IGF-1R gene copy number status in a panel of NSCLC cell lines that were previously examined using Affymetrix 250K SNP arrays. 4 and 12 of our sensitive and resistant lines, respectively, happened to be included in the SNP array analysis that was performed as part of a separate study IGF-1R were significantly enriched for R1507-sensitive cell lines . We did not perform additional in situ hybridization to confirm these findings. Notably, none of the 5 sensitive lines harbored any mutations in IGF-1R (data not shown).We also interrogated . Because they also harbor the L858R mutation of EGFR associated with sensitivity to EGFR tyrosine kinase inhibitors like erlotinib . Pre-treatment of cells with erlotinib abolished EGFR phosphorylation, while pre-treatment with R1507 abolished phosphorylation of IGF-1R. The latter was accompanied by degradation of total IGF-1R.11–18 cells displayed the most sensitivity to R1507 . Because. These data indicate that the IGF-1R pathway is both necessary and sufficient for AKT activation in 11–18 cells. Similar results were obtained with extracts from R1507-sensitive HCC15 cells treated in an analogous manner .Ligand binding to IGF-1R and EGFR initiates a series of phosphorylation events that can lead to activation of both the MAPK pathway involved in cell proliferation and the PI3K/AKT signaling pathway involved in cell survival . These d. In HCC95 cells, AKT remained phosphorylated despite serum starvation, and R1507 treatment could not abolish AKT activation . Similar results were obtained in R1507-resistant PC-9 cells (data not shown). Collectively, these data demonstrate that in R1507-sensitive cells, AKT is solely dependent on IGF-1R for activation, whereas in R1507-resistant cells, AKT can be activated by other pathways or remain constitutively activated.By contrast, R1507-resistant cell lines displayed different signaling properties. For example, in H226 cells treated in an analogous manner, both EGF and IGF-1 activated AKT, and suppression of the IGF-1R pathway by R1507 alone was not sufficient to eliminate AKT phosphorylation . In HCC9. Consistent with results obtained using R1507 treatment, IGF-1R siRNAs dramatically decreased AKT phosphorylation without affecting ERK phosphorylation in 11–18 cells. Cells treated with control siRNAs against GAPDH did not display these changes.To verify further that AKT was required for IGF-1R signaling in 11–18 cells, we examined the effect on AKT phosphorylation of knocking down IGF-1R protein expression using gene-specific short interfering RNAs (siRNAs) . Compared to either single agent alone, the combination further inhibited cell growth. Similar results were obtained with the four other R1507 sensitive lines, even though only 11–18 cells harbored a drug-sensitive EGFR mutation. By contrast, there was no additive effect in adding erlotinib to R1507 in the lines already resistant to R1507 alone. We were unable to calculate a combination index for the drug combination, as dose response curves derived from treatment of cells with R1507 did not fit into a Hill-type of curve . Analogous results were also obtained by combining R1507 with the chemotherapeutic agent, paclitaxel .We examined the effects of adding erlotinib to R1507 on the growth of 11–18 cells . Single agent R1507 at 25 micrograms/milliliter had little effect as well. However, co-treatment with both erlotinib and R1507 induced an increase in annexin-V-positive cells, both at 100 nanomolar (8.9% to 17.5%) and 500 nanomolar (8.9% to 25.4%) erlotinib concentrations . Based upon the analysis of IGF-1R/EGFR signaling , R1507 contributes to apoptosis induction most likely by eliminating survival-related AKT phosphorylation. Consistent with this notion, we also observed enhanced apoptosis in 11–18 cells using a combination of erlotinib and an experimental PI3K inhibitor (data not shown).We studied further in 11–18 cells the effect of the combination of R1507 and erlotinib on inducing apoptosis, as measured by standard annexin-V/propidium iodide (PI) assays. Although 11–18 cells harbor a drug-sensitive EGFR L858R mutation, these cells did not die after 24 h of exposure to erlotinib, even at a drug concentration of 500 nanomolar . Single ( and ). Whereas sensitive lines with high levels of total IGF-1R reacted strongly with G11 in a membranous staining pattern, resistant lines with low levels of the protein did not. We next performed IHC analysis on 4 existing tissue microarrays (TMAs) comprised of primary NSCLC tumors from 270 patients. Clinical characteristics of the patients are summarized in , see Having identified total IGF-1R protein levels as a potential biomarker of NSCLC cell line sensitivity to R1507, we sought an independent, more clinically applicable assay to assess IGF-1R status in human primary NSCLCs. We obtained a monoclonal antibody (G11) that recognizes the C-terminus of both unphosphorylated and phosphorylated forms of the IGF-1R beta chain. The antibody does not cross react with the insulin receptor (data not shown). We then stained sections made from formalin-fixed NSCLC cell lines and found a striking correlation between IHC staining of the lines and levels of total IGF-1R as determined by ELISAs and . WIGF-1R overexpression was strongly associated with squamous cell carcinoma, as 79.2% of squamous cell lung cancer displayed high IGF-1R staining versus 35.4% of adenocarcinomas . However, by contrast, the growth of The lack of significant growth inhibition induced by R1507 in NSCLC cells can be further explained by two additional factors. First, IRs and IGF-1Rs can form hybrid receptors, and it is not yet clear how specific anti-IGF-1R-targeted therapies will interfere with signals emanating from hybrid receptors. However, use of agents that target both IR and IGF-1R was not the focus of this study. Second, we only assessed the effect of R1507 in vitro. R1507 does not elicit antibody-dependent cytotoxicity, but the antibody could still induce more dramatic responses in vivo.Through analysis of EGFR, IGF-1R, and downstream MAPK and AKT signaling pathways, we found in the R1507-sensitive but not -resistant lines that IGF-1R activation is sufficient and required for AKT activation. Activation of IGF-1R by IGF-1 or inhibition of IGF-1R by R1507 had minimal effect on ERK activation in either R1507 sensitive or resistant lines. Thus, IGF-1R appears to be the major driving force for AKT activation in drug-sensitive cells. The dependence of AKT on IGF-1R signaling was further verified by knockdown of IGF-1R using siRNAs. Consistent with this finding, previous studies have shown in rhabdomyosarcoma cells that phospho-AKT status is controlled predominantly by IGF-1R activation Through analysis of TMAs with another anti-IGF-1R antibody (G11), we found evidence for high total IGF-1R expression in primary human NSCLCs. 39.3% of tumors had medium to high levels of IGF-1R expression (scores of 2+ to 3+), while 16.7% had high IGF-1R expression (scored as 3+). Higher levels of expression were associated with the squamous cell carcinoma subtype. There was no correlation with gender, smoking history, or overall survival. These results are consistent with a previous study with fewer numbers of patients , a majority of squamous cell lung cancers displayed high levels of IGF-1R expression. These data suggest that squamous cell lung cancers may be more sensitive than lung adenocarcinomas to IGF-1R targeted therapy. However, in our growth inhibition assays, only 1 out of 9 squamous cell carcinoma lines versus 4 out of 11 adenocarcinoma lines displayed moderate sensitivity to R1507. This discrepancy could possibly be explained by phenotypic changes that occur in squamous cell lines when they are grown in vitro. Lung adenocarcinoma cell lines and primary tumors were recently shown to be genetically and transcriptionally similar In a randomized phase II study of patients with advanced treatment-naïve NSCLC, disease in those with squamous cell carcinoma responded to the combination of paclitaxel, carboplatin and an anti-IGF-1R antibody better than those with other histological subtypes In summary, characterization of 22 NSCLC cell lines has lead to identification of high expression levels of total IGF-1R as a predictive marker of relative sensitivity to R1507, an anti-IGF-1R antibody. In such sensitive cells but not resistant cells, additive effects were observed by combining R1507 with other anti-cancer agents. Immunohistochemical analysis of primary NSCLC tumors with an anti-IGF-1R antibody demonstrated that a significant fraction display high levels of the receptor. These results lay groundwork for rationally designing clinical trials to enrich for lung cancer patients that might benefit from treatment with anti-IGF-1R antibodies like R1507.Figure S1R1507 binds to human IGF-1R. 500 micrograms of total protein from H3255 and 11–18 cell lysates were incubated with 5 µg R1507 overnight followed by 2 hours incubation with protein A/G beads (Santa Cruz Biotechnology). Immunoprecipitates (IP) were separated by SDS/PAGE and then subjected to immunoblotting with a commercial antibody against the beta chain of IGF-1R. WCL - whole cell lysates.(0.93 MB TIF)Click here for additional data file.Figure S2The single agent activity of R1507 in sensitive lines. Various cell lines were treated with R1507 for 72 hours, and growth inhibition was measured using CellTiter Blue reagents.(0.72 MB TIF)Click here for additional data file.Figure S3The combinatorial effect of R1507 and paclitaxel in NSCLC cell lines. R1507 enhances paclitaxel-induced growth inhibition in R1507 sensitive cell lines. Various cell lines were treated with increasing concentrations of paclitaxel in the absence or presence of 25 µg/ml R1507 for 72 hours, and growth inhibition was measured by CellTiter Blue reagents. Data represent the mean ± standard deviation of triplicates.(0.23 MB TIF)Click here for additional data file.Table S1IGF-1R cDNA sequencing primers.(0.06 MB PDF)Click here for additional data file. |
Food and Drug Administration, Environmental Protection Agency, and Consumer Product Safety Commission. The acceptance was based on recommendations made by ICCVAM after an extensive evaluation of the methods.In a significant step forward for alternative safety test methods designed to reduce, refine, or replace the use of live test animals, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recently announced the regulatory acceptance of two new The United States tallies an estimated 125,000 eye injuries in the home each year caused by accidental exposure to common household products such as bleach and oven cleaner, according to the American Academy of Ophthalmology. Proper identification and labeling of substances that can damage the eye is one way to combat such injuries. Several agencies require manufacturers to test new products for their potential to cause temporary or permanent blindness, irritation, or other eye injuries.Working with the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), ICCVAM evaluated and recommended the bovine corneal opacity and permeability (BCOP) and the isolated chicken eye (ICE) test methods—the first nonanimal ocular safety test methods to be accepted by the regulators. In both cases, the animal eyes used for the tests are slaughterhouse waste, so no animals are euthanized specifically to obtain these tissues. The assays have been in development since the early 1990s.Now that the BCOP and ICE assays have earned regulatory acceptance, they must be considered as the first option for ocular safety testing under the Animal Welfare Act, which requires the consideration of alternative methods before animals are used for procedures that may cause more than slight or momentary pain or distress. “If you get a positive result in either of these assays, you can use that as a positive for the purposes of classifying and labeling [a material] as a severe irritant,” says Marilyn Wind, chair of ICCVAM and a deputy associate executive director with the Consumer Product Safety Commission. “If it’s negative, then [manufacturers] have to go to the next step and test in animals. This eliminates the most corrosive and severe chemicals from having to be tested in animals, so there is a reduction in potential pain and distress.”in vitro methods for ocular safety, hoping to eventually eliminate altogether the need for in vivo testing in this realm.Although precise numbers are not available for the use of live animals in ocular testing, William Stokes, director of NICEATM and executive director of ICCVAM, estimates that based on the relative distribution of adverse effects, use of the two assays could reduce the use of live animals for eye safety testing by 10% or more. “The overall goal is to come up with an integrated testing strategy using several nonanimal tests that will accurately predict whether chemical products have the potential to damage the eye or not,” he says. ICCVAM and NICEATM are in the process of evaluating other In the near term, ICCVAM is working with its counterparts in Europe and Japan to expedite approval of the BCOP and ICE assays at the international level by the 30-member Organisation for Economic Co-operation and Development. This group includes the United States, Canada, Japan, and most of the European Union, where a ban on live animal testing of cosmetic ingredients takes effect in March 2009 and the newly implemented REACH legislation will require testing of thousands of chemicals by 2018. |
Levamisole, which has immunostimulant activity, is now being used to treat some forms of cancer. We report that the drug enhances granulocyte colony formation. The mechanism of action appears to be partly through modulation of molecules on cell membranes. The molecular content of colony-stimulating activity (CSA) released into leucocyte-conditioned medium by cells of leukaemic and some preleukaemic patients can be quantitatively altered by levamisole, both in vitro and in vivo, but the CSA produced is qualitatively similar to that released by normal cells. The demonstrated levamisole enhancement of colony formation, and altered CSA types detected in leucocyte-conditioned medium, makes this drug a promising candidate for treatment of selected leukaemic states and in preleukaemia. |
Odocoileus virginianus) and mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose . A leucine variant at position 132 (132L) in prion protein of Rocky Mountain elk confers a long incubation time with CWD, but not complete resistance. However, variants in regulatory regions outside the open reading frame of PRNP have been associated with varying degrees of susceptibility to prion disease in other species, and some variants have been observed in similar regions of Rocky Mountain elk PRNP. Thus, additional genetic variants might provide increased protection, either alone or in combination with 132L.Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids including white-tailed the number of known variants in this region. A haplotype-tagging approach was used to reduce the number of genetic variants required to survey this variation in the PRNP gene region of 559 Rocky Mountain elk. Eight haplotypes were observed with frequencies over 1.0%, and one haplotype was present at 71.2% frequency, reflecting limited genetic diversity in the PRNP gene region.This study provided genomic sequence of all exons for PRNP region (P > 0.05).The presence of 132L cut odds of CWD by more than half , which was similar to a previous report. However after accounting for 132L, no association with CWD was found for any additional variants in the Odocoileus virginianus and O. hemionus, respectively), Rocky Mountain elk (Cervus elaphus nelsoni), and moose [Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids including white-tailed and mule deer . This ins alces) . There is alces) ,3.d) that is protease resistant and infectious [c) is expressed in neurons of all mammals studied to date [d is capable of recruiting the normal cellular form of the protein (PrPc) into the same misfolded conformation [The TSEs are believed to be caused by a misfolded prion protein in prion protein confers a long incubation time with CWD, but not complete resistance -11. HowePRNP gene region - whether previously known, discovered in this study, or as yet undiscovered - that may provide increased protection from CWD in Rocky Mountain elk.The many variants in any gene region are arranged in only a limited number of haplotypes, or linear arrangements of genetic variants as they exist on chromosome segments in a population. To find an important genetic variant, one need only genotype a limited set of variants that "tag" all the major haplotypes in a gene region to capture all or most of the information contained in the complete set of genetic variants in a region . This std detection using an automated monoclonal antibody immunohistochemistry assay [d. Because the time between infection and appearance of detectable PrPd is unknown [d were not defined as CWD negative; these animals were defined as herd-matched controls.A total of 559 captive and free-ranging Rocky Mountain elk were sampled from herds following positive CWD diagnosis in 6 states including Colorado, Montana, Minnesota, Nebraska, Oklahoma, and South Dakota. All animal procedures used were exempt by the Institutional Animal Care and Use Committee of Washington State University as no live animal use was involved; all samples were from depopulation programs approved by federal and state regulatory bodies to control the spread of CWD. In total, 120 animals tested positive and 439 tested negative for CWD by immunohistochemical methods as previously described . Brieflyry assay . The staGenetic variant discovery was performed on a group of 20 animals chosen for geographic diversity, including animals from Colorado, Montana, Minnesota, and Oklahoma. PCR was performed using primers as shown . Association analysis of other markers while accounting for 132L was performed on markers with greater than 5% minor allele frequency using the logistic procedure of SAS 9.2 with a model including the presence/absence of 132L in addition to presence/absence of the variant of interest. Additionally, similar logistic models were used to test full genotypes while accounting for 132L by performing exact conditional tests for genotypes of 132L and the variant of interest. Fisher's exact was used to perform specific comparison of CWD frequency among L132-bearing haplotypes.PRNP gene region, assessed haplotype structure in this gene region in Rocky Mountain elk, and tested common haplotype variants for association with CWD.While it is clearly possible to have strong genetic resistance to TSEs, Rocky Mountain elk have only been shown to have the 132L substitution which confers extended incubation time with CWD but not complete resistance ,10. AddiPRNP gene region of Rocky Mountain elk. As depicted in Figure PRNP gene of Rocky Mountain elk. The total sequence was over 7 kb from a region spanning approximately 63 kb, of which over 40% was not previously reported to the best of our knowledge . The 132L variant was underrepresented among CWD cases (P = 0.0031), occurring in cases less than half as often as the predominant genotype 132 MM . However, after accounting for 132L no other variants showed significant association with CWD even on a nominal basis (P > 0.05), before any correction for multiple testing. The statistical tests specifically included comparison of CWD frequency among carriers of two haplotypes (2 and 7) that harbor L132, but no significant differences were observed (P = 0.99). Furthermore, all genotypes with any appreciable frequency showed CWD positive animals, suggesting that there is no complete resistance to CWD on the basis of common Katherine I. O'Rourke is the inventor of patented monoclonal antibody 99.SNW conceived the study, carried out the statistical analyses, and drafted the manuscript. TRS participated in sample collection and performed diagnostic pathology. JOR participated in study design, genotyping, and analysis. KIO participated in study design, sample collection, genotyping, and analysis. All authors read and approved the final manuscript.Amplification primers and conditions. Primer sequences, strand directions, base positions relative to reference sequences, annealing temperatures, and included region descriptions.Click here for fileSequencing primers. Primer sequences, strand directions, base positions relative to reference sequences, and descriptions of regions sequenced using each primer.Click here for file |
Streptococcus pneumoniae has increased in the United States over the past decade. Considerable geographic variation in susceptibility necessitates regional resistance tracking. Traditional active surveillance is labor intensive and costly. We collected antibiogram reports from North Carolina hospitals and assessed pneumococcal susceptibility to multiple agents from 1996 through 2000. Susceptibility in North Carolina was consistently lower than the national average. Aggregating antibiogram data is a feasible and timely method of monitoring regional susceptibility patterns and may also prove beneficial in measuring the effects of interventions to decrease antimicrobial resistance.Antimicrobial resistance to penicillin and macrolides in Streptococcus pneumoniae is a leading cause of community-acquired illness, resulting in an estimated 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and 7 million cases of otitis media each year in the United States guidelines for susceptibility cut points.This study was conducted in North Carolina from April to September 2001. A study packet was mailed in April 2001 to the directors of clinical microbiology laboratories at all 114 North Carolina hospitals identified by the Centers for Medicare and Medicaid Services (CMS). Hospitals subsequently identifying themselves as specialty hospitals were excluded from all analyses. The packet included a letter describing the project, a questionnaire on hospital characteristics and laboratory testing methods, a request for submission of From the antibiograms, the numbers of pneumococcal isolates tested and numbers of isolates testing susceptible were added across all hospitals for each antimicrobial agent for each year of data to create statewide summary totals. Data for drugs that predictably elicit the same susceptibility result were combined: penicillin/oxacillin, cefotaxime/ceftriaxone, and levofloxacin/ofloxacin. Antibiogram data from each hospital for each year of data were assessed for inclusion. Data from antibiograms reporting 1) testing results cumulative over >1 year, 2) percentages of susceptible isolates without providing the total number of isolates tested, 3) more than one susceptibility value for the same drug for the same period, or 4) results by nursing unit or named patient were all excluded for the year and drug in question. We also excluded data for drugs other than penicillin if more isolates were tested for penicillin susceptibility than for the other drugs, and the antibiogram did not clearly indicate that the subgroup selected for additional testing was based on the source of the specimen . Testing only penicillin-nonsusceptible isolates for susceptibility to other drugs could yield misleading results because penicillin-resistant isolates are more likely to be resistant to other drugs as well by the county the respondent listed on the questionnaire. The Committee on the Protection of the Rights of Human Subjects, University of North Carolina School of Medicine, granted Institutional Review Board approval.S. pneumoniae susceptibility. Data from published reports were included if they covered a period of no more than 12 months and identified the source of isolates as respiratory, invasive, or both. If the surveillance period overlapped two calendar years while covering one respiratory season, the data were classified by the latter year. For instance, isolates collected from October 1999 through April 2000 were labeled as year 2000 data.North Carolina’s pneumococcal susceptibility pattern from 1997 through 2000 was compared to patterns shown by national surveillance systems tracking <0.05 was considered statistically significant. Trend tests were performed by using SAS version 8.1 . Exact binomial 95% confidence intervals were calculated for proportions by using Stata version 7.0 .We used the Cochran-Armitage trend test, which tests for trends in binomial proportions across levels of an ordinal covariate, to evaluate temporal patterns in the data. A two-sided p-value Overall, 60 of the 110 (55%) potentially eligible hospitals replied to the survey, although ultimately fewer hospitals were able to contribute pneumococcal susceptibility data. Thirty of the 114 CMS-identified hospitals responded to the initial request for information within the first month. After follow-up telephone calls to the remaining 84 hospitals, 30 additional hospitals responded. Four hospitals were excluded: one listed under two different names, one psychiatric hospital, one orthopedic hospital, and one alcohol treatment center. North Carolina hospitals not enumerated on the CMS list, and hence not invited to participate in this study, included five military, four Veterans Affairs, and two prison hospitals, as well as several long-term care and rehabilitation centers. Additionally, small hospitals that were part of a health-care system dominated by one large hospital were frequently omitted from the CMS list. These noninvited hospitals were primarily rural, community facilities.>1,000). The central region contained most of the state’s large, academic hospital centers as well as most of its urban counties. No discernable difference was evident between the 50 potentially eligible hospitals that did not participate and the 60 that did, except that all major academic centers participated.The proportion of responding hospitals was similar across the three regions of the state: 17/30 (57%) hospitals in the west, 27/51 (53%) in the central region, and 16/29 (55%) in the east. The average number of beds was 257 seven antibiograms provided data for a period of more than 12 months, 2) four antibiograms reported susceptibility rates without numbers of isolates tested,3) one antibiogram listed more than one susceptibility result for the same drug for the same period, 4) two antibiograms provided results by nursing unit or named patient, and 5) 12 antibiograms reported more isolates tested for penicillin susceptibility than for susceptibility to other drugs without explaining the drop-off in isolate number, therefore, requiring exclusion of the nonpenicillin data. After accounting for the aforementioned exclusions, the number of antibiograms with usable data for any single drug for a given 1-year period ranged from 1 to 42 .Eleven of the 60 (18%) hospitals responding to the study request did not provide antibiogram data. Of these hospitals, nine did not perform on-site susceptibility testing, and two gave no explanation for not submitting antibiograms. One additional hospital reporting off-site testing made antibiogram information available through a reference laboratory. Although 49 hospitals contributed antibiogram data for at least one drug for at least 1 year, not all of the data from these antibiograms qualified for inclusion in the analyses. Susceptibility data were excluded for at least one class of drugs for Although most hospitals submitted susceptibility testing results for 2000, fewer did so for earlier years. The numbers of hospitals providing data on penicillin susceptibility were 18 hospitals for 1996, 24 hospitals for 1997, 33 hospitals for 1998, 36 hospitals for 1999, and 42 hospitals for 2000 . The numS. pneumoniae isolates testing susceptible to penicillin decreased (p<0.001) . Althougp<0.001) . From 19A subanalysis of the 15 hospitals for which usable information on penicillin susceptibility was available for each year produced a comparable trend. Although with this reduced amount of data susceptibility was higher in 1996 (69%) than in the full analysis (65%) (chi square p=0.03), the trend over time remained consistent (p<0.001). By 2000, 54% of the isolates reported at these 15 hospitals were susceptible to penicillin, compared to 52% in the entire study group (chi-square p=0.13).Among penicillin-nonsusceptible isolates, the proportions intermediate and resistant were available from 5 hospitals (419 isolates) in 1996, 6 hospitals (592 isolates) in 1997, 11 hospitals (849 isolates) in 1998, 12 hospitals in 1999, and 11 hospitals in 2000. These represented between one-quarter and one-third of all isolates tested for penicillin susceptibility, depending on the year. During the 5-year period, the proportion of susceptible isolates appeared to decrease, the proportion of resistant isolates increased, and the proportion of intermediate isolates showed little change .S. pneumoniae isolates susceptible to cefotaxime decreased 8%. Although third-generation cephalosporins did not show a consistent decrease in susceptibility each year, the decline during the 5-year period was still significant (p<0.001). Susceptibility to quinolones and vancomycin remained high, despite the fact that two hospitals, one for two different years, reported a total of five isolates as vancomycin-resistant. The low level of levofloxacin susceptibility in 1998 (92%) was based on only 237 isolates. Larger numbers of isolates available in subsequent years did not support a pattern of greatly reduced susceptibility.Macrolide susceptibility decreased from 78% in 1996 to 61% in 2000 (p<0.001). From 1996 to 2000, the proportion of Aggregating hospital antibiogram data from the state of North Carolina appears to be a feasible, practical method for monitoring trends in pneumococcal susceptibility. Large numbers of isolates are available for annual comparisons with consistent reporting on penicillin, albeit less consistent reporting on other drugs. Susceptibility to clinically important antibiotics was shown to decrease significantly.The observed progression of both penicillin and macrolide resistance is of particular concern. The increase in penicillin resistance appears to correlate with an increase in high-level rather than intermediate-level resistance and high-level penicillin resistance has been associated with worse outcomes for pneumococcal infections and the support of one graduate student. Chin et al. spent $52,000 for 1 year of active surveillance in 12 hospitals. The antibiogram surveillance had several potential limitations, however. First, a 55% response rate may be adequate for certain surveys, but full participation from all N.C. microbiology laboratories would be the best way to ensure that surveillance data accurately reflect susceptibility patterns. Furthermore, all participating hospitals did not submit an antibiogram for each year nor did all data on each antibiogram meet inclusion criteria. Yet our data included many more hospitals in this study locale than any previously published surveillance system. Second, many hospitals were unable to access data from past years for a variety of reasons, including changes in testing and computer systems. Collecting antibiogram reports on a yearly basis should allow more hospitals to more easily contribute their data. Third, specimens are increasingly sent to referral hospitals or reference laboratories. For instance, 9 of the 60 participating hospitals did not have antibiogram data, and we were able to get results from a reference laboratory for only a single hospital. The lack of availability of antibiograms at hospitals that use reference laboratories is disconcerting since information needed to guide local antibiotic decisions is not accessible. Lastly, testing and reporting procedures were inconsistent, such as drugs tested, identification of specimen source, breakdown of intermediate and high-level resistance, and period covered by the antibiogram. We hope that providing N.C. microbiology laboratories with these study findings will encourage participation in continued surveillance activities as well as more uniform testing and reporting procedures.The submitted data appeared to be consistent despite the fact that the population under study changed from year to year as more data became available. The results of the penicillin sub-analysis, consisting solely of those hospitals providing usable information for each year from 1996 to 2000, yielded results comparable to those found in the overall study. Additionally, the observed susceptibility results generally support known resistance patterns, such as the correlation of penicillin and ceftriaxone susceptibility, lower levels of macrolide than clindamycin susceptibility, and near universal vancomycin susceptibility (Streptococcus pneumoniae, both in North Carolina and within regions of the state. To further develop this antibiogram surveillance system, we are partnering with the North Carolina State Laboratory of Public Health to establish an electronic network of microbiology laboratories to enhance interlaboratory communication. We hope to share practices, encourage testing consistent with current NCCLS guidelines, and support standardized, efficient, annual, electronic submission of antibiograms. We also hope that knowledge of N.C. resistance patterns will both guide treatment decisions and motivate judicious antibiotic prescribing behavior.Combining hospital antibiogram data appears to be an effective method of tracking antimicrobial susceptibility among Judicious use of antibiotics is essential for their continued effectiveness. Not only have regional trends in antibiotic resistance been linked to antibiotic use ( |
Age-related macular degeneration (AMD), the leadingcause of blindness in the elderly, targets the retinal pigment epithelium(RPE), a monolayer of cells at the back of the eye. As AMD progresses, itcan develop into two distinct forms of late AMD: "dry," atrophic AMD,characterized by RPE senescence and geographic RPE loss, and "wet,"neovascular AMD, characterized by RPE activation with abnormal growth ofchoroidal vessels. The genetic and molecular pathways that lead to thesediverse phenotypes are currently under investigation. We have found thatbone morphogenetic protein-4 (BMP4) is differentially expressed in atrophicand neovascular AMD. In atrophic AMD, BMP4 is highly expressed in RPE, andmediates oxidative stress induced RPE senescencein vitro via Smadand p38 pathways. In contrast, in neovascular AMD lesions, BMP4 expressionin RPE is low, possibly a result of local expression of pro-inflammatorymediators. Thus, BMP4 may be involved in the molecular switch determiningwhich phenotypic pathway is taken in the progression of AMD. Age-related maculardegeneration (AMD) is the leading cause of irreversible blindness in theelderly -2. ConsiRecently, we reported that bonemorphogenetic protein (BMP)4 is prominently expressed in the RPE and adjacentextracellular matrix of patients with the dry or atrophic form of AMD whencompared to controls Figure , B. Herein vitro [WAF1/ cip1, and to decrease phospho-Rb. Importantly,BMP4-mediated RPE senescence can be inhibited by Chordin-like, a BMP4antagonist, and SB203580, a phospho-p38 inhibitor. Our findings not onlydisclose a molecular pathway linking oxidative stress with RPE senescence, butalso provide a novel therapeutic target for treatment of atrophic AMD.BMP4 is an important regulator ofdifferentiation, senescence and apoptosis in many different cells and tissues,10. We rin vitro ,and thaRecently, Demidenko et al. evaluateINK4a and p21WAF1/ cip1.BMP4 and other BMP signaling pathways were also found to participate insenescence of multiple cancer cell types or in the inhibition of tumor cellgrowth [WAF1/ cip1 up-regulation, and Rbdephosphorylation, while in glioblastoma, BMP4 and its cognate receptors cantrigger the Smad signaling cascade to reduce the proliferation of tumor cells[Interestingly, BMP4 has beenfound to be involved in chemotherapeuticagent-induced premature senescence of cancer cells . Adriamylgrowth ,15. For or cells. Togethe WAF1/ cip1 and thedown-regulation of phosphorylated Rb and that blockade of TGF-β signaling by specific TGF-β antibody can impede RPE senescence [Transforming growth factor (TGF)-β has been extensively reported to beinvolved in mediating oxidative stress induced premature senescence offibroblasts -19. Recenescence .This fiA variety of intrinsic andextrinsic stress signals can activate the p53 pathway, which then triggerseither cellular senescence or apoptosis ,22. We fIt remainsunanswered why some patients develop atrophic AMD while others develop theneovascular form of the disease. The switch between dry and wet AMD may berelated to differences in the microenvironment created by senescent RPE cells,which secrete a number of cytokines and growth factors . The defIn vitro culture of human fibroblasts results in a fraction ofcells senescing at every population doubling. The senescent cells have shortertelomeres than their cycling counterparts. Thus, it was concluded that the maincause of intrinsic heterogeneity of senescent fibroblasts was the cell to cellvariation of telomere shortening [It has been previously observed thattissues in aged individuals may exhibit the paradoxical juxtaposition ofatrophy and hyperplasia within the same tissue or even within the same celltype . This reortening . Using portening .Much remains to be learned about thegenetic and environmental factors mediating the progression of early AMD to itslate forms. Our finding of differential expression of BMP4 in geographicatrophy and neovascular AMD and the interactive roles of oxidative stress,inflammation and senescence in the regulation and functional effects of thisgrowth factor, suggests the possibility that BMP4 may be playing a part in themolecular switch determining which phenotypic pathway is taken in theprogression of AMD.This work was supported by National Institutes ofHealth grants EY01545 and EY03040 and by the Arnold and Mabel BeckmanFoundation. |
Plasmodium vivax, a parasite that is almost impossible to cultivate in vitro, and Anopheles aquasalis is an important malaria vector. Understanding the interactions between this vector and its parasite will provide important information for development of disease control strategies. To this end, we performed mRNA subtraction experiments using A. aquasalis 2 and 24 hours after feeding on blood and blood from malaria patients infected with P. vivax to identify changes in the mosquito vector gene induction that could be important during the initial steps of infection. A total of 2,138 clones of differentially expressed genes were sequenced and 496 high quality unique sequences were obtained. Annotation revealed 36% of sequences unrelated to genes in any database, suggesting that they were specific to A. aquasalis. A high number of sequences (59%) with no matches in any databases were found 24 h after infection. Genes related to embryogenesis were down-regulated in insects infected by P. vivax. Only a handful of genes related to immune responses were detected in our subtraction experiment. This apparent weak immune response of A. aquasalis to P. vivax infection could be related to the susceptibility of this vector to this important human malaria parasite. Analysis of some genes by real time PCR corroborated and expanded the subtraction results. Taken together, these data provide important new information about this poorly studied American malaria vector by revealing differences between the responses of A. aquasalis to P. vivax infection, in relation to better studied mosquito-Plasmodium pairs. These differences may be important for the development of malaria transmission-blocking strategies in the Americas.Malaria affects 300 million people worldwide every year and is endemic in 22 countries in the Americas where transmission occurs mainly in the Amazon Region. Most malaria cases in the Americas are caused by Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae while the main vectors are Anopheles darlingi, Anopheles aquasalis and some species of the Anopheles albitarsis complex Plasmodium to drugs and of insects to insecticides indicates the need for discovering new strategies to combat this disease. In some Anopheline species studies aiming at blocking malaria transmission have been successful Malaria affects 300 million people worldwide every year. Among the endemic countries 22 are in the Americas, where transmission occurs mainly in the Amazon region. Brazil had an estimated 1.4 million malaria cases in 2006, over half of the total for the Americas, while Colombia had an estimated 408,000 cases (“WHO/HTM/GMP/2008.1”). The malaria parasites that circulate in this region are P. vivax. The mosquito A. aquasalis is an important American malaria vector that breeds in brackish coastal marsh habitats from Nicaragua to Southern Brazil Plasmodium sp. life cycle in the insect vector is very complex. During 14 to18 days the parasite passes through various stages of development, and interacts with different tissues of the insect. The study of the reproduction and development of these parasites in their vector is essential for the development of new malaria control strategies. However, almost all previous studies are based on African and Asian anopheline species such as Anopheles gambiae infected with P. falciparum or Plasmodium berghei and Anopheles stephensi infected with P. bergheiThe parasite responsible for the majority of malaria cases in the Americas is P. vivax infection on A. aquasalis gene expression. Due to the lack of a practical continuous cultivation system for P. vivaxP. vivax malaria patients.The main goal of this study is to analyze the effect of A. aquasalis genes should lead to a better understanding of this vector-parasite relationship and to the identification of targets for blocking malaria transmission.Different subtraction mRNA libraries were constructed and analyses of these libraries revealed numerous mosquito genes that are up and down–regulated by infection. The analysis of these parasite induced Plasmodium infection can induce genes responsible for immunity, as well as more general aspects of stress, we constructed mRNA subtraction libraries with the aim of identifying genes regulated by this challenge. A. aquasalis were infected with P. vivax through artificial feedings with blood of malaria patients. All infected insect samples utilized in this study were tested for P. vivax infection by PCR and all of them amplified the expected 84 bp band (Based on the precedent from anopheline studies bp band .Subtraction cDNA libraries were constructed for these studies from RNAs obtained from insects at 2 and 24 hours post-feeding on uninfected blood (F) or on infected blood (I). Four libraries were constructed using cDNAs from 2 and 24 hours infected minus non-infected insects and 2 and 24 hours non-infected minus infected insects . The 2 and 24 hour timepoints were chosen for library generation since they represent the first stages of infection prior to parasite differentiation into oocysts, and thus should provide data most relevant to development of transmission blocking strategies. At 2 hours after infection (AI) the gametocytes differentiate and fertilization and zygote formation occur, and at approximately 24 hours AI the ookinetes pass through the midgut epithelium, a crucial and traumatic step in infection.Plasmodium database, and since these were related to very conserved sequences, they could be of mosquito origin. This absence of Plasmodium sequences can be explained by the low parasite load observed in this natural vector-parasite pair or the early times of infection, before sporozoites amplification.The amplicons obtained from these libraries were cloA. aquasalis or represent untranslated regions of genes poorly conserved among species. Also, 11% of the sequences coded for unknown conserved proteins, normally related with other mosquito sequences which might represent conserved genes useful in ample spectrum control strategies.After annotation, sequences were divided into 13 different categories based on their biological functions , S3, S4.Plasmodium infection on the mosquito reproductive fitness One major category of genes (59%) identified in the 24hI-F library codes for unknown proteins . These gA. stephensi infected with P. bergheiA. gambiae-P. falciparumA. aquasalis in the early steps of P. vivax infection could be responsible for the success of colonization and transmission of this malaria parasite. These differences in the biology of Old and New World parasite-vector interactions could well be due to genetic/evolutionary factors since, albeit belonging to the same genus, these species have evolved in distant parts of the world, submitted to very different environmental pressures. Still, we cannot exclude completely the possibility of the subtraction approach missing some immune genes. Future analyses using more sensitive mRNA abundance assays will be used to address this issue when genome sequences become available.Unexpectedly, only 3% of all ESTs (15 sequences) were related to immunity , and no To validate differential expression results a subset of the genes identified in the subtraction libraries were selected for analysis by real time PCR (RTPCR). In some cases, the expression of some of these genes was investigated in relation to blood-feeding, development, and comparing males and females. The majority of RTPCR experiments corroborated the subtraction library results. The few inconsistencies observed are related to the nature of the subtraction screening methodology. Nevertheless, these did not invalidate this approach, but rather confirmed its utility as a technique to identify differentially expressed sequences and rare transcripts Aedes aegypti was more closely related to actin5 of aegypti . The exp aegypti . In fema aegypti , which m aegypti . Most imhours AI . This coBased on the importance of digestive enzymes for insect reproduction and in premises that these enzymes may affect positively or negatively the parasite development and insect vector competence, the expression of two digestive enzymes, chymotrypsin-like serine protease and carboxypeptidase were evaluated.P. vivax infection decreased the expression of this gene were found only in the 2hI-F library. This gene was more closely related to carboxypeptidase A than B based on phylogenetic analyses . ExpressF and AI . This isA. aquasalis, mainly in first instar larvae (L1) and pupae and 2hI-F (ficolin and fibronectin-like). The expression of the first one (GR486377) related to mosquito and horse-crab techylectins , a molecnd pupae and the nd pupae . No signnd pupae . NeverthA. gambiae (GR486800) was identified in the 2hI-F library. Phylogenetic analysis showed the deduced peptide to be more related with BRP2 than BRP1 of gambiae . BRP prohours AI indicateA. aquasalis cecropin protein with high similarity (96%) to cecropin were found in the 2hF-I library. These two ESTs generated the complete open reading frame for an protein . Phyloge gambiae . Males p gambiae . In fema gambiae . This inhours AI , at a tiPlasmodium in the insect A. aquasalis serpin to group with inhibitory proteins more related to the A. gambiae serpin 4 and lower levels of the negative immune regulator serpin than females, indicating that these insects are immunologically ready for the ingestion of contaminated sugar or other fluids. On the other hand, prior to blood feeding, females presented higher levels of mRNA for the digestive enzymes chymotrypsin-like serine protease and carboxypeptidase, possibly involved with digestion of blood necessary for egg maturation, than males.RTPCR experiments revealed high expression of mRNAs for some digestion and immune genes in sugar-fed female and male Although blood was obtained from many different donors, and RNA was extracted from pools of insects, we cannot totally exclude the possibility, albeit improbable, that some of the gene expression differences observed were due to heterogeneity of the blood ingested by the insects or to anemic state commonly found in malaria patients A. aquasalis and its natural parasite P. vivax. Subtraction experiments and RTPCR showed that the early steps of P. vivax infection did not activate the A. aquasalis immune system as powerfully as observed in other mosquito-Plasmodium pairs. In the case of A. aquasalis, the presence of the parasite in insect haemolymph 36 hours after infection, rather than its presence in the midgut or during passage through its epithelium 24 hours after infection, appeared to correlate with the induction of a potent anti-microbial immune response. This information could contribute for the development of new strategies intended to interrupt the P. vivax cycle within its vector. Differences detected in relation to others malaria vectors might help direct new malaria transmission-blocking strategies specific for A. aquasalis. Garver et al.A. gambiae to P. falciparum and P. berghei, and also revealed some common aspects of the immune response of three anopheline species, A. gambiae, A. stephensi and Anopheles albimanus to a single Plasmodium species, P. falciparum. These observations demonstrate the importance of obtaining specific information on the various malaria parasite and vector pairs, since it apparently is impossible to predict their responses and outcome of infection.Our present studies revealed new aspects of interactions between the important American vector P. vivax infected human blood, eight patients were selected among the people visiting the Health Center looking for malaria diagnosis and treatment during outbreaks. Diagnosis was performed by Giemsa stained blood smear. After positive diagnosis and visualization of gametocytes, patients were interviewed and inquired about the possibility of volunteer donation of a small amount of blood for research purposes. After verbal agreement, a term of consent was first read to the potential volunteers, with detailed verbal explanation, and, after final consent, signed by the patient. After this, one 200 µl sample of venous blood was drawn from each patient and placed in heparinized tubes. Blood samples were kept under refrigeration in an ice box (at approximately 15°C) for about 15 minutes, taken to the laboratory and used to feed A. aquasalis. Patient selection criteria were: to be P. vivax positive, to have about 4–8% of circulating gametocytes, determined by the National Institutes of Health international protocols, and to consent to be part of the research , written approval number 3726).For the acquisition of A. aquasalis were reared at 27°C and 80% humidity. Insect infections were performed in an endemic area of Manaus, Amazonas state. Malaria patients were diagnosed by microscopic examination of Giemsa-stained blood smears. Infected or control blood was offered to the insects by artificial feeding at 37°C constant temperature, maintained using a water circulation system, to prevent exflagellation of microgametocytes. After the experimental feeding, the mosquitoes were kept in cages and given 20% sucrose ad libitum. Whole mosquitoes were separated in pools of 25 insects for subtraction experiments at 2 and 24 hours AF and AI, and at 2, 24 and 36 hours AF and AI were separated in pools of 5 insects for RTPCR. Infection was evaluated by PCR using a specific Plasmodium 18 s rRNA gene A. aquasalis fed on human blood infected or not with P. vivax was extracted with TRIzol (Invitrogen), messenger RNA (mRNA) was purified using the NucleoTrap® mRNA Mini Purification Kit (Clontech) and cDNAs used for subtractions were synthesized using the PCR-Select™ cDNA Subtraction Kit (Clontech). Equal amounts of cDNA from each sample were pooled to construct the four libraries using cDNAs from 2 and 24 hours infected minus non-infected insects and 2 and 24 hours non-infected minus infected insects. After two PCR rounds, the amplicons were cloned into pGEM®-T Easy Vector (Promega) and utilized to transform high efficiency DH5-α E. coli. Sequencing of 2,138 selected clones was performed using an ABI 3700 sequencer (Applied Biosystems) in the PDTIS/FIOCRUZ Sequencing Platform.RNA from http://stingray.biowebdb.org/). Vector and primer sequences were trimmed and quality evaluated by Phred, Phrap and Consed programs. The sequences were clustered using the CAP3 program and clusters were searched for similarity using the BLASTN and BLASTX algorithm with sequences of insects, plasmodia and different databases . The cutoff e-values utilized were ≥10-5 for tBLASTX similarity and 10-10 for BLASTN. Sequences were annotated and grouped in functions. Nucleotide sequences have been submitted to Genbank and their respective accession numbers are indicated in The sequences obtained were submitted to the STINGRAY platform and utilized for cDNA synthesis. RTPCR reactions were performed using the SyberGreen fluorescent probe in an ABI 7000 machine (Applied Biosystems). The PCR cycles used were 50°C 2 min, 95°C 10 min, 95°C 15 sec and 63°C 1 min for 35 times for all reactions. Primer sequences and amplicon lengths are listed in http://www.ebi.ac.uk/Tools/clustalw2/) and presented with BOXSHADE programs. For phylogenetic analyses, the sequence alignments were examined with the Mega program The sequences were aligned using the ClustalW - pool of 25 P. vivax infected insects and I (1) - one P. vivax infected insect.(0.69 MB TIF)Click here for additional data file.Figure S2Differentially expressed products amplified after different subtractions . W - molecular weight marker, F-I - cDNA after feeding minus after infection and I-F - cDNA after infection minus after feeding. h - Hours of feeding or infection. bp - base pairs.(0.82 MB TIF)Click here for additional data file.Table S1List of sequences from the 2 hours non-infected minus infected insects library. Sequences with significant similarity on BLASTN or BLASTX were grouped based on the function of the homologous protein.(0.29 MB DOC)Click here for additional data file.Table S2List of sequences from the 2 hours infected minus non-infected insects library. Sequences with significant similarity on BLASTN or BLASTX were grouped based on the function of the homologous protein.(0.28 MB DOC)Click here for additional data file.Table S3List of sequences from the 24 hours non-infected minus infected insects library. Sequences with significant similarity on BLASTN or BLASTX were grouped based on the function of the homologous protein.(0.13 MB DOC)Click here for additional data file.Table S4List of sequences from the 24 hours infected minus non-infected insects library. Sequences with significant similarity on BLASTN or BLASTX were grouped based on the function of the homologous protein.Sequences with significant similarity on BLASTN or BLASTX were grouped based on the function of the homologous protein.(0.12 MB DOC)Click here for additional data file.Table S5Primers used for quantitative PCR.(0.03 MB DOC)Click here for additional data file. |
However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin at postnatal day 9 (P9). Bromodeoxyuridine was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between −/−Vim−/−GFAP and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN+ neurons in the ischemic and contralateral hemisphere was comparable between −/−Vim−/−GFAP and wild-type mice. In wild-type mice, the number of S100+ astrocytes was lower in the ipsilateral compared to contralateral hemisphere . In the −/−Vim−/−GFAP mice, the number of S100+ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, −/−Vim−/−GFAP mice showed an increase in NeuN+BrdU+ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice , but a comparable number of S100+BrdU+ (surviving newly born) astrocytes.We subjected Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons. The central nervous system (CNS) contains abundance of astroglial cells which induce formation of neuronal synapses and support neurons structurally and metabolically −/−Vim−/−GFAP) showed that up-regulation of astrocyte IF is a key step in activation of astrocytes and that reactive gliosis is important for the wound healing process −/−Vim−/−GFAP mice are unable to form any cytoplasmic IFs since neither nestin nor synemin can self-polymerize or co-polymerize in the absence of both GFAP, and vimentin −/−Vim−/−GFAP mice show attenuated reactive gliosis with reduced hypertrophy of astrocyte processes and increased synaptic loss in the acute stage of the injury −/−Vim−/−GFAP mice, which suggested protective role of reactive astrocytes in adult brain ischemia.Mice lacking the IF proteins GFAP and vimentin on GFAP expression in wild-type mice, we measured relative GFAP mRNA expression immediately after, 6 hours, 24 hours, 3 days, 7 days and 21 days after HI. Twenty four hours after HI, GFAP mRNA expression in the cortex was substantially increased compared to 6 hours or 3 days after HI , there was no difference in the volume of the ipsilateral hemisphere between −/−Vim−/−GFAP and wild-type mice . The volume of the contralateral hemisphere was comparable in −/−Vim−/−GFAP and wild-type mice (13.5±3.1 mm3vs. 12.2±3.5 mm3) or the contralateral hemisphere (46.0±8.5 mm3vs. 44.2±7.1 mm3) between −/−Vim−/−GFAP and wild-type mice at P31, i.e. 22 days after HI or at P31 . Furtherafter HI . We did ype mice .+ neurons in the subventricular zone region, striatum and cortex. As a measure of HI-induced neurogenesis, we counted NeuN+BrdU+ cells (surviving newly born neurons) in the same brain regions. We did not find any difference in the number of NeuN+ neurons in the ipsilateral or contralateral hemisphere between −/−Vim−/−GFAP and wild-type mice at P31 , striatum and cortex . In −/−Vim−/−GFAP mice, a higher number of NeuN+ neurons in contralateral compared to the ipsilateral hemisphere was observed in cortex . Compared to the ipsilateral hemisphere, the wild-type mice had a higher number of NeuN+BrdU+ neurons in the contralateral subventricular zone and cortex , striatum and cortex .To assess the effect of HI on neuronal survival outside the infarct, we counted the NeuNe at P31 . In wild+ astrocytes in the subventricular zone region, striatum and cortex. As a measure of HI-induced astrogenesis, we counted S100+BrdU+ cells (surviving newly born astrocytes) in the same brain regions. In −/−Vim−/−GFAP mice, the number of S100+ astrocytes did not differ between the ischemic and contralateral hemisphere at P31 , both in subventricular zone and in the cortex . The −/−Vim−/−GFAP and wild-type mice did not differ in number of S100+BrdU+ cells (surviving newly born astrocytes) .Astrocytes respond to hypoxia-ischemia (HI) by morphological and functional changes as early as 8 hours after the insult and have been implicated to play a role in the pathophysiological events that lead to the brain damage after HI −/−Vim−/−GFAP mice responded to HI by increased production of new neurons. Interestingly, the wild-type mice showed a lower number of surviving newly born neurons in the ischemic subventricular zone and the cortex compared to the contralateral hemisphere, whereas in the −/−Vim−/−GFAP mice the neurogenic response to ischemia was the same in the two hemispheres. Since neuronal cell loss in the ischemic hemisphere was comparable between the wild-type and −/−Vim−/−GFAP mice, it is conceivable that the absence of reactive gliosis results in a niche that is more permissive to neurogenesis and the survival of newly formed neurons after ischemia. Indeed, we have earlier shown that survival and integration of neuronal grafts is increased in the CNS of −/−Vim−/−GFAP mice −/−Vim−/−GFAP mice We have also found that both the wild-type and the + astrocytes in the ischemic hemisphere of wild-type but not −/−Vim−/−GFAP mice. The −/−Vim−/−GFAP and wild-type mice did not differ in the number of surviving newly formed astrocytes. These findings suggest that the astrocytes lacking GFAP and vimentin might be less vulnerable to ischemic stress. Indeed, the dynamics of the response of −/−Vim−/−GFAP astrocytes to hypotonic stress, a component of HI, might be altered. It was previously shown that −/−Vim−/−GFAP astrocytes in vitro reacted to hypotonic stress by releasing less taurine than wild type astrocytes −/−Vim−/−GFAP astrocytes exhibited more prolonged changes in extracellular space diffusion parameters than wild-type astrocytes during cell swelling evoked by hypotonic stress or high concentration of potassium Remarkably, we found that HI led to the loss of S100In summary, our data suggest that in contrast to its protective role in adult focal brain ischemia, reactive gliosis does not seem to play a major role in the protection of brain tissue against HI injury in the immature brain. Thus, the role of reactive astrocytes in the pathogenesis of ischemic brain injury might be profoundly affected by age at the time of insult.The animal experiments were approved by the local Animal Ethics Committee at the University of Gothenburg .−/−Vim−/−GFAP and wild-type mice were on a mixed C57BL6/129SV/129Ola genetic background The 2O2 for 10 minutes, in 2 M HCl for 1 hour and then 30 minutes in PBS with 0.2% Triton X-100 and 3% BSA.The mice were deeply anesthetized with thiopenthal (50 mg/ml) i.p. on P12 or P31. The mice were perfusion fixed intracardially with 0.9% NaCl followed by 5% paraformaldehyde . The brains were removed and post-fixed in 5% paraformaldehyde for 24 hours at 4°C, placed in 70% ethanol for 24 hours at room temperature (RT), dehydrated and embedded in paraffin. The brains were cut in 8 µm coronal sections using a water microtome . For immunohistochemical staining, sections were deparaffinised in xylene and alcohol and boiled in 0.01 M citric acid buffer (pH 6.0) for 10 minutes, incubated in phosphate buffered saline (PBS) with 3% HFor brain infarction area evaluation, the primary monoclonal mouse anti-microtubule-associated protein-2 (MAP-2) antibody (Sigma-Aldrich) 1∶2000 and anti-pan-neurofilament protein (SMI 312) antibody (Covance) 1∶2000 were used with the secondary horse-anti-mouse, BA-2001 antibody (Vector Laboratories) 1∶250. Sections were exposed to avidin-biotin enzyme complex ABC-Elite (Vector Laboratories) for 1 hour and visualized with 3,3-diaminobenzidine and nickel sulphate (15 mg/ml).The following antibodies were used for fluorescent staining at the following dilutions: primary monoclonal mouse anti-BrdU antibody (Dako) 1∶100 with the secondary goat anti-mouse Alexa Fluor 594 antibody (Invitrogen) 1∶500, primary monoclonal mouse anti-NeuN biotin conjugated antibody (Chemicon) 1∶100 with the secondary Streptavidin Alexa Fluor 488 antibody (Invitrogen) 1∶500, primary polyclonal rabbit anti-S100 antibody (Dako) 1∶200 with the secondary goat anti-rabbit Alexa Fluor 488 antibody (Invitrogen) 1∶500. The primary antibody solution was left on the sections over night, in a dark room at 4°C. The sections were washed in PBS and the secondary antibody solution was added for 1 hour at RT. Finally, the sections were washed in PBS and mounted with Dako fluorescent mounting medium.A CCD camera (Olympus DP50) connected to a microscope was used to obtain images of the MAP-2 stained brain sections for calculation of the infarct volume and hemisphere volume. Olympus Micro Image, v4.0 software was used to delineate MAP-2 positive and negative areas of contralateral and ipsilateral hemisphere, on sections with 320 µm interval, throughout the brain. The infarct volume was assessed as the MAP-2 negative area in the ipsilateral hemisphere and the ipsilateral hemisphere volume was assessed as the MAP-2 negative area plus the ipsilateral MAP-2 positive area.Images of brain sections double stained with fluorescent antibodies were captured through 3-dimensional Z-series consecutive scans with fluorescent signals detected and processed by a confocal scanning laser microscope . Three images, representing three horizontal brain regions, were sampled at the level of the striatum, starting medially from the subventricular zone (SVZ), through the dorsal striatum and laterally to the cortex. ImageJ 1.40 software was used to analyse the Z-series stacks. Fluorescent positive cells were counted on 8 µm thick stacks of confocal images within the 450 µm×300 µm optical fields in the respective brain regions. The length of the longest cellular processes of cortical astrocytes around the infarct area was measured using a Leica DM 6000B microscope with a 40× objective and Stereoinvestigator 9 on 8 µm thick S100 immunostained sections, as previously described Gfap_fwd: AACCGCATCACCATTCCT and Gfap_rev: CGCATCTCCACAGTCTTTACC. Reference genes were evaluated using the Mouse Endogenous Control Gene Panel (TATAA Biocenter) and NormFinder Pgk1 and B2m. Formation of correctly sized PCR products was confirmed by agarose gel electrophoresis (2%) for all assays and melting curve analysis for all samples. Data analysis was performed as previously described Wild-type mice were killed at 0 hours, 6 hours, 24 hours, 3 days, 7 days and 21 days after hypoxia-ischemia. The hippocampus, striatum and cortex were dissected out and were quickly frozen on dry-ice and store at -80°C. Total RNA was extracted using RNeasy Lipid Tissue Mini Kit, including DNase treatment (Qiagen). RNA concentrations were measured with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies) and RNA integrity was checked on some randomly picked samples using the Agilent 2100 bioanalyzer (Agilent Technologies). Reverse transcription was performed with SuperScript III (Invitrogen) according to the instructions of the manufacturer using a mixture of 2.5 µM oligo (dT) and 2.5 µM random hexamers (both Invitrogen) as primers. Temperature profile for reverse transcription was 25°C for 5 minutes, 50°C for 60 minutes, 55°C for 15 minutes, and 70°C for 15 minutes. Real-time PCR measurements were performed using a LightCycler 480 (Roche Diagnostics). Temperature profile of real-time PCR was 95°C for 3 minutes, followed by 50 cycles at 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. Ten-microliter reactions contained iQ SYBR Green Supermix (Bio-Rad) and 400 nM of each primer (Eurofins MWG Operon). The following primer sequences were used: <0.05.The Student t-test and Mann Whitney test was used for statistical analysis between different time points or between the animal groups (GraphPad Prism). The Wilcoxon signed rank test was used to compare data between brain hemispheres from the same animal. Results are given as mean ± standard deviation (SD). Statistical differences were considered significant if p |
The five-membered imidazole ring is disordered over two positions with the major component having a site occupancy of 0.712 (4); the N-bound methyl substituents are ordered. The imidazole ring is approximately perpendicular to the six-membered phenylene ring .The title imidazolium-based ionic-liquid salt, C DOI: 10.1107/S1600536809026324/tk2494Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: |
While the higher criticism method suggests that there is some evidence for modest effects, interpreting individual single-nucleotide polymorphisms with significant higher criticism statistics is of undermined value. The goal of higher criticism is to alert the researcher that genetic effects remain to be discovered and to promote the use of more targeted and powerful studies to detect the remaining effects.In high-dimensional studies such as genome-wide association studies, the correction for multiple testing in order to control total type I error results in decreased power to detect modest effects. We present a new analytical approach based on the higher criticism statistic that allows identification of the presence of modest effects. We apply our method to the genome-wide study of rheumatoid arthritis provided in the Genetic Analysis Workshop 16 Problem 1 data set. There is evidence for unknown bias in this study that could be explained by the presence of undetected modest effects. We compared the asymptotic and empirical thresholds for the higher criticism statistic. Using the asymptotic threshold we detected the presence of modest effects genome-wide. We also detected modest effects using 90 Another explanation of this deviation from the expected distribution could be the presence of undetected, and therefore unexplained, modest effects. It has been reported that even for a larger data set that contains the provided data as a subset, the power to detect a disease-associated allele with population frequency of 0.2 and an odds ratio of 1.3 is only 13%, while for an odds ratio of 1.5 the power is 90% [Multiple genetic association studies of rheumatoid arthritis (RA) have reported inconsistent results . It is hThe data consist of 545,080 SNPs genotyped for 868 cases from the North American Rheumatoid Arthritis Consortium (NARAC) and 1194 controls. This is a subset of the Stage 1 data previously analyzed by Plenge et al. , after rp < 10-5). Because no information on the ancestry was provided, we assume that all related subjects and subjects with non-Europian ancestry were removed [We performed quality-control filtering of SNPs following the procedures in Plenge et al. . We remo removed .f1 and null effects have pdf f0. Then the pdf for the mixture distribution is f = ε f1 + (1-ε)f2 The HC test for presence of modest effects is equivalent to testing H0: ε = 0.We applied the refined version of the HC statistic of Donoho and Jin to test p-values and is implemented as followsThe HC test utilizes individual p(1) <p(2) <...<pn) , which is similar to one in Plenge et al. [We obtained a in PLINK . Accordiin PLINK there isin PLINK based onin PLINK . Approxiin PLINK , we did in PLINK , we chose et al. .p-values corresponding to chi-square statistics from the logistic regression model described above; these were also corrected for the residual inflation by dividing by The HC test evaluates evidence of modest effects that could be present in the data in addition to the significant effects already identified. Therefore, we applied the HC test genome-wide after removing known significant effects, which were defined as regions identified in the previous studies as associated with RA on a genome-wide level. Excluded SNPs were from the extended MHC region from HISth, 95th, and 99th percentile of We compared the use of the asymptotic threshold for the HC statistic as in Cayon et al. , i.e., ,i ≤ 0.05n, and pi) (≥ 1/n conditions and was equal to 3.333 while the genome-wide asymptotic threshold was 2.268. Figure HCn, i statistics for the region over which maximum HC was computed. It also shows the asymptotic threshold. These results indicate presence of modest effects on a genome-wide level. Thus, after SNPs with known significant effects have been removed there is still evidence for association with RA that has not been explained. The highest p-value for which HC exceeds the threshold is 9.19 × 10-4, while the maximum of HCn, i corresponds to p = 7.46 × 10-6. There were 282 HCn, i statistics exceeding the asymptotic threshold, indicating modest effects.There were 488,126 SNPs remaining after quality control filtering and removing regions with previously identified significant effects. Maximum HC was computed for 24,402 SNPs that satisfied 1 ≤ th, 95th, and 99th percentiles of th and 95th percentiles of th percentile is selected as threshold, seven HC statistics exceed the threshold, indicating the presence of modest effects. The p-values corresponding to these statistics range between 6.81 × 10-6 and 6.21 × 10-4. There is no evidence of modest effects at the 95th and 99th percentile levels.The values of 90ely. Jin suggesteThe HC statistic using the asymptotic threshold indicates the presence of modest effects on a genome-wide level. However, this threshold is not a boundary for a significance test, but rather a large-sample analytical result that applies to any data set, and gives the expectation of the HC statistic under the null hypothesis. It gives a crude idea of what values of HC start to be interesting, and displays the effect of the number of tests. Using an asymptotic threshold in this application may not be appropriate due to dependency between the individuals. Asymptotic assumptions discussed elsewhere ,4 are noHCn, i statistics and p-values in a sense that smaller p-values produce higher HCn, I values. In fact, the maximum of HCn, i corresponds to p = 7.46 × 10-6, which is not significant after correction for multiple testing using Benjamini-Hochberg rule [pBH = 0.364. Thus, this effect is not significant enough to be detected by traditional approaches, which supports the usefulness of the HC statistic for detection of the presence of modest effects in the context of multiple hypothesis testing.The HC graph in Figure HCn, i statistics exceed the asymptotic threshold does not include the statistics corresponding to the smallest p-values, this is the region of interest. The region of HCn, i statistics above the threshold can be used to identify the range of p-values that could contain modest effects because it contains larger frequency of p-values in a specific range than expected by chance [p-values does not have a higher frequency of p-values than expected by chance. Therefore, the hypothesis is that the unidentified modest effects could be found in the range of p-values for which the HCn, i statistics exceed the threshold, while the conventional approach of considering only most extreme p-values up to a certain threshold may lead to missing modest effects. HC results could be used to alert a researcher that there is another range of larger p-values and smaller effect sizes that could be of interest and to promote the use of more targeted and powerful studies to detect the remaining genetic effects. Because the HC test is a global test of the presence of modest effects, caution should be exercised when trying to interpret individual SNPs with p-values in the range of interest.In addition, although the region where y chance . On the HC: Higher criticism; NARAC: North American Rheumatoid Arthritis Consortium; PDF: Probability density function; RA: Rheumatoid arthritis; SNP: Single-nucleotide polymorphism.The authors declare that they have no competing interests.EP performed the statistical analysis and drafted the manuscript. All authors participated in the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript. |
Therapeutic interventions are made in the best interests of the patient taking into account the risks and benefits believed to be true at the time. However, adverse effects associated with the treatment may only become apparent much later, sometimes long after the anticipated survival of the patient.A lady in her late sixties presents with multiple pathologies of the head and neck that appear to be a direct consequence of irradiation performed as a young child when she was not expected to live for long. Some deformities resulted early after exposure, but most of the dozen pathologies in the affected area have evolved during the subsequent fifty years.This case acts as a potent reminder to consider the long term effects of therapeutic ionising radiation, especially if there is a possibility of long term survival post treatment. It also demonstrates the effects of a single physical modality on multiple and wide ranging tissue types, highlighting a continuing need in medical education for the teaching of clinical anatomy and an understanding of histological pathology. This case demonstrates the serious clinical problems that can develop later in life resulting from deep XR treatment applied to a young child for a skin lesion.A-sixty eight-year old Caucasian lady of British Jewish descent presents with the long term sequelae of deep XR treatment administered at the age of six months to the right side of her face. Information regarding the childhood treatment is limited to family testimony as the hospital has closed and the original notes are no longer available. We have no other details regarding dose and frequency of the irradiation. Subsequent health developments and interventions are well documented in the case notes within both the General Practice setting and the hospital departments she has visited since. Relatives, one of whom I interviewed, have told the lady that during the London Blitz, she was treated for an extensive mole on her face and neck that was present at birth, and that being a frail child, she was not expected to live.As a child, right facial asymmetry became progressively more apparent due to arrested development of the skull bones and teeth. Figure At the aDuring a four year period from the age of 53, she was diagnosed as having neurofibromata of the left jaw and right cheek; sialadenitis of the left parotid resulting in the gland's excision (as seen in Figure From the age of 60, there were further developments, some more painful and sinister than had occurred previously - right sided trigeminal neuralgia; multiple basal cell carcinomata affecting the right temple (scarring from their excision seen in Figure As an adult, this lady worked in a leading West End department store until retirement at the normal age. As would be expected, there have been a succession of age related problems, including hypertension, hallux valgus, lumbar spine degeneration, lymphoedema and recurrent varicose veins. At present, she remains a cheerful retired lady, troubled by unsteadiness and gradually worsening headaches, She is kept busy with numerous hospital appointments to attend. She also cares for an older step-sister who suffers from dementia.This lady presents with a catalogue of disorders, many of them unusual, affecting a wide set of structures and tissue types, arising predominantly on the right side of the head and neck, corresponding to the area of irradiation in early childhood. Although specific information regarding the original lesion and its treatment is lacking, there is evidence of the use of similar treatment for extensive cutaneous haemangiomata in young children in pre-War Europe, and the subsequent development of clinical pathology . The incThis case demonstrates how well intended treatment may have severe sequelae that only develop decades later, causing us to reflect on the adequacy of the evidence base for treatment that may be in vogue at any given time. It also reminds us that any health benefits achieved in the short term may be outweighed by subsequent problems that may themselves become life threatening. The unusual nature of the case also emphasises the importance of teaching clinical pathology at a time when many medical schools are dropping the subject from the curriculum, as it demonstrates a single external cause as responsible for the pathological changes in a multiplicity of tissue types.I can confirm that informed written consent was obtained from the patient for publication of the manuscript and figures. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The author declares he has no competing interests. |
Euphausia superba (the Antarctic krill).Little is known about the genome sequences of Euphausiacea (krill) although these crustaceans are abundant components of the pelagic ecosystems in all oceans and used for aquaculture and pharmaceutical industry. This study reports the results of an expressed sequence tag (EST) sequencing project from different tissues of E. superba sequences identified 69 microsatellite containing ESTs. Clusters, consensuses and related similarity and gene ontology searches were organized in a dedicated E. superba database .We have constructed and sequenced five cDNA libraries from different Antarctic krill tissues: head, abdomen, thoracopods and photophores. We have identified 1.770 high-quality ESTs which were assembled into 216 overlapping clusters and 801 singletons resulting in a total of 1.017 non-redundant sequences. Quantitative RT-PCR analysis was performed to quantify and validate the expression levels of ten genes presenting different EST countings in krill tissues. In addition, bioinformatic screening of the non-redundant E. superba based on functional categorization among the examined tissues. The analyses of annotated transcripts showed a higher similarity with genes from insects with respect to Malacostraca possibly as an effect of the limited number of Malacostraca sequences in the public databases. Our catalogue provides for the first time a genomic tool to investigate the biology of the Antarctic krill.We defined the first tissue transcriptional signatures of Euphausiacea (krill) are small shrimplike crustaceans that are abundant in the pelagic ecosystems of all oceans. There are about 85 species of Euphausiacea, making this one of the smallest orders in the class of Malacostraca . PhylogeIn the Southern Ocean, krill is a critical link between primary productivity and most of the predators at higher trophic levels such as birds, fish, seals, squid and whales . The kriEuphausia superba, Dana 1852) has a circumpolar distribution with the highest concentrations in the Atlantic sector of the Southern Ocean. It is a key species of the Antarctic ecosystem and plays an important role both as feeder of algae, bacteria and micro-zooplancton and as a prey of vertebrates [E. superba displays a large daily vertical migration that occurs generally within the upper 200 m water column making a significant amount of biomass available as food for predators near the surface at night and in deeper waters during the day [E. superba only 69 nucleotide and 17 amino acid sequences have been obtained; they identify key proteins and enzymes of oxidative phosphorylation and of phototransduction (opsin). In the subphylum Crustacea there are 33 complete (or nearly complete) mitochondrial DNA sequences: 4 Branchiopoda, 8 Maxillopoda, 18 Malacostraca, and one of Ostracoda, Cephalocarida and Remipedia .The Antarctic krill dissected from specimens of Five independent tissue-specific cDNA libraries, named K01 and K05 (head), K06 (abdomen), K07 (photophores) and K09 (thoracopods), were produced from total RNA pools. For head we have sequenced only K05 cDNA library because it presented more recombinant clones compared to K01. Recombinant bacterial clones from each library were randomly picked and the EST were sequenced from the 5'-end. The average insert size for all libraries was estimated to be 412 bp.A total of 2,046 ESTs were initially analyzed for sequence quality and vector sequences were recognized and deleted. Two-hundred-seventy-six low quality ESTs were removed and 1,770 (86.5%) high-quality ESTs were further processed. These ESTs assembled by similarity into 216 clusters and 801 singletons, resulting in a total of 1,017 non-redundant (consensus) sequences. A list of the sequencing trend for each cDNA library is presented in Table The number of ESTs in each cluster varies from 2 (93 clusters) to 88 (1 cluster). The average length of a cluster is 436 bp with the longest assembled sequence being 1,417 bp and the shortest 153 bp. The 91% of cluster consensus contains the 3'-end region of mRNAs as demonstrated by the presence of a poly-adenylation signal.e-value cut off of < e-40 and < e-10, respectively. These values were empirically chosen considering the low amount of sequences data available for Euphausiacea and similar shrimp species and the need of stringency in providing a reliable catalogue of Antarctic krill genes. All annotations were further manually examined, in order to assign the best describing text to the correspondent cluster.Each non redundant sequence was searched in the nucleotides database and UniProtKB database using Blast-N and Blast-X with an Euphausia genes or sequences showing significant similarity to genes from arthropods (63.4%) and other species (36.6%), such as Homarus americanus (3.2%), Aedes aegypti (12.0%), Drosophila melanogaster (3.9%), Bombyx mori (3.6%), Brachydanio rerio (4.9%), Mus musculus (3.9%), Rattus norvegicus (3.9%), Homo sapiens (4.5%). Antarctic krill sequences generally show a greater similarity to genes of insects (about 36%) than to genes of Malacostraca (about 23%) and only 7% were similar to the known sequences of Euphausiacea. This result could be due to the limited number of Malacostraca gene and protein sequences available in the public databases respect to insects . The total collection of 1,770 E. superba 3'-EST has been deposited in the EBI-GenBank-DBJ database (GenBank accession numbers from ES542703 to ES544472).Overall, 70% of non-redundant sequences , identified by about 50% of total produced ESTs, showed no or poor similarity matches, and they probably represent completely unknown Antarctic krill transcripts that could be characterized in future studies. Additional file E. superba mitochondrial transcripts and about 5% ESTs identified the large mitochondrial 16S rRNA gene. In future experiments, to avoid the repetitive sequencing of this abundant mRNA, we plan to introduce, during cDNA insert amplification, interference primers specifically designed for the 16S rRNA [One of the most noticeable features of our EST catalogue is that mitochondrial transcripts are quite abundant : 84 ESTs (about 4.7%) matched with nine different 16S rRNA . MoreoveE. superba mitochondrial gene sequences are very similar to those commonly found in the mitochondrial genomes of other arthropods, including 13 protein-coding genes, 19 tRNA and 2 rRNA genes [et al. [ND1, ND4L, ND4 and ND5 genes, while two ribosomal RNA genes are encoded by L strands.The NA genes . Machida [et al. have demmyosin light chain that is highly represented in a strictly committed tissue such as skeletal muscle localized in abdomen (library K06). Moreover, we found 3 unknown transcripts among the 20 most expressed genes: KRC00118 (10 ESTs), KRC00407 (9 ESTs) and KRC00101 (7 ESTs) that could be interesting for future functional studies.We did not find highly represented tissue-specific mRNAs, while some ribosomal proteins like L22 (31 ESTs), S25 (31), P1 (16), L37A (15), L24 (13), P2 (13), S14 (9), S14 (10), L34 (9) were expressed at the same level in all analyzed krill tissues. The only genuine tissue-specific mRNA appears to be E. superba mRNAs.Since very few abundant transcripts were found that could hamper the identification of rare transcripts, it seems plausible that random sequencing of our Antarctic krill libraries would continue to represent an effective strategy for identifying novel translation factor SUI1,initiation factor 4A and 3, in the translational elongation like elongation factor 1α, 1β, 2 and a specific tail muscle elongation factor 1γ. Other abundant E. superba sequences fall into gene categories related to cell structure, cell motility and functional homeostasis. For instance, genes involved in the mechanisms of DNA transcription (1.47%), transport (1.97%), skeletal muscle contraction (3.4%), and in amino acid, fatty acid and carbohydrate metabolism (4.03 %) are comprised in this category. This class includes also SEC61 β-subunit, an important transport protein that plays a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum and cellular retinoic acid/retinol binding protein (RBP1), involved in the transport of retinol from the digestive gland to peripheral tissues [In order to facilitate functional genomic studies in Antarctic krill, 309 consensus sequences showing similarity with known genes or proteins were grouped into 14 functional categories and peptidylprolyl isomerase B (cyclophilin B). Cyclophilines are members of the immunophilin protein family, which play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. ESTs with good similarity to hemocyanin are present in our collection: this protein has been recently reported to have antifungal and antiviral activities [Other interesting krill transcripts that we were able to annotate are those involved in stress responses, proteolysis and immunoresponse 3.24%) like oteinase , peptidytivities ,24.% like otIctarulus punctatus [Mytilus galloprovincialis [E. superba histone transcripts deserves a more detailed analysis. In fact, Eirin-Lopez et al. [Chironomus thummi [D. melanogaster [Chaetopterus variopedatus [M. galloprovincialis [Some krill ESTs identify histone 2A KRC00431) and histone 3.3A (KRC00024) indicating the presence of unexpected polyadenylated histone transcripts displaying the polyadenylation signal and tail. In vertebrates, these evolutionary conserved housekeeping mRNAs are not polyadenylated, and this has been related to the high turnover of these transcripts in the dividing cells. Interestingly, polyadenylated H2A and H3 histone sequences were detected also in the systematic sequencing of 3'-end cDNA libraries obtained from brain and kidney of channel catfish unctatus ,26 and fincialis . The prez et al. have recs thummi , D. melanogaster , Chaetop and histincialis and Crusincialis . Althougincialis .Gene expression profiling depends on the functional specificity of cells composing different tissues. So, the systematic sequencing of EST from unbiased cDNA libraries is a suitable approach for analyzing the gene expression profile of a given tissue . In factE. superba, annotated ESTs obtained from the four tissue-specific cDNA libraries were separately grouped in 13 functional categories and two subunits of the troponin complex , a key regulator of muscle contraction. About 10% of sequences produced from head and thoracopods libraries fall in functional categories related to metabolic processes . Interestingly, about 6% and 4% of ESTs respectively sequenced from head and thoracopods libraries identified structural constituents of cuticle . This reflects the presence of cuticle traces in the head and thoracopods samples. In photophores and thoracopods transcripts displaying putative identity with ribosomal sequences are more abundant compared to other tissues , indicating a relevant activity of the translation machinery.To define tissue transcriptional signatures of es Table a furtheE. superba (GenBank accession no. DQ852576–DQ852580) show a spectral sensitivity with short wavelength and cannot be aligned with our consensus [We have also identified from the head cDNA library a novel opsin sequence , a light-sensitive membrane-bound G protein-coupled receptors mediating the conversion of a photon of light into an electrochemical signal in the visual transduction cascade. In insects there are at least four main spectral classes: long-wavelenght-sensitive (LWS), middle-wavelenght-sensitive (MWS) and two short-wavelenght-sensitive (SWS) groups. The opsin sequences available for onsensus .compound eye opsin BCRH1, myosin light chain, myosin heavy chain, arthrodial cuticle protein AMP16.3, tail muscle elongation factor 1 gamma, cellular retinoic acid/retinol binding protein, eukaryotic initiation factor 4A, transport protein SEC61 subunit gamma, chromodomain helicase DNA binding protein and voltage-dependent calcium channel) representative of different levels of transcript abundance.Quantitative RT-PCR analysis was performed to quantify and validate the expression level of some genes presenting different EST countings in krill tissues. We selected ten genes consensus sequences containing ESTs were identified by using MISA software. Twelve of these consensus present 2 distinct simple sequence repeats interrupted by more than 100 bp for a total of 69 identified microsatellites (SSR). The majority of these sequences (72%) fall into the 3 bp repeat type class with a preponderance of GAA and GAT. After a manual inspection of redundancy, raw sequence, data quality and the presence of sufficient flanking sequences we designed 9 pairs of specific PCR primers. We obtained successful amplifications for 6 of these 9 pairs of primers. Assessment of polymorphism information content (PIC), observed and expected heterozygosity and other population genetics analysis will be performed in the near future. These markers will increase the currently available Euphausiacea SSR markers. In fact, only five microsatellite loci isolated from the northern krill d so far . Since oE. superba tissues has allowed us to establish an EST database containing 1,017 unique sequences. Over 65% of the Antarctic krill sequences resulted in no BLAST matches with published sequences and they probably represent novel genes that could be functionally characterized. We have defined the transcriptional signatures of krill tissues and performed qRT-PCR to validate the level of expression of ten representative genes. All sequencing data have been deposited on the E. superba EST database available from our web site [Since genome sequencing and BAC libraries of Antarctic krill are not yet available, EST sequencing from randomly selected cDNA clones represents a powerful approach to identify large numbers of transcripts that could be used in gene expression and functional genomics studies . The sysweb site . In addiOur EST catalogue could provide a source for the design of microarray platform that will allow the study of the transcriptional responses of this abundant marine organism to environmental challenges .Euphausia superba) were fished from the Ross Sea in the January 2004 during the XIX Italian Antarctic Expedition. Specimens were collected at different time of the day , over a complete 24-hour cycle. Samples were frozen at -40°C in RNA stabilization solution . For each fishing, selected tissues from five animals were dissected individually in RNA later ice solution (Ambion). After dissection, tissues were rapidly rinsed in sterile water, weighed, frozen in Trizol reagent (Invitrogen) and stored at -80°C. A large excess of Trizol (15 ml for 0.5–1.5 g. of sample) was used in order to prevent RNA degradation by endogenous RNAse. Frozen tissues were minced and homogenized for 3–5 min using an ultra-turrax-T8.01 blender (IKA-Werke). Total RNA was isolated using the Trizol reagent (Invitrogen) following the manufacturer's instruction and further purified with LiCl in order to remove glucidic contaminants. All RNA samples were checked for quality by capillary electrophoresis . For each tissue , equal amounts of total RNA (2 μg) extracted from every collection were pooled.Antarctic krill , K06 (abdomen), K07 (photophores) and K09 (thoracopods), were constructed.20VN-3') used for first strand synthesis include an attB1 and attB2 recombination site respectively.We have developed a new method using a combination of SMART protocol (Clontech), ensuring almost full-length cDNA, and Gateway technology (Invitrogen), allowing unidirectional cloning without enzymatic digestion. In this protocol, only fully-transcribed first strand cDNA (ss cDNA) is tagged with a short sequence complementary to a modified SMART oligo Fig. . The SMAE. coli cells. Recombinant colonies were selected on agar SOB medium plus kanamicin. Individual library colonies were arrayed by manual picking on 96 well plates in liquid selective SOB medium plus 7.5% of glycerol for independent growth [First strand cDNA synthesis was performed from 1.5 μg of total RNA in a 15 μl reaction. Then, the reaction was then diluted 1:5 ratio and incubated at 72°C for 2 min. Second strand reaction mix was added to 1 μl of diluted first strand cDNA to give a final concentration of 1× BD Advantage 2 PCR reaction buffer (Clontech), 0.2 mM dNTPs, 120 nM primers (attB1-8T3: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTAATTAACC-3' and attB2: 5'-GGGGACCACTTTGTACAAGAAAGCTGGG-3') and 1× of Advantage2 DNA polymerase mix (Clontech) in a volume of 50 μl. This second strand reaction mixture was incubated for 21 cycles of 15 sec 95°C, 30 sec 66°C and 3 min 68°C. Only those ss cDNAs having a SMART anchor sequence at the 5' end were used as template and exponentially amplified. The second strand reaction was glass fibre column purified and cDNA was size selected by Sepharose CL-4B SPUN COLUMN . The cDNA was inserted in the cloning vector by a recombination reaction performed at 25°C for 18 h with about 35 ng of attB-cDNA, 150 ng of pDONR221 (Invitrogen) and 2 μl of BP Clonase II (Invitrogen) in 10 μl final volume. 1/5 of the purified reaction was used to transform electrocompetent DH10B t growth .After lysis of the bacterial colonies, cDNA inserts were directly amplified with universal primers M13 forward (5'-TGTAAAACGACGGCCAGTCTTA-3') and M13 reverse (5'-CAGGAAACAGCTATGACCATGT-3'). The polymerase chain reaction (PCR) profile consisted of: (1) initial denaturation for 5 min at 95°C, (2) 35 cycles of 40-s denaturation at 95°C, 40-s annealing at 60°C and 1-min elongation at 72°C and (3) final extension for 5 min at 72°C; samples with a size over 0.5 Kb were selected for sequencing. Single pass DNA sequencing from plasmids was performed by using the vector specific primer attB1_seq (5'-CTTTGTACAAAAAAGCAGGCT-3') and a modified Sanger dideoxy terminator cycle sequencing chemistry, the ABI BigDye kit version 3.1, on a ABI 3730 48-capillary sequencer and 36 cm capillaries .Trace2dbest and Partigene were useEach consensus, converted in FASTA format, was searched locally in nucleotides database, downloaded from NCBI and UniP-10 for protein (Blast-X) and e-40 for nucleotide (Blast-N) were considered as poorly informative. Moreover, for each UniProt ID, taken from Blast-X description field, we associated specific Gene Ontology annotation, that integrates information about process, function, and component. Clusters, consensus and related similarity and gene ontology searches were electronically organized and stored in a dedicated PostgreSQL database.Each cluster annotation in our database was further manually examined to assign the best describing text to the correspondent cluster: matches with expectations values greater than eThe unique sequences were screened for microsatellites by using the MISA software . Only diQuantitative RT-PCR was conducted for some genes using the same tissues tested to confirm the integrity and robustness of EST sequencing.® Software (Applera) to amplify fragments of 120–180 bp in length, close to the 3'-end of the transcript. To avoid the amplification of contaminant genomic DNA, we treated total RNA samples with DNase I (Qiagen). The dissociation curve was used to confirm the specificity of the amplicon. PCR reactions were performed in a 7500 Real-Time PCR System (Applied Biosystems). Thermal cycling conditions were as follows: 15 min denaturation at 95°C; followed by 40 cycles of 30 sec denaturation step at 95°C, annealing and elongation steps for 1 min each at 60°C and a final 3 min elongation at 72°C. To evaluate differences in gene expression a relative quantification method was chosen where the expression of the target gene is standardized by a non-regulated reference gene; consequently, three replicates of each sample and endogenous control were amplified. 18S rRNA was used as an endogenous control because the level of rRNA remains essentially constant from sample to sample . To calculate the relative expression ratio, the 2-ΔΔCt method implemented in the 7500 Real Time PCR System software [compound eye opsin BCRH1, myosin light chain, myosin heavy chain, arthrodial cuticle protein AMP16.3, tail muscle elongation factor 1 gamma, cellular retinoic acid/retinol binding protein, eukaryotic initiation factor 4A, transport protein SEC61 subunit gamma, chromodomain helicase DNA binding protein and voltage-dependent calcium channel) which displayed differential expression in the head, thoracopods and photophores, compared with the abdomen.Three μg of total RNA from each tissue was used to perform three independent cDNA syntheses in a final volume of 10 μl, using random decamers and SuperScript II reverse transcriptase (Invitrogen). 1 μl aliquot of diluted first-strand cDNA was PCR amplified in 10 μl volume using SYBR Green chemistry, according to the manufacturer's recommendations (Finnenzymes). Gene-specific primers were designed using Primer Expresssoftware was usedEuphausia superba);Project name: Systematic sequencing of mRNA from the Antarctic krill : Debian GNU/Linux;Programming language: PHP;Licence: none;Any restrictions to use by non-academics: none.CDP performed total RNA sample preparation, construction and systematic sequencing of the cDNA libraries, annotation of ESTs, qRT-PCR and drafted the manuscript. CB conceived the study, carried out ESTs analysis and drafted the manuscript. GMM and GR participated in systematic sequencing of cDNA libraries, in design of the study and revision of the manuscript. FB performed bioinformatic analysis of cDNA libraries sequence data, clustering of ESTs and annotation of ESTs and identification of microsatellite containing ESTs. BDN and AP participated in development of cDNA libraries production method and identification of microsatellite containing ESTs. GL supervised the study, participating in the design and coordination of the work, the interpretation of the results and revision of the manuscript. RC supervised the study, participating in the design and coordination of the work, the interpretation of data and manuscript writing. All Authors read and approved the final version of the manuscript declaring that they have no potential conflicts of interests.E. superba genes or sequences showing significant similarity with genes from arthropods and other species. These transcripts have been grouped into 13 different functional categories.This table lists 309 non-redundant sequences identifying known Click here for fileQuantitative RT-PCR validation. Forward and reverse primer pairs for ten tested transcripts are detailed at the top. Relative expression values resulting from the quantitative real-time PCR are in the middle and related data plots are also reported. For each transcriptional profiling we have associated the EST counting obtained from the cDNA libraries systematic sequencing.Click here for file |
Fistula formation in patients with Crohn’s disease is a common complication during the course of the disease. Perianal and enteroenteric are the most common forms of fistulas, whereas the involvement of the upper gastrointestinal tract with gastrocolic and duodenocolic fistulas represents an extremely unusual condition. Moreover, hyperthyroidism in association with Crohn’s disease has been rarely described.We present here a rare case of a 25-year-old male with simultaneous onset of hyperthyroidism and fistulizing Crohn’s disease. Crohn’s disease was complicated with intra-abdominal fistulas involving the upper gastrointestinal tract and an intra-peritoneal abscess formation in the lesser sac. We describe the clinical presentation and therapeutic management of the patient including both medical treatment and surgical intervention. Despite intense medical treatment with total parenteral nutrition, antibiotics, aminosalicylates and corticosteroids the clinical course of the disease was suboptimal. Finally, the patient underwent laparotomy and right hemi-colectomy with ileo-transverse anastomosis performed, with simultaneous drainage of the abdominal abscess and primary closure of the upper gastrointestinal tract openings at one stage operation. Although the surgical approach definitively cured the perforating complications of the disease (fistulas and abscess), the luminal disease in the colon remnant was still active and steroid-refractory. The subsequent successful treatment with infliximab, azathioprine and mesalazine resulted in the induction and maintenance of the disease remission. Thyrotoxicosis was successfully treated with methimazole and the hyperthyroidism has definitely subsided.The management of intra-abdominal fistulas in Crohn’s disease is a complex issue, requiring a multi-disciplinary approach and ‘tailoring’ of the treatment to the individual patient’s needs. Probably, a sensible approach involves early surgical intervention with prior optimization of the patient’s general condition when feasible. Common autoimmune mechanisms are probably involved in thyroid dysfunction associated with Crohn’s disease. Moreover, diagnosis and treatment of coexisting thyroid disorder in patients with Crohn’s disease has a favorable impact in disease prognosis. Crohn’s disease (CD) is a chronic inflammatory disease of unknown etiology characterized by a chronic, granulomatous, segmental transmural inflammation that may affect any part of the gastrointestinal (GI) tract.The transmural nature of inflammation in CD predisposes to the formation of fistulas. Fistulas are a common and often serious complication of CD, occurring in 30% to 50% of CD patients during the course of their disease .In general, fistulas are classified as external and internal depending on their location and their connection with adjacent structures. External fistulas connect a diseased part of the bowel with the skin , whereas internal fistulas connect either two parts of the bowel or a diseased part of the bowel and other adjacent organs .Perianal fistulas are the most common type of fistulas in CD patients, followed by internal enteroenteric fistulas, while the involvement of the upper gastrointestinal tract with gastrocolic and duodenocolic fistulas is very rare and reported in few cases in the literature so far ,3.The coexistence of thyroid disease and inflammatory bowel disease (IBD) is uncommon. Thyroid dysfunction has been described more commonly in patients with ulcerative colitis (UC). On the contrary, its presence in patients with Crohn’s disease is extremely rare ,5. We reoC) and crampy abdominal pain. His medical history was remarkable for hyperthyroidism due to a thyrotoxic goiter diagnosed 4 months before. However, it was left untreated and the patient mentioned a gradual body weight loss of 10 Kg during the last four months. His family history was unremarkable.A 25-year-old, non-smoker, Caucasian male was admitted to our department with a 10-day history of vomiting, watery diarrheas, high fever , thrombocytosis (PLT 540 × 103/μl), mild normocytic anemia , hypoalbuminemia (2.8 g/dl) and significantly elevated markers of inflammation . Thyroid function tests included low thyroid stimulating hormone (TSH) , high serum free T4 (FT4) and normal serum free T3 (FT3) . Anti-thyroid antibodies were also elevated; the anti-thyroperoxidase antibody (anti-TPO) was 385.8 and the anti-thyroglobulin antibody (anti-TG) was 88.5 .On admission the patient was febrile and looked severely ill. His height and weight were 186 cm and 95 Kg respectively (BMI 27.5 Kg/cm), and his vital signs included a temperature of 3999mTc scan of the thyroid showed a moderate thyroid gland enlargement with increased thyroid gland total mass and increased homogenous parenchymal uptake of technetium pertechnetate . Treatmend day) the patient had bloody diarrheas without signs of hypovolemic shock and endoscopy of the upper and lower GI tract was performed. Colonoscopy to the terminal ileum demonstrated mild cecal inflammation with edematous, erythematous, non-friable and non-ulcerated mucosa without specific features on histology, while the upper GI endoscopy showed a normal esophagus, stomach and duodenum except for two fistulous tract openings, one in the greater curve of the stomach scan of the abdomen and pelvis showed an abscess formation without well-defined margins (phlegmon) in the lesser sac , thickenIn the context of the findings above, treatment for severe active fistulizing Crohn’s disease (CDAI score: 452) complicated with an abdominal abscess was initiated with the institution of total parenteral nutrition (TPN) and the intravenous (IV) administration of high-dose prednisolone (75 mg/day), ciprofloxacin (400 mg bid), metronidazole (500 mg tid), and pantoprazole (40 mg bid), along with oral mesalamine (3 g/day), and azathioprine (2 mg/kg/day).During the next 2 weeks, a gradual improvement of the patient’s clinical and laboratory findings was noted and barium upper GI series with a small-bowel follow-through study were performed. The small bowel series showed a colo-duodenal fistula between the ascending colon and the second portion of the duodenum and multiple fistulas between the jejunum and the lesser sac , whereasst hour, CRP 60 mg/dl, low grade temperature, clammy skin, heart rate of 90 beats per minute), despite adequate control of the thyroid function and a CDAI score of 172. A second colonoscopy was performed and revealed a stenotic area in the transverse and ascending colon with erythematous, edematous, and friable mucosa with ulcers and a fistulous tract opening at the proximal transverse colon near to the hepatic flexure and the patient was clammy and tachycardiac with a heart rate of 90 beats per minute despite good control of his thyroid function and a CDAI score of 105. Computed tomography (CT) scan of the abdomen and pelvis was unremarkable for intraperitoneal abscess or mass. At that point we decided to start anti-TNFa therapy with infliximab and taper-off corticosteroids, since the latter were proved insufficient in controling the disease. Infliximab infusions (5 mg/kg) were well tolerated and induced a sustained to date (36 months postoperatively) endoscopic, clinical and biochemical response. Azathioprine and mesalamine are also continued as maintenance treatment. The thyroid function maintains normal without any treatment.In the light of these findings and because of the suboptimal clinical course of the disease, despite the intense medical treatment, surgical intervention was decided to be performed. Right hemi-colectomy with ileo-transverse anastomosis, drainage of the abdominal abscess and primary closure of the upper GI tract openings were performed at one stage operation. The postoperative medical management included oral steroids with gradual tapering, azathioprine, mesalamine, and antibiotics with gradual introduction of oral feeding. Still though, 3 weeks postoperatively, the markers of inflammation were high (ESR 76 mm/1The involvement of the upper GI tract in fistulizing Crohn’s disease with gastrocolic and duodenocolic fistulas is very rare (<1%). In most cases the fistulas arise from the diseased small or large bowel. Pain located in the epigastrium in association with nausea and vomiting are the most common symptoms, while fecal vomiting is rare ,6,7. ConIn our case the predominant symptoms , were suggestive for colonic involvement, whereas the presence of nausea and non-fecaloid vomiting was not specific. Endoscopy of the upper and the lower GI identified the fistulus openings in the stomach and the duodenum, and the disease in the proximal colon, respectively. Furthermore, histologic examination of the biopsies confirmed the disease in the large bowel, while the upper GI was uninvolved except for the fistulus openings. On the other hand, the barium upper GI series with small-bowel follow-through study identified the course of the fistulus tracts.The internal fistulas are thought to be indicative of a more aggressive subtype of Crohn’s disease and often require urgent and repeated surgical intervention. Although new immunosuppressive agents have been used for the treatment of fistulas, the majority of data refer to perianal fistulas, while controlled data for the efficacy of medical treatment in healing of internal fistulas are lacking -10.The presence of abscess formation is an important issue and must be aggressively sought in patients with fistulizing Crohn’s disease. While a small abscess may be successfully treated with antibiotics, larger abscesses require intervention with percutaneous or surgical drainage ,12. The Our patient presented intra-abdominal fistulas involving the upper GI tract, accompanied by an abdominal abscess formation in the lesser sac. His nutritional status and his general clinical condition were quite poor on admission. We followed an aggressive medical management with hyper-alimentation (TPN), antibiotics, aminosalicylates and immunosuppressive agents (corticosteroids and azathioprine). Infliximab was not considered as the best approach both because it could complicate the intra-abdominal abscess with sepsis and because of the reported low efficacy in healing internal fistulas . We coulIn general, gastrocolic and duodenocolic fistulas represent indications for surgery. Resection of the diseased segment of the bowel containing the fistula (primary site of the disease), resection of the fistula and primary repair of the affected organ, has been proven so far to be the most effective treatment method of intra-abdominal fistulas and numerous operative approaches have been suggested ,6,10,14.While the surgical approach in our patient definitively cured the perforating complications of the disease (fistulas and abscess), the luminal disease in the colon remnant was still active. The on-going inflammation post-operatively, that was eventually steroid-refractory, necessitated the use of anti-TNFa, that was no longer contraindicated after the draining of the abscess, simultaneously with continuous use of azathioprine and mesalazine .Thyroid involvement in Crohn’s disease has also been described in a few cases ,5. TheseThe management of intra-abdominal fistulas in Crohn’s disease is a complex issue, which requires a multi-disciplinary approach and ‘tailoring’ of the treatment to the individual patient’s needs. Probably a sensible approach involves early surgical intervention with prior optimization of the patient’s general condition when feasible. Continuous immunosuppressive and anti-inflammatory treatment post-operatively is necessary to maintain the disease in remission and to prevent relapses. Thyroid involvement in Crohn’s disease is also possible and thyroid gland status should not be overlooked when assessing an IBD patient with active inflammation. |
Arabidopsis, auxin distribution along the central root axis has several maxima: in the root tip, in the basal meristem and at the shoot/root junction. The distal maximum in the root tip maintains the stem cell niche. Proximal maxima may trigger lateral or adventitious root initiation.In plant roots, auxin is critical for patterning and morphogenesis. It regulates cell elongation and division, the development and maintenance of root apical meristems, and other processes. In reflected flow mechanism for the formation of the auxin maximum in the root apical meristem. The mechanism is based on auxin's known activation and inhibition of expressed PIN family auxin carriers at low and high auxin levels, respectively. Simulations showed that these regulatory interactions are sufficient for self-organization of the auxin distribution pattern along the central root axis under varying conditions. The mathematical model was extended with rules for discontinuous cell dynamics so that cell divisions were also governed by auxin, and by another morphogen Division Factor which combines the actions of cytokinin and ethylene on cell division in the root. The positional information specified by the gradients of these two morphogens is able to explain root patterning along the central root axis.We propose a reverse fountain has not yet been formed or has been disrupted. In addition, the proximal maxima are formed under the reflected flow mechanism in response to periods of increasing auxin flow from the growing shoot. These events may predetermine lateral root initiation in a rhyzotactic pattern. Another outcome of the reflected flow mechanism - the predominance of lateral or adventitious roots in different plant species - may be based on the different efficiencies with which auxin inhibits its own transport in different species, thereby distinguishing two main types of plant root architecture: taproot vs. fibrous.We present here a plausible mechanism for auxin patterning along the developing root, that may provide for self-organization of the distal auxin maximum when the Plant architecture is formed by the activities of meristems, which comprise stem cells and their derivatives, giving rise to various cell types. The root apical meristem (RAM) is formed at the earliest stages of embryogenesis and is localized to the root apex after germination . DependiAuxin concentration maxima in plant tissues are mainly formed due to active auxin transport between cells -9. Polarin silico is provided for by a reflux of auxin from the basipetal flow back to the acropetal flow all along the meristem, which transports auxin in a loop.Understanding of the role of active transport in the formation of auxin concentration gradients is a topical problem in developmental biology. Computer modeling approaches are also used for solving this problem. In particular, Jonsson et al. (2006), Smith et al. (2006), and de Reuelle et al. (2006) have studied models for phyllotaxis mechanisms in the shoot -15; Stomreverse fountain mechanism is based on a pre-assigned positioning and levels of PINs in RAM. Such a mechanism, whereby tissue patterning predetermines the morphogene distribution, could be defined as a "structural mechanism". Structural mechanisms describe well the processes of auxin distribution in a mature root - in pre-established RAM structure as the ai+1 -ai gradient increases. Due to the boundary position of the cell i = N, the auxin gradient is not defined in this cell, so there is no synthesis of Division Factor in the cell.where constant Division Factor degradationDivision Factor degradation d, DivFV(a) in the cell depends on the auxin concentration within it defines the positional information that governs the profile of cell divisions along the root.In Eq. (16), D and 2D minimal models was integrated using the MGSModeller software package [minimal models was solved using Gear's method [D extended model [Additional file Mathematica, a computer system for symbolic mathematics [D extended model in Mathematica was solved using Runge-Kutta 4th order method, the results of the 1D minimal and 1D extended models with the same sets of parameters and cell numbers were similar (data not shown).The nonlinear system of equations of 1 package . The mins method . The muls method . The 1D hematics -33. The PIN expression and auxin dynamics in the cell [Additional file The parameters were estimated using published experimental data on the mechanisms involved in the regulation of DR5::GUS plants, for example in [DR5::GUS along the central root axis were reconstructed by processing the images using the ImageJ software package [The proposed model is based on published data. The experimental data on the auxin distribution in the root are represented by images of roots from transgenic ample in ,7,8. Expample in . The plo package see for package .D and 2D minimal mathematical models that take into account auxin and PIN1 concentrations dynamics (see the Methods section). The 1D minimal model (7) describes the auxin distribution (from a source in the shoot) over a linear array of non-dividing cells located along the central root axis ; 4: I). : I. D an3q), auxin starts to inhibit PIN1 expression. This leads to a decrease in the active auxin transport towards cell 1 and a shift in auxin maximum from cell 1 to cell 2. Subsequently the same processes occur in cell 3, cell 4, etc. leading to a corresponding shift of the auxin maximum away from the end of the root. This shift continues until the acropetal and reflected auxin flows becomes balanced. For definiteness, we named this mechanism of auxin maximum self-organization the reflected flow mechanism.The mechanism of auxin distribution self-organization found in the resulting stationary solutions is the following. In cells with low auxin concentration, the positive regulation of PIN1 expression by auxin provides for self-enhancement of the acropetal auxin flow. This results in a rapid auxin accumulation at the root end (cell number 1). This is followed by the increase of auxin concentration in the neighboring cell 2 whereto high amount of auxin moves by diffusion from the cell 1 (reflected flow of diffusion). As soon as the auxin concentration in cell 2 exceeds the threshold for auxin-dependent PIN1 degradation . The cell layout of the 2D model is a rectangular array that consists of four "provascular" inner layers and two "epidermal" outer layers on each side of the provascular layers an auxin maximum in the provascular layers at a distance from the root end and (2) auxin gradients in the epidermal layers from the root end towards the root base root cap cells where PIN proteins redistributes auxin in all directions; (2) basipetal auxin flow in the epidermal layers; (3) lateral auxin transport at the edge between the vascular cylinder and epidermal layers, that accounts for the auxin reflux from the basipetal flow back to acropetal flow in the reverse fountain mechanism. In the reflected flow mechanism, the auxin maximum in the root tip arises from the dynamics of the level of PIN1 in an auxin concentration-dependent manner . For the 1D extended model, we estimated the values of additional parameters that were introduced when expanding 1D minimal model. In this way, we obtained a parameter set that guaranteed that the 1D extended model also provides for formation and maintenance of the auxin distribution pattern in the in silico growing root [Additional files Vα) The root growth in the 1 D extended model with the basic set of parameters was simulated starting from three cells, and specifying the initial auxin maximum in the second cell, which corresponds to the auxin distribution pattern in the embryonic RAM of A. thaliana [Vα. This effect can be avoided by adding to the model processes of self-regulated auxin synthesis (data not shown). Consistent with this in silico observation, in vivo at the early stages of seedling development the shoot is the main source of auxin to root, but as the root develops the auxin synthesized in the root becomes of dominant importance [D extended model has demonstrated that cell growth and division in the root do not interfere with the formation and maintenance of the distal auxin maximum.To investigate whether cell divisions might disrupt or destroy the auxin distribution pattern that arises under the thaliana . When sithaliana ; these cthaliana . In a roportance . Thus, sD extended model to simulate more realistic cell-level dynamics along the central root axis . When calculating the 1D extended model, the concentration maxima of the morphogens auxin and Division Factor are localized to the neighboring cells at an approximately constant distance from the root tip with the characteristics of cell types located in vivo along the central root axis.An additional morphogen was introduced to the 1s Figure , includip Figure . Charactin silico the following structure Three to four nondividing cells with a high auxin concentration are located in the root end. Their characteristics match the characteristics in vivo of columella cells;(ii) The next cell is rarely dividing and adopts the global maximum of auxin concentration. Its characteristics match those of the root cap initials;(iii) The next cell is almost nondividing and corresponds in its characteristics to the QC;(iv) The next cells in the in silico root are rarely dividing cells with a low auxin concentration. These characteristics are typical of the vascular initials;(v) The actively dividing cells with a low auxin concentration correspond to the meristematic zone of RAM; and(vi) Finally, the nondividing cells with a low auxin concentration correspond to the differentiation zone of the vascular system.in silico in the cells by the distributions of auxin and Division Factor forms characteristics that match cell fate specifications along the central axis in A. thaliana roots. The possible candidates for the role of Division Factor are considered in the Discussion.Thus, the positional information established The model analysis has demonstrated that the distal auxin maximum, found in the fifth cell of provascular layers, is formed for different sets of parameters and the model tolerates their slight variation. However, more significant changes in the parameter values led to a shift in the maximum position. Moreover, additional maxima of auxin concentration appear along the root axis [Additional files D extended model, the intensity of auxin flow from the shoot (Vα) changes exogenously in time as Vα = α0 + kt [Additional file k is small enough, the growth in Vα is insufficient to compensate for the dilution of auxin concentration in the root caused by its growth (which is reached through increase in cell size and number). Consequently, the total auxin concentration in the root decreases, thereby leading first to the shift of the distal auxin maximum to the root end, a1 ≈ a2 ≈... ≈aN. On the other hand, if a high growth in the auxin flow from the shoot to the root is specified, then the total auxin content in the in silico growing root will increase with time, thereby leading to periodic formation of additional auxin maxima near the first one results in formation of an auxin maximum at the root base in addition to fluctuating inner maxima. The oscillatory solutions found in the 1D minimal model are replaced in the 1 D extended model with solutions displaying periodic formation of additional auxin maxima. These maxima match the experimentally observed oscillation of auxin concentrations in the basal meristem that precede lateral root initiation [in silico under varying auxin flow from the shoot, may predetermine in vivo adventitious root initiation [Arabidopsis. The same calculations with the robust set of parameters showed that the additional auxin maximum forms in the stationary solutions by splitting of the proximally shifted auxin maximum into two separated maxima.We used the 1n method , we studn Figure , while a1 Figure . The Vα t Figure . Further0 Kin the 1 D minimal model. When calculating 1D minimal model with a moderately (no more than twofold) decreased 0 Kvalue, we observed a shift of the distal auxin maximum from the root tip towards its middle and a decrease in this maximum, which fit the experimental data. In addition, several low amplitude auxin concentration maxima appeared at 0 K= 0.08-0.05 , stationary solutions with two auxin maxima at the root base and at the root tip were formed preserving intact the cells proximal to the QC [DR5 activity in vivo and auxin concentration in silico in the cells neighboring the destroyed QC were observed immediately after "QC ablation" in the corresponding experiments . In the model, this increase continued until the auxin concentration in the second cell from the ablated QC exceeded the threshold level j ∈ {3, M -2}), when an increased PIN1 degradation commenced; in this case, a new auxin maximum was formed in cell 2 by the reflected flow mechanism . In the in vivo experiments, the formation of the new DR5 activity maximum near the ablated QC was also connected with a decrease in PIN1 expression in first and second cells from the ablated QC on the x axis [in silico until a balance was reached between acropetal and reflected auxin flows around the forming maximum, which suggest the regeneration of the overall RAM structure.After cutting off the root tip or ablating the QC by laser, the RAM gradually returns to its normal structure by forming a new QC from vascular initials ,21,42. WC Figure . Comparim Figure , curve 3s Figure . The re-s Figure , in the A. thaliana seedlings does not qualitatively change the auxin distribution pattern in the root. This suggests the presence of a homeostatic mechanism that maintains auxin distribution [A. thaliana root are observable only after treatment with high doses of exogenous auxin [in silico by changing the auxin concentration i avalues in a cell, in the initial data of 1D minimal model [Additional file cu. The only effect of fluctuations above 4 cu is the shift in the auxin maximum position to the middle part of the root, l ≤ 11. In the 1D extended model, the shift of the auxin maximum may be compensated by increasing the number of cells in the array so that the auxin maximum position maintains. Correspondingly, we inferred that the regulation mechanism for acropetal auxin transport described in the model at the basic set of parameter values provides resistance of the auxin distribution pattern to a stochastic variation of auxin concentrations in the cells, which agrees with experimental observations [IAA treatment of ribution . Substanus auxin . This tyrvations .1D extended model with different sets of parameters will describe the auxin distribution in the roots of different plant species, reflecting both the common and individual characteristics of distinct transport systems. It is of undoubted interest from this standpoint to study the 1D extended model at different sets of parameters. Supplementary [Additional files 1D extended model with the "robust set" of parameters [Additional file h2 = 10 specifies a pronounced inhibition of the PIN1 expression when intracellular auxin concentration reaches the threshold value 3 q= 3.26. In the 1D extended model with the robust set of parameters, the maximum of auxin concentration is maintained during root development. Yet the position of the distal maximum does not occupy a stringently constant position, "floating" in a certain range k ≤ 25. Characteristic of the model with the robust parameter set is also a greater diversity of stationary auxin distributions in the root, which display additional inner auxin maxima [Additional file The mechanisms of auxin transport in higher plants are highly conserved , yet thereflected flow mechanism is based on the auxin-dependent regulation of auxin acropetal flow auxin distribution pattern formation in the root tip with the maximum at a certain distance form the root end; (2) maintenance of the auxin maximum in growing root; and (3) restoration of the auxin distribution pattern after the RAM damages. The models adequately reproduced experiments on root treatment by auxin transport inhibitors, exogenous auxin or after QC laser ablation. In simulation results we observed an additional effect of the dual dose-dependent regulation of PIN1 expression by auxin: increase of auxin flow from the shoot results in additional auxin maxima formation at the inner root cells or at the root base. We now discuss the biological impact of the simulation results and a set of testable predictions.In this work, we propose and substantiate a plausible mechanism for self-organization of the auxin distribution pattern along the central root axis, and its effect on establishment of the RAM. The w Figure : low auxproteins . To tests Figure . The 1D s Figure . In numeflux-based polarization mechanism relies on the canalization hypothesis proposed by Sachs [concentration-based polarization mechanism provides for auxin distribution in tissue where the auxin flux towards the neighboring cell depends on the auxin concentration in the neighboring cell [concentration-based polarization mechanism was used for explanation of auxin pattern formation in the shoot apical meristem [structural mechanisms, which imply that tissue structure determines the auxin distribution. The mechanism was first implemented by Grieneisen et al., (2007) [reverse fountain concept [structural mechanism also has also been used to explain of auxin maxima formation in curved root regions [At least four main mechanisms of auxin distribution pattern formation have been suggested. The by Sachs and descby Sachs and by Gby Sachs . These mby Sachs ,49 and fby Sachs . The coning cell ,14. The meristem ,14,50 asmeristem . The thi, (2007) , who stu concept in proce regions .activator-inhibitor mechanism. The activator-inhibitor mechanism explains morphogenetic pattern formation under a positive and a negative regulation. This mechanism was first implemented in the reaction-diffusion model by Turing [activator-inhibitor mechanism. In the reflected flow mechanism presented here, auxin is both the activator and the inhibitor of the expression of its carrier (PIN1). The results described in the present work demonstrate that the dual regulation of polar auxin transport is a sufficient condition for self-organizing and maintenance of auxin distribution pattern in root tip during plant development.Another well-known mechanism that can describe auxin distribution patterning but hasn't been implemented for this purpose before is the y Turing and theny Turing . These wreflected flow mechanism is a plausible alternative to the reverse fountain mechanism for auxin pattern formation in the root tip. The model of Grieneisen et al. (2007) [reverse fountain mechanism explains the generation of the auxin distribution pattern in the root tip based on a specific RAM structure in which each cell has a specified set of directions of auxin efflux that impx Figure . In the reverse fountain mechanism that shows its advantages over the reflected flow mechanism. First, the auxin pattern generated under the reverse fountain mechanism is extremely robust to the parameters variation whereas in our simulations are sensitive to changes in parameters value [Additional files reverse fountain mechanism provides for formation of the gradient from the distal auxin maximum towards the root base, whereas the reflected flow mechanism does not. Third, the model [reverse fountain mechanism more accurately explains the auxin distribution pattern in a mature root, where the structural layout is already formed.There are at least three effects provided exclusively by the he model reproducreflected flow mechanism over the reverse fountain mechanism becomes evident by comparison of the in silico experiments on root tip cut or QC ablation. In the model [in vivo observations, where the restoration of the auxin maximum at a distance of the ablated QC precedes the restoration of both the QC and overall RAM structure [D minimal model we observed restoration of the auxin maximum at a distance from the new root end that matches experimental data [in silico experiment reproduced step-by-step the changes in DR5 and PIN1 expressions observed in vivo in course of RAM regeneration [reflected flow mechanism provides a better explanation of auxin pattern formation in developing RAM or RAM recovering after damage. In addition, unlike the prespecified RAM structure with five types of cells assumed in the model [2 D minimal model with the only two cell types after root treatment with auxin and its analogs, (2) in response to increase of auxin flow from the shoot while plant is growing, or (3) to fluctuations in auxin flow caused by circadian rhythms.By comparison with the reverse fountain and the reflected flow mechanisms are complementary. In particular, the reflected flow mechanism commences operating from the very early stages of root development. At later developmental stages, an anatomical structure forms and provides for the functioning of the reverse fountain mechanism that serve for more robust maintenance of the auxin maximum in the RAM. However, the reflected flow mechanism does not disappear even after formation of the RAM structure. Its role becomes less evident in the background of the structural mechanism of the reverse fountain, but morphogenes continue to function, revealing themselves if structure is disrupted or the environment changes.We suggest that the in silico experiments demonstrated that upon increase in auxin flow from the shoot to root an additional maximum was formed periodically in the provascular cells along with the distal auxin maximum [Additional file During plant growth, the acropetal auxin flow from the aboveground plant part to the root increases during development and plays a key role in regulating elongation of the main root and development of lateral roots ,40. IncrD extended model cell divisions are regulated by auxin and a hypothetical morphogen Division Factor. The Division Factor combines the functions of cytokinin and ethylene in regulation of cell division rates in root - inhibition and activation, respectively . The synthesis and degradation of the Division Factor depends on auxin . The distributions of two morphogens form the positional information fields in the root patterning. In the model, we observed the appearance of individual cell characteristics, such as auxin concentration, relative localization on the axis, and mitotic activity, which could together correspond to the characteristics of various cell types located along the central root axis a repressor of cell division, which is synthesized in the root cap and spreads over the root tissue from the root tip towards its base, forming a decreasing gradient , and (2) an activator of cell division, which is synthesized in the QC cells and anisotropically diffuses in root. The activator distribution matches to the domain of the ethylene precursor ACC expression [The same profile of mitotic activity along the central root axis could be observed (data not shown) if we replace pression , whereaspression or auxinpression distribuA. thaliana [A. thaliana [in silico by varying the parameters of the model. Both the "basic" and "robust" sets of parameters provide for generating and maintaining the distal auxin maximum, although the detailed model behavior differed depending on which set was used [Additional files Auxin transport in plants is a highly conserved mechanism: auxin distribution patterns with the maximum in the stem cells of the RAM have been demonstrated for thaliana , rice [5thaliana , and maithaliana . The corthaliana , rice [5thaliana , and maithaliana . Thus, sD extended model. Second, additional auxin maxima arose in stationary solutions when we (1) increased auxin flow from the shoot, (2) decreased the rate of auxin transport; (3) modified the coefficients for auxin-dependent PIN1 expression [Additional files in silico growing roots under different sets of parameters [Additional file First, the model with the robust set of parameters is sensitive to auxin fluctuations--additional auxin concentration maxima appeared in the middle of the root and at its base in simulations of root treatment with rather low doses of exogenous auxin. Moreover, once formed, these additional maxima were stably retained during root growth in the 1PIN1 expression) agrees more closely with the pattern of auxin transport in the taproot system, while the model with robust set of parameters (high inhibition efficiency) better simulates the transport system of fibrous roots [Additional file Analysis of the model's behavior using two sets of parameters suggests a key role for the auxin transport inhibition mechanisms in the formation of different root system types. The model's behavior with the basic set auxin distribution in the root according to DR5 reporter activity from Sabatini et al. (1999) [. (1999) ; (b) TheClick here for fileThe model parameters. The table containing all model parameters.Click here for fileSimulation of root growth along the central root axis. In the figure, (a) Distribution of auxin (red), Y (blue) and rates of cell division (gray columns) in conventional units along the central root axis. The curves were calculated in the 1 D minimal model with basic set of parameters. (b-d) The extended model solutions: (b) The mitotic activity along the central root axis; (c-f). Auxin and substance Y distribution. The green curve indicates the growth mode of cells . c. the model was started from three cells; d. 10 cells; e. 20 cells; (f.) more than 100 cells. Cells of different types can be distinguished by considering both auxin concentration in the cell and its mitotic activity (see the main text for more details): QC - quiescent center; RCI - root cap initial; RC- root cap; MZ- meristematic zone; DZ- differentiation zone.Click here for filein silico growing root under normal conditionMaintenance of auxin maximum at the root tip in the . The movie 1 simulated using the 1D extended model [Additional file α = 0.3+1.7*10-5t, auxin (red line), substance Y (blue line), cell phase .Click here for fileD minimal model with basic set of parametersAnalysis of the 1. The figure showing changes in the auxin distribution pattern at N = 50 in response to variations in: (a-b). α value; (c). diffusion rate (D) value; (d). 0 Kvalue; (e). 1 qvalue; (f). 2 qvalue; (g). 3 qvalue; (h). Kd value, where other parameters were defined as in [Additional file minimal model analysis in the STEP+ package [th cells obtained by the method of continuation with respect to parameter α. The number of crossings of the selected component with a vertical line at α = 1 corresponds to the total number of stationary solutions (stable and unstable), with the same set of parameters. k. The stationary solutions of the model estimated on figure (e.). j. Oscillation of auxin concentration in the ith cells in time (tu). In all plots, the y axis specifies auxin concentration in concentration units (cu). package . e. The Click here for fileD minimal model with robust set of parametersAnalysis of the 1. The figure showing changes in the auxin distribution pattern at N = 50 in response to variations in: a. α value; b.-c. diffusion rate (D) value; d. 0 Kvalue; e. 1 qvalue; f. 2 qvalue; g. 3 qvalue; h. Kd value, where other parameters were defined as in [Additional file minimal model analysis with the robust set of parameters in the STEP+ package [th cells are obtained by the method of continuation with respect to parameter α. The number of crossings (102) of the selected component with a vertical line at α = 1 corresponds to the total number of stationary solutions (stable and unstable), with the same set of parameters. In all plots, the y axis specifies auxin concentration in concentration units (cu). package . The staClick here for filein silico growing root under increased rates of auxin flow from the shootPeriodic formation of additional inner auxin maximum in the . The movie 2 simulated using the 1D extended model [Additional file α = 0.4+1.7*10-5t, auxin (red line), substance Y (blue line), cell phase .Click here for fileComparison of the models behavior with basic and robust sets of parameters. The table showing the differences in the minimal and extended models behavior with basic and robust sets of parameters.Click here for file |
The authors investigated the current practice of intensive psychotherapy by residents in the department of psychiatry.We mailed a questionnaire to 126 fourth-year psychiatry residents in order to obtain data on their clients' sociodemographic characteristics, the settings in which psychotherapy is being conducted, the effects of psychotherapy, the difficulties associated with psychotherapy, the state of supervision and the level of clients' satisfaction.Approximately 51.5% of the residents completed the questionnaires. The average number of clients was 4.9±3.8, the average number of psychotherapy sessions was 26.2±20.1, and 69.4% of the residents had performed insight-oriented psychotherapy. Approximately 69.8% of the fourth-year residents had received some form of supervision, and 58.7% agreed to increase the frequency of supervision. Approximately 74.2% of the cases were supervised. The average number of supervisions per case was 9.2±10.5.The setting in which psychotherapy is conducted, number of clients, and type of supervision varied greatly among the training institutes surveyed. Based on these findings, we expect to create better psychotherapy training programs for psychiatric residents. The introduction of modern Western psychotherapy in Korea began immediately after the Korean War.7Although psychiatry has rapidly developed in various fields in recent years, the importance of psychotherapy in psychiatry has been proven in numerous studies. Mohl et al.Therefore, the authors investigated how psychiatric residents are conducting psychotherapy and how they are being supervised. Due to the lack of domestic investigation data regarding these issues, the authors surveyed the current situation and intended to reflect the study result in the establishment of a resident training policy after comparing the current result with those of previous studies.The current study collected data by mailed the final version of a questionnaire to 126 fourth-year residents working at 72 training hospitals around the nation between June and July 2007. Sixty-five (51.5%) of the 126 residents responded to the survey. Among the respondents, 85% worked at university hospitals, and 8.5% and 6.3% of them were training in hospitals specializing in psychiatry and general hospitals, respectively. The non-respondents were called, sent mailings and e-mailed repeatedly up to three times. Anyone who did not respond the questionnaires after the third try was defined as a 'rejecter'. The rejection rates were 46% for University hospitals, 14.2% for general hospitals and 73.6% for hospitals specializing in psychiatry, respectively. The rejection rates in each region were 43.4% in Seoul, 49.7% in Gyeonggi, 71.5% in Gangwon, 58.4% in Chungcheng, 58.4% in Gyeongsang, and 44.5% in Jeolla.A senior researcher and collaborative researchers held a preliminary meeting to prepare the first questionnaire after reviewing the previously reported data on the status of psychotherapy and investigating the related questionnaires. The first survey was conducted on five fourth-year residents in psychiatry by using the first preparation, and problems were dealt with in order to develop the final version of the questionnaire. The questionnaire asked about the following: 1) Sociodemographic and clinical characteristics of clients, including the clients' sex, age, education level, socioeconomic status, and diagnosis; 2) Clinical variables of clients, including the start of therapy, types of psychotherapy practiced, frequency of preliminary interviews, total frequency, whether or not therapy was terminated, situation at the time of therapy termination, causes of therapy termination after negotiation, reason for stopping therapy, important difficulties faced at the time of psychotherapy, combination of drug therapy or not, major substance of drug therapy, route of asking in psychotherapy, and effects of psychotherapy; 3) the settings in which psychotherapy was conducting, including weekly frequency of psychotherapy, estimation of psychotherapy fee, time point at which the fee for psychotherapy is paid, fee when a client does not arrive on time, whether or not the psychotherapy is recorded, and whether or not the psychotherapy is memorized; and 4) the supervision of psychotherapy, including the method of supervision, place of supervision, and frequency of supervision and level of client satisfaction. The type of psychotherapy used during the study was limited to intensive psychotherapy, which includes analytical psychotherapy and supportive psychotherapy. Cognitive-behavioral therapy was not included in this study.Among several variables, continuous variables were calculated to obtain mean values and standard deviations, and frequency analyses were performed for nominal scales. Analysis of variance (ANOVA) was used for comparisons among the investigated items. The nominal scales were analyzed using cross analysis. Statistical analyses of all evaluation data were performed using the SPSS 13.0 program operated in Windows.Each resident treated 4.9±3.8 psychotherapy clients (ranging from one to 22 cases) during their resident training.The mean age of the clients was 28.4±8.5 years, and most of the clients were female (66.7%). Investigations into the education levels of the clients indicated that 39.6% of them graduated from high school, and 51.7% of them had a degree equal to or beyond a college degree. The distribution of socioeconomic status from the most prevalent to the least was III, II, IV, I, and V. Socioeconomic status was higher than Group III in 79% of the total clients. Cases diagnosed with Axis-I disorders comprised 40.8% of the clients, while 6.6% of the clients had Axis-II diagnoses. The rest of the cases had both Axis-I and Axis-II diagnoses. Depressive disorder, anxiety disorder, and adjustment disorder were frequently found in clients with Axis-I diagnoses. In the clients with Axis-II diagnoses, 2.1% were cluster A, 72.5% were cluster B, and 22.5% were cluster C as demonstrated by the prevalence of borderline personality disorder, followed by histrionic personality disorder, and avoidant personality disorder.Although 2.1% of the respondents said they began studying psychotherapy during the first semester of their first year of resident training, 23% responded that the start of psychotherapy was in the first semester of the second year of resident training, and 24.6% responded that the start of psychotherapy during the first semester of their third year of resident training, thus showing a gradually increasing trend as the year of resident training increased.When asked about the type of psychotherapy practiced, 67.4% responded by saying they practiced insight-oriented psychotherapy, 28.5% reported practicing supportive psychotherapy, and 4.2% report practicing psychoanalysis. In the initial stages of psychotherapy, 69.5% of the clients receiving the psychotherapy were outpatients, and 30.5% received the treatment during hospitalization.Regarding the means by which psychotherapy was requested, 39.9% of the cases were requested by outpatients visiting physicians, 28.1% were requested by other physicians at the same hospital, 26.3% were requested by the attending physicians of a hospital department, and 5.7% were requested by other hospitals.The average frequency of preliminary meetings was 2.2±2.7 sessions. The average frequency of psychotherapy was 26.2±20.1 times per case, with a minimum of 1 and a maximum of 150 times. The case in which 150 sessions of psychotherapy were recorded was excluded from the analysis in order to reduce the deviation. For each session of psychotherapy, the average treatment time was 47.8±5.5 minutes.Psychotherapy was terminated in 71.7% of the cases. In most cases (57.9%), the psychotherapy was terminated due to the client's failure to show up for therapy. The reasons for discontinuing therapy were failure to overcome transference resistance in 49.2% of the cases, high expectations for a treatment effect in 13.8%, economical burdens in 6.2%, and noncooperation of guardians in 3.8%.Successful completion of the therapy was reported by 17% of the respondents. Successful completion of therapy could be attributed to the client's motivation in 51.5% of the cases, psychopathology of the client in 27.3%, and appropriate supervision in 12.1%. The greatest difficulty experienced by the resident while conducting psychotherapy was a lack of client understanding, followed by lack of supervision, lack of time, lack of understanding by the client's family, and lack of economical capacity, which showed the trend of highly accounting the causes from the therapists' point of view.Although psychotherapy was only conducted in 31.7% of the cases, 68.3% of the cases were treated using a combination of drug therapy and psychotherapy, and the drugs used to treat 56.1% of the combined drug therapy cases were prescribed by the physician administering the therapyWhen asked about the treatment time, 61.3% of the respondents said they conducted the therapy during regular business hours. The average frequency of psychotherapy sessions was 1.1±0.4 times a week. The expenditure for one session differed according to the calculation methods of the training hospital, and the average cost was 28,029±10,007 won (Ca 30±10 US dollars) for each session of therapy. Most of the sessions were paid in full after each treatment. Even if clients did not arrive on time, half of the hospitals did not charge the expenditure at the next session. When conducting psychotherapy, 59.2% of the respondents recorded the session, and 47.5% memorized the therapy.When asked about the supervision of psychotherapy, 74.2% of respondents reported receiving some form of supervision, and 38.8% reported receiving individual supervision . The aveThe need to increase the amount of psychoanalysis and psychotherapy conducted during residency training was recognized by 58.7% of the respondents recognized, and 41.3% believed that the current training level is appropriate. None of the respondents believed there was a need to reduce the amount of therapy conducted during the training.In the case of female clients, many cases of psychotherapy were successfully terminated without showing statistical significance, and no difference was observed. Individual supervision was mainly conducted in university and general hospitals, while group supervision was conducted more frequently in hospitals specializing in psychiatry, but this difference was not significant. Third- and fourth-year residents began conducting insight-oriented psychotherapy more frequently than first- and second-year residents (p=0.045). The total duration of treatment was longer when the residents started the therapy during their first or second year of residency (p=0.042), and the frequency of receiving supervision was also high (p=0.026), but no statistically significant difference was found when comparing the cases in which therapy was terminated. The difference was thought to be attributable to numerous cases of currently progressing psychotherapies for third- and fourth-year residents.Consistent with the study by Stirman et al.,In clients with Axis-II diagnoses, most of the psychotherapies were performed in clients with cluster B characteristics, and borderline personality disorder was the most prevalent disorder, comprising 39.4% of the single diagnoses. This finding was similar to that published by Rhee and Rhee,Many of the residents performed psychotherapy during their second and third years of residency training, and the proportion of residents starting psychotherapy for hospitalized clients were found to be low, at just 30.5%. This is considered to be attributable to the fact that first-year residents are mainly responsible for the treatment of hospitalized clients, and residents in the later years of training usually see outpatients and clients with personality disorders. However, because the continuation of psychotherapy training is circumstantially difficult after finishing the residency program, it is desirable to start the psychotherapies during the early years of the resident training program in order to experience long-term progression of psychotherapy. This is related to the direction of training provision suggested by the Korean Neuropsychiatric Association. The resident's psychotherapy evaluation categories listed on the resident training regulation status evaluation formThe quality and evaluation of the effect of psychotherapy during the supervision of a psychotherapy session are uncertain. The result obtained in the current study could be considered to reflect such a general level of evaluation categories. Although ten residents selected psychoanalysis as a type of psychotherapy practiced, the frequency at which they actually conducted psychotherapy was limited to once a week. Therefore, none of the respondents were conducting psychoanalysis in their regular practice.Regarding the frequency of therapy, there was no case requiring more than three sessions a week. Considering that a lack of time was deemed to be one of the main difficulties associated with psychotherapy, the heavy workloads given to residents could be considered to be a large obstacle in the performance of psychotherapy.In the current study, residents reported treating a client for about three to seven months. Previous studiesIn a study conducted by KangIn a study by Rhee and Yoon,The clinical effects of psychodynamic psychotherapy have already been proven, and it is known to be as effective as cognitive behavioral therapy in patients with specific psychiatric diseases.The importance of supervision during psychotherapy training has been emphasized with educational analysis.Although it has been expected to be related to total therapy hours, therapy type, therapy success rate, cost of psychotherapy, and supervision frequency as the therapy time is delayed, no such relationship was reported in actual circumstances. However, it is thought that this result alone could not be used to indicate a lack of correlation between the psychiatric training period and psychotherapy skill. It is therefore necessary to establish an evidence-based training system in order to determine whether the start of psychotherapy training during residency is indeed appropriate. If so, the year that resident training has to be started, the number of cases that have to be experienced, and how the supervisor training itself has to be managed must be established.The current study has some limitations. One limitation of this study is that it only included fourth-year residents. In addition, since most of the respondents had been training at university hospitals, it was difficult to draw any conclusion that would be representative of the entire population of psychiatric residents. Probably due to the complexity of the questionnaires, the reply rate was only limited to 51.5%, even though the residents were contacted three times and encouraged to participate. The possibility of that some of the residents skipped their education in psychotherapy cannot be excluded. Since the current investigation was conducted in the form of a survey, the limitation of questions and the issue of reliability should be also considered. For instance, some respondents reported conducting psychoanalysis, even though they only conducted psychotherapy. The reported rate at which psychotherapy was conducted in inpatients was 30.5%, which was far from reality. Furthermore, although about half of the treatment failure was due to unsolved transference resistance, these conclusions were based on the interpretation of therapists alone (if they did not receive supervision). There was a difference between the total number of clients receiving psychotherapy and the number of psychotherapy clients analyzed, which might be have resulted from respondents failing to report all of the psychotherapy cases they treated due to the large amount of time required to prepare the data.Despite the above limitations, the current study could find meaning from its systematic attempt to determine the status of intensive psychotherapy in psychiatric residents by conducting a survey. The objective of psychotherapy education has been stated as being to cultivate basic grounds for the understanding and practice of psychotherapy, and not to create experts.In summary, the current study investigated the status of intensive psychotherapy training by conducting a nationwide survey of current fourth-year residents in psychiatry. The results of the study showed that psychiatric residents experienced various types of cases but needed in-depth educational programs concerning psychodynamics as well as intensive supervision. Investigating and analyzing the current status of psychotherapy conducted during the resident training programs in psychiatry will provide fundamental data for establishing a better training environment. |
Sir,The recent publications of On a further point, |
Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.low targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses. Ovarian cancer is the deadliest of the gynecological malignancies. Eighty percent of the 14,000 cases of ovarian cancer that are diagnosed each year are of epithelial cell origin. Epithelial ovarian cancer is associated with the formation of a large amount of peritoneal fluid and is extremely metastatic. Immune regulation plays an important role in controlling ovarian tumor growth. Infiltration of T cells within the tumor is strongly associated with an increase in 5-year survival of ovarian cancer patients ,3. RecogOvarian tumors, however, have developed elaborate mechanisms to counter immune recognition and attack. Factors produced by the tumor can alter the expression of important activating molecules on immune cells present in the peritoneal cavity. In one study, a 10-14 kDa protein produced by the tumor cells was shown to downregulate the expression of the key signaling molecule CD3ζ . MacrophWe have studied the effects of one particular factor produced in high quantities by the tumor cells, MUC16, and its effect on the cytolytic function of human NK cells ,11. MUC1sMUC16 is a potent inhibitor of the cytolytic ability of NK cells . IncubatStaphylococcus aureus adhesion on the corneal epithelial cell layer [To date, csMUC16 has not been studied for its potential role in protecting ovarian tumor cells from immune attack. csMUC16, similar to sMUC16, may directly interact with NK cells and inhibit their ability to lyse tumor targets. Alternatively, csMUC16 may also protect ovarian tumor cells from NK attack by a different mechanism. Mucins are known to possess both adhesive and anti-adhesive properties . csMUC16ll layer .Because NK cells form immune synapses with their target cells -30, whicHere, we provide evidence that the expression of csMUC16 attenuates the interactions between ovarian cancer cells and NK cells. Our data indicates that NK cells are unable to form immune synapses with the ovarian tumor targets, regardless of the expression of activating or inhibitory ligands on the surface of the tumor cells. This results in another redundant molecular mechanism that epithelial ovarian tumor cells utilize to avoid immune attack.low, csMUC16medium, and csMUC16high based on fluorescent intensity as visualized by microscopy of NK cells. Therefore, HLA class I expression protects tumor cells from NK cell attack. However, although comparable levels of HLA class I were expressed on the csMUC16d Figure . NK cellC Figure .pos-OVC and csMUC16neg-OVC expressed HLA class I antigens that could contribute to their protection from NK cell mediated lysis. Therefore, another effector cell model was required where the protection provided by HLA class I antigens would not be as pronounced as with the primary NK cells from healthy donors.As shown in Figure pos-OVC and csMUC16neg-OVC sublines and allows better isolation of the immunoprotective properties of csMUC16.The NK cell leukemia cell line, NKL, does not express the prominent KIR (Killer Immunoglobulin-like Receptors) CD158a, CD158b, and CD158e Figure . These Kneg-OVC compared to csMUC16pos-OVC and csMUC16neg-OVC as compared to csMUC16pos-OVC was also used in these experiments. This protocol for the generation of this additional cell line has been reported [neg-OVC/NKL and csMUC16neg-OVC-A/NKL co-cultures , indicating Siglec-9-csMUC16 binding may not play a role in the observed inhibition of cytotoxicity.We have shown previously that sMUC16 binds specifically to a subset of NK cells in epithelial ovarian cancer patients. Our current data indicates that the receptor for sMUC16 is the inhibitory receptor Siglec-9 . The binding of csMUC16 to Siglec-9 could be a mechanism by which csMUC16 is causing inhibition of NK cells. We have shown using cytotoxicity assays that NKL cells are also inhibited by MUC16 present on csMUC16An alternate explanation for NK cell protection afforded by csMUC16 lies in the extraordinary biochemical properties of the molecule itself. Mucins are known to exhibit both adhesive and anti-adhesive properties. In cancer, the loss of apical expression of these large molecules and the subsequent distribution over the entire cell membrane has consequences for cell-cell interactions . The larMUC16 has a protein backbone of approximately 24,000 amino acids and a hipos-OVC and csMUC16neg-OVC, these mucins did not contribute to the differences in conjugation and lysis that were seen between these two sublines.Other large molecules have been shown to interfere with cell-cell contact because of the bulky nature of their extracellular domains. CD43, which is abundantly expressed on T cells and is heavily glycosylated, is removed from the immune synapse before synapse formation between T cells and APCs . Other rlow ovarian tumor cells by NK cells may result in the selective survival of csMUC16high cancer cells. Such a selective increase in csMUC16high ovarian cancer cells is supported by our observation that csMUC16pos-OVC that were resistant to NKL attack expressed a higher level of csMUC16 . Selective immunoediting of csMUC16high ovarian tumors may therefore lead to tumors that have a higher potential to withstand immune attack and also to metastasize and proliferate in the peritoneal environment.Our data may represent an example of an immunoediting mechanism that allows progression and proliferation of ovarian tumors. NK cells may selectively eliminate csMUC16l cavity . The surWe have conclusively shown that the expression of csMUC16 blocks the conjugation step of immune synapse formation of NK cells with ovarian cancer targets. This blockage prevents NK cell lysis of cancer cells. Immune protection afforded by csMUC16 should therefore be considered as another important mechanism that promotes tumor growth.neg-OVC and csMUC16neg-OVC-A and control subline csMUC16pos-OVC were obtained as described in our recent studies [Unless otherwise stated, all chemicals were purchased from either Sigma or Fisher. NK cells were isolated from healthy donors using the RosetteSep (Stem Cell Technologies) NK Cell Isolation Kit protocol. Purity of the isolated NK cells was between 80 and 90% based on flow cytometry using anti-CD3, CD16, and CD56 antibodies. Antibodies against MUC1, CD2, CD3, CD16, CD56, CD158a, CD158b, CD158e, and HLA Class I (w6/32), were from BD Biosciences. Anti-LFA-1 and anti-MUC4 antibodies were from eBiosciences and Zymed Laboratories, respectively. OVCAR-3 cells were obtained from ATCC . OVCAR-3 csMUC16 knockdown sublines csMUC16 studies ,35.pos-OVC, and csMUC16neg-OVC cells were cultured in the conditions prescribed for OVCAR-3 cells by ATCC. Target cells (7.5 × 104 cells) were mixed with NK cells at a 1:1 ratio in a total volume of 400 μl. The cells were centrifuged for 5 minutes at 100 × g and I incubated at 37°C at 5% CO2 for 25 minutes. The media was aspirated and cells were resuspended in PBS without calcium and magnesium and placed on poly-L-lysine coated coverslips. All subsequent procedures were performed at room temperature. Cells were fixed with 3% paraformaldehyde for 15 min, washed twice with 150 mM glycine, permeabilized with 0.1% Triton-X for 4 min, washed with PBS, and blocked overnight with PBS containing 5% goat serum.NK-target synapse experiments were conducted according to previously established protocols ,50,51. OCells were stained with murine antibodies against LFA-1, CD2, or MUC16 (VK8 antibody kindly provided by Dr. Beatrice Yin) in 5% goat serum for 30 min. Coverslips were washed with 1 mL PBS, stained with a cocktail of goat anti-mouse FITC (Jackson ImmunoResearch), and rhodamine conjugated phalloidin (Invitrogen) for 30 min. Cells were washed with PBS, dipped in deionoized water, and mounted with DAPI containing mounting media (Invitrogen). After drying overnight, cells were visualized using the Bio-Rad Radiance 2100 MP Rainbow confocal microscope. Fifty conjugates between NK and tumor targets were counted on each coverslip. Conjugates were defined as an effector (NK cell) and target (cancer cell line) in contact with one another. Each conjugate was scored for polarization of the activating synapse markers LFA-1 and F-actin or CD2 and F-actin. Polarization was determined using two criteria, both of which had to be met to be defined as a synapse. These criteria were as follows: if greater than 70% of the fluorescence (of CD2 and F-actin or LFA-1 and F-actin) was noted to be at the interface between target and effector cell, and if there was a noticeable "flattening" of the membranes at the contact interface, than the synapse was deemed an activating synapse. Percent synapse formation was determined as: .pos-OVC or csMUC16neg-OVC were blocked with Goat IgG (BD Biosciences) for 15 min., washed, and 15 μg/mL of either DNAM-Fc or NKG2D-Fc (R&D Systems) was added for 30 min on ice. Goat anti-human-Fc FITC (Jackson ImmunoResearch) was added for 30 min and washed. To determine mucin and HLA class I expression cells were treated with the primary antibodies. Where appropriate, cells were stained with goat anti-mouse PE . Immediately before data acquisition on an LSRII (Beckton Dickinson) flow cytometer, the viability indicator PI or DAPI were added to each sample. Automatic compensation was applied. FlowJo software was used for analysis of the raw flow cytometry data, and comparison and statistical analysis (student t-test) of the data was done using GraphPad Prism software .For flow cytometric analysis, cells were washed two times in PBS containing 1% BSA by centrifugation at 300 ×g for 10 minutes at 4°C. Cells were labeled with primary and secondary antibodies for 30 min on ice, and washed with PBS containing 1% BSA in between antibody staining and prior to flow cytometry. In the DNAM-1-Fc and NKG2D-Fc binding experiments, OVCAR-3, csMUC16neg-OVC, csMUC16pos-OVC, or csMUC16pos-OVC-R were labeled with 51Cr in suspension. The targets were mixed with NK cells isolated from 4 healthy donors in 96-well plates at different effector:target ratios. After 4 h incubation, 51Cr released from the targets was measured and percent lysis was determined as described previously [csMUC16eviously . Comparineg-OVC or csMUC16pos-OVC cells. After 24 h incubation, the media and the adherent cells were harvested using trypsin-EDTA, transferred to a tube, stained with PI, and analyzed for percentage of live target cells by flow cytometry on a BD LSR-II instrument. Comparison and statistical analysis (student t-test) of the data was done using GraphPad Prism software .CellTracker Green (Invitrogen) labeled NKL cells were added to wells of 6-well plates that contained confluent csMUC16neg-OVC and csMUC16pos-OVC cells by NKL cells was also measured by counting the number of surviving adherent tumor targets. These experiments were set up similarly to the flow cytometry based cytotoxicity assays except that NKL cells were not stained. Following co-culture, the floating NKL cells and dead tumor cells were removed by gentle washing and the number of adherent (considered live) cells and colonies were counted on an inverted microscope. The cell counts from three independent experiments were pooled and the averages (n = 15) were plotted. csMUC16pos-OVC cells that survived NKL challenge were further cultured to confluence and re-challenged with NKL cells. Tumor cells surviving this second NKL treatment were harvested and analyzed by flow cytometry for amount of csMUC16 expression.Lysis of adherent csMUC162. Concurrently, csMUC16neg-OVC and csMUC16pos-OVC cells were trypsinized, washed, and dyed with 1.25 pM CellTracker Green (Invitrogen) in the same conditions. After washing off excess dye with PBS, cells were resuspended in PBS containing 1% BSA solution. NK cells were placed in flow tubes with either csMUC16neg-OVC or csMUC16pos-OVC cells at a 1:1 ratio and centrifuged for 2 minutes at 100 ×g. Tubes were incubated for 25 minutes at room temperature, and vortexing was avoided. Immediately before data acquisition on an LSRII (Beckton Dickinson) flow cytometer, viability indicator DAPI (BD Biosciences) was added to each sample. Comparison and statistical analysis (student t-test) of the data was done using GraphPad Prism software .Freshly isolated naïve NK cells were labeled with 0.5 μM CellTracker Red (Invitrogen) for 25 minutes at 37°C in 5% COneg-OVC and csMUC16pos-OVC were plated at 100,000 cells/well of a 96-well plate and allowed to grow for 48 hours to confluence. On the day of the assay, NKL cells were dyed with calcein AM in 1% PBS-BSA for 30 min at 37°C, washed, and resuspended in media containing FBS at a concentration of 50,000 or 100,000 NK cells/50 uL. Fifty μL of dyed NKLs were added to each well containing the previously plated csMUC16neg-OVC or csMUC16pos-OVC. The plate was centrifuged at 400 ×g for 5 minutes and placed in the incubator for 25 minutes. The plate was washed gently with 1% PBS-BSA three times to remove non-adherent cells and then read using the Victor V-3 plate reader (Perkin Elmer). Comparison and statistical analysis (student t-test) of the data was done using GraphPad Prism software .csMUC16The authors declare that they have no competing interests.JAAG conducted the majority of the experiments and helped in designing experiments, data analysis, and preparing the manuscript. MF and JAB conducted some of the flow cytometry experiments. SH conducted the cell binding experiments. HH and SP assisted in maintaining the cell lines and isolation of blood samples. MM and CR developed the MUC16 knockdown cell lines. AK assisted in monitoring MUC16 expression by the knockdown cell lines. JPC assisted in designing the experiments and data interpretation. MSP is the corresponding author and helped in study design, data interpretation and preparation of the manuscript. All authors read and approved the final manuscript.neg-OVCMUC16 is not detected in the lysates of csMUC16. 1. Lysates of OVCAR-3, csMUC16pos-OVC, and csMUC16neg-OVC cells were analyzed by western blotting. MUC16 was detected by using VK-8 (anti-CA125) as the primary antibody. Actin was used as loading control.Click here for file |
The mature miRNA sequence binds to more or less specific target sites on the mRNA. Both their small size and sequence specificity make the detection of completely new miRNAs a challenging task. This cannot be based on sequence information alone, but requires structure information about the miRNA precursor. Unlike comparative genomics approaches, ab initio prediction of miRNAs by genome scanning that only relies on (predicted) secondary structure to distinguish miRNA precursors from other similar-sized segments of the human genome. We apply a machine learning technique, called linear genetic programming, to develop special classifier programs which include multiple regular expressions (motifs) matched against the secondary structure sequence. Special attention is paid to scanning issues. The classifiers are trained on fixed-length sequences as these occur when shifting a window in regular steps over a genome region. Various statistical and empirical evidence is collected to validate the correctness of and increase confidence in the predicted structures. Among other things, we propose a new criterion to select miRNA candidates with a higher stability of folding that is based on the number of matching windows around their genome location. An ensemble of 16 motif-based classifiers achieves 99.9 percent specificity with sensitivity remaining on an acceptable high level when requiring all classifiers to agree on a positive decision. A low false positive rate is considered more important than a low false negative rate, when searching larger genome regions for unknown miRNAs. 117 new miRNAs have been predicted close to known miRNAs on human chromosome 19. All candidate structures match the free energy distribution of miRNA precursors which is significantly shifted towards lower free energies. We employed a human EST library and found that around 75 percent of the candidate sequences are likely to be transcribed, with around 35 percent located in introns.MiRPred is a novel method for ab initio miRNA discovery. In doing so, it requires less previous knowledge about miRNA precursor structures while programs and motifs allow a more straightforward interpretation and extraction of the acquired knowledge.Our motif finding method is at least competitive to state-of-the-art feature-based methods for With the discovery of miRNAs the traditional view of RNA as pure helper molecules in protein biogenesis has changed radically. MiRNAs belong to a class of single-stranded, non-coding RNA (ncRNA) with only 21–25 nt in sequence length. They are directly involved in downregulation of gene expression at the post-transcriptional level, i.e., they act as negative regulators of translation, in multi-cellular animals and plants, and also appear in viruses transcript is cleaved into a 60–70 nt long precursor sequence by the Drosha/Pasha complex. The pre-miRNA is transported into the cytoplasm by Exportin 5 and cleaved by Dicer into the mature miRNA. In the RISC (RNA-induced silencing complex) these molecules regulate expression of target genes by binding to complementary sites on the messenger RNA (mRNA). This causes either cleavage and degradation of the mRNA or just suppresses its translation into a protein.loops or bulges by unpaired bases and continuous segments of base pairings (stems) as secondary structure components.The miRNA precursor sequence folds into the typical hairpin stem-loop structure . Because motif matching is position-independent, there is also no need to preselect candidate hairpins. The overall method for finding miRNA structures by scanning genome regions, named miRPred, may be summarized in five steps:We apply linear genetic programming (GP) to devel(1) A fixed-length window is shifted in regular steps over the input sequence and its secondary structure sequence is predicted.(2) Non-miRNA structures are filtered out based on free energy and number of base pairings .(3) The core method applies several classifier programs to a windowed structure sequence and combines their predictions by voting with threshold.(4) Double matches by overlapping sequence windows are detected and removed.(5) The predicted miRNA candidates are filtered using a stricter threshold, e.g., on the number of successive matches around their genome location .The general motivation to combine multiple predictions is to increase confidence. Here an ensemble of 16 classifiers was trained on known miRNAs in the human genome. This number was necessary to achieve a specificity of 99.9 percent if all classifiers have to agree on a positive decision of the ensemble. A low false positive rate is preferable to a low false negative rate, when scanning wider genome regions for new miRNAs. A higher proportion of candidate hairpins, instead, might come along with too many false positive predictions.fixed-length example sequences (see Methods).Machine learning methods derive classification models or rules from known positive and negative examples. Our predictors were trained and are applied on The positive set is composed of all 474 human miRNA hairpin sequences reported in miRBase (release 9.0) ,21. All For the negative examples, we first select 20,000 random locations in the human genome. At each position we shift a 100 nt wide window 5 times in sense direction – using a constant step size of 5 nt – and extract the subsequence of each window. The resulting 100,000 fixed-length and partly overlapping sequences simulate the input situation for the predictors when applied for scanning genome regions. A higher number of negative samples is needed because of the higher diversity of non-miRNA structures. Moreover, it is relatively unlikely to include a significant proportion of real miRNAs in this way, also because of their assumed low frequency in the genome.The RNA secondary structure (stem-loop sequence) of all extracted nucleotide sequences is predicted using RNAfold at dBoth data sets are prefiltered to rule out structures which are quite likely non-miRNAs. A candidate structure must meet three relatively weak conditions: (1) minimum 18 base pairings in the stem arm of the hairpin structure including the wobble pair G-U; (2) maximum -15 kcal/mol free energy. These thresholds derive from the lowest number of base pairing and the highest free energy found among known human pre-miRNAs. (3) Both conditions must be met by the structure of the central 70 nt sequence in the scanning window (100 nt). This is approximately the size and location of the miRNA precursor, ignoring the 15 nt extensions from the primary sequence. In doing so, only 5 (~1 percent) of the positive examples (human miRNAs) but 79,097 (~80 percent) negative examples are excluded.Note that prefiltering is not essential for the application of our motif-based GP classifiers. Actually, we found that it affects the final set of predicted candidates only slightly. That is, the classifiers have learned to reject the prefiltered structures even though they have not been exposed to such during training. Nevertheless, prefiltering is important for a reduction and better selection of the training examples. Different arguments may be found for using a scanning window of 100 nt: (1) The ~70 nt long pre-miRNA sequence may not always be perfectly aligned in the window center. (2) Short single-stranded extensions from the primary transcript flanking both ends of the precursor are argued to carry relevant structural information for Drosha processing ,25. The The motif-based genetic programs (see Methods) act as predictors that return a binary decision about whether an input sequence/structure contains a potential pre-miRNA or not. Predictions from multiple classifiers (16 here) are combined by voting with threshold, i.e., the minimum number of individual decisions required for an overall positive decision. Higher thresholds yield fewer structures predicted as miRNAs, but with a higher confidence.The ROC curve in Figure When searching wider genome regions for new miRNAs, however, a higher specificity is desirable to keep the hit rate and number of potentially false positives low. Voting with maximum threshold (16/16) requires that all individual classifiers must vote "yes" and achieves a specificity of above 99.9 percent. This results in 87 (140) positive predictions out of 100,000 negative structures, excluding (including) multiple matches of directly successive windows. The small increase in specificity is paid by a more significant drop in sensitivity to around 82 percent here. A certain amount of missed true positives is acceptable, if new miRNAs are predicted with a higher confidence instead.The performance of the individual classifiers (1/1) is at around 99.1 percent specificity and 95 percent sensitivity. However, this also means an almost 10 times higher rate of positive predictions.In a separate experiment five-fold cross-validation was applied to verify the performance of the classifier programs. Because GP is a probabilistic method, 5 independent GP runs were performed for each of the 5 positive training sets and the best program on each set was selected. The negative training sets were selected as described in Methods. On average, sensitivity remains at 95 percent over all human miRNAs and drops down to 90 percent on the respective set of miRNAs left out for testing. Specificity remains at an average of 99.1 percent over the 100,000 negative test examples.ab initio methods, including both pioneering approaches [et al. [Both values – specificity and sensitivity – compare well to what has been previously reported for other comparable proaches ,16 and sproaches ,18,27,28 [et al. ). These MiRNAs of the same family are given names with the same (integer) number but different types of suffixes that relate to the similarity of the mature sequence . NumericTo validate our ensemble classifier on different sets of positive examples, we extracted fixed-length windows centered around known miRNA sequences in mouse and rat, as described above for human.There are 363 (228) mouse (rat) miRNA hairpins reported in miRBase 9.0 – with known genome location and without double sequences. We exclude 54 (42) which have an identical precursor in human. These have been found by matching the central 70 nt – the approximate precursor sequence – against the full human windows.Our method correctly predicts 74 (81) percent of all miRNAs in mouse (rat) with the highest confidence, i.e., 100 percent agreement between all classifiers. If minimum 50 percent agreement is required for a positive decision the classification rate increases to 91 (98) percent. Only 97 (22) mouse (rat) miRNAs have no member of the same family in human, due to the high sequence conservation of (known) miRNAs. 71 (76) percent of these are found with maximum voting threshold and 87 (100) percent with majority voting. This also demonstrates that our classifiers while trained on human data are able to generalize to miRNAs of other species.In addition, we test the performance of our combined classifier on 60 new human miRNA sequences in miRBase 10.0 of which 44 are founding members of new miRNA families. From the first set, 73 percent are found with maximum confidence and 90 percent with confidence ≥50 percent. For the second set, these numbers drop to 68 and 86, respectively.Comparing the new structure sequences by their string edit distance reveals Most miRNA genes are located in DNA regions which previously have been considered as non-coding regions, including intergenic regions and introns. Especially intronic miRNAs seem to be much more frequent than previously thought . Many miAs a more realistic test scenario, we scan regions on chromosome 19 which has the highest number of validated miRNAs among all human chromosomes. The 66 miRNAs are grouped into 16 clusters with a minimum distance of 20 kilobases. The 100 nt wide scanning window is moved in 5 nt steps, starting 10 kb upstream of each cluster and ending 10 kb downstream.The prefilter already identifies 56,142 (63 percent) from the total 88,808 structure sequences as negatives. The combined classifier finds 295 positive predictions (with 100 percent agreement) which is about twice as many as for random locations used above, indicating a higher number of true positive miRNAs. After removing double matches by overlapping windows these reduce to 173, of which 56 are rediscovered known miRNAs (85 percent of 66). The remaining 10 were not filtered out beforehand and all lie on the sense strand (which is scanned). This leaves 117 candidates as potentially new miRNAs. × 88, 808/100, 000 = 77 are likely false positives, while the remaining 173 - 77 = 96 are potentially real. Excluding the found known miRNAs leaves around 40 to be true positives.We can use the results for the random locations – which are more likely true negatives – to estimate the number of real miRNAs out of all positive matches here. Of the 173 hits approximately 87Because it would be counterintuitive to scan regions around known miRNAs without using their information for finding new ones, miRNAs on chromosome 19 are part of the training set here. For comparison reasons, we trained another ensemble classifier without using these 66 miRNAs. Its sensitivity is only a little lower on chromosome 19 (52/66) and slightly better on all human miRNAs. One has to note here that neither the composition nor the size of the ensembles are particularly optimized.Additional criteria may be applied on the positive predictions to obtain less miRNA candidates, that are more likely true. We focus on structural aspects again, including the free energy of matching structures and the number of directly successive matches of the scanning window.MiRNA structures are known to have lower free energy, i.e., to be more stable, than other non-coding or random structures . Figure left) shows the frequency distribution over the number of successive matches by the sequence window, separately for the 117 unknown hits and the 56 known hits. Only full matches are counted, meaning that the corresponding structure must be predicted positively by all 16 classifiers. Known (true) miRNA locations match more frequently during scanning than unknown locations . More than 60 percent of the unknown candidates match only once, while this is true for only around 20 percent of the known candidates. On the other hand, around 40 percent known ones, but only 5 percent unknown ones match three times or more. This shows that miRNA structures are more independent from their position in the scanning window than other structures and is another indicator for their above-average stability, besides a lower free energy.The number of directly successive sequence windows is measured in two different ways. Figure Performance on other miRNA data sets). That is, clearly more miRNAs are predicted with at least three matches than with only one match.Here we only note that we found similar distributions of successive matches (1) over the 52 miRNAs redetected by the ensemble not trained on miRNAs from chromosome 19 and (2) when scanning over the 60 new miRNAs in miRBase 10.0 (see Section right) where the distributions also include partial matches by any number of classifiers. Depending on the proportion of positive predictions, full matches count 1 and partial matches < 1. These values are summed up from three window shifts before to three window shifts after a full match or a series of full matches. According to this distribution, 40 percent of the unknown predictions fall below 3 counts, but less than 5 percent of the known ones do.A more detailed picture is given in Figure How similar are the different measures of structural stability? Figure One may derive different thresholds from these distributions to further select the 117 unknown predicted structures. For instance, a higher maximum free energy threshold of -27 would leave only 71 candidates. When applying an additional minimum threshold of 3 on the number of matches (second measure) the number of candidates reduces to 47. This is about the number of true positives estimated in the previous section. Also note that both thresholds are still passed by the vast majority (45 from 56) of known predicted miRNAs on chromosome 19.A necessary precondition for a miRNA candidate to be true is that its genomic location is transcribed. A large number of transcripts is known not to be translated into proteins, but not all of these are necessarily functional. Non-coding RNAs not only occur between (protein-)coding genes, but often overlap with coding regions on the same or the opposite strand .Expressed Sequence Tags (ESTs) are relatively short (about 300–600 nt) and inaccurate (around 2 percent errors) subsequences of transcribed and spliced cDNA, synthesized from mature mRNA. More than 8 million human ESTs are now available in the GenBank dbEST database (release 062207) [Alignment of nucleotide sequences to an EST library offers one way to identify coding and non-coding genes. 062207) . This liWe use BLAST with staMicroRNA identification during genome scanning) to the EST library. If an EST alignment is split up into several discontiguous segments, we extract the whole sequence between the two outermost alignments, provided that these are longer than 60 nt. Then the intermediate regions are most likely introns which have been spliced out. A miRNA candidate is identified as being intronic, if it aligns to an extended EST, but not to the original (spliced) EST. In total we found 35 percent (41/117) of the candidates to match EST-defined introns and more than 75 percent ((48 + 41) / 117) to be transcribed. By comparison, only 12 out of the 66 known miRNAs on chromosome 19 match ESTs while 44 match introns.To find out how many candidate sequences lie in introns, we first align all sequence regions that have been scanned on human chromosome 19 is not enough to capture all information held in a miRNA precursor and matches the structure sequence only in parts . Figure The terminal loop region (approximately in the window center) is matched clearly less frequently than the stem regions. In fact, only few expressions match over the full hairpin loop and even fewer contain subexpressions which determine loop sizes precisely. The matching frequency is even lower for the flanking sequences (~15 nt from each window end). Apparently, these regions are at least less important for finding characteristic structure motifs. This result is in line with the most distinctive features of miRNA precursors and predicted non-miRNA structures derived from secondary structure clustering in , which aOne explanation may be found in the conservation profile over miRNA precursor sequences which is saddle-like ,13 and rab initio method for miRNA discovery in human that is purely based on finding distinctive motifs in predicted RNA secondary structure. In doing so, we favored a specificity-sensitivity tradeoff that is shifted towards higher specificity. Combining the predictions from multiple classifiers by voting with maximum threshold (100 percent agreement on a positive decision) has turned out to be a simple but effective means to increase confidence in and reduce the number of positive predictions and potentially false positives. 99.9 percent specificity requires an ensemble of 16 classifiers here. Sensitivity is still maintained at a level sufficient for genome scanning applications.We introduced a new Another means to increase confidence is to postfilter the predicted miRNA candidates by imposing additional criteria. The robustness of the secondary structure against the position of the scanning window has been proposed as a structural criterion for miRNA identification. The sliding sequence window is more frequently identified as a match over a miRNA location than over a non-miRNA location.Finally, we collected different statistical and empirical evidence to validate the correctness of our predictions. All 117 miRNA candidates structures match the free energy distribution of known miRNA precursors that is significantly shifted towards lower free energies, compared to the distribution of non-miRNA structures. Approximately one third was estimated to be true positives, i.e., real miRNAs or other small RNAs of similar structure. By aligning the candidate sequences to a human EST database, we found that above 75 percent are most likely transcribed and that around 35 percent are located in introns. ab initio miRNA identification. More general advantages over SVM-based methods include:The performance of our motif-based GP approach has been found at least competitive to presently existing methods for (1) Less assumptions are made about how a pre-miRNA structure has to look like. The patterns (motifs) used for classification are not predefined, but are automatically derived from positive and negative examples during training (see Methods). SVMs, instead, requires certain "rules" to be set up which determine how a structure sequence is transformed into a feature vector.(2) A preselection of candidate hairpins is not required since regular expressions are relatively independent from the absolute position of matching substructures. SVM classification, instead, requires hairpin-like structures to measure certain quality features.(3) Because GP is a probabilistic method, a gain in performance is possible by combining multiple classifiers even if these have been trained on the same data. Deterministic SVMs only produce a single solution in this case.(4) Our motif-based programs give a more direct and unbiased insight into what rules have been learned. Mostly the stem region of hairpin structures is used for miRNA prediction, in contrast to loop region and flanking sequences. A more detailed analysis of programs and structure motifs is a subject of our ongoing research.ab initio miRNA finding, by definition, does not apply homology search or conservation. For instance, we did not prefilter the input sequences by scanning only regions conserved between human and, e.g., mouse or rat. Only 4 (3) of the potential miRNA sequences found in human are highly conserved in mouse (rat), i.e., with above 90 percent sequence identity over an alignment of at least 80 nt.As indicated above, In general, it may be doubted whether one computational method alone is sufficient to estimate reliably how many miRNAs actually exist in the human genome. 99.9 percent specificity is, of course, not the end of the story. The number of predictions produced is still large when scanning the complete genome and it may be questioned if these can be all real miRNAs. To find the true positives may still require additional support by other computational and/or experimental approaches, testing other criteria than the precursor structure. Nevertheless, identification methods like the one presented here serve as an important step toward filtering potential pre-miRNA candidates.de novo and are built from maximum 6 characters including parentheses and dots. Each character is followed by a {min, max} quantifier which specifies its allowed number in a structure sequence presented in dot-bracket notation , a machine learning technique that automatically develops imperative computer programs in an artificial evolutionary process . MultiplAlready the probabilistic nature of GP makes that two training runs can produce different results (programs). In order to make the combination of multiple programs more efficient, we use a different negative training set in each run including around 10 times as many structures as there are positive examples (468 human miRNA structures after prefiltering). Such a relation reflects very roughly the distribution in the genome and the much higher variety of non-miRNA structures.Prediction performance is calculated during training as the mean of positive classification error rate and negative classification error rate:P (N) denotes the total number of positive (negative) examples, while TP (TN) is the number of true positive (true negative) predictions.to balance the influence of the differently sized sets. MB designed the method, carried out the experiments, and wrote the manuscript. CW participated in the design of the study and in the statistical analysis. Both authors read and approved the final manuscript.Example of an individual classifier program (in C notation). Each program consists of a matching part with regular expressions and a calculation part. Continuous output is mapped to Boolean value (yes/no decision). Unused regular expressions and code lines not shown.Click here for file |
Laparoscopic nephrectomy has assumed a central role in the management of benign and malignant kidney diseases. While laparoscopy is less morbid than open surgery, it still requires several incisions each at least 1-2 cm in length. Each incision carries morbidity risks of bleeding, hernia and/or internal organ damage, and incrementally decreases cosmesis. An alternative to conventional laparoscopy is single access or keyhole surgery, which utilizes magnetic anchoring and guidance system (MAGS) technology or articulating laparoscopic instruments. These technical innovations obviate the need to externally space trocars for triangulation, thus allowing for the creation of a small, solitary portal of entry into the abdomen. Laboratory and early clinical series demonstrate feasibility as well as safe and successful completion of keyhole nephrectomy. Future work is necessary to improve existing instrumentation, increase clinical experience, assess benefits of this surgical approach, and explore other potential applications for this technique. Open nephrectomy has historically been the gold standard therapy for the management of benign and malignant kidney disease. Despite evolution of open kidney surgery, considerable morbidity and delayed convalescence occurs from the muscle splitting flank incision. Since the first laparoscopic nephrectomy by Clayman and colleagues in 1991, minimally invasive urologic surgery has gained significant momentum.3 While t47Natural orifice transluminal endoscopic surgery (NOTES) has been described as the next surgical frontier with the objective of incision-free abdominal surgery. NOTES approaches abdominal surgery through natural orifices thus obviating external abdominal scars. Contemporary laboratory investigation is investigating the infectious and immunologic implications of NOTES. Indeed, the concept of a purposeful viscerotomy either using the gastric or vaginal route raises concerns of intrabdominal contamination. The immunologic impacts, however, may actually be favorable for NOTES surgery. McGee and colleagues demonstrated lower levels of tumor necrosis factor-α (TNF-α) after NOTES peritoneoscopy compared to conventional laparoscopic exploration. Hence, Net al. presented their experience with single-port NOTES transvaginal nephrectomy and encountered similar difficulty until a purpose-built multilumen operating platform was utilized.[Animal models have been used to demonstrate the potential applications of NOTES, including transgastric and transvesical peritoneoscopy, transvaginal tubal ligation, hysterectomy, and cholecystectomy. Preliminutilized. Even witPotential drawbacks to NOTES nephrectomy are not insignificant. Operative duration is longer than conventional laparoscopy, specialized equipment is necessary, and there is a steep learning curve.Triangulation is one of the fundamental concepts of conventional laparoscopic surgery. An alternative to conventional laparoscopy and NOTES is single access or keyhole surgery utilizing a magnetically anchored guidance system, articulating laparoscopic instruments, and/or specialized trocars.et al. have subsequently described the first successful completion of two nonsurvival porcine nephrectomies via a single 15 mm transumbilical trocar using a prototype MAGS camera and a magnetically anchored robotic arm cauterizer.[Park and colleagues have recently developed a novel adjunct laparoscopic system consisting of a moveable, “lockable” platform that is positioned intraabdominally and stabilized by an external permanent magnet on the abdominal skin. MAGS canPrior to widespread adoption of the MAGS platform, both clinical and engineering limitations must be addressed. Surgeons must become familiar with the MAGS components both in a dry laboratory and in animal models. As with all new technology, there will be a learning curve and it will be incumbent on surgeons to develop new “MAGS techniques” by modifying traditional laparoscopic modalities. The coupling strength of magnetics (electromagnetic or permanent magnets) decreases as a decaying exponential with respect to the distance between the source magnet and its target. Currently, tissue thicknesses in excess of 1.5 cm limit the effectiveness of the paddle retractor, while the camera can be supported up to tissue thicknesses of 2.5 cm. As such, present day clinical utilization of MAGS technology would be restricted to thin or pediatric patients. Future directions are needed to develop electromagnets capable of generating stronger magnetic fields. Finally, additional work is needed to develop a more robust MAGS camera system. Current laboratory work has been limited by fogging of the camera and a lack of sufficient lighting (despite on-board LED). Some cases have required laparoscope and flexible endoscope assistance for visualization. Purpose-built modifications in camera design are necessary to obviate the need for additional lighting sources.MAGS technology is still currently in evolution and is not commercially available for clinical uses.An alternative to MAGS for single access surgery involves using articulating instrumentation via a single large caliber trocar or small, adjacent trocars. Advances in technology have led to the development of new laparoscopic access ports capable of allowing multiple instruments to be inserted through different cannulas of a single port. Alternatively, adjacent 5 mm trocars can be utilized with skin incisions connected at the time of specimen extraction . The latArticulating instrumentation allows for triangulation to occur intracorporeally despite trocars being adjacent to one another through the same skin incision. Currently, articulating laparoscopic graspers , endoshears (Cambridge Endo), and laparoscopic needle drivers (Cambridge Endo) are commercially available for clinical use. Optimal use of instrumentation requires crossing intracorporeally such that tissue manipulation, traction, and cautery are performed with the contralateral hand compared with conventional laparoscopy. Such differences and collision of instrumentation creates an inherent learning curve during initial procedures; though, this curve is significantly less steep than for NOTES surgery.In conjunction with articulating instrumentation, the development of novel intrabdominal retractors will further facilitate evolution of laparoscopic procedures. One such device is the padron endoscopic exposing retractor (PEER) which can be deployed intracorporeally through a 5- or 10-mm port. Adequate and stable positioning of the intrabdominal retractor provides excellent and secure visualization of the operative field during laparoscopic procedures.Another important component is the selection of an appropriate laparoscope to optimize visualization while minimizing collision with working instruments. Anecdotally, we have found that laparoscopes using right angle light sources to be problematic due to collision with working instruments. More recently, we have used 45° 5-mm rigid laparoscope with an end light source or a 5-mm deflectable tip video laparoscope .Early laboratory and clinical experience with single access umbilical nephrectomy with articulating instrumentation is promising. Raman and colleagues recently reported their initial experience with keyhole nephrectomy in a porcine model and in human subjects. In theirThe attractiveness of keyhole umbilical nephrectomy is multifaceted. First, it improves cosmesis by allowing for a single umbilical incision. Second, it is within a surgeon's comfort range since specimen extraction occurs via the abdomen. This may be a significant consideration as vaginal or gastric incisions may present complications. Third, the learning curve appears to be much shorter than for NOTES. This is attributable to instrumentation that is similar to conventional laparoscopic devices. Finally, keyhole umbilical surgery provides a “familiar” anatomical view of the kidney which may be lost during the evolution of transvaginal, transgastric, or transcolonic surgery.Although the early experience for keyhole umbilical surgery is promising, experienced laparoscopic skill is essential for the safe and effective completion of the procedure. As such, coordination between the surgeon and the camera driver is essential. Single port umbilical surgery does permit the introduction of other transabdominal conventional laparoscopic ports to aid completion of the surgical procedure if failure of progression occurs.Future work with keyhole umbilical surgery is multifaceted. Evolution of MAGS technology and articulating instrumentation are necessary to improve the ergonomics and visualization of the surgical procedure. Proponents of single access surgery suggest that in addition to benefits in cosmesis, there is the possibility of less perioperative pain and morbidity. Comparison of short-term measures of convalescence to that of NOTES and traditional laparoscopic surgery are needed to better address this issue. To date, in addition to our experience with keyhole nephrectomy, we have also completed three single access pyeloplasties and a single access adrenalecomy. Other gr20Single access umbilical nephrectomy is feasible. Using varied instrumentation and technology, several groups have demonstrated safe and successful completion in both a porcine model and in human patients. Future work will need to assess benefits of keyhole surgery and explore other potential applications for this novel approach. |
Classical non-insulin antihyperglycemic drugs currently approved for the treatment of type 2 diabetes mellitus (T2DM) comprise five groups: biguanides, sulfonylureas, meglitinides, glitazones and alpha-glucosidase inhibitors. Novel compounds are represented by the incretin mimetic drugs like glucagon like peptide-1 (GLP-1), the dipeptidyl peptidase 4 (DPP-4) inhibitors, dual peroxisome proliferator-activated receptors (PPAR) agonists (glitazars) and amylin mimetic drugs. We review the cardiovascular effects of these drugs in an attempt to improve knowledge regarding their potential risks when treating T2DM in cardiac patients. Metformin may lead to lethal lactic acidosis, especially in patients with clinical conditions that predispose to this complication, such as recent myocardial infarction, heart or renal failure. Sulfonylureas exert their effect by closing the ATP-dependent potassium channels. This prevents the opening of these channels during myocardial ischemia, impeding the necessary hyperpolarization that protects the cell. The combined sulfonylurea/metformin therapy reveals additive effects on mortality in patients with coronary artery disease (CAD). Meglitinides effects are similar to those of sulfonylureas, due to their almost analogous mechanism of action. Glitazones lower leptin levels, leading to weight gain and are unsafe in NYHA class III or IV. The long-term effects of alpha-glucosidase inhibitors on morbidity and mortality rates is yet unknown. The incretin GLP-1 is associated with reductions in body weight and appears to present positive inotropic effects. DPP-4 inhibitors influences on the cardiovascular system seem to be neutral and patients do not gain weight. The future of glitazars is presently uncertain following concerns about their safety. The amylin mimetic drug paramlintide, while a satisfactory adjuvant medication in insulin-dependent diabetes, is unlikely to play a major role in the management of T2DM.Summarizing the present information it can be stated that 1. Four out the five classical oral antidiabetic drug groups present proven or potential cardiac hazards; 2. These hazards are not mere 'side effects', but biochemical phenomena which are deeply rooted in the drugs' mechanism of action; 3. Current data indicate that the combined glibenclamide/metformin therapy seems to present special risk and should be avoided in the long-term management of T2DM with proven CAD; 4. Glitazones should be avoided in patients with overt heart failure; 5, The novel incretin mimetic drugs and DPP-4 inhibitors – while usually inadequate as monotherapy – appear to be satisfactory adjuvant drugs due to the lack of known undesirable cardiovascular effects; 6. Customized antihyperglycemic pharmacological approaches should be implemented for the achievement of optimal treatment of T2DM patients with heart disease. In this context, it should be carefully taken into consideration whether the leading clinical status is CAD or heart failure. Diabetes mellitus threatens to become a global health crisis; treating diabetes and its complications is going to dominate future health care expenditures. Type 2 diabetes mellitus (T2DM) accounts for about 90% of the total diabetic population, and coronary artery disease (CAD) is the most common cause of morbidity and mortality. Cardiovascular deaths are increased up to fourfold in diabetics compared with their nondiabetic counterparts . More thWhen oral antidiabetic monotherapy does not achieve the glycemic goal, combination treatment is implemented. A sulfonylurea – usually glibenclamide – plus metformin constitute the most widely used antihyperglycemic combination in clinical practice . Howeverper se may lead to a further derangement of cardiovascular status. Five types of classical oral antihperglycemic drugs are currently approved for the treatment of diabetes: biguanides, sulfonylureas, meglitinides, glitazones and alpha-glucosidase inhibitors. The novel antihyperglycemic compounds are represented by the incretin mimetic drugs, the dipeptidyl peptidase (DPP-4) inhibitors, the dual peroxisome proliferator-activated receptors (PPAR) agonists (glitazars) and the amylin mimetic drugs.Insulin resistance represents the background of a series of common factors for the development of both diabetes and heart disease. These factors include genetics, hypertension, obesity, hyperglycemia, dyslipidemia, prothrombotic state, aging, physical inactivity. Once both diseases are clinically established, antidiabetic therapy We will briefly review the cardiovascular effects of the most commonly used antidiabetic drugs within these types, in an attempt to improve knowledge and awareness regarding their influences and potential risks when treating cardiac patients.The biguanides were launched in the 1950's, and metformin is the only drug belonging to this class currently available in most parts of the world. It reduces blood glucose levels through suppression of gluconeogenesis, stimulation of peripheral glucose uptake by tissue in the presence of insulin, and decreased absorption of glucose from the gastrointestinal tract. It has no direct effects on beta-cells, does not produce hypoglycemia, reduces glycohemoglobin (HgA1c) and improves both blood lipid profile and fibrinolytic activity. In contrast to other antidiabetic medications, metformin does not cause weight gain and appears to be the drug of choice in obese patients.Despite these beneficial effects, metformin presents potential disadvantages that may influence the cardiovascular system. Gastrointestinal disturbances such as diarrhea are frequent, and the intestinal absorption of group B vitamins and folate is impaired during chronic therapy . This deThese compounds are available nearly half a century. Today, sulfonylureas continue to represent a mainstay of therapy in patients with T2DM; their hypoglycemic potency is directly related to baseline plasma glucose values . At the It is important to stress that not all the undesirable effects on cardiovascular outcome reported by the UGDP for the first-generation sulfonylureas such as tolbutamide can be aMeglitinides are insulin secretagogues. The first drug of this group, repaglinide, a benzoic acid derivative, was introduced in the USA in 1998. The second, nateglinide, is a d-phenylalanine derivative. Like sulfonylureas, these compounds act by closing the ATP-dependent potassium channels. However, its mechanism of action seem to be more complex since possibly three meglitinide receptor binding sites have been found on the beta-cells .Despite a common basic mechanism of action, the insulinotropic effects of the two approved agents can be differently influenced by ambient glucose, leading to dissimilar responsiveness. Nateglinide may exert a more physiologic effect on insulin secretion – i.e. a glycemia-dependent response – than repaglinide, presenting less propensity to elicit hypoglycemia in vivo . On the The cardiovascular safety of these insulin secretagogues is still uncertain. Increased morbidity, particularly acute ischemic events, was observed for repaglinide after 1 year compared with glibenclamide. Nevertheless, patients on repaglinide appeared to have had more severe CAD at baseline than those in the glibenclamide group, and when adjustments were made the relative risk declined . Thus, wRegarding nateglinide, it appears to have less affinity for the potassium channels than repaglinide and it iThis group of drugs, called also thiazolidinediones, was introduced in 1997 and includes antidiabetic medications such as troglitazone, pioglitazone, and rosiglitazone, the chemical structure and mechanism of action of which are very different from those of the other groups. Chemically, they are thiazolidinediones having chroman moieties; some of the analogues may present an aminoalkyl group as a linker between the chroman ring and the phenoxy moiety. Troglitazone, which was the first agent in this class to receive labeling approval, was withdrawn from clinical use in the US due to hepatotoxicity .These drugs are insulin sensitizers, and they bind to the PPAR gamma, leading to increased glucose transporter expression. Sensitivity to insulin – especially in adipocytes, muscle and liver – is improved, and an additional major effect is the inhibition of hepatic gluconeogenesis . It shouRosilitazone monotherapy is only modestly effective in reducing glucose and HgA1c levels. Plasma triglycerides are reduced by 10–20%, and high density lipoprotein (HDL) cholesterol levels increase by 5–10%, since it also stimulates the isoform PPAR-alpha that regulates lipid metabolism. These favorable effects are counterbalanced by a 10–15% increase in low density lipoprotein (LDL) cholesterol . Edema hIt was stressed that rosiglitazone reduces urinary albumin excretion in T2DM and may even mildly reduce blood pressure . NontradPioglitazone treatment in patients with advanced T2DM at high risk for cardiovascular events participating in the PROactive Study, yielded significant risk reductions in major adverse events composite end points at 3 years ,42. The Thus, glitazones exhibit a broad landscape of complex clinical effects, in part favorable and in part detrimental for the cardiovascular system. The concluding balance between these effects requires further elucidation. In this context, it should be mentioned that only few head-to-head comparisons of drug regimens containing glitazones versus drug regimens containing insulin were found . This leThe primary mechanism of action of antidiabetic drugs like acarbose, voglibose and miglitol is grounded on competitive inhibition of several enzymes of the alpha-glucosidase group . These are membrane-bound enzymes that hydrolyze oligosaccharides and disaccharides to glucose in the brush border of the small intestine Thus, by delaying digestion of carbohydrates, these compounds shift their absorption to more distal parts of the small intestine and colon, and defer gastrointestinal absorption of glucose. Their hypoglycemic potency is less than that of biguanides and sulfonylureas and, unlIt is well established that impaired fasting glucose concentrations in nondiabetic patients with ischemic heart disease are a marker for a worse prognosis ,47. AcarInterestingly, and additional metabolic pathway for these compounds was recently described. Chronic treatment with voglibose stimulates GLP-1 secretion and decreases plasma DPP-4 activity by reducing its circulating levels ,49. SimiCombined therapy is based on the premise that pharmacological agents acting via different mechanisms and presenting differing side effects permit the design of individualized antidiabetic regimens. This approach reflects the plausibility that monotherapy with any currently available medication is likely to fail over time in some patients, and this type of pharmacological diabetes management is widely used. Findings from the United Kingdom Prospective Diabetes Study (UKPDS) showed that after 3 years, approximately 50% of patients could attain satisfactory glucose levels with monotherapy; by 9 years this had declined to only 25% . Long-teHence, the combined antihyperglycemic treatment with classical drugs leads to a peculiar entanglement since sulfonylureas and metformin are 1) the most powerful antiabetic drugs; 2) those presenting the most unfavorable cardiac effects; 3) the most frequently employed combination in routine clinical practice ,57.Taking into consideration the intimate interrelationship between diabetes and heart disease, the American Heart Association has coined in 1999 the phrase 'diabetes is a cardiovascular disease' . DiabetiThe progressive impairment of beta cell function and increased insulin demand as tissue becomes insulin resistant are core pathophysiologic defects in the development of hyperglycemia in T2DM ,60. AnywThus, incretin mimetic drugs are nowadays extensively investigated. A key role for intestinal peptides in the regulation of postprandial insulin secretion and glucose levels was proposed, based on the observation that insulin responses to an oral glucose load exceeded those measured after intravenous administration of an equivalent amount of glucose . This phPhysiological actions of incretins were extensively defined in animal studies with exogenous GLP-1 and GLP-1 receptor antagonists, highlighting its role as a meal-stimulated factor with potent glucose-lowering activity. Of significant clinical relevance is that exogenous GLP-1 has the potential to normalize fasting plasma glucose concentrations in patients with T2DM. In several studies in subjects with diabetes, GLP-1- whether administered by intravenous or subcutaneous infusion – normalized both fasting and postprandial glycemia by enhancing glucose-mediated insulin secretion, as well as by suppressing glucagon secretion -69.Additional studies in animals and humans have demonstrated glucose-lowering effects of GLP-1. GLP-1 slows gastric emptying to decrease the rate of nutrient absorption, which results in more synchronous nutrient delivery with endogenous insulin action. Significant acute reductions in appetite and food intake after intravenous administration of GLP-1 in both healthy individuals and in patients with T2DM have also been demonstrated -72.The mechanism through which the incretin hormones elicit their cytoprotective effects on the beta cell has attracted significant attention because preservation and restoration of beta-cell mass may contribute to the therapeutic potential of the incretins for the treatment of both type 1 and T2DM. Endoplasmic reticulum stress within the beta cell, possibly occurring as the result of the overproduction or misfolding of insulin, may be a contributing factor to the increased beta-cell apoptosis and loss of islet mass observed in diabetic patients .Exenatide was first in the new class of incretin mimetics for the treatment of patients with T2DM. Several short-term phase 2 clinical trials have reported that subcutaneos exenatide acutely lowered both fasting and postprandial plasma glucose concentrations. The rate of gastric emptying was also slowed in patients treated with exenatide. Large-scale clinical trials designed to assess the safety and efficacy of twice daily subcutaneos exenatide over a six-month period were completed in subjects with T2DM who were unable to attain glycemic control with oral sulfonylureas, metformin, or both . A novelper se as a therapeutic agent. However, prolonged activity is achieved by chemical manipulation, reaching to 11–15 hours and making it suitable for once-daily dosing. This is attained by attaching a fatty acid molecule at one position of the GLP-1 molecule, enabling it to bind to albumin within the subcutaneous tissue and bloodstream. The active GLP-1 is then released from albumin at a slow, consistent rate. Binding with albumin also results in slower degradation and reduced elimination of liraglutide from the circulation by the kidneys compared to its natural form [Another incretin mimetic compound, liraglutide, is a once-daily GLP-1 derivative in development for the treatment of T2DM. GLP-1, in its natural form, is short-lived in the body , so it is not very useful ral form ,78. A reral form .Additional long-acting incretin mimetic drugs, like albiglutide and taspoglutide are currently under investigation, presenting encouraging results ,81.X-proline or X-alanine. It is a complex molecule that exists as a membrane-spanning cell-anchored protein that is expressed on many cell types, and as a soluble form in the circulation; both forms have proteolytic activity.While discovered in 1967, serine protease DPP-4 was only subject of intensive research during recent years. DPP-IV is ubiquitously expressed and exhibits postproline or alanine peptidase activity, thereby generating biologically inactive peptides via cleavage at the N-terminal region after Because both GLP-1 and GIP have an alanine residue at position 2, they are substrates for DPP-4. DPP-4 inhibitors like sitagliptin are orally administered drugs that improve glycemic control by preventing the rapid degradation of incretin hormones, thereby resulting in postprandial increases in levels of biologically active intact GLP-1 and GIP ,83.Sitagliptin is an orally-bioavailable selective DPP-4 inhibitor – the first one approved by the FDA – that was discovered through the optimization of a class of beta-aminoacid-derived DPP-4 inhibitors. It lowers DPP-4 activity in a sustained manner following once daily administration, preserves the circulating levels of intact GIP and GLP1 following meals in both acute and chronic studies and reduces blood glucose levels without significant increases in hypoglycemia . Thus, tSeveral additional DPP4 inhibitors, like saxagliptin, vildagliptin and alogliptin are currently investigated. Vildagliptin is the second DPP-4 inhibitor approved in Europe. Similarly to sitagliptin, vildagliptin has pharmacokinetic properties that support a once daily dosing regimen. Alogliptin in combination with pioglitazone, in an experimental model, improves glycemic control, lipid profiles, and increases pancreatic insulin content . WhereasIn comparison to DPP-4 inhibitors, incretin mimetic agents have more pharmacological specificity but require subcutaneous injections. As with DPP-4 inhibitors, improvements in glycemic control were achieved with either no weight gain or with weight loss. Considering the impact of obesity on diabetes, along with weight gain that generally accompanies the use of insulin, insulin secretagogues, and insulin sensitizers, interventions with favorable effects on weight are likely to become increasingly important.It should be pinpointed that there is a risk of potential adverse effects of DPP-4 inhibitors, especially on the immune system: an increased relative risk of 34% for all-cause infections after sitagliptin treatment was observed. Although, the risk of increased infection appears small, its consequences when translated into clinical practice with millions of T2DM patients treated could be considerable . Thus, wThere are three PPARs subtypes which are commonly designated PPAR alpha, PPAR gamma and PPAR beta/delta. PPAR alpha activation increases HDL cholesterol synthesis, stimulates "reverse" cholesterol transport and reduces triglycerides. PPAR gamma activation results in insulin sensitization and antidiabetic action. Until recently, the biological role of PPAR beta/delta remained unclear. However, treatment of obese animals by specific PPAR delta agonists results in normalization of metabolic parameters and reduction of adiposity. Combined treatments with PPAR gamma and alpha agonists may potentially improve insulin resistance and alleviate atherogenic dyslipidemia, whereas PPAR delta properties may prevent the development of overweight which typically accompanies "pure" PPAR gamma ligands. Clearly, an optimal PPAR agent with improved safety profile that provides both effective glycemic and lipid control is needed. Compounds that affect both PPAR-alpha and PPAR-gamma, particularly with an optimized balance of agonist activity, might prove especially beneficial for patients with T2DM .The old and well known lipid-lowering fibric acid derivative bezafibrate is the first clinically tested pan- PPAR activator. It is the only pan-PPAR activator with more than a quarter of a century of therapeutic experience with a good safety profile. Therefore, bezafibrate could be considered as an "archetype" of a clinically tested pan-PPAR ligand. Bezafibrate leads to considerable raising of HDL cholesterol and reduces triglycerides, improves insulin sensitivity and reduces blood glucose level, significantly lowering the incidence of cardiovascular events and new diabetes in patients with features of the metabolic syndrome. It attenuates the progression of insulin resistance, defers the onset of overt T2DM, enhances adiponectin levels and reduces the incidence of myocardial infarction in patients with metabolic syndrome during long-term follow-up -95. HoweSeveral novel and potent dual PPAR-alpha/gamma agonists (glitazars) have been clinically developed. These agents have a major effect on peripheral and hepatic insulin sensitivity, with HbA1c reductions of 0.5–2%. On the basis of their mode of action, it is expected that these agents could modulate cardiovascular risk by improving endothelial reactivity, reducing blood pressure, and improving lipid profiles . HoweverThe recently reported SYNCHRONY study aimed toAmylin is a synergistic partner to insulin, with which it is cosecreted from pancreatic beta cells in response to meals. Deficient amylin secretion is a well-recognized phenomenon in type 1 diabetes and in a later-stage in T2DM, in whom pancreatic insulin production is markedly reduced. Its physiological effects mimic in part those of GLP-1. Amylin suppresses glucagon – a pancreatic hormone that regulates the production of glucose by the liver – secretion from pancreatic alpha cells, thereby attenuating hepatic glucose production. It also delays gastric emptying and likely possesses a central effect to enhance satiety .Pramlintide is a synthetic hormone for parenteral (subcutaneous) administration, resembling human amylin effects. It reduces the production of glucose by the liver by inhibiting the action of glucagon and diminishes postprandial glucose fluctuations. The drug was approved by the FDA in March 2005. While it seems to be a satisfactory adjuvant medication in insulin-dependent diabetes, it is unlikely to play a major future role in the management of T2DM .While he main physiologic benefits demonstrated from exenatide therapy have been on indexes of glycemic control, cardiovascular effects have also been described. In experimental models, GLP-1 receptors have been demonstrated in cardiac myocytes and in certain regions of the brain that regulate autonomic function . In someRegarding DPP-4 inhibitors, few data are available concerning cardiovascular markers or clinical outcomes. Given the preliminary data they might be considered in individuals with impaired ventricular function. However, no clinical trials using these agents have yet been reported in this or any other group of patients with cardiovascular disease. Glitazars may yield some reduction in blood pressure . The amyOur pharmacological armamentarium is nowadays increasingly complex, offering a wide array of drugs, both as monotherapy or in combination. It is therefore frequently difficult to determine the best therapeutic option for a given patient. A common problem arises when a drug is known to give a prompt and beneficial effect in the short term, but data regarding long-term outcome and safety are either lacking or insufficient. This is particularly true regarding antihyperglycemic drugs in patients with CAD.Comprehensive risk reduction is mandatory for diabetic patients with CAD. General measures should comprise diet, physical activity, complete cessation of smoking, and weight and lipid profile management. However, fewer than 10% of patients achieve acceptable long-term glycemic values with non-pharmacological therapy only . SpecialEvidence is available that long-term maintenance of normal or near-normal glucose levels using pharmacological means is protective in diabetic patients, improving microvascular disease and reducing both morbidity and mortality ,109. Is What should the policy be regarding the widely used sulfonylurea-metformin combined treatment? Following approval of a given therapy for a chronic condition, large prospective, randomized, placebo-controlled trials designed to check its long-term safety and effectiveness require many years to be completed, and sometimes such studies are not performed at all. This is the case with this combined treatment in CAD patients. The data from observational studies available at present indicate increased mortality in patients receiving this therapy -55 suggeBased on current data, stringent guidelines or strategies regarding non-insulin antidiabetic pharmacotherapy in T2DM patients with heart disease cannot yet be outlined. Anyhow, summarizing the available information it can be stated that1. Four out the five classical oral antidiabetic drug groups present proven or potential cardiac hazards.2. These hazards are not mere 'side effects', but biochemical phenomena which are deeply rooted in the drugs' mechanism of action.3. Current data indicate that the combined glibenclamide/metformin therapy seems to present special risk and should be avoided in the long-term management of type 2 diabetics with proven CAD.4. Glitazones should be avoided in patients with overt heart failure.5, The novel incretin mimetic drugs and DPP-4 inhibitors – while usually relatively ineffective as monotherapy – appear to be satisfactory adjuvant drugs due to the lack of known undesirable cardiovascular effects.6. Customized antihyperglycemic pharmacological approaches should be implemented for the achievement of optimal treatment of T2DM with heart disease. In this context, it should be carefully taken into consideration whether the leading clinical status is CAD or heart failure.CAD: coronary artery disease; DPP-4: dipeptidyl peptidase 4; FDA: US Food and Drug Administration; GIP: glucose-dependent insulinotropic polypeptide; GLP-1: glucagon like peptide-1; HbA1c: glycohemoglobin; HDL: high density lipoprotein; LDL: low density lipoprotein; PPAR: peroxisome proliferator-activated receptor; T2DM: type 2 diabetes mellitus; UGDP: University Group Diabetes Program; UKPDS: United Kingdom Prospective Diabetes Study.The authors declare that they have no competing interests.Both authors have equally contributed in the conception and drafting of the manuscript. |
Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ∼10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using For BAC by BAC sequencing, tiling BACs into contiguous linear arrays is usually achieved by high throughput fingerprinting www.barleygenome.org). Under such circumstances, the prime objective is to arrange the contigs into an order that accurately reflects their position along the respective linkage group.At present, genome sequencing projects typically apply “BAC by BAC” approaches Assembly of the genome sequence is facilitated by paired end sequencing to generate larger scaffolds BAC HAPPY MAPPING (BAP mapping) that is designed to overcome these limitations. BAP mapping combines the principles of the BAC landing method HAPloid DNA samples analysed using the PolYmerase chain reaction) mapping uses panels of sheared genomic DNA diluted to 0.7x haploid genome equivalents and generates a map based on the premise that linked pairs of single copy STS markers (HAPPY markers) will co-segregate significantly more frequently than unlinked markers and in a manner that is proportional to their physical proximity Here we describe a new procedure called A. thaliana as an exemplar to validate the BAP mapping approach; a linkage map and a BAC tiling path of a 1.8 Mbp region of chromosome 4 were created. This approach merges for the first time genetic and physical mapping in one pipeline and generates a “genomic” map in an easy and fast procedure at low cost. Our approach bears the potential for universal applications and will play an important role in sequencing, mapping, and assembly of many large genomes for biomedical and agricultural purposes.Here, we used The principle of the BAP mapping method is illustrated in The BAP map is generated in two steps: the first uses a panel created by mixing BAC clones; the second step uses a long range panel comprised of genomic DNA. For the first step, a genomic BAC library is pooled in a three-dimensional manner to creatThe BAC-range panel contains DNA fragments of a mean size of ∼100 Kbp and allows detection of linkage between markers up to ∼80 Kbp apart and so provides fine resolution ordering of STS markers. At the same time, the relatively small size of the BAC clones also is a shortcoming of maps created from this panel: the short span of each member within the pool limits its scope to traverse gaps (>100 Kbp) caused by incomplete marker coverage, by marker clustering, or through the presence of large repetitive regions of DNA . It is tMultiplex Tandem (MT) and High Resolution Melting (HRM) PCR was used to ensure reliable amplification of STS markers from sub-genome template concentrations. This technique first requires a multiplex partial pre-amplification step of the panel with external primers, typically for only 20 thermo-cycles, so that PCR amplification is arrested in the log-linear phase and template concentration ratios are broadly retained A. thaliana We demonstrate the value of BAP mapping for merging linkage and physical maps into one pipeline re-using 40 out of 107 markers from the FCA region of Analysis of all mapping data showed that the mean DNA presentation of the BAC range-panel was 0.67x genomes per aliquot (GPA). The proportion and pattern of aliquots that are positive or negative for all markers reflected the relative assortment of these markers. Eight small linkage groups together containing positive BAC IDs for each typed marker were generated at a very stringent threshold of LOD = 6.0 . The BACIf two adjacent markers are more distant from each other than the size of the BAC clones (∼80 Kbp), they will segregate independently. Since the BAC library E. coli DNA. (ii) By converting the BAC landing data into a physical linkage map, already existing sources of mapped STS markers may be used to accelerate BAP mapping. Conversion of the existing BAC landing data into physical linkage map bears the potential to support ongoing genome physical mapping and sequencing projects , wheat (http://www.wheatgenome.org/), potato (http://www.potatogenome.net/), etc…) by accelerating the joining of small contigs. Based on the list of positive BAC IDs for 40 STS markers . The aliquots not containing any positive BAC clone are scored as negative ( = 0). Analysis of the scoring results yielded 8 BAC linkage contigs, each with a marker order fitting perfectly to the previous BAP mapping data.One motivation behind our development and evaluation of BAP mapping was to convert existing BAC landing data into physical linkage mapping data. Integrating BAC landing data into the BAP mapping concept offers several advantages: (i) For species with large genomes, it is easier and faster to land STS markers by screening a BAC library than to work with a BAC-range panel. For instance, 290 to 300 384-well plates of a genomic BAC library with 100 kb average insert size are needed to be pooled together to get aliquots of 0.7 fold of the 16,000 Mbp wheat genome. It could be difficult to get reliable amplification of STS markers when using such BACs directly pooled from glycerol stocks due to excessive dilution of the template sequence(s) by markers , we geneWe have demonstrated that BAP mapping is a viable strategy for merging linkage and physical maps into one pipeline. The approach has several advantages over other mapping methods .Mimicking recombination between genetic loci by creating artificial recombination points, BAP mapping has the potential to yield a high resolution physical map for entire chromosomes, without the limitation of insufficient supply of polymorphic loci. This strategy enables one to make a physical linkage map and BAC contigs even for chromosome regions of suppressed recombination.Unlike conventional mapping technologies that generate physical maps by anchoring genetic maps to contig maps through BAC landing Arabidopsis thaliana, although the application of more markers to the same pools could equally be used (or re-used) to generate genome-wide and/or more intensive genome coverage. Thus, we have demonstrated that BAP mapping is a simple technology with universal applications. A physical linkage map of Mbp range can be constructed with a resolution in the Kbp range and the capability to bridge ∼2 Mbp gaps that might arise by tandem repeats or other un-cloneable genomic sequences. The flexibility of the BAP mapping allows conversion of BAC landing data into BAC linkage contigs, utilizable for ongoing physical mapping and sequencing projects, especially for organisms with a large genome size. The 1.8 Mbp high resolution physical linkage map reported here for A. thaliana chromosome 4 demonstrated the potential of BAP mapping as a powerful tool in genomic map construction and sequence assembly.In addition to a high resolution based on the average BAC insert size, the BAP mapping method has the advantage of generating extended maps as the Mb-sized genomic DNA panel can bridge the gaps between BAC contigs. This ability is important for physical mapping and sequencing projects especially for large genomes where numerous blocks of highly repetitive sequences cause gaps in the maps due to the absence of unique markers. By mapping markers at both ends of the short linkage contigs, the order and orientation of these contigs are rapidly assembled in the long range physical linkage maps. In our study, the long range DNA panel of ∼500 Kbp was sufficient to bridge and assemble 8 BAC-range linkage contigs into a complete 1.8 Mbp physical linkage map with high resolution. Previous work using HAPPY mapping We used the Arabidopsis BAC library Mi/P1 A. thaliana plants by grinding 2 g of leaf material into a fine powder in liquid nitrogen before transfering into 200 mL of ice-cold wash buffer Polyvinylpyrrolidone-40; 0.5 M sucrose; 0.5% (v/v) Triton X-100; 0.15% (v/v) β-mercaptoethanol; pH 9.3) and stirring gently for 10 min on ice. The suspension was then filtered through three layers of Miracloth to remove tissue debris. The suspension containing nuclei was then subjected to three successive rounds of centrifugation in 40 mL ice-cold wash buffer. The pelleted nuclei were re-suspended in 1 mL of homogenization buffer and warmed to 45°C for 5 min. Nuclei were then embedded into an equal volume (1 mL) of 1% w/v low melting-point agarose solution (45°C) in glass capillaries using a wide-bore pipette tip. The final concentration within the agarose strings was ∼106 nuclei/mL. After 15 min at 4°C, the embedded agarose strings were transferred to 40 mL lysis buffer sodium lauryl sarcosine; 0.1 mg/mL proteinase K; pH 9.3) and incubated for 48 h at 45°C under continuous gentle shaking (50 rpm). Strings were then washed in 50 mL EDTA for 1 h at 50°C; in 50 mL EDTA for 1 h on ice and finally 3 times in TE (Tris∶EDTA = 20 mM∶50 mM) for 1 h on ice. The washed strings were stored in TE (20∶50) at 4°C.Nuclei were isolated from 4-week-old Saccharomyces cerevisiae chromosomal markers (Bio-Rad), and subjected to pulsed-field gel electrophoresis . After electrophoresis, the gel segments containing the chromosomal markers was excised and stained with ethidium bromide to visualize DNA under UV light. The central gel portion was incubated for 3 h in 375 mL 0.1xTE at 4°C (TE buffer re-newed every hour). After removing the gel from 0.1x TE, a glass capillary was used to remove agarose plugs from the gel region in the size range of ∼500 Kbp, indicated by the S. cerevisiae markers. One plug was transferred into each well of a 96-well PCR plate and overlaid with ∼30 µl of light mineral oil (Sigma).An agarose string was γ-irradiated to randomly break the nuclear DNA, and loaded across a single well in a gel of 1% w/v chromosomal grade agarose (Bio-Rad) in 0.5x TBE, together with e([T-N]/T), where T is the total number of aliquots analysed, and N is the number of aliquots positive for the marker .Before typing all markers, the DNA content of the mapping panel needed to be checked to make sure that each well harbours 0.5–0.7 genomes per aliquot (GPA). This calculation was based on the analysis of the first few markers using the formula GPA = –logprimer extension pre-amplification (PEP) with a random 15-mer primer 2, 200 µM of each dNTP, 10 µM N15 primer , 1 U Taq polymerase in addition to the agarose plugs. The PCR cycling conditions used were: 93°C for 5 min; then 50 cycles of 94°C for 30 s, 37°C for 2 min, 37–55°C ramp over 3 min, and 55°C for 4 min. PEP products were then diluted to 150 µL in HPLC-grade water and stored at 70°C until needed as templates for multiplexed pre-amplification.We used three steps for marker typing: a whole genome PCR amplification, a multiplexed pre-amplification and hemi-nested marker-specific amplification. The whole genome amplification of the panel consists of a 2 and 200 µM of each dNTP. PCR cycling conditions used were: 93°C for 10 min; then 22 cycles of 94°C for 20 s, 55°C for 30 s and 72°C for 1 min. Amplification products were diluted in HPLC grade water to a final volume of 250 µL and stored at −20°C.The multiplex marker pre-amplification reaction (step2) contained 5 µL of the diluted PEP product, 0.25 µM of each primer , 1x AmpliTaq Gold reaction buffer, 1 unit AmpliTaq Gold polymerase (PE Applied Biosystems), 4 mM MgClPlus SYBR (Quantace) and 5 µM of each forward and reverse primer. The MT-PCR-HRM was performed in a Rotor-Gene 6000 using the following conditions: 95°C for 10 min, followed by 35 cycles at 95°C for 20 s, 57°C for 30 s and 72°C for 50 s; the detection option ‘green’ was used to monitor fluorescence during each cycle at 72°C. High resolution melting analysis was performed at a ramp from 65°C to 90°C, increasing by 0.3°C each step, holding for 90 s prior to each melt, and also holding for 2 s after each melt.Individual markers are then typed by ‘Multiplex PCR and High Resolution Melt’ in 10 µl reactions consisting of 2 µL of the diluted pre-amplification product, 5 µl of SensiMixDGmap software Pairwise LOD scores, calculated using the Lodulator program, are entered in the Lontig program to attribute the markers to linkage groups |
Nicotine plays an important role in cigarette-smoke-associated airway disease. The present study was designed to examine if nicotine could induce airway hyperresponsiveness through kinin receptors, and if so, explore the underlying mechanisms involved.1 receptor agonist, des-Arg9-bradykinin, and B2 receptor agonist, bradykinin, were monitored with myographs. The B1 and B2 receptor mRNA expressions were semi-quantified using real-time PCR and their corresponding protein expressions assessed with confocal-microscopy-based immunohistochemistry. Various pharmacological inhibitors were used for studying intracellular signaling pathways.Murine tracheal segments were cultured for 1, 2 or 4 days in serum-free DMEM medium in presence of nicotine (1 and 10 μM) or vehicle (DMSO). Contractile responses induced by kinin B1 and B2 receptor-mediated airway contractions, without altering the kinin receptor-mediated relaxations. No such increase was seen at day 1 or day 2. The airway contractile responses to 5-HT, acetylcholine and endothelin receptor agonists remained unaffected by nicotine. Two different neuronal nicotinic receptor antagonists MG624 and hexamethonium blocked the nicotine-induced effects. The enhanced contractile responses were accompanied by increased mRNA and protein expression for both kinin receptors, suggesting the involvement of transcriptional mechanisms. Confocal-microscopy-based immunohistochemistry showed that 4 days of nicotine treatment induced activation (phosphorylation) of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. Inhibition of JNK with its specific inhibitor SP600125 abolished the nicotine-induced effects on kinin receptor-mediated contractions and reverted the enhanced receptor mRNA expression. Administration of phosphodiesterase inhibitors (YM976 and theophylline), glucocorticoid (dexamethasone) or adenylcyclase activator (forskolin) suppressed the nicotine-enhanced airway contractile response to des-Arg9-bradykinin and bradykinin.Four days of organ culture with nicotine concentration-dependently increased kinin B1 and B2 receptors, an effect mediated via neuronal nicotinic receptors. The underlying molecular mechanisms involve activation of JNK- and PDE4-mediated intracellular inflammatory signal pathways. Our results might be relevant to active and passive smokers suffering from airway hyperresponsiveness, and suggest new therapeutic targets for the treatment of smoke-associated airway disease.Nicotine induces airway hyperresponsiveness via transcriptional up-regulation of airway kinin B In vivo studies in guinea pigs have demonstrated that chronic exposure to tobacco smoke selectively increases airway reactivity to bradykinin and capsaicin, without altering responses to methacholine or histamine : Forward: 5'-CCA TAG CAG AAA TCT ACC TGG CTA AC-3'; Reverse: 5'-GCC AGT TGA AAC GGT TCC-3'Kinin B2 receptor [Accession Number: NM_009747]: Forward: 5'-ATG TTC AAC GTC ACC ACA CAA GTC-3'; Reverse: 5'-TGG ATG GCA TTG AGC CAA C-3'Kinin BGAPDH [Accession Number: XM_001473623]: Forward: 5'-CAT GGC CTT CCG TGT TCC TA-3'; Reverse: 5'-TGC TTC ACC ACC TTC TTG ATG-3'® Green PCR kit in the Smart Cycler® II system . The system automatically monitors the binding of a fluorescent dye SYBR® Green to double-stranded DNA during each cycle of PCR amplification. The real-time PCR was prepared in 25 μl reaction volumes and carried out with heating 95°C for 15 min followed by touchdown PCR i.e. denature at 94°C for 30 sec and annealing at 66°C for 1 min for the first PCR cycle, thereafter, a 2°C decrease for the annealing temperature in every cycle until 56°C. Finally, 40 thermal cycles with 94°C for 30 sec and 55°C for 1 min were performed. The data were analyzed with the threshold cycle (CT) method and the specificity of the PCR products was checked by the dissociation curves. A blank (no template) was included in all the experiments as negative control. The relative amount of mRNA was expressed as the CT values of mRNA for kinin B1 or B2 receptor in relation to the CT values for the house-keeping gene GAPDH in the same sample.Real-time PCR was performed with QuantiTect™ SYBRAfter organ culture, the tracheal segments were immersed in a fixative solution consisting of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h at 4°C. After fixation, the specimens were dehydrated in 20% sucrose in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C, then frozen in Tissue-Tek and stored at -80°C. Sections were cut to 10-μm-thick slices in a cryostat and mounted on SuperFrost Plus slides .1 receptor , kinin B2 receptor , phospho-SAPK/JNK (Thr183/Tyr185) , phospho-p38 MAPK (Thr180/Tyr182) and phospho-ERK1/2 MAPK (Thr202/Tyr204) . The appropriate secondary antibodies, goat anti-rabbit IgG H&L conjugated to fluorescein isothiocynate (FITC) or Texas Red or Alexa Fluor® 488 donkey anti-goat IgG H&L was used for fluorescence microscopic imaging, respectively. In the control experiments, either the primary antibody or the secondary antibody was omitted. The stained specimens were examined under a confocal microscope . The fluorescence intensity was measured and analysed by Image J software http://rsb.info.nih.gov/ij.Immunohistochemistry were carried out using standard protocols, i.e. the sections were incubated with the primary antibody overnight at 4°C and the secondary antibody for 1 h at room temperature in darkness. Primary and secondary antibodies as well as the dilutions used were as following: kinin BTo avoid systemic errors, the nicotine-treated specimen and the corresponding control are always cultured, fixated, stained, examined and scanned at the same time as the same batch, and the setting of the confocal microscope is kept unchanged throughout. This ensures comparability between the groups. The measurements are repeated for each specimen at 6 preset randomly selected sections, at each section the florescence density was measured at 6 areas, and the mean florescence density was obtained from 6 experiments. All measurements are checked and confirmed by another senior researcher.9-bradykinin, sarafotoxin 6b and sarafotoxin 6c were purchased from Neosystem S.A., Strasbourg, France. SP600125 pyrazol-6(2H)-one) was from Calbiochem, Bad Soden, Germany. Nicotine, dexamethasone, indomethacin, 5-HT, carbachol, acetylcholine, YM976, theophylline, forskolin, hexamethonium, MG624, DMEM and Krebs-Henseleit buffer were from Sigma, St. Louis, MO, U.S.A. The stock solutions of bradykinin, des-Arg9-bradykinin, sarafotoxin 6b and sarafotoxin 6c were prepared in 0.1% bovine serum albumin. Nicotine, YM976, SP600125, MG624 and forskolin were dissolved in DMSO. Theophylline, hexamethonium, 5-HT, carbachol and acetylcholine were dissolved in distilled water, and indomethacin in 95% ethanol. All agonists were serially diluted with physiological saline prior to experiments.Bradykinin, des-Argmax) and pEC50 . Contractile responses to agonists are all expressed in mN. Concentration-effect curves obtained from myograph studies were compared using two-way analysis of variance (ANOVA) with Bonferroni's post-test. Unpaired student's t-test with Welch's correction was used when two groups were compared. P ≤ 0.05 was considered to be statistically significant.All data were expressed as mean ± S.E.M. Agonist concentration-effect curve data from individual segments were fitted to the Hill equation using an iterative, least-squares method to provide estimates of maximal contraction or vehicle. A tendency towards an increased airway contractile response to des-Argy 4 Fig. , Table 19-bradykinin and bradykinin. Culture with 10 μM of nicotine significantly increased the Emax for both agonists. Although a tendency towards an increased pEC50 can be seen, it did not reach statistical significance did not significantly increase contractile responses to des-Argnce Fig. , Table 1 μM did nnce Fig. or endotnce Fig. .9-bradykinin can also produce relaxant effects on preconstricted tracheal segments. This relaxation is dependent on the airway epithelium as well as on COX activity and EP receptors [1 nor B2 receptor-mediated relaxations are affected by nicotine or hexamethonium (1 or 10 μM). Results show that MG624 completely revoked the enhanced contractions caused by nicotine for both kinin receptors without altering the contractile response in the control group (0.1% DMSO) at all increased the mRNA expression for both receptors, compared to control of JNK, ERK1/2 and p38 signal molecules were studied with confocal-microscopy-based immunohistochemistry. After 4 days of organ culture with nicotine (10 μM), an activation of JNK was observed in the airway epithelial and in smooth muscle cells compared to control Fig. . This in1 and B2 receptors, a specific JNK inhibitor SP600125 (10 μM) was added together with nicotine during the 4 days of culture. Pharmacological inhibition of JNK abolished the nicotine-enhanced kinin B1 and B2 receptor-mediated contractions together with nicotine in the organ culture for 4 days almost completely abolished the nicotine-enhanced airway contractions to both des-Argnin Fig. and bradnin Fig. .1 receptor-mediated contractions for 4 days in the absence or presence of nicotine (10 μM). Results show that forskolin suppresses contractions induced by both bradykinin and des-Arg9-bradykinin, and this is regardless of the presence or absence of nicotine induced no significant effects. Neither did nicotine treatment affect airway contractions mediated by 5-HT, cholinergic or endothelin receptors. The increase in maximal contraction, without significant change of pEC50, seen after 4 days of nicotine treatment suggests an increase in kinin receptor protein expression rather than alteration of receptor sensitivity. This conclusion is further supported by the discovery of an up-regulated protein expression for both B1 and B2 receptors using confocal microscopy. In addition, real-time PCR reveals a parallel increase in B1 and B2 receptor mRNA suggesting the involvement of transcriptional mechanisms in nicotine's effects. The neuronal nicotinic receptor antagonists MG624 and hexamethonium both abolish the nicotine-enhanced kinin effect, signifying the participation of nicotinic receptors in the start of the process. Further, the intracellular cascade related to the kinin receptor up-regulation seems to involve JNK- and PDE4-related intracellular signal pathways.Cigarette smoke is associated with chronic airway inflammation, AHR, increased asthma severity and to a certain degree, asthma development in children -7. Chronin vivo . The preNeuronal nicotinic receptors in non-neuronal cells have been proposed to be mediators of tobacco toxicity since they are considered to have a "hormone-like" function . Our res1 and B2 receptor-mediated airway contractions, leaving 5-HT, cholinergic and endothelin receptor-mediated contractions completely unaffected. This suggests that nicotine acts on specific targets within the airways. Thus, the effects observed are neither the result of a general hyperresponsiveness nor due to alteration of down-stream G-protein signaling processes. This idea is further strengthen by our findings of a simultaneous up-regulation of receptor function, mRNA and protein expression. It is known that bradykinin acts as a potent bronchoconstrictor in asthmatic patients, but has no effect in normal individuals [2 receptor gene has been found to be associated with asthma prior to the age of 4 [Many GPCRs are involved in the regulation of the contractile state of airway smooth muscle, including 5-HT, bradykinin, endothelin (type A and type B) and M3 muscarinic acetylcholine receptors. Bradykinin, endothelin and M3 muscarinic receptors are Gq-coupled while 5-HT receptors are Gi-coupled . The preividuals . Many stividuals -34. Furtage of 4 . Our res9-bradykinin-induced relaxations were unaffected. This might be due to involvement of different pathways. Stimulation of kinin B1 and B2 receptors on the airway smooth muscle directly activates the inositol 1,4,5-trisphosphate (IP3) pathway increasing intracellular Ca2+ levels which subsequently activates the cellular contractile machinery [9-bradykinin activate COX and stimulate the release of PGE2 from airway epithelial cells which induce airway relaxation through EP receptor activation [Stimulation of the kinin receptors can cause both bronchoconstriction and epithelium-dependent relaxations in the airways. It is interesting to note that though kinin receptor protein expression was increased both on the epithelium and smooth muscle, bradykinin- and des-Argachinery . Kinin rtivation . Therefotivation . This mi1 and B2 receptors in airways [9-bradykinin elicits only negligible contractile responses in fresh segments and the culture procedure per se causes an up-regulation of the kinin receptors [1 and B2 receptor-mediated contractions were nearly completely abolished by SP600125, suppressing the contractile response to a level similar to that seen in fresh segments [1 and B2 receptor mRNA and protein expression after organ culture with nicotine strengthens the evidence for an alteration at the receptor level rather than a down-stream process. Furthermore, SP600125 up to 30 μM causes no alteration in carbachol-elicited contractile responses [JNK, ERK1/2 and p38 are the classical members of the MAPK family. They are known to play key roles in the regulation of gene expressions. A recent study with human lung macrophages revealed an increase in MAPK phosphorylation and activation of the MAPK/AP-1 pathway caused by cigarette smoke . In anot airways . Both breceptors . The B1 segments . This suesponses , which e1 and B2 receptor-mediated effects in murine airways. It is interesting to note that the effect of dexamethasone appears to be very similar to those of SP600125. Dexamethasone is classically thought to exert its effects via the inhibition of the pro-inflammatory transcription factors activator protein-1 (AP-1) and NF-κB [Dexamethasone reduces inflammation and hyperreactivity in asthmatic airways ,41, inhind NF-κB . The JNKnd NF-κB and its nd NF-κB . In addind NF-κB ,47. It ind NF-κB and postnd NF-κB ,49.1 and B2 receptor-mediated airway contractions. Moreover, YM976 also suppresses nicotine-enhanced kinin receptor mRNA expression. PDE4 is expressed in airway smooth muscle cells and increases intracellular concentration of the second messenger cAMP [2 receptors [YM976 is a selective PDE4 inhibitor shown to possess powerful anti-inflammatory and direct broncho-relaxant effects in combination with low emetogenicity . The latger cAMP . Inhibitger cAMP , inhibitger cAMP . The mecger cAMP . When ineceptors . HoweverOur results show the simultaneous involvement of both JNK and PDE4/cAMP-mediated pathway in nicotine's effects on kinin receptors. Supporting this, there have been many reports on the "cross-talk" between cAMP and JNK pathway. cAMP has the ability to inhibit JNK activation in human airway smooth muscle cells , and in 9-bradykinin-induced airway contractions without affecting their relaxations. The nicotine effect is mediated by activation of airway neuronal nicotinic receptors which results in a transcriptional up-regulation of kinin B1 and B2 receptors. The whole process depends on the activation of JNK- and PDE4-related intracellular signal pathways Thus, our findings might provide new therapeutic targets for future treatment of tobacco smoke-associated AHR.To conclude, nicotine has been shown to have the ability to enhance bradykinin- and des-ArgThe authors declare that they have no competing interests.YX carried out the experiments and data analysis, participated in design of the study and drafted the manuscript. YZ conceived and designed the study, helped with performing the immunohistological studies and drafting the manuscript. LOC supervised the work and provided intellectual input in the study. All authors contributed in writing the manuscript and approved the final version. |
To investigate psychological well-being and substance abuse among medical students in Pakistan.A cross-sectional questionnaire-based survey was conducted in six medical colleges across Pakistan. Final-year medical students were interviewed by either a postgraduate trainee in psychiatry or a consultant psychiatrist.A total of 540 medical students were approached; 342 participated and the response rate was 64.5%. Mean age was 23.73 years (SD 2.45 years); 52.5% were male and 90% single. Two out of every five respondents reported that work/study at medical school affected their personal health and well-being. A considerable proportion of students were aware of alcohol and smoking as coping strategies for stress in medical students. The main factors causing stress were heavy workload (47.4%), relationship with colleagues (13.5%) and staff (11.9%). A total of 30% reported a history of depression and 15% among them had used an antidepressant. More than half were aware of depression in colleagues. The majority of respondents said that teaching provided on substance misuse in the areas of alcohol and illegal drugs, management/treatment of addiction, and models of addiction was poor. There was significant association (p = 0.044) between stress and awareness about alcohol as a coping strategy for stress among medical students. A significant negative association was also found between medical colleges in public sector (p = 0.052), female gender (p = 0.003) and well-being.The majority of the medical students reported a negative impact of heavy workload on their psychological well-being. Significant numbers of medical students think that substance misuse is a coping strategy for stress. Teaching on addiction/addictive substances is poor at undergraduate level in Pakistani medical colleges. Medicine has been a gratifying profession and held in high esteem since dawn of the history. It not only requires commitment, enthusiasm and altruism, but physicians are also expected to show care, compassion and a dedication to their profession.et al. [Few studies have looked at stress related to medical education. Studies conducted in medical schools in the US and UK show a negative impact on student's physical and mental health -3. A revet al. showed tA study from Newcastle, UK, showed that college students as a whole have a higher prevalence of alcohol drinking and alcohol use disorders than non-college youth . MedicalStudies from Pakistan -10 have Final-year medical students were approached in six medical colleges of four provinces in Pakistan to participate in the study. These include two from North-West Frontier Province (NWFP), two in Sind Province and one each in Baluchistan and Punjab.In total 343 students participated in the survey.We used a questionnaire which has already been used by British Medical Association (BMA) in survey of medical student's well-being in UK .The questionnaire was in the English language, which is the medium of instruction in all medical colleges in Pakistan. The questionnaire consisted of seven items with multiple responses, covering psychological well-being of the students, their awareness about substance misuse, and depression. We collapsed some of the responses for the purpose of simplicity.A demographic extraction sheet containing age, gender, marital status and background was used to record the demographic variables. The data was collected by either a postgraduate trainee in psychiatry or consultant psychiatrist, and the anonymity of the respondent was insured. The study was approved by departmental ethical committee.Data was entered in SPSS version 16.0 and the frequencies of responses calculated. We also used the chi square test to assess the significance between different variables.Out of a total of 540 medical students from the 6 medical colleges, 342 participated in the survey, yielding response rate of 64.5%. The mean age of respondents was 23.73 years (standard deviation (SD) 2.45 years), 52.5% were male and 90% were single.With regard to ethnic distribution, 19.9% were Punjabis, 29.5% Pukhtuns, 11.5% Urdu speaking, 22.5% Sindis, and 7.6% Balochi. The number of students from rural and urban backgrounds was almost equal. The overwhelming majority (91%) were Muslims.Tables Heavy workload (47.4%), followed by relationship with colleagues 13.5%) and staff (11.9%) appeared to be the main sources of stress as shown in figure 3.5% and While 30% of respondents reported that they have suffered from depression, only 15% have used antidepressant medications. More than half were aware of their colleagues suffering from depression and were using antidepressant medication.The majority of the respondents were of the opinion teaching provided on substance misuse in the areas of alcohol, illegal drugs, management/treatment of addiction, and models of addiction is poor as shown in table The association between demographic variables and well-being is shown in Table This study is part of a larger project covering well-being and bullying of medical students in Pakistan. Other findings will be reported separately.The training period for medical students is a constantly changing environment of 5 to 6 years, to ensure that graduates gain sufficient skills. Some aspects of the training have been found to have negative effects on the student's life, which manifest in the form of stress, depression and burn out.An important finding of this survey is the prevalence of stress among medical students; about 65% of them found the training period stressful. This finding is in agreement with other studies conducted in Pakistan and elseAn alarming result of this study is the awareness of a significant number (39.2%) of students about smoking and alcohol 22.8%) as coping mechanisms for stress in their colleagues. This finding lends support to previous research from Pakistan, showing that 90% of medical students perceived academic stress as being responsible for drug use among medical students % as copi. MoreoveWith regard to the main factors responsible for causing stress, the majority of the students attribute it to heavy workload 47.4%) followed by relationship with colleagues (13.5%) and staff 11.9%). This finding is consistent with a BMA survey in the UK .9%. This though s7.4% follAlmost 40% (40.1%) of participants reported a history of depression, while more than 50% were aware of depression among their fellow students. This is the most striking finding of our study. Similar findings have been reported in other research from Pakistan; for example, one study reported suicidal ideation in a third of Pakistani medical students . In partVarious studies from US have identified a high frequency of depression and suicidal tendencies among medical students; in fact, suicide ranks second among the leading causes of death in medical students . In a UKThe medical students in this survey reported poor teaching on substance abuse. This could be explained by the fact that psychiatry is not taught as a major subject at undergraduate level in Pakistan except in a few medical colleges. As such it is understandable that students will be less likely to know a great deal about substance abuse problems.Female medical students and government-run institutions have been found to be negatively associated with health and well-being of medical students. This finding is consistent with studies conducted previously in Pakistan and elseA significant association between stress and awareness of alcohol use as a coping strategy for stress is an important finding of this study. This finding necessitates the need for further research to study actual alcohol use in stressed medical students.This is a preliminary study concerning medical students and substance use, with obvious limitations. Direct figures were not obtained on actual patterns of substance misuse among medical students, and associations were observed. Additionally, no attempts were made to interview students for current depressive symptoms. For psychological well-being, self-report was used instead of standard scale measurements.Our sample may also not be representative of the population; for example, only one medical college was selected from Punjab, which is the largest province of Pakistan.High levels of distress have been reported by medical students in Pakistan, and a significant proportion reported that their well-being has been affected by stress. The vast majority of medical students reported that they know of colleagues who use alcohol and smoking to cope with stress. Moreover, workload was cited by the majority of students as the source of stress.Depression among medical students is high, as reported by the students in this survey. There is general agreement amongst medical students that the teaching of substance abuse in medical schools is over all poor.The authors declare that they have no competing interests.AWY Conceived the idea and also took part in collection and analysis of data and wrote the initial draft of the article. SA took part in data analysis and manuscript writing. ES: did literature search and reviewed the manuscript while NB collected the data and retrieved the relevant references. SI: Analyze the data, and reviewed the manuscript. MNS: Reviewed the manuscript. MZ: entered and analyzed the data. |
In particular, cell-cell interactions in the Peyer's patches involving CD40 and/or CD80/CD86 have been implicated in germinal-center formation and IgA B-cell development. Also soluble factors, such as IL-4, IL-5, IL-6, and TGFβ may be critical for IgA B-cell differentiation in vivo. Here we report on some paradoxical findings with regard to IgA B-cell differentiation and specific mucosal immune responses that we have recently made using gene knockout mice. More specifically, we have investigated to what extent absence of CD4+ T cells, relevant cytokines, or T-cell-B-cell interactions would influence IgA B-cell differentiation in vivo. Using CD4– or IL- 4-gene knockout mice or mice made transgenic for CTLA4Ig, we found that, although specific responses were impaired, total IgA production and IgA B-cell differentiation appeared to proceed normally. However, a poor correlation was found between, on the one hand, GC formation and IgA differentiation and, on the other hand, the ability to respond to T-celldependent soluble protein antigens in these mice. Thus, despite the various deficiencies in CD4+ T-cell functions seemingly intact IgA B-cell development was observed.It is thought that IgA B-cell differentiation is highly dependent on activated CD4 |
With the 22 December 2008 collapse of a Tennessee Valley Authority (TVA) ash pond in Kingston, Tennessee, and the arrival of the Obama administration the following month, the regulatory ground is shifting in regards to coal combustion waste (CCW), the millions of tons of waste left over each year from burning coal for electricity. The U.S. Environmental Protection Agency (EPA) is pursuing a host of initiatives that could directly or indirectly affect the disposition of CCW. States, too, are revisiting their regulations. The goal of these initiatives is to ensure public health; the challenge is to do so without compromising a recycling industry that the American Coal Ash Association (ACAA) says keeps about 56 million tons of CCW per year out of the landfilled waste stream.CCW is created in the process of burning coal to generate steam in electric power plants. The approximately 440 coal-fired power plants in the United States are located primarily in the East, but can be found in virtually every state. These plants generated roughly 131 million tons of CCW in 2007, according to the ACAA.This waste takes the form of fly ash, bottom ash, boiler slag, and flue gas desulfurization (FGD) materials. Fly ash is captured in the chimneys of coal-fired power plants, while the heavier bottom ash and boiler slag are collected from the bottom of the furnace. FGD materials are produced by emission control systems in which an alkaline agent, primarily limestone, is sprayed into the smoke stream to remove acid gases and some heavy metals. The resulting FGD sludge is dried before reuse.EHP by William Alexander Hopkins and colleagues, have documented adverse effects, including developmental and behavioral abnormalities, in fish and amphibians exposed to CCW constituents that have been released to aquatic ecosystems.CCW contains varying levels of the same potentially toxic elements that are found in coal. These include arsenic, chromium, lead, cadmium, selenium, and mercury. Numerous studies, such as work published in the May 2006 issue of About 43% of the CCW produced each year is recycled, primarily as a replacement for portland cement in concrete mix and, in the case of FGD gypsum, as a replacement for virgin gypsum in wallboard. These uses conserve landfill space, reduce the demand for virgin materials, and reduce carbon dioxide emissions caused by the extraction of virgin materials and the manufacture of finished products. A recent analysis by the Electric Power Research Institute (EPRI) and the Recycled Materials Resource Center at the University of New Hampshire suggests that such “beneficial uses” saved about 160 trillion BTUs of energy, 11 million tons of carbon dioxide equivalent, and 32 billion gallons of water in 2007. CCW that is not recycled typically is stored on-site in landfills or stored long-term in ponds (wet storage). Ash ponds, which are often built behind earthen dams or retaining walls, can pose a direct threat to human health and the environment should their bounds fail. This was what happened at TVA’s Kingston Fossil Plant in a spill that spread coal ash over 300 acres and damaged a dozen homes.Following that event, the EPA requested information from electric utilities around the nation to assess the structural integrity of their ash ponds. The survey responses, published on the Special Wastes section of the EPA website, identified 584 impoundments, 49 of which were considered to have “high hazard potential” according to Federal Emergency Management Agency classifications for dams—that is, the volume and/or siting of the dam make it “probable” that human death and possibly major environmental damage will occur if the dam were to fail . Another 60 impoundments were rated as having “significant hazard potential.” In this case, the concern is primarily economic and environmental loss; human deaths are not expected.However, says John Suttles, a senior attorney at the Southern Environmental Law Center (SELC), “Many utilities in the Southeast U.S. refused to provide complete data such as size and volume of waste ponds, citing confidential business information. Most other utilities divulged this information and made no such claims of confidentiality.”The EPA has followed up its survey with onsite assessments of all units having a potential rating of “high” or “significant” hazard. Reports on the first 17 units were published in September 2009; the EPA rated the structural integrity of 7 of these impoundments as “satisfactory,” 9 as “fair,” and 1 as “poor.” All assessments are to be completed by the end of the year.TVA, for its part, reported to the EPA that its Kingston Fossil Plant had been rated as having “low hazard potential” at its last assessment in October 2008. However, cleanup of the site, currently under way, will cost an estimated $1 billion.Environmental Science & Technology by a consortium of Duke University scientists and engineers, identified elevated levels of arsenic, selenium, lithium, and boron in a tributary of the Emory River that had been dammed by the spilled ash. Concentrations of dissolved arsenic in this pond reached 86 μg/L, compared with upstream water concentrations of 0.1–0.4 μg/L. Mercury concentrations of 92–130 μg/kg in sediments downstream of the spill were almost as high as those in the CCW itself.A preliminary assessment of the ecologic consequences of the Kingston spill, published in the 1 August 2009 issue of Concentrations of these elements were significantly lower at sampling sites downstream on the Emory and Clinch Rivers but still were above background concentrations. Levels of radioactivity were slightly higher than those in ambient soil at the site, although according to the U.S. Geological Survey, CCW radioactivity generally is comparable to that of natural sources including some shales and granitic and phosphate rocks.Coal Combustion Waste Damage Assessments, identified 24 cases of proven damage and 43 cases of potential damage to ground or surface water adjacent to CCW disposal sites. Damage was “proven” when exceedances of health-based standards were documented in water far enough from the disposal site “to indicate that hazardous constituents have migrated to the extent that they could cause human health concerns,” according to the report.Aside from the direct danger of failing dams, CCW disposal sites can pose a potential threat to human health and the environment by virtue of the leaching of toxic constituents into groundwater when appropriate protective measures are not in place. These chemicals may then be conveyed to nearby drinking water wells and surface waters. A 2007 EPA study, Human and Ecological Risk Assessment of Coal Combustion Wastes, the authors modeled elevated cancer risk from arsenic for people who drank water from wells near unlined or clay-lined surface impoundments. They also modeled elevated noncancer risks from molybdenum, boron, lead, cadmium, and cobalt. The report has remained unpublished in final form for two years, but EPA spokeswoman Latisha Petteway says it has now been peer reviewed and that a final version is scheduled for publication in December 2009.The year before, the EPA commissioned Research Triangle Institute to perform an analysis of the health risks of landfills and storage ponds containing CCW. The authors assessed soil and aquifer data within a 5-km radius of each of 181 selected coal-fired power plants. In a draft report titled Contaminated water is not the only problem associated with CCW storage. Fine ash particles also can be transported by wind and may be inhaled. Moreover, some scientists and advocacy groups believe certain “beneficial uses” of CCW, for instance as fill in unlined mines, may pose these same water and air quality threats.As far back as 1980, the EPA has debated designating CCW as a hazardous waste under Subtitle C of the Resource Conservation and Recovery Act (RCRA). A Subtitle C designation would mean CCW must be managed as a hazardous waste “from cradle to grave.” Under Subtitle C, federal-level regulations govern the generation, transportation, and treatment, storage, and disposal of hazardous wastes. RCRA’s Subtitle D, in contrast, addresses nonhazardous solid wastes including household garbage, sludge from industrial and municipal wastewater plants and pollution control facilities, and certain hazardous wastes that are exempt from Subtitle C regulations (such as household hazardous waste). State and local governments are responsible for regulating Subtitle D wastes, with guidance from the EPA.But in two separate regulatory determinations, the EPA determined CCW should not be regulated under Subtitle C, nor under Subtitle D, for that matter. Instead, regulation has been left to the states, which have generally included disposed CCW under municipal solid waste or industrial waste regulations, if they regulate CCW at all (36 states have permit programs for CCW landfills).The EPA is now pushing forward on a number of policy fronts, including stricter regulation of CCW disposal sites, as well as another foray into possibly classifying CCW as a hazardous waste under RCRA. Matthew Hale, director of the EPA Office of Resource Conservation and Recovery, says, “EPA is considering a range of options, including regulation of coal combustion under Subtitle D authority, under Subtitle C authority, and combined options that involve a mixture of Subtitle C and Subtitle D authorities. EPA . . . will propose one option as the preferred approach, but will take comment on a full range of options.”RCRA offers two main avenues by which a material can be classified as a hazardous waste under Subtitle C. The first avenue is to specifically list CCW in the act, which assumes that all CCW is of a uniform nature as far as toxic constituents go. RCRA also has provisions that define a listed waste as hazardous but allow certain specified uses of the waste that are unlikely to pose a threat. The second is through a set of technical analyses known as the toxicity characteristic leaching procedure. If, during testing, the material leaches more than a specified concentration of certain constituents, it is deemed “hazardous by characteristic.”Very little CCW sampled has failed the toxicity characteristic leaching procedure. However, in an 8 September 2008 presentation at the Global Waste Management Symposium, EPA scientist Susan Thornloe and colleagues reported that efforts are currently under way to incorporate more reliable CCW leach tests into the EPA’s SW-846, a compendium of accepted test methods for evaluating solid waste. Past CCW testing, they noted, has not always considered field conditions known to influence leaching. Furthermore, assessments have sometimes considered initial but not final site conditions, although changes at a site over time could change the propensity of a material to leach.The distinction ultimately chosen for CCW will have tremendous implications for recycling and disposal. Few would disagree that some of the constituents in CCW can be toxic under certain conditions . At the same time, many assert that classification of all CCW as “hazardous” under Subtitle C is unjustified and would severely constrain, if not altogether end, recycling of the material. “The marketplace would not choose to use something designated hazardous waste when they have other options,” says Tom Adams, ACAA executive director.If classified as hazardous, utility companies would be left with more than 50 million additional tons of CCW to dispose of each year under strict regulations. This waste would have to be transported, in some cases long distances, to landfills specifically designed and permitted to handle hazardous wastes. EPRI, using a cost estimate prepared for the Utilities Solid Waste Action Group of the Edison Electric Institute, modeled a tenfold increase in disposal costs, which are currently around $1 billion annually. In addition, existing facilities might have to be closed or, if possible, retrofitted.For this reason, electric utility and coal industry officials are strongly opposed to Subtitle C classification. So, too, is the Association of State and Territorial Solid Waste Management Officials (ASTSWMO), which voiced its opinion in an April 2009 letter to Hale. “Coal combustion byproducts rarely if ever fail the criteria by which materials are determined to be hazardous waste,” the authors wrote. “To artificially classify them as hazardous waste will needlessly limit the management options for both [CCW] and other waste classified as hazardous, which will be competing with [CCW] for limited hazardous waste disposal capacity, while not providing any greater degree of environmental protection. . . . The prospect of adding a significant new waste stream to be managed by severely underfunded State hazardous waste programs is unconscionable unless a significant amount of new sustained funding is included.”ASTSWMO is also opposed to a hybrid Subtitle C and D designation in which disposed material is classified as hazardous whereas recycled material is classified as solid waste. “The uncertainty that a presumed hazardous waste material could be deemed hazardous as a result of a determination that a generator failed to follow Subtitle D requirements will create too much uncertainty and liability concerns for the beneficial user,” the group wrote in its letter. Instead, the organization encourages EPA to classify all CCW under Subtitle D, with implementation left to the states and enforcement by the federal government under RCRA.Environmental groups do not want to end all genuinely beneficial uses of CCW, but they do want stiffer requirements for disposed waste than are called for under Subtitle D, and they also have serious concerns about unconsolidated land-based uses such as structural fills and agricultural applications, says Chandra Taylor, a senior attorney at the SELC. “There’s no question that CCW has hazardous constituents, and the EPA should promulgate rules in consideration of that fact,” says Taylor. “Certainly, we want at least minimum federal standards for disposal—a dual liner for landfills, a leachate collection system, testing for contaminants in the groundwater supplies, and phaseout of wet storage.”Industry spokespeople say the EPA is indeed likely to call for a phaseout of wet storage. TVA has already announced that it will phase out wet storage within eight years at an estimated cost of $1.5–2.0 billion to pay for new ash-handling equipment and storage facilities. But industry groups remain opposed to any mandatory closure of existing ponds.“Mandatory pond closure is not necessary to ensure safe management of [CCW],” says James Roewer, executive director of the Utilities Solid Waste Action Group. “The ‘nonhazardous’ regulations we are advocating would allow those ponds that are managing [CCW] without adverse environmental impact to continue to do so. Why should a disposal facility that is safely managing [CCW] be forced to close? The costs of conversion of ponds to landfills are huge—as much as $39 billion across the industry.”“Whatever EPA decides, it’s going to require a lot of new landfill space,” Adams says. “And new space is hard to identify and hard to permit.”Irrespective of federal classification, states are moving toward stricter regulation of CCW used in various applications, most notably structural fills. This practice, a popular use of CCW in many states, involves depositing the waste at the core of earthen fills that are subsequently built upon as recreational, commercial, or industrial sites. The key to the environmental safety of these structural fills is preventing water from leaching through the core and mobilizing toxic constituents of the CCW.Most states do not require monitoring of groundwater around most structural fills, so there is little information on the potential for leaching from these facilities. But several sites with environmental concerns have been cited, and at least one case of potentially serious concern has been detected. In Chesapeake, Virginia, heavy metals have been detected in the groundwater surrounding a golf course built on 1.5 million tons of fly ash. Residents are suing Dominion Virginia Power Company to have the ash removed and public water and sewer brought to their neighborhoods. The EPA continues its study and monitoring in this case; the extent of the damage and a definitive link to the golf course have not been established, according to Petteway.Virginia’s Department of Environmental Quality has formed an advisory committee to look at strengthening the regulations for structural fills using CCW. “There are going to be some new requirements to limit the permeability of fills,” says W. Lee Daniels, a professor of crop and soil environmental sciences at Virginia Tech and a member of the committee. “New regulations would not allow a fill to be built in the 100-year floodplain.”Other federal-level policy initiatives may impact the future of CCW by imposing pollution control technologies that affect the quality of fly ash. The EPA is currently in the process of developing standards under the Clean Air Act for reduction of mercury and other pollutants emitted by oil- and coal-fired power plants. The EPA’s new rules would supplant a plan passed by the Bush administration in 2006 that would have allowed power companies to avoid controls by purchasing emission “credits” from power plants in other parts of the country. A trading system can make sense for greenhouse gases that are widely dispersed, but experts agree it is not appropriate for mercury, which deposits locally.The EPA is currently acquiring data from utilities that have implemented various control technologies at their power plants to determine what levels of reduction are feasible for mercury and other toxics included in the list of 187 hazardous air pollutants the agency is required to regulate under the Clean Air Act. The EPA has already found that coal-burning power plants emit 67 of these pollutants, notes the SELC’s Suttles. Some technologies, such as activated carbon injection, have proven highly effective in reducing mercury emission.However, says Suttles, a great deal of pollution that is removed from power plant emissions—and thus goes into CCW—ends up being emitted into the air anyway from cement plants when these plants use CCW as a feedstock. This was the thinking behind a New York State announcement on 13 October 2009 that cement manufacturer Lafarge North America will no longer be allowed to use fly ash in its Ravena cement plant. The announcement follows the measurement of elevated levels of mercury in the soil and wildlife surrounding the plant.Moreover, depending on how activated carbon injection is incorporated, it can alter the air-entrainment characteristics of fly ash, which are important to helping concrete resist the stress of freeze/thaw cycles. These alterations render the fly ash potentially less desirable for use as a concrete feedstock.The latter “may not be a showstopper,” says Ken Ladwig, EPRI senior program manager. EPRI and other groups are researching technologies for collecting fly ash separately from the injected carbon and for treating fly ash to minimize the effects of the injected carbon on concrete. “Any of these are going to raise the cost [of using fly ash in concrete],” says Ladwig. But because incorporation as a concrete component is the largest recycling use of CCW at more than 14 million tons per year, any practice that reduces this option would also increase CCW disposal costs and amounts.EPA has yet to finalize any of its proposed rules, so it is too early to say what requirements will be placed on individual power plants and what technologies the utilities will use to achieve those requirements.Suttles, a staunch advocate of strict controls on power plant emissions, is cognizant of the impacts that new regulations may have on disposal costs and recycling of CCW. But he does not believe such impacts are justification for compromising public health and safety.“We are starting to see the true cost of using coal as an energy source,” he says. “Yes, we may be taking pollution that would have gone into the air and creating a land disposal problem, [but] our position is that you have to account for all these risks. The result is that using coal is not cheap. We need to be pushing energy efficiency and renewable energy.” |
Various studies have identified risk factors associated with decreased breastfeeding duration. The aim of this study was to investigate whether there is an association between oral contraceptive (OC) use before pregnancy and breastfeeding duration.In 1994/95, as part of a 3-year epidemiologic follow-up study of school children, reproductive interviews were conducted with their mothers. The study population consists of 663 women residing in Hesse, Central Germany; 575 provided information on their reproductive history. The interview included retrospective ascertainment of OC use, its timing before pregnancy, and duration of breastfeeding. To estimate its effect on duration of breastfeeding, survival analysis was applied controlling for maternal age, socio-demographic characteristics, smoking during pregnancy, age at menarche, planning of the pregnancy and birth order. Hazard ratios and median breastfeeding duration were estimated.The mean age of the women at delivery was 27.3 years. Among participants, 34.9% had high school education or less, 10.4% had more than 2 children, and 30.1% smoked during pregnancy. In total, oral contraceptive use in the 12 months before conception was reported by 40.4% of the women, within 3 months of conception by 18.4%. 81.4% (468/575) of women initiated breastfeeding. Compared to those who did not use OC in the 12 months preceding pregnancy, mothers who used OC during the 3 months before conception had a shorter duration of breastfeeding , as did mothers who stopped OC use 4–12 months before conception . Smoking during pregnancy and lower education were also significantly associated with shorter duration of breastfeeding.The results suggest that OC use during the 12 months prior to conception may affect breastfeeding duration. These findings may be due to the endocrine disrupting effect of OC. Alternatively, both OC use and shorter duration of breastfeeding may represent lifestyle-related conditions. Breastfeeding is the perfect way to nourish infants and protect them from illness . DocumenIn addition it has been reported that maternal burden of dichlorodiphenyldichloroethylene (DDE), a metabolite of the pesticide dichlorodiphenyltrichloroethane (DDT), is related to a shorter breastfeeding duration possibly by an endocrine disruptive effect -12. An eIn 1994/95, a 3-year follow-up study of school children was initiated to monitor environmental health risk -19. PareTrained personnel conducted face-to-face interviews with the mothers, retrospectively ascertaining health and living conditions. The interview questionnaire was based on the standardized questionnaire used in the European Studies on Fertility and Subfecundity . The folProportions and average values were used for description. To estimate effects on duration of breastfeeding, we used proportional hazard regressions to assess hazard ratios (HR) and their 95% confidence intervals. The HR reflects the relative risk of stopping breastfeeding conferred by each independent variable. The survival analyses also provided estimates of median breastfeeding duration for individual risk factors.The risk factor of interest was OC use. To estimate HR, the following confounders ware taken into account: maternal age, socio-demographic characteristics, smoking during pregnancy, maternal age at menarche, planning this pregnancy and birth order. Since risk factors related to shorter duration of breastfeeding may change in the course of the reproductive history, we additionally stratified for birth order (one versus two and more).Information on breastfeeding was collected only from women who breastfed. However, some mothers may have categorized themselves as non-breastfeeders if they had early difficulties and stopped breastfeeding in the few days following birth. We assumed that a combined analysis that included non-breastfeeding as zero duration in a time-to-stop breastfeeding analysis may partially overcome this misclassification. However, in addition, we also tested the association of the risk factors for breastfeeding excluding non-breastfeeders. The SAS statistical package version 9.1 was used to conduct the data analysis .In the index school children, 671 of 1,091 participated in the study (61.5%). A total of 663 women were interviewed; 639 provided information about their reproductive history including oral contraceptive use; 37 were not the natural mother of the index child and were not asked about breastfeeding. Of the remaining 602, information on breastfeeding duration was gathered from 575 mothers. In the following, we focused on total duration of breastfeeding. At delivery, the age range of women was 15 to 38 years with a mean age of 27.3 years , the HR for using OC during the 3 months preceding conception was stronger for the first pregnancy than for pregnancies with a higher order . Contrary to this finding, OC use in the 4–12 months before conception was no longer statistically significant for the first pregnancy, but was important for later pregnancies . The association of maternal education and duration of breastfeeding did not change after stratification by birth order. Maternal smoking during pregnancy showed no statistically significant hazard ratio for breastfeeding after the first pregnancy , but an increased hazard ratio for smoking during pregnancy with higher parity . To stratify for planning this pregnancy, we grouped the variable into planning and no planning; the latter included undecided women and women with no intention to conceive. Interestingly, OC use showed no effect on duration of breastfeeding in women who did not plan their pregnancy or who were undecided. However, the associations between OC use and breastfeeding duration in women who planned their pregnancy did not differ from the findings in the total sample , only minor changes occurred in estimated hazard ratios (data not shown). The association between OC use during the 3 months preceding conception and breastfeeding duration became stronger , whereas the hazard ratio for OC use in the 4–12 months before conception did not substantially change . However, participating women were more highly educated: 57% of the non-participants did not have high school education compared to only 35% of the participating women. Since the educational level was related to breastfeeding initiation, it is possible that the increased proportion of better educated women has augmented the proportion of breastfeeding women and the duration of breastfeeding. Such an educational difference is often seen in surveys . HoweverThe proportion of breastfeeding initiation identified in our study (81.4%) is comparable to findings by the German Health Interview and Examination Survey for Children and Adolescents. Across all age groups, 76.7% of German children were breastfed . Based oRegarding education and smoking during pregnancy, our findings are in agreement with prior reports ,8,26,29.Only in women who planned their pregnancy (n = 421) the HRs for OC use in the 4–12 months before conception and OC use immediately before conception (0–3 months) were statistically significantly elevated. This finding suggests that the effect of OC use on the duration of breastfeeding is not explained by the status of pregnancy planning. On the contrary, the use of OC seems to be more frequent in women who planned their pregnancy. The reason may be that OC use provides a better control option. Hence, the stratification put forward the notion that the diminishing effect of OC use on duration of breastfeeding is not a shared intentional characteristic of a life style, but an unintentional effect.Regarding smoking, in agreement with other studies we found that a lower proportion of mothers who had smoked during pregnancy initiated breastfeeding Table 3,8,26],8,263,8,A study from Saudi-Arabia showed that OC (type of OC not specified) use after delivery diminish the duration of breastfeeding . Briend Our results add to the evidence that oral contraceptives may reduce breastfeeding duration. It is possible that oral contraceptive act via endocrine disruption. This proposition requires the initiation of long-term changes in sex steroid hormone levels by oral contraceptive use before pregnancy. In support of a long-term effect, Keski-Nisula et al showed that oral contraceptive use within one year before pregnancy increased estradiol (non-significant) and progesterone levels during pregnancy . Hence, Our findings are in agreement with a prior report that endocrine disruption, for instance by DDE in maternal serum may be related to lower initiation rates and shorter duration of breastfeeding -12. An aThese results suggest that in mothers who used oral contraceptives in the 12 months before conception, duration of breastfeeding was shorter compared to mothers who did not. Other risk factors for breastfeeding cessation were smoking during pregnancy and low level of maternal education. In the light of the established benefits of breastfeeding for mother-infant dyad, there is a need to better understand and prevent adverse effects of xenoestrogens and/or lifestyle choices on lactogenesis.The authors declare that they have no competing interests.NSR conducted data analysis and drafted the manuscript. WK was responsible for project design, data analysis and drafting of the manuscript. |
Serum protein profiling seems promising for early detection of breast cancer. However, the approach is also criticized, partly because of difficulties in validating discriminatory proteins. This study's aim is to validate three proteins previously reported to be discriminative between breast cancer cases and healthy controls. These proteins had been identified as a fragment of inter-alpha trypsin inhibitor H4 (4.3 kDa), C-terminal-truncated form of C3a des arginine anaphylatoxin (8.1 kDa) and C3a des arginine anaphylatoxin (8.9 kDa).Serum protein profiles of 48 breast cancer patients and 48 healthy controls were analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Differences in protein intensity between breast cancer cases and controls were measured with the Mann-Whitney U test and adjusted for confounding in a multivariate logistic regression model.m/z) 4276, 4292, 8129 and 8941, were found that were assumed to represent the previously reported proteins. M/z 4276 and 4292 were statistically significantly decreased in breast cancer cases compared to healthy controls (p < 0.001). M/z 8941 was decreased in breast cancer cases (p < 0.001) and m/z 8129 was not related with breast cancer (p = 0.87). Adjustment for sample preparation day, sample storage duration and age did not substantially alter results.Four peaks, with mass-to-charge ratio (M/z 4276 and 4292 both represented the previously reported 4.3 kDa protein and were both decreased in breast cancer patients, which is in accordance with the results of most previous studies. M/z 8129 was in contrast with previous studies not related with breast cancer. Remarkably, m/z 8941 was decreased in breast cancer cases whereas in previous studies it was increased. Differences in patient populations and pre-analytical sample handling could have contributed to discrepancies. Further research is needed before we can conclude on the relevance of these proteins as breast cancer biomarkers. In the search for new breast cancer biomarkers several studies have been performed comparing serum protein profiles of breast cancer cases with those of healthy controls using surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) -5. In thFor instance, in 2002 Li et al. performem/z (mass-to-charge ratio) of 4286 and 4302) were both statistically significantly decreased in breast cancer cases compared to non-cancer controls . Two peaks that could possibly represent the 8.9 kDa protein (m/z of 8919 and 8961) were both statistically significantly increased in breast cancer cases compared to non-cancer controls , 17% [IQR: 7–47], 11% [IQR: 5–22] and 13% [IQR: 5–23] for The relations between peak intensities and day of sample preparation, sample storage duration and age were estimated in the control group. The results are presented in Table m/z of 4276, 4292 and 8129. The intensities of these peaks were statistically significantly higher in control samples prepared on day 1 than those prepared on day 2. We also found a statistically significant trend in intensity over the four storage duration categories for these three peaks, with lower intensities in samples that were stored for a longer time. Unintentionally, samples stored for less than 18 months were all prepared on day 1 and samples stored for 18 months or more on day 2. Therefore, it is difficult to disentangle the effects of day of preparation and storage duration. Age was not significantly related to the intensity of any of the peaks as shown in Table A clear relation between peak intensity and day of sample preparation was found for the peaks with an m/z of 4276, 4292 and 8941, women with a low peak intensity were statistically significantly more often affected with breast cancer than women with a high peak intensity (reference group). The crude OR's with 95% CI for these three peaks were 6.0 [95% CI: 2.0–18.2], 7.6 [95% CI: 2.4–24.3] and 13.0 [95% CI: 3.3–50.8], respectively. Women with an intermediate peak intensity for any of these three peaks were not more likely to be affected with breast cancer than women in the reference group. The intensity of the peak with an m/z of 8129 was not related to the presence of breast cancer. After adjustment for day of sample preparation, sample storage duration and age results remained essentially the same.The relationships between the intensities of the four peaks and the presence of breast cancer, before and after adjustment for day of sample preparation, sample storage duration and age are listed in table M/z 8129 did not statistically significantly contribute to the model. M/z 4276, m/z 4292 and m/z 8941 all contributed statistically significantly to the discrimination of cases and controls (data not shown).Subsequently, we performed a backward logistic regression analysis in which we simultaneously included the four peaks (continuous). m/z of 4276 and 4292 in cases without lymph node involvement were higher than in cases with lymph node involvement . No statistically significantly relations were observed between the intensities of the four peaks and any of the other tumor characteristics.The relations between the intensities of the four peaks and tumor stage, tumor size, lymph node involvement and tumor differentiation are shown in Table m/z 8129, a statistically significantly decrease in intensity was observed in postmenopausal cases compared to premenopausal cases . In cases with p53– tumors compared to cases with p53+ tumors, the decrease in intensity for m/z 8129 was borderline statistically significantly . No other statistically significant relation was found.The relations between the intensities of the four peaks and menopause status, hormone receptor status and HER2/neu and p53 expression are listed in Table m/z 4276 and m/z 4292 indeed represent the 4.3 kDa fragment of the ITIH4 protein and its oxidized form. We found three other peaks in this study which masses highly corresponded with the theoretical masses of three other fragments of ITIH4 previously described by Villanueva et al. [m/z 3270 and m/z 3965 were highly correlated with the intensities of m/z 4276 and m/z 4292 . This high correlation in intensity is only expected when all these proteins originated from the same protein. Since masses of all these proteins have very high resemblances with the theoretical masses of the previously reported ITIH4 fragments and these proteins were all detected under the same conditions, we can assume that all these peaks represent fragments of ITIH4.The assumption that the proteins found in this study indeed represent the previously reported proteins -4 was baa et al. . The intm/z of 8941 was actually previously identified in our laboratory in a breast cancer serum sample as C3adesArg. The method of protein identification was similar to that performed in the validation study by Li et al. [desArg, 61% sequence coverage), a protein with theoretical mass 8939.46 Da and pI 9.54. This identity was confirmed by an immunoassay, for which ProteinA beads were loaded with a C3a polyclonal antibody .The peak with an m/z within the mass range of the three previously reported proteins [m/z of 4276, 4292 and 8941 were all statistically significantly decreased in cases compared to controls. The intensity of the peak with an m/z of 8129 was not different between cases and controls. After splitting our data of the two duplicates into two groups, results were similar. This together with CV's within an acceptable range indicates that our results are robust.In this study including 48 breast cancer cases and 48 healthy controls we discovered four peaks with an m/z of 4276 and 4292 were assumed to represent a fragment of ITIH4 (the 4.3 kDa protein) and its oxidized form. The decrease in intensity of the 4.3 kDa protein in breast cancer cases reported in the study by Li et al. [The peaks with an desArgΔ88[The 8.1 kDa protein, previously identified as C3adesArgΔ88 was incrdesArgΔ88. This codesArgΔ88.desArg was increased in breast cancer cases. This could not be replicated in our study. In our study, this protein was decreased in cases compared to controls, which thus appears to be an exception. Remarkably, a decrease of this protein was also found in two other breast cancer sample-sets analyzed by our group (publication in preparation). Furthermore, it is also remarkable that this protein was the best discriminating protein between breast cancer cases and healthy controls found in this study. The 8941 m/z peak found in our study was previously identified by our group as C3adesArg, which is thus in agreement with the identity of the 8.9 kDa protein found in the validation study by Li et al. [In the three previous studies -4, the 8Li et al. suggesteAnother factor that is important for storage is the temperature at which samples are stored. Although in several studies no major differences in serum protein profiles were observed after a storage period of 1–3 months at -20°C, -80°C or liquid nitrogen -12, EngwAnother, possibly more likely explanation for the discrepant results found in the several studies is differences in pre-analytical sample handling. Several studies have shown that the time between venipuncture and centrifugation as well as the temperature at which samples are held meanwhile is of major influence on protein profiles ,12-16. WIn this study we also examined the relationships between a number of patient and tumor characteristics and peak intensities in order to find possible explanations for the inconsistencies in the literature. Most characteristics appeared to be unrelated to peak intensity, however we did find a relation between the intensity of one of the peaks and menopause status. The previous studies did not provide information on menopause status ,4 or didm/z 8129. It might be possible that differences in ratio of the several molecular subtypes of breast cancer between the studies have led to inconsistencies in the results. However, no information about p53 expression was available in the previous studies [We also investigated the relations between the intensities of the several peaks and HER2/neu and p53 expression and ER and PR status. Since breast cancer is such a heterogeneous disease, factors causing this heterogeneity, like HER2/neu and p53 expression and ER and PR status , should studies .In conclusion, in this study we were able to detect of all three previously reported proteins -4. Most The authors declare that they have no competing interests.AWJW, MCWG and CHG conducted the general design of the study and AWJW and MCWG performed the protein profiling analysis. AWJW and CHG were involved in the data-analysis and drafted the manuscript together with JHB. EJThR included the breast cancer patients. MCWG, DEG and PHMP participated in editing and reviewing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:m/z. Peaks with statistically significantly different intensities (p < 0.01) in cases compared to controls in order of An overview of the peaks found in this study that were statistically significantly different in intensity between cases and controls.Click here for file |
This report describes the case of a woman with treatment-resistant schizophrenia who became pregnant while being treated successfully with clozapine. Possible risks associated with continuation of clozapine during pregnancy are discussed. Increasing use of clozapine in the treatment of psychotic illness has associated caveats. One of these being increased chances of pregnancy both desired and unwanted in woman taking clozapine. Sufficient data are not available to psychiatrists to guide the management of such cases. The following case adds to the existing data on the use of clozapine during pregnancy.Ms S, a 20-year-old unmarried woman from middle socio-economic status, presented with a history of 2 years' duration suggestive of schizophrenia according to the DSM-IV guidelines. She had been treated with various typical and atypical antipsychotics. She showed no improvement but experienced side-effects. So treatment with clozapine was considered. Under regular leucocyte monitoring, the dose of clozapine was gradually increased to a maximum of 250 mg/day. She showed remarkable improvement and got married in the next 6 months. Despite repeated advice to practice contraception, Ms S disclosed about being pregnant at the end of the first trimester. She and her husband were counselled on the possible harm to the foetus by clozapine, but they decided to continue with the pregnancy as well as with the treatment at the same dose fearing that she might relapse with dose reduction. Besides regular antenatal check-up, blood sugar and leucocyte counts were monitored regularly. She delivered a healthy female child without any complications. She was advised not to breastfeed. The child has been followed up for 2 years and has shown no developmental delays. The patient is also maintaining well.Data regarding the potential teratogenicity of clozapine are not unanimous. There are reports of no congenital abnormalities with clozapine in animals and human beings.5However, in our patient clozapine was continued at the same dose throughout the gestational period with no hyperglycaelmia or any complication during labour and delivery. The child also showed no congenital anomaly. Breastfeeding was not advised considering the high concentration of clozapine in breast milk and exposing the child further to clozapine.7Since the introduction of atypical antipsychotics particularly clozapine, increasing number of patients with treatment-resistant schizophrenia are benefiting in terms of remission and better social functioning. Unlike typical antipsychotics, clozapine does not cause hyperprolactinaemia, thus increasing the chances of a woman taking clozapine to conceive. Though the patient described here had an uneventful pregnancy while on clozapine, it needs to be emphasized that like other psychotropics clozapine should be used with caution during pregnancy. This is more so with clozapine as there is insufficient knowledge regarding clozapine-induced agranulo-cytosis in foetus/neonate. |
The CDC estimates more than 76 million new cases of foodborne illness resulting in 5,000 deaths and 325,000 hospitalizations occur each year in the United States.The Produce Safety Project of The Pew Charitable Trusts estimated in March 2010 that foodborne illnesses cost the United States $152 billion each year and each citizen an average of $1,850 per case. The report, available at http://www.epa.gov/oppt/newchems/pubs/invntory.htm.The EPA announced 15 March 2010 it will now provide free online access to the Toxic Substances Control Act Chemical Substance Inventory, which provides data on thousands of industrial chemicals. Until now, this consolidated set of information was available only by purchase. The move is part of the agency’s stated priority of making chemical information more accessible to the public and follows a January announcement that the EPA is seeking to reduce some confidentiality claims on the identity of chemicals . The inventory is available at www.epa.gov/getleadsafe.Effective 10 April 2010 all renovations of housing constructed before 1978 and of child-occupied facilities (such as schools) must be performed by certified renovators using specific lead-safe work practices. “Renovation” is defined broadly under the EPA rule to include window repair, weatherization, and modification of painted doors. The new regulation goes beyond earlier tenant notification stipulations by requiring that renovators post warning signs at the remodeling site to inform workers and occupants of lead hazards. Contractors also must now follow lead dust containment and waste management procedures. The EPA provides more information for contractors, certification trainers, homeowners, and landlords at Recycling—From E-Waste to Resources, released 22 February 2010, UNEP estimates the number of discarded computers shipped to some developing countries could increase by as much as 500% by 2020. The informal recycling of electronics is a lucrative but highly hazardous cottage industry in many developing countries. The UNEP report therefore offers guidance for countries to build successful and safer e-waste management systems.The world’s stockpile of e-waste—discarded computers, mobile phones, and other electronic devices—is growing by an estimated 40 million tons per year with little sign of stopping. In Quarterly Review of Biology assesses the factors contributing to increases in malaria cases worldwide. The researchers report that climate change, human migration, and land-use changes all are causing malaria to spread into highland areas of East Africa, Indonesia, Afghanistan, and elsewhere. They systematically show how climate affects multiple biological components of malaria transmission and highlight the need for research to better understand the transmission dynamics of this disease and how to sustainably control or eliminate it.A review by Luis Fernando Chaves and Constantianus Koenraadt in the March 2010 Journal of Environmental Quality, show stormwater runoff was the main pathway, contributing about half the harbor’s PAH load, and atmospheric deposition was an important contributor of smaller PAH compounds. The results suggest that minimizing the flow of PAHs into waterways may require tweaking stormwater management plans to control runoff.Polycyclic aromatic hydrocarbons (PAHs), chemicals released during combustion of biomass and fossil fuels, are ubiquitous in the environment. Lisa Rodenburg and colleagues undertook a 4-year study to identify the primary routes by which PAHs end up in New York/New Jersey Harbor. Their findings, reported in the March–April 2010 |
DSS-18. Their structures were established on the basis of detailed spectroscopic analyses, including 1D-, 2D-NMR and HR-ESI MS techniques.Three new polyketides, phaeochromycins F ( Streptomyces was isolated from deep sediment collected from the west Pacific.Marine microorganisms are widely recognized as rich sources of novel natural products –2. In re1), G (2) and H (3), from Streptomyces sp. DSS-18 under constant agitation (240 rpm). After filtration of the harvested culture broth, the culture filtrate was extracted exhaustively with ethyl acetate. The ethyl acetate extract was purified by column chromatography to afford three new polyketides.1) was obtained as a brown powder. The IR absorption at 3424 and 1653 cm−1 indicated the presence of OH groups and aryl ketone. The 1H NMR spectra of 1 (2 moieties , five aromatic CH groups . The 13C NMR (DEPT) spectrum of 1 , 7.57 , 7.39 suggested the presence of a trisubstituted aromatic ring. The HMBC correlations from H-C(8) to C(5), C(6), C(7), C(9), C(10), C(11), C(12) and C(13), and H-C(9) to C(7), C(8), C(10), C(11) and C(12), and from H-C(10) to C(7), C(8), C(9), C(11), C(12) and C(13), and from H-C(14) to C(12), C(13), C(15) and C(16), along with 1H,1H-COSY correlations, and the connection from C(11) to C(15) via oxygen was confirmed by downshift of their chemical shift, established the presence of fragment 1a (benzopyranone). The assignments of the benzopyranone carbons are in good agreement with those of SEK34b + peak at m/z 603.1282, establishing the molecular formula C33H24O10, which further supported the symmetrical structure. Therefore, from those data, the structure of 1 was elucidated as 5-{[4-hydroxy-3-({4-hydroxy-6-[(2-methyl-4-oxo-4H-chromen-5-yl)methyl]-2-oxo-2H-pyran-3-yl}methyl)-2-oxo-2H-pyran-6-yl]methyl}-2-methyl-4H-chromen-4-one, named phaeochromycin F.Phaeochromycin F was isolated as an amorphous powder. Its molecular formula was determined as C13H12O3 based on HR-ESI mass spectra and NMR data. The IR absorption at 1660 cm−1 indicated the presence of aryl ketone. Inspection of the NMR spectral data (2 had the same fragment 1a. The long-range correlations from H-C(2) to C(3) and C(4) and H-C(4) to C(3), C(5), C(6) and C(10) suggested the presence of the acetonyl group connected to C(5). Again, NMR assignments were in excellent agreement with the literature -2-oxo-2H-pyran-3-yl}methyl)-2-oxo-2H-pyran-6-yl]methyl}-2-methyl-4H-chromen-4-one 1), brown, amorphous powder. UV (MeOH): 301.5; [α]D20 = 0 ; IR (KBr): 3424, 2974, 1653, 1391, 1089, 1050; 1H and 13C NMR: see m/z 603.1282 .: 248, 300; [α]D20 = 0 ; IR (KBr): 1660, 1603, 1391, 1118; 1H and 13C NMR: see m/z 217.0897 ., white powder; UV: 230, 249.5, 302; [α]D20 = +13.2 ; IR: 3438, 2971, 1712, 1644, 1605, 1392, 1366, 1118; 1H and 13C NMR: see m/z 283.2717 [M + Na]+ calc. 283.2749).[5-(4-hydroxy-2-oxopentyl)-2-methyl-4File 11–3NMR spectra of compounds |
The preoperative assessment of the patient for neurosurgical and endovascular procedures involves the understanding of the neurological disease and its systemic presentation, and the requirements of the procedure. There is a wide spectrum of different neurosurgical disorders and procedures. This article provides an overview of the preoperative evaluation of these patients with respect to general principles of neuroanesthesia, and considerations for specific intracranial and vascular neurosurgical and interventional neuroradiological procedures. The preoperative assessment of any patient is an integral part of safe anesthesia practice. Neurosurgery covers a wide spectrum of well-established procedures, but there are also areas that are rapidity expanding such as functional neurosurgery, awake craniotomy and interventional neuroradiology. The purpose of the preoperative assessment is for the anesthesiologist to review the patient's medical history including the neurological events, to obtain an understanding of the disease and its systemic manifestations, to ensure optimization of all comorbidities, and to prepare and plan for an appropriate and safe anesthetic. This article will cover the general preoperative approach for neurosurgical patients as well as considerations for specific intracranial and vascular neurosurgical and interventional neuroradiology procedures.Neurosurgical patients vary widely in their presentation, from an alert and coherent patient presenting for an elective procedure to a patient with depressed neurological status due a devastating neurological insult for an emergent procedure. Hence, the diagnosis, preoperative physical and detailed neurological status, and the urgency of the procedure are important preoperative considerations to formulate an appropriate anesthetic plan . The timIn addition to the routine assessment and preparation of any preoperative patient, emphasis should be placed on the review of the neurological system and comorbidities of the disease and of the patient. A neurological history is mandatory and should include the type and location of the lesion, symptoms, and medications related to the neurological problem, as well as the plan for treatment which may involve both surgery and/or endovascular therapy. Questions that will help illicit further information include a history of seizures, neurological deficits, signs and symptoms of raised intracranial pressure (ICP) such as headaches, nausea, vomiting, confusion, and a history of transient ischemic attacks (TIA) or stroke. Neurological examination should include the level of consciousness and the neurological physical examination which involves the status of the sensory and motor systems and evaluation of cranial nerves. Preoperative cardiovascular disturbances are common in patients undergoing neurosurgical interventions and include blood pressure fluctuations, electrocardiographic abnormalities, arrhythmias, and myocardial ischemia or failure. They can occur as consequences of central neurogenic effects on the myocardium and the autonomic nervous system or concurrently associated medical conditions. Preexisting cardiac disease should be identified, both symptomatic and asymptomatic. The decision to perform further diagnostic evaluations should follow established guidelines . MultiplReview and optimizing of the respiratory system is important to ensure adequate oxygenation and ventilation intra and postoperatively. Patients with acute and chronic pulmonary disease need to have their disease stabilized preoperatively. Patients with neurological disorders may have respiratory complications such as aspiration of gastric contents and pneumonia, which can adversely affect neurologic outcome and survival. Neurogenic pulmonary edema may occur in patients with brain injury, subarachnoid hemorrhage (SAH), and stroke. History of smoking and cessation of smoking should be part of the preoperative evaluation , though Other medical disorders such as diabetes, renal impairment, and hepatic disease will also affect the anesthetic management of the patient and need to be optimized. There is an adverse association of hyperglycemia and neurosurgical outcome . In a prIn the preoperative period neurosurgical patients often present with rapid changes in intravascular volume precipitated by hemorrhage, dehydration, diuretics and mannitol administration, and fluid restriction. Fluid management in the neurosurgical patient takes into consideration the restoration of intravascular volume, the maintenance of cerebral perfusion pressure and the avoidance of hyperglycemic and hypotonic fluids . Iso-osmAll current ongoing medications and allergies need to be reviewed. The preoperative continuation of most ongoing medications is usually straightforward but continuing of medications specific for neurological disorders will depend on recommendations from the neurosurgeon or neurologist. Patients on antiepileptic medications are known to have adverse effects from these medications as well as intraoperative pharmacokinetic interactions. Patients who have been placed on preoperative dexamethasone may present with elevated blood glucose levels which require careful monitoring , 20. PatThe presence of preoperative anxiety is high among neurosurgical patients. A recent study found that there was an increased need for information, especially about surgery, in patients with high levels of preoperative anxiety . PreoperPhysical examination of the patient needs to emphasize the neurological, as well as the cardiac and respiratory systems. Documentation of the assessment of neurological system is helpful for continued patient care. This includes patient's preoperative cognitive function , and language . The Glasgow Coma Scale (GCS) is a standard means of assessment of the neurological state 26]. Th. Th26]. The establishment of a preoperative blood pressure level is helpful for intra- and postoperative decisions for appropriate blood pressure management , 28. TheA thorough examination of the airway must be completed and documented using standard assessment guidelines . The uppThe ASA practice advisory for preanesthesia evaluation supports that routine preoperative investigations should not ordered, but rather that the tests should be selected based on type of procedure and the accompanying co morbidities, for the purpose of guiding and optimizing the preoperative management . The tesPreoperative assessment also includes a thorough discussion with the patient and family of the techniques of anesthesia, requirements for the specific procedure such as invasive monitoring, the risks associated with anesthesia and with the patient's medical comorbidities, and the plans for the postoperative care, such as postoperative ventilation and pain management. Some procedures are performed as day surgery and the assessment of the ability of the patient and family to do this, plus instructions need to be clarified during their preoperative interview . ApproprThe presentation of the patient with a supratentorial tumor will vary depending on the type of tumor, its location and size, and proximity to vital structures. The documentation of any altered level of consciousness, signs, and symptoms of raised ICP, sensory or motor deficits and seizures is essential. Tumors operated upon are usually meningiomas, gliomas, ventricular colloidal cysts or basal cistern epidermoids. Meningiomas can be technically demanding operation and is often associated with significant blood loss, while gliomas often undergo debulking with little risk of bleeding. It is also useful to review the CT and MRI imaging to ascertain characteristics of the tumor such as vascularity, size, location, and its proximity to a sinus and presence of increased ICP. Other aspects to evaluate are the hydration and volume status of the patient, especially those scheduled for emergency surgery. This can be assessed in terms of fluid intake, history of nausea and vomiting, diuresis with mannitol therapy for increased ICP, and the presence of inappropriate secretion of anti diuretic hormone syndrome (SIADH), or cerebral salt wasting. Preoperative management of the electrolyte imbalances may be needed. For patients who receive preoperative steroids, dexamethasone, and antiepileptic such as phenytoin or fosphenytoin, the drugs should be continued preoperatively. Dexamethasone will reduce tumor cerebral edema and this often leads to substantial symptomatic relief . HoweverAwake craniotomy for tumor surgery is often used when the lesion is adjacent to eloquent cortex, such as speech or motor function . BenefitTumors of the pituitary gland are heterogeneous. Anesthetic care requires an understanding of the complex pathophysiology secondary to the patient's dysfunction of their endocrine system . PituitaPosterior fossa surgery provides unique concerns and problems for the anesthesiologist. Patients may present various symptoms such as dysphagia, loss of gag reflex, laryngeal nerve dysfunction, and altered states of consciousness. Preoperative documentation of these is important for assessment of the neurological status at emergence and for planning of postoperative care. Patients with a diminished gag reflex may require continued intubation postoperatively. Positioning is more complicated and may be park bench, prone or sitting. In the lateral and prone position patients are at increased risk for peripheral nerve injury, eye injury and postoperative blindness . Review The extent of the preoperative assessment of a patient with a cerebral aneurysm will be determined by their clinical presentation. Patients with intact aneurysms may be completely asymptomatic, in contrast to the patient with an acute SAH . The neuPatients with an AVM present with an SAH, neurological deficits, seizures, headaches or may be completely asymptomatic. A cerebral aneurysm may also be associated with the AVM. Many of the anesthetic issues involved in treatment of cerebral aneurysms are also applicable to the AVM group. The important consideration in surgical AVM resection is attentiveness to perioperative blood pressure management. Induced hypotension is frequently used intraoperatively to reduce bleeding and postoperatively strict control of the blood pressure is often needed to minimize complications such as bleeding and hyperperfusion syndrome . AvailabPatients scheduled for a carotid endarterectomy can present with TIA, completed stroke or may be asymptomatic. Common symptoms include changes in vision (amaurosis fugax), speech, or weakness of the extremities. Physical examination may reveal a high pitch bruit at the origin of the internal carotid artery, changes in the retina, sensory, and motor deficits. Radiological imaging should be reviewed. The occurrence of bilateral carotid artery disease should be noted. Preoperative evaluation needs to emphasize the neurological system but also the cardiac system, as the risk of myocardial ischemia is similar to the risk of neurological injury postoperatively , 59. TheExtracranial-intracranial (ECIC) bypass surgery is performed for cerebral revascularization in patients with history of cerebral ischemia such as patients with cerebral vascular occlusive disease and moyamoya, and for replacement of blood flow where sacrifice of a vessel is required during complex aneurysm or tumor surgery , 69. TheThe endovascular treatment of many neurosurgical patients includes the coiling and stenting of aneurysms, embolisation of AVM, arteriovenous fistula, and vascular tumors, and stenting of carotid artery stenosis . The aneEndovascular therapy with the use of coils and stents is an accepted mode of treatment of many cerebral aneurysms, though there is controversy . The preFunctional neurosurgery with the use of deep brain stimulators (DBS) began and was initially successful in patients with Parkinson's disease. Its applications have since expanded to treat disorders such as dystonia, tremors, epilepsy, and chronic pain. The preferred technique of anesthetic management in many centers is local anesthesia with monitored care . HoweverPatients with Parkinson's disease are often elderly with a classic triad of resting tremor, muscle rigidity, and bradykinesia with loss of postural reflexes and multPatients with intractable epilepsy not controlled with medical therapy are candidates for surgical resection of their seizure foci . A numbeAnesthesia for the patient undergoing a neurosurgical and endovascular procedure presents special considerations. The brain is a highly vascular organ enclosed within a rigid skull. Tolerance of the brain to disruption of substrate delivery is minimal. Anesthetic and physiological parameters controlled by the anesthesiologist have profound effects on the cerebral homeostasis. The appropriate preoperative evaluation and preparation of the patient is critical to the success of the outcome. An understanding of the pathophysiological disturbances, the systemic manifestations associated with the disease state, the procedure and its special requirements form the essence of a through preoperative assessment. Communication with the patient and family, as well as the neurosurgeon and neuroradiologist, will ensure a good understanding of the anticipated events and allow for a well-prepared patient. |
This is a welcome addition to the list of books available in the discipline of Preventive Medicine/Community Medicine/Public Health. The book covers both conventional and advanced public health issues with equal vigor and enthusiasm. The author has arbitrarily categorized all the reviews into ten units. Fundamental questions asked by new entrants to the aforementioned disciplines are given in unit 1 of the book, aptly named as, “The Basics.” Important issues of public health/preventive and social medicine such as “Essential Medicines”, “International Health Regulations”, as well as “Poverty and Health” which are usually not covered in conventional text books, find a place in this book. Newer topics like “Genetically Modified Foods and Health” and “The Air Ticket Levy” are placed appropriately in the book. The flagship program of the Government of India, the “National Rural Health Mission” is covered in detail. The unit on “Social Medicine” covers a mixed group of topics staring from “Qualitative Research Methods” to management issues like “Medical Audit and Social Marketing.” The chapter on “District Level Household Survey 2002–04” is arbitrarily placed in the “Social Medicine” unit, while “National Family Health Survey-III” is placed within the “Health” unit. The chapter on “Health Economics: The basics” covers little less than the basics, which need elaboration. Certain chapters like “Health Economics,” “Small Pox,” “Health Sector Reforms,” “Design Effect,” “Social Audit and Untied Fund,” and “Indian Public Health Standards” do not have any bibliography which might put the reader at some disadvantage.Overall, the book has a good layout and makes good reading. The book has a good index spreading over 12 pages. Considering the nature of the contents, the book requires periodic revision. The size of the book stands between that of a pocket book and a standard textbook. Priced modestly, the book can be an additional resource material for students of community medicine, public health, and the social sciences. |
High predictive validity – that is, a strong association between the outcome of peer review and the scientific quality of a manuscript submitted to a journal (measured as citations of the later published paper) – does not as a rule suffice to demonstrate the usefulness of peer review for the selection of manuscripts. To assess usefulness, it is important to include in addition the base rate and the selection rate (the proportion of submissions accepted).Angewandte Chemie International Edition (AC-IE), we present a general approach for determining the usefulness of peer reviews for the selection of manuscripts for publication. The results of our study show that peer review is useful: 78% of the submissions accepted by AC-IE are correctly accepted for publication when the editor's decision is based on one review, 69% of the submissions are correctly accepted for publication when the editor's decision is based on two reviews, and 65% of the submissions are correctly accepted for publication when the editor's decision is based on three reviews.Taking the example of the high-impact journal The paper points out through what changes in the selection rate, base rate or validity coefficient a higher success rate (utility) in the AC-IE selection process could be achieved. Reputable scientific journals only publish manuscripts that have been subjected to peer review – that is, critical scrutiny by scientific experts. When a manuscript is submitted, reviewers who are researching and publishing work in the same field (peers) are asked to evaluate the content of the manuscript and recommend to the editor that the manuscript be published, revised and then published, or rejected Angewandte Chemie International Edition (AC-IE). AC-IE is a chemistry journal with a higher annual Journal Impact Factor (JIF) than the JIFs of comparable journals . It is a journal of the German Chemical Society (Gesellschaft Deutscher Chemiker (GDCh), Frankfurt am Main, Germany) and is published by Wiley-VCH . The journal introduced peer review in 1982, primarily in conjunction with one of the document types published in the journal, “Communications,” which are short reports on works in progress or recently concluded experimental or theoretical investigations. What the editors of AC-IE look for most of all is excellence in chemical research. Submissions that reviewers deem to be of high quality are selected for publication.We have examined in three publications For the investigation of the predictive validity of the AC-IE publication decisions citation counts for the accepted and rejected (but published elsewhere) manuscripts were used. In the absence of other operationalizable criteria, a conventional approach is to use citation counts as a proxy for research quality, since they measure the international impact of scientific work not have the scientific potential for publication and rejected it, when it actually did – as reflected in a high scientific impact subsequent to publication. We found that the AC-IE editors' decisions regarding 15% of the manuscripts demonstrate a type I error (accepted manuscripts that did not perform as well as or worse than the average rejected manuscript) Using an approach developed by Bornmann et al. In the present study, we expand the approach that we used in previous investigations of the predictive validity of the manuscript selection process at AC-IE to include the model developed by Taylor and Russell The importance of the base rate and selection rate in assessment of a selection process was established by Taylor and Russell The model consists of four parameters xyr indicates the strength of the relationship between the predictor value and the criterion value .The validity coefficient The base rate is the percentage of submissions that are ‘qualified‘ submissions. Qualified submissions are papers that are fundamentally suitable for publication. After publication they are comparatively frequently cited.The selection rate is the percentage of submissions that are accepted for publication.The success rate is the percentage of submissions accepted that are qualified submissions. This percentage can either be computed from the available data, as shown in the following, or taken from the tables provided by Taylor and Russell xyr>0), the points of all submissions produce a point cloud in the form of an ellipse. However, if xyr = 1 or xyr = −1 the result is a line , and if xyr = 0, the result is a circle .The three graphs in Areas AQ and RN in the three graphs of Based on the number of submissions found in each of the four areas, it is possible to calculate the base rate, selection rate, and success rate:If there is no correlation between the selection process and the success rate (then the point cloud is a circle) and the base and selection rates are 50% , only one-half of the accepted submissions will be qualified , as a result there would be more qualified than non-qualified submissions among the accepted submissions. Another possible way to increase the success of a manuscript selection process is to reduce the selection rate. However, this measure is only successful if there is a correlation between the outcome of peer review and the scientific quality of a submission . If the vertical line (the predictor cutoff) in graph B in However, the success of a manuscript selection process can also be increased by raising the base rate and thus the number of submissions fundamentally suitable for publication. This can be achieved, for example, through successful submission policy . Even if a manuscript selection process is not valid (xyr = .5 (see graph B in Taylor and Russell xyr = .5), in graph C submissions will hardly be selected that later turn out to be non-qualified . According to the corresponding Taylor-Russell table, with a base rate of 80%, a selection rate of 30%, and a validity coefficient of xyr = .5, 94% of submissions accepted for publication are correctly accepted. However, as graph C shows, also many submissions are rejected that would have been fundamentally suitable for publication.In contrast to graph B, in graph C in A manuscript submitted to AC-IE is usually subject to internal and external review. First, editors at the journal evaluate whether the manuscript contributes to the development of an important area of research . If the editors find this to be the case, the submitted manuscript is sent to usually three independent reviewers ; the reviewers are asked to review it using an evaluation form and a comment sheet. The reviewers know the authors' identities, but their reviews are not signed (single blinding). In the year 2000 the AC-IE evaluation form for reviewers contained the following four questions (in 2008 this was changed to five questions): (1) ‘How important do you consider the results?’ ; (2) ‘Do the data obtained by experiment or calculation verify the hypothesis and conclusions?’ ; (3) ‘Is the length of the manuscript appropriate to its contents?’ ; (4) ‘Do you recommend acceptance of the Communication?’ . If reviewers find a manuscript unsuitable for AC-IE, they are asked to name another journal in which the study findings might more suitably be published. Once they have received the reviewers' reports, the editors make the decision to accept or reject a manuscript for publication.Our previous findings show that in general a manuscript is published in AC-IE only if two reviewers rate the results of the study as ‘important’ or ‘very important’ (on question 1 above) and also recommend publication in the journal (do not answer ‘no’ to question 4 above) Decision rules like the AC-IE's ‘clear-cut rule’ have become a new research topic in recent publications on the journal peer review process n = 878) were accepted for publication in AC-IE, and 54% (n = 1021) were rejected. A search in the literature databases Science Citation Index (SCI) (Thomson Reuters) and Chemical Abstracts (CA) revealed that of the 1021 rejected manuscripts, 959 (94%) were later published in 136 other (different) journals. For accepted manuscripts and manuscripts that were rejected (but published elsewhere), we determined the number of citations for a fixed time window of three years after the publication year. “Fixed citation windows are a standard method in bibliometric analysis, in order to give equal time spans for citation to articles published in different years, or at different times in the same year” http://www.cas.org/).For the investigation of the manuscript selection process at AC-IE, we used information on all 1899 manuscripts that went through internal and external review in the year 2000. Of the 1899 manuscripts, 46% ” w) (where W refers to ‘world’) relates the number of citations obtained by the set of papers evaluated to the number of citations received by a same number of papers published in journals dedicated to the respective discipline, field or subfield” w for the manuscripts in this study, specific reference standards were used that refer to the subject areas of CA w in this study, the number of citations for accepted or rejected (but published elsewhere) manuscripts were divided by the (arithmetic) mean number of citations of all publications in a corresponding subject area w quotient allows determination of whether the citation impact of the accepted and rejected (but published elsewhere) manuscripts is far below (Rw<0.5), below (Rw = 0.5–0.8), approximately the same as (Rw = 0.8–1.2), above (Rw = 1.2–1.5), or far above (Rw>1.5) the international (primarily the Western world) citation impact baseline for the corresponding subject areas. With Rw values above 1.5, the probability of identifying excellent contributions is very high To find out whether a publication has a high or low citation impact, its performance is compared with international scientific reference standards Of all 1837 manuscripts published in the AC-IE (accepted manuscripts) or another journal (rejected manuscripts), 906 could be included in the analysis of this study. The reduced number was due to missing values for one or more of the variables included in this study. Reviewer's ratings were not available for all manuscripts; for example, some reviewers filled out only the comment sheet and did not fill out the evaluation form with the 2 questions . In addition, for some manuscripts, no citation counts and/or reference values were available w>1.5 (criterion cutoff). Hence, all submissions with Rw>1.5 were categorized as qualified and all submission with Rw≤1.5 as non-qualified .If the usefulness of peer reviewing for the manuscript selection process is determined using the model developed by Taylor and Russell w criterion cutoff is plotted as a red line starting from the y-axis. As the Rw values were logarithmized (log(x+1)) in order that the distribution of data more likely approximates a normal distribution one review; for the submissions in graphs B and C the editor made the publication decisions based on two and three reviews, respectively. In all three graphs in In the three graphs in The three graphs show accepted manuscripts as blue circles and rejected manuscripts as red circles. The distributions of the blue and red circles in the graphs make it clearly visible that for nearly all of the manuscripts, values for the reviewers' ratings above a certain predictor cutoff led to acceptance by the AC-IE editor and values below the cutoff led to rejection. When the publication decision is based on one review, the predictor cutoff is 3; when the publication decision is based on two and three reviews, this value is 6 and 8.5, respectively. As the reviewers usually recommend that a submission should be published if it is in their opinion important, the above-mentioned “clear-cut rule” at AC-IE results in most cases in an unambiguous assignment of the reviewers' ratings to the editorial decisions. For only a few manuscripts is this not the case. As presented in Bornmann and Daniel w) cutoffs, we determined for the submissions in the three graphs of xyr = .34 (with one review), xyr = .26 (with two reviews), and xyr = .17 (with three reviews). Given the typical use of two or three reviewers assigned to manuscripts submitted to a journal, we would expect to see that the use of more reviewers per manuscript yields more valid recommendations and thus greater utility. But, the validity coefficients actually drop with more reviews per manuscript. This unexpected result is due to the usual decision process of the AC-IE editors: They wait for further reviews when a publication decision does not seem possible on the received reviews w) for one review (first review) and two reviews (first and second review) for those 205 manuscripts that have received three reviews. This analyses show the expected order of coefficients: xyr = .05 (with one review), xyr = .11 (with two reviews) and xyr = .17 (with three reviews).On the basis of the predictor (reviewers' ratings) and criterion (RIf Eq. (3) is used for calculating the success rates for AC-IE, 78% of the submissions accepted are correctly accepted for publication (with a predictor cutoff>3) when the editor's decision is based on one review, 69% of the submissions are correctly accepted for publication (with a predictor cutoff>6) when the editor's decision is based on two reviews, and 65% of the submissions are correctly accepted for publication when the editor's decision is based on three reviews (with a predictor cutoff>8.5). As the values in xyr = .2 and xyr = .3, respectively, it is difficult, to achieve considerably better success rates than base rates using the predictor (the reviewers' ratings). Using the tables in Taylor and Russell xyr = .55 – for example by reviewers having precise information on what makes a manuscript a high quality manuscript and information on how high the selection rate at AC-IE is – then according to the Taylor-Russell table, with a base rate of 60% and a selection rate of 40%, there would be 81% submissions accepted that are qualified submissions – an approximately 20 percentage-point gain in utility over the base rate. About the same gain could be achieved through lowering the selection rate to about 5% – with an unchanged validity coefficient and unchanged base rate.Independently of the number of reviews on which the editor's decision is based, the percentage of submissions accepted that are qualified submissions is above the base rates; however, the differences between the base rates and success rates are not large. But with base rates of approximately 60% in the AC-IE selection process and validity coefficients of In this paper we presented an approach that in addition to validity points to the importance of the base rate and selection rate for determining the utility of peer review for editors' publication decisions. High predictive validity (meaning a high correlation between the outcome of peer review and the scientific impact of the later publication) does not inevitably mean that the ratings of the reviewers are very useful for the selection of submissions. In addition to considering the base rate and selection rate when evaluating a manuscript selection process, according to Cascio The Taylor and Russell Atmospheric Chemistry and Physics (ACP) conducts an access review The second step should be pre-selection using a pre-screening procedure, so that the later time-consuming and costly process is carried out only for those submissions that seem potentially suitable. A successful pre-selection increases the base rate for the subsequent selection process. For example, the journal Following similar procedures to those used at ACP, editors and reviewers at other journals could conduct initial pre-screenings of submitted papers to check whether the manuscripts are basically suitable for publication in the journal and whether there are compelling arguments against publication in the journal. The task of the actual review process with extensive commenting would then be to mainly formulate recommendations for improvement of the submitted manuscript and if necessary to recommend a more suitable journal for publication of the manuscript.The usefulness of peer review for editors' decisions to accept submissions for publication cannot be fully determined using Taylor and Russell's t = 1) to determine the ex-ante suitability of a paper (at t = 0). Yet, there are many things that could have happened between t = 0 and t = 1 that could have contributed to the citation count, without the paper being ‘good.’ For instance, the journal could have risen in attractiveness or ranking, thereby motivating more scholars to cite papers of this journal. Or, the topic of a paper could have been on the peak of a “fashion wave”, i.e., a hot topic w> and <1.5 to measure ‘suitability.’ This is an the ‘experience-based’ assignment There are some limitations of our study that we would like to point out: First, we used post-publication above average citation counts as the measure for deeming a submission ‘suitable for publication.’ It can be questioned, whether citation counts is a measure of publication suitability? Recently, Straub |
Sulforaphane (SFN), an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC). The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC.SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose)-polymerase (PARP). Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB) and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex.SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB) phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest. Epithelial ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stage. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the overall survival ,2. CytorEpidemiological observations indicate that cruciferous vegetables such as broccoli, cabbage, cauliflower and brussels sprouts have been shown to offer protection against various cancers ,6. PhytoRecently it has been shown that SFN inhibits the growth of the epithelial ovarian cancer cell (EOC) line SkOV-3 by down-regulating AKT activity . RelatedThe retinoblastoma protein (RB) is a well-known regulator of G1-S phase cell cycle transition . NegativSulforaphane was dissolved in DMSO and Stock solutions were freshly prepared and added to the cell cultures to obtain the indicated final concentrations. The DMSO concentration was used at 0.01% and the same concentration was used as a vehicle. DMSO alone (0.01%) was found to have no significant effect on cellular function. Cell proliferation assays were conducted using Cell Counting Kit-8 (CCK-8) . Cell cycle analysis was conducted using Cell Cycle Phase Determination Kit . Human fibroblasts were similarly treated as cancer cells to display differential cytotoxicity at any given dose. Retinoblastoma and E2F-1 antibodies were purchased from Millipore . Cyclins, CDKs, poly (ADP-ribose) polymerase (PARP) and β actin antibodies were purchased from Santa Cruz Biotechnology, .2 at 37°C. Trypsin (0.25%)/EDTA solution was used to detach the cells from the culture flask for passing the cells.Ovarian cancer cell lines, MDAH 2774 and SkOV-3 were propagated in McCoy's 5A medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin . Cells were cultured in a humidified atmosphere with 5% COStandard prototype growth curves and number of viable cells were determined for each cell line (treated and control groups) in triplicate experiments according to the CCK-8 manufactures' instructions. Growth curves were plotted as a percentage of the value of DMSO-treated controls minus the value of untreated cells on day 0. Day 3 values were considered for the determination of the 50% cell proliferation inhibition (IC50) for a given treatment. In some cases parallel manual count was also performed with trypan blue and counting by exclusion method using a Hemocytometer. The findings confirmed CCK-8 assay results.6 cells/ml) were mixed with annexin V-biotin and medium-binding reagent, and incubated in the dark for 15 min at room temperature. Cells were then centrifuged and medium was replaced with 1× Binding Buffer containing FITC-streptavidin. Propidium iodide was added to discriminate early apoptotic cells from late apoptotic or necrotic cells. A portion of cell suspension (50 μl) was placed onto a glass slide, covered with a cover slip, and viewed immediately using a fluorescence microscope equipped with FITC (green) and propidium iodide (red) Filters .Apoptotic cells were analyzed by using Annexin V FITC apoptosis detection kit according to the manufacturers instructions. The DNA content of the cells was analyzed by flow cytometer and sub G1 population was considered to represent apoptotic cells. For fluorescent microscopic image analysis of apoptotic fraction of the cells, treated and control . Samples were analyzed in FL2 channel of flow cytometer with a 488 nm excitation laser.MDAH-2774 cells were seeded at 10These assays were performed according to our earlier publication. AntibodBriefly, PANC-1 cells untreated or treated with 15 μM ritonavir for 48 h, were harvested and total RNA was isolated utilizing an RNeasy kit as described by the manufacturer. Total RNA was sent to MOgene company for GEP analysis.To determine if SFN had potential to induce growth arrest in ovarian cancer cells, we conducted cell proliferation assays using two human ovarian cancer cell lines, MDAH-2774 and SkOV-3. Cells were grown as sub-confluent monolayer cultures and propagated under standard conditions. Cell lines were treated with serial dilutions of SFN dissolved in DMSO. Human fibroblasts were similarly treated to display differential cytotoxicity (data not shown). SFN treatment resulted in a concentration-dependent inhibition of the proliferation of MDAH 2774 Fig and SkOV2+ -dependent protein with high affinity for PS [Inhibition of cell cycle progression in tumor cells may be associated with a concomitant activation of cell death pathways such as apoptosis. We, therefore, examined the contribution of apoptosis in SFN-treated MDAH-2774 cells. Apoptosis induces changes on the cell surface, which results in translocation of phosphatidylserine (PS) from the inner layer of the plasma membrane to the outer layer ,19. AnneIn order to determine the causes of the growth inhibition observed by cell proliferation assays, we assessed the cell cycle progression of the cells in the presence of SFN. Serum starved MDAH-2774 cells were treated with either vehicle or 10 μM SFN for 12 or 24 hrs with complete growth medium. The cells were then washed, fixed, and cell cycle phase determination was performed utilizing flow cytometry and a cell cycle phase determination kit. The results demonstrated that SFN induces G1 arrest in a time-dependent manner Fig. and 3F iSince we observed cell cycle inhibition with SFN treatment at G0/G1 phase of the cells, we evaluated genes that influence the cell cycle progression into S phase. Under phosphorylated tumor suppressor retinoblastoma proteins sequesters cell cycle promoting E2F-1 transcription factors. Gene expression analysis of RB proteins revealed a 1.5 and 2.0 fold decrease of RB and p130, respectively; as well as, a 2 folds increase in p107 levels Fig. . The E2FUnder-phosphorylated active retinoblastoma protein (RB) family of proteins, RB, p107 and p130 tumor suppressor proteins control cell cycle progression through the late G1 phase to the S phase by inhibiting E2F family of transcription factors ,20,21. WCell motility following wound generation showed a greater cell migration in control cells compared with SFN treated cells. After 20 hrs, we observed almost complete closure of the wound in control cells which was inhibited by 50% and 75% by 5 μM and 10 μM SFN, respectively Fig . Cell miSFN mediating cell growth arrest has been documented in colon, prostate and several other cancers -8. ChaudCombination therapy with paclitaxel is known to increase overall survival , but conSFN treatment resulted in cleavage of Poly (ADP-ribose) polymerase-1 (PARP-1) in dose dependent manner indicating the induction of apoptosis that was further confirmed by annexin V staining. Induction of apoptosis by SFN in different cellular systems is associated with Bax protein expression . SimilarPhosphorylated RB cannot interact with E2F-1, thus leaving large pool of free E2F-1 transcription factors driving the G1/S cell cycle transition. E2F-1 has been shown to have growth promoting activity in EOC and is over expressed in roughly half of the ovarian cancers . SimilarGene profile analysis showed down regulation of these CDKs with SFN treatment corroborating the under phosphorylated status of RB. Although SFN treatment is known to causes induction of p21 in prostate cancer , in our Invasion followed by degradation of basal membrane is hallmark of tumor metastasis where proliferating tumor cells infiltrate into other tissues . Wound hin vitro by contributing to the modulation of cell cycle regulatory proteins and by increasing the apoptosis. These effects may be correlated to the observed inhibition of cell migration. These observations highlight the possibility that SFN may be a good candidate for combination therapy of EOC with paclitaxel.SFN induces growth arrest and apoptosis in EOC cells by inhibiting RB phosphorylation and reduction in the levels of free E2F-1. In summary, we have provided evidence that SFN suppresses growth of EOC cells SFN: Sulforaphane; EOC: Epithelial Ovarian Cancer; RB: Retinoblastoma.The authors declare that they have no competing interests.All authors read and approved the final manuscript. RBB designed the work and wrote the initial drafts of the manuscript. MP and CS supervised cell cycle analyses and contributed to data interpretation. CSB and SK carried out growth curves and paclitaxel combination experiments. JP and SC carried out RB-E2F-1 co-immunoprecipitation experiments. JS and SS performed most of the western blot analysis. JP carried out the western blots of caspase substrate cleavage. MS made original observations leading to this work and contributed to the critical revision of the manuscript. AMQ and MH conducted flow cytometry experiments and helped generate graphs from flow cytometry analysis. MBL provided valuable reagents and contributed to the critical revision of the manuscript. RM, AS and RBP contributed to the experimental design and critical revision of the manuscript. DWW contributed to the conception and design of the entire study and the final editing of the manuscript. |
The first wave of pandemic influenza A(H1N1)2009 (pH1N1) reached New South Wales (NSW), Australia in May 2009, and led to high rates of influenza-related hospital admission of infants and young to middle-aged adults, but no increase in influenza-related or all-cause mortality.To assess the population rate of pH1N1 infection in NSW residents, pH1N1-specific haemagglutination inhibition (HI) antibody prevalence was measured in specimens collected opportunistically before and after the 2009 winter, and before the introduction of the pH1N1 monovalent vaccine. Age- and geographically-weighted population changes in seroprevalence were calculated. HI antibodies against four recent seasonal influenza A viruses were measured to assess cross-reactions. Pre- and post-pandemic pH1N1 seroprevalences were 12.8%, and 28.4%, respectively, with an estimated overall infection rate of 15.6%. pH1N1 antibody prevalence increased significantly - 20.6% overall - in people born since 1944 (26.9% in those born between 1975 and 1997) but not in those born in or before 1944. People born before 1925 had a significantly higher pH1N1 seroprevalence than any other age-group, and against any seasonal influenza A virus. Sydney residents had a significantly greater change in prevalence of antibodies against pH1N1 than other NSW residents (19.3% vs 9.6%).Based on increases in the pH1N1 antibody prevalence before and after the first pandemic wave, 16% of NSW residents were infected by pH1N1 in 2009; the highest infection rates (27%) were among adolescents and young adults. Past exposure to the antigenically similar influenza A/H1N1(1918) is the likely basis for a very high prevalence (49%) of prepandemic cross-reacting pH1N1 antibody and sparing from pH1N1 infection among people over 85 years. Unless pre-season vaccine uptake is high, there are likely to be at least moderate rates including some life-threatening cases of pH1N1 infection among young people during subsequent winters. The first wave of infection due to pandemic influenza A (H1N1) 2009 - pH1N1 - in Australia began in May, 2009 It is difficult to estimate the true rates of pH1N1 infection or differences between geographic areas and age-groups from limited epidemiological data, but information about population prevalence of infection and immunity is needed to inform vaccine distribution policy and planning for subsequent waves of pH1N1 infection. Serosurveys are used extensively to supplement laboratory notification, hospitalisation and mortality data for many vaccine preventable diseases The aim of this study was to determine the prevalence of subtype-specific influenza A pH1N1 haemagglutination inhibition (HI) antibodies in a broadly-based sample of children and adult residents of NSW, before and after the first pandemic wave, using opportunistically collected plasma or serum specimens.Clinical chemistry laboratories in NSW were asked to provide serum or lithium heparin-treated plasma specimens which had been submitted for diagnostic testing in August or September, 2009 and would otherwise have been discarded. This period was 3–11 weeks after the first epidemic wave peaked in NSW and before the monovalent pH1N1 vaccine became available. The sample size was calculated to provide power to detect a difference in seroprevalence of 10–15% between age groups with a worst case 95% confidence interval of ±7%. We aimed to test approximately equal numbers of specimens from NSW residents in each of seven age-groups , providing a total sample size of ∼1200 specimens. Specimens which represented both urban and rural NSW populations were retrieved using postcode of residence. Specimens were given a unique identifying code and then de-identified; only the sex, age or date of birth and patient postcode were recorded.To estimate the level of pre-existing antibodies to pH1N1, we also tested stored sera from NSW residents which had been submitted for non-influenza serological testing during 2007 and 2008. The sample size for prepandemic sera was determined largely by availability but we aimed to test approximately 50 in each age-group.The specimens used for testing had been submitted for diagnostic testing and would otherwise have been discarded. Consistent with longstanding practice in the performance of national serosurveys, informed consent was not obtained from subjects for the use of their specimens in this study. However, all specimens were deidentified before testing and only the age or date of birth and address postcode were recorded. This study, including the waiver of informed consent, was approved by the Sydney West Area Health Service Human Research Ethics Committee.The antigen used for HI testing was a gamma-irradiated preparation of influenza A/California/07/2009 (pH1N1) virus, provided by the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia. Antigens derived from four recent seasonal influenza A viruses – Brisbane/59/2007/H1N1, New Caledonia/20/1999/H1N1, Brisbane/10/2007/H3N2, and Wisconsin/67/2005/H3N2 - were used to determine levels of antibody to these viruses in the same specimens.A pH1N1 subtype-specific HI assay was developed using established methods. As initial evaluation demonstrated that lithium heparin used as anticoagulant did not significantly affect antibody titres in plasma (compared with serum) whereas EDTA plasma specimens gave less consistent results, only lithium heparin-treated plasma and serum specimens were used.Vibrio cholerae receptor destroying enzyme and incubated overnight at 37°C to remove inhibitors, then diluted 1/2 in citrate and heat inactivated at 56°C. Serial doubling dilutions (and appropriate controls) were reacted with antigens in 96-well V-bottom microtitre trays for 1 hour at room temperature before a 1% v/v suspension of human group O red blood cells was added. After 1–2 hours (or when the cell control had fully haemagglutinated), endpoints were read by two independent operators as the last dilution showing complete inhibition of haemagglutination. Titres of ≥40 were determined to be “positive” for the purpose of this serosurvey.Briefly, specimens were diluted 1/5 in Geometric mean titres (GMTs) were calculated by assigning a titre of 5 to specimens in which no HI antibody was detected (titre<10). Seroprevalence of pH1N1 antibodies was calculated as the percentage with antibody titres≥40, after weighting for age, sex and geographic region.The post-pandemic sample size was designed with a disproportionately greater representation of very young and very old persons and with broad coverage of all geographic areas of NSW by using postcode as a stratification variable. As postcodes in NSW are not grouped geographically, there was some disproportionate representation by region. The initial design of the sample required design weights to be calculated to account for the non-uniform probability of selection between age and postcode strata.In order to compensate for under- and over-sampling from sections of the NSW population, post-stratification weights for both the pre- and post-pandemic samples were also created to balance the sample by five-year age group and by geographic region Differences in weighted proportions were tested using the Rao-Scott Chi-square test. Confidence intervals for differences in proportions between the pre-pandemic and post-pandemic samples were estimated using bootstrapping with 10,000 replications A total of 474 “pre-pandemic” specimens collected in 2007–2008 were tested. Antibodies were detected at titres of ≥40 in only one of 53 from children born in 1998 or later. Overall, 12.8% of patients had pre-existing pH1N1 antibody at titres≥40. Differences in pre-pandemic prevalence of pH1N1 antibody titres≥40 across the five youngest birth-cohorts (born after 1944), were not statistically significant but the pre-pandemic prevalence was significantly higher in the 1925–44 birth cohort and higher again in those born before 1925 .A total of 1247 “post-pandemic” specimens collected in August and September 2009 were tested. The overall prevalence of pH1N1 antibody titres≥40 in the NSW population, after the first wave of pH1N1 infection, was 28.4% . Post-pandemic seroprevalence was highest in those born in the periods 1975–1991 (40.1%), 1992–1997 (40.0%) and pre 1925 49.4%; .The overall increase in seroprevalence between pre- and post-pandemic periods was 15.6% and differences were statistically significant in birth cohorts after 1944 , and greatest in the 1992–1997 and 1975–1991 birth cohorts. The increase did not reach statistical significance in the 0–4 and 5–11 age-groups, separately, but when these two groups were combined the increase, from 1.6% (95% CI 0.0–4.7) to 13.7% (95% CI 9.9–17.6); difference 12.1% was highly significant. Apparent decreases in seroprevalence in the 1925–44 and pre-1925 birth cohorts (65–85 and ≥85 year age-groups) were not statistically significant .Seroprevalence of pH1N1 antibodies in pre- and post-pandemic specimens were compared between the Sydney region (estimated resident population 4.5 million) and the rest of NSW categories. Seroprevalence was highest (30.2%) in the most accessible (urban) region, but differences by accessibility classification were not significant (p = 0.30). Finally, seroprevalence was compared across socioeconomic indices for areas (SEIFA) quintiles, based on postcode of residence. Seroprevalence varied from 25.5% to 33.3% by socioeconomic status, but differences were not statistically significant (p = 0.58).In addition to pH1N1 antibody, subtype/strain-specific HI antibodies against four recent seasonal influenza A virus antigens were measured in postpandemic specimens. The highest seroprevalences (at titres≥40) for different viruses were in different age-groups/birth cohorts: 5–11 (1998–2004) and 12–17 year olds (1992–1997) for Brisbane/59/2007/H1; 5–11 (1998–2004), 12–17 (1992–1997) and 18–34 year olds (1975–1991) for New Caledonia/20/1999/H1; 12–17 (1992–1997) for Wisconsin/67/2005/H3 and 5–11 (1998–2004) for Brisbane10/2007/H3. There were significant differences in seroprevalence and GMTs across age groups for each of the five viruses .The proportions of specimens with HI antibody titres≥40 against seasonal influenza A subtypes were compared for specimens with pH1N1 antibody titres≥40 vs <40 . There wDuring the southern hemisphere winter of 2009, in Australia, the pH1N1 epidemic period lasted about 18 weeks in all In view of these unusual features, estimates of true infection rates in different age-groups and geographic areas are needed to inform immunisation policy and planning for the predicted second wave of infection. Clinical and epidemiological data alone are inadequate, since mild or asymptomatic infections were not recorded and the clinical presentation and rates vary in different age groups. Laboratory diagnostic strategies also varied across States and at different times within the pandemic period. Although pH1N1 was the most common influenza virus detected, seasonal influenza A/H3N2 and A/H1N1 (and other respiratory viruses) were circulating at the same time, at least early in the pandemic Influenza A serosurveillance is complicated by cross-reactions between different influenza A subtypes, variable and often relatively short-lived influenza antibody responses, repeated previous infections and the technical challenges of HI and viral neutralization assays th century pandemic influenza A viruses Genomic and proteomic studies show that pH1N1 is most like the North American swine A/H1N1 and pandemic A/H1N1(1918) viruses and distinct from recent seasonal A/H1N1 and other 20Similarities between pH1N1 and influenza A/H1N1(1918) have been reflected in a number of surveys of sera collected before the 2009 pandemic This is supported by the results of a serosurvey in an unvaccinated population in southern China, in which no cross-reacting pH1N1 antibody was detected in prepandemic sera in subjects aged ≥60 years, in a largely unimmunised population; pH1N1 antibody levels did not increase after administration of seasonal influenza vaccine. The authors suggested that the presence of cross-reactive pH1N1 antibody in western populations could be due to repeated seasonal influenza vaccination, rather than exposure to older, seasonal H1N1 influenza viruses The significantly higher proportions of subjects over 85 years with cross-reacting antibodies in pre-pandemic specimens in our study, are consistent with results of recent studies from England and Finland et al showed comparable age-related differences in seroprevalence increases in London and the West Midlands, where infection rates were highest, but increases only in children under 15 years in other areas We demonstrated an overall increase in the seroprevalence of pH1N1 antibodies with titres≥40 (corresponding with infection rates) of nearly 16% in NSW residents following the southern hemisphere 2009 winter. Increases were restricted to age-groups less than 65 years, particularly the 12–17 (1992–7 birth cohort) and 18–34 (1975–91) year age groups with increases of 34.5% and 24.3%, respectively. In a similar study, Miller In addition to pH1N1-specific antibodies, we also studied the prevalence, in post-pandemic specimens, of antibodies to four recent seasonal viruses, including two A/H1N1 and two A/H3N2 strains. In general the highest seroprevalence rates and GMTs were in the same young age-groups, in which pH1N1 antibodies were most prevalent, although there was some variation between strains. However, a major difference was the relatively high seroprevalence of pH1N1 antibody compared with the other four recent seasonal influenza A viruses in those aged over 85 years (and to a lesser extent in the 65–85 year group). This supports the hypothesis that repeated infection by seasonal influenza A viruses or vaccination boost responses to the first influenza A virus to which these older people were exposed when very young - which is likely to have been A/H1N1(1918)-like viruses - rather than to more recent seasonal viruses. The proposition that recent seasonal influenza A virus vaccine can provide modest protection against pH1N1, despite antigenic differences, has been suggested previously In summary, we have shown that the highest rates of infection were among children over 12 years and young to middle-aged adults. The elderly were largely spared, confirming clinical and epidemiological data. As expected, infection rates were higher in Sydney than in smaller cities and rural areas of NSW. The study provided evidence of previous exposure to an antigenically similar influenza A virus in the oldest age-groups (>85 years and 65–85 years). The overall infection rates in NSW indicate that pH1N1 infections are likely to recur with at least moderate frequency in younger age-groups during the 2010 winter influenza season, unless there is high pH1N1 immunisation uptake. |
Fungi are the main organisms responsible for the degradation of biopolymers such as lignin, cellulose, hemicellulose, and chitin in forest ecosystems. Soil surveys largely target fungal diversity, paying less attention to fungal activity.−2 y−1 in form of sodium nitrate pellets since 1994, which led to increased accumulation of organic matter in the soil. Our aim was to assess, in samples taken from all six plots, transcript-level expression of fungal genes encoding lignocellulolytic and chitinolytic enzymes. For this we collected RNA from the forest soil, reverse-transcribed it, and amplified cDNAs of interest, using both published primer pairs as well as 23 newly developed ones. We thus detected transcript-level expression of 234 genes putatively encoding 26 different groups of fungal enzymes, notably major ligninolytic and diverse aromatic-oxidizing enzymes, various cellulose- and hemicellulose-degrading glycoside hydrolases and carbohydrate esterases, enzymes involved in chitin breakdown, N-acetylglucosamine metabolism, and cell wall degradation. Among the genes identified, 125 are homologous to known ascomycete genes and 105 to basidiomycete genes. Transcripts corresponding to all 26 enzyme groups were detected in both control and nitrogen-supplemented plots.Here we have focused on the organic horizon of a hardwood forest dominated by sugar maple that spreads widely across Eastern North America. The sampling site included three plots receiving normal atmospheric nitrogen deposition and three that received an extra 3 g nitrogen mMany of these enzyme groups are known to be important in soil turnover processes, but the contribution of some is probably underestimated. Our data highlight the importance of ascomycetes, as well as basidiomycetes, in important biogeochemical cycles. In the nitrogen-supplemented plots, we have detected no transcript-level gap likely to explain the observed increased carbon storage, which is more likely due to community changes and perhaps transcriptional and/or post-transcriptional down-regulation of relevant genes. They provide essential ecosystem services, such as decomposing organic matter, nutrient cycling, and in the case of mycorrhizal species, also nutrient transfer to plants −1) −2 y−1 to ambient deposition), slowed the decay of plant detritus, thereby significantly increasing soil carbon storage in a temperate forest dominated by sugar maple (Acer saccharum Marsh.), that spreads widely across Eastern North America To elucidate the roles that fungi play in carbon sequestration and ecosystem functioning, there remains much to be learned about their contribution to biopolymer degradation and biogeochemical cycling. A problem that received considerable attention is the link between carbon cycling and nitrogen availability. Additional nitrogen can stimulate early-stage decomposition of plant litter and soil organic matter, but it suppresses this activity at later stages, when humus and lignin are abundant One cause of this nitrogen-supplementation-induced slowing of plant detritus decomposition might be that single biodegradation steps involving important lignocellulolytic enzymes are “switched off”, leaving gaps in the degradative carbon cycle. Accumulating intermediates might then participate in negative feedback loops affecting other genes. It is known, for instance, that the expression of fungal genes required for cellulose biodegradation is subject to regulations such as catabolic repression Here we have focused on this same sugar-maple-dominated forest site, with the intention of identifying transcriptionally expressed fungal genes encoding key lignocellulolytic, chitinolytic, and related enzymes. For this, we have isolated total RNA from the forest soil, reverse-transcribed it, and synthesized cDNAs using long-distance PCR (LD PCR), thus providing templates for subsequent detection of relevant transcripts by PCR. As few primers are available for accessing such genes, our first goal was to develop molecular tools for detecting transcripts encoding a wide range of fungal enzymes that are both ecologically interesting and potentially useful in biotechnology Twenty-three degenerate primer pairs were developed for PCR-based detection of transcripts related to biopolymer degradation in the organic horizon of the above-mentioned forest site . We further detected transcripts of aromatic-oxidizing enzymes, including aromatic peroxygenases Among the enzyme groups highlighted, 7 are involved directly or indirectly in the breakdown or conversion of lignin or in the oxidation of aromatic derivatives. Lignin has a complex three-dimensional structure based on phenyl propane units, and provides structural rigidity in woody plants Phanerochaete chrysosporium, that catalyzes key steps in the degradation pathway of vanillate, an intermediate in general lignin breakdown, suggests a contribution to soil turnover processes involving fungi 3+ ions Fungal tyrosinases probably act mostly intracellularly, e.g. in pigmentation or detoxification processes, however there remains a need to investigate their contribution to extracellular phenol or lignin conversions in soils. Interestingly, the expression of a specific fungal tyrosinase was recently monitored in a forest soil, suggesting its potential importance Enzymes related to the degradation of cellulose and hemicellulose, the most abundant biopolymers in terrestrial ecosystems http://www.cazy.orgCellulose, a linear polymer consisting of D-glucose monomers linked by β-1,4-glycosidic bonds, is the major structural component of cell walls in woody plants. To degrade cellulose and hemicellulose, fungi use a panoply of glycoside hydrolases (GH) and carbohydrate esterases (CE) Hemicellulose, another major component of forest soil inputs from wood and leaves, is a frequently branching polymer with a heterogeneous composition. Its building blocks are mainly pentoses, hexoses, hexuronic acids, and deoxyhexoses N-acetyl-D-glucosamine monomers linked via β-1,4-glycosidic bonds N-acetylhexosaminidase . Interestingly, we also amplified cDNA corresponding to the hydrolysis of nitrogen-rich S-formylglutathione.Lastly, our study highlighted six groups of chitinolytic or related enzymes . Chitin,In this study we have looked only at transcript-level gene expression in the soil horizon, so our data can tell us nothing about post-transcriptional regulation. It should be stressed, however, that many of the enzyme activities highlighted here have been detected with various classical enzymatic substrates in soil extracts from the organic horizon of this research site vs. ectomycorrhizal basidiomycetes to decomposition and soil cycling in forest soils vs. saprotrophic basidiomycetes vs. saprotrophic ascomycetes) are most important in soil cycling processes.How different kinds of fungi contribute to soil turnover processes is a major question in forest soil ecology. Basidiomycetes are regarded as major degraders of wood resources and are characterized as white-rot fungi decaying preferentially lignin components or brown-rot fungi decaying primarily cellulose −2 y−1 to ambient deposition). This means increased carbon storage in the soil of a temperate forest spreading widely across Eastern North America Much debate centers on the mechanisms governing carbon sequestration in the globally important carbon sink constituted by the forests of the northern hemisphere −2 y−1 applied to the forest floor in equal increments over the growing season In the above-mentioned field-based experiment, replicate plots continuously received either ambient or simulated increased atmospheric nitrogen deposition over a 14-yr period beginning in 1994. The simulated deposition treatment consisted of 3 g sodium nitrate mAs transcripts corresponding to all 26 enzyme groups highlighted here were found in both nitrogen-supplemented and control soils , our datFungi provide essential ecosystem services, even in a changing environment. Future research should aim to understand molecular regulatory mechanisms in soils in order to draw conclusions about effects on ecosystems. Some of the tools provided here may help to establish links between fungal communities, their enzymatic activities in soils, and the consequences for ecosystems. Many of the fungal enzymes highlighted in this work receive much attention in applied biotechnological research, and the molecular tools developed here may find further use in both basic and applied research.Acer saccharum Marsh. This is the southernmost site of a zone involved in a long-term, 500-km climatic and nitrogen-deposition gradient study begun in 1994 −2 yr−1, lowest amount in the northernmost site, highest in the southernmost site). At the Oceana site, three 30-m×30-m plots receive ambient atmospheric nitrogen deposition and three 30-m×30-m plots receive simulated increased atmospheric nitrogen deposition. The simulated atmospheric nitrogen deposition treatment was initiated in 1994 and consists of six equal applications of sodium nitrate (NaNO3) delivered as dry pellets to the forest floor over the growing season. The soil is sandy, mixed, a mesic Entic Haplorthod. Soil sampling was done in November 2007 after leaf senescence. In each of the six plots, 10 random 0.1 m×0.1 m litter samples were collected, composited, and homogenized (by cutting the litter in 1–2 cm2 pieces and mixing them in a plastic container), in order to ensure plot coverage and representation of all overstory tree species. The homogenized samples were immediately transferred to liquid nitrogen for RNA extraction.Soil samples were taken from a long-term study site in Oceana County, Michigan, USA , a northern hardwood forest dominated by via 17 cycles of a long-distance PCR (LD-PCR) using the SMART™ PCR cDNA Synthesis Kit .A previously published protocol http://www.cazy.orghttp://www.cazypedia.org) or FOLy . All sequences were submitted to GenBank and are available under accession numbers FJ040216-FJ040219, FJ040222-FJ040225, GU734340-GU734565.Degenerate primer pairs for amplifying coding sequences corresponding to fungal ligninolytic, cellulolytic, hemicellulolytic, and chitinolytic enzymes were developed on the basis of reference protein sequences from curated databases like CAZy Click here for additional data file.Table S2Transcribed genes giving a blastp match, putative fungal phylum , derived ambient (A) or nitrogen-amended (N) plots, and accession number.(0.50 MB RTF)Click here for additional data file. |
The octahedral [PtCl6]2− dianion is located on an inversion centre. π–π interactions between neighboring acridinium cations produce stacks along the a axis; the shortest distance between the centroids of the six-membered rings within the cations is 3.553 (9) Å. In the crystal, two independent intermolecular O—H⋯Cl hydrogen bonds, both involving the same Cl atom of the anion as acceptor, give rise to chains also running along the a axis; in addition each water molecule, as a hydrogen-bond acceptor, is linked to the acridinium N—H group.The asymmetric unit of the title compound, (C DOI: 10.1107/S1600536810009566/ya2119Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: |
In this study, we extend these investigations asking specifically whether the TNF-α effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-α on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-α significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-α were inhibited by the general protease inhibitor α2 macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-α on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF- Proteolytic degradation of the basement membrane and the extracellular matrix (ECM) and movement through it are regarded as essential steps in the cascade of events leading to successful tumour cell invasion and metastasis. Studies on human melanoma have provided evidence that the plasminogen activator system and the matrix metalloproteinase (MMP) enzyme systems play an important role opposed cell attachment to ECM substrates and cell invasion through fibronectin, both of which would be consistent with TNF-α promoting metastasis and α-MSH providing partial protection against the action of TNF-α.Studies on the effects of TNF-α on melanoma cell migration and degradative enzyme activity.In the current study, we extend our investigations into the role of inflammation in melanoma invasion by studying the effects of TNF-α on melanoma cell general proteolytic enzyme expression and expression/activation of MMPs -2 and -9 and studied the extent to which degradative enzymes are involved in the TNF-α induced cell responses by looking at the effect of the general protease inhibitor α2 macroglobulin on TNF-α stimulated cell migration and invasion.Specifically, we examined the effect of TNF-α and α2 macroglobulin from Roche Diagnostics (Germany); and fluorescent BODIPY® casein (EnzChek® Protease Assay Kit-green fluorescence) from Molecular Probes Inc. . Tissue culture plastics were purchased from Corning Costar Corporation . All other chemicals were of analytical grade.Culture medium, additives and antibiotics were obtained from Gibco BRL and Sigma Chemicals Ltd. . Foetal calf serum (FCS) was purchased from GlobePharm Limited ; newborn calf serum (NBCS) from Sigma Chemicals Ltd. ; trypsin/ethylenediaminetetraacetic acid (EDTA) and phosphate-buffered saline (PBS) tablets from Oxoid Ltd. . Human fibronectin was purchased from Sigma Chemicals Ltd. ; human recombinant TNF-−3 mol l−1 glutamine, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin sulphate.The human cutaneous melanoma cell line HBL was established in Professor Ghanem's laboratory from a lymph node metastasis of a nodular malignant melanoma , 0.2% (w/v) D-glucose solution, 0.1% (w/v) bovine serum albumin (BSA), 2 × 10−3 mol l−1 glutamine, 100 IU ml−1 penicillin, 100 μg ml−1 streptomycin sulphate and the relevant concentration of TNF-α was added. This serum-free medium was used for the fibronectin invasion assays as described later (serum-free invasion assay medium – SFIAM) and subsequently in all the assays where a 24-h preincubation period was required in order to keep the experimental culture conditions the same.For the experiments where a 24-h preincubation was required, the cell culture medium was changed to RPMI-1640 medium (Gibco) supplemented with 20 ng mlα and α2 macroglobulin on the invasion of the melanoma cells through a layer of human fibronectin. Briefly, a suspension of 1.8 × 105 HBL melanoma cells (contained in 500 μl SFIAM with or without the appropriate drug concentration as required) was added to Nunc™ tissue culture inserts containing a polycarbonate filter with 8 μm diameter pores covered with fibronectin (100 μg ml−1). Cells were incubated for 20 h under standard culture conditions. At the end of the incubation period, the cells that had invaded through the filter (cells attached to the bottom of the well and to the undersurface of the filter) were collected separately from the cells that had not invaded (cells remaining attached to the upper surface of the filter). A combined analysis of the cell counts was used to calculate the percentage of the total population of cells that had invaded through the fibronectin monolayer.A modification of the method we first described in α 200 U ml−1 for the last 24 h before passaging and adding to the Nunc™ tissue culture inserts.When a preincubation period was required prior to the invasion assay, the cells were cultured in SFIAM with or without TNF-α2 macroglobulin was used as a general protease inhibitor after initially confirming its activity against trypsin prior to using it to inhibit proteolytic activity in the cell culture medium during the fibronectin invasion assay.α-stimulated cells (incubated with TNF-α 200 U ml−1 for 24 h) were detached from the tissue culture flask and were lysed using cell extraction buffer for 15 min at 4°C. The cell conditioned medium was also collected for gelatin zymography analysis (following centrifugation at 2000 g for 5 min to remove any cellular debris).Cell extraction of MMPs was performed when cell cultures were 70–80% confluent. Control and TNF-3 cells per well with or without TNF-α for 24 h. At the end of the preincubation period, the conditioned medium was collected for measurement of proteolytic activity, and extraction from the cells was performed according to the protocol described above for extraction of MMPs.For the purpose of extraction of general proteolytic activity, the melanoma cells were cultured in a 24-well plate at a density of 50 × 10s-indacene-3-propionic acid (BODIPY® FL) . BODIPY® FL casein is a conjugate consisting of casein labelled with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-μg ml−1 was used in each experiment to demonstrate proteolytic degradation of BODIPY® casein. The above concentration was selected after initial experiments to confirm that the fluorescence emitted by the cleaved substrate was proportional to the concentration of trypsin. The following controls were also included in each assay: BODIPY® casein alone, fresh culture medium (SFIAM) with BODIPY® casein and extraction buffer with BODIPY® casein. Cell extracts were diluted 1 : 20 in assay buffer before being added to the fluorescent substrate as experimentation showed this to be the optimum dilution to minimise the effect of the extraction buffer itself on the fluorescent BODIPY® substrate.As an internal control for the assay, trypsin at a concentration of 3.3 α treated cell extracts (24-h cell preincubation in SFIAM containing TNF-α 200 U ml−1 as described above) and conditioned medium (collected at the end of the 24-h preincubation period). Proteins were separated in nonreducing, nondenaturing conditions using a 10% resolving polyacrylamide gel containing 0.5% gelatin substrate. Internal human purified MMP-2 and MMP-9 standards were included at concentrations of 20 ng per lane. After electrophoresis, the zymographs were developed, fixed and stained according to the protocol described by Zymography was carried out on control (nontreated) and TNF-A modified ‘scratch wound’ migration assay was used for the assessment of the migration of the melanoma cells. The method was previously described by 5 cells per ml per well and were incubated for 24 h under standard culture conditions. After 24 h, when the cells had attached to the bottom of the tissue culture dish, the confluent cell monolayer was scratched with a plastic pipette tip to create a cell free zone in the well. The culture medium was removed and was replaced with an equal volume of fresh SFIAM with or without TNF-α 200 U ml−1 or α2 macroglobulin. The cells were incubated for a further 24 h under standard culture conditions. During this period, photographs of the scratched cell monolayer were taken at predetermined time points using an Apple McIntosh video microscope (Open Lab 3.0.4 software) and measurements of the distance between the two edges of the scratch were recorded for each different time point. The percentage reduction of the distance between the scratch edges at the different time points represented the migration rate of the melanoma cells. Triplicate wells were used for each experimental condition.Cells were plated in a 24-well culture plate in serum containing Ham's F10 culture medium at a density of 2 × 10vs stimulated cell samples were analysed using Student's paired t-test for fibronectin invasion assays, migration assays and for general protease activity assays.Control α ranging from 100 to 500 U ml−1 on HBL melanoma cell invasion through fibronectin over 20 h. Unstimulated (control) cells showed a mean invasion rate of 24.6±2.7% (n=4 experiments). Stimulation with TNF-α 100 U ml−1 significantly increased the invasion rate of the cells to 33.3±3.5% (P<0.05), which represents an increase of +35% above the control level of invasion. Stimulation with higher concentrations of TNF-α (250 and 500 U ml−1) was less effective and the increases in invasion did not reach statistical significance.Figure 1Aα, cells were preincubated with TNF-α for 24 h prior to the invasion assay and were also subsequently stimulated with TNF-α again during the period of the invasion assay (a further 20 h). Preincubation with TNF-α resulted in a 43% increase in invasion above control level . Further addition of TNF-α to the invasion assay did not increase any further the invasiveness of these preincubated cells (results not shown). On the basis of these data, we concluded that the biological effects of TNF-α on invasion had occurred fully within 24 h.To determine the optimum period of exposure for maximum response to TNF-α on melanoma cell migration. Cells stimulated with TNF-α 200 U ml−1 (cytokine present during the 24-h assay) had a significantly higher migration rate compared to the control (nonexposed to TNF-α) cells. The migration rate (represented by the percent reduction of the scratch width) of the TNF-α stimulated cells was 53.2±4.4% by 24 h, whereas the migration rate of the control cells at the same time point was 43.8±2.9%. This represented a 21% increase in migration as a result of TNF-α stimulation, P<0.05 based on eight experiments (as illustrated in α stimulated) at 8 or 24 h.A ‘scratch wound’ model was used to examine the effect of TNF-α 200 U ml−1for 24 h prior to cell extraction and collection of cell conditioned medium for gelatin zymography . α2 macroglobulin at a concentration of 2 U ml−1 was used for future experiments as this achieved 84% inhibition of trypsin activity.To confirm that the fluorescence emitted by cleavage of the BODIPY® casein substrate was representative of proteolytic enzyme activity, initial experiments used serial dilutions of trypsin and the lower chamber (invading cells) was sampled at the end of the 20-h assay period and the fluorescence of the sample, representing protease activity, was measured. TNF-α did not increase general protease activity in the conditioned media in these experiments (results not shown).To measure noninvasively whether cell surface bound proteolytic activity was modified by TNF-α on proteolytic activity of HBL melanoma cells, it was still conceivable that levels may have been beneath the level of detection of these biochemical assays and that proteases played some role in the invasion of these cells through fibronectin. Accordingly, we used α2 macroglobulin, an inhibitor of all classes of endoproteases, to examine its effect on control and TNF-α stimulated HBL melanoma cells. α2 macroglobulin inhibits proteases present in the cell conditioned medium or on the cell surface, but it cannot enter the cell or penetrate rapidly into the extracellular matrix due to its large size (720 kDa tetrameric glycoprotein).Although we had failed to show any effect of TNF-α2 macroglobulin (2 U ml−1) was added to the cell suspension in the upper chamber with or without TNF-α 200 U ml−1 for the 20-h culture period of the assay. Controls included cells with no stimulants and cells where only TNF-α was added during the invasion assay. In three separate experiments, α2 macroglobulin had no significant effect on cell invasion through fibronectin (−6% of the control level of invasion), as summarised in Figure 4AP<0.05) in invasion observed following stimulation with TNF-α (+21% of the control) was completely inhibited by the addition of α2 macroglobulin.Using the model of fibronectin invasion, α2 macroglobulin (2 U ml−1) was added to cells alone or in combination with TNF-α (200 U ml−1) for the 24-h duration of the migration assay. The migration rate of the non-TNF-α stimulated cells decreased by −26% when α2 macroglobulin was added was completely inhibited by the addition of the protease inhibitor insults. As such, it is invariably associated with the early phases of wound healing and persists to a certain degree, until regeneration of the injured tissues and restoration of function have been achieved. .vs narrow margin excisions were compared and significant inhibition by α-MSH in all the cell types examined within 24 h of TNF-α exposure for the HBL melanoma cells expression by the malignant cells . Upregulα stimulation, we examined the expression and activation of MMPs -2 and -9 using gelatin zymography, and the general protease activity of the melanoma cells using a quenched fluorescent substrate assay. As maximal upregulation of cell invasion was achieved with TNF-α between 100 and 250 U ml−1 and previous data from our group demonstrated maximal activation of NFκB in these cells with 200 U ml−1 of TNF-α in SK-MEL-109 melanoma cells. This suggests that TNF-α may be a potent regulator of degradative enzyme activity during malignant cell invasion. More importantly, this study demonstrates that the TNF-α cell response can be modulated (both upregulated and downregulated) at a postreceptor level via activation of different second-messenger systems suggesting that the effect of TNF-α on various target cells can be modified inside the cell to produce the cell's final response to the cytokine.The literature also reports that TNF-α on the functional biology of different human malignancies including melanoma (Although our current study reports observations made on a single cell line, our data are in agreement with previous reports from the literature on the effects of TNF-melanoma and exteα upregulates malignant melanoma invasion and migration in vitro. Taken together, the results of our current study combined with the data we have previously published (α may exert its proinvasive effect on HBL melanoma cells via an integrin-dependent mechanism as well as a modest upregulation of degradative enzyme activity not readily detected in general protease assays.In conclusion, we report that the proinflammatory cytokine TNF- |
An increasing number of genetic variants have been identified for many complex diseases. However, it is controversial whether risk prediction based on genomic profiles will be useful clinically. Appropriate statistical measures to evaluate the performance of genetic risk prediction models are required. Previous studies have mainly focused on the use of the area under the receiver operating characteristic (ROC) curve, or AUC, to judge the predictive value of genetic tests. However, AUC has its limitations and should be complemented by other measures. In this study, we develop a novel unifying statistical framework that connects a large variety of predictive indices together. We showed that, given the overall disease probability and the level of variance in total liability (or heritability) explained by the genetic variants, we can estimate analytically a large variety of prediction metrics, for example the AUC, the mean risk difference between cases and non-cases, the net reclassification improvement (ability to reclassify people into high- and low-risk categories), the proportion of cases explained by a specific percentile of population at the highest risk, the variance of predicted risks, and the risk at any percentile. We also demonstrate how to construct graphs to visualize the performance of risk models, such as the ROC curve, the density of risks, and the predictiveness curve (disease risk plotted against risk percentile). The results from simulations match very well with our theoretical estimates. Finally we apply the methodology to nine complex diseases, evaluating the predictive power of genetic tests based on known susceptibility variants for each trait. Recently many genetic variants have been established for diseases, and the findings have raised hope for risk prediction based on genomic profiles. However, we need to have proper statistical measures to assess the usefulness of such tests. In this study, we developed a statistical framework which enables us to evaluate many predictive indices analytically. It is based on the liability threshold model, which postulates a latent liability that is normally distributed. Affected individuals are assumed to have a liability exceeding a certain threshold. We demonstrated that, given the overall disease probability and variance in liability explained by the genetic markers, we can compute a variety of predictive indices. An example is the area under the receiver operating characteristic (ROC) curve, or AUC, which is very commonly employed. However, the limitations of AUC are often ignored, and we proposed complementing it with other indices. We have therefore also computed other metrics like the average difference in risks between cases and non-cases, the ability of reclassification into high- and low-risk categories, and the proportion of cases accounted for by a certain percentile of population at the highest risk. We also derived how to construct graphs showing the risk distribution in population. Genome-wide association studies have succeeded in uncovering many common genetic variants underlying complex diseases, raising hope for individualized risk prediction based on genomic profiles. Although the effect size of a single genetic marker is typically very modest and unlikely to be useful in risk prediction, prediction based on a collection of susceptibility variants may be promising. Several commercial companies are already offering disease risk estimates based on genomic profiles of susceptibility variants. However, it remains controversial whether such tests will be useful clinically. This calls for appropriate measures to evaluate the performance of genetic risk prediction models. It should be noted that a high level of statistical significance does not equate to good predictive power A very popular method of assessing the discriminatory ability of test is the AUC . AUC can be defined as the area under the receiver operating curve, which is a plot of sensitivity versus 1-specificity. AUC is also equal to the probability that the test score or predicted probability is higher for a case than a non-case. A few previous studies have investigated the use and performance of AUC in genetic tests. Janssens et al. Despite its widespread use, the ROC curve and AUC are not without limitations and they are not the only ways to assess the performance of a prediction model. For instance, it has been pointed out that that the AUC is not directly related to the absolute disease risks (i.e. probability of disease given test result), which is often of great clinical interest In this study, we propose a novel unifying statistical framework that connects different measures of predictive power together. The framework is based on the liability threshold model, which assumes an underlying liability that is normally distributed. Affected individuals have a liability above the threshold. We show that given the overall disease prevalence and total variance in liability explained by the set of susceptibility variants, it is possible to estimate analytically the aforementioned prediction indices and construct graphs to visualize the performance of risk models.Here we establish links to various measures of predictive power within our variance explained framework. We will derive analytic expressions to evaluate different prediction indices.m) by known variants (or other risk factors) and the corresponding liability score for each genotype will be presented elsewhere and the other comprising other risk factors yet to be found. The measurable liability is normally distributed with mean 0 and variance equal to the total variance (or heritability) explained by the known variants (z) is normally distributed with mean z and variance 1−m and Our statistical framework is based on the liability threshold model. The methods to derive total variance in liability explained (Vc. The absolute disease risk (R) is given by the chance that the overall liability, conditioned on the measurable liability, exceeds the threshold. R can be expressed as T is the liability threshold. The liability threshold is related to the overall probability of disease in the population (K), with K can also be the probability of disease in a given period of time, say the 5-year or 10-year risk of disease. It is easy to see that the measurable liability is monotone increasing with the predicted risk, thus ROC curve constructed using either criterion will be identical.Consider a 2×2 table with disease status and test result . Test isc. Pr(Disease +ve|test+ve) is given by the average risk of people whose percentiles of measured liability exceeds the given cut-off c:z denotes the liability score at a certain percentile from the distribution of measurable genetic factors. p is a random variable representing the percentile of the measurable liability. Note that we need to integrate over all the percentiles above the cut-off c.To construct the ROC curve, we need to evaluate specificity and sensitivity at different percentile cut-offs. Test positive can be defined as having a measured liability higher than a certain percentile cut-off z can also be expressed as the inverse normal of p, i.e. in the whole population. The distributions of the measurable liabilities conditioned on the affection status are also usually close to normal curves. By the Pearson-Aitken (PA) formula The binormal ROC curve is a classic example in ROC methodologies. In this model, we assume the test results are normally distributed in the affected and unaffected individuals. In our case, the test results are the measurable liability scores. We have already assumed the measurable liability follows a normal distribution A and affected individuals, the mean of aσ2 and the variance of σ2 [1−(1−b) σ2].We consider the overall and measurable liability of an individual, denoted by d = unaffected subjects, the mean of cσ2 and the variance of σ2[1−(1−d) σ2].Similarly, selection for unaffected individuals changes the overall liability to a truncated normal distribution (this time the truncation is from above) with mean The measurable liabilities in the affected and unaffected groups can be assumed to follow normal distributions:p to be the subject of formula,p) of them have predicted risks above the risk threshold R and will be classified as “high-risk”. The true positive rate (TPR) is simply 1−p. By the same argument, we can also compute the approximate false positive rate (FPR) ( = 1- specificity), byBy assuming normality in the measurable liability distributions in cases and controls, we can also provide simple formulas to approximate the TPR (i.e. sensitivity) and FPR (i.e. 1-specificity) given a risk threshold. The idea is to convert the absolute risk to its corresponding risk percentile within cases or controls. Within cases, we haveA major purpose of risk prediction is to stratify individuals into different risk categories of clinical importance. The ROC curve, however, does not typically display risk thresholds. As shown by Pepe et al. Suppose we are going to predict disease risks based on a set of known susceptibility genes or variants. What will be the distribution of the predicted risks in the whole population?R can be represented byT is the liability threshold, p is the percentile of measurable risk derived from known genetic factors.The predicted absolute risk of disease g(R)] is derived by differentiation of the cdf, or dp/dR. The detailed mathematical derivations and results may be found in the supplementary methods (The graph of cumulative density function (cdf) of the predicted absolute risk can be obtained by plotting the risk percentiles against the predicted risks. The probability density function (pdf) is worse than erroneously moving up the risk category of a healthy individual [the component P(up|D = 0)] and treating the person. This may be true if the morbidity or mortality of the disease is much greater than the side-effects from treatment. To tackle this problem one can for example incorporate different loss functions for the components. This may be done within our framework by weighing the four components differently. In a similar vein, Gail We have used NRI to assess how a new test will improve the re-classification of subjects into risk categories. The NRI may also be viewed as the difference in expected loss between the new test and the old. Recalling the formula for NRI,Another noteworthy limitation is that if there exists more than one risk bin, NRI does not consider the number of risk categories changed upon adding the new marker. For example, say there are three risk categories, placing a case in the lowest risk bin and the second-lowest bin will have the same effect on NRI. To correct this problem, we may consider giving numeric scores that reflect the number of categories shifted, as suggested in in the specified period of time should be provided to compute the correct variance explained. The rest of the calculations remain unchanged. Concepts of absolute risk estimation were discussed for example in We have mainly considered lifetime risk when calculating predictive indices. In clinical practice the risk in a given period of time may be more relevant than the lifetime risk. For example, the ATP III guideline quoted above relies on 10-year risk of coronary heart disease. The use of tamoxifen for breast cancer prevention is dependent on the 5-year risk of disease We have not considered the variability of the predictive metrics in this study. It should be noted that the disease probability and the variance explained by known variants are both subject to variations, and so will be the predictive metrics derived from these two quantities. One should be careful in interpretation of the results if the disease probability or effect sizes of variants are estimated from small sample sizes.We noticed a recent study on AUC estimation in the context of genetic risk prediction https://sites.google.com/site/honcheongso/software/pred-metrics.Evaluation of a risk prediction model is no simple task, and inevitably we cannot perfectly deal with every complexity involved. Nevertheless, we hope the current study will stimulate more thoughts on the proper assessment of prediction models and provide a convenient and useful methodology for researchers to assess the predictive ability of sets of susceptibility loci. Programs (written in R) to implement the methodology presented in this paper are available at Figure S1Graphs showing risk distribution and predictive power of known susceptibility variants for bipolar disorder.(0.47 MB TIF)Click here for additional data file.Figure S2Graphs showing risk distribution and predictive power of known susceptibility variants for prostate cancer.(0.24 MB TIF)Click here for additional data file.Figure S3Graphs showing risk distribution and predictive power of known susceptibility variants for coronary artery disease.(0.48 MB TIF)Click here for additional data file.Figure S4Graphs showing risk distribution and predictive power of known susceptibility variants for Crohn's disease.(0.47 MB TIF)Click here for additional data file.Figure S5Graphs showing risk distribution and predictive power of known susceptibility variants for type 1 diabetes mellitus.(0.47 MB TIF)Click here for additional data file.Figure S6Graphs showing risk distribution and predictive power of known susceptibility variants for type 2 diabetes mellitus.(0.47 MB TIF)Click here for additional data file.Figure S7Graphs showing risk distribution and predictive power of known susceptibility variants for schizophrenia.(0.47 MB TIF)Click here for additional data file.Figure S8Graphs showing risk distribution and predictive power of known susceptibility variants for systemic lupus erythematosus (SLE).(0.48 MB TIF)Click here for additional data file.Table S1List of the risk allele frequencies (RAF) and odds ratios (OR) of 30 established susceptibility variants for prostate cancer.(0.05 MB DOC)Click here for additional data file.Table S2Predictive indices from simulations (sim) compared to theoretical estimates (theo).(0.07 MB DOC)Click here for additional data file.Table S3NRI and its components in simulations and theoretical estimates.(0.05 MB DOC)Click here for additional data file.Text S1Supplementary methods.(0.11 MB PDF)Click here for additional data file. |
Neurology is regarded as a difficult component of the medical curriculum. This has been so marked that the term neurophobia and its effects are being investigated. Given the impact of neurological disorders worldwide, neurophobia has the potential to affect the diagnosis and management of such cases."What is your current level of interest in the following medical specialties?"; "What is your current level of knowledge in the given medical specialties?"; "Do you think the subject is easy or difficult?" and "Why do you think neurology is difficult?" Students were required to answer using a Likert scale and results were tabulated into mean scores and standard errors.A cross-sectional survey was done among clinical fourth and fifth year students at the Faculty of Medical Sciences, University of the West Indies, St. Augustine, Trinidad & Tobago. A survey tool successfully used in other schools was adapted to assess perceived level of difficulty, knowledge and interest in various medical sub-specialties including, neurology, cardiology, psychiatry, geriatrics, endocrinology, respiratory medicine, gastroenterology and pediatrics. Questions asked included: The response rate was 65% (167/255). Neurology was identified as the subject which students found most difficult (score 3.89 ± 0.068) and had least knowledge of (2.32 ± 0.075). These scores were significantly different from those observed for the other disciplines (p < 0.001). The need to know basic neuroscience was identified as the biggest contributor to the difficulty associated with neurology (3.89 ± 0.072) followed closely by the complex clinical examination associated with neurology (3.69 ± 0.072). Greater clinical and practical exposure, more time being spent on the subject, and improved teaching skills of lecturers were put forward as suggestions for improving the teaching of neurology.This study provides empirical evidence that 'neurophobia' may indeed exist among the student population of the school. It suggests the need to re-visit the approach to neuroscience and neurology education and is consistent with similar trends worldwide. Historically, neurology and neurosciences in general have been regarded as one of the more difficult components of the traditional medical curriculum. While such perceptions were anecdotal for many years, the last 10–15 years have seen attempts to test and describe these assertions.'the fear of neural sciences and neurology' among medical students and even doctors [In 1994 the term neurophobia was coined by Jozefowicz to describe doctors . Similar doctors .The reasons for neurophobia are not clear. It has been suggested that the manner in which neuroscience and neurology are taught may be the cause. Another UK study seeking to explore this observed no evidence of neurophobia when: (i) a modified delivery platform that focused upon increasing the length of time spent on neurology was used and (ii) focusing of the course deliverables took place . Other fNeurophobia, if it does exist, has profound implications for the practice of health care in any nation. The World Health Organization (WHO) estimates that neurological conditions contribute approximately 6.3% to the global health burden and 28% Another consequence of neurophobia may be its impact on the number of persons who chose to specialize in neurology and other neuroscience based sub-specialties. In the UK there are less neurologists practising than in other disciplines and though hard evidence is lacking in the Caribbean, a review of the yellow pages in Trinidad & Tobago tends to suggest this may also be the case. Interestingly a recent report from Trinidad & Tobago observed that first year medical students are least inclined to chose psychiatry as their preferred specialization .Given the evidence of neurophobia from other medical schools, and its implication for public health, we have sought to assess the extent of neurophobia among the clinical medical students at our school, their perceptions towards neurology and suggestions for improvements in teaching neurology and neuroscience.This study was approved by the Faculty Ethics Committee. It was designed as a cross-sectional study to assess neurophobia among fulltime fourth and fifth year medical students of the Faculty of Medical Sciences, The University of the West Indies at the St. Augustine Campus, Trinidad & Tobago.A questionnaire was designed based upon that developed and successfully used in other studies ,3. The q• What is your current level of interest in the following medical specialties? Scored on 6 pt scale: 0 = not known/other; 1 = little or no interest; 2 = some interest; 3 = moderate interest; 4 = quite interested; 5 = very interested• What is your current level of knowledge in the given medical specialties? Scored on a 6 pt scale: 0 = not known or other; 1 = little or none; 2 = some; 3 = moderate; 4 = fair; 5 = great.• Do you think the subject is easy or difficult? Scored on a 6 pt scale: 0 = not known or other; 1 = very easy; 2 = quite easy; 3 = moderate; 4 = quite difficult; 5 = very difficult.et al (2002) and students were also asked to assess how the following factors contributed to the difficult nature of neurology: (i) The need to know basic neuroscience; (ii) The complex clinical examination; (iii) Neurology having a reputation as a difficult subject; (iv) Neurology covering such a large number of diagnoses and (v) Neurology being badly taught.Eight specialties were included in the original questionnaire but geriatrics was subsequently excluded from the analysis due to the fact that it is not focused upon as a sub-discipline at any point during the training of our medical students. The final part of the instrument focused on reasons why neurology may be perceived as difficult and ways in which improvements can be made. Questions were based upon the results of Schon Questionnaires were distributed to students in their clinical clerkship groups and students were then allowed to complete the questionnaire off site. Completed questionnaires were then collected at a later date. In all 167 completed questionnaires were collected from 90 fourth year students and 77 fifth year students. We cannot guarantee that every registered student received a questionnaire but using total enrollment as our total population the response rate for fourth year was 90/124 (73%) and for fifth year 77/131 (59%). Total response rate was 65%.Data was analyzed using the SPSS Version 12.0. Means and standard errors were calculated for questions where applicable and comparison of means done using a Student's t-test. The α-error was set at p < 0.05.When students were asked to assess the difficulty of various medical sub-specialties neurology was rated as the most difficult with a mean score of 3.89 ± 0.068. This result was statistically significant p < 0.001) and 0.52 points higher than the subject considered second most difficult, cardiology 3.37 ± 0.063 . Cardiology and psychiatry were ranked second and third respectively . Other responses clustered around 4 areas. The first was building more clinical or practical exposure into the teaching . Twenty two students (13%) felt more time was necessary to be devoted to the teaching of the subject, and 16 students (10%) felt there was a need for improved teaching tools, in particular audio-visual equipment. Finally 27 students (16%) identified the need for the actual lecturing skills of the teachers to be improved or teaching needing to be better organized.'the fear of neural sciences and neurology among medical students and doctors' [Neurology has traditionally been perceived as one of the more difficult clinical sub-specialties and as such the term neurophobia was coined to describe doctors' . Though doctors' -11.Our medical school was established in 1989 and currently has an intake of approximately 200 medical students annually. Its curriculum has followed a traditional British model with a preclinical phase lasting 3 years and a clinical phase lasting 2 years before graduation and internship. Since its inception it has inculcated Problem Based Learning (PBL) as a core teaching methodology, especially during the preclinical years, designed to give students early introduction to clinical scenarios and improve their critical thinking and decision making skills. This was hoped to bear fruit in the clinical years and one recent study notes that it has at least 'broken even' .We report that despite a PBL approach in the pre-clinical years our students still ranked neurology as the most difficult of seven medical sub-specialties by a fairly substantial margin, with approximately one quarter of all students indicating they found neurology very difficult. Similar results have been reported in other studies out of the United Kingdom and Ireland ,3. Interet al (2002) were able to extend their study to include junior doctors and it was noted that they had least confidence when dealing with neurological cases 'at the bedside' [These results were borne out when students were asked to assess the subject that they felt they had least knowledge in and neurology again scored unfavorably, having the lowest mean. Coupled with the perceived difficulty associated with neurology these findings may have significant implications. Schon bedside' . This suet al (2007) examined the possible effects of neurophobia in clinical practice within the United Kingdom. They noted that neurological diseases are increasing among the general population and this coupled with a lack of confidence among general practioners often results in over referrals to specialists [Ridsdale cialists . At the The onus therefore must be on improved training of our medical graduates. In fact we observed that neurology was not recorded as the discipline in which students had least interest coming ahead of respiratory medicine and psychiatry (though it is worth noting that the latter is a neuroscience based discipline) a finding similar to other studies . When stWithin our context basic neuroscience is taught as an eight week course in the second year of medical school. Though heavily drawing on PBL it seems to suggest that this not enough time for the students to assimilate the material required. Neurology is taught in the clinical years but not as an independent clerkship; rather it is bundled inside of the larger medicine clerkship. This arrangement for teaching neuroscience and neurology may lack focus and based on our results does not allow enough time for assimilation of the material taught.Data from the recently formed GKT Medical School in London suggest that if more time is allocated to teaching of neurology and neuroscience and this is coupled to focused course objectives, neurophobia is reduced. Students though, still perceived neurology as a difficult subject and lacked confidence in approaching problems . Other aThese interventions are clearly not complete solutions in themselves but represent movement in the right direction. They suggest that modifications in curriculum and teaching methodology can have a positive effect upon learning and such practices should be considered elsewhere. They also highlight that in our context, though PBL has brought with it some success, there is still a need to evaluate and bring refinements consistent with latest pedagogical data. Ultimately though, neurology as a sub-specialty may indeed be more difficult than other subjects and teachers must simply take the time and use all resources available to ensure adequate learning takes place."the neurologist is one of the great archetypes: a brilliant, forgetful man with a bulging cranium....who....talks with ease about bits of the brain you'd forgotten existed, adores diagnosis and rare syndromes, and – most importantly – never bothers about treatment." Schon et al even suggests that such a reputation is possibly enjoyed and encouraged by neurologists who like the notion that neurology is a discipline for which only 'young Einsteins need apply' [st century.Finally it may be worthwhile considering the perception students have of various subjects upon entering medical school. A recent study in the Caribbean noted a definite bias against psychiatry . In a sid apply' . Such stThis study highlights that neurophobia is indeed a problem among our students but having identified the problem solutions need to be considered. The students themselves have highlighted the need for increased clinical exposure and this must be considered. The Medical School in Mona, Jamaica has introduced bedside teaching from year one and it would be interesting to compare attitudes to neurology among those students as compared to ours. In addition given the general feeling that basic neuroscience is difficult, it is easy to suggest that more time be allocated to the subject. However, this is not readily achievable and is also the desire of almost all other disciplines. The solution perhaps lies in identifying topic areas that have most relevance to doctors in training and streamlining the curriculum; efforts along these lines are in fact in train across the faculty. Along these lines there is also a push to increase vertical integration throughout the curriculum which would increase clinical exposure in the early years of training and also allow the revisiting of basic science concepts during the clinical years.One obvious limitation of this study was the response rate of 65%. During the clinical years our students function in small group clerkships and rarely come together as a whole group. Given this limitation we distributed the questionnaires within the clerkships and allowed the students to complete them off site. Greater efforts could have been made to follow up with individual students to ensure a higher response rate but this was limited by manpower and the students being spread across four hospitals in different parts of the country. This meant that only the more motivated and perhaps more conscientious students returned the questionnaires, hence the response rate of sixty five percent. However it is to be expected that this population would probably also have been more focused in their attempts to 'come to grips' with neurology and so an increased response rate may have been expected to further highlight the problem of neurophobia.In conclusion we have demonstrated that neurophobia does indeed exist among our student population. While the use of PBL is still to be encouraged, the same principles that underlie its success need to be fully embraced throughout the entire medical curriculum and combined with the latest findings in medical education, to create a more focused and practical approach to neuroscience/neurology education. We suggest these results add to the growing body of data that highlights an increasing awareness for the need to modify neuroscience and neurology training to better meet the needs of the general population.The author declares that they have no competing interests.FFY was responsible for the conceptualization, coordination, design and statistical analysis of this study. FFY was responsible for writing the manuscript.The pre-publication history for this paper can be accessed here: |
Microglia are the resident macrophages in the central nervous system. In the spinal cord dorsal horn, microglia stay in resting condition during physiological sensory processing, and are activated under pathological conditions such as peripheral nerve injury. In cases such as this, the nearby resting microglia increase their motility and accumulate at the site of injury. However, direct evidence to support that nerve activity can enhance the motility of microglia has not yet to be reported. In this study we investigated whether the activation of spinal microglia under in vivo nerve injury may be mimicked by neuronal activity in the spinal cord slice preparation. We found that local application of spinal excitatory neurotransmitters, such as glutamate and substance P did not cause any change in the motility of microglial cells in the spinal cord dorsal horn. The motility of microglial cells is unlikely modulated by other transmitters, neuromodulators and chemokines, because similar applications such as GABA, serotonin, noradrenaline, carbachol, fractalkine or interleukin did not produce any obvious effect. Furthermore, low or high frequency stimulation of spinal dorsal root fibers at noxious intensities failed to cause any enhanced extension or retraction of the microglia processes. By contrast, focal application of ATP triggered rapid and robust activation of microglial cells in the spinal dorsal horn. Our results provide the first evidence that the activation of microglia in the spinal cord after nerve injury is unlikely due solely to neuronal activity, non-neuronal factors are likely responsible for the activation of nerve injury-related microglial cells in the spinal dorsal horn. Microglial cells are the principal immune-response cells in the central nervous system (CNS) ,2. In phThe spinal cord dorsal horn is a key area for nociceptive transmission and modulation . In the in vitro spinal cord slices is similar to those in spinal dorsal horn in vivo. Under the confocal microscope, the processes of ramified microglia in brain slices were very dynamic, showing rapid extensions and retractions with a speed of 1 μm/min.To investigate the microglial cells in spinal cord dorsal horn, we used transgenic mice with GFP-labeled microglia cells as previously reported ,18-20. Sp > 0.05).Glutamate is the major excitatory neurotransmitter in the spinal cord dorsal horn . To testWe also applied AMPA and NMDA at different doses 1 or 10 mM) to examine if microglial cells may be activated. Similar to the results of glutamate, neither AMPA nor NMDA application affect the status of microglial cells in the spinal cord dorsal horn . These results suggest that enhancing spinal inhibitory transmission did not affect the motility of microglia.GABA and glycine are two major inhibitory transmitters in the spinal cord dorsal horn. If microglia can be regulated by synaptic activity, we expect that application of GABA or glycine may reduce the motility of microglia. After local application of all of these drugs 1 or 10 mM), no significant change in motility was observed Figs. . The rat or 10 mMp > 0.05).Spinal nociceptive transmission is regulated by multiple neurotransmitters released locally or from descending projection fibers, including serotonin (5-HT), noradrenalin (NA), acetylcholine and opioid peptide -27. BecaP > 0.05) Fig. .Peripheral nerve injury can also increase the number of microglia in spinal dorsal horn ,13,17. IChemokines, especially MCP-1 and FNK, have been suggested to contribute to microglia activation ,31,32. HIn spinal cord dorsal horn, activated microglia can release some chemokines like NGF, BDNF, IL-1β, IL-6 and TNF-α. These factors may spread and activate adjacent microglia and neurons ,5,17,33.ATP is known to be a key regulator for microglia during the injury. Microglia expresses both ionotropic (P2X) and metabotropic (P2Y) ATP receptors. Under both in vivo and in vitro brain slice conditions, it has been reported that ATP induced an obvious enhancement of the motility of microglia and caused a chemotaxis effect ,12,19,20In this study, by combining a time lapse confocal imaging and histological observation, we demonstrated that motility of microglia in the spinal dorsal horn is not driven by neuronal activity directly. Stimulation of sensory afferent fibers at noxious intensities did not cause any activation of microglial cells. Furthermore, stimulation protocols that are known to induce long-term plasticity in spinal cord dorsal horn neurons failed to trigger any alteration in microglia. These results are important as they provide the first evidence that spinal microglia are not likely regulated by sensory inputs under physiological or pathological conditions. Although recent studies confirmed the activation of spinal microglia by nerve injury using the same transgenic mice , we suggIn physiological conditions, the majority of microglia are found to be in a resting state. They have ramified processes with continuous extending and retracting reaction for detecting changes ,19. Our Despite the expression of AMPA, NMDA, GABA, opioid and adrenergic receptors on cultured microglia -11, the Chemokines are one kind of signal involved with microglia activation. Among them, MCP-1 and FKN are considered as neuronal-microglia signals. They are reported to be expressed on DRG neurons and spinal dorsal horn neurons after nerve injury -39. MCP-In the present study, ATP application but not dorsal root stimulation can cause increased motility of microglia. This finding confirms that microglial cells in spinal cord slice preparation are similar to those in vivo in term of ATP sensitivity. Considering ATP can be released from efferent terminals after dorsal root stimulation , we thinAlthough no enhanced motility of microglia was observed, it does not exclude the possibility that biochemical changes may happen after drug application or LTP induction within microglial cells. After peripheral nerve injury, the first changes in spinal microglia are the withdrawal processes and enlarged soma , which iin vivo may be due to the combined effects of the co-release of several signals and removal of inhibitory factors.In summary, our work on the spinal cord dorsal horn indicates that the motility of spinal microglia cannot be enhanced by dorsal root stimuli or local application of neurotransmitters, neuromodulators and chemokines, which is consistent with our previous works on brain slices. These results suggest that resting microglia in the CNS may not be directly related to synaptic transmission and plasticity. Activation of microglia in a cultured dish or GFP/+ mice were used for all the experiments [Heterozygous Cx3cr1eriments . All mic2-5% CO2) artificial cerebrospinal fluid (ACSF) solution containing (in mM) 124 NaCl, 25 NaHCO3, 2.5 KCl, 1 KH2PO4, 2 CaCl2, 2 MgSO4, and 10 glucose. After removal of the dura mater, all ventral and dorsal roots, with the exception of the L3 or L4 dorsal root on one side, were cut near the root entry zone. The pia-arachnoid membrane was then removed, sparing the area around the site of insertion of the preserved dorsal root. The spinal cord, together with the attached dorsal root, was placed in a shallow groove formed in an agar block. The spinal cord was immersed in cold ACSF solution and a transverse slice (500-700 μm) with attached dorsal root was cut with a Vibratome according to the protocols reported before [2, 5% CO2 at room temperature for 1 hour and then transferred to a recording chamber and perfused with oxygenated ACSF solution at 3-4 ml/min at 34 ± 1°C. The dorsal root of the spinal cord slice was then sucked into a suction electrode for electronic stimuli.Adult male or female mice (6-10 wks old) were anesthetized with 1-2% urethan, and body temperature was kept in the range 35-37°C with an infrared lamp. A lumbosacral laminectomy was performed and a 1-2.0 cm length of spinal cord with attached dorsal roots was excised. The spinal cord was placed in cold (0-4°C) oxygenated were imaged by confocal microscope . The laser with a wavelength of 488 nm was used for GFP excitation, and 633 nm was used for DIC images. The image of microglia was collected for 8-10 consecutive focal steps of 2 μm once every 2 min using × 40, 0.8 numeric aperture water-immersion objectives. XYZT image galleries were collected, and Z projections were made for the quantification.All drugs were obtained from Sigma except monocyte chemoattractant protein-1 (MCP-1), fractalkine (FKN), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α . A picopump was used to apply neurotransmitters, neuromodulators and cytokines to induce microglial chemotaxis according to our previous reports . The diaTo study the effect of electronic stimuli on microglial motilities, the 40 volt, 0.5 ms low frequency or high frequency electronic stimuli, which were proved to induce LTP effect in different groups of spinal dorsal horn neurons, were delivered through the electrode ,28,29. TAfter the procedure for dorsal root stimuli and confocal imaging, spinal cord slices with dorsal root would be kept staying in ACSF for one hour and then removed to fixation solution containing 4% paraformaldehyde in 0.01 M phosphate buffered saline for 24 hrs. Subsequently, the spinal cords were placed into 30% (w/v) sucrose in 0.01 M PBS overnight at 4-8°C. After making a pinhole in the ventral part of the sample contralateral to the dorsal root, the fixed spinal cord was cut serially into 30 μm-thick frontal sections on a freezing microtome . Then the serial sections were mounted onto glass slides, air dried, cover-slipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine in 0.01 M PBS, and observed with the confocal microscope .The microglial motilities were analyzed by using Image-Pro Plus software . The number, distance, and volume of extending and retracting microglial processes were calculated every 2 min. Microglia quantification was performed on sections of 30 μm thickness. Only the cell bodies were taken into account. Cell counting was performed by using confocal microscope under 20× objective through IPP 5.0 software. Layers I-V in ipsilateral and controlateral sites of dorsal root were examined for each of the three randomly selected sections per mouse. Data were expressed as mean ± SEM. Statistical comparisons were performed with the Student's t-test. In all cases, P < 0.05 was considered statistically significant.The authors declare that they have no competing interests.TC carried out electrophysiological, imaging and histological experiments and drafted the manuscript. KK participated in electrophysiological and histological experiments. XYL helped with imaging experiments. MZ designed and finished the final draft of the manuscript. All authors read and approved the final manuscript. |
Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking. The following protocols will explain how to optimize the acquired video images (Part 1) and run two types of basic visuo-olfactory experiments (Part 2 & 3).The OMT is an adaptation of a magnetic tether system Optimizing the video image is crucial for the quality of the acquired data. Follow these steps for cleaner images.Place a freshly tethered fly in the arena using a pair of forceps.Position the firewire board camera (Fire-I 1394store.com) under the clear acrylic vacuum chamber such that the camera view is directly upward through the chamber, through a glass tube, to the ventral aspect of the tethered fly. Be sure that there is no IR filter coating on the lens of the video system.To illuminate the fly, use a circular array of infrared LEDs positioned just under the LED panels. Focus each LED on the fly’s body individually for uniform illumination.To reduce glare from the panel system and to minimize visual cues observed by the fly, it is useful to coat all reflective surfaces within the camera view with flat black paint. Room lights should be switched off to reduce glare and brightness modulations detectable by the fly’s eye.Visually inspect the camera image. The fly should be bright white against a black background. If the image is blurred the lens may need to be focused. If the fly is dark, the LED array may be unfocused or insufficiently powered for proper illumination. If the background contains bright objects, mount a disk of black flock paper just above the fly to minimize background reflectance.We acquire video with custom subroutines written in Matlab using the Image Acquisition toolbox.One simple experiment in the OMT is observing a hungry (starved) fly locating and then actively tracking a plume of attractive food odor. Manipulating the visual surroundings influences the fly’s ability to robustly track an appetitive food odor. Note that experiments are typically run in a random block format to minimize any bias in experimental order. All external hardware are controlled by custom software routines written in Matlab (Mathworks).Place a freshly tethered fly in the arena using a pair of forceps.3 to ensure smooth motion around the entire 360 yaw axis. If the fly does not spin smoothly discard it. If the problem persists, the likely cause is misaligned magnets. Note: If the distribution of arena heading for several trajectories during the spin trial shows significant peaks (i.e. is not flat over 360 degrees), there is likely an orientation bias due to misaligned magnets.Before every experiment, using the visual display, rotate the fly for several revolutions Present the fly with the following sample experimental conditions in random order: i) high-contrast striped pattern with water vapor emanating from the odor port; ii) high-contrast striped pattern with attractive odor vapor; iii) a single vertical stripe offset 90 degrees from the water vapor nozzle; and iv) a single 90 degree offset stripe with odor vapor. In our experience, 30 second intervals work well for experiments where flies are challenged to locate the odor source on their own. We acquire video at 30 Hz for the duration of each experimental condition and analyze the images offline using custom software routines written in Matlab keep the flies actively engaged in the experiment and to (ii) keep flies from remaining in one spot. An example experiment may have the following order: i) twenty second initial diagnostic spin; ii) five second intermediate spin; iii) twenty second condition pair #1; iv) five second intermediate spin; v) twenty second condition pair #2.A second basic experiment in the OMT is to visually drag a fly into an odor plume, instantaneously change the visual conditions, and then measure its ability to remain in the plume. Again, note that all experiments are done in a random block format to minimize any bias in experimental order.Place a freshly tethered fly in the arena using a pair of forceps.Part 2.2).Visually rotate (spin) the fly several times around the arena before every experiment as a diagnostic check to ensure that the animal has the capacity to orient in every direction and visually “drag” the fly into the plume by oscillating a small vertical stripe at the position of the plume. This takes advantage of the powerful stripe fixation behavior observed in flies [4].Fig. 1A); ii) uniform background with water vapor ; iii) high contrast panoramic stripes with odor vapor ; iv) uniform background with odor vapor . Acquire video as in 2.3. An example experiment may have the flowing order: i) twenty second initial diagnostic spin; ii) five second plume stripe oscillation; iii) twenty second condition pair #1; iv) five second plume stripe oscillation; v) twenty second condition pair #2.Now that the fly is oriented toward the plume, remove the oscillating stripe and present the following sample visual conditions: i) high contrast panoramic stripes with water vapor to rotate several revolutions in the arena to ensure a smooth and complete range of motion. In Part 2 a fly is challenged to find an odor plume on its own. To keep the fly engaged in the experiment, to reduce the effect of residual odor release, and to keep flies from maintaining a single heading throughout the experiment, it is useful to visually rotate the fly for a few seconds between trials. A fly should never localize a water vapor plume to a significant degree, and should always localize an odor plume in the presence of rich panoramic visual cues. In Part 3 a fly is visually dragged directly into an odor plume and challenged to maintain its heading within the plume against a variety of stationary visual backgrounds. If the fly is dragged into a water vapor plume, it should quickly turn away. If the fly is dragged into an odor plume and there are sufficient visual cues to mediate robust tracking, the fly will remain in the plume.Part 1 explains how to optimize the OMT video images. The video image should have a clear view of the illuminated fly on a dark black background. Overly bright images can be improved by reducing background interference, reducing the intensity of the IR LEDs, or by turning off the room lights. If the fly is improperly illuminated, adjusting the focus of the IR LEDs or modulating the intensity of the IR LEDs may fix the problem. 1. Furthermore, this system could easily be modified to include real-time video tracking. The example experimental protocols described here meant to be a starting point to ensure the proper operation of an OMT. It is possible to experiment with different visual displays and even different odors.The image acquisition for this system utilizes an inexpensive firewire board camera and software written in Matlab. Clear images are crucial for obtaining accurate data traces. The strategies described above are useful for cleaning up images in this system. Other video tracking procedures for this system have been described |
In each independent molecule, one of two terminal ethyl groups is disordered over two conformations: the occupancies of major components were fixed at 0.53 and 0.64 in the two molecules. In the crystal structure, weak intermolecular C—H⋯O hydrogen bonds link molecules into chains propagating along [10The asymmetric unit of the title compound, C Å b = 23.6608 (5) Å c = 17.3769 (3) Å β = 96.826 (1)°V = 5475.72 (19) Å3 Z = 8 Kα radiationMo −1 μ = 0.10 mmT = 296 K 0.43 × 0.25 × 0.17 mm Bruker APEXII CCD detector diffractometer74220 measured reflections10790 independent reflectionsI > 2σ(I)6912 reflections with R int = 0.041 R[F 2 > 2σ(F 2)] = 0.060 wR(F 2) = 0.184 S = 1.02 10790 reflections709 parameters10 restraintsH-atom parameters constrainedmax = 0.51 e Å−3 Δρmin = −0.41 e Å−3 Δρ APEX2 used to solve structure: SIR97 (Altomare et al., 1999SHELXL97 (Sheldrick, 2008PLATON (Spek, 2009publCIF (Westrip, 2010Data collection: 10.1107/S1600536810010512/cv2702sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536810010512/cv2702Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials: |
Sera from rabbits injected with BCG and then with endotoxin contain a factor (tumour-necrosis factor TNF) which, even at high dilutions, is cytotoxic in vitro for mouse L cells and some other cell lines. Using a 51Cr-release assay, cytotoxicity was detected as early as 7-8 h after addition of TNF serum to L cells and cell death was evident microscopically by 24 h. TNF was cytotoxic at 37 degrees C but not at 21 degrees C or 4 degrees C, and acted on both dividing and non-dividing cells. The antimetabolites sodium azide and dinitrophenol partially protected L cells from TNF, suggesting that actively metabolizing cells are the most sensitive. Treatment of L cells with trypsin did not delay cytotoxicity nor was cytotoxicity inhibited in the presence of various saccharide derivatives of cell-surface glycoproteins. Rabbit TNF was remarkably stable with a mol. wt. of 40-50,000. It was eluted with the more acidic serum proteins on ion-exchange chromatography, but precipitated in 50%-saturated ammonium sulphate. Sensitivity to TNF could not be correlated with tumourigenicity of several animal and human lines tested nor with the production of C-type viruses. |
However, data about the influence of TGFβ1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFβ1, TGFβ1 protein level has been measured by enzyme-immoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFβ1 with a range of 0–684 pg mg−1 protein. In the overall population, an increase of tumoral TGFβ1 was observed in premenopausal patients when compared to postmenopausal subgroup (P=0.0006). When patients were subdivided according to nodal status, TGFβ1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P=0.040). Multivariate analysis revealed that, after lymph node status (P=0.0002) and urokinase-type plasminogen activator (P=0.004), TGFβ1 was an independent prognostic marker for DFS (P=0.005) in the overall population. In the node-negative population, TGFβ1 was the prominent prognostic factor (P=0.010). In the same population, Kaplan–Meier curves demonstrated that high TGFβ1 level was correlated with a shorter disease-free survival (P=0.020). These data suggest that the measurement of tumoral TGFβ1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.Transforming growth factor-beta (TGF These patients were previously included in a retrospective multicenter study , 29.6% presented one to three axillary invaded nodes (N+) and 21.7% had more than three invaded nodes (N++). Among the 193 tumours graded according to Scarff, Bloom and Richardson classification (SBR), 25.5% were classified grade I, 52.8% were grade II, and 21.7% were grade III. Ductal carcinomas were diagnosed in 75% of patients, and invasive lobular carcinomas in 25% of patients.The primary treatment was tumorectomy or quadrantectomy (92%) or modified radical mastectomy (8%) with axillary dissection, followed by radiotherapy in 98% of cases. Among the 121 postmenopausal patients, 21 received no adjuvant treatment whereas 33 received hormone therapy, 33 were treated with chemotherapy, and 34 received both treatments. Among the 72 premenopausal patients, 36 received no adjuvant therapy and 36 were treated as follow: 10 with chemotherapy, 22 with hormone therapy and four patients with both treatments. The median follow-up was 94 months . At the cutoff date of this study, 16 local recurrences, 42 metastasis and 28 deaths had been recorded. Tumour samples and clinical informations were obtained under Institutional Clinical Board approval.M Tris-HCl pH 7.4, 1.5 mM EDTA, 10 mM Na2MoO4, 0.5 mM DTT and 10% glycerol. The suspension was centrifuged for 60 min at 105 000 g at 4°C. The high-speed supernatants (cytosols) were collected and stored in liquid nitrogen. For all samples, cytosolic protein concentration was determined using BCA assay . ER and PR levels were determined by enzyme immunoassay as described previously (Tumour tissues were stored in liquid nitrogen and routinely assayed for estrogen (ER) and progesterone receptors (PR) levels, according the recommendations of the European Organization for Research and Treatment of Cancer (EORTC), as previously described (β1.The remaining cytosols were frozen and stored in liquid nitrogen until used for the determination of thymidine kinase (TK) enzyme activity, urokinase-type plasminogen activator (uPA), type-1 plasminogen activator inhibitor (PAi-1), and TGFTK enzyme activity was measured using the Prolifigen TK Radioenzymatic Assay , with the modifications recommended by the EORTC Receptors and Biomarkers Study Group , according to the instructions of the manufacturer. Inactive and active forms of uPA are all recognised by the uPA ELISA kit, as is receptor-bound uPA and uPA complexed with PAi-1 and PAi-2. PAi-1 ELISA detects latent and active forms of human PAi-1 and PAi-1 complexes. The assay is insensitive to PAi-2.β1 levels in breast tumour cytosols were measured by ELISA. This assay used monoclonal antibody as capture antibody and biotinylated polyclonal antibody as detection antibody. The assay specifically measures active TGFβ1 forms. To measure total TGFβ1 present in tumour samples, biologically latent TGFβ1 was activated by acid-treatment. For this purpose, cytosols were diluted with four volumes of DPBS buffer . Samples were then incubated for 15 min at room temperature in the presence of 0.02 vol of 1 N HCl, then neutralised with equal volume of 1 N NaOH. ELISA analysis was performed in 96-well plates following the instructions of the manufacturer . Recombinant human TGFβ1 was used as standard at 0–1000 pg ml−1. A preliminary evaluation was performed to assess the buffer compatibility and the parallelism of sample dilutions. The inter- and intra-assay (n=10) CVs of a pool of tumour extracts with mean value of 134.1 pg TGFβ1 per mg protein were 7.5 and 3.9% respectively.TGFβ1 with other variables was tested with Spearman rank correlation. The associations of TGFβ1 (used as continuous variables) with other variables (used as grouping variables) were examined using Mann–Whitney U test (two categories), or in the case of more than two ordered categories by Kruskal–Wallis test. Survival curves were generated using the method of Kaplan and Meier and the log-rank test for trend was used to examine survival data. For the univariate survival analysis, DFS time and OS time were used as follow-up parameters. P-values ⩽0.05 were considered as significant.The strength of the associations of TGFβ1 in the overall or N+ or N− populations. Multivariate analysis was performed with variables eliminated in a step-down fashion. Variables with a P⩽0.05 were retained in the final multivariate models. Hazard ratios (HR) derived from the estimated regression coefficients, are presented with their 95% confidence intervals (CI).Cox multivariate regression analysis was used to evaluate the prognostic value of TGFvs postmenopausal), pathological nodal status , histologic grade , and histologic type .Variables were categorised as follows: age , pathological tumour size (⩽20 mm or >20 mm), menopausal status , all tumours were considered to be estrogen receptor-negative (ER−) if ER values <15 fmol mg−1 protein; for the premenopausal population, tumours with ER 15–205 fmol mg−1 protein (75th percentile) were classified ER+, whereas tumours of postmenopausal patients were considered as ER+ when ER level was 15–377 fmol mg−1 protein (75th percentile). In both pre- and post-menopausal populations, ER++ represents tumours with ER values exceeding the 75th percentile. In all cases, the tumours were considered to be PR-positive if values exceeded 20 fmol mg−1 protein. For all others biological parameters, cutpoints corresponded to the 25th and 75th percentiles of the distribution and from 0.27 to 54 ng mg−1 protein , respectively. TGFβ1 was detectable in 94.3% of samples and its concentration ranged from 0 to 684 pg mg−1 protein, with a median at 86.7 pg mg−1 protein.The distribution of biological factors in breast cancer samples are listed in β1 and each of the others parameters was examined in the overall population, no significant correlation could be observed between TGFβ1 and the biological and clinicopathological variables, except the hormonal status. Thus, premenopausal patients were found to express higher TGFβ1 levels than postmenopausal patients (not shown). When patient population was subdivided according to pathological nodal status, TGFβ1 remained correlated to the hormonal status in both node-negative (P=0.012) and node-positive (P=0.008) subgroups (β1 and PAi-1 (P=0.040) was observed in the node-negative population.When the correlation between TGFubgroups . Moreoveβ1 on OS and DFS was determined in the overall population and node-negative/node-positive subsets. When 25th and 75th percentiles of the distribution were used as cutoff values, TGFβ1 appeared significant (P=0.020) for DFS in the overall population (β1 levels (<42 pg mg−1 protein), 72% for the intermediate group (42–148 pg TGFβ1/mg protein) and 61% for patients with TGFβ1⩾148 pg mg−1 protein. The patients were then dichotomised according to their nodal status. While TGFβ1 was found to have no significant impact on DFS in the node-positive subgroup (not shown), high TGFβ1 levels were significantly associated with poor DFS in the node-negative population (P=0.02) were observed, whereas 17 and 38% relapses were observed for patients with intermediate and high TGFβ1 expression levels, respectively. Unlikely, the level of TGFβ1 had no impact on OS, neither in the overall population nor in the node-positive/node-negative groups (not shown).The impact of TGFpulation . The 10-(P=0.02) . Thus, aβ1 might significantly add to the contribution of the traditional prognostic factors. A significance level of 5% in the univariate analysis was chosen as the criterion for entering variables (not shown). The analysis was conducted in the overall population and node-negative/node-positive subgroups (P=0.0003) and SBR grade (P=0.0008) as independent parameters in the overall population. The prominent predictor for OS was SBR grade (P=0.004) and ER (P=0.020) in the node-positive population, and uPA (P=0.010) in the node-negative subgroup. In addition of nodal status (P=0.0002) and uPA (P=0.004), TGFβ1 was independently associated to poor DFS in the overall population (P=0.005). Whereas PAi-1 appeared as the prominent independent predictor for the node-positive patients (P=0.019), the parameter associated with DFS in the population without node-infiltration was TGFβ1 (P=0.010).A Cox multivariate analysis was performed to evaluate whether TGFubgroups . The anaβ in some tumour systems appears to involve multiple mechanisms, including loss of functional TGF-β receptor proteins . These data are in apparent opposition with some published studies, indicating that estradiol decreased the production of TGFβ1 by breast cancer epithelial cells in vitro . The activity of PAi-1 is tightly regulated on the transcriptional level, and TGFβ1 is the major regulator of PAi-1 expression and in turn of local PAi-1 activity and uPA (P=0.004), TGFβ1 was an independent prognostic marker for DFS (P=0.005) in the overall population. Furthermore, TGFβ1 remained the prominent prognostic factor in the node-negative population (P=0.010). In this late population, Kaplan–Meier curves further demonstrated that high level of TGFβ1 was correlated with a shorter disease-free survival (P=0.020). Conversely, TGFβ was not a prognostic factor for OS in the node-negative population. However, it has to be mentioned that at the cutoff date of the study, three deaths had been recorded in the node-negative subgroup. This is probably insufficient to distinguish a potential influence of TGFβ on overall survival, in this population. Whereas clinical studies in breast cancers have led to conflicting results, our data suggest that TGFβ1 has the potential to promote metastasis and recurrence for patients with breast carcinomas. It has to be noted that patients included in this study have not received modern chemotherapy, which could influence the outcomes. The fact that prognostic value of TGFβ1 was observed in node-negative population strongly suggests that TGFβ1 interferes at early stages of tumour progression, probably by making cell environment favorable for metastatic spread.The increased expression of uPA has been reported to be associated with poor prognostic for patients with breast cancer has been measured in our study, it would be helpful to determine the respective role for latent and active TGFβ as prognostic markers in breast cancers.Although the lymph node status is one of the best prognostic factors in breast cancer, it is not sufficiently accurate to predict the clinical course of the disease. Indeed, 20–30% of node-negative breast cancer patients will experience disease recurrence and metastatic dissemination. Whereas numerous predictive factors have been characterised thus far, early prognostic markers that interfere at the beginning of tumour progression are scarce. The prognostic significance of high TGF |
Subchondral fracture of the femoral head is an uncommon entity and usually occurs as an insufficiency fracture associated with poor bone quality or as a fatigue fracture in young military recruits. This condition should be considered in the differential diagnosis of acute hip pain in young patients along with transient osteoporosis and avascular necrosis of the hip. We report a case of acute onset hip pain in an asymptomatic healthy adult in which the diagnosis was made by magnetic resonance imaging and the patient responded well to conservative treatment. Subchondral fracture of the femoral head is an uncommon entity and usually occurs as an insufficiency fracture associated with poor bone quality or as a fatigue fracture in young military recruits.A 27-year-old healthy male presented to out patient department with complaints of pain in the right hip for the past 1 month. Pain was more pronounced in the front of the hip, gradual in onset, and aggravated on walking for long periods. There was no history of trauma, alcohol abuse, steroid use, or any recent increase in physical activity. There was no back pain or any radiation of pain to the knee. There were no other systemic symptoms.On physical examination, the patient had painful terminal limitation of flexion and internal rotation. There was no limb length discrepancy and the contralateral hip had a normal examination. X-rays of the pelvis with both hips in anteroposterior view and crosThe possible differential diagnosis was avascular necrosis, transient osteoporosis, and subchondral fracture. The treatment options were discussed with him and he was offered a trial of conservative treatment with the explanation that he would need a repeat MRI. At this time, the patient was put on non weight bearing crutch walking and was also put on Alendronate 70 mg once in 2 weeks.The patient followed-up at 8 weeks revealed a normal clinical examination and absence of symptoms. There was no pain on flexion and internal rotation at this time. A repeat MRI performeThe patient did not have any symptoms at 1 year follow-up.et al., who reported their observations on five asymptomatic young adults that healed spontaneously with conservative treatment.4The diagnosis in our case was established with MRI, which showed marrow edema along with the fracture line. To the best of our knowledge, there has been only one previous report on healthy adults by Kim et al., Subchondral stress fracture of the femoral head has been attributed to insufficiency fracture associated with poor bone quality 1MRI in fatigue fracture shows a serpiginous line parallel to the articular surface with a high signal on fat suppression proximal to the fracture line. The two important differential diagnoses are osteonecrosis and transient osteopenia of the hip. MRI in all the three conditions reveals bone marrow edema.Osteonecrosis is usually seen in men above the age of 40 with a history of excessive alcohol intake or chronic steroid administration.9Transient osteoporosis of the hip on clinicoradiological findings mimic that of subchondral fracture albeit the fracture line, but it usually occurs in healthy middle-aged people not involved in sports or similar activities. X-rays do not reveal focal bone loss or subchondral collapse.5To summarize, subchondral stress fractures of the femoral head in healthy adults are uncommon and in the absence of femoral head collapse they have a good prognosis. The advances in MRI technology allow the clinician to pick up this uncommon entity and also follow it up to see if it responds to conservative treatment. |
Analysis of plastid transporter proteins in Arabidopsis suggests a host origin and provides new insights into plastid evolution. It is generally accepted that a single primary endosymbiosis in the Plantae , and glaucophyte algae) common ancestor gave rise to the ancestral photosynthetic organelle (plastid). Plastid establishment necessitated many steps, including the transfer and activation of endosymbiont genes that were relocated to the nuclear genome of the 'host' followed by import of the encoded proteins into the organelle. These innovations are, however, highly complex and could not have driven the initial formation of the endosymbiosis. We postulate that the re-targeting of existing host solute transporters to the plastid fore-runner was critical for the early success of the primary endosymbiosis, allowing the host to harvest endosymbiont primary production.Arabidopsis thaliana. Of 137 well-annotated transporter proteins that were initially considered, 83 that are broadly distributed in Plantae were submitted to phylogenetic analysis. Consistent with our hypothesis, we find that 58% of Arabidopsis transporters, including all carbohydrate transporters, are of host origin, whereas only 12% arose from the cyanobacterial endosymbiont. Four transporter genes are derived from a Chlamydia-like source, suggesting that establishment of the primary plastid likely involved contributions from at least two prokaryotic sources.We tested this model of transporter evolution by conducting a comprehensive analysis of the plastid permeome in Chlamydia-like bacteria likely co-resident in the first algae.Our results indicate that the existing plastid solute transport system shared by Plantae is derived primarily from host genes. Important contributions also came from the cyanobacterial endosymbiont and Taken tion, see ).2:CO2 ratio is present in the vicinity of Rubisco. Mutants lacking these transporters are unable to survive in ambient CO2 concentrations [Arabidopsis mutants deficient in both AtNTT1 and AtNTT2 develop necrotic lesions when grown under short days, accumulate H2O2, and, strikingly, show constitutive expression of CuZnSOD2 and ascorbate peroxidase [2 levels or low phosphate availability). This may explain why the genes encoding these four plastid transporters have been retained in the Arabidopsis genome.In summary, it is surprising that bacteria not putatively involved in the endosymbiosis contributed 8% of the transporters that we have identified. When one considers the functions of these transporters, the chlamydial contribution becomes more important. HMA1 increases copper and/or zinc transport into the plastid under conditions of high light, facilitating the production of copper/zinc superoxide dismutase (CuZnSOD), which protects the plant from superoxide radicals produced under high light conditions ,43. PHT2trations ,45,46. Ftrations . Arabidoroxidase . The pheroxidase . The impChlamydia-like' genes entered into the Plantae ancestor is unclear but it is possible that both the cyanobacterial endosymbiont and chlamydial parasites may have co-existed in the cell. Many environmental Chlamydia are known today that are broadly distributed in animals and protists [How the 'protists . The co-protists and contprotists .Thermococcus and Pyrococcus clade). Although the prokaryotic source of this gene in Plantae is unclear with the available data, the eukaryotic clade is clearly monophyletic, which is consistent with a single gene origin in the Plantae ancestor and, thereafter, transfer to chromalveolates via secondary endosymbiotic gene transfer [We were unable to conclusively determine the origin of 18 transport proteins. Fourteen of these data sets resulted in PHYML trees in which the Plantae transporters were rooted within prokaryotes but without bootstrap support for a specific affiliation. An excellent example is provided by At1g32080 Figure , which itransfer . The unrtransfer , it is lKarlodinium micrum, which, as described above for Dit2, likely has resulted from a HGT.Of the remaining transporters, four fell in the 'Plantae-specific' category because they lacked identifiable homologs outside of this supergroup and may simply be too divergent to determine their origin. This includes At5g24690 nuclear genome sequence [Here we determined the phylogeny of 83 sequence will allsequence -4,7, thaA. thaliana predicted or confirmed chloroplast solute transporters.As a starting point for the compilation of a conservative set of predicted or confirmed plastid envelope membrane transporters, we used a previously published list of 137 plastid-targeted membrane proteins that was based on predicted plastid localization and classification by the transporter classification system . This liArabidopsis Information Resource website [Cyanidioschyzon merolae Genome Project website [Galdieria sulphuraria Genome Project website [Arabidopsis thaliana, Oryza sativa, Physcomitrella patens, Chlamydomonas reinhardtii, Ostreococcus tauri, Ostreococcus lucimarinus, Cyanidioschyzon merolae, Galdieria sulphuraria, Cyanophora paradoxa, Dictyostelium discoideum, Strongylocentrotus purpuratus, Xenopus laevis, Danio rerio, Mus musculus, Canis familiaris, and Homo sapiens. In addition, we included at least one insect, three fungal species, and a broad range of Bacteria and Archaea in our analysis. The BLAST searches used an e-value cut-off < 10-5. If a translated EST sequence was not available, the nucleotide sequence was translated over six frames using the ExPASy translate tool [The sequence for each protein was obtained from The website . These p website , the pla website , the Cya website , the Gal website , and Draate tool . The resArabidopsis and other Plantae.We used the ClustalW feature included with BioEdit V7.0.5.3 to generate protein alignments . AlignmeChlamydia-like' origin was inferred if the Plantae sequence formed a monophyletic group with protein sequences from either of these lineages with strong bootstrap support. Other bacterial or eukaryotic sequences could (not necessarily) be in these trees but there had to be a robust separation of the Plantae + cyanobacteria or Chlamydia-like clade from all other homologs. We had two other categories of gene origin that likely reflected a lack of phylogenetic resolution or pervasive HGT among taxa that defied a clear inference of origin. The first was the 'Other' category in which the Plantae transporter formed a well-supported monophyletic clade but its position relative to available bacterial data was unresolved, thereby not allowing us to identify the donor taxon. The second, 'Plantae-specific', was for transporters that had no significant hits to sequences in GenBank or other databases and appeared to be limited to the Plantae. All of the transporter PHYML bootstrap trees are available in Additional data file 1. The protein alignments are available in the download section of the Bhattacharya Lab website [The designation of gene origin was done as follows. When the Plantae solute transporter formed a well-supported monophyletic group with homologs in opisthokonts and secondarily with other eukaryotes such as excavates and chromalveolates (if present), then it was classified as having a 'Host' origin. Under this scheme, no bacterial sequences interrupted the eukaryotic domain. 'Cyanobacterial' or ' website .Arabidopsis thaliana folate transporter 1, 268 amino acids); 'Cyanobacterial' - At1g19800 , At5g64940 ; 'Chlamydia-like' - At1g15500 , At4g37270 ; 'Other' - At1g32080 ; 'Plantae-specific' - At5g24690 . These detailed analyses used a random starting tree (one round of taxon addition) and the rapid hill-climbing algorithm . To generate bootstrap values for these phylogenies, we used RAxML with the same settings and 100 replications. In addition, we used Bayesian inference (MrBayes V3.0b4) [Chlamydia-like' genes in Plantae to assess the topologies. We incorporated a representative diversity of available sequences in all of these trees.For eight representative transporters from the five categories described above we inferred a maximum likelihood phylogeny using RAxML and the V3.0b4) with eacA. thaliana folate transporter; AtNTT, A. thaliana ADP/ATP translocase; DiT, dicarboxylate translocator; EST, expressed sequence tag; HGT, horizontal gene transfer; HMA, heavy metal ATPase; TGD, trigalactosyldiacylglycerol.AtFOLT, Arabidopsis plastid transporter data for downstream bioinformatic analyses. HMT did the subsequent database searches and built the initial phylogenetic trees. DB was responsible for the final phylogenetic trees presented in the manuscript figures. HMT wrote the initial draft of the manuscript. APMW and DB conceived of and supervised this study and prepared the final manuscript. All authors read and approved the final manuscript.APMW and ML gathered and prepared the The following additional data are available with the online version of this paper. Additional data file Chlamydia-like', 'Other', and 'Plantae-specific' origin found in our study.PHYML bootstrap trees of plastid targeted solute transporters of putative 'Host', 'Cyanobacterial', 'Click here for file |
The aim of this study was to further describe the potential neurotrophic actions of RXR ligands and, for this and future purposes, develop a suitable in vitro-platform using mouse embryonic stem cells (mESCs).Parkinson's disease (PD) is caused by degeneration of dopamine (DA) neurons in the ventral midbrain (vMB) and results in severely disturbed regulation of movement. The disease inflicts considerable suffering for the affected and their families. Today, the opportunities for pharmacological treatment are meager and new technologies are needed. Previous studies have indicated that activation of the nuclear receptor Retinoid X Receptor (RXR) provides trophic support for DA neurons. Detailed investigations of these neurotrophic effects have been hampered by the lack of readily available DA neurons 2O2) or the excitotoxic agent kainic acid (KA). The neurotrophic effect is selective for DA neurons. DA neurons from rat primary vMB and mESCs behaved similarly, but the mESC-derived cultures contained a much higher fraction of DA cells and thus provided more accessible experimental conditions.We studied the potential neurotrophic effects of the RXR ligand LG100268 (LG268) and the RXR-Nurr1 ligand XCT0139508 (XCT) in neuronal cultures derived from rat primary vMB and mESCs. RXR ligands protect DA neurons from stress, such as that induced by the PD-modeling toxin 6-hydroxy dopamine (6-OHDA) and hypoxia, but not from stress induced by oxidative hydrogen peroxide (Hin vitro-platform for studying PD inducing toxins and potential trophic agents.RXR ligands rescue DA neurons from degeneration caused by the PD simulating 6-OHDA as well as hypoxia. Thus, RXR is a novel promising target for PD research. mESC-derived DA cells provide a valid and accessible PD is caused by progressive degeneration of dopaminergic neurons in the substantia nigra of the vMB -3. The rNuclear hormone receptors (NRs) are emerging as interesting factors in PD research. Most NRs are regulated by small, lipophilic ligands that easily enter the cell nucleus to control transcription. RXR (NR2B1-3) is activated by the synthetic ligand LG268 . Interesin vitro, where it causes DA cell death and neurotransmitter depletion [In vivo, its uptake is selective for DA cells through the DA transporter, and when inside the neuron, 6-OHDA produces oxidative stress [2O2 [The origin of DA cell degeneration in PD is largely unknown, but it is suggested to be caused by agents causing oxidative damage and energy depletion in the brain -16. The epletion . In vivoe stress ,19 as wee stress . Severale stress , the oxiess [2O2 ,23, and ess [2O2 -26.in vitro studies using DA cells derived from mESCs. Overexpression of the homeobox domain containing transcription factor Lmx1a under the control of the neuroprogenitor specific Nestin enhancer (NesE) induces formation of high numbers of bona fide vMB DA neurons in culture [Primary neuronal cultures provide data of high biological relevance. However, their use is diminished by low yields and high technical demands. Recently, we developed a platform for culture . These n culture .2O2. The protective effects are only seen in Nurr1-expressing DA cells. To conclude, RXR ligands and mESC-derived DA cells represent promising platforms in the search for novel PD therapies.Here we have used DA neurons derived from primary vMB cultures as well as mESCs to further establish the neurotrophic role of RXR activity. We can show that RXR ligands selectively protect DA neurons from stress caused by 6-OHDA and hypoxia, but not from KA and Hin vitro (DIV) the cell death was negligible, whereby the cultures could be maintained for weeks . After To simulate PD-like neurodegeneration, primary vMB cultures of three to five DIV were exposed to the neurotoxic DA analogue 6-OHDA. RXR ligands were added three hours prior to the toxin, and after 20 minutes of 6-OHDA exposure the medium and ligands were replaced to avoid unspecific stress from oxidized 6-OHDA. After additional 24 hours, cells were fixed, stained and scored. 6-OHDA produced clear signs of neurodegeneration in the TH-positive population, with evident neuronal loss, leaving cell debris and damaged neurites Figure . The deg2, 5% CO2). Ligands were added three hours prior to the hypoxic insult and kept throughout the experiment. Cells were fixed, stained and scored after different time intervals. In control culture conditions there is no decrease in the number of DA cells over time. However, hypoxia induced a time dependent decrease in DA cells Figure . Simulta) Figure . Further Figures and 2D. in vitro studies, since they provide a very high number of the correct cell type and homogeneous cultures. Stable NesE-Lmx1a mESCs (Lmx1a cells) were induced for neuronal differentiation as monolayer cultures in medium supplemented with the DA instructive cues Sonic Hedgehog (Shh) and Fibroblast growth factor (FGF) 8 [+/TH- neuronal population (data not shown). Also the selective Nurr1-RXR ligand XCT fully rescued the vMB DA neurons from 6-OHDA-induced degeneration 8 . After 1 (FGF) 8 ). Cells ) Figure , suggestPD inflicts considerable suffering for the affected and their families, and it constitutes a great cost for the society. Today, the opportunities for pharmacological treatment are meager and mainly consist of substitution with the DA precursor Levodopa. However its medical potential decreases severely over time . The dev2O2, respectively. Thus, the effects of ligand activation is not generally neuroprotective, but rather likely to affect individual cell death pathways.NR ligands are small and lipophilic, and they easily pass the blood-brain-barrier, providing excellent targets for the pharmaceutical industry. Indeed, the characterization of novel NR-mediated signaling pathways during the last decade has resulted in development of novel drugs used in the treatment of metabolic disease and cancer. Specifically, RXR ligands have limited toxicity in humans and selein vitro-based PD research.Ligands activating RXR and the RXR-Nurr1 heterodimer selectively protect DA neurons from stress induced by the PD-modeling toxin 6-OHDA and hypoxia. Thus, the regulation of RXR activity holds promises to contribute to a novel, alternative strategy in PD treatment. Furthermore, mESC-derived cultures offer accessible platforms of DA neurons and may provide novel means to conduct valid 2 and 99% humidity unless otherwise stated.All experiments were performed in accordance with guidelines from the Swedish National Board for Laboratory Animals. Primary vMB cultures were obtained as previously described . Briefly2-mercaptoethanol (Sigma). For generation of stable ESC lines, 2 × 10^6 cells were nucleofected with 7 μg linearized NesE-mLmx1a-PGK-neo vector according to protocol , selected with G418, replated on gelatinized dishes and induced to differentiate in N2B27 differentiation medium [2 and 99% humidity throughout all experiments.DA cells were derived from E14.1 mESCs as previously described ,28. Brien medium suppleme2 and 0-1% O2 from one to 36 hours. The excitotoxic glutamate analogue KA and the oxidative agent H2O2 were added over night, in the range of 100 to 500 μM and 80 to 110 μM, respectively. To stress mESC-derived DA cells, 150 to 500 μM 6-OHDA in 0.1% ascorbic acid were added three hours before medium and ligand exchange. The cultures were left for another 36 hours in the incubator.Experiments were performed in triplicates. The ligands , were diluted to working dilutions in culture medium and added to the cultures for two to three hours prior to neurodegenerative stressors. Final concentrations were 0.1 and 1 μM for LG268 and XCT, respectively. The DA analogue 6-OHDA was given to the vMB primary neuron cultures at 5-15 μM for 20 minutes before medium and ligand replacement. Hypoxic stress was induced by placing cultures in a modular incubator chamber at 37°C filled with 5% COParaformaldehyde fixed cultures were incubated overnight with TH , TuJ1 antiserum in PBS containing 5% fetal calf serum and 0.3% triton X-100. Following rinses, cultures were incubated with FiTC- and Cy3 conjugated secondary antibodies for direct detection or with biotinylated secondary antibodies followed by detection of immuno-staining using the ABC immunoperoxidase kit from Vector .Analysis, imaging and cell counting were performed on Eclipse E1000M and Eclipse TE300 microscopes (both Nikon) coupled to the Spot2 camera . Scoring was performed by cell counting, and counts were made blind to avoid observation bias. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test when appropriate.in vitro; FGF: Fibroblast growth factor; GDNF: glial cell line-derived neurotrophic factor; H2O2: hydrogen peroxide; KA: Kainic acid; LG268: LG100268; mESCs: mouse embryonic stem cells; NesE: Nestin Enhancer; ns: non-significant NRs: Nuclear hormone receptors; PD: Parkinson Disease; RXR: Rexinoid × receptor; Shh: Sonic Hedgehog; TH: tyrosine hydroxylase; vMB: ventral midbrain; XCT: XCT139508.6-OHDA: 6-hydroxy dopamine; ANOVA: one-way analysis of variance; DA: dopamine; DIV: days SF constructed the stable Lmx1a mESC line, performed cell culture, experiments and analyses and participated in manuscript writing. MB participated in experiments and analyses. SK conceived of and designed the study, performed cell culture, experiments and analyses and drafted the manuscript. All authors read and approved the final manuscript. |
In the current study we investigated 12 patients, six de novo and six familial, with apparently balanced translocations and mental retardation and/or congenital malformations by applying 1 Mb resolution array-CGH. In all de novo cases, only the patient was a carrier of the translocation and had abnormal phenotype. In five out of the six familial cases, the phenotype of the patient was abnormal, although the karyotype appeared identical to other phenotypically normal carriers of the family. In the sixth familial case, all carriers of the translocations had an abnormal phenotype.Carriers of apparently balanced translocations are usually phenotypically normal; however in about 6% of de novo and one familial . The nature and type of abnormalities differed among the cases. In the first case, what was identified as a de novo t, a complex rearrangement was revealed involving a ~6.1 Mb duplication on the long arm of chromosome 9, an ~10 Mb deletion and an inversion both on the long arm of chromosome 15. These imbalances were located near the translocation breakpoints. In the second case of a de novo t, an ~6.6 Mb deletion was identified on the short arm of chromosome 7 which is unrelated to the translocation. In the third case, of a familial, t, two deletions of ~4.3 Mb and ~2.3 Mb were found, each at one of the two translocation breakpoints. In the remaining cases the translocations appeared balanced at 1 Mb resolution.Chromosomal and FISH analyses suggested that the rearrangements were "truly balanced" in all patients. However, array-CGH, revealed cryptic imbalances in three cases , two de novo and familial apparently balanced translocations unlike other relatively large studies which are mainly focused on de novo cases. This study provides additional evidence that cryptic genomic imbalances are common in patients with abnormal phenotype and "apparently balanced" translocations not only in de novo but can also occur in familial cases. The use of microarrays with higher resolution such as oligo-arrays may reveal that the frequency of cryptic genomic imbalances among these patients is higher.This study investigated both The great majority of cases with apparently balanced translocations are usually not associated with abnormal phenotypes. Apparently balanced rearrangements in general, represent an interpretational and counseling dilemma when detected in cases with abnormal phenotypes and/or mental retardation.de novo and familial balanced translocations. Warburton et al., 1991 analyses of this patient showed an apparently balanced de novo translocation between chromosomes 9 and 15. The initial karyotype was designated as 46,XY,tdn. Array-CGH though revealed an ~6.1 Mb duplication on chromosome 9 at 9q34.2-q34.3 and deletions on three regions of chromosome 15 adding to ~10 Mb .ish dup(9)(q34.1q34.3),del(15)(q21.2q21.3)(RP11-32N2-),del(15)(q22.31q23),del(15)(q25.1q25.2)(RP11-2E17-), inv(15)(q21.1q21.2)dn.The patient, a 22 years old male, has a history of mild mental and growth retardation, motor dyspraxia and myoclonic seizures. He presents antimongoloid palpebral fissures, microtia, high-arched palate, low-set and dysplastic ears, strabismus, bulbous nose, short webbed neck, low posterior hairline, scoliosis, lordosis, short nails and broad first toes bilaterally.de novo translocation between chromosomes 4 and 9. The initial karyotype was designated as 46,XY,tdn. Array-CGH however showed an ~6.6 Mb deletion on chromosome 7 which is unrelated to the translocation analyses of this patient showed an apparently balanced Y,tq5;q21.2dnRevised karyotype after FISH confirmation of the array results:46,XY,t.ish del(7)(p12.3p13)dn.Case 3 involves a familial translocation between chromosomes 4 and 7 which was inherited from the mother. The above family has 3 children, two daughters that are both carriers of the translocation and share the same clinical phenotype and one son with normal karyotype and phenotype. The older daughter is currently 16 years old and the younger is 15. They were both born after uneventful pregnancies and deliveries. As infants, they had feeding problems and delayed motor milestones. They both have moderate mental retardation, seizures, attention deficit hyperactivity syndrome and severe learning difficulties. They attend school for children with special needs. They have no dysmorphic features or other congenital abnormalities. The mother has also mental retardation but in milder form.Chromosomal and FISH (subtelomeric and wcp probes) analyses of the mother and her daughters revealed apparently identical karyotypes: 46,XX,tq13.3;p15.3). Array-CGH revealed an ~4.3 Mb and an ~2.3 Mb deletion on chromosome 4 and 7, respectively in all carriers .ish del(4)(q13.3q13.3)(RP11-373J21-), del(7)(p15.3p21.1)mat.de novo cases (33.3%) and in one of the six familial cases (16.6%). The remaining of the cases appeared to be simple and balanced at 1 Mb resolution.Array-CGH revealed cryptic genomic imbalances at 1 Mb resolution in three out of the twelve cases studied (25%) with an apparently balanced translocation and abnormal phenotype, in two of the six de novo cases. Usually, familial cases are reported as individual case studies. Only one other study has been performed with array-CGH by [de novo and two familial of patients with balanced translocations and abnormal phenotype.The present study is to our knowledge the first where a relatively large number of patients with familial balanced translocations and abnormal phenotype were investigated for cryptic imbalances using array-CGH, unlike most of the studies that are focused on y-CGH by Ciccone ede novo cases is in accordance with the largest systematic study performed to date by De Gregori et al 2007 [de novo balanced translocation cases with abnormal phenotype. Although the sample sizes are still small, we can safely assume by combining the two previous studies with the supporting evidence of the current study that about 30–50% of the de novo apparently balanced translocations with abnormal phenotype are associated with causative cryptic imbalances. In the remaining 50–70% of the patients the phenotype might me caused by other mechanisms such as interruption of a dosage sensitive gene, or by uniparental disomy or by unmasking of a recessive mutated gene on the homolog chromosome or finally by position effect with variable expression of a gene(s) near the translocation breakpoint. However a disruption of a recessive gene might occur at the breakpoints of the translocation without a phenotypic effect. Baptista et al., 2008 [The frequency of imbalances detected in the al 2007 , where 2 al 2007 using 1 l., 2008 studied de novo case and in both patients with familial translocations suggesting that cryptic imbalances in familial cases may be common. Therefore more familial cases need to be analyzed in order to draw final conclusions. It is possible that higher resolution arrays such as high-density oligo-arrays may reveal smaller aberration that could have been missed in the current study due to lower resolution. It is also possible, that other mechanism are involved in inherited cases like production of a recessive mutation by the translocation that is expressed when a mutation in the same gene is inherited from the other parent or the phenotype might simply be coincidental and unrelated to the translocation.Only in one case (Case 3), out of the six familial cases analyzed, additional aberrations were identified, which involved a family where all translocation carriers shared the same abnormal phenotype. The deletions detected at the breakpoints of the translocation were present in all carrier family members and absent in the phenotypically normal members of the family. In the remaining five families in which only the patients had an abnormal phenotype while all the other carriers were phenotypically normal no additional aberrations were detected at the resolution of 1 Mb. Results obtained in the current study from the familial cases suggest that cryptic copy number changes at least at the resolution of 1 Mb, do not constitute a major cause for the phenotypic abnormalities in patients with familial apparently balanced translocations. However, the study by Ciccone et al 2005 [It is interesting that in a study published by our group in 2004 , in whicThe nature, location and size of the abnormalities differed among the three cases. Among the imbalances detected three were deletions, one was a duplication and one an inversion and their size ranged from 2.3 to 10 Mb. The locations of these rearrangements relative to the translocation breakpoints were also different from case to case. In the first case, the imbalances were on the same chromosomes involved in the translocation but were not directly associated with the breakpoints, in the second case a deletion was identified on a chromosome, unrelated to the translocation and in the third case imbalances were found at the translocation breakpoints. The above results show that cryptic aberrations are not always related to the rearrangements breakpoints. Similar results were previously reported by other studies ,13,14.de novo cases, it should also be investigated with FISH assays since more breaks usually involve higher risk. In addition, in cases of de novo translocations, array-CGH should be performed as subtle copy number changes may be present not only at the breakpoints but anywhere else in the genome. Although, array-CGH is well established method and is widely used for the evaluation of patients with mental retardation and congenital abnormalities, its implementation in prenatal diagnosis is still very limited. A major factor for the limited used of array-CGH in prenatal diagnosis is the presence of benign copy number variations (CNVs) [Balanced translocations remain a challenge for geneticists especially when they are detected prenatally. In the case of familial translocations, the translocation present in the parent should always be investigated with FISH assays to determine whether it is a simple two way translocation or it involves more chromosomes so that more accurate diagnosis in the fetus is performed. In s (CNVs) ,17 whichde novo "apparently balanced" translocations. In contrast to previous investigations both de novo (six cases) and familial (six cases) cases were analyzed by array-CGH and imbalances were identified in both de novo and familial cases. These imbalances were either linked (near or at the breakpoints of the translocation) or were unrelated to the rearrangement. These findings further highlight the necessity of whole genome approaches for "apparently balanced" rearrangements.The current study provides additional evidence and confirms previous studies which showed that cryptic genomic imbalances are common (33.3%) in patients with abnormal phenotype and de novo cases, only one member of each family was a carrier of a translocation and had an abnormal phenotype while all the other members had normal karyotypes and normal phenotypes. In five out of the six familial cases the patients of each family had an abnormal phenotype but their karyotype appeared identical to other phenotypically normal translocation carriers of the family. In one familial case, all the carriers of the translocations had abnormal phenotype.Twelve patients with apparently balanced translocations familial or de novo and abnormal clinical phenotype were included in the study. In all Family history and clinical information of each patient were recorded. Patients were included in the study only if chromosomal and FISH analyses suggested that the rearrangement was "truly" balanced.Chromosomal analyses were performed from peripheral blood samples using conventional GTG-banding techniques at the 550-band level.FISH analyses were performed using commercially available subtelomeric specific probes and whole chromosome paints (wcp) to confirm that the translocations were "truly balanced". FISH procedures were performed according to the manufacturer's protocols .. Labelling and FISH procedures were performed according to the manufacturer's protocols FISH was also carried out using BAC-probes for confirmations of the array-CGH findings. Location of the clones was obtained from NCBI DNA was isolated using the PUREGENE DNA extraction kit according to the supplier's protocol. The Cytochip 1-Mb resolution chip was used for microarray-CGH analysis. Array-CGH was carried out according to the recommendations of the manufacturer. In brief, DNAs were labelled by random priming using Bio Prime labelling kit with cyanine 3 and cyanine 5 fluorescent dyes. DNAs were co-hybridized on two microarrays as dye swap was used. Pooled genomic DNA from Promega (UK) was used as reference. Data was analysed with the BlueFuse for microarrays software package .CS drafted the manuscript and carried out the array-CGH assays, MI and CC performed the FISH assays SKT, VA, GS, EP, ZKA and EK performed the clinical evaluation of the patients, PP conceived the study, participated in its design and coordination and also approved the manuscript. |
Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains. Clostridium perfringens, an anaerobic Gram-positive bacterium, is ubiquitous in the intestinal flora of human and animals, and is also commonly isolated from environmental materials such as soil and water C. perfringens is an extremely important pathogen of human and domestic animals. In a commonly used classification scheme, C. perfringens is divided into five toxinotypes (A to E) based on the production of four toxins ; however, this bacterium also produces ten other toxins such as C. perfringens enterotoxin (CPE), beta2 toxin, and theta toxin cpe) is highly conserved, although the cpe gene can be present on the chromosome or on a large plasmid(s) in type A strains cpe-carrying strains can cause C. perfringens food poisoning outbreaks, but chromosomal cpe isolates are responsible for the majority of foodborne illnesses cpe isolates. In addition, there are several reports of biological differences, such as heat resistance and other traits cpe isolates and plasmid cpe isolates that may suggest these two types of strains possess different genetic backgrounds.Of these many toxins, CPE is extremely important for human gastrointestinal diseases such as food poisoning and antibiotic-associated diarrhea C. perfringens has been published C. perfringens isolates share a similar chromosomal genetic organization, although this genome sequencing has tested only a limited number of strains. Multilocus sequence typing (MLST) represents an alternative approach capable of investigating the genetic characterization of many strains of a given species. A MLST approach has been applied to many bacteria, but not yet to compare the genetic relatedness of large numbers of chromosomal cpe isolates vs. plasmid cpe isolates from varied sources To investigate the genetic background of a bacterium, total genome sequence analysis has been performed in many pathogens. To date, the complete genome sequence of three pathogenic type A strains of C. perfringens strains, firstly using a conventional PCR survey for representative virulence genes on the chromosome or for genes borne on toxin plasmid(s), as well as for several housekeeping genes. After confirming selected PCR results by Southern blot, a MLST analysis based on those PCR/Southern blot results was then applied to characterize the genetic backgrounds of chromosomal cpe isolates vs. plasmid cpe isolates.This study sought to characterize the genetic background of enterotoxigenic C. perfringens isolates, a PCR survey was first performed to evaluate the carriage of selected genes including chromosomal toxin genes (plc and pfoA), several chromosomal housekeeping genes, plasmid maintenance genes, and genes related to plasmid transfer . Similarly, PCR reactions for three chromosomal genes failed to amplify PCR products from chromosomal cpe strains that originated in Europe, Japan, or the USA, as well as from some plasmid cpe-positive strains and cpe-negative strains. These results indicate that eno, virS or pfoA genes are either missing from these strains or there is nucleotide diversity at the primer binding site(s).The carriage of twelve known (from genome sequencing) C. perfringens plasmids, including two cpe-encoding conjugative plasmids (pCPF4969 and pCPF5603) cpe- plasmid genes from all surveyed plasmid-cpe positive strains, including sporadic diarrhea strains, food poisoning strains, and food strains, as well as from several isolates originating from feces of healthy humans, and several food isolates. However, PCR assays for these genes did not produce a positive reaction from any surveyed chromosomal cpe strain, except for food strain P-1/09/03. In contrast, a PCR survey for three genes present on pCP13 amplified a positive reaction for ten of eleven chromosomal cpe strains and for eleven of twelve plasmid cpe strains. In PCR assays for genes present on both cpe plasmids and pCP13, a cpb2 product was not amplified from any chromosomal cpe strains, but a cna product was obtained for two chromosomal cpe food strains. A PCR assay for the bacteriocin gene (bcn) present on pIP404, was positive for 16 of 58 investigated C. perfringens strains (including both cpe-positive and cpe-negative strains).PCR assays were then performed to detect the carriage of representative genes present on four completely sequenced eno, virS, pfoA tcpH, cna, and soj ORFs Southern blot assays were performed .To definitively establish the presence or absence of erformed using teeno and virS), Southern blot assays showed a positive reaction with all tested C. perfringens strains and ATCC13124 pfoR gene and a gene downstream of pfoA that encodes a conserved hypothetical protein are present on the chromosome of both strains .To further investigate the strains . These rcpe strains might share a common genetic background. To further compare the genetic backgrounds of chromosomal cpe strains versus plasmid cpe strains collected from various geographical origins or sources , MLST analysis was performed with eight representative housekeeping genes.The results from our PCR survey and Southern blot assay suggested that chromosomal pe strains and thirteen plasmid cpe strains. This analysis identified twelve main groups, designated Cluster I to XII, (cpe-negative type strain), while SM101 is a derivative of European food poisoning isolate NCTC8798. M-07 and M-08 strains were isolated from the same meat sample so these pairs of strains might also share the same genetic backgrounds. However, thirty-seven strains gave unique individual MLST patterns in this study. Collectively, these results confirm that MLST is useful to investigate the genetic relationship between C. perfringens strains.Phylogenetic analysis by the Clustal W program was then performed on our MLST results for fifty-eight strains, including eleven chromosomal c to XII, . Severalcpe strains were assigned to Cluster I obtained from retail foods not associated with a food poisoning outbreak. These genetic findings showing relatedness between chromosomal cpe nonoutbreak food strains and chromosomal cpe food poisoning strains were consistent with previous findings indicating that food strains carrying cpe on the chromosome form similarly heat resistant spores as do food poisoning strains pgk, sod, gyrB), two (nadA), or four amino acid substitutions. Overall, our MLST results strongly suggest that, regardless of their geographical origins, date of isolation, or origination from nonoutbreak foods or food poisoning outbreaks, type A chromosomal cpe strains in Japan, Europe, and USA share a common genetic background and belong to a cluster distinct from most other C. perfringens isolates. The one exception was that Cluster I also includes the type C cpe-positive NCTC8081 strain that was isolated from human necrotizing enteritis in Europe.Nine chromosomal luster I . These scpe isolates localizing to Cluster I, three isolates from a 2001 food poisoning outbreak in Toyama area , each carrying a plasmid with an IS1151 sequence downstream of the cpe gene cpe strains, with a downstream IS1151 sequence, which had been obtained from human sporadic diarrhea cases occurring in Europe cpe isolates with downstream IS1151 sequence share a common genetic relationship. However, this Cluster VI also included two cpe-negative strains.In contrast to the chromosomal 1470-like sequence downstream of the cpe gene, belonged to Cluster VII, a neighboring cluster to Cluster VI (1470-like cpe plasmid (F4969 and TM138). These two other cpe plasmid strains have the exact same sequence for the eight investigated housekeeping genes as found in the three Toyama food poisoning strains belonging to this cluster. This finding strongly suggested that strains in Cluster VII also share a similar genetic background.Three food poisoning isolates from a 1980 outbreak occurring in the Toyama area of Japan , each carrying a plasmid with an ISuster VI . This Clcpe strains belonged to different clusters. This genetic variability may have resulted from the conjugative nature of cpe- plasmids, with this plasmid transferring into unrelated C. perfringens strains.Another three plasmid cpe. Six of those eight isolates belonged to Cluster II, one isolate (M-04) aligned with Cluster VI, and the final isolate (TM111C6)belonged to Cluster X. While six of these food isolates were assigned to Cluster II, no plasmid cpe strains from food belonged to Cluster II or VI. This finding further suggested that plasmid cpe-positive strains often share a similar genetic background.Out of eleven nonoutbreak food isolates from Wakayama city in Japan that were investigated in this study , eight iC. perfringens diseases were assigned to Cluster III isolated from European animals suffering from ster III . Clustera cpe plasmid carrying a downstream IS1470-like sequence was assigned to Cluster V, two isolates belonged to Cluster VI, MR2-14 aligned with Cluster IX, MR1-1 was assigned to Cluster X, MR2-12 belonged to Cluster XI, MR2-3 to Cluster XII. These findings suggested that healthy humans carry C. perfringens strains with various genetic backgrounds.While many strains from each source belonged to the same cluster, eleven fecal strains isolated in 2000 from healthy people in Japan belonged to eight different clusters . Two isoClostridium perfringens strains producing enterotoxin (CPE) are the causative agent for several human GI diseases, including food poisoning, antibiotic-associated diarrhea, and sporadic diarrhea cpe) is found in a small population of type A C. perfringens (approximately 1 to 5%) cpe on the chromosome usually possess higher resistance properties against heat, cold, and nitrates than strains carrying cpe on a plasmid cpe strains typically grow faster at optimal temperature, and have a broader growth temperature range, compared to plasmid cpe strains or other C. perfringens isolates cpe isolates and other C. perfringens isolates.C. perfringens, by surveying fifty-eight cpe-positive and cpe-negative strains from various sources. Previous genetic studies have identified four groups of type A enterotoxigenic C. perfringens: i) food and food poisoning isolates that carry cpe on the chromosome cpe gene with a downstream IS1470-like sequence cpe gene with a downstream IS1151 sequence cpe gene but produced no PCR product with a cpe-genotyping PCR assay Therefore, the current study investigated the genetic background of type A enterotoxigenic plc, colA, pfoA), remarkably found the θ toxin gene (pfoA) is missing from all ten surveyed chromosomal cpe strains and from five of twelve plasmid cpe strains. A study conducted back in the 1960's C. perfringens, but localization of the cpe gene to the chromosome or plasmids was not possible at that time. Our finding that most chromosomal cpe isolates are pfoA-negative, significantly extends the genome sequencing observation that chromosomal cpe isolate SM101 is pfoA-negative The results of our PCR survey and Southern blot assay for toxin genes , but not a plasmid related to cpe plasmids such as pCPF4969.Concerning housekeeping genes, our initial PCR survey for ot assay , Fig. 1.C. perfringens food poisoning, the relationships among C. perfringens isolates have primarily been investigated by pulsed-field gel electrophoresis (PFGE) C. perfringens strains using MLST analysis have been reported C. perfringens animal disease isolates (type B to E) C. perfringens, isolates, including a limited number of cpe-positive food poisoning strains and suggested that food poisoning isolates form a distinct cluster of C. perfringens isolates.In epidemiological studies of cpe gene, as well as only three plasmid cpe isolates, all nonfoodborne GI disease isolates from Europe. Since that earlier study, it has become clear that some food poisoning isolates carry their cpe gene on large transferable plasmids rather than on the chromosome and that chromosomal cpe isolates can be recovered from some retail meats. Therefore, MLST analysis using housekeeping genes present on the chromosome and cpe plasmid was employed in the current study to explore i) the relatedness among a larger collection of cpe-positive isolates, including many from Japan and ii) the similarity of these isolates to cpe-negative isolates. The cpe location, ability to produce CPE and spore heat resistance of all cpe-positive strains included in this study had been previously determined cpe-positive C. perfringenssod, and groEL) and propagation in foods, and genes related to spore formation (sigK), which can also contribute to survival in foods.However, that previous study examined only a few food poisoning isolates, mostly American and all carrying a chromosomal cpe strains share a common genetic background and belong to the distinct Cluster I. In particular, these surveyed chromosomal cpe strains all possess three common features, 1) absence of the pfoA gene, but retention of neighboring sequences in the pfoA locus, 2) lack of plasmid-borne major-toxin genes (including cpe) cpe-positive and/or cpe-negative strains.From our MLST analysis, the most remarkable finding was that, regardless of their source their geographic origin, or date of isolation, all surveyed chromosomal cpe and was isolated from a patient suffering from necrotizing enteritis (Pigbel) in Europe. Human necrotizing enteritis is a rare disease and not fully understood with respect to its pathogenesis, although the plasmid-encoded β-toxin clearly plays a major role in the enteric virulence of type C isolates cpe-positive type C strains are available from strain collections in Japan, USA, and Europe. Further investigation, if possible, using more cpe-positive type C strains should evaluate the genetic relationship between type A chromosomal cpe strains and type C cpe-positive strains.Interestingly, cluster I also included type C strain NCTC8081, which carries 1151 sequences located downstream of the cpe gene. These isolates included three food poisoning strains from an outbreak in Japan in 2000 1151 cpe-genotype strains share a similar genetic background that facilitates their ability to cause human GI disease.Cluster VI included five strains carrying a plasmid with IS1470-like sequences downstream of the cpe gene, five strains belonged to Cluster VII. However, the other three strains belonged to Cluster V or Cluster IX. These results suggested that while IS1470-like cpe-genotype strains often share a similar genetic background, they are more variable than IS1151-genotype plasmid strains.Of eight surveyed strains carrying a plasmid with IScpe isolates that, 1) food poisoning outbreaks involving plasmid cpe strains often involve clonal expansion rather than plasmid transfer, 2) IS1151 cpe genotype strains are closely related, but can also share a genetic relationship with some cpe negative isolates which may have lost the cpe plasmid or which represent potential future hosts for the IS1151 cpe plasmid, 3) IS1470-like cpe genotype strains also share genetic relationships but are more variable than the IS1151 cpe genotype strains. In addition, the current results conclusively demonstrate that chromosomal cpe isolates, whether originating from food poisoning or nonoutbreak-associated foods, do not share a close genetic linkage with plasmid cpe food poisoning strains (or other plasmid cpe strains).Collectively, our results suggest for plasmid cpe-positive human strains (even from a type C human strain), and also from cpe-positive and cpe-negative food strains. Further MLST analyses of non-type A strains is warranted to confirm these conclusions.Strains isolated from feces of healthy humans were found to distribute into many clusters, i.e. these strains have varied genetic backgrounds. This variability of fecal strains might be attributable, in part, to dietary differences or personal factors such as age or economic status. The investigated type B to E livestock origin strains all formed one cluster, which was distinguished from cpe isolates provides one explanation for previous phenotypic studies cpe isolates versus other C. perfringens isolates. A shared genetic background by most or all chromosomal cpe isolates is also consistent with our previous studies identifying a variant small acid soluble protein, that is made by most chromosomal cpe isolates cpe isolates, it has been proposed 5565 onto the C. perfringens chromosome. If so, our MLST findings could suggest this chromosomal transposon integration occurred only a limited number of times (perhaps only once) in a C. perfringens type A isolate(s) possessing a genetic background favorable for growth and survival in the food environment. Acquiring the ability to produce a potent enterotoxin, upon Tn5565 integration, thus created a formidable food poisoning agent. Since C. perfringens isolates multiply to very high levels in the GI tract during food poisoning, t is possible that acquiring the ability to produce CPE is advantageous by facilitating the dissemination of these bacteria so the infectious cycle can be repeated in other hosts.Finally, the common and distinct genetic background of chromosomal C. perfringens were included in this study, including type A cpe-positive strains, type A cpe-negative strains and type B to E strains. A breakdown of C. perfringens strains with their various origins is shown in cpe-positive strains from food poisoning outbreaks occurring in Japan cpe-positive strains were isolated from food poisoning outbreaks in Japan Fifty-eight strains of C. perfringens strain was inoculated into 5 ml of fluid thioglycolate medium(FTG [Becton Dickinson]) and then incubated overnight at 37°C. An aliquot of that overnight FTG culture was inoculated into 10 ml of TGY broth and then incubated overnight at 37°C. DNA for PCR and multilocus sequence typing analysis was prepared from 200 µl of overnight TGY culture with the InstaGene matrix kit [Bio-Rad] according to the manufacture's instructions. To reduce the chance of cross-contamination, DNA templates was prepared with the InstaGene matrix kit, because it requires only two tubes with three steps for preparing DNA templates. DNA materials for Southern blot assays were prepared according to methods described previously An aliquot of a cooked meat medium [Difco] stock culture of each plc, a ubiquitous gene of C. perfringens), DNA gyrase B gene (gyrB), one of the sporulation sigma factors , heat shock protein gene , genes encoding enzymes involved in energy production from glucose , a nucleotide metabolism gene , a collagenase gene , a regulator gene and also theta toxin gene (pfoA).For this PCR survey, eleven housekeeping genes were selected, including: phospholipase C gene (cpe) and the beta2 toxin gene (cpb2)), a putative collagen adhesion protein gene (cna) cpe-carrying and antibiotics-resistant gene-carrying plasmid rep) on transferable plasmids soj, parB, top) present on pCP13, which is found in the first completely sequenced strain, strain 13 bcn) on pIP404 C. perfringens shuttle vectors were derived.For plasmid-encoded genes, this PCR survey tested for: two toxin genes . The reaction mixtures, with a total volume of 50 µl, were placed in a thermal cycler and subjected to the following amplification conditions: 1 cycle at 94°C for 2 min; 35 cycles at 94°C for 30 s, 55°C for 60 s, 68°C for 60 s, and a single extension of 68°C for 8 min. PCR products were then electrophoresed on a 1.5% agarose gel, which were stained with ethidium bromide.PCR primer pairs were principally designed based on genes annotated in the cpe strains, six plasmid cpe food poisoning strains, three plasmid cpe food strains, two human sporadic diarrhea strains, were investigated by Southern blot analyses (cpe human feces isolate (MR2-4) cpe-negative human feces isolate (MR2-12) and food isolate in Japan (M-08) were also examined. As a negative control, Clostridium tertium isolate, identified based on 16 rRNA gene sequence, from Japanese retail food was used.To further investigate PCR-negative results, ten chromosomal analyses . Plasmideno, virS, pfoA, cna, tcpH, and soj. For Southern blot assays, DNA sample of each strains, prepared with the methods previously described PstI overnight at 37°C and then electrophoresed on a 1% agarose gel with 8 mA constant current, for 16 to 18 hours and then transferred to nylon membranes [Roche] with a vacuum blotter [Bio-Rad] with manufactures' instructions. The membranes were hybridized with one of the gene probes as described previously DNA was prepared from strains F4969 or F5603 and that DNA was used with a PCR DIG-labeling kit [Roche] cpe strains and five plasmid cpe strains do not carry pfoA, the genetic organization of upstream and downstream region of pfoA in pfoA-negative strains was investigated. First, a bioinformatic investigate was performed on the complete genome sequenced pfoA-positive and pfoA-negative strains, ATCC13124 and SM101 cpe-positive SM101 and cpe-negative ATCC13124 carry upstream pfoR and downstream hypothetical protein gene on the pfoA region. To investigate whether this simple pfoA-specific deletion might be common among pfoA-negative, cpe-positive strains, primer pairs were constructed inside of the upstream pfoR ORF and a downstream hypothetical protein ORF, based on sequence information for ATCC13124 and SM101. In this pfoA-genotype assay, the estimated size of a PCR product should be 2,375 bp for a pfoA-positive strain (such as ATCC13124) but only 280 bp for a pfoA-negative strain (such as SM101). In this pfoA-genotype assay, PCR was performed with twelve pfoA-negative, cpe-positive, type A strains under the same conditions as cpe-genotying assay Taq polymerase, PrimeSTAR GXL DNA polymerase [Takara], that is suitable for long-range PCR.Since PCR survey and Southern blot assay results indicated that all ten tested chromosomal C. perfringens strains, MLST was performed using eight housekeeping genes, related to survival in foods, bacterial proliferation in foods, or spore formation (CPE is formed during sporulation). The genes used in our MLST analysis contained genes for toxin genes , stress response , sigma factor for sporulation (sigK), putative metabolism genes and genes in DNA replication (gyrB). PCR products were purified with a QIA quick PCR purification kit [QIAGEN], and then sequenced with ABI PRISM®BigDye™terminator Cycle Sequencing Ready Reaction Kits (Version 1.1 and 3.1) according to the manufacture's instructions. All sequence data were concatenated to produce an in-frame 5,274 bp, according to genome arrangement of strain 13, plc, colA, nadA, sodA, pgk, sigK, groEL, and gyrB, (approximately 0.17% of strain 13 whole genome). Sequence information of these eight genes from the three completely sequenced C. perfringens strains, i.e., Strain13,ATCC13124,SM101 (derivative from NCTC8798), were also included in this survey. Concatenated sequence data were applied to phylogenetic analysis with Clustal W format by using Lasergene software Ver. 6 [DNASTAR].To thoroughly investigate the genetic background of enterotoxigenic The sequences determined in this study have been deposited in the GenBank under accession AB477535-AB477966.Whole genome sequence information for strain 13, SM101, and ATCC13124 is available according to the following accession numbers, NC003366, CP000312, NC008261, respectively. |
P=0.012) while high nuclear Id-1 expression could predict development of distant metastasis within 1 year of oesophagectomy (P=0.005). In addition, high levels of Id-2 expression in both cytoplasmic and nuclear regions predicted longer patient survival (P=0.041). Multivariate analysis showed that high-level expression of Id-2 in both cytoplasmic and nuclear regions and lower level of nuclear Id-1 expression were independent favourable predictors of survival in our ESCC patients. Our results suggest that Id-1 may promote distant metastasis in ESCC, and both Id-1 and Id-2 may be used for prognostication for ESCC patients.Id protein family consists of four members namely Id-1 to Id-4. Different from other basic helix–loop–helix transcription factors, they lack the DNA binding domain. Id proteins have been shown to be dysregulated in many different cancer types and their prognostic value has also been demonstrated. Recently, Id-1 has been shown to be upregulated in oesophageal squamous cell carcinoma (ESCC). However, the prognostic implications of Id proteins in ESCC have not been reported. We examined the expression of the Id proteins in ESCC cell lines and clinical ESCC specimens and found that Id protein expressions were dysregulated in both the ESCC cell lines and specimens. By correlating the expression levels of Id proteins and the clinicopathological data of our patient cohort, we found that M1 stage tumours had significantly higher nuclear Id-1 expression ( Oesophageal cancer is the eighth most common cancer and the sixth most common cause of cancer-related death worldwide with a 20-fold higher risk being observed in China when compared to other low-risk areas .Id protein family is a group of helix–loop–helix transcription factors that have been implicated in different steps in carcinogenesis such as tumorigenesis, differentiation and metastasis . Id-1 haα-induced apoptosis in ESCC cell lines . ESCC cell lines EC18, EC109 and KYSE70 were maintained in RPMI-1640 (Invitrogen) with 10% FBS. HKESC2 was maintained in MEM (Invitrogen) with 20% FBS. KYSE30 and KYSE150 were maintained in 1 : 1 RPMI-1640/Ham's F12 medium with 10 and 2% FBS, respectively.Western blotting was performed as previously described (Tissue microarray (TMA) was constructed with a Beecher Instruments tissue microarraryer . Three representative areas of each specimen, including 33 non-neoplastic oesophageal epithelium and 84 ESCC specimens, were selected by a pathologist (K-W Chan) and transferred to a TMA block using a 0.6 mm core diameter needle. Haematoxylin- and eosin-stained sections from each TMA block were checked by K-W Chan to ensure that adequate targeted areas had been included. The sample numbers varied slightly among staining results due to a variation in the number of interpretable specimens on TMA sections.Immunohistochemical staining using Dako EnVision+ system-HRP was carried out according to the manufacturer's instructions. Polyclonal anti-Id-1 (C-20), Id-2 (C-20), Id-3 (C-20) and Id-4 (H-70) antibodies were applied at a 1 : 600, 1 : 800, 1 : 200 and 1 : 50 dilution, respectively, for overnight incubation at 4°C. For peptide-blocking analysis used to verify antibody specificity, five-fold (w/w) excess of blocking peptide was preincubated with the corresponding primary antibody overnight at 4°C before applying them to the TMA slides.The stained sections were reviewed by two independent observers (K-W Chan and Y-P Chan) who had no prior knowledge of the clincopathological data of the patient cohort. The ‘hot spot’ method was employed in evaluation of the staining results. All three cores of each specimen in the TMA were individually scored, and the highest score was selected for later statistical analysis. The extent and intensity of cytoplasmic staining was graded by an arbitrary scale ranged from 0 to 3, representing negative (0), weak (1), moderate (2) and strong (3) staining, respectively. Negative and weak cytoplasmic staining was classified as low expression, while moderate and strong cytoplasmic staining was classified as high expression. Nuclear staining was scored by the percentage of nuclei positively stained in the whole core with at least 200 nuclei counted. Specimens with nuclear staining score lower than the mean percentage were classified as low expression and vice versa. An overall grade of expression was then assigned by combining both cytoplasmic and nuclear staining results such that only specimens having both high cytoplasmic and nuclear staining scores were graded as high overall staining. Staining patterns other than that were classified as low overall expression.χ2 test, Mann–Whitney U-test or Fisher's exact test where applicable. The association between expression level and the risk to develop distant metastasis within 1 year of oesophagectomy was estimated by odds ratio and 95% confidence intervals using binary logistic regression analysis. Survival curves between low- and high-level expressions of the four Id proteins were estimated by Kaplan–Meier analysis and compared by log-rank test. Cox regression analysis was performed to identify independent factors that predict patient survival. All factors with P-value <0.10 in the univariate analysis were allowed entry to the multivariate analysis. Backward stepwise selection was used with removal limits of P-value <0.05. A P-value <0.05 was considered significant in all the statistical analyses.Statistical analysis was performed using SPSS 14.0 software. Differences in expression level among different clinicopathological stages were analysed by As shown in As shown in In contrast, expressions of Id-3 and Id-4 did not differ much between non-neoplastic epithelium and tumour tissues. Id-3 was positively detected in 94% (31 out of 33) of non-neoplastic specimens and in 90% (72 out of 80) of ESCC specimens. While 17 out of 33 (52%) non-neoplastic oesophageal epithelium samples expressed a low level of Id-4, 36 out of 80 (45%) ESCC specimens showed similar low expression. Id-3 was mainly expressed in the nucleus and sometimes in cytoplasm of non-neoplastic oesophageal epithelium, but its expression in ESCC was generally both cytoplasmic and nuclear. Id-4 was mainly detected in the nucleus of non-neoplastic oesophageal epithelium samples, while its expression in cytoplasm is more frequent in ESCC.P=0.012) nuclear expression of Id-1 (16.13±7.82%) than those at M0 stage (4.76±1.55%). Binary logistic regression was then employed to estimate the odds ratio for patients having high-level nuclear expression of Id-1 in their primary ESCC to develop distant metastasis within 1 year. Primary ESCC tumours with more than 11.86% (mean percentage of nuclei stained positive in ESCC specimens) of nuclei stained positive for Id-1 were regarded as having high and others as having low nuclear expression. The results showed that patients with high-level nuclear expression of Id-1 in their primary ESCC were at a higher risk to develop distant metastasis within 1 year : 1.596−13.348, P=0.005).As shown in P=0.045) associated with T4 tumour.In contrast, we observed that 59% of primary ESCC at the T4 stage showed a high cytoplasmic Id-1 expression, whereas only 30% of ESCC at the lower T stages with similar staining extent . By statP=0.013). While 30% of poorly differentiated ESCC expressed low-level cytoplasmic Id-2, the same was observed in only 7% of well or moderately differentiated tumours excess of corresponding blocking peptide, the signal was almost completely abolished . These rP=0.041) predicted better survival in our cohort (P=0.007), M1 stage (P=0.026) and low overall Id-2 expression (P=0.045) were significant predictors of poor survival in univariate analysis , male sex (P=0.019), increasing nuclear Id-1 expression (P=0.011) and low overall Id-2 expression (P=0.020) individually contributed significantly to poor prognosis as shown by the multivariate analysis . A high level of cytoplasmic Id-1 expression was significantly associated with T4 stage tumour (P=0.045). In contrast, low-level cytoplasmic Id-2 expression was significantly associated with poorly differentiated tumours (P=0.013). In addition, high level of overall Id-2 expression in primary tumour predicted better survival of ESCC patient in Kaplan–Meier analysis (P=0.041). By multivariate Cox-regression analysis, we were able to demonstrate that increasing nuclear Id-1 expression (P=0.011) and low overall Id-2 expression (P=0.020) were independent predictors of poor overall survival in our ESCC patients.Id proteins have been recently demonstrated to promote cancer progression and metastasis and are potential prognostic factors in different types of cancer. In this study, we aimed to investigate how this knowledge could be applied to further our understanding of the pathogenesis and progression of ESCC. The expression levels of Id proteins in ESCC and non-neoplastic oesophageal epithelium in corresponding oesophagectomy specimens were studied by immunohistochemistry and the results were correlated with clinicopathological parameters. We observed that primary tumours at M1 stage had a significantly higher nuclear Id-1 expression than M0 stage tumours (in vitro (P=0.012) higher nuclear expression of Id-1 than the M0 stage tumours. In addition, by binary logistic regression, we observed that a high nuclear expression of Id-1 in primary ESCC tumours significantly predicted the development of distant metastasis of the corresponding patients within 1 year of oesophagectomy . In addition, a high level of cytoplasmic Id-1 expression was significantly (P=0.045) correlated with T4 stage tumours. These results suggest that upregulation of Id-1 in ESCC might promote distant metastasis and cancer progression. In vitro experiments are being undertaken to investigate the functional role of Id-1 in metastatic progression of ESCC.Id-1 has been shown to promote metastasis in several different types of cancer. Overexpression of Id-1 promotes mammary epithelial cell invasion (P=0.013) associated with poor tumour differentiation. Moreover, high level of both nuclear and cytoplasmic expression of Id-2 provides favourable prognosis for our ESCC patients, with mean survival of 28.9 (95% CI: 16.7–41.1) months when compared to those having low overall Id-2 expression (16.8 (95% CI: 12.3–21.1) months).Id-2 has been suggested to promote cellular differentiation in normal murine mammary epithelial cells and low-level overall expressions of Id-2 (P=0.020) were independent predictors for poor prognosis in our cohort of ESCC patients.To test whether Id-1 and Id-2 might act as independent prognostic factors in our patient cohort, Cox-regression with backward stepwise selection was carried out. The results showed tIn summary, our results suggest that overexpression of Id-1 can promote neoplastic transformation of normal oesophageal epithelium, ESCC progression and the development of distant metastasis. In contrast, expression of Id-2 might inhibit the emergence of a more aggressive phenotype of ESCC. We provide new evidence that Id-1 and Id-2 may be useful prognostic indicators of metastatic potential of ESCC and survival of patients, respectively. |
Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.We have identified novel miRNAs from S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.Authentic miRNAs were identified for the first time in Noncoding RNAs (ncRNAs) of approximately 22 nucleotides (nt) in length are becoming increasingly recognized as important regulators of gene expression in animals, plants, and viruses C. elegans http://microRNA.sanger.ac.uk), which includes animal, plant, even virus miRNAs, contained 6396 entries at the time this manuscript was prepared. Indeed, the total number of miRNAs is estimated to represent approximately 1% of all genes in each genome, implying that a myriad of miRNAs and their roles in cellular processes such as development and size control of organisms, remain to be discovered The first characterized endogenous miRNAs were lin-4 and let-7, both of which were shown to act in the pathway controlling the timing of larval development in Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. In China, Schistosomiasis japonica remains a major public health problem. Humans can become infected by wading or bathing in contaminated water. Schitosomes have a complex life cycle, alternating between the definitive mammalian host and an intermediate freshwater molluscan host.The platyhelminthe schistosome parasites (flatworms) cause schistosomiasis, which seriously affects 200 million people worldwide. The schistosome species that infect humans include S. mansoni was characterized Schistosomes have haploid genomes estimated to be approximately 270 MB, arrayed on seven pairs of autosomes and one pair of sex chromosomes S. japonicum. To this end, we constructed a library of size-fractionated RNAs from S. japonicum adult worms and analyzed 227 RNA sequences from a total of 106 insert-containing clones randomly selected from the library. From the abundant and diverse population of small RNAs observed in S. japonicum, potential miRNAs with flanking sequences that were predicted to fold into a stem-loop precursor structure were further detected by Northern blot. We further utilized a highly sensitive and specific stem-loop RT-PCR for quantitative analysis of the expression patterns of these novel miRNAs across the life span of S. japonicum.In light of the evidence implicating small RNA species in gene regulation in flatworms, here we investigated potential miRNAs within S. japonicum were isolated from the hepatic portal system and mesenteric veins of infected rabbits or mice at 6–7 weeks post infection. Male and female adult worms were manually separated under a light microscope. Hepatic schistosomula were isolated from the portal system and from mesenteric veins of infected mice at 2 weeks post-infection. All procedures performed on animals within this study were conducted in accordance with and by approval of the Internal Review Board of Tongji University School of Medicine. Cercariae of S. japonicum were shed from the lab-infected intermediate host, the snail O. hupensis hupensis, with hatched miracidia provided by National Institute of Parasitic Disease, Chinese Center for Disease Control and Prevention. After collection, all freshly isolated samples were washed three times with 1×PBS (pH 7.4) and were immediately used for extraction of total RNA or stored in liquid nitrogen until being subjected to further analysis.Adult worms and eggs of S. japonicum were extracted using Trizol® reagent (Invitrogen) according to the manufacturer's protocol with one modification. Namely, after addition of isopropanol, the RNA extract was incubated for at least 2 h at −20°C (instead of 5 min at room temperature) to enhance precipitation of low-molecular-weight (LMW) RNAs. Following a wash with 80% ethanol, RNA was re-suspended in DEPC-treated water or formamide (Sigma) and stored at −80°C.Total RNA from samples from the 6 different life cycle stages of 5′-pacuGTAGGCACCATCAAx-3′) RNA/DNA adaptor and then to a 5′ (5′-ATCGTaggcacctgaaa-3′) adaptor. For the ligation, the short RNAs were first dephosphorylated using Shrimp Alkaline Phosphatase (Takara), and then ligated to the 3′ RNA/DNA adaptor using T4 RNA Ligase (Takara) according to the manufacturer's instructions. Further ligation to the 3′ end of the 3′ adaptor was blocked by the C6-NH2 group on the 3′ end of the adaptor oligonucleotide. After PAGE purification of the ligated products and rephrosphorylation of the 5′ terminus, ligation of the 5′ DNA adaptor was performed. The final ligation product was ethanol precipitated, resolved on denaturing 15% polyacrylamide, eluted from the gel, ethanol precipitated, and used as a template in a reverse transcription reaction using an RT primer (TTGATGGTGCCTAC-3′5′-A) which is complementary to part of the 3′ adaptor sequence (underlined). Resulting cDNAs were amplified by PCR using antisense (GGTGCCTACAG-3′5′-ATTGAT) and sense (GGCACCTGAAA-3′5′-ATCGTA) primers containing internal BanI restriction sites (underlined), based on the sequences of the 5′ adaptor and the RT primer. Resulting PCR products were purified, digested with BanI, concatenated, then ligated into pGEM-T Easy vector (Promega). Following transformation, plasmids were purified from ampicillin-resistant colonies and sequenced.The cloning and analysis of small RNAs was performed essentially as described previously S. japonicum small RNAs library, were used in BLAST searches to identify candidate S. japonicum miRNAs. We screened the databases of S. japonicum transcriptome (http://www.ncbi.nih.gov/Genbank/index.html), and entire collection of unassembled genomic sequence reads (http://lifecenter.sgst.cn/sj.do). The S. mansoni genome database (http://www.sanger.ac.uk/Projects/S_mansoni) was also used for the supplementary annotation analysis of cloned sequences by using WU-BLAST with default parameters and a non-stringent cutoff of E<1.8S. japonicum miRNA candidate sequences with approximately 100 bp of flanking sequence were extracted and analyzed using RNA-fold software such as Mfold (http://www.bioinfo.rpi.edu/applications/mfold) and Vienna RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) for the potential to adopt a stem-loop conformation typical of pre-miRNAs with a folding free energy of at least 25 kcal/mole . The candidate sequences were then screened against a database of known miRNAs (http://www.sanger.ac.uk/Software/Rfam/mirna) to compare our candidate S. japonicum miRNAS to miRNAs in other species. In order to further verify candidate miRNAs, mature miRNAs and pre-miRNAs from S. japonicum and S. mansoni genomes were subjected to family alignment processing using the ClustalW2 program .After masking of vector and adaptor sequences and removal of redundancy, the inserts of sizes between 18 and 26 nt obtained from the S. japonicum adult worms were separated by electrophoresis on a 15% polyacrylamide gel under denaturing conditions, and transferred to Hybond-N nylon membrane . After a brief rinse in 2×SSC, the filter was blotted dry, and the RNA was uv-crosslinked to the filter. The filter was pre-hybridized for 2 h at 37°C in hybridization solution , then incubated with approximately 100 ng/ml biotin-labeled oligonucleotide probe in hybridization solution for 8–12 h at 37°C. We probe sequences that were complementary to the candidate miRNAs identified from the S. japonicum small RNA library. Each oligo were labeled one biotin moieties in 5′-end . After hybridization, filters were washed twice in 2×SSC and 0.1% SDS at room temperature for 10 min and twice in 0.1×SSC and 0.1% SDS at 45°C for 20 m. Filters were incubated in blocking buffer (Pierce) for 15 min, then streptavidin-HRP conjugate (Pierce) was added to the blocking buffer (Pierce) at a 1∶300 dilution and incubated for 15 min at room temperature. Filters were washed 4 times with washing buffer (Pierce) for 5 min, then incubated in equilibration buffer (Pierce) for 5 min. For detection, the filters were incubated in enhanced luminal-based chemiluminescent substrate (Pierce) for 15 min. Finally, the membranes were covered with plastic wrap and exposed to X-ray film for 20 min at room temperature for signal detection.As a verification step and to provide size and sequence confirmation of candidate miRNAs, total RNA or LMW RNAs extracted from a mixture of male and female S. japonicum. The primers for these reactions were designed according to Chen et al. S. japonicum adult worm. The RT product was then amplified using a miRNA-specific forward primer and a universal reverse primer. The primers used in the RT and subsequent quantitative PCRs are specified in Because of their small size, detection of these miRNAs by traditional methods is technically demanding. The stem-loop reverse transcription primers provide more specificity and sensitivity than linear primers owing to the base stacking and spatial constraints of the stem-loop structure. Therefore, stem-loop real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate miRNA expression across the life cycle of S. japonicum, 50 nM each individual stem-loop RT primer, 2 U RNase inhibitor, 5 U M-MLV reverse transcriptase (Takara), and 0.5 µM dNTP. The reaction parameters were as follows: 16°C for 30 min, 42°C for 30 min, 70°C for 15 min, and holding at 4°C. To generate the cDNA template for the endogenous control quantitative PCR reactions, first strand cDNA was synthesized using oligo(dT) in the presence of 2 µg of total RNA isolated from the tissue samples from each of the six life cycle stages. The synthesis reaction conditions were as follows: 42°C for 30 min, 70°C for 15 min, then hold at 4°C.The RT reactions contained 2 µg total RNA isolated from the tissue samples from each of the six different life cycle stages of 5′-GTACATGTTGGTCAAGCTGGTGT-3′ and reverse 5′-AGTTCGCACTTCATCCACTACAGT-3′. The quantitative RT-PCR reaction conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. The threshold cycle (Ct) was defined as the cycle number at which the fluorescence intensity passes a pre-determined threshold. In our analyses, quantification of each miRNA relative to α-tubulin was calculated using the following equation −ΔCt, ΔCt = CtmiRNA−Ctα-tubulin. Dissociation curves were generated and 8% PAGE electrophoresis was performed for each real-time RT-PCR to verify the amplification of only the desired product.Real-time quantitative PCR was performed using an Applied Biosystems 7300 detection system in a 15 µl reaction. All reactions were carried out in duplicate. For quantitation of the three miRNAs, the PCRs were carried out in a total volume of 15 µl, including 1 µl of RT product for each miRNA, 1×SYBR Green I Mastermix, 0.5 µM specific forward primer, and 0.5 µM common reverse primer. For the endogenous control, α-tubulin, 1 µl of cDNA synthesized with oligo(dT) was used as the template. The α-tubulin primers were as follows: forward S. japonicum, we generated two libraries of small (18–26 nt) RNA species, one from cercaria and the other from adult worm. When the cercaria library sequences were further analyzed using the annotated gene database at GenBank and http://lifecenter.sgst.cn/sj.do, all sequences proved to be fragments of rRNA or tRNA sequences (data not shown). Therefore, we proceeded to analyze only the library cloned from S. japonicum adult worm.In order to identify the miRNAs in S. japonicum and S. mansoni indicated that among the isolated small RNAs, 65.63% were rRNA, 3.52% were mRNA, and 24.24% were ncRNAs. The 55 ncRNAs were further analysed for the potential miRNAs.A total of 106 insert-containing clones randomly selected from the library yielded 227 unique sequences, which indicated that each concatamer contained ∼2 small RNA sequences. The sequences ranged in length from 18 to 26 nt, which is consistent with the size of known miRNAs processed by Dicer. As shown in S. japonicum are actual miRNAs, we first removed contaminating rRNA, tRNA, and EST sequences. A nonredundant set of 12 sequences out of the 55 ncRNA clones were predicted to be capable of forming stem-loop structures characteristic of miRNA precursors. These 12 candidate sequences were further examined by Northern blot to confirm their size and sequence. Northern blots of total RNA from a mixture of male and female S. japonicum adult worms were hybridized with biotin-labeled probes of the twelve candidate sequences. Five of these twelve probes gave a hybridization signal that was characteristic of miRNA (∼22 nt mature miRNA and /or ∼70 nt pre-miRNA) . Unless e-miRNA) appears S. mansoni and S. japonicum showed high conservation , miRNA precursors (60–100 nt) and mature miRNAs (18–26 nt). Each of isolated templates was purified from 5 µg total RNA by denaturing in a 15% polyacrylamide gel. Similar yields of amplification product were obtained from the reactions using total RNA and LMW RNA after 22 and 30 cycles. The amplification product was not detectable in the reactions after 22 cycles, but a small amount of product was observed after 30 cycles using isolated miRNAs precursors as a template genomics and transcriptomics studies have revealed that several ESTs in schistosomes have homology to Dicer and Argonaut that are protein components of the miRNA silencing pathway S. japonicum by constructing and screening parasite cDNA library of size-fractionated RNAs: sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1. Analysis of sja-let-7, sja-miR-71 and sja-bantam expression by stem-loop RT-real time PCR revealed highly stage-specific expression patterns. Importantly, the expression of sja-miR-71 and sja-bantam peaked in cercaria and then decreased quickly to their lowest levels in schistosomulum, following host infection. These findings suggest that these miRNAs are involved in schistosome infection, growth and development. The identification and examination of the expression and functions of these miRNAs opens up a new field of study in this important parasite pathogen.In this study, we firstly identified five authentic miRNAs in S. japonicum miRNA expression. Our experiments demonstrated that this more cost-efficient method enables high specificity, sensitive detection of miRNAs in S. japonicum. Importantly, it provides precise discrimination between S. japonicum and host miRNAs. A similar method recently described by Varkonyi-Gasic et al. The quantification of miRNAs at various life cycle stages is essential for elucidating their functions in gene regulation. Conventional methods such as Northern blot may not have sufficient sensitivity to detect low abundance miRNAs. A stem-loop Taqman miRNA RT-PCR assay was recently developed for miRNA detection based upon a RT reaction initiated by a stem-loop primer S. japonicum library severely hinder discovery of miRNAs. As shown in S. japonicum. Although we repeated the cloning process several times, the very high percentage of molecules of rRNA origination remained (data not shown). The low percentage of molecules of mRNA origination rules out the possibility of external degradation during the RNA isolation and cloning procedures. The phenomenon known as the large rRNA subunit “nick in vivo” has previously been reported in schistosome S. japonicum adult worm showed only one major band slightly larger than 18S, which was distinct from the two major bands (28S and 18S) typically seen in other species. This degradation of the 28S large rRNA subunit may be the source of the large proportion of rRNA fragments cloned into the small RNA library.The over-representation of degraded rRNAs in the pool of short RNAs in the S. japonicum.miRNAs regulate a variety of developmental and physiological processes. Most miRNAs are expressed in a developmental or organ-specific manner, or both, which provide a few hints about their functions C. elegans and D. melanogaster C. elegans, promoting the transformation from larva to adult, and is also highly expressed in the late third instar larval stage of D. melanogaster when a pulse of the ecdysone triggers puparium formation and onset of metamorphosis S. japonicum indicates that sja-let-7 might take part in the transformation from miracidium to sporocyst in the snail intermediate host. Bantam miRNAs are known regulators of both proliferation and apoptosis, and target the proapoptotic gene hid hid-induced apoptosis in the eye S. japonicum by regulating cell proliferation and apoptosis. Thus far, no studies concerning the function of miR-71 family miRNAs have been reported. It has been difficult to construct a detailed 3′-UTR library from the schistosoma transcriptome because of an incomplete EST sequence, which has greatly inhibited extensive computational prediction of these miRNAs targets.The role of let-7 during metamorphosis in Drosophila and in C. elegans development is well documented for S. japonicum. Untangling S. japonicum physiology can have important ramifications for efforts to control schistosmiasis, which remains a substantial challenge in endemic areas due to the difficulty of eradicating the snail intermediate hosts. Clarifying the role of miRNAs in the parasite's growth, development and ability to infect mammalian hosts will be particularly important given its complex life cycle, involving several distinct developmental stages of vertebrate and invertebrate hosts and a unique repertoire of genes expressed at different life cycle stages. Elucidation of schistome gene regulation will enable dissection of the biological basis of antigenic diversity, infectivity, and pathology, and will provide the best prospects for identifying new drug targets and vaccine candidates.Further work aimed at identifying the target mRNAs of these novel miRNAs will be needed to elucidate the functions of newly identified miRNAs in Table S1Sequences of the primers used for stem-loop RT-PCR.Stem-loop RT primers were designed according to Chen et al. (0.03 MB DOC)Click here for additional data file.Table S2http://www.ncbi.nih.gov/Genbank/index.html), the entire collection of unassembled genomic sequence reads (http://lifecenter.sgst.cn/sj.do) and the S. mansoni genome (http://www.sanger.ac.uk/Projects/S_mansoni). a, the sequence of 18 clones contained 5 miRNAs that matched the criteria of miRNA and were verified by Northern blot. b, sequences of the clones can form stem-loop structures of miRNA precursors but were not verified by Northern blotting. c. Sequences of the clones contain more than three mismatches with the reported genome and transcriptome sequence in GenBank, Sanger center, Shanghai LSBI libraries.Composition in percentage of the 227 total sequences analyzed from the library of S. japonicum small RNAs. The annotation was based on information from the databases of S. japonicum transcriptome ((0.03 MB DOC)Click here for additional data file.Figure S1Predicted stem-loop structures for the mir-bantam and mir-125 precursors of S. japonicum and S. mansoni using Vienna RNAfold method. Mir-125 precursor from both S. japonicum and S.mansoni genome showed acceptable bulges in their secondary structure by Vienna RNAfold method. The bugle in the secondary structure of bantam precursor from S.mansoni was disappeared while the bugle from S. japonicum genome remains.(0.92 MB TIF)Click here for additional data file.Figure S2Alignments of S. japonicum miRNAs precursors with S.mansoni. The sequences of mature microRNAs are boxed.(1.19 MB TIF)Click here for additional data file.Figure S3Sequence alignments of pre-miRNAs in each miRNA family. Abbreviation is the same as (4.50 MB TIF)Click here for additional data file.Figure S4Phylogeny analysis of four miRNAs precursors. Abbreviation is the same as (0.74 MB TIF)Click here for additional data file.Figure S5The specificity of stem-loop RT-PCR for S. japonicum miRNA quantitation. (A) Electrophoresis of the miRNA RT-PCR products on 8% PAGE showing bands of the anticipated size (65 bp). (B) Electrophoresis of the miRNA RT-PCR products on 8% PAGE showing no amplification using host RNA template. Lane 1: total RNA isolated from total blood cells of rabbit, lane 2: total RNA isolated from total blood cells of mice, lane 3: total RNA isolated from S. Japonicum, lane S: stem-loop RT-PCR reactions using the RNA of S. Japonicum adult worm with the mice mmu-let-7c specific primers. (C) Dissociation curves for the three duplicate RT-PCR reactions showing specificity of the reactions. (D) The specificity of the stem-loop RT-PCR assay in detecting mature miRNA expression. Four different RNA templates (lanes 1–4) were subjected to stem-loop RT PCR using the indicated stem-loop RT primer, then electrophoresed on 8% PAGE. Lane 1, 5 µg total RNA, lane 2, low molecular weight (LMW) RNA purified from 5 µg total RNA , lane 3, miRNA precursors (60–100 nt) isolated from 5 µg total RNA on denaturing 15% polyacrylamide gel, lane 4, mature miRNA (18–26 nt) isolated from 5 µg total RNA on denaturing 15% polyacrylamide gel. PCR cycle numbers are indicated to the left.(3.95 MB TIF)Click here for additional data file.Figure S6Dynamic range and sensitivity of sja-miR-71 RT-QPCR assay using synthetic sja-miR-71 miRNA. (A) Amplification plot of synthetic sja-miR-71 miRNA over three orders of magnitude. Synthetic RNA input ranged from 1pM to 1000pM in PCR. (B) Standard curve of the sja-miR-71 miRNA.(0.94 MB TIF)Click here for additional data file.Figure S7Dynamic range of sja-miR-71 RT-QPCR assay using adult S. japonicum total RNA. (A) Amplification plot of sja-miR-71 miRNA over three orders of magnitude. Total RNA input ranged from 0.016 to 16 ng per RT reaction. (B) Correlation of total RNA input to the threshold of cycle (CT) values of sja-miR-71 miRNA assays.(0.96 MB TIF)Click here for additional data file.Figure S8The amplification plot of sja-let-7, sja-mir-71, sja-bantam and alpha tubulin. The cDNAs for QPCR were obtained from the RT reactions that used the same cercaria sample. The RT reactions and consequent real-time quantitative PCR were performed at the same conditions. 1, 2, 3 and 4 were the amplification plot of sja-mir-71, sja-bantam, sja-let-7 and alpha tubulin, respectively, the RT product of cercaria RNAs. 5, 6, 7 and 8 were the amplification plot of sja-let-7, sja-mir-71, alpha tubulin and sja-bantam, respectively, using the no-RT control.(2.30 MB TIF)Click here for additional data file. |
C) converts into a misfolded isoform (PrPSc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrPC; in older flies, PrP misfolds, acquires biochemical and structural properties of PrPSc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrPSc-specific conformational epitopes. In contrast to PrPSc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrPSc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrPSc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrPSc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP Creutzfeldt-Jakob disease is a type of dementia caused by the deposition of the prion protein in the brain. This disorder belongs to a unique class of degenerative diseases that includes mad-cow disease in bovine and scrapie in sheep. An abnormal form of the prion protein is not only responsible for the disease in several mammals, but is also an infectious agent that can transmit the disease within or across species. To shed light on how the prion protein changes from its normal to the disease-causing form, we expressed the prion protein from hamster in transgenic flies. We observed that the prion protein progressively converts to the pathological form and induces neuronal loss in the brain. Thus, the prion protein experiences its typical transition from normal to disease-causing form in flies. This behavior gave us the opportunity to investigate whether other proteins can regulate such transition. We found that the stress-related protein Hsp70 prevents the accumulation of abnormal prion protein and prevents neuronal loss. We also determined that Hsp70 directly interacts with the prion protein in specific membrane domains. Overall, our studies provide new insight into the mechanisms that regulate the accumulation of abnormal prion protein. This discovery could have therapeutic applications in treating these devastating disorders. C) into its pathological scrapie isoform (PrPSc) seems to be the fundamental process underlying the pathogenesis of prion diseases Sc can be distinguished from PrPC by its partial resistance to heat, denaturing agents and protease digestion, its insolubility in non-ionic detergents, and its fibrillar aggregation Sc in the brain is associated with cerebral damage, including spongiform degeneration and neuronal loss. According to the “protein-only” hypothesis, PrPSc transmits the disease by propagating its abnormal conformation using PrPC as a substrate by autocatalytic mechanisms The prion protein (PrP) appears to be an essential element in the pathogenesis of an incurable class of neurological disorders called transmissible spongiform encephalopathies (TSE) or prion diseases. These protein deposition disorders can present with sporadic, inherited or infectious origins and lead to dementia, motor dysfunction, and inevitably, death C converts into typical protease-resistant PrPSc by mechanisms largely unknown. It has been accepted, though, that intrinsic biochemical properties encoded into the amino acid sequence of PrP are the basis for its conformational changes. Indeed, transgenic mice overexpressing wild type PrP display neuronal loss, astroglyosis, and PrP deposition Sc in transmission has been thoroughly documented, it is not clear whether PrPSc is the neurotoxic isoform of PrP. PrP conformers that do not share all the biochemical properties of PrPSc may be responsible for neuropathology in TSE toxic or PrPL , has been challenging, so far.The unique infectious aspects of prion diseases have received substantial attention due to the scare of the “mad cow’ epidemics of the 1980's. However, the sporadic disease accounts for 80–85% of all prion disorders in humans Given the interest in the infectious aspects of prions, the elucidation of the cellular mechanisms involved in spontaneous PrP misfolding and PrP-dependent neurotoxicity has progressed at a slower pace. Recent studies suggest that both endoplasmic reticulum (ER) and cellular stress may play an important role in prion diseases Sc has accumulated over the last 20 years. Since flies do not posses a PrP orthologue, this is a good host system to understand the consequences of expressing mammalian PrP in a genetically tractable model. However, modeling prion diseases in flies has proved challenging Drosophila neurons progressively misfolds, acquires biochemical features of PrPSc and induces spongiform degeneration of brain neurons. Remarkably, the molecular chaperone Hsp70 directly interacts with PrP, prevents the accumulation of misfolded isoforms and reduces its neurotoxicity in neurons of the fly brain. These results suggest that Hsp70 upregulation might be of therapeutic interest in prion diseases.In this paper, we have focused on three relevant aspects of PrP biology: Can wild type PrP spontaneously convert in vivo? What is the nature of the neurotoxic PrP species? And, do molecular chaperones play a role in PrP misfolding and aggregation? To answer these questions we expressed wild type PrP from Syrian Golden Hamster (HaPrP) in transgenic flies. The initial isolation of PrP came from hamsters C from a healthy hamster due to the presence of two facultative N-glycosylation sites that yield di-, mono- and unglycosylated PrP fractions . To determine the ability of wild type mPrP to induce degenerative phenotypes, we tested mPrP and HaPrP lines in the same conditions. First, we determined the relative expression levels of the mPrP and HaPrP transcripts by quantitative RT-PCR. We identified mPrP lines expressing slightly lower levels (P1) and twice as much (J1) than our strong HaPrP line (The progressive neurotoxicity of wild type HaPrP in motor neurons seemed to disagree with a previous report in which wild type PrP from mouse (mPrP) did not induce neurodegeneration PrP line . These mPrP line . While tDrosophila brain neurons, a hallmark of prion neuropathology.Spongiform degeneration is the neuropathological hallmark of TSE. In the brain of scrapie-infected hamsters, spongiosis is the consequence of vacuolation of cell bodies and processes . To inveC into new conformers that are insoluble in non-ionic detergents ScC from a healthy hamster was only detected in the sarkosyl/NaPTA soluble fraction, while PrPSc from a scrapie-infected hamster accumulated in the insoluble fraction that detects common epitopes in fibrils of different types of amyloids Sc from hamster to denaturing agents. PrPSc is highly resistant to heat, urea and guanidine thiocyanate; however, high concentration of chemical denaturants can destabilize the PrPSc conformation, rendering it soluble. We subjected extracts from scrapie-infected hamsters and heads from old flies to a gradient of guanidine thiocyanate and then tested for PrP solubility. Both PrPSc from hamster and Tg-PrP from old flies exhibited remarkable resistance to high concentrations of guanidinium (up to 1.5 M) . InteresC) from disease-specific (PrPSc) conformations in bovine, sheep, rodents and human CJD and the production of a protease resistant core of smaller size (PrP27–30) Sc, our results are consistent with other PrP conformations that might be relevant in disease One of the most typical features of infectious PrPAmong the chaperone proteins Hsp70 has the exceptional ability to correct the misfolding of several amyloidogenic proteins involved in neurodegenerative diseases We followed these experiments by assessing the ability of Hsp70 to protect against PrP-dependent neurotoxicity. First, we wondered if Hsp70 could protect against the vacuolar degeneration of brain neurons induced by Tg-PrP. For this, we created flies that exhibited an intermediate spongiform phenotype by using a moderate PrP line. The rationale for this moderate phenotype was to provide sensitive conditions to detect Hsp70 neuroprotection. Flies expressing moderate PrP levels in all neurons showed a mixed phenotype at day 30, where 44% of cells were undergoing vacuolar changes, while the rest exhibited preserved cytoplasm . The nucNext, we analyzed the mushroom bodies, which are the dorsal (α lobe) and medial (β and γ lobes) projections of the Kenyon cells . As expeFinally, we tested the protective activity of Hsp70 in functional assays. For this, we established a moderate locomotor phenotype by inducing a ubiquitous, albeit weak, expression of PrP. Under these conditions, the flies expressing only PrP displayed a steady decline in climbing ability over 20 days, with a 50% climbing activity at day 7 . Flies eTo better understand how Hsp70 exerts its chaperone activity on PrP, we evaluated the possibility that Hsp70 could interact directly with PrP. For this, we performed pull-down and co-immunoprecipitation assays in flies co-expressing PrP and Hsp70. For the pull-down we prepared active His-Hsp70 coated beads in a spin column and tested the binding of Tg-PrP from brain homogenates of young and old flies. Then, we resolved the interacting fraction in western blot and assessed PrP Immunoreactivity. Tg-PrP from both young and old flies interacted with Hsp70 in the column, but the amount of PrP recovered from older flies was several fold higher . This reThese intriguing results presented a clear problem: where does the interaction between Hsp70 and PrP take place? Hsp70 is the major cytosolic chaperone, while PrP is a secreted glycoprotein attached to the extracellular membrane. Hence, the chances for these two proteins to physically interact seemed limited. Therefore, we wondered if we could identify the subcellular domain where Hsp70 and PrP interact. Recent reports show that Hsp70 exhibits a remarkable ability to translocate to different cellular compartments, including the extracellular space A relevant cellular microdomain for PrP biology is the lipid raft or detergent-resistant membrane (DRM), an specialized plasma membrane domain involved in critical cellular functions, such as trafficking, signalling and protein sorting. Moreover, the lipid raft has been proposed as the site for PrP conversion Sc-specific conformational antibody. These new features suggest that wild type PrP progressively misfolds and accumulates in a conformational state that shares several properties with PrPSc. However, this PrP conformer is protease sensitive, which clearly distinguishes it from prototypical PrPSc. Hence, Tg-PrP acquires a conformation consistent with PrP isoforms previously described in both experimental animals and patients toxic, PrPL or protease-sensitive PrPSc conformers have been interpreted as PrPSc byproducts or, alternatively, they could be immature metabolic intermediaries of PrPScSc conformation (conversion factor) or the presence of molecules that prevent the formation of PrPSc (anti-conversion factor). It is not clear, thus, why flies accumulate this specific PrP conformer or whether flies could produce PrPSc through genetic modification of the cellular environment. These relevant aspects of PrP biology can be further studied in Tg-PrP flies through genetic studies.Considerable attention has been devoted in the last 25 years to define the chemical nature of prions and their transmissibility. However, less is known about the cellular mechanisms that participate in PrP misfolding, how prions actually damage the central nervous system and how this process can be prevented. To understand the mechanisms regulating spontaneous PrP misfolding, we described how the biochemical properties of wild type PrP change over time in transgenic flies. Early on, Tg-PrP is mostly soluble and accumulates very little misfolded conformers. In contrast, Tg-PrP from older flies is mostly insoluble, fibrillar, resistant to high concentrations of guanidinium and is recognized by a PrPC to PrPSc is central to prion pathogenesis because Prnp null mice and mice in which PrP expression is knocked-out after infection are resistant to disease Sc because significant pathology and/or clinical dysfunction can develop with little accumulation of protease-resistant PrPSc in rodent models of TSE L conformers induce deleterious effects by gain-of-function mechanisms since neurotoxicity in flies correlates with the progressive accumulation of novel, protease-sensitive PrPSc-like conformers C to PrPL and a low rate of maturation of PrPL to PrPSc would favor the accumulation of neurotoxic conformers L, resulting in strong neurotoxicity. These results also suggest that neurotoxic PrP can form independently of the typical PrPSc pathway and may represent a stable conformer with its own kinetics. Once the PrPL conformers accumulate, they can exert neurotoxicity by sequestration of cellular proteins, inhibition of the cellular clearance machinery , and/or induction of ER stress and the UPR, among other mechanisms. These deleterious effects of wild type PrP in flies are consistent with the brain and muscle defects observed in transgenic mice that overexpress wild type PrP We describe here the formation of a neurotoxic PrP conformer that leads to typical spongiform vacuolation of brain neurons. But, what have we learned about the mechanisms of PrP neurotoxicity? Conversion of PrPDrosophila be considered prions? Based on the classic definition, which includes transmissibility, PrP* does not share all properties of prions since it is not protease resistant, an important feature for PrP infectivity. However, transmissibility has been achieved with protease sensitive material in some instances So, can these new PrP* conformers generated in Sc-like conformers, and involves the direct binding of Hsp70 and PrP. Supporting this idea, the direct interaction of Hsp70 to cytosolic PrP (cytPrP) prevents apoptosis in cultured neurons Hsp70 is one of the most potent molecular chaperones and has been shown to prevent misfolding of α-Synuclein and expanded polyglutamine proteins in transgenic flies It is possible that the Hsp70/PrP interaction is mediated by Hsp70 co-chaperones, such as Hsp40. Hsp40 directly binds substrates and presents them to the catalytic site of Hsp70 Prnp gene was isolated by PCR amplification from genomic DNA. EcoRI and NotI restriction sites were included in the primers (5′-GAATTCATCATGGCGAACCTTAGCTACTG-3′ and 5′-GCGGCCGCTCATCCCACCATCAGGAAGATG-3′) to facilitate cloning into the Drosophila pUAST vector yw embryos and seven single-insertion lines were created.The open reading frame of the Syrian golden hamster Drosophila Hsc4-dn (HSC4-K71S), the reporter strains UAS:LacZ and UAS:CD8-GFP, and the mushroom body , motor neuron and ubiquitous drivers were obtained from the Bloomington Drosophila Stock Center. Two strong mPrP strains were provided by S. Supattapone UAS flies expressing human Hsp70 (HSPA1L), Flies expressing PrP throughout the brain under the control of da-GAL4 were collected at 1 and 30 days after eclosion, along with sibling control flies. Plastic embedding was prepared as described TS were raised at 18°C and the adults were placed at either 25°C (no expression) or 30°C (high PrP expression) upon eclosion Flies carrying HaPrP-M6, HaPrP-M9, mPrP-P1, mPrP-J1 or control constructs were crossed with the BG380-Gal4 driver and the progeny was subjected to climbing assays To quantify the levels of PrP transcripts expressed from hamster or mouse PrP transgenes, we performed real-time RT-PCR assays using the SYBR green fluorescent reagent. Total RNA was isolated from fly heads expressing PrP under the control of the OK107 driver. DNA traces were eliminated with Turbo DNAse (Ambion). Real-time PCR reactions were done using the ABI PRISM 7700 system (Applied Biosystems) and the relative amounts of mRNAs were calculated by amplifying RNA Pol II mRNA in the same reactions. For Hsp70 experiments, PrP transcripts were quantified in GFP/PrP or Hsp70/PrP flies. Plotted values were obtained from three independent reactions and arbitrarily normalized against one of the lines tested.Ten to twenty fly heads from each relevant genotype were used for brain extracts. Fly heads were homogenized in 30 µl of PBS containing 150 mM NaCl, 1% Triton X-100, 4 mM EDTA and Complete Protease Inhibitors (Roche). 10% brain homogenates (w/v) from healthy and sick hamsters were prepared as described NaPTA precipitation was conducted as outlined Homogenates from young (day 1) and old (day 30) flies were incubated with PK concentrations from 0 to 5 µg/ml for 30 min at 4°C. The digestions were stopped by adding 2 mM PMSF and the samples were resolved in 12% SDS gels, transferred to a .2 µm nitrocellulose membrane and stained for 3F4 immunoreactivity.Sc-specific conformations were detected in hamster and fly brain extracts using the 15B3 immunoprecipitation kit PrPg for 5 h at 4°C. Ten 300 µl fractions were collected from the top and analyzed by western blotting after methanol precipitation. The anti-α Subunit of the Na+/K+ ATPase and Syntaxin (1∶ 50) antibodies were obtained from the Developmental Studies Hybridroma Center.For microsome preparations, forty heads were homogenized in 70 µL of BIB buffer . Samples were centrifuged 30 min at 5,000 rpm at 4°C to eliminate debris. Supernatants were subjected to a second centrifugation for 1 h at 20,000 rpm at 4°C. Supernatants (cytoplasmic fraction) were recovered and pellets (microsomes) were resuspended in BIB buffer to run a Western blot. Detergent-resistant membranes (DRMs) or lipid rafts were prepared as described The significance of the differences between PrP and Hsp70/PrP flies in climbing assays was determined by Chi-square with 7 degrees of freedom (8 points per day). The average speed was tested by a two-tailed t-student. Statistical significance was considered below 1% of chance.Figure S1Wild type mouse PrP induces locomotor dysfunction. (A) The relative expression of PrP transcripts induced by two wild type mPrP lines was compared with moderate (M9) and strong (M6) HaPrP lines by quantitative RT-PCR. The mPrP-P1 line induces slightly lower expression than HaPrP-M6, but mPrP-J1 induces twice as much PrP transcripts. (B) Wild type mPrP expression induces locomotor dysfunction. Expression of the strong (P1) and very strong (J1) mPrP transgenes in motor neurons induce early locomotor dysfunction. When compared to HaPrP-M6, the strength of these phenotypes correlate with the expression levels shown in A. Expression of LacZ is used as control.(0.46 MB TIF)Click here for additional data file.Figure S2The Solubility of cytosolic β-Galactosidase is not affected by age. Separation of sarkosyl/NaPTA soluble (S) and insoluble (P) fractions from head extracts of 1 or 40 day-old flies expressing bacterial LacZ. β-Galactosidase is only detected in the soluble fraction in both young and old flies, indicating that its solubility does not change over time.(0.76 MB TIF)Click here for additional data file. |