The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe3O4 nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.Magnetic nanoparticles (Fe Magnetic materials have been used with grain sizes down to the nanoscale for longer than any other type of material . This is3O4) nanoparticles can be attracted by a magnetic field and are easily separable in solution. Similarly, substances to which they have been attached can be separated from a reaction medium, or directed by an external magnetic field to site specific drug delivery targets [Due to their magnetic character, magnetite  that produces hydrogen peroxide according to the reaction 1.Cholesterol + O2 → 4 - Cholesten - 3 - one + H2O2   (1)3O4 magnetic nanoparticles will contribute to the basic understanding of its activity and function.The structure of cholesterol oxidase reveals deeply buried active sites occupied by water molecules in the absence of its substrate steroids . Cholest3O4 magnetic nanoparticles. The sizes and structure of the nanoparticles were characterized using TEM and FTIR spectroscopy. The stability, activity, and kinetic behavior of bound cholesterol were also examined.The present study proposes to investigate the direct binding of cholesterol oxidase to Fe3O4 particle. It could also be envisaged that CHO molecules formed aggregates to bind several magnetic nanoparticles. Another possible factor in the agglomeration process is the centrifugation process involved in the separation of the supernatant from the Fe3O4-CHO. It is obvious that the centrifugation tend to bring particles together as a compact material. The effect of agglomeration at this stage can be reduced by separating the Fe3O4-CHO by an external magnetic field. Since the particles are released after removal of the magnetic field, they may fall separately apart from each other, and are less likely to agglomerate.TEM micrographs of "bare" magnetic nanoparticles and CHO-functionalized magnetic nanoparticles are shown in Figure 3O4 nanoparticles used were 14.4, 17.2 and 20 mg/mL, corresponding to CHO/Fe3O4 weight ratios of 0.01, 0.08 and 0.007, respectively. These results show that in all the binding operations, there were sufficiently available amount of particles to bind the enzymes till complete saturation. In a previous study [3O4 nanoparticles, that is reducing the weight ratio of CHO to Fe3O4 below 0.033 caused an increase in lipase binding up to 100%. This was not observed in this study, possibly because of the difference in the binding mechanism, due to differences in the structure of the enzyme. However, the percentage of bound CHO (98–100%) shows that the binding process was successful.The unbound enzyme was determined by assaying the protein content in the supernatant. It was found that the percentage of cholesterol oxidase bound was between 98 and 100%, irrespective of the amount of particles. The amounts of Fe3O4, Fe3O4-CHO, and CHO in water, respectively. A characteristic band of NH2 was observed at 1618 cm-1 in the "bare" Fe3O4nanoparticles. The NH2 group can be associated with NH stretch at 3400 cm-1 which is not visible here, because of a possible hindrance by OH stretch from water. However, this band was not apparent in the spectra of Fe3O4-CHO suggesting that the binding of CHO to the nanoparticles involved this amino group and the carboxylic groups of CHO after being activated by Carbodiimide, as suggested by [-1 and 1445 cm-1 are more visible in Figure 3O4-CHO) and could be assigned to traces of residual ammonium hydroxide. The characteristic bands of proteins at 1647 and 1541 cm-1, and 1645 and 1541 cm-1, in the spectra of Fe3O4-CHO, and CHO, respectively shows that cholesterol oxidase was effectively present in the samples, confirming the binding of cholesterol oxidase to Fe3O4 nanoparticles. The negative peak at 3400-2799 cm-1 is possibly due to a reduced amount of water in the sample compared to the water used for background subtraction. The characteristic bands of proteins in the Fe3O4-CHO spectra were very weak compared to those in the spectra of cholesterol oxidase in water. The weakness of the peaks is due to the limited amount of CHO bound to the nanoparticles, in comparison to the amount dispersed in water.The binding of CHO to magnetic nanoparticles was confirmed by FTIR analysis. Figure Vmax and Km for CHO were determined to be 0.67 μmol/min mg and 2.08 mM for the free enzyme and 1.64 μmol/min mg and 0.45 mM for the immobilized enzyme, respectively. The Vmaxvalue of the bound CHO was 2.4 fold higher than that of the free, and the Km value of the bound CHO was 4.6 fold lower than that of the free CHO. The low Km reflects the high affinity to substrate [3O4 nanoparticles contributed to an enhancement of the activity of the enzyme.The kinetic parameters of the enzymatic reactions estimated by the Lineweaver-Burk plots of the initial rates of cholesterol oxidase from experimental data are presented in Figure ubstrate , the reaThe effect of pH on the activities of the free and bound CHO was investigated in the pH range of 6–8.5 at 25°C and presented in Figure k) at temperatures where the inactivation experiments were observed. The rate constants increased with increasing temperature and were higher for the free CHO than for bound CHO. As stated above, the binding to nanoparticles suggests a better resistance of the enzyme to temperature. We hypothesize that the bound enzyme could possibly undergo a conformational change and a spatial rearrangement that could slow down the folding process and denaturation of the enzyme.The thermal stability of free and bound CHO was investigated after 40 min of storage in the temperature range of 25–70°C Figure . There wThe effect of temperature on the activity of the free CHO was examined by measuring its relative activity when stored at various temperatures Figure . It can 3O4 nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO providing a better stability to the enzyme.The stability and activity of the enzyme are naturally reduced during storage. Figure Nocardia sp. was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide reagent, Triton X-100, TRIS (Hydroxymethyl) aminomethane HCL, 4-cholesten-3-one, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 %, and iron (III) chloride hexahydrate 99% were obtained from Sigma-Aldrich, St Louis (USA). The Biorad Protein Assay Dye Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Acetonitrile was obtained from EMD Chemicals, (New Jersey, USA).Cholesterol oxidase (EC 1.1.3.6), 3O4) were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in a solution of ammonium hydroxide followed by a treatment under hydrothermal conditions [4OH solution (30%), at a control pH (10–10.4). The suspensions were heated at 80°C for 35 min under continuous mixing and separated by centrifuging several times in water and then in ethanol at 2800 rpm. The purification step was used to remove impurities from Fe3O4 nanoparticles. The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction to a magnetic rod.Magnetic nanoparticles Fe3O were prenditions . Iron (I3O4 nanoparticles and Fe3O4bound cholesterol oxidase (Fe3O4-CHO) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, 0.1 M NaCl and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles [50–70 mg of magnetic nanoparticles was added to 1 mL of phosphate buffer (0.05 M. pH 7.4). The mixture was sonicated for 15 min after adding 0.5 mL of carbodiimide solution (0.02 g/mL in phosphate buffer (0.05 M. pH 7.4). Following the carbodiimide activation, 2 mL of cholesterol oxidase (0.25 mg/mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath and the mixture was centrifuged at 3000 rpm . The preThe amount of protein in the supernatant was determined by a colorimetric method at 595 nm using the Biorad Protein Assay Reagent Concentrate with bovine serum albumin (BSA) as the protein standard. The amount of bound enzyme was calculated from:A = (Ci - Cs)*V   (2)A is the amount of bound enzyme, Ci and Cs is the concentration of the enzyme initially added for attachment, and in the supernatant, respectively (mg-mL-1), V is the volume of the reaction medium (mL).Where 3O4 nanoparticles and Fe3O4-CHO was characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL USA) and structure by Fourier Transform Infrared (FTIR) spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared by placing a drop of the magnetic nanoparticles dispersed in nanopure water onto a copper grid and evaporated in air at room temperature. Before preparing a sample onto the copper grid, the dispersed solution was sonicated for 4 min to obtain better particle dispersion. The binding of CHO onto the magnetic nanoparticles was investigated using FTIR. CHO and Fe3O4-CHO samples in phosphate buffer and Fe3O4 particles were dissolved in nanopure water for FTIR analysis.The size of Fe3O4-CHO, and mixed by vortex. A solution of free CHO of the same concentration was used to evaluate the activity of the free enzyme. The solution was incubated at various temperatures (25–70°C) at specific intervals of time (1 h) and centrifuged at 3000 rpm for 5 min to separate the supernatant from Fe3O4-CHO. 10 μL aliquots of the supernatant were then taken and the concentration of 4-cholesten-3-one was assessed. Before measuring the amount of 4-cholesten-3-one in a sample, the activity of the free enzyme was stopped by adding an equal volume of acetonitrile to the reacting solution [The activity of bound CHO was determined by measuring the initial oxidation rates of cholesterol by cholesterol oxidase at given temperature following the increase of 4-cholesten-3-one concentration at 240 nm, using a Beckman Du Spectrometer. A solution of cholesterol was prepared by dissolving 4.8 g of cholesterol in 10 mL of 2-propanol. A phosphate buffer solution (0.05 M. pH 7.4) containing 4% of Triton-100 was added to the mixture to result in a 0.26 M cholesterol solution. The mixture was gently heated until the solution was clear. To start the enzymatic reaction, 5 ml of cholesterol solution was added to 15 mL centrifuge test tubes containing Fesolution . Each ki3O4-CHO were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (25–70°C) in phosphate buffer (0.05 M, pH 7.4) for 40 min. Aliquots of the reacting solution were taken at time intervals (every 30 min for 7 hours) and assayed for enzymatic activity as described above. The first order inactivation rate constant, k was calculated from the equation:The thermal stability of free and FeA = In A0 - kt   (3)In A0 is the initial activity, A is the activity after a time t (min), k is the reaction constant.where 3O4-CHO was estimated by determining the concentration of 4-cholesten-3-one in samples at various temperatures. A solution of cholesterol was added to the various centrifuge test tubes containing bound or free enzymes. The test tubes were stored in a water bath at specific temperatures (25, 37, 50, 60, and 70°C). At time intervals, the concentration of 4-cholesten-3-one was determined by spectrophotometric analysis.The effect of temperature on the free CHO and Fe3O4-CHO or free enzyme solution were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 30 days. Thereafter, 5 mL of cholesterol was added. The storage stability of the free and bound cholesterol oxidase was determined by assaying for their residual activity.The storage stability was evaluated by determining the concentration of 4-cholest-en-3-one at room temperature at time intervals (5 days). Test tubes containing Fe3O4-CHO, Km and Vmax were determined by measuring initial rates of oxidation of cholesterol (1.3–5.2 mM) by CHO (0.25 mg/mL) in phosphate buffer pH 7.4 at 25°C.The kinetic parameters of free CHO and Fe3O4 magnetic nanoparticles reduced the energy barrier for CHO activity. As a result of the binding to the magnetic nanoparticles, the storage stability of CHO was considerably enhanced. This higher stability of the Fe3O4-CHO is attributable to its possible fixation on the surface of the particles preventing auto-digestion and thermal inactivity. In addition, the binding on Fe3O4 nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO to provide better stability to the enzyme. The overall improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound cholesterol oxidase. The study may be useful in improving the stability and activity of cholesterol oxidase, and will contribute to more efficient use of this enzyme.Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The binding of CHO to the particles was confirmed by FTIR spectroscopy and the size characterized by TEM. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of CHO were significantly improved upon binding to nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy indicated that the binding of CHO onto FeCHO: Cholesterol oxidaseTEM: Transmission electron microscopyFTIR: Fourier Transform InfraredBSA: Bovine serum albuminDrs Gilles K Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept, experimental plan, and analysis. Dr Gregory McCarty was the secondary author and contributed to the overall effort.


In the microarray experiment, many undesirable systematic variations are commonly observed. Normalization is the process of removing such variation that affects the measured gene expression levels. Normalization plays an important role in the earlier stage of microarray data analysis. The subsequent analysis results are highly dependent on normalization. One major source of variation is the background intensities. Recently, some methods have been employed for correcting the background intensities. However, all these methods focus on defining signal intensities appropriately from foreground and background intensities in the image analysis. Although a number of normalization methods have been proposed, no systematic methods have been proposed using the background intensities in the normalization process.In this paper, we propose a two-stage method adjusting for the effect of background intensities in the normalization process. The first stage fits a regression model to adjust for the effect of background intensities and the second stage applies the usual normalization method such as a nonlinear LOWESS method to the background-adjusted intensities. In order to carry out the two-stage normalization method, we consider nine different background measures and investigate their performances in normalization. The performance of two-stage normalization is compared to those of global median normalization as well as intensity dependent nonlinear LOWESS normalization. We use the variability among the replicated slides to compare performance of normalization methods.For the selected background measures, the proposed two-stage normalization method performs better than global or intensity dependent nonlinear LOWESS normalization method. Especially, when there is a strong relationship between the background intensity and the signal intensity, the proposed method performs much better. Regardless of background correction methods used in the image analysis, the proposed two-stage normalization method can be applicable as long as both signal intensity and background intensity are available. After being reverse-transcribed into cDNA and labeled using red (Cy5) and green (Cy3) fluorescent dyes, two target mRNA samples are hybridized with the arrayed DNA sequences or probes. Then, the relative abundance of these spotted DNA sequences can be measured. The ratio of the fluorescence intensity for each spot represents the relative abundance of the corresponding DNA sequence.In cDNA microarray experiments, there are many sources of systematic variation. Normalization is the process of removing such variation that affects the measured gene expression levels. The main idea of normalization is to adjust for artifact differences in intensity of the two labels. Such differences result from differences in affinity of the two labels for DNA, differences in amounts of sample and label used, differences in photomultiplier tube and laser voltage settings and differences in photon emission response to laser excitation. Although normalization alone cannot control all systematic variations, normalization plays an important role in the earlier stage of microarray data analysis.et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [Many normalization methods have been proposed by using the statistical regression models. Kerr et al.  and Kerr [et al.  suggeste [et al.  proposed [et al.  proposed [et al.  used a l [et al.  summariz [et al. ). Workma [et al.  proposed [et al.  suggeste [et al.  presenteAfter performing two dye cDNA microarray experiments, we get foreground and background intensities from red channel and green channel, respectively. Although a complex modeling approach can be used, the signal intensity is usually computed by subtracting the background intensity from the foreground intensity. Thus, the noise in the background intensity may have a large effect on the signal intensity.et al. [et al. [et al. [et al. [et al. [Several approaches have been proposed for decreasing the background noises in image analyses . Kim et [et al.  found ou [et al.  and Edwa [et al. . As poin [et al. , the sigMethods section. Also, Methods section describes the proposed two-stage normalization methods. Results section describes the results from NCI 60 cDNA microarray experiment, which illustrates the effects of background intensities . In add [et al. ) and tho [et al. ). The peM; at the second stage, we correct bias on M caused by other sources of systematic variation.We propose a two-stage normalization method for the cDNA microarray data analysis using background intensities. At the first stage, we adjust for the effect of background intensities on gfi and gbi be the means(or medians) of the i th foreground and background intensity of green channel, respectively; rfi and rbi be the corresponding means(or medians) of red channel, respectively. Then for each spot, we have two pairs of intensities: (gfi, gbi) and (rfi, rbi), i = 1,..., p, where p is the number of spots in a slide.  - log(gf - gb)] = [log(R) - log(G)],     (2)In cDNA microarray experiments, there are red and green background intensities. It would be desirable to consider the background intensities that are more closely related with the signal intensities. We consider nine possible background measures from red channel, green channel, and both channels as follows:(a) Red channelY1 = log(rb),     (3)Y2 = log(rf / rb),     (4)Y3 = log(rf)/log(rb),     (5)(b) Green channelY4 = log(gb),     (6)Y5 = log(gf / gb),     (7)Y6 = log(gf)/log(gb),     (8)(c) Both channelsFor each category, there are three types of background measures. The first one is log-transformed background intensities. The second one is the log-transformed ratio of foreground and background intensities. The third one is the ratio of the log-transformed values of foreground and background intensities. Here we used log base 2, but any logarithmic base can be used as desired. Figure Yk (k = 1,...,9) be an appropriate background intensity defined by red channel (a), green channel (b), or two channels (c). Then, fit the nonlinear LOWESS curve as follows:For simplicity, let Mk = C(1) (Yk),     (12)Mk(1) = Mk - C(1) (Yk), for k = 1,...,9     (13)C(1)(·) represents the LOWESS curve and Mk(1) is the residual from the curve. Note that Mk(1) is the log-ratio of relative intensities after removing the effect of background intensities. For these ratios, we can perform the usual MA normalization at the second stage.where In the second stage, we perform the normalization process as follows:Mk(1) = C(2) (A),     (14)Mk(2) = Mk(1) - C(2) (A),     (15)C(2) (·) is the LOWESS curve and Mk(2) is the residuals from the curve in the second stage.where Note that at the second stage any normalization method can be applied including a simple global normalization method.. The National Cancer Institute's Developmental Therapeutic Programs  has been studying a large number of anti- cancer drug compounds and molecular targets on the 60 cancer cell lines . In par [et al.  performeM versus Yk, where M = log(R/G) and Yk, k = 1,...,9 are background measures described in Methods section. The correlation coefficients between M and Yk's (k = 1,...,9) are 0.2025, 0.6238, 0.6256, -0.0184, 0.5065, 0.5096, 0.1291, 0.5707, and 0.5729, respectively. Background measures Y2 and Y3 tend to have higher correlations than others.From ten slides we choose one slide to illustrate the proposed method. Figure M(1) versus Yk, where M(1) is the residual in equation (13) demonstrate some reduction in variability, which can be seen more clearly by comparing Figure M(1) and Yk have values close to zero. Using M(1), we carry out the second stage normalization.Figure quation 1 demonstrMA plots. The first row shows the MA plots for original , after global normalization, and after nonlinear LOWESS normalization, respectively (from left to right), The second row shows M(2) versus A plots after two-stage normalization for Y1, Y2, and Y3, respectively. The third row shows MA plots for Y4, Y5, and Y6 respectively, and the bottom row shows MA plots for Y7, Y8, and Y9, respectively. We can see that the two-stage normalization methods using Y2 and Y3 have the effect of reducing the variability among Ms and perform better than global and non-linear LOWESS normalization methods.Figure et al. [MSEl (l = 1,..., number of gene) which is the variance estimator for each gene derived from replicated slides. The main idea is that the better the normalization method, the smaller the variation among the replicated observations. Here, we use three different sets of microarray data: colorectal cancer data of NCI 60 , cortic [et al. ), and mo [et al. ).The goal of cortical stem cells study is to identify genes that are associated with neuronal differentiation of cortical stem cells. In this experiment, there are 3,840 genes in each slide from two experimental groups for comparison measured at six different time points (12 hrs, 1 day, 2 days, 3 days, 4 days, 5 days). All experiments were replicated three times, thus we have 36 slides for the analysis.et al. [The objective of mouse gene expression study of Pritchard et al.  is to aset al. [et al. [In this comparative study, all five microarray data sets were used: colorectal cancer data set from NCI 60, cortical stem cells data set from Park et al. , and thr [et al. . Since rY2 and Y3 in two-stage normalization methods always yield better performances than the nonlinear normalization method, while the other background measures yield comparable results to those of the nonlinear normalization. Thus, we suggest either Y2 or Y3 as background measures in the two-stage normalization.In this study, the performances of two-stage normalization using nine background measures are compared to global normalization and intensity dependent nonlinear LOWESS normalization. Figure et al. [In microarray studies, many undesirable systematic variations are commonly observed. Normalization becomes a standard process for removing some of the variation that affects the measured gene expression levels. One major source of variation is the background intensities. Recently, some methods have been employed for adjusting for the background intensities. However, all these methods focus on defining signal intensities appropriately from foreground and background intensities during the image analysis . AlthouM in most subsequent analysis, the noise caused by background intensities may still remain in M even after normalization. The two-stage normalization may be quite effective especially when there is a high correlation between M and background measures such as Y2 and Y3.In this paper, we propose two-stage normalization for adjusting for the effect of background intensities systematically. The motivating idea is that the noise caused by background intensities may increase the variability in signal intensities. Although we use the log-transformed ratio of two channels denoted by Y2 and Y3 based on the results of the comparative studies. For these two background measures, we show that the two-stage normalization method always performs better than the global normalization methods and the nonlinear LOWESS normalization method.Among nine background measures, we recommend two background measures Y2 and Y3 is due to low intensities. We investigated this problem for NCI 60 data after removing spots with low intensities. The spots whose ratio of foreground and background intensities were smaller than 1.5 were removed in the analysis. This new data set also provided quite similar results.We wondered if the relative good performance of two-stage normalization using Y2 and Y3 perform better than other backgrounds are as follows. The background fluorescence might be relatively strong in the Cy5 channel due to interaction between the slide substrate and the hybridization materials. This effect is weaker in the Cy3 channel. It might be also possible that the background fluorescence in the Cy5 channel inflates the values of rb without correspondingly inflating the values of rf. This means that for weakly-responding spots, the rf and rb values are similar. This produces very low values in M, Y2 and Y3 for these spots. Note values under 5 for log(R) but not for log(G) in Figure M in all panes in Figure M are partially corrected by regressing against Y2 or Y3. However, the effectiveness of the proposed method for other data may depend on the background fluorescence artefacts.The main reasons why background measures The comparison is based on the variability measures derived from the replicated microarray samples. These variability measures can be easily derived from any replicated microarray experiment. Although we have studied only a limited number of data sets, our findings are quite consistent.Y2 and Y3 yielded better results than other background measures.For these data sets, we also conduct a similar analysis to see the effect of print-tips in normalization process. The results were consistent to those in Figure M: the original one (O), after the global normalization (G), after LOWESS normalization (L), and two-stage normalization using Y2 and Y3 (T2 and T3, respectively). The density plot is in Figure M for T2 and T3 are quite similar to that of L. This simple empirical investigation shows that the proposed method does not always cause a bigger overcorrection than the LOWESS normalization. However, we must be careful for over-fitting which may overcorrects the biological findings of interest.One major concern for normalization is about over-fitting which may cause overcorrection of real biological significance. In fact, every normalization method has a possibility of overcorrecting the data and removing some existing biological significance in the data. For the NCI60 data, for example, it is not easy to find out whether such negatively low expression genes are biologically significant genes or not. We investigate the possibility of overcorrecting by drawing the density plot of In addition, we performed the hypothesis test for M equal to zero and counted the number of genes that have different expression level between two channels. The number of significant gene with different expression value is 909 for the original data before normalization, 326 for the data after global normalization, 302 for the data set after LOWESS normalization, and 297 for the data after two-stage normalization. The numbers of significant genes do not differ much among normalization methods.rf - rb) for the red channel. Our method was illustrated using the usual subtraction (rf - rb). Although our method starts with a most common approach based on the usual subtraction, it can be applied to any other models for defining spot intensity r = f(rf,rb), where r is the signal intensity, as long as a background intensity is available. Our method can be applicable as long as the image analysis provides the signal intensity and background intensity. For example, our method using Y2 builds on the relationship between r = f(rf,rb) and rb.The proposed method can be applicable when both background and foreground intensities are available after the image analysis. Many image software provides both signal intensity as well as background intensity of spots. In most cases, the signal intensity is defined by simple subtraction such as (Y2 and Y3. The performance of the two-stage normalization method tends to depend on the correlations between background measures and M. That is, if there is a strong relationship between them, our method has a large effect. Thus, for the experimentalists it might be important to tell when to use the two-stage normalization method. In order to answer this question, we compute correlations between M and all background measures from Y1 to Y9. Figure M and Yks for each data set. For example, Figure Y2 and Y3 are relatively higher than those from other background measures. We also think that is why Y2 and Y3 have better performances than the other background measures.We recommend that experimentalists examine their data carefully and consider applying the two-stage normalization methods using Y2 and Y3 are higher than 0.5 for both CCD and SCD, while those for KD are smaller than 0.5. Note that CCD and SCD have more reduction in variability than KD in the two-stage normalization. For lower correlations of Yks do not reduce variability much compared to the usual LOWESS normalization.Figure


A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. However, there is very little known about how inputs across the body mid-line are processed within SII.Optical intrinsic signal imaging was used to evaluate the response of primary somatosensory cortex (SI and SII in the same hemisphere) to 25 Hz sinusoidal vertical skin displacement stimulation ("skin flutter") applied contralaterally, ipsilaterally, and bilaterally to the central pads of the forepaws. A localized increase in absorbance in both SI and SII was evoked by both contralateral and bilateral flutter stimulation. Ipsilateral flutter stimulation evoked a localized increase in absorbance in SII, but not in SI. The SII region that responded with an increase in absorbance to ipsilateral stimulation was posterior to the region in which absorbance increased maximally in response to stimulation of the contralateral central pad. Additionally, in the posterior SII region that responded maximally to ipsilateral stimulation of the central pad, bilateral central pad stimulation approximated a linear summation of the SII responses to independent stimulation of the contralateral and ipsilateral central pads. Conversely, in anterior SII , bilateral stimulation was consistently less than the response evoked from the contralateral central pad.The results indicate that two regions located at neighboring, but distinctly different A-P levels of the anterior ectosylvian gyrus process input from opposite sides of the body midline in very different ways. The results suggest that the SII cortex, in the cat, can be subdivided into at least two functionally distinct regions and that these functionally distinct regions demonstrate a laterality preference within SII. There is general agreement that in cats and monkeys (and presumably in humans) the spike discharge activity a mechanical stimulus sets up in rapidly adapting (RA), slowly adapting (SA), and Pacinian (PC) skin mechanoreceptors is projected centrally, at short latency and with relatively minor transformation, to primary somatosensory cortex (both SI and SII) in the contralateral hemisphere. There is no consensus, however, about the way in which the stimulus-evoked response in the ipsilateral hemisphere contributes to cerebral cortical somatosensory information processing and somatosensation. A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. Similar to SI, SII possesses a clear topographic organization , but unlFigure To more accurately characterize the spatial disparity between SII loci activated by contralateral vs. ipsilateral stimulation, the absorbance values were obtained and plotted along the posterior-anterior axis of SII. Figure A more comprehensive view of the SII response to the contralateral and bilateral stimulus conditions can be better appreciated with a multi-dimensional surface plot of the data. Figure To directly compare the time course of the response of the anterior and posterior regions of SII to the three stimulus conditions, we determined the time course of the absorbance changes in each region under each stimulus condition Figure . The ploCluster plots were used to directly compare the response of SII to the different conditions of ipsilateral and contralateral stimulation. In each plot in Figure 2 boxel surrounding the peak. In anterior SII, the majority of the responses to contralateral stimulation are stronger than the responses to bilateral stimulation – hence, the majority of the points plotted fall below the reference line (which has a slope of 1). In the posterior region, the majority of the points plotted are above the reference line – indicating that the response to the bilateral stimulus was greater than the response to the contralateral stimulus.The contralateral and bilateral stimulus conditions can also be compared using cluster analysis. However, because the difference between the contralateral and ipsilateral responses is more robust than the difference between the contralateral and bilateral responses, Figure Thus far, the results suggest that the anterior and posterior regions of SII are differentially activated by contralateral, ipsilateral and bilateral stimulation. To determine the across-subject consistency of these findings, the average absorbance values evoked by the 3 different stimulus conditions were determined for all 6 of the subjects Figure . ClearlyThe findings of this study demonstrated clearly that the anterior and posterior regions of SII process bilateral inputs very differently. At the locus of the maximal OIS response evoked in the posterior region by an ipsilateral stimulus, bilateral stimulation evoked a response that was, on average, about 25% larger than that evoked from the contralateral stimulus site. Conversely, at the locus of the maximal OIS response evoked by contralateral stimulation in the anterior region, bilateral stimulation evoked a response that was, on average, 35% lower than the activity evoked by a contralateral stimulus. This discrepancy between the optical responses of the anterior and posterior regions could be related to neurophysiological observations reported in earlier studies. For example, Carreras and Andersson  found inThe main, although not the only, route for ipsilateral input to SII is through the corpus callosum, from cells located in SI and SII of the opposite cerebral hemisphere ,7,8,11. The neurons in the distal limb regions of SII do receive substantial callosal connections, but these neurons have been reported to lack ipsilateral RFs , indicatA number of interactions between stimuli applied to both hands have been demonstrated in human psychophysical studies. Gilson  found thA recent report  demonstrOne question that the results suggest is whether or not SII can be segregated by laterality preference, in a manner similar to that observed in other sensory systems. Laterality has been demonstrated in the primary sensory cortex of both the visual system and the auditory system of both primates and cats, and the data in this report strongly suggest that there are cortical areas within SII that exhibit preference to ipsilateral or contralateral inputs. In terms of processing information from simultaneous contralateral and ipsilateral stimuli, there could be further similarities between the somatosensory, auditory and visual systems that have yet to be described. Future investigations will aim to further clarify the role of SII in integration of information from inputs across the body midline.The responses evoked by contralateral and ipsilateral flutter stimulation of the central pad of the cat forepaw define functional subdivisions in SII: the two modes of stimulation maximally activate cortical regions that are anterior and posterior to one another, respectively. Bilateral stimulation, or providing simultaneous contralateral and ipsilateral stimulation, reveals, additionally, that the two adjacent cortical areas process bilateral inputs differently. In the posterior region, where ipsilateral stimulation evokes a maximal response, bilateral stimuli evoke a response that is greater than the response evoked by either the individual ipsilateral or contralateral response. In the anterior region of SII, where the contralateral stimulus evokes a maximal response, bilateral stimuli evoke responses that are smaller in magnitude than the responses evoked by the contralateral stimulus.Adult cats  were subjects. All surgical procedures were carried out under deep general anesthesia . After induction of general anesthesia the trachea was intubated with a soft tube and a polyethylene cannula was inserted in the femoral vein to allow administration of drugs and fluids (5% dextrose and 0.9% NaCl). For each subject, a 1.5 cm diameter opening was made in the skull overlying somatosensory cortex, a chamber was mounted to the skull over the opening with dental acrylic, and the dura overlying anterior parietal cortex was incised and removed. Following the completion of the surgical procedures all wound margins were infiltrated with long-lasting local anesthetic, the skin and muscle incisions were closed with sutures, and each surgical site outside the recording chamber was covered with a bandage held in place by adhesive tape.2 between 3.0 – 4.0%; EEG and autonomic signs  were monitored and titrated (by adjustments in the anesthetic gas mixture) to maintain levels consistent with light general anesthesia. Rectal temperature was maintained (using a heating pad) at 37.5°C.Subjects were immobilized with Norcuron and ventilated with a gas mixture  delivered via a positive pressure respirator 1–3 hours prior to the data acquisition phase of the OIS imaging experiments. Respirator rate and volume were adjusted to maintain end-tidal COEuthanasia was achieved by intravenous injection of pentobarbital (45 mg/kg) and by intracardial perfusion with saline followed by fixative . Following perfusion fiducial marks were placed to guide removal, blocking, and subsequent histological sectioning of the cortical region studied. All procedures were reviewed and approved in advance by an institutional committee and are in full compliance with current NIH policy on animal welfare.Results were obtained during stimulation of the contralateral central pad of the forepaw and/or the ipsilateral central pad of the forepaw. The stimuli always consisted of sinusoidal vertical skin displacements  and were applied using a servocontrolled transducer (Cantek Enterprises, Canonsburg, PA) that is capable of delivering sinusoidal stimuli in the range of 1–250 Hz at amplitudes in the range of 0–1000 microns. The stimuli were delivered independently to the ipsilateral and contralateral skin sites, and also were applied simultaneously to both sites . The stimulus probes were positioned 500 microns beyond the point at which skin contact was detected (via force transducer on the Cantek). The bilateral stimulus protocols reported in this paper were synchronized to start and stop at the same time. The contralateral, ipsilateral and bilateral stimuli were interleaved on a trial-by-trial basis. This approach was used to control for temporal changes in cortical "state" unrelated to stimulus conditions which, if unrecognized, might obscure or modify any differences between the optical responses evoked by the contralateral, ipsilateral and bilateral stimulus conditions.Near-infrared  OIS imaging was carried out using an oil-filled chamber capped with an optical window . Images At the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 μm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 μm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries  identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria .A-P = anterior-posteriorRA = rapidly adaptingSA = slowly adaptingPC = PacinianRF = receptive fieldOIS = optical intrinsic signalIR = near infraredEEG = electro-encephalogramMEG = magneto-encephalogramBW and OF participated in the design of the experiments, the data collection, and drafting of the manuscript. SS, JC, and VT made significant contributions to the data collection and the analysis of the data. MT played a major role in all aspects of the development of the manuscript.


Drosophila fruitfly has a remarkably sophisticated immune system. Granted, it can't customize an immune response by grooming cells to “remember” and target specific pathogens. But it can rally the less specialized tools of innate immunity to fight disease and infection, and in so doing draws on several aspects of blood cell development —the foundation of the cellular immune response—also found in vertebrates.For a three-millimeter invertebrate, the In fruitflies, as in vertebrates, hematopoiesis occurs in distinct stages and locations, with nascent cell populations migrating to establish new hematopoietic frontiers. A population of progenitor cells generates all the blood cell types in the organism. These cells arise in two distinct waves and in two distinct locations, with their progeny differentiating into the specialized tissues and organs of the hematopoietic system. In mammals, these organs include the liver and bone marrow, an ongoing source of blood cells after embryogenesis. In fruitflies, the definitive hematopoietic organ is the lymph gland, which churns out three types of blood cells: plasmatocytes and crystal cells, which are also produced by embryonic hematopoietic precursors, and lamellocytes. Plasmatocytes account for up to 95% of circulating fruitfly blood cells and act much like their mammalian counterpart, the macrophage, engulfing substances deemed foreign and dangerous. Crystal cells account for most of the rest and are involved in melanization reactions, which trigger mechanisms involved in containing and killing invading microbes. Unlike plasmatocytes and crystal cells, lamellocytes appear en masse and only under certain conditions, such as the unwelcome appearance of parasitic wasp eggs. Lamellocytes encapsulate and neutralize the invader.Molecular factors involved in determining the fate of hematopoietic cells have been identified for plasmatocytes and crystal cells but not for lamellocytes—until now. In a search for genes that might precipitate lamellocyte differentiation, Marie Meister, Alain Vincent, and colleagues homed in on a protein, called Collier (Col), that is expressed in lymph glands at the end of embryogenesis. Col is quite similar to a mammalian protein, called Early B-cell Factor (EBF), that controls B-cell development in mice. Both proteins are transcription factors, exerting control by initiating gene transcription.To investigate Col's part in lamellocyte development, the researchers had to get around the fact that mutations that render Col nonfunctional eventually kill the embryo. Using tricks of the genetics trade, Meister and colleagues generated fly larvae that survive loss-of-function mutations in the gene that encodes Col, allowing them to study its role in hematopoiesis.The mutants had normal amounts of circulating plasmatocytes and crystal cells, but when exposed to parasitic wasp eggs, could not muster the requisite response: lamellocyte production. With no lamellocytes, fly larvae had no means of protection against encroaching wasp eggs, which, uncontested, developed into parasitic larvae. The flies with normal Col levels had no such problem, producing considerable numbers of lamellocytes. In these flies, Col expression was restricted to a lymph region called the posterior signaling center (PSC). Col's influence on lamellocyte fate was strong enough that forcing Col expression in precursor blood cells induced lamellocyte differentiation even in the absence of wasp infestation. Based on these findings, Meister and colleagues propose a model for Col-mediated lamellocyte differentiation in which wasp infestation activates Col-expressing cells in the PSC, which then instructs immature blood cells in the lymph gland to become lamellocytes and dispatch the gathering threat.Col's role in fruitfly hematopoiesis closely parallels that of its mammalian ortholog in white blood cell development, EBF. Both are required to generate specialized populations of cells in response to a particular immune threat, and both confer an extra line of defense when faced with special circumstances—key features of vertebrate adaptive immunity. Could it be that building blocks of adaptive immunity were already in place some 550 million years ago, when flies and vertebrates parted ways? Researchers will have to investigate the molecular agents of immunity in intervening species to find out.


The recruitment of cohesins to pericentric chromatin in some organisms appears to require heterochromatin associated with repetitive DNA. However, neocentromeres and budding yeast centromeres lack flanking repetitive DNA, indicating that cohesin recruitment occurs through an alternative pathway. Here, we demonstrate that all budding yeast chromosomes assemble cohesin domains that extend over 20–50 kb of unique pericentric sequences flanking the conserved 120-bp centromeric DNA. The assembly of these cohesin domains requires the presence of a functional kinetochore in every cell cycle. A similar enhancement of cohesin binding was also observed in regions flanking an ectopic centromere. At both endogenous and ectopic locations, the centromeric enhancer amplified the inherent levels of cohesin binding that are unique to each region. Thus, kinetochores are enhancers of cohesin association that act over tens of kilobases to assemble pericentric cohesin domains. These domains are larger than the pericentric regions stretched by microtubule attachments, and thus are likely to counter microtubule-dependent forces. Kinetochores mediate two essential segregation functions: chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions. We suggest that the coordination of chromosome movement and biorientation makes the kinetochore an autonomous segregation unit. Kinetochores mediate chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions The proper segregation of replicated chromosomes, or sister chromatids, to daughter cells during mitosis requires that sister chromatids establish stable attachments to microtubules emanating from opposite spindle poles, known as chromosome biorientation, and that sister chromatids move to opposite poles of the cell during anaphase. Chromosome biorientation is made possible by the cohesion of replicated sister chromatids, which occurs along the entire length of the chromosome and is especially robust in large centromere-flanking or “pericentric” domains . The cenGiven the importance of cohesion in pericentric regions, it is critical to understand how these large functional domains are assembled. An important clue came with the discovery of a group of proteins that mediate cohesion and are conserved in organisms from the yeasts to vertebrates . This grSchizosaccharomyces pombe have provided evidence for the role of repetitive heterochromatic DNA in pericentric cohesion. In this organism, the recruitment of high levels of cohesion factors to centromeric regions is dependent on Swi6 (SPAC664.01c), the fission yeast homolog of the heterochromatin protein HP1  . Furthermore, this binding within troughs is unlikely to reflect poor shearing of the pericentric chromatin by sonication, as ChIPs performed using antibodies specific for the kinetochore protein Mif2p showed an enrichment within centromeric DNA that decreased 5-fold in flanking regions only approximately 245 bp away from CEN3  . We obse–279 kb) A–3C. The–279 kb) D (Blat a–279 kb) . The pearom CEN3 . Lastly,While all pericentric regions were enriched for Mcd1p and Smc3p binding, we found that the pattern of cohesin subunit association was different within each pericentric region A–3C. ForS. pombe likely occurs through an interaction with the heterochromatin constituent HP1 bound to repetitive DNA sequences, the absence of repetitive DNA flanking budding yeast centromeres indicates that some other mechanism is used for the recruitment of cohesin throughout large pericentric domains. One possibility is that the high density of cohesin within pericentric chromatin is dependent on the centromere–kinetochore complex. To test this possibility, we examined Mcd1p association in the centromere-flanking regions of chromosomes in the presence and absence of the centromere. For these experiments, the endogenous centromeres on CHRIII and CHRXIV were replaced by CEN3 and CEN14 sequences, respectively, flanked by site-specific recombination target sites for the R recombinase from Zygosaccharomyces rouxii . This observation is consistent with two alternative explanations. First, the centromeric enhancer may form a gradient of activity that dissipates with distance from the kinetochore. This model is unlikely, however, since the fold increases in cohesin association adjacent to the ectopic centromere were similar throughout the flanking approximately 37-kb region. Alternatively, the length of pericentric chromatin that can be influenced by the centromeric enhancer may be constrained by ntromere . Interesntromere . It is pntromere .trans factor that either recruits cohesin to pericentric chromatin or maintains high levels of pericentric cohesin binding. One candidate for such a trans factor could be a histone-modifying enzyme. The centromeric enhancer would augment the recruitment of the histone-modifying enzyme and enhance cohesin association. Indeed, results from S. pombe indicate that a histone methyltransferase is targeted to centromere-proximal heterochromatin, and this modification is important for cohesin recruitment (In this report we have provided insights into the control of cohesin binding within pericentric chromatin in budding yeast. We have demonstrated that the centromere–kinetochore complex behaves as an enhancer for cohesin association in pericentric chromatin and appears to be largely responsible for the increased levels of pericentric cohesin association at both endogenous and ectopic locations. Presumably, the enhancer functions by activating a ruitment . Since bOur results suggest that the coordination of microtubule attachment and pericentric cohesin recruitment by the budding yeast kinetochore generates an autonomous segregation unit that ensures sister kinetochore biorientation and, consequently, the maintenance of genomic integrity. The integration of these activities by the kinetochore was likely key for the first identification of budding yeast centromeric DNA, since microtubule attachment in the absence of pericentric cohesion is unlikely to have increased the mitotic stability of minichromosomes, the assay used for centromere DNA identification . SimilarBAR1 wild-type and mutant strains, respectively. Cells were then released from the G1 arrest by two washes in the appropriate growth medium containing 0.1 mg/ml Pronase (Sigma, St. Louis, Missouri, United States) to proteolyze the αF. Cells were then allowed to grow in medium containing 0.1 mg/ml Pronase and then arrested in mitosis using either 15 μg/ml nocodazole (Sigma) resuspended in 1% DMSO, or a temperature-sensitive cdc16 mutation, as indicated. Mitotic arrest, as determined by a large-budded cell morphology, was generally reached after 2.5–3 h of growth. The R-site-specific recombinase from Zygosaccharomyces rouxii used for centromere excision events was induced in G1 cells by the addition of galactose  to rich medium containing 2% raffinose.The genotypes of the yeast strains used in this study are listed in Zygosaccharomyces rouxii  containing CEN14 and approximately 200 bp of flanking DNA was inserted between head-to-tail-oriented recombination target sites. A URA3-marked version of this construct was used to replace the endogenous centromere on CHRXIV by one-step gene replacement, and the chromosomal context of CEN14 was then restored using the same strategy outlined above for CEN3. The construction of the CEN3 replacement of CEN1 DNA was performed similarly, except that URA3 was placed adjacent to the centromere between the recombination target sites. Replacement of CEN1 sequences with the CEN3-URA3 cassette resulted in the deletion of 558 bp containing CEN1 (CHRI, SDG coordinates 151263-151820), and was confirmed by Southern analysis of genomic DNA (unpublished data). To obtain PMY318, a strain having the centromere at an ectopic location on CHRIII, strain 1829-15B was transformed with DNA encoding a CEN6-URA3 cassette flanked by regions homologous to the TRX3 locus. The TRX3 ORF and a small amount of flanking sequences, encompassing SGD coordinates 259521-260061, were deleted by the integration of the CEN6-URA3 cassette. Prior to transformation, 1829–15B cells were grown in rich medium containing raffinose, and then plated on medium containing galactose to induce excision of endogenous CEN3 and approximately 200 bp of the flanking region by site-specific recombination, as described above.Centromeres were excised from endogenous chromosomes by site-specific recombination events using a galactose-inducible R recombinase from s rouxii . A 320-beviously . URA3 walacement , selectihttp://www.uchsc.edu/sm/bbgn/megee.html  gels containing 0.15 μg/ml ethidium bromide. Digital images of ethidium bromide–stained gels were quantitated using ImageQuant software . The details of oligonucleotide primer pairs used for PCR analyses are available upon request. In general, PCR primer pairs amplified approximately 300-bp sequences and were separated from neighboring pairs by an average of approximately 1.5 kb. In the CHRIII pericentric region, the mean size of the 37 PCR products used in the ChIP analysis was 304 ± 68 bp. For CHRXIV, the mean size of the 41 PCR products used in the analysis was 309 ± 36 bp. In the CHRI pericentric region, the mean size of the 22 PCR products used in the analysis was 306 ± 31 bp. ChIP experiments were performed at least twice, and representative data from one experiment are presented.Protocol S2 in the accompanying paper by http://microarrays.org). The fluorescence in each spot on the microarray was detected using an Axon 4000B laser scanner/detector , and the ratio of the two signals was determined using GenePix 4.0 software (Axon Instruments). This ratio indicates the enrichment for a given sequence in the ChIP. The AMAD database was used to normalize and store microarray data. The microarrays used in this study contained all the ORFs and intergenic regions in the yeast genome as individual spots .Click here for additional data file.Dataset S2File cdc16_Mcd1-6HA_A364a.(957 KB TXT).Click here for additional data file.Dataset S3File Mcd1-6HA_S288c_NZ.(449 KB TXT).Click here for additional data file.Dataset S4File Mcd1-6HA_ncd10-42_S288c_NZ.(418 KB TXT).Click here for additional data file.Dataset S5cdc16-ts Mcd1-6HA ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S6cdc16-ts Mcd1-6HA ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S7cdc16-ts Mcd1-6HA ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S8cdc16-ts Mcd1-6HA ChIP in A364a.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S9cdc16-ts Mcd1-6HA ChIP in A364a.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S10Cy3 = Mcd1-6HA ChIP in nocodazole-arrested S288C background; Cy5 = genomic DNA.(4.6 MB XLS).Click here for additional data file.Dataset S11ndc10-42 Mcd1-6HA ChIP in nocodazole-arrested S288C background; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S12Cy3 = Mcd1-6HA ChIP in nocodazole-arrested S288C background; Cy5 = genomic DNA.(4.6 MB XLS).Click here for additional data file.Dataset S13cdc16-ts Smc3-6Myc ChIP in A364a; Cy5 = genomic DNA.Cy3 = (4.6 MB XLS).Click here for additional data file.Dataset S14cdc16-ts Smc3-6Myc Mcd1-6HA A364a strain; Cy5 = genomic DNA.Cy3 = ChIP of Mcd1-6HA in (4.6 MB XLS).Click here for additional data file.Dataset S15cdc16-ts SMC3-6Myc MCD1-6HA A364a strain; Cy5 = genomic DNA.Cy3 = ChIP of SMC3-6Myc in (4.6 MB XLS).Click here for additional data file.Dataset S16cdc16-ts Smc3-6Myc ChIP in A364a.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S17Cy3 = genomic DNA; Cy5 = Mcd1-6HA ChIP in nocodazole-arrested S288C background.(4.6 MB XLS).Click here for additional data file.Dataset S18ndc10-42 Mcd1-6HA ChIP in nocodazole-arrested S288C background.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S19Cy3 = genomic DNA; Cy5 = Mcd1-6HA ChIP in nocodazole-arrested S288C background.(4.6 MB XLS).Click here for additional data file.Dataset S20ndc10-42 Mcd1-6HA ChIP in nocodazole-arrested S288C background.Cy3 = genomic DNA; Cy5 = (4.6 MB XLS).Click here for additional data file.Dataset S21cdc16-ts Smc3-6Myc Mcd1-6HA A364a strain.Cy3 = genomic DNA; Cy5 = ChIP of SMC3-6Myc in (4.6 MB XLS).Click here for additional data file.Dataset S22Cy3 = genomic DNA; Cy5 = Mcd1-6HA ChIP in nocodazole-arrested S288C background.(4.4 MB XLS).Click here for additional data file.Saccharomyces Genome Database (http://www.yeastgenome.org/) accession numbers for the genes and gene products discussed in this paper are Irr1p/Scc3p (SGDID S0001288), Mcd1/Scc1p (SGDID S0002161), Mif2p (SGDID S0001572), Pds5p (SGDID S0004681), Smc1p (SGDID S0001886), and Smc3p (SGDID S0003610). The S. pombe Genome Project (http://www.sanger.ac.uk/Projects/S_pombe/) accession number for Swi6 is SPAC664.01c.The


Browne et al. [Browne, MacCallum, Kim, Andersen, Glaser: When fit indices and residuals are incompatible. Psychol Methods 2002] employed a structural equation model of measurements of target cell lysing by natural killer cells as an example purportedly demonstrating that small but statistically significant ill model fit can be dismissed as "negligible from a practical point of view".Reanalysis of the natural killer cell data reveals that the supposedly negligible ill fit obscured important, systematic, and substantial causal misspecifications.A clean-fitting structural equation model indicates that measurements employing higher natural-killer-cell to target-cell ratios are more strongly influenced by a progressively intrusive factor, whether or not the natural killer cell activity is activated by recombinant interferon γ (rIFN γ). The progressive influence may reflect independent rate limiting steps in cell recognition and attachment, spatial competition for cell attachment points, or the simultaneous lysings of single target cells by multiple natural killer cells.If the progressively influential factor is ultimately identified as a mere procedural impediment, the substantive conclusion will be that measurements of natural killer cell activity made at lower effector to target ratios are more valid. Alternatively, if the individual variations in the progressively influential factor are modifiable, this may presage a new therapeutic route to enhancing natural killer cell activity. The methodological conclusion is that, when using structural equation models, researchers should attend to significant model ill fit even if the degree of covariance ill fit is small, because small covariance residuals do not imply that the underlying model misspecifications are correspondingly small or inconsequential. Browne, MacCallum, Kim, Andersen and Glaser  employed51Cr-release measures of natural killer cell lysis were obtained using effector (NK cell) to target cell (K562 human myeloid cell) ratios of 100:1, 50:1, 25:1 and 12.5:1. Following Browne et al. [Browne et al.  analysedr = 0.852), and that the four rIFNγ enhanced NK measures also correlate highly with one another (averaging 0.960). However, the low correlations between the sets of NK and rIFNγ measurements (averaging only .111) indicate that the two sets of measurements reflect relatively distinct aspects of natural killer cell functioning. Browne et al. [2 = 103.59, degrees of freedom (df) = 13, and probability p < 10-15). The small but significant residual differences between the data correlations and the correlations implied by the two-factor model were dismissed by Browne et al.[The correlations reported in Browne et al.'s  Table 1 51Cr and aliquoted with the effector cells (either the NK, or the rIFNγ activated NK cells) in the ratios reported above. The cell mixture was centrifuged to ensure cell surface contact, and incubated to provide an opportunity for the NK cells to bind and rupture the target cells, thereby releasing the radioactive target cell cytoplasm. Gamma radioactivity of the supernatant collected from a second centrifuging indicated the effectiveness of the NK or rIFNγ-activated-NK cells at lysing the target cells, with larger measurements corresponding to more effective NK cell activity.Andersen, Farrar, Golden-Kreutz, Kutz, MacCallum, Courtney & Glaser  provide Browne et al.  modelledThese considerations led to the model of E:T-progressive interference depicted in Figure 2 = 11.97, df = 17, p = 0.802) and an estimate of -80.1, rather than a strictly proportional value of -100. The alternative of constraining the offending measurement error variance to be non-negative while maintaining the -100.0 loading, also results in a fitting model (χ2 = 14.72, df = 18, p = 0.681) having very similar estimates, so whether the interfering effects are "nearly proportional" or "strictly proportional" is equivocal. The progressive and nearly-proportional model , eight measurement error variances (one per indicator), and the single variance applied or assigned to both the separate interfering factors. This model fits, but a negative measurement error variance estimate for NK100 suggested a ceiling had been reached for the largest E:T ratio. Freeing the loading for the NK100 indicator results in clean fit (χ2 = 62.5, df = 17, p < 0.001) and thereby informs us that even the seemingly cleaner IFN measurements are influenced by some progressively interfering factor.Additional models were estimated in attempts to further characterize the entity providing the progressive interference. The lowest inter-item correlations, and the greatest ill-fit of the Browne et al.  model, a2 = 75.5, df = 17, p < 0.001) and thereby speaks against the progressive entity being something connected to each case as a whole. That is, no single feature common to the full set of measurements (e.g. the time of blood sampling, or the person's age, or their cancer progression, or mistaken cell counts), could be progressively applied across both measurement series to account for the data. Such factors might constitute the entity spanning the items within one series, but the other measurement series would have to be progressively influenced by something else.Are two similar, yet separate, factors required for the NK and IFN measurement series, or might it be possible that one E:T-coordinated progressive factor spans all eight measurements? That is, could the progressive interfering factor be something common to an individual, rather than something set for each individual once within the NK measurement series and reset independently (or be some other interference) within the IFN measurement series? To check this, we specified a model having a single progressive methods factor leading to all eight indicators. The same E:T ratio dictated loadings were used, and the NK100 loading freed. This model also fails convincingly , we attempted to check whether the progressive methods factors were necessarily independent of the corresponding true NK activity factors, by freeing the corresponding covariances . This also resulted in signs of underidentification. Hence these data should be heard as being consistent with, but not necessarily as demanding, the independence of the methods factors from the corresponding true NK activity factors. Forcing even a modest correlation between true NK activity and progressive methods factors results in substantial suppression of effects, and standardized effects exceeding 1.0 – which is not "impossible" but which would certainly "confront" anyone inclined to postulate a coordination between true NK activity and the progressive methods factors on substantive rather than "exploratory-statistical" grounds.It might be tempting to interpret these underidentified models as signs of insufficient power due to the rather small N of 72 provided by Browne et al.  but we tWe have basically two sets of estimates to consider: the estimates for the failing Browne et al.  two-factIt is no coincidence that the weakest loading estimates appear where the progressive interference is the greatest, namely for the highest E:T ratio measurements. As more variance in a measure is accounted for by the progressively interfering factor, less variance is left to be accounted for by the true NK or IFN activity factor. According to the fitting progressive model, only about half the variance in the NK100 indicator arises from true variability in NK activity, while the "other half" of the variance arrives primarily from the progressive methodological factor, with a minimal amount of error variance contributed by features unique to the NK100 measurement.Given that the latent variables have variance 1.0, the variance the NK activity factor contributes to an NK indicator can be calculated as the square of the appropriate loading. The Browne et al. [2  and the correlation between NK25 and NK12 (0.930). The Figure  and the Notice that while the correlations are not radically different (0.902 vs 0.930) the contribution to the correlation provided by the causal actions of true NK activity differ substantially (0.600 versus 0.912). A substantial portion of the correlation between the NK100 and NK50 measurements is being provided by the progressively interfering factor, and when this is taken into account, there is a substantial reduction in the degree of coordination that can be attributed to both these measures causally responding to true NK activity. This is the classic distinction between reliability and validity. The NK100 and NK50 measures seem to possess substantial reliability (the basic 0.902 correlation) but much less validity since a substantial portion of the stability, or inter-measure reliability, is arising from a stable, and in this instance progressively-influential interfering entity.2 fold, to 0.695, while the other estimates remain unchanged.The small variance estimate for the progressively interfering factor (0.0000695) is partially an artifact of the large absolute values used in setting the proportional methods effects (-100, -50, -25, etc.). If each of these effects is rescaled by dividing by 100, the effects become -1.0, -0.5, -0.25, -0.125 for NK and -0.5, -0.25, -0.125 and -0.0625 for IFN, and the proportionality of the effects is preserved but the estimated variance of the progressive factor is increased 100One additional model estimate is worth noting. The Figure 51Cr "credit" for having lysed a target cell will be shared among the multiple attacking NK cells, and hence will reduce the seeming per-NK-cell effectiveness of the NK cells. Individual differences in the mechanisms of cell recognition, strength of attachment, delay in NK cytoplasmic reorganization, or energy supply, which are separate from whatever rate-limit constitutes "true NK cell activity", could provide individual differences constituting the variance in the "progressive factor". From this perspective, the independent progressive factor within the rIFNγ series might constitute a rIFNγ induced switch to a different rate-limiting component associated with multiple NK lethal attachments.Let us first consider features capable of producing progressive interference within each series. Multiple simultaneous lysings of a single target cell provide several possibilities. With higher E:T ratios it becomes progressively more likely that any given target cell will be simultaneously attacked by more than one NK cell. The 51Cr-producing effectiveness of that cell – again a phenomena which should coordinate with the E:T ratio.Alternatively, the progressive interference might arise from the blocking of some effector NK cells by physical presence of scrimmage-line NK or lymphocyte cells. If an NK cell is obstructed or delayed in making contact with a target cell by: a) the physical obstruction created by other cells between this NK cell and the target, or b) the NK cell wasting time discovering that the adhered cell is merely another NK or lymphocyte cell rather than a valid target, this progressively reduces the apparent The "multiple simultaneous attacks" and "blocking" scenarios might be supplemented by individual differences in the ability of NK cells to lyse multiple sequential targets. At higher E:T ratios fewer pristine targets are available and hence fewer NK cells have the opportunity to deliver second-lethal-doses, or may end up sharing their second-dose credits. Or, if lethal doses from multiple NK cells reduce the time to ion-gradient-induced cell membrane rupture, multiple-simultaneous-NK activity might instantiate the positive-valued model reported above.Yet other possibilities arise from NKT-cells and T-cell suppression of NK cells. At higher E:T ratios, there may be greater suppression of NK cells by higher concentrations of suppressor chemical signals. Similarly, NKT-cells may become progressively activated or deactivated by E:T-concentration-dependent signals from other T cells in the medium.51Cr by NK cells or other lymphocytes following its release from lysed target cells. At higher E:T ratios more cells are present to re-uptake 51Cr released from lysed target cells, and hence less 51Cr will appear in the centrifuged supernatant. Clearly, there are multiple possibilities for what might be providing the E:T ratio coordinated variations in lysing ability, and any independent pairing of these possibilities potentially constitute the interference in the NK and IFN measurement series.If an individual's NK cells are not uniformly active, but rather display a within-individual gradient of activity (some NK cells being more active than others), and if this gradient is set independently of the features underlying "true NK activity" this would provide another form of explanation. Yet another possibility arises from the uptake of 51Cr reuptake.This study was prompted by fortuitous use of NK cell activity measurements in a debate over the testing of structural equation models. According to Browne et al. , even thOne important consequence of the fitting model is that it provides evidence indicating the lowest E:T measures provide the most valid measures of NK and rIFNγ induced lysing activity. The NK or rIFNγ measurements made at higher E:T ratios correlate highly with one another, but a substantial portion of these correlations appears to result from the progressive interfering factor and not "true" NK and rIFNγ activity. The higher E:T ratio measures remain reliable in the sense of being stable, but they are not as valid as measurements of "true" lysing activity. Given that nearly half the variance in the NK100 measurements is connected to the progressive factor, we have encountered something that is substantial and probably routinely noticed in practice, and is just being mislabeled or overlooked.A second important consequence of the Figure The fact that the progressive factors are tightly connected to E:T ratios makes differential NK activity at various E:T ratios an obvious point of investigative departure. The fact that one of the progressive factors contributes about half the variance in the highest E:T ratio NK measurements implies we are not confronting issues at the limits of measurement, but rather are confronting issues of measurement confounding. Incorporating measures of variables connected to the "candidate explanations" in an expanded version of Figure The author(s) declare that they have no competing interests.LAH developed and ran the structural equation models, and prepared multiple drafts of the manuscript. HPR discussed, and suggested changes to, the various manuscript drafts; prepared the figures, and arranged for consultation with outside experts. GGC discussed, and suggested changes to, the various drafts; and arranged for consultation with outside experts. MJDL discussed, and suggested changes to, the various drafts; and prepared the table. MAB discussed, and suggested changes to, the nearly final manuscript drafts. All the authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


The closure of the abdomen after median laparotomy is still a matter of debate among surgeons. Further well designed and performed randomised controlled trials determining the optimal method of abdominal fascial closure are needed.This is a three armed, multi-centre, intra-operatively randomised, controlled, patient blinded trial. Over 20 surgical departments will enrol 600 patients who are planned for an elective primary abdominal operation. The objective of this study is to compare the frequency of abdominal incisional hernias between two continuous suture techniques with different, slowly absorbable monofilament materials and an interrupted suture using an absorbable braided suture material at one year postoperatively.This trial will answer the question whether the continuous abdominal wall closure with a slowly absorbable material with longitudinal elasticity is superior to the continuous suture with a material lacking elasticity and to interrupted sutures with braided thread. Median laparotomy is the most common technique of abdominal incisions because it is simple, provides adequate exposure to all four quadrants, is rapid to open and usually bloodsparing . A majorWhereas patient related factors such as age, gender, body mass index (BMI), underlying disease, co-morbidities, prior surgical procedures and life-style factors (e. g. smoking) cannot be controlled or standardised, the decisive chance to lower the incidence of incisional hernias is to optimise the surgical technique. Therefore, a great variety of suture materials and needles has been developed to provide an adequate closure of the fascia and thus the abdominal wall. Thousands of patients have been included in trials in order to answer the question which is the optimal method in abdominal fascia closure and today a number of reviews and a meta-analysis are available. However, the reliability of the existing evidence is compromised by the low number of relevant randomised controlled trials (RCT's) -8.Therefore the discussion regarding the optimal technique of abdominal fascia closure continues and most surgeons practice according to their own experience rather than acting evidence-based. This attitude resulted in an unchanged frequency of incisional hernias over the last decades . None ofWe therefore conclude that there is a lack of data from a truly well-designed long-term trial performed in the daily practice of surgery. This has led us to develop a large randomised controlled trial comparing different surgical techniques of abdominal closure after median laparotomy. INSECT is a multi-centre, intraoperatively randomised controlled trial comparing three different standardised surgical techniques with certain needle/suture combinations on the occurrence of incisional hernia in patients with elective primary midline laparotomy. Two groups will use running sutures with different longitudinal elasticity (one group PDS™ and the other MonoPlus™) combined with an atraumatic needle and one group interrupted sutures (Vicryl™) with a traumatic needle. A three-group parallel equivalence design was selected because due to the results of the latest published meta-analysis  the supeINSECT is the first large-scale trial that started after a detailed theoretical and practical training of the participating surgical centers in March 2004 in order to reduce surgical bias in the study. INSECT will provide internal valid data for an adequate surgical technique of abdominal closure. Although we are well aware that this study cannot answer all open questions, the results should help to further improve evidence based surgery.INSECT has been designed and carried out by the Study Centre of the German Surgical Society (SDGC). The SDGC is an independent research group of the German Surgical Society and the Medical School of the University of Heidelberg that has to design, conduct and analyse large randomised surgical trials in order to improve daily surgical practice. The role of the sponsor (BBD Aesculap) is limited to material supply and local first-level-support. The sponsor is not involved in the database management and has no access to the randomisation code.), database management, quality assurance including monitoring, reporting and for the scientific program of all trial related meetings.The trial is coordinated by the SDGC, which is responsible for overall trial management, trial registration , Patients will be recruited by over 20 surgical centres in Germany. All investigators are hospital-based surgeons with a focus on general surgery. In order to obtain a representative trial result hospitals of all levels of care and education (county/community, private and university centres) are participating in this trial.This committee consists of 3 surgeons and decides on the final diagnostic classification of critical clinical events. For all serious adverse events the documentation and relevant patient data are verified by co-ordinating personnel of each centre before submitting the data to the Adverse Events Committee for diagnostic classification.Adverse Event covers any sign, symptom, syndrome or illness that appears or worsens in a patient during the period of observation in the clinical trial and that may impair the well-being of the patient. The term also covers laboratory findings or results of other diagnostic procedures that are considered to be clinically relevant. A Serious Adverse Event is any adverse event that occurs at any time during the period of observation, that results in death, is immediately life-threatening, requires or prolongs hospitalisation, results in persistent or significant disability or incapacity.Burst abdomen, pulmonary infection and wound infection are secondary endpoints, but are also defined as Adverse Events  guidelines. The data monitoring for this trial will be performed by an independent study nurse who is not involved in the trial or in completion of the case report form (CRF). The surgical monitoring will be done by independent surgeons being not involved in conducting this trial.The final protocol was approved by the ethics committee of the University of Heidelberg, Medical School. Secondary approval is gathered from all local ethics committees responsible for the participating centres. Informed consent will be obtained from each patient in oral and written form before inclusion in the trial.INSECT focuses on hospitalised patients over 18 years of age who are planned for an elective primary abdominal operation and are eligible for a vertical abdominal incision in order to perform the planned surgical procedure. A detailed overview of all eligibility criteria is given in Table The primary objective of this study is to compare the frequency of incisional hernias between three different abdominal fascia closure methods after one year postoperatively: two continuous slowly absorbable monofilament suture materials with and without longitudinal elasticity respectively (MonoPlus™ USP 1, 150 cm loop, with a HRT-48 needle, BBD Aesculap Tuttlingen, Germany and PDS II™ USP 1, 150 cm loop, Ethicon Norderstedt, Germany) and a interrupted suture with an absorbable braided suture material (Vicryl™ USP 2, 6 × 45 cm, non-needled plus a traumatic needle, Ethicon Norderstedt, Germany).Secondary objectives are the frequencies of early and late onset complications such as burst abdomen, postoperative pulmonary complications, wound infections and incisional hernias after three years postoperatively. Additionally a set of surgical and non-surgical parameters related to the operation will be analysed as secondary objectives such as the frequencies of various complications, the lung function and the postoperative length of hospital stay. A qualitative analysis is included in the study to assess the relevance of the primary endpoint from the patient's and the surgeon's perspective. The following aspects are ranked in a descending order from 1 (= most important) to 9 (= least important): postoperative complication, intraoperative complication, length of hospital stay, onset of enteral nutrition, death, postoperative pain, postoperative fatigue, convalescence of the complete physical maximum resilience and cosmetic result . The ranA block-randomisation-list is generated via computer system (SAS Version 8.2, SAS Institute Inc., Cary, USA) and stratified for the individual centre. Each centre will contribute 30 patients (10 to each group). The sealed randomisation list is stored in the investigator file. Patients are randomised using sealed opaque envelopes in the operation theatre after the surgical procedure has been started and before the abdominal wall will be closed.Major challenge in this surgical trial compared to pharmaceutical trials is the standardisation of the surgical technique. All patients undergo a skin incision using electric cautery. Upon completion of the surgical procedure, closure of the abdominal wall is performed in all three groups in a standardised manner: four sharp Mikulicz-Clamps are placed at the corners of the incision and in the middle of the edges of the abdominal fascia and then the closure technique will be performed according to randomisation. Three groups are available: a continuous, all-layer closure technique with either two monofilament loops or an interrupted technique using a braided material . In all three groups suturing is initiated at both ends of the incision towards the middle, whereas the continuous suture line is overlapping at the centre for at least 2 cm to secure each other (for details see figure th – 6th, 2004, AESCULAPIUM, Tuttlingen, Germany) all participating centres have been trained in the required techniques using abdominal wall models of mini-pigs. An evaluation of the investigator meeting was performed by all participants and will be published shortly. Investigators being unable to attend were trained on-site using the same training materials as in the investigator meeting. Training materials were supplemented by videos demonstrating the abdominal wall closure techniques (both continuous and interrupted) in the in vivo situation and in the animal model giving all sub-investigators good insight into the required techniques. Furthermore, a trial manager of the SDGC is available and responsible for all trial related issues and questions.In an investigator meeting before trial initiation . These are arranged for each visit time-point and contain instructions and relevant definitions. All treatments are recorded in treatment logs. Standard adverse events forms are used to document (serious) adverse events and relevant clinical procedures that have been carried out. After verification of the data entered according to "Good Clinical Practice" (GCP), one copy of each completed CRF is sent by mail to the SDGC. A concurrent database is maintained there. All incoming CRF are scanned to be electronically archived. Data are entered in a specially developed relational data base management system. The data entry module contains on-line range and logical checks. For data that are found missing, illegible or inconsistent, data clarification forms are generated which are sent to the on-site monitor for resolution.Certain events must be reported immediately by the investigator by fax on preprinted forms directly to the coordinating centre. Examples are: informed consent and randomisation form, serious adverse events and premature withdrawal form the trial. The reporting of serious adverse events complies with national regulatory requirements.Statistical methods are used to assess the quality of the data, homogeneity of treatment groups, endpoints and safety of the three different techniques.The analysis is performed on the basis of an intention to treat (ITT) population and with respect to ITT principles. A patient belongs to the ITT population after the randomisation. The primary endpoint will also be analysed on the basis of a "per protocol" population.1, R2, and R3. These hypotheses H12: R1 = R2, H13: R1 = R3, and H23: R2 = R3, are all contained in the global null hypothesis H123: R1 = R2 = R3. Sample size estimation, however, will be based on the elementary hypotheses, thus leading to sufficient power in the global test. Bauer had recommended that "sample sizes should be chosen large enough to give a high chance of jumping over the initial hurdle." [To enable multiple comparisons in this three-armed study the closed testing procedure will be used. All thrhurdle." .®, incisional hernias were seen in about 13% of patients. This number is primarily based on the four-armed trial by Wissing et al. [®, MonoPlus® or Maxon®, lower herniation rates were found, but this holds true only for continuous suturing. So far, only one trial has compared fast and slowly absorbable materials for interrupted sutures [The calculation of sample size is based on literature data, as summarised most recently by van 't Riet et al . With ing et al. . With th sutures .® groups over the first postoperative year. Smaller differences are also unlikely to be of clinical relevance.The comparison of interrupted rapidly absorbable versus continuous slowly absorbable sutures has been the aim of four previous studies. The results of these studies showed a non-significant tendency towards a lower rate of incisional hernia. Taking this data and the improved suture properties into consideration, a hernia incidence of 4% could be reasonably expected in one or both of the non-Vicryl). To account for an estimated 10% loss to follow-up and 2% surgical non-compliance with treatment allocation, sample size should be increased to 200 patients per group. In summary, the trial should recruit a total of 600 patients, with equal randomisation into the three groups. Thus a total of 720 patients have to be screened according to the CONSORT statement . We used the PS power and sample size program of Dupont and Plummer (Version 1.0.17) for sample size estimation  use the same alpha level of 0.05, because closed test procedures in three-armed designs do not require alpha level adjustment. If, however, the first global hypothesis cannot be rejected at alpha level, the family of elementary hypotheses will not be tested at all, except for exploratory reasons.In the primary intention-to-treat analysis, testing will start with the global null hypothesis HTo control for possible differences with regard to surgical procedures , centre, and surgical expertise (board-certified surgeon vs. assistant surgeon), logistic regression will be used. We expect that the use of logistic regression will not essentially comprise power assumptions when compared to univariate testing. The inclusion of time-to-event data in the primary statistical analysis (e.g. by applying Kaplan-Meier-curves or Cox-regression) does not confer specific advantages and seems unwarranted, also because an incisional hernia causes similar consequences for the patient irregardless of whether the hernia occurred after 3, 6 or 9 months.The analysis of the secondary endpoints and demographic variables will be descriptive.The description of continuous variables includes at least: numbers of observations, mean, standard deviation, median, minimum and maximum. The description of categorical variables  includes at least the number and percentage of patients belonging to the relevant categories in the trial population as well as in each treatment group.The description of the ranking of parameters of interest (patient's view before, after operation (day of discharge and 12 months later) and surgeon's view) includes the mean and median for each category in the trial population. There will be a listing for all measurements taken for the patients. In order to improve the presentation of the observed data, graphical methods will be applied.The clinical database including all information until 12 months after the operation will be closed six months after the last visit, complete documentation of all cases and resolution of all queries. At this time the primary endpoint of the trial will be ascertained. The information of the final follow-up three years after the operation will be added later. This database will be closed three months after the last telephone three years after the last operation.th and 6th, 2004. In June 2004, following completion of contracts and approval of local ethics committees, the first centres were initiated and the first patient was recruited in July 2004. Currently 23 centres are in an active status recruiting the required 30 patients per centre. Further 6 centres are processed and will be activated within the next months. Assuming an enrolment of 5 patients per month and centre the end of recruitment is assumed to be in October 2005.The initial idea and hypothesis for the study was developed in March 2003. After a systematic review of the literature according to Cochrane standards has been performed the study protocol was completed in October 2003. The study protocol was approved by the local ethics committee of the University of Heidelberg in December 2003. Preparation of all study and instructional materials (including video clips) was completed in February 2004 and the first investigator meeting was held in Tuttlingen, Germany on March 5The strategy to publish study protocols in surgery and thus to enhance transparency in a clinical trial might be relatively new in surgery, but has been practised in other medical fields for several years ,20. ThisINSECT is designed to help answering the question which is the optimal method for abdominal wall closure. Although there are a substantial number of randomised studies and several meta-analyses examining different techniques of abdominal fascia closure the optimal and definite method for closing the abdomen has not yet been found ,7,11. ThSeveral design features of INSECT were discussed before the trial started. A two armed or a three armed study design was debated before writing the protocol. As we wanted to compare the clinically most relevant and most evidence-based suture materials and suture techniques for closure of midline abdominal incisions the most recent and most relevant meta-analysis as a data-base for choosing the different groups in our trial was used . Two difAn important issue for a good trial is to define adequate and well designed end-points. Regarding the optimal method for abdominal closure a great variety of objectives has to be considered: wound infection, wound dehiscence, incisional hernia, suture sinus, wound pain, etc. The socio-economic and surgical most relevant objective is the incidence of incisional hernias . BecauseA major challenge in this INSECT trial compared to other studies, e. g. pharmaceutical trials, is the standardisation of the surgical technique. Lack of standardisation in surgical trials is often an argument of opponents who state that the achieved trial results would not be transferable to surgical routine. Therefore all patients in the INSECT trial undergo a standardised skin incision and opening of the abdominal cavity as well as the closure of the abdominal wall is performed in all three groups in a standardised manner. To provide an overall highly standardised surgical procedure in every study site all participating centres have been trained in the required techniques using abdominal wall models of mini-pigs during an investigator meeting before trial initiation. For investigators who were unable to attend the meeting training materials and videos demonstrating the different abdominal wall closure techniques (both continuous and interrupted) used in the study were provided. For further quality assurance there will be a regular surgical monitoring during the trial. Additionally, randomisation is stratified for each individual centre and the trial results will be therefore comparable among the different participating centres.Over the last years, surgeons' attitude towards randomised surgical trials has changed. It becomes more and more evident that surgical procedures could also be tested in randomised studies. Surgical expertise is largely a personal conviction and an apprenticeship with surgical techniques passed from one surgical generation to the next ,22. MostAWC Abdominal wall closureCRF Case Report FormGCP Good Clinical PracticeICH International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human UseIEC Independent Ethics CommitteeITT Intention-to-treat-analysis® Polydioxanone PDSRCT Randomised Controlled TrialSDGC Study Centre of the German Surgical SocietyThe author(s) declare that they have no competing interests.Hanns-Peter Knaebel and Moritz Koch designed and wrote the manuscript, Stefan Sauerland prepared bio-statistics of the trial and is in charge of the data management, Markus K Diener searched and assessed the literature, Markus W Büchler sponsors the trial and provides necessary infrastructure, Christoph M Seiler searched and assessed the literature and overlooked the completion of the manuscript.The pre-publication history for this paper can be accessed here: